Research ArticlePhytochemical Screening and Antidiabetic Antihyperlipidemicand Antioxidant Properties of Anthyllis henoniana (Coss)Flowers Extracts in an Alloxan-Induced Rats Model of Diabetes

Ameur Ben Younes 1 Maryem Ben Salem2 Hanen El Abed 3 and Raoudha Jarraya1

1Department of Chemistry Laboratory of Organic Chemistry (LR17ES08) Chemistry of Natural Substances TeamFaculty of Sciences BP 1171 3000 Sfax Tunisia2Laboratory of Pharmacology Faculty of Medicine University of Sfax 3000 Sfax Tunisia3Department of Biology Laboratory of Vegetal Biotechnology Applied in Agriculture Improvement Faculty of SciencesUniversity of Sfax 3000 Sfax Tunisia

Correspondence should be addressed to Ameur Ben Younes amerbenyouneslivefr

Received 29 March 2018 Accepted 20 May 2018 Published 24 June 2018

Academic Editor Luigi Milella

Copyright copy 2018 Ameur Ben Younes et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background This study investigates the biological activities of Anthyllis henoniana flowers extracts Materials and MethodsAntioxidant activity and the in vitro inhibitory effect of key digesting enzymes related to postprandial hyperglycemia weredetermined Diabetic rats were orally and daily given the best extract from flowers of Anthyllis henoniana at a dose of acarbosefor one month Results Among the extracts the ethyl acetate one displayed remarkable antioxidant activity including DPPH (IC50= 234mgmL) and was more effective in inhibiting 120572-glucosidase (IC50 = 17 120583gmL) than 120572-amylase (IC50 = 920 120583g mL) activitiesIn vivo the results proved that ethyl acetate extract at doses of 400 mgkg bw decreased significantly the blood glucose leveland lipid profile levels and increased the activities of antioxidant enzymes These protective impacts of Anthyllis henoniana ethylacetate flowers extract were confirmed by histological results Conclusion This study demonstrates for the first time that Anthyllishenoniana flowers ethyl acetate extract is effective in inhibiting hyperglycemia and oxidative stress caused by diabetes

1 Introduction

Nowadays diabetes mellitus (DM) has increased becauseof sedentary lifestyle and nutrition behavior changes inmany countries especially developing ones This diseasecomprising almost 90 of all cases of DM is anticipatedto rise regarding the number of diabetic people which isalso expected to increase badly and reach above 80 milliondiabetes case by 2025 [1] DM is a chronic metabolic irreg-ularity caused by an imprecise balance of glucose home-ostasis [2] that may cause death to humans The diges-tive mechanism combines two steps it starts by using the120572-amylase enzyme that hydrolyzes the polysaccharides tooligosaccharides such as sucrose and then the interventionof the other enzymes (such as 120572-glucosidase maltase andsucrase) catalyzes the final step in carbohydrate hydrolysis to

discharge the absorbable monosaccharides such as glucosePrevious researches have proved that 120572-glucosidase and 120572-amylase inhibitors are apt to DM [3] The degradation ofpolysaccharides is carried on in a fast way and conductsto an ascent of the postprandial hyperglycemia levels Theadjustment of this activity has been demonstrated in the smallintestine by human pancreatic 120572-amylase and it can be usedas an aspect of DM therapy [4] Otherwise preceding studieshave displayed that the oxidative stress is contributed as aresult of persistent hyperglycemia causing a decrease in theenzymes antioxidant activity and the excessive productionof free radicals [5 6] and the substances that are capable ofreducing oxidative stress and postprandial hyperglycemia canbe therapeutic for diabetes [7] Furthermore various phe-nolic compounds were able to inhibit in vitro 120572-glucosidaseactivity [8 9] and to protect the cellular system against

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 8516302 14 pageshttpsdoiorg10115520188516302

2 Evidence-Based Complementary and Alternative Medicine

the oxidation damage [10] thus what leads a survey aboutthe secondary metabolic content The main issues behinddrugs treatment like acarbose are the side effects such asflatulence abdominal distention meteorism and possiblydiarrhea [11] This threat increases the demand for the useof natural substances containing antipostprandial hyper-glycemia activity which certainly provide less or withoutside effects than the use of oral hypoglycemic and insulinPrevious studies supported and showed the link between theinhibitory potential of key carbohydrate-digesting enzymesthe oxidative stress and the secondary metabolites contentsfrom natural sources such as the crude extracts of marineseaweed Turbinaria ornata avocado pears leaves and fruit[12 13] the hydromethanolic extract of leaves ofEmblica offic-inalis Gaertn [14] and also numerous extracts of these plants(Cinnamomum zeylanicum Crataegus oxyacantha Hibiscussabdariffa Morus alba Portulaca oleracea Rubus fruticosusSyzygium aromaticum Teucrium polium Trigonella foenum-graecum and Vaccinium arctostaphylos) [15] With the inten-tion to identify new plant species in this antihyperglycemiacategory through specific inspection the unexplored plantspecies Anthyllis henoniana (Coss) has been investigatedMoreover there are no specific scientific reports nor specificreferences to Anthyllis henoniana constituents influence onthe carbohydrate-digesting enzymes It is a woody Saharanplant located in the big desert of North Africa and belongsto the genus Anthyllis (Fabaceae) It is appropriate for therehabilitation of deteriorating area [16] and one of the poten-tial candidates interesting for protection against wind erosionand improvement of the pastoral value [17] This research isfocused on the valorization of this unstudied Saharan plantand the investigation of its phenolic compounds potentialantioxidant antidiabetic activity and its protective effectsagainst liver and kidney toxicities in diabetic rats

2 Materials and Methods

21 Collection and Extraction of Plant Material Anthyllishenoniana flowers were collected from the Sahara of BeniKhedache Medenine Tunisia approximate to this GPS coor-dination 33∘13101584009610158401015840N 10∘12101584046210158401015840E The botanical identifi-cation was done in the botany laboratory of the Facultyof Sciences University of Sfax Tunisia by Dr ZouhaierNOUMIA voucher specimen (N∘ LCSN 140)was created anddeposited at the Laboratory ofOrganicChemistry Chemistryof Natural Substances herbarium Department of ChemistryFaculty of Sciences of Sfax

100 g of Anthyllis henoniana flowers was dried milledinto a thin powder and got extracted sequentially in theorder with hexane ethyl acetate and methanol All extractswere filtered and evaporated using a rotavapor at 40∘Cto identify extraction yields Thereafter the three collectedextracts hexane extract (HEx) ethyl acetate extract (EAEx)and methanol extract (MEx) were kept at low temperatureuntil analysis

22 Preliminary Phytochemical Screening Phytochemicalscreening was done to identify the presence of secondarymetabolites in according to the Harborne methods [18]

23 In Vitro Study

231 Total Phenol Total Flavonoid andTanninContents Thetotal phenol content of Anthyllis henoniana flowers extractswas resolved using the method of Folin-Ciocalteu presentedby Singleton andRossi [19] 05mL of Folin-Ciocalteu reagentand 5 mL of Na2CO3 (20) were added to 01 mL of eachextract After 30 minutes the absorbance of each mixturewas measured at 727 nm Gallic acid was used as a standardThe phenol content in the extracts was expressed in termsof gallic acid equivalent GAE (mg of GAg of extract) Thethree extracts were analyzed in triplicate The total flavonoidcontent ofAnthyllis henoniana flowers extracts was identifiedby the aluminum chloride method reported by Zhishen et al[20] 03mL ofNaNO2 (5) and 4mL of water were added to1 mL of each extract Themixture allowed standing for 5 minfollowed by the addition of 03 mL of AlCl3 (10 ) then 2 mLofNaOH (1M)Themixturewas adjusted to 10mLwithwaterAbsorbance wasmeasured at 510 nm and Quercetin was usedas a standard The three extracts were analyzed in triplicate

Condensed tannin content was assessed using the vanillinassay [21] and 50 120583L of each extract was added to 15mL of vanillin (4 ) and 750 120583L of HCl After 20 min atroom temperature the absorbance was calculated at 500 nmResults were expressed as milligram Catechin equivalents pergram of extractThe three extracts were analyzed in triplicate

232 Antioxidant Capacity Estimation

(1) DPPH Inhibition Assay This assay was achieved by usingthe method of Ak and Gulcin [22] with slight modification1 mL of each concentration of test substances in ethanol(625-1250-2500ndash5000 120583gmL) was added to 2 mL of the0004 (vv) solution of DPPH in ethanol by dissolving 4mg of DPPH in 100 mL ethanol and the mixture was stirredvigorously After 30minutes of incubating the tubes in dark atroom temperature the absorbance was taken at 517 nm Thedata were compared with the controls which contained 1 mLof 95 ethanol instead of the extract and vitamin E was usedas positive control The inhibition percentage was measuredby

Inhibition = [1 minus (A sampleA control

)] times 100 (1)

(2) Reducing Power Assay (FRAP) The reducing power ofAnthyllis henoniana flowers extracts was identified usingthe Oyaizu et alrsquos method [23] Briefly extracts (1mg) weredissolved in 1 mL of distilled water and added to a mixture of25 mL of potassium ferricyanide [K3Fe(CN6)] (1 wv) and25 mL phosphate buffer (02 M) After 20 min of incubationat 50∘C 25 mL of a (10 wv) trichloroacetic acid was addedto the mixtures followed with centrifugation the samplesat 3000 rpm for 10 min Finally 25 mL of the supernatantsolution was merged with 05 mL of the 01 (wv) solutionof ferric chloride (FeCl3) and 25 mL of distilled water Theabsorbances were analyzed spectrophotometrically at 700nm and vitamin E was the chosen standard Otherwisethe detected increase in absorbance of the three samplesdemonstrated the reducing power elevation

Evidence-Based Complementary and Alternative Medicine 3

233 Total Antioxidant Capacity (TAC) The evaluation oftotal antioxidant capacity was assessed by the Prieto et alrsquosmethod of [24] The absorbance was determined at 695 nmagainst a blank after cooling the reaction at room tempera-ture Total antioxidant capacity was expressed as equivalentsof vitamin E which was used as a standard

234 Determination of 120572-Amylase Activity In Vitro by aCNPG3Method The 120572-amylase inhibition analysis was ana-lyzed according to the method mentioned by Hui et al [25]400 120583L of 01M sodium phosphate buffer and 500 120583L ofstarch solution 1 were added to 50 120583L of sample solutionor standard (acarbose) and 50 120583L of enzyme solution (4UmL) The incubation of this mixture was carried out at37∘C for few minutes and then 3 mL of 35-dinitro salicylicacid was included as a reagent This mixture was heatedto boiling for 10 min before the addition of 20 mL ofdistilled water Absorbance measurement was done at 540nm and the inhibition percentage was calculated utilizing thisformula

Inhibition = [1 minus (A sampleA control

)] times 100 (2)

IC50 values were resolved from the scheme of inhibi-tion percentage against sample concentration and acarbosewas used as a specific standard All tests were made intriplicate

235 The Determination In Vitro of 120572-Glucosidase Activity(1) Isolation According toNishioka et al [26] the crudemiceintestinal extract was prepared with minor modificationsAfter the dissection of 6 mice small intestines were takenand then were washed with a normal saline solution (sodiumchloride 09 ) and then with 01M potassium phosphatebuffer with 5mMEDTAThemucosa was scraped from thesesmall intestines and was mixed with potassium phosphatebuffer before centrifugation at 21000g for 1h The precipitatewas stirred during 30min after the complete dissolving in 01M potassium phosphate buffer and 1 TritonX-100 solutionThe mixture has been centrifuged at 20000 g for 60 min andthe supernatant was dialyzed in 001M potassium phosphatebuffer for 24 h

(2) 120572-Glucosidase Inhibitory Activity Assay According toRanilla et al [27] the enzyme activity was carried onusing PNP-glycoside as a substrate in 01M potassium phos-phate buffer This activity was identified by calculating p-nitrophenol released from PNP-glycoside at 400 nm Allreactions were performed at 37∘C for a period of 30 min withthree replicates

Inhibition = [1 minus (A sampleA control

)] times 100 (3)

IC50 values were resolved from the scheme of inhibi-tion percentage against sample concentration and acarbosewas used as a specific standard All tests were made intriplicate

24 In Vivo Study

241 Experimental Animals The analysis of the currentstudy was carried out with 32 adult healthy male Wistar ratsweighing 180plusmn20 g Rats were received from animal houseof the local Central Pharmacy Tunisia The animals wereretained and conditioned in a breeding chamber with thenatural controlled system (room temperature and a natural12 h-12 h light-dark cycle) A standard diet and a tap waterwere furnished

242 Acute Toxicity Test Theprocedure of acute oral toxicitytest was carried out using the classic procedure of the acutetoxic The animals were branched into six groups of sevenrats each and were given the ethyl acetate flowers extract ofAnthyllis henoniana at the doses of 100 200 500 3000 1000mgkg and 2000 orally in a volume of 10 mLkg and theanimals All rats were maintained under regular observationfor mortality and behavioral changes during 24 h at 72 h [28]

243 Induction of Diabetes Diabetes was treated by anintraperitoneal injection of alloxan (150mgKg bw) dissolvedin normal saline The alloxan injection may produce afatal hypoglycemia as a result of a high reactive release ofpancreatic insulin that is why the rats were also orally given5ndash10 mL of a 20 glucose solution after six hours These ratswere then kept with free access to a 5 glucose solution forone day to avert the intense hypoglycemia The developmentof diabetes has been checked by measuring serum glucoselevel after four days of alloxan injection DM was confirmedby elevated fasting serum glucose over 300 mgdL [29]

244 Experimental Design The experimental animals werebranched into 4 groups (n=8) as follows

Group 1 normal control rats (cont) which were given abalanced diet (Cont)

Group 2 untreated diabetic control rats (Diab)Group 3 diabetic rats treated with the ethyl acetate

flowers extract of Anthyllis henoniana extract at a dose(400 mgkg bwday) in 1 mL by oral gavage for 28 days(Diab+EAEx)

Group 4 diabetic rats provided and treated with thestandard diabetic drug (acarbose 12 mgKg bwday) in 1 mLby oral gavage for 28 days (Diab+Acar)

After 28 days of treatment all rats fromeach experimentalgroup were weighted and after an overnight fast Their trunkblood was collected from the spots of decapitation for theserum preparation The kidney liver and pancreas weredetached and managed to be fat-free Pieces from theseorgans were maintained in a Bouin solution for one day andthen planted in paraffin 5-120583msections were taken to performhematoxylin-eosin staining The slides were photographedoptically with an Olympus CX41 microscope equipped witha camera

245 Time Course of Changes in Blood Glucose LevelsBlood glucose concentrations of 12h fasted rats groups weremeasured at 800 am before starting the feeding programwith glucose oxidase-peroxidase method The fasting blood

4 Evidence-Based Complementary and Alternative Medicine

glucose levels were determined on 1 7 14 21 and 28 daysregularly

246 Measurements of Body Weights All measurements ofthe body weight were estimated in 1 7 14 21 and 28 daysregularly Rats were weekly weighed individually using aprecision balance to measure the weekly body weight gains

247 Oral Glucose Tolerance Test (OGTT) According toKolsi et al [30] this test demonstrates the capacity to replysuitably to a glucose challengeOral glucose tolerance test wasperformed in control and treated rats groups 24 h before theirdecapitation A glucose load (2 gkg bw) was given to eachrat from all consisting groups by gastric gavage path Bloodsampleswere assembled from the tail vein at 0 30 60 120 and240min regularly and their glucose levels were automaticallymeasured using a one-touch glucometer

248 Analysis of Lipid Profile The concentrations of serumlipids level of triglycerides TG total-cholesterol T-Chlow-density lipoprotein-cholesterol LDL-c and high-densitylipoprotein-cholesterolHDL-cweremeasured using the stan-dard commercial kits (Biolabo) on an automatic biochemistryanalyzer (BS-300) at the biochemical laboratory of Hospitalof Hedi Chaker of Sfax Low-density lipoprotein-cholesterolconcentration was identified basing on Friedewald et alformula [31]

249 Biochemical Analysis Theanalysis of serum lipase pan-creatic120572-amylase alanine aminotransferase (ALT) aspartateaminotransferase (AST) creatinine and urea rates weremeasured using commercial kits from Biolabo (France) Themanufacturerrsquos protocols and instructions have been followedto perform the current assays

2410 Antioxidant Activity All different used organs tissues(Kidney pancreas and liver) were measured grinded andhomogenized in 5 mL of phosphate buffer and centrifugedat 3500 rpm for 30 min at 4∘C After the collection ofthe supernatant the amount of thiobarbituric acid reactivesubstances (TBARS) was measured according to Yoshioka etal method [32] and expressed as nmol MDAmg protein andAOPP oxidation of protein levels was identified using Kayaliet al method [33] and expressed as nmolmg protein

The activity of superoxide dismutase (SOD) was exam-ined byMarklund and Marklund method [34] and expressedas Umg protein Reduced glutathione (GSH) activity wasestimated according to the described method by Paglia andValentine [35] and expressed as ug GSHmg protein Thecatalase (CAT) activity was analyzed colorimetrically at 240nm and expressed as mmoles of H2O2 consumedminmgprotein as described by Aebi [36] The determination of totalprotein level was processed through the procedure of Lowryet al [37] using BSA (bovine serum albumin) as the standardat 660 nm

2411 Histopathological Study After removing liver pan-creas and kidney small pieces of these organs were fixed inBouin solution for one day After an aqueous wash the tissues

Table 1 Phytochemical screening of Anthyllis henoniana flowersextracts

Hexaneextract (HEx)

Ethyl acetateextract (EAEx)

Methanolextract (MEx)

Flavonoids minus ++ ++Alkaloids minus minus minusQuinones minus minus minusSterols ++ + minusTropolones minus minus minusTerpenoids ++ + minusTannins minus + +(+)Weak presence (++) presence (minus) absence

were dehydrated using an ascendant gradient of ethanol andthen were cleared utilizing xylene or toluene Finally thetissues were embedded in paraffin before being sliced intosections of 3-5 mm thickness and stained with hematoxylinand eosin

25 Statistical Analysis Results are presented as mean plusmnSDfrom three replicates of each experiment Statistical analysiswas performed using one-way analysis of variance (SPSSsoftware) and the significant differences between means weredetermined by Studentrsquos t-test The values of p lt 005 wereconsidered significantly different Pearson correlation coef-ficient was calculated to establish the relationship betweenantioxidant activity and polyphenols flavonoids and con-densed tannins contents

3 Results

31 Phytochemical Screening Phytochemical screening of allAnthyllis henoniana flowers extracts proved the absence ofalkaloids quinones and tropolones (Table 1) However bothof methanol and ethyl acetate extracts tested positive for theexistence of flavonoids and tannins Sterols and terpenoidsmarked their presence in hexane and ethyl acetate extracts

32 Total Phenol Flavonoid and Tannin Contents Themain secondary metabolites including phenols flavonoidsand tannins contents in the three extracts (ethyl acetatemethanol and hexane) are shown in Figure 1 Phenolscontent was measured using Folin-Ciocalteu reagent in termsof gallic acid equivalent (standard curve equation y = 9221x +02 r2= 0955)The ethyl acetate extract possessed the highestcontent of total phenols recorded of value 52108 plusmn 094mg ofGAEg as compared tomethanol extract of value 4919 plusmn 096mg of GAEg However hexane extract was recorded as 1606plusmn 076 mg of GAEg Total flavonoid amount was explicitlyreferred in terms of Quercetin equivalent (the standard curveequation y = 1257x + 0106 r2 = 0996) the ethyl acetateextract exhibited the maximum of flavonoid content andcorresponded to 6720 plusmn 097 mg of Querceting followed bymethanol and hexane extracts as showed respectively 5231plusmn 185 mg of Querceting and 289 plusmn 068 mg of QuercetingThe condensed tannins contents of the three extracts were

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

Phenols (mg AGEg ofextract)

Flavonoids (mg ofQuerceting of extract)

Tanins (mg of Cateching ofextract)

HExEAExMEx

Figure 1 Total phenols flavonoids and tannins contents of flowersextracts ofAnthyllis henoniana All the values aremeans plusmn SD (n=3)HEx hexane extract EAEx ethyl acetate extract MEx methanolextract

0

20

40

60

80

100

120

0625 125 25 5

HExEAEx

MExVIT E

IP

Concentration (mgml)

Figure 2 Antioxidant activity by DPPHmethod of flowers extractsof Anthyllis henoniana at different concentrations All the values aremeans plusmn SD (n=3) HEx hexane extract EAEx ethyl acetate extractMEx methanol extract

expressed in terms of Catechin equivalent (the standard curveequation y = 7026x ndash 00191 r2 = 0999)Anthyllis henonianaflowers extracts contained a low quantity of the condensedtannins present respectively in the ethyl acetate extract (657plusmn 056 mg of Cateching) methanol extract (629 plusmn 129mg of Cateching) and hexane extract (318 plusmn 055 mg ofCateching)

33 Antioxidant Activity by DPPHAssay As evident from theFigure 2 the radical scavenging activity of the extracts andthe standard increased with the concentration elevation atthe maximum concentration (5000 120583gmL) all the extractsgave lower inhibition percentages than the vitamin E (9832

Table 2 Antioxidant activity ofAnthyllis henoniana flowers extractsusing DPPH method and total antioxidant capacity (TAC)

Extracts DPPH IC50(mgmL)

TACmg of equivalents ofVitamin E g extract

HEx - 613 plusmn 153EAEx 234 5061 plusmn 190MEx 158 5933 plusmn 166Vitamin E 02 -Values are expressed as the means plusmn SD of three independent assays (n=3)

0

005

01

015

02

025

03

035

04

045

05

0 00125 0025 005 01

HExEAEx

MExVIT E

Abso

rban

ce 7

00 n

m

Concentration (mgml)

Figure 3 Reducing power of flowers extracts ofAnthyllis henonianaAll the values are means plusmn SD (n=3) HEx hexane extract EAExethyl acetate extract MEx methanol extract

plusmn 082)Themethanol extract had the highest DPPH radicalinhibition activity (8774plusmn 119) followed by the ethyl acetateextract (758 plusmn 115) and hexane extract (2148 plusmn 063)Otherwise the comparison of IC50 (mgmL) which is thenecessary amount of each sample to decline by 50 theabsorbance of DPPH is already presented in Table 2 in thisdecreasing order vitamin E (IC50 = 02 mgmL) gtmethanolextract (IC50 = 158 mgmL) gt ethyl acetate extract (IC50 =234 mgmL) However the hexane extract with a maximuminhibition percentage at a maximum concentration did notreach 50 of DPPH radical inhibition this result may be dueto the low antioxidant activity of this extract

34 Ferric-ReducingAntioxidant Power Assay (FRAP) FRAPis used to assess the ability of an antioxidant to give anelectron and prevent from peroxidation of linoleic acidwhich is due to the existence of reductones Figure 3 showsthe reducing powers of the three extracts At 1 mgmL con-centration the methanol extract reduced the (Fe3+) to ferrousions (Fe2+) more effectively (absorbance = 0449plusmn0016) thanethyl acetate extract (absorbance = 0427plusmn0017) and hexaneextract (absorbance = 0233plusmn0018) The reducing powerof the extracts and the standard increased with increasingconcentration Among all the three extracts only the hexane

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Correlation coefficients (r2values) of total polyphenolflavonoid and condensed tannins concentration with DPPH activ-ity expressed as IC50 values TAC expressed as mg of antioxidant gof extract

Correlation R2

DPPH versus Polyphenols 0722DPPH versus Flavonoids 0712DPPH versus Tannins 0710TAC versus Polyphenols 0679TAC versus Flavonoids 0420TAC versus Tannins 0685

extract has shown the lowest reducing power Besides theethyl acetate extract andmethanol extract have demonstratedhighermaximum absorbance than vitamin Ewhich is used asa reference

35 Total Antioxidant Capacity TAC TAC was measured bya spectrophotometric method using a phosphomolybdenummethod which was based on the reduction of Mo6+ to Mo5+by the sample analytes and the subsequent formation of greenphosphate Mo5+ compound with a maximum absorption at695 nm Sharp absorbance values proved the possession ofsignificant antioxidant activity TAC was identified using thestandard curve of vitamin E (equation y = 2046x + 0043r2= 0991) and revealed that the methanol extract (5933 mgvitamin Eg extract) is endowed with the highest antioxidantcapacity followed by ethyl acetate extract (5061 mg vitaminEg extract) and hexane extract (613mg vitamin Eg extract)respectively (Table 2)

36 Correlation between Phenolics Contents and AntioxidantActivity To prove the contribution and the role of pheno-lic flavonoid and tannins contents in Anthyllis henonianaflowers extracts on antioxidant activity the Pearson cor-relation coefficient (1199032) was demonstrated in Table 3 Theresults showed the presence of a significant linear correlationbetween antioxidant activities determined by using DPPHand TACmethods and phenols flavonoids and tannins con-tents respectively The strongest correlative value is obtainedwith DDPH and phenols contents (1199032 = 072) These resultspresented a good correlation between phenolic compoundswith antioxidant activities of Anthyllis henoniana flowersextracts

37 In Vitro Antipostprandial Hyperglycemia Studies

371 120572-Amylase and 120572-Glucosidase Inhibitory Potentials Theresults demonstrated that the screening of all three flowersextracts of Anthyllis henoniana had been performed in orderto identify which one(s) have an antiamylase activity byusing pancreatic amylase as a model Antiamylase activitywas expressed as inhibition percentage of three extracts thehexane extract has the lowest result (7) then the methanolextract (184) and the highest result was established forthe ethyl acetate extract by 7245 Thus the focus was

0

20

40

60

80

100

120

0 05 1 15 2 25 3Concentration [mgml]

Resid

ual

-am

ylas

e act

ivity

()

EAExAcarbose

Figure 4 120572-amylase activity () of the ethyl acetate flowers extract(EAEx) ofAnthyllis henoniana at different concentrations comparedwith acarbose All the values are means plusmn SD (n=3)

0

20

40

60

80

100

120

0 20 40 60 80 100 120Resid

ual

-glu

cosid

ase a

ctiv

ity (

)

Concentration [gml]

EAExAcarbose

Figure 5 120572-glucosidase activity () of the ethyl acetate flowerextract (EAEx) of Anthyllis henoniana at different concentrationscompared with acarbose All the values are means plusmn SD (n=3)

directed to the EAEx The inhibition analysis of 120572-amylaseand120572-glucosidase was performed using acarbose as a positivecontrol The120572-glucosidase inhibition assay presented that theenzyme was potently inhibited by ethyl acetate extract withthe IC50 value of 17 120583gmL in resemblance with acarbose (125120583gmL) (plt0001) Interestingly in the 120572-amylase inhibitionassay the inclusion of the Anthyllis henoniana EAEx withdifferent concentrations reduced successfully the residual 120572-amylase activity which was less effective in inhibiting 120572-glucosidase than acarbose drug EAEx showed an IC50 valueof 920 120583gmL in resemblance with acarbose (IC50 of 220120583gmL) (plt0001)The important in vitro inhibitory potentialof ethyl acetate extract compared to the other two extractsguides to its use for further in vivo study (Figures 4 and 5)

38 Acute Toxicity Studies The toxicity studies demonstratedthat ethyl acetate extract from Anthyllis henonianarsquos flowersdoes not mark any sign of toxic reactions or mortality up to a

Evidence-Based Complementary and Alternative Medicine 7

Table 4 Measurement of body weights of different experimental groups of rats

Groups Day1 Day7 Day 14 Day 21 Day 28Cont 17956plusmn 06 18123 plusmn 012 18523plusmn 012 19550 plusmn 123 20412 plusmn 223Diab 18026 plusmn 123 17812 plusmn 236lowastlowastlowast 17356 plusmn 112lowastlowastlowast 17366 plusmn 112lowastlowastlowast 15523plusmn 120lowastlowastlowastDiab+EAEx 18421 plusmn 22 18856 plusmn 074 19645 plusmn 012 20533 plusmn374 21056 plusmn004Diab+Acar 1854 plusmn 312 18923 plusmn 115 19523 plusmn 12 20045 plusmn 123 20316 plusmn 046Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The valuesaremeans plusmnSD (n=8 for each group) Values differ significantly at lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and p lt 0001as considered significant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

Table 5 OGTT levels of different experimental groups of rats

Groups 0min 30min 60 min 120 min 240 minCont 963 plusmn 251 1806 plusmn 321 130 plusmn 5 98 plusmn 26 823 plusmn 251Diab 205 plusmn 1 245 plusmn 15 267 plusmn 125 258 plusmn 76 238 plusmn 74Diab+EAEx 140 plusmn 5 170 plusmn 5 198 plusmn 264 155 plusmn 5 125 plusmn 5Diab+Acar 1063 plusmn 45 124 plusmn 321 125 plusmn 404 123 plusmn76 1113 plusmn 321Control group (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkg bw per day for 28 daysDiab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The values are means plusmnSD (n=8 for each group)

dose of 2000 mgkg bwThe observation of rats behavior wasmade at an interval of every 8 h during 72 h

39 Effect of Anthyllis henoniana Flowers Extracts on BodyWeight and Blood Glucose Levels As shown in Table 4and Figure 6 diabetic rats presented significant decreases(plt0001) in body weights by -315 as compared to controlrats However treated diabetic rats by ethyl acetate extract (atdose 400 mgkg bw) and with acarbose displayed significantincreases in body weights by + 3528 and +3850 respec-tively Moreover we had noticed significant increases (plt0001) in blood glucose in diabetic rats by +5447 comparedwith the control groups Otherwise the administration ofethyl acetate extract at doses of 400 mgkg bw demonstrateda significantly (plt0001) decrease in blood glucose level by -4008 as compared to diabetic rats

310 Effect of Anthyllis henoniana Flowers Extraction OGTTin Diabetic Rats OGTT has been performed in responsivefasted rats after oral administration of ethyl acetate extract toprove its outcome and affirm the potential inhibitory effect ofdigestive enzymes on carbohydrates digestion

The results clearly showed that acute oral administrationof EAEx (400 mgkg bw) presented an important decrease ofglucose concentration peak 60 min after glucose administra-tion as compared to diabetic rats (Table 5)

311 Effect of Anthyllis henoniana Flowers Extract on 120572-Amylase and Lipase Activity in Serum As comparing thediabetic rats with a control group in both Figures 7 and8 the activities of both 120572-amylase and lipase pancreatic inthe serum of diabetic rats presented significant elevationby +5703 and +4071 accordingly However the ethylacetate flowers extract of Anthyllis henoniana administrationto diabetic rats presented a remarkable contraction in serum

0

05

1

15

2

25

3

35

4

Day 1 Day 7 Day 14 Day 21 Day 28

ContDiab

Diab+EAExDiab+Acar

Glu

cose

leve

l (g

L)

Figure 6 Blood glucose levels of different experimental groupsof rats Control group (Cont) and diabetic control (Diab) Diab+EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days The values aremeans plusmnSD (n=8 for each group)

120572-amylase and lipase activities by -2770 and by -3833respectively

312 Effect of Anthyllis henoniana Flowers Extracts in LipidProfile This study showed an important rise in lipid profileparameters (T-CH TG LDL-c and HDL-c) in diabetic ratsas compared to control group (Table 6) The treatment withethyl acetate flowers extract of Anthyllis henoniana (400mgkg bw) revealed significant decreases in T-CHby -1243TG by -7127 and LDL-c by -3939 and an increase inHDL-c by +152 as compared to diabetic rats

8 Evidence-Based Complementary and Alternative Medicine

Table 6 Effect of ethyl acetate flowers extract of Anthyllis henoniana on lipid profile levels of experimental groups of rats

Lipid profile Control Diab Diab+ EAEx Diab+AcarT-Ch (mmolL) 068 plusmn017 172 plusmn128lowastlowastlowast 155 plusmn 058lowastlowastlowast 060plusmn017lowastlowastlowastTG (mmolL) 145plusmn026 188 plusmn046lowastlowastlowast 054 plusmn012lowastlowastlowast 138plusmn056LDL-Ch (mmolL) 050 plusmn019 099 plusmn 008lowastlowastlowast 06 plusmn023 058 plusmn 028lowastHDL-Ch (mmolL) 065 plusmn 017 025 plusmn 008lowastlowastlowast 055 plusmn 007lowastlowast 038 plusmn 084lowastValues represent mean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkgbw per day for 28 days Valuesdiffer significantly atlowastp lt005lowastlowastp lt001 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 andp lt 0001 as consideredsignificant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

0

500

1000

- A

myl

ase a

ctiv

ity (U

IL)

1500

2000

2500

Cont Diab Diab+ EAEx Diab+ Acar

lowastlowastlowast

Figure 7 120572-amylase activity in serum of different experimentalgroups of rats Values represent mean plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastlowastlowastplt 0001 as considered significant comparedto control groups plt005 p lt 0001 as considered significantcompared with diabetic ratsp lt005 as considered significantcompared with diabetic rats treated with acarbose

020406080

100120140160

Cont

Panc

reat

ic li

pase

(UI

L)

Diab Diab+ EAEx Diab+ Acar

lowastlowast

lowast

lowast

Figure 8 Lipase pancreatic level in serum of different experimentalgroups of rats Values represent means plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastp lt005lowastlowastlowastp lt 0001 as considered significantcompared to control groupsplt005

plt001 and p lt 0001

as considered significant compared with diabetic rats plt005plt001 and p lt 0001 as considered significant comparedwith diabetic rats treated with acarbose

313 Effect of Anthyllis henoniana Flowers Extract in HepaticDysfunction The results showed increases in markers ofoxidative stress as TBARS and AOPP by + 6419 and+7611 in diabetic rats as compared to control groups(Table 8)The indices of hepatic dysfunction parameters ALTand AST activities obtained in serum of diabetic rats werealso increased by +3458 +4063 respectively (Table 7)Theresults presented a fascinating decrease in the CAT SODand GSH activities in hepatic tissues of diabetic animalscompared to controls groups (Table 8) Diabetic rats givenethyl acetate flower extract of Anthyllis henoniana at doses of400mgkg bw showed an apparent protective effect in hepaticfunction by decreasing significantly (plt0001) in AST andALT by -2950 and -4768 as compared with diabetic ratsand aimed to adjust the antioxidant effectiveness of hepaticfunction also to fix the transformed values by reaching thenormal levels again

All of these findings were associated with the histologicalsections of the liver which showed a tolerable vacuolation ofhepatocytes in diabetic rats (Figure 9(b)) while the treatmentwith ethyl acetate extract (400 mgkg bw) and acarboseshowed a protective effect (Figures 9(c) and 9(d))

314 Effect of Anthyllis henoniana Flowers Extract in PancreasDysfunction In diabetic rats TBARS and AOPP levels wereelevated by +6491 and +4344 respectively However theantioxidant enzymes activities such as CAT SOD and GSHhad endured important decreases by -1036 -14661 and-23184 as compared with the control group (Table 8)

By oral administration of the ethyl acetate extract (400mgkg bw) and acarbose the antioxidant ability of pancreaswas regulated and the concerned values reached the normallevels The histological study confirmed these results anddisclosed the protective action of the pancreas 120573-cells ofdiabetic rats given by the administration of the ethyl acetateextract (400 mgkg bw) (Figure 10(c))

315 Effect of Anthyllis henoniana Flowers Extract in RenalDysfunction In diabetic rats the SOD CAT and GSHactivities in the kidney decreased by -22845 -1036 and-13611 respectively as compared to control groupMoreoveran important increase in TBARS and AOPP in kidney andalso renal dysfunction parameters observed in creatinine andurea level in plasma by +3434 and +4593 respectivelywere acquired in alloxan-treated rats (Tables 7 and 8) Therenal SOD CAT and GSH antioxidant activities increased

Evidence-Based Complementary and Alternative Medicine 9

Table 7 Plasma hepatic and renal parameters of experimental groups of rats

Hepatic Renal parameters Cont Diab Diab+EAEx Diab+AcarAST (UIL) 1166plusmn 120 17825plusmn563lowastlowastlowast 125plusmn515 15166 plusmn 317ALT (UIL) 3933plusmn578 6625plusmn 118lowastlowastlowast 3466plusmn 568 4775plusmn 600Urea (mmolL) 486 plusmn 857 899 plusmn 226lowastlowastlowast 556plusmn188 700 plusmn 526Creatinine(umolL) 1854 plusmn 324 2824 plusmn 317lowastlowastlowast 1913plusmn45 1521 plusmn 525Values representmean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllishenoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly atlowastlowastlowastp lt 0001 as considered significant compared to control groupsp lt 0001 as considered significant compared with diabetic ratsp lt0001 as considered significant compared with diabetic rats treated with acarbose

Table 8 Effect of the ethyl acetate flowers extract of Anthyllis henoniana on oxidative stress markers of different experimental groups of rats

Groups SOD (Umgprotein)

CAT (umoleH2O2minmg

protein)

GSH (ugmgprotein)

TBARS (nmolMDAmgprotein)

AOPP(nmolmgprotein)

LiverCont 7012plusmn130 170plusmn132 4012plusmn125 1100plusmn025 016plusmn125Diab 6001 plusmn120lowast 080plusmn002 123plusmn002lowastlowastlowast 2850plusmn121lowastlowastlowast 067plusmn105lowastlowastlowastDiab+ EAEx 6455plusmn739 144plusmn224 2707plusmn19 1638plusmn936 052plusmn030Diab+ Acar 5022plusmn 139 15plusmn002 3000plusmn04 1447plusmn613 032plusmn002

ReinCont 3659plusmn570 125plusmn044 1785plusmn397 2012plusmn103 011plusmn156Diab 1824plusmn170lowastlowastlowast 011plusmn082lowastlowastlowast 906plusmn119lowastlowastlowast 2400plusmn023lowastlowastlowast 030plusmn1231lowastlowastlowastDiab+ EAEx 3016plusmn003 040plusmn083 1312 plusmn506 1981plusmn538 015plusmn008Diab+ Acar 2036plusmn236 079plusmn012 1900plusmn017 1831plusmn150 017plusmn025

PancreasCont 9771plusmn158 105plusmn014 717plusmn013 1000plusmn075 024plusmn012Diab 3962plusmn223lowastlowastlowast 010plusmn102lowastlowastlowast 321plusmn002lowastlowastlowast 2581plusmn170lowastlowastlowast 087plusmn014lowastlowastlowastDiab+ EAEx 5363plusmn106 289plusmn112 850plusmn610 1037plusmn319 033plusmn013Diab+Acar 6515plusmn492 081plusmn112 253plusmn100 1271plusmn173 025plusmn023

Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12mgkg bw per day for 28 days Values differsignificantly atlowastp lt005 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and plt001p lt 0001 as considered significantcompared with diabetic rats plt005 plt001 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

after administration of ethyl acetate extract (400 mgkg bw)as well as acarbose and showed also a decrease in TBARSand AOPP amounts (Table 8) and renal dysfunction indiceslevels in plasma Observation of the histological sections ofthe kidneys of all experimental rats showed that the kidneysof the diabetic rats presented a structural alteration of thecortical zone affecting the Bowman capsules These affectedcapsules showed an increase in Bowmanrsquos capsule space(Figure 11(b)) Kidney protection was remarkable due to thetreatment with the ethyl acetate extract (400 mgkg bw) andthe drug acarbose which preserved the structure of the cortexand corrected the nephropathic damage compared to diabeticrats (Figures 11(c) and 11(d))

4 Discussion

DM is one of the most common metabolic diseases it isknown as an increase of the blood glucose level and impairedmetabolism of proteins and lipids Currently diabetes is

considered as one of the most critical issues in the worldMuch research performed more efforts to seek new naturalantioxidant molecules which are considered to be relativelysafer and with less or without side effects Recently theattention is focused on plants which contain high concentra-tions of phytochemical compounds because of their potentialhealth-promoting effects [38] This study has investigatedfor the first time the potential antidiabetic and antioxidanteffects of Anthyllis henoniana flowers extracts in metabolicdisorders in alloxan-induced diabetic rats

As compared to others extracts the ethyl acetate flowersextract of Anthyllis henoniana possessed the maximum ofphenols and flavonoids contents which can be attributed toits good solubility low toxicity medium polarity and highextraction capacity Likewise ethyl acetate tends to extract away better than ethers and hexane and is known to be morenatural and safer than other chemicals because it occurs innature by contributing to the characteristic aroma of severalfruits [39]The extraction of phytochemical constituents may

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

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Evidence-Based Complementary and Alternative Medicine 3

233 Total Antioxidant Capacity (TAC) The evaluation oftotal antioxidant capacity was assessed by the Prieto et alrsquosmethod of [24] The absorbance was determined at 695 nmagainst a blank after cooling the reaction at room tempera-ture Total antioxidant capacity was expressed as equivalentsof vitamin E which was used as a standard

234 Determination of 120572-Amylase Activity In Vitro by aCNPG3Method The 120572-amylase inhibition analysis was ana-lyzed according to the method mentioned by Hui et al [25]400 120583L of 01M sodium phosphate buffer and 500 120583L ofstarch solution 1 were added to 50 120583L of sample solutionor standard (acarbose) and 50 120583L of enzyme solution (4UmL) The incubation of this mixture was carried out at37∘C for few minutes and then 3 mL of 35-dinitro salicylicacid was included as a reagent This mixture was heatedto boiling for 10 min before the addition of 20 mL ofdistilled water Absorbance measurement was done at 540nm and the inhibition percentage was calculated utilizing thisformula

Inhibition = [1 minus (A sampleA control

)] times 100 (2)

IC50 values were resolved from the scheme of inhibi-tion percentage against sample concentration and acarbosewas used as a specific standard All tests were made intriplicate

235 The Determination In Vitro of 120572-Glucosidase Activity(1) Isolation According toNishioka et al [26] the crudemiceintestinal extract was prepared with minor modificationsAfter the dissection of 6 mice small intestines were takenand then were washed with a normal saline solution (sodiumchloride 09 ) and then with 01M potassium phosphatebuffer with 5mMEDTAThemucosa was scraped from thesesmall intestines and was mixed with potassium phosphatebuffer before centrifugation at 21000g for 1h The precipitatewas stirred during 30min after the complete dissolving in 01M potassium phosphate buffer and 1 TritonX-100 solutionThe mixture has been centrifuged at 20000 g for 60 min andthe supernatant was dialyzed in 001M potassium phosphatebuffer for 24 h

(2) 120572-Glucosidase Inhibitory Activity Assay According toRanilla et al [27] the enzyme activity was carried onusing PNP-glycoside as a substrate in 01M potassium phos-phate buffer This activity was identified by calculating p-nitrophenol released from PNP-glycoside at 400 nm Allreactions were performed at 37∘C for a period of 30 min withthree replicates

Inhibition = [1 minus (A sampleA control

)] times 100 (3)

IC50 values were resolved from the scheme of inhibi-tion percentage against sample concentration and acarbosewas used as a specific standard All tests were made intriplicate

24 In Vivo Study

241 Experimental Animals The analysis of the currentstudy was carried out with 32 adult healthy male Wistar ratsweighing 180plusmn20 g Rats were received from animal houseof the local Central Pharmacy Tunisia The animals wereretained and conditioned in a breeding chamber with thenatural controlled system (room temperature and a natural12 h-12 h light-dark cycle) A standard diet and a tap waterwere furnished

242 Acute Toxicity Test Theprocedure of acute oral toxicitytest was carried out using the classic procedure of the acutetoxic The animals were branched into six groups of sevenrats each and were given the ethyl acetate flowers extract ofAnthyllis henoniana at the doses of 100 200 500 3000 1000mgkg and 2000 orally in a volume of 10 mLkg and theanimals All rats were maintained under regular observationfor mortality and behavioral changes during 24 h at 72 h [28]

243 Induction of Diabetes Diabetes was treated by anintraperitoneal injection of alloxan (150mgKg bw) dissolvedin normal saline The alloxan injection may produce afatal hypoglycemia as a result of a high reactive release ofpancreatic insulin that is why the rats were also orally given5ndash10 mL of a 20 glucose solution after six hours These ratswere then kept with free access to a 5 glucose solution forone day to avert the intense hypoglycemia The developmentof diabetes has been checked by measuring serum glucoselevel after four days of alloxan injection DM was confirmedby elevated fasting serum glucose over 300 mgdL [29]

244 Experimental Design The experimental animals werebranched into 4 groups (n=8) as follows

Group 1 normal control rats (cont) which were given abalanced diet (Cont)

Group 2 untreated diabetic control rats (Diab)Group 3 diabetic rats treated with the ethyl acetate

flowers extract of Anthyllis henoniana extract at a dose(400 mgkg bwday) in 1 mL by oral gavage for 28 days(Diab+EAEx)

Group 4 diabetic rats provided and treated with thestandard diabetic drug (acarbose 12 mgKg bwday) in 1 mLby oral gavage for 28 days (Diab+Acar)

After 28 days of treatment all rats fromeach experimentalgroup were weighted and after an overnight fast Their trunkblood was collected from the spots of decapitation for theserum preparation The kidney liver and pancreas weredetached and managed to be fat-free Pieces from theseorgans were maintained in a Bouin solution for one day andthen planted in paraffin 5-120583msections were taken to performhematoxylin-eosin staining The slides were photographedoptically with an Olympus CX41 microscope equipped witha camera

245 Time Course of Changes in Blood Glucose LevelsBlood glucose concentrations of 12h fasted rats groups weremeasured at 800 am before starting the feeding programwith glucose oxidase-peroxidase method The fasting blood

4 Evidence-Based Complementary and Alternative Medicine

glucose levels were determined on 1 7 14 21 and 28 daysregularly

246 Measurements of Body Weights All measurements ofthe body weight were estimated in 1 7 14 21 and 28 daysregularly Rats were weekly weighed individually using aprecision balance to measure the weekly body weight gains

247 Oral Glucose Tolerance Test (OGTT) According toKolsi et al [30] this test demonstrates the capacity to replysuitably to a glucose challengeOral glucose tolerance test wasperformed in control and treated rats groups 24 h before theirdecapitation A glucose load (2 gkg bw) was given to eachrat from all consisting groups by gastric gavage path Bloodsampleswere assembled from the tail vein at 0 30 60 120 and240min regularly and their glucose levels were automaticallymeasured using a one-touch glucometer

248 Analysis of Lipid Profile The concentrations of serumlipids level of triglycerides TG total-cholesterol T-Chlow-density lipoprotein-cholesterol LDL-c and high-densitylipoprotein-cholesterolHDL-cweremeasured using the stan-dard commercial kits (Biolabo) on an automatic biochemistryanalyzer (BS-300) at the biochemical laboratory of Hospitalof Hedi Chaker of Sfax Low-density lipoprotein-cholesterolconcentration was identified basing on Friedewald et alformula [31]

249 Biochemical Analysis Theanalysis of serum lipase pan-creatic120572-amylase alanine aminotransferase (ALT) aspartateaminotransferase (AST) creatinine and urea rates weremeasured using commercial kits from Biolabo (France) Themanufacturerrsquos protocols and instructions have been followedto perform the current assays

2410 Antioxidant Activity All different used organs tissues(Kidney pancreas and liver) were measured grinded andhomogenized in 5 mL of phosphate buffer and centrifugedat 3500 rpm for 30 min at 4∘C After the collection ofthe supernatant the amount of thiobarbituric acid reactivesubstances (TBARS) was measured according to Yoshioka etal method [32] and expressed as nmol MDAmg protein andAOPP oxidation of protein levels was identified using Kayaliet al method [33] and expressed as nmolmg protein

The activity of superoxide dismutase (SOD) was exam-ined byMarklund and Marklund method [34] and expressedas Umg protein Reduced glutathione (GSH) activity wasestimated according to the described method by Paglia andValentine [35] and expressed as ug GSHmg protein Thecatalase (CAT) activity was analyzed colorimetrically at 240nm and expressed as mmoles of H2O2 consumedminmgprotein as described by Aebi [36] The determination of totalprotein level was processed through the procedure of Lowryet al [37] using BSA (bovine serum albumin) as the standardat 660 nm

2411 Histopathological Study After removing liver pan-creas and kidney small pieces of these organs were fixed inBouin solution for one day After an aqueous wash the tissues

Table 1 Phytochemical screening of Anthyllis henoniana flowersextracts

Hexaneextract (HEx)

Ethyl acetateextract (EAEx)

Methanolextract (MEx)

Flavonoids minus ++ ++Alkaloids minus minus minusQuinones minus minus minusSterols ++ + minusTropolones minus minus minusTerpenoids ++ + minusTannins minus + +(+)Weak presence (++) presence (minus) absence

were dehydrated using an ascendant gradient of ethanol andthen were cleared utilizing xylene or toluene Finally thetissues were embedded in paraffin before being sliced intosections of 3-5 mm thickness and stained with hematoxylinand eosin

25 Statistical Analysis Results are presented as mean plusmnSDfrom three replicates of each experiment Statistical analysiswas performed using one-way analysis of variance (SPSSsoftware) and the significant differences between means weredetermined by Studentrsquos t-test The values of p lt 005 wereconsidered significantly different Pearson correlation coef-ficient was calculated to establish the relationship betweenantioxidant activity and polyphenols flavonoids and con-densed tannins contents

3 Results

31 Phytochemical Screening Phytochemical screening of allAnthyllis henoniana flowers extracts proved the absence ofalkaloids quinones and tropolones (Table 1) However bothof methanol and ethyl acetate extracts tested positive for theexistence of flavonoids and tannins Sterols and terpenoidsmarked their presence in hexane and ethyl acetate extracts

32 Total Phenol Flavonoid and Tannin Contents Themain secondary metabolites including phenols flavonoidsand tannins contents in the three extracts (ethyl acetatemethanol and hexane) are shown in Figure 1 Phenolscontent was measured using Folin-Ciocalteu reagent in termsof gallic acid equivalent (standard curve equation y = 9221x +02 r2= 0955)The ethyl acetate extract possessed the highestcontent of total phenols recorded of value 52108 plusmn 094mg ofGAEg as compared tomethanol extract of value 4919 plusmn 096mg of GAEg However hexane extract was recorded as 1606plusmn 076 mg of GAEg Total flavonoid amount was explicitlyreferred in terms of Quercetin equivalent (the standard curveequation y = 1257x + 0106 r2 = 0996) the ethyl acetateextract exhibited the maximum of flavonoid content andcorresponded to 6720 plusmn 097 mg of Querceting followed bymethanol and hexane extracts as showed respectively 5231plusmn 185 mg of Querceting and 289 plusmn 068 mg of QuercetingThe condensed tannins contents of the three extracts were

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

Phenols (mg AGEg ofextract)

Flavonoids (mg ofQuerceting of extract)

Tanins (mg of Cateching ofextract)

HExEAExMEx

Figure 1 Total phenols flavonoids and tannins contents of flowersextracts ofAnthyllis henoniana All the values aremeans plusmn SD (n=3)HEx hexane extract EAEx ethyl acetate extract MEx methanolextract

0

20

40

60

80

100

120

0625 125 25 5

HExEAEx

MExVIT E

IP

Concentration (mgml)

Figure 2 Antioxidant activity by DPPHmethod of flowers extractsof Anthyllis henoniana at different concentrations All the values aremeans plusmn SD (n=3) HEx hexane extract EAEx ethyl acetate extractMEx methanol extract

expressed in terms of Catechin equivalent (the standard curveequation y = 7026x ndash 00191 r2 = 0999)Anthyllis henonianaflowers extracts contained a low quantity of the condensedtannins present respectively in the ethyl acetate extract (657plusmn 056 mg of Cateching) methanol extract (629 plusmn 129mg of Cateching) and hexane extract (318 plusmn 055 mg ofCateching)

33 Antioxidant Activity by DPPHAssay As evident from theFigure 2 the radical scavenging activity of the extracts andthe standard increased with the concentration elevation atthe maximum concentration (5000 120583gmL) all the extractsgave lower inhibition percentages than the vitamin E (9832

Table 2 Antioxidant activity ofAnthyllis henoniana flowers extractsusing DPPH method and total antioxidant capacity (TAC)

Extracts DPPH IC50(mgmL)

TACmg of equivalents ofVitamin E g extract

HEx - 613 plusmn 153EAEx 234 5061 plusmn 190MEx 158 5933 plusmn 166Vitamin E 02 -Values are expressed as the means plusmn SD of three independent assays (n=3)

0

005

01

015

02

025

03

035

04

045

05

0 00125 0025 005 01

HExEAEx

MExVIT E

Abso

rban

ce 7

00 n

m

Concentration (mgml)

Figure 3 Reducing power of flowers extracts ofAnthyllis henonianaAll the values are means plusmn SD (n=3) HEx hexane extract EAExethyl acetate extract MEx methanol extract

plusmn 082)Themethanol extract had the highest DPPH radicalinhibition activity (8774plusmn 119) followed by the ethyl acetateextract (758 plusmn 115) and hexane extract (2148 plusmn 063)Otherwise the comparison of IC50 (mgmL) which is thenecessary amount of each sample to decline by 50 theabsorbance of DPPH is already presented in Table 2 in thisdecreasing order vitamin E (IC50 = 02 mgmL) gtmethanolextract (IC50 = 158 mgmL) gt ethyl acetate extract (IC50 =234 mgmL) However the hexane extract with a maximuminhibition percentage at a maximum concentration did notreach 50 of DPPH radical inhibition this result may be dueto the low antioxidant activity of this extract

34 Ferric-ReducingAntioxidant Power Assay (FRAP) FRAPis used to assess the ability of an antioxidant to give anelectron and prevent from peroxidation of linoleic acidwhich is due to the existence of reductones Figure 3 showsthe reducing powers of the three extracts At 1 mgmL con-centration the methanol extract reduced the (Fe3+) to ferrousions (Fe2+) more effectively (absorbance = 0449plusmn0016) thanethyl acetate extract (absorbance = 0427plusmn0017) and hexaneextract (absorbance = 0233plusmn0018) The reducing powerof the extracts and the standard increased with increasingconcentration Among all the three extracts only the hexane

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Correlation coefficients (r2values) of total polyphenolflavonoid and condensed tannins concentration with DPPH activ-ity expressed as IC50 values TAC expressed as mg of antioxidant gof extract

Correlation R2

DPPH versus Polyphenols 0722DPPH versus Flavonoids 0712DPPH versus Tannins 0710TAC versus Polyphenols 0679TAC versus Flavonoids 0420TAC versus Tannins 0685

extract has shown the lowest reducing power Besides theethyl acetate extract andmethanol extract have demonstratedhighermaximum absorbance than vitamin Ewhich is used asa reference

35 Total Antioxidant Capacity TAC TAC was measured bya spectrophotometric method using a phosphomolybdenummethod which was based on the reduction of Mo6+ to Mo5+by the sample analytes and the subsequent formation of greenphosphate Mo5+ compound with a maximum absorption at695 nm Sharp absorbance values proved the possession ofsignificant antioxidant activity TAC was identified using thestandard curve of vitamin E (equation y = 2046x + 0043r2= 0991) and revealed that the methanol extract (5933 mgvitamin Eg extract) is endowed with the highest antioxidantcapacity followed by ethyl acetate extract (5061 mg vitaminEg extract) and hexane extract (613mg vitamin Eg extract)respectively (Table 2)

36 Correlation between Phenolics Contents and AntioxidantActivity To prove the contribution and the role of pheno-lic flavonoid and tannins contents in Anthyllis henonianaflowers extracts on antioxidant activity the Pearson cor-relation coefficient (1199032) was demonstrated in Table 3 Theresults showed the presence of a significant linear correlationbetween antioxidant activities determined by using DPPHand TACmethods and phenols flavonoids and tannins con-tents respectively The strongest correlative value is obtainedwith DDPH and phenols contents (1199032 = 072) These resultspresented a good correlation between phenolic compoundswith antioxidant activities of Anthyllis henoniana flowersextracts

37 In Vitro Antipostprandial Hyperglycemia Studies

371 120572-Amylase and 120572-Glucosidase Inhibitory Potentials Theresults demonstrated that the screening of all three flowersextracts of Anthyllis henoniana had been performed in orderto identify which one(s) have an antiamylase activity byusing pancreatic amylase as a model Antiamylase activitywas expressed as inhibition percentage of three extracts thehexane extract has the lowest result (7) then the methanolextract (184) and the highest result was established forthe ethyl acetate extract by 7245 Thus the focus was

0

20

40

60

80

100

120

0 05 1 15 2 25 3Concentration [mgml]

Resid

ual

-am

ylas

e act

ivity

()

EAExAcarbose

Figure 4 120572-amylase activity () of the ethyl acetate flowers extract(EAEx) ofAnthyllis henoniana at different concentrations comparedwith acarbose All the values are means plusmn SD (n=3)

0

20

40

60

80

100

120

0 20 40 60 80 100 120Resid

ual

-glu

cosid

ase a

ctiv

ity (

)

Concentration [gml]

EAExAcarbose

Figure 5 120572-glucosidase activity () of the ethyl acetate flowerextract (EAEx) of Anthyllis henoniana at different concentrationscompared with acarbose All the values are means plusmn SD (n=3)

directed to the EAEx The inhibition analysis of 120572-amylaseand120572-glucosidase was performed using acarbose as a positivecontrol The120572-glucosidase inhibition assay presented that theenzyme was potently inhibited by ethyl acetate extract withthe IC50 value of 17 120583gmL in resemblance with acarbose (125120583gmL) (plt0001) Interestingly in the 120572-amylase inhibitionassay the inclusion of the Anthyllis henoniana EAEx withdifferent concentrations reduced successfully the residual 120572-amylase activity which was less effective in inhibiting 120572-glucosidase than acarbose drug EAEx showed an IC50 valueof 920 120583gmL in resemblance with acarbose (IC50 of 220120583gmL) (plt0001)The important in vitro inhibitory potentialof ethyl acetate extract compared to the other two extractsguides to its use for further in vivo study (Figures 4 and 5)

38 Acute Toxicity Studies The toxicity studies demonstratedthat ethyl acetate extract from Anthyllis henonianarsquos flowersdoes not mark any sign of toxic reactions or mortality up to a

Evidence-Based Complementary and Alternative Medicine 7

Table 4 Measurement of body weights of different experimental groups of rats

Groups Day1 Day7 Day 14 Day 21 Day 28Cont 17956plusmn 06 18123 plusmn 012 18523plusmn 012 19550 plusmn 123 20412 plusmn 223Diab 18026 plusmn 123 17812 plusmn 236lowastlowastlowast 17356 plusmn 112lowastlowastlowast 17366 plusmn 112lowastlowastlowast 15523plusmn 120lowastlowastlowastDiab+EAEx 18421 plusmn 22 18856 plusmn 074 19645 plusmn 012 20533 plusmn374 21056 plusmn004Diab+Acar 1854 plusmn 312 18923 plusmn 115 19523 plusmn 12 20045 plusmn 123 20316 plusmn 046Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The valuesaremeans plusmnSD (n=8 for each group) Values differ significantly at lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and p lt 0001as considered significant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

Table 5 OGTT levels of different experimental groups of rats

Groups 0min 30min 60 min 120 min 240 minCont 963 plusmn 251 1806 plusmn 321 130 plusmn 5 98 plusmn 26 823 plusmn 251Diab 205 plusmn 1 245 plusmn 15 267 plusmn 125 258 plusmn 76 238 plusmn 74Diab+EAEx 140 plusmn 5 170 plusmn 5 198 plusmn 264 155 plusmn 5 125 plusmn 5Diab+Acar 1063 plusmn 45 124 plusmn 321 125 plusmn 404 123 plusmn76 1113 plusmn 321Control group (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkg bw per day for 28 daysDiab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The values are means plusmnSD (n=8 for each group)

dose of 2000 mgkg bwThe observation of rats behavior wasmade at an interval of every 8 h during 72 h

39 Effect of Anthyllis henoniana Flowers Extracts on BodyWeight and Blood Glucose Levels As shown in Table 4and Figure 6 diabetic rats presented significant decreases(plt0001) in body weights by -315 as compared to controlrats However treated diabetic rats by ethyl acetate extract (atdose 400 mgkg bw) and with acarbose displayed significantincreases in body weights by + 3528 and +3850 respec-tively Moreover we had noticed significant increases (plt0001) in blood glucose in diabetic rats by +5447 comparedwith the control groups Otherwise the administration ofethyl acetate extract at doses of 400 mgkg bw demonstrateda significantly (plt0001) decrease in blood glucose level by -4008 as compared to diabetic rats

310 Effect of Anthyllis henoniana Flowers Extraction OGTTin Diabetic Rats OGTT has been performed in responsivefasted rats after oral administration of ethyl acetate extract toprove its outcome and affirm the potential inhibitory effect ofdigestive enzymes on carbohydrates digestion

The results clearly showed that acute oral administrationof EAEx (400 mgkg bw) presented an important decrease ofglucose concentration peak 60 min after glucose administra-tion as compared to diabetic rats (Table 5)

311 Effect of Anthyllis henoniana Flowers Extract on 120572-Amylase and Lipase Activity in Serum As comparing thediabetic rats with a control group in both Figures 7 and8 the activities of both 120572-amylase and lipase pancreatic inthe serum of diabetic rats presented significant elevationby +5703 and +4071 accordingly However the ethylacetate flowers extract of Anthyllis henoniana administrationto diabetic rats presented a remarkable contraction in serum

0

05

1

15

2

25

3

35

4

Day 1 Day 7 Day 14 Day 21 Day 28

ContDiab

Diab+EAExDiab+Acar

Glu

cose

leve

l (g

L)

Figure 6 Blood glucose levels of different experimental groupsof rats Control group (Cont) and diabetic control (Diab) Diab+EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days The values aremeans plusmnSD (n=8 for each group)

120572-amylase and lipase activities by -2770 and by -3833respectively

312 Effect of Anthyllis henoniana Flowers Extracts in LipidProfile This study showed an important rise in lipid profileparameters (T-CH TG LDL-c and HDL-c) in diabetic ratsas compared to control group (Table 6) The treatment withethyl acetate flowers extract of Anthyllis henoniana (400mgkg bw) revealed significant decreases in T-CHby -1243TG by -7127 and LDL-c by -3939 and an increase inHDL-c by +152 as compared to diabetic rats

8 Evidence-Based Complementary and Alternative Medicine

Table 6 Effect of ethyl acetate flowers extract of Anthyllis henoniana on lipid profile levels of experimental groups of rats

Lipid profile Control Diab Diab+ EAEx Diab+AcarT-Ch (mmolL) 068 plusmn017 172 plusmn128lowastlowastlowast 155 plusmn 058lowastlowastlowast 060plusmn017lowastlowastlowastTG (mmolL) 145plusmn026 188 plusmn046lowastlowastlowast 054 plusmn012lowastlowastlowast 138plusmn056LDL-Ch (mmolL) 050 plusmn019 099 plusmn 008lowastlowastlowast 06 plusmn023 058 plusmn 028lowastHDL-Ch (mmolL) 065 plusmn 017 025 plusmn 008lowastlowastlowast 055 plusmn 007lowastlowast 038 plusmn 084lowastValues represent mean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkgbw per day for 28 days Valuesdiffer significantly atlowastp lt005lowastlowastp lt001 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 andp lt 0001 as consideredsignificant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

0

500

1000

- A

myl

ase a

ctiv

ity (U

IL)

1500

2000

2500

Cont Diab Diab+ EAEx Diab+ Acar

lowastlowastlowast

Figure 7 120572-amylase activity in serum of different experimentalgroups of rats Values represent mean plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastlowastlowastplt 0001 as considered significant comparedto control groups plt005 p lt 0001 as considered significantcompared with diabetic ratsp lt005 as considered significantcompared with diabetic rats treated with acarbose

020406080

100120140160

Cont

Panc

reat

ic li

pase

(UI

L)

Diab Diab+ EAEx Diab+ Acar

lowastlowast

lowast

lowast

Figure 8 Lipase pancreatic level in serum of different experimentalgroups of rats Values represent means plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastp lt005lowastlowastlowastp lt 0001 as considered significantcompared to control groupsplt005

plt001 and p lt 0001

as considered significant compared with diabetic rats plt005plt001 and p lt 0001 as considered significant comparedwith diabetic rats treated with acarbose

313 Effect of Anthyllis henoniana Flowers Extract in HepaticDysfunction The results showed increases in markers ofoxidative stress as TBARS and AOPP by + 6419 and+7611 in diabetic rats as compared to control groups(Table 8)The indices of hepatic dysfunction parameters ALTand AST activities obtained in serum of diabetic rats werealso increased by +3458 +4063 respectively (Table 7)Theresults presented a fascinating decrease in the CAT SODand GSH activities in hepatic tissues of diabetic animalscompared to controls groups (Table 8) Diabetic rats givenethyl acetate flower extract of Anthyllis henoniana at doses of400mgkg bw showed an apparent protective effect in hepaticfunction by decreasing significantly (plt0001) in AST andALT by -2950 and -4768 as compared with diabetic ratsand aimed to adjust the antioxidant effectiveness of hepaticfunction also to fix the transformed values by reaching thenormal levels again

All of these findings were associated with the histologicalsections of the liver which showed a tolerable vacuolation ofhepatocytes in diabetic rats (Figure 9(b)) while the treatmentwith ethyl acetate extract (400 mgkg bw) and acarboseshowed a protective effect (Figures 9(c) and 9(d))

314 Effect of Anthyllis henoniana Flowers Extract in PancreasDysfunction In diabetic rats TBARS and AOPP levels wereelevated by +6491 and +4344 respectively However theantioxidant enzymes activities such as CAT SOD and GSHhad endured important decreases by -1036 -14661 and-23184 as compared with the control group (Table 8)

By oral administration of the ethyl acetate extract (400mgkg bw) and acarbose the antioxidant ability of pancreaswas regulated and the concerned values reached the normallevels The histological study confirmed these results anddisclosed the protective action of the pancreas 120573-cells ofdiabetic rats given by the administration of the ethyl acetateextract (400 mgkg bw) (Figure 10(c))

315 Effect of Anthyllis henoniana Flowers Extract in RenalDysfunction In diabetic rats the SOD CAT and GSHactivities in the kidney decreased by -22845 -1036 and-13611 respectively as compared to control groupMoreoveran important increase in TBARS and AOPP in kidney andalso renal dysfunction parameters observed in creatinine andurea level in plasma by +3434 and +4593 respectivelywere acquired in alloxan-treated rats (Tables 7 and 8) Therenal SOD CAT and GSH antioxidant activities increased

Evidence-Based Complementary and Alternative Medicine 9

Table 7 Plasma hepatic and renal parameters of experimental groups of rats

Hepatic Renal parameters Cont Diab Diab+EAEx Diab+AcarAST (UIL) 1166plusmn 120 17825plusmn563lowastlowastlowast 125plusmn515 15166 plusmn 317ALT (UIL) 3933plusmn578 6625plusmn 118lowastlowastlowast 3466plusmn 568 4775plusmn 600Urea (mmolL) 486 plusmn 857 899 plusmn 226lowastlowastlowast 556plusmn188 700 plusmn 526Creatinine(umolL) 1854 plusmn 324 2824 plusmn 317lowastlowastlowast 1913plusmn45 1521 plusmn 525Values representmean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllishenoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly atlowastlowastlowastp lt 0001 as considered significant compared to control groupsp lt 0001 as considered significant compared with diabetic ratsp lt0001 as considered significant compared with diabetic rats treated with acarbose

Table 8 Effect of the ethyl acetate flowers extract of Anthyllis henoniana on oxidative stress markers of different experimental groups of rats

Groups SOD (Umgprotein)

CAT (umoleH2O2minmg

protein)

GSH (ugmgprotein)

TBARS (nmolMDAmgprotein)

AOPP(nmolmgprotein)

LiverCont 7012plusmn130 170plusmn132 4012plusmn125 1100plusmn025 016plusmn125Diab 6001 plusmn120lowast 080plusmn002 123plusmn002lowastlowastlowast 2850plusmn121lowastlowastlowast 067plusmn105lowastlowastlowastDiab+ EAEx 6455plusmn739 144plusmn224 2707plusmn19 1638plusmn936 052plusmn030Diab+ Acar 5022plusmn 139 15plusmn002 3000plusmn04 1447plusmn613 032plusmn002

ReinCont 3659plusmn570 125plusmn044 1785plusmn397 2012plusmn103 011plusmn156Diab 1824plusmn170lowastlowastlowast 011plusmn082lowastlowastlowast 906plusmn119lowastlowastlowast 2400plusmn023lowastlowastlowast 030plusmn1231lowastlowastlowastDiab+ EAEx 3016plusmn003 040plusmn083 1312 plusmn506 1981plusmn538 015plusmn008Diab+ Acar 2036plusmn236 079plusmn012 1900plusmn017 1831plusmn150 017plusmn025

PancreasCont 9771plusmn158 105plusmn014 717plusmn013 1000plusmn075 024plusmn012Diab 3962plusmn223lowastlowastlowast 010plusmn102lowastlowastlowast 321plusmn002lowastlowastlowast 2581plusmn170lowastlowastlowast 087plusmn014lowastlowastlowastDiab+ EAEx 5363plusmn106 289plusmn112 850plusmn610 1037plusmn319 033plusmn013Diab+Acar 6515plusmn492 081plusmn112 253plusmn100 1271plusmn173 025plusmn023

Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12mgkg bw per day for 28 days Values differsignificantly atlowastp lt005 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and plt001p lt 0001 as considered significantcompared with diabetic rats plt005 plt001 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

after administration of ethyl acetate extract (400 mgkg bw)as well as acarbose and showed also a decrease in TBARSand AOPP amounts (Table 8) and renal dysfunction indiceslevels in plasma Observation of the histological sections ofthe kidneys of all experimental rats showed that the kidneysof the diabetic rats presented a structural alteration of thecortical zone affecting the Bowman capsules These affectedcapsules showed an increase in Bowmanrsquos capsule space(Figure 11(b)) Kidney protection was remarkable due to thetreatment with the ethyl acetate extract (400 mgkg bw) andthe drug acarbose which preserved the structure of the cortexand corrected the nephropathic damage compared to diabeticrats (Figures 11(c) and 11(d))

4 Discussion

DM is one of the most common metabolic diseases it isknown as an increase of the blood glucose level and impairedmetabolism of proteins and lipids Currently diabetes is

considered as one of the most critical issues in the worldMuch research performed more efforts to seek new naturalantioxidant molecules which are considered to be relativelysafer and with less or without side effects Recently theattention is focused on plants which contain high concentra-tions of phytochemical compounds because of their potentialhealth-promoting effects [38] This study has investigatedfor the first time the potential antidiabetic and antioxidanteffects of Anthyllis henoniana flowers extracts in metabolicdisorders in alloxan-induced diabetic rats

As compared to others extracts the ethyl acetate flowersextract of Anthyllis henoniana possessed the maximum ofphenols and flavonoids contents which can be attributed toits good solubility low toxicity medium polarity and highextraction capacity Likewise ethyl acetate tends to extract away better than ethers and hexane and is known to be morenatural and safer than other chemicals because it occurs innature by contributing to the characteristic aroma of severalfruits [39]The extraction of phytochemical constituents may

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

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4 Evidence-Based Complementary and Alternative Medicine

glucose levels were determined on 1 7 14 21 and 28 daysregularly

246 Measurements of Body Weights All measurements ofthe body weight were estimated in 1 7 14 21 and 28 daysregularly Rats were weekly weighed individually using aprecision balance to measure the weekly body weight gains

247 Oral Glucose Tolerance Test (OGTT) According toKolsi et al [30] this test demonstrates the capacity to replysuitably to a glucose challengeOral glucose tolerance test wasperformed in control and treated rats groups 24 h before theirdecapitation A glucose load (2 gkg bw) was given to eachrat from all consisting groups by gastric gavage path Bloodsampleswere assembled from the tail vein at 0 30 60 120 and240min regularly and their glucose levels were automaticallymeasured using a one-touch glucometer

248 Analysis of Lipid Profile The concentrations of serumlipids level of triglycerides TG total-cholesterol T-Chlow-density lipoprotein-cholesterol LDL-c and high-densitylipoprotein-cholesterolHDL-cweremeasured using the stan-dard commercial kits (Biolabo) on an automatic biochemistryanalyzer (BS-300) at the biochemical laboratory of Hospitalof Hedi Chaker of Sfax Low-density lipoprotein-cholesterolconcentration was identified basing on Friedewald et alformula [31]

249 Biochemical Analysis Theanalysis of serum lipase pan-creatic120572-amylase alanine aminotransferase (ALT) aspartateaminotransferase (AST) creatinine and urea rates weremeasured using commercial kits from Biolabo (France) Themanufacturerrsquos protocols and instructions have been followedto perform the current assays

2410 Antioxidant Activity All different used organs tissues(Kidney pancreas and liver) were measured grinded andhomogenized in 5 mL of phosphate buffer and centrifugedat 3500 rpm for 30 min at 4∘C After the collection ofthe supernatant the amount of thiobarbituric acid reactivesubstances (TBARS) was measured according to Yoshioka etal method [32] and expressed as nmol MDAmg protein andAOPP oxidation of protein levels was identified using Kayaliet al method [33] and expressed as nmolmg protein

The activity of superoxide dismutase (SOD) was exam-ined byMarklund and Marklund method [34] and expressedas Umg protein Reduced glutathione (GSH) activity wasestimated according to the described method by Paglia andValentine [35] and expressed as ug GSHmg protein Thecatalase (CAT) activity was analyzed colorimetrically at 240nm and expressed as mmoles of H2O2 consumedminmgprotein as described by Aebi [36] The determination of totalprotein level was processed through the procedure of Lowryet al [37] using BSA (bovine serum albumin) as the standardat 660 nm

2411 Histopathological Study After removing liver pan-creas and kidney small pieces of these organs were fixed inBouin solution for one day After an aqueous wash the tissues

Table 1 Phytochemical screening of Anthyllis henoniana flowersextracts

Hexaneextract (HEx)

Ethyl acetateextract (EAEx)

Methanolextract (MEx)

Flavonoids minus ++ ++Alkaloids minus minus minusQuinones minus minus minusSterols ++ + minusTropolones minus minus minusTerpenoids ++ + minusTannins minus + +(+)Weak presence (++) presence (minus) absence

were dehydrated using an ascendant gradient of ethanol andthen were cleared utilizing xylene or toluene Finally thetissues were embedded in paraffin before being sliced intosections of 3-5 mm thickness and stained with hematoxylinand eosin

25 Statistical Analysis Results are presented as mean plusmnSDfrom three replicates of each experiment Statistical analysiswas performed using one-way analysis of variance (SPSSsoftware) and the significant differences between means weredetermined by Studentrsquos t-test The values of p lt 005 wereconsidered significantly different Pearson correlation coef-ficient was calculated to establish the relationship betweenantioxidant activity and polyphenols flavonoids and con-densed tannins contents

3 Results

31 Phytochemical Screening Phytochemical screening of allAnthyllis henoniana flowers extracts proved the absence ofalkaloids quinones and tropolones (Table 1) However bothof methanol and ethyl acetate extracts tested positive for theexistence of flavonoids and tannins Sterols and terpenoidsmarked their presence in hexane and ethyl acetate extracts

32 Total Phenol Flavonoid and Tannin Contents Themain secondary metabolites including phenols flavonoidsand tannins contents in the three extracts (ethyl acetatemethanol and hexane) are shown in Figure 1 Phenolscontent was measured using Folin-Ciocalteu reagent in termsof gallic acid equivalent (standard curve equation y = 9221x +02 r2= 0955)The ethyl acetate extract possessed the highestcontent of total phenols recorded of value 52108 plusmn 094mg ofGAEg as compared tomethanol extract of value 4919 plusmn 096mg of GAEg However hexane extract was recorded as 1606plusmn 076 mg of GAEg Total flavonoid amount was explicitlyreferred in terms of Quercetin equivalent (the standard curveequation y = 1257x + 0106 r2 = 0996) the ethyl acetateextract exhibited the maximum of flavonoid content andcorresponded to 6720 plusmn 097 mg of Querceting followed bymethanol and hexane extracts as showed respectively 5231plusmn 185 mg of Querceting and 289 plusmn 068 mg of QuercetingThe condensed tannins contents of the three extracts were

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

Phenols (mg AGEg ofextract)

Flavonoids (mg ofQuerceting of extract)

Tanins (mg of Cateching ofextract)

HExEAExMEx

Figure 1 Total phenols flavonoids and tannins contents of flowersextracts ofAnthyllis henoniana All the values aremeans plusmn SD (n=3)HEx hexane extract EAEx ethyl acetate extract MEx methanolextract

0

20

40

60

80

100

120

0625 125 25 5

HExEAEx

MExVIT E

IP

Concentration (mgml)

Figure 2 Antioxidant activity by DPPHmethod of flowers extractsof Anthyllis henoniana at different concentrations All the values aremeans plusmn SD (n=3) HEx hexane extract EAEx ethyl acetate extractMEx methanol extract

expressed in terms of Catechin equivalent (the standard curveequation y = 7026x ndash 00191 r2 = 0999)Anthyllis henonianaflowers extracts contained a low quantity of the condensedtannins present respectively in the ethyl acetate extract (657plusmn 056 mg of Cateching) methanol extract (629 plusmn 129mg of Cateching) and hexane extract (318 plusmn 055 mg ofCateching)

33 Antioxidant Activity by DPPHAssay As evident from theFigure 2 the radical scavenging activity of the extracts andthe standard increased with the concentration elevation atthe maximum concentration (5000 120583gmL) all the extractsgave lower inhibition percentages than the vitamin E (9832

Table 2 Antioxidant activity ofAnthyllis henoniana flowers extractsusing DPPH method and total antioxidant capacity (TAC)

Extracts DPPH IC50(mgmL)

TACmg of equivalents ofVitamin E g extract

HEx - 613 plusmn 153EAEx 234 5061 plusmn 190MEx 158 5933 plusmn 166Vitamin E 02 -Values are expressed as the means plusmn SD of three independent assays (n=3)

0

005

01

015

02

025

03

035

04

045

05

0 00125 0025 005 01

HExEAEx

MExVIT E

Abso

rban

ce 7

00 n

m

Concentration (mgml)

Figure 3 Reducing power of flowers extracts ofAnthyllis henonianaAll the values are means plusmn SD (n=3) HEx hexane extract EAExethyl acetate extract MEx methanol extract

plusmn 082)Themethanol extract had the highest DPPH radicalinhibition activity (8774plusmn 119) followed by the ethyl acetateextract (758 plusmn 115) and hexane extract (2148 plusmn 063)Otherwise the comparison of IC50 (mgmL) which is thenecessary amount of each sample to decline by 50 theabsorbance of DPPH is already presented in Table 2 in thisdecreasing order vitamin E (IC50 = 02 mgmL) gtmethanolextract (IC50 = 158 mgmL) gt ethyl acetate extract (IC50 =234 mgmL) However the hexane extract with a maximuminhibition percentage at a maximum concentration did notreach 50 of DPPH radical inhibition this result may be dueto the low antioxidant activity of this extract

34 Ferric-ReducingAntioxidant Power Assay (FRAP) FRAPis used to assess the ability of an antioxidant to give anelectron and prevent from peroxidation of linoleic acidwhich is due to the existence of reductones Figure 3 showsthe reducing powers of the three extracts At 1 mgmL con-centration the methanol extract reduced the (Fe3+) to ferrousions (Fe2+) more effectively (absorbance = 0449plusmn0016) thanethyl acetate extract (absorbance = 0427plusmn0017) and hexaneextract (absorbance = 0233plusmn0018) The reducing powerof the extracts and the standard increased with increasingconcentration Among all the three extracts only the hexane

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Correlation coefficients (r2values) of total polyphenolflavonoid and condensed tannins concentration with DPPH activ-ity expressed as IC50 values TAC expressed as mg of antioxidant gof extract

Correlation R2

DPPH versus Polyphenols 0722DPPH versus Flavonoids 0712DPPH versus Tannins 0710TAC versus Polyphenols 0679TAC versus Flavonoids 0420TAC versus Tannins 0685

extract has shown the lowest reducing power Besides theethyl acetate extract andmethanol extract have demonstratedhighermaximum absorbance than vitamin Ewhich is used asa reference

35 Total Antioxidant Capacity TAC TAC was measured bya spectrophotometric method using a phosphomolybdenummethod which was based on the reduction of Mo6+ to Mo5+by the sample analytes and the subsequent formation of greenphosphate Mo5+ compound with a maximum absorption at695 nm Sharp absorbance values proved the possession ofsignificant antioxidant activity TAC was identified using thestandard curve of vitamin E (equation y = 2046x + 0043r2= 0991) and revealed that the methanol extract (5933 mgvitamin Eg extract) is endowed with the highest antioxidantcapacity followed by ethyl acetate extract (5061 mg vitaminEg extract) and hexane extract (613mg vitamin Eg extract)respectively (Table 2)

36 Correlation between Phenolics Contents and AntioxidantActivity To prove the contribution and the role of pheno-lic flavonoid and tannins contents in Anthyllis henonianaflowers extracts on antioxidant activity the Pearson cor-relation coefficient (1199032) was demonstrated in Table 3 Theresults showed the presence of a significant linear correlationbetween antioxidant activities determined by using DPPHand TACmethods and phenols flavonoids and tannins con-tents respectively The strongest correlative value is obtainedwith DDPH and phenols contents (1199032 = 072) These resultspresented a good correlation between phenolic compoundswith antioxidant activities of Anthyllis henoniana flowersextracts

37 In Vitro Antipostprandial Hyperglycemia Studies

371 120572-Amylase and 120572-Glucosidase Inhibitory Potentials Theresults demonstrated that the screening of all three flowersextracts of Anthyllis henoniana had been performed in orderto identify which one(s) have an antiamylase activity byusing pancreatic amylase as a model Antiamylase activitywas expressed as inhibition percentage of three extracts thehexane extract has the lowest result (7) then the methanolextract (184) and the highest result was established forthe ethyl acetate extract by 7245 Thus the focus was

0

20

40

60

80

100

120

0 05 1 15 2 25 3Concentration [mgml]

Resid

ual

-am

ylas

e act

ivity

()

EAExAcarbose

Figure 4 120572-amylase activity () of the ethyl acetate flowers extract(EAEx) ofAnthyllis henoniana at different concentrations comparedwith acarbose All the values are means plusmn SD (n=3)

0

20

40

60

80

100

120

0 20 40 60 80 100 120Resid

ual

-glu

cosid

ase a

ctiv

ity (

)

Concentration [gml]

EAExAcarbose

Figure 5 120572-glucosidase activity () of the ethyl acetate flowerextract (EAEx) of Anthyllis henoniana at different concentrationscompared with acarbose All the values are means plusmn SD (n=3)

directed to the EAEx The inhibition analysis of 120572-amylaseand120572-glucosidase was performed using acarbose as a positivecontrol The120572-glucosidase inhibition assay presented that theenzyme was potently inhibited by ethyl acetate extract withthe IC50 value of 17 120583gmL in resemblance with acarbose (125120583gmL) (plt0001) Interestingly in the 120572-amylase inhibitionassay the inclusion of the Anthyllis henoniana EAEx withdifferent concentrations reduced successfully the residual 120572-amylase activity which was less effective in inhibiting 120572-glucosidase than acarbose drug EAEx showed an IC50 valueof 920 120583gmL in resemblance with acarbose (IC50 of 220120583gmL) (plt0001)The important in vitro inhibitory potentialof ethyl acetate extract compared to the other two extractsguides to its use for further in vivo study (Figures 4 and 5)

38 Acute Toxicity Studies The toxicity studies demonstratedthat ethyl acetate extract from Anthyllis henonianarsquos flowersdoes not mark any sign of toxic reactions or mortality up to a

Evidence-Based Complementary and Alternative Medicine 7

Table 4 Measurement of body weights of different experimental groups of rats

Groups Day1 Day7 Day 14 Day 21 Day 28Cont 17956plusmn 06 18123 plusmn 012 18523plusmn 012 19550 plusmn 123 20412 plusmn 223Diab 18026 plusmn 123 17812 plusmn 236lowastlowastlowast 17356 plusmn 112lowastlowastlowast 17366 plusmn 112lowastlowastlowast 15523plusmn 120lowastlowastlowastDiab+EAEx 18421 plusmn 22 18856 plusmn 074 19645 plusmn 012 20533 plusmn374 21056 plusmn004Diab+Acar 1854 plusmn 312 18923 plusmn 115 19523 plusmn 12 20045 plusmn 123 20316 plusmn 046Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The valuesaremeans plusmnSD (n=8 for each group) Values differ significantly at lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and p lt 0001as considered significant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

Table 5 OGTT levels of different experimental groups of rats

Groups 0min 30min 60 min 120 min 240 minCont 963 plusmn 251 1806 plusmn 321 130 plusmn 5 98 plusmn 26 823 plusmn 251Diab 205 plusmn 1 245 plusmn 15 267 plusmn 125 258 plusmn 76 238 plusmn 74Diab+EAEx 140 plusmn 5 170 plusmn 5 198 plusmn 264 155 plusmn 5 125 plusmn 5Diab+Acar 1063 plusmn 45 124 plusmn 321 125 plusmn 404 123 plusmn76 1113 plusmn 321Control group (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkg bw per day for 28 daysDiab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The values are means plusmnSD (n=8 for each group)

dose of 2000 mgkg bwThe observation of rats behavior wasmade at an interval of every 8 h during 72 h

39 Effect of Anthyllis henoniana Flowers Extracts on BodyWeight and Blood Glucose Levels As shown in Table 4and Figure 6 diabetic rats presented significant decreases(plt0001) in body weights by -315 as compared to controlrats However treated diabetic rats by ethyl acetate extract (atdose 400 mgkg bw) and with acarbose displayed significantincreases in body weights by + 3528 and +3850 respec-tively Moreover we had noticed significant increases (plt0001) in blood glucose in diabetic rats by +5447 comparedwith the control groups Otherwise the administration ofethyl acetate extract at doses of 400 mgkg bw demonstrateda significantly (plt0001) decrease in blood glucose level by -4008 as compared to diabetic rats

310 Effect of Anthyllis henoniana Flowers Extraction OGTTin Diabetic Rats OGTT has been performed in responsivefasted rats after oral administration of ethyl acetate extract toprove its outcome and affirm the potential inhibitory effect ofdigestive enzymes on carbohydrates digestion

The results clearly showed that acute oral administrationof EAEx (400 mgkg bw) presented an important decrease ofglucose concentration peak 60 min after glucose administra-tion as compared to diabetic rats (Table 5)

311 Effect of Anthyllis henoniana Flowers Extract on 120572-Amylase and Lipase Activity in Serum As comparing thediabetic rats with a control group in both Figures 7 and8 the activities of both 120572-amylase and lipase pancreatic inthe serum of diabetic rats presented significant elevationby +5703 and +4071 accordingly However the ethylacetate flowers extract of Anthyllis henoniana administrationto diabetic rats presented a remarkable contraction in serum

0

05

1

15

2

25

3

35

4

Day 1 Day 7 Day 14 Day 21 Day 28

ContDiab

Diab+EAExDiab+Acar

Glu

cose

leve

l (g

L)

Figure 6 Blood glucose levels of different experimental groupsof rats Control group (Cont) and diabetic control (Diab) Diab+EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days The values aremeans plusmnSD (n=8 for each group)

120572-amylase and lipase activities by -2770 and by -3833respectively

312 Effect of Anthyllis henoniana Flowers Extracts in LipidProfile This study showed an important rise in lipid profileparameters (T-CH TG LDL-c and HDL-c) in diabetic ratsas compared to control group (Table 6) The treatment withethyl acetate flowers extract of Anthyllis henoniana (400mgkg bw) revealed significant decreases in T-CHby -1243TG by -7127 and LDL-c by -3939 and an increase inHDL-c by +152 as compared to diabetic rats

8 Evidence-Based Complementary and Alternative Medicine

Table 6 Effect of ethyl acetate flowers extract of Anthyllis henoniana on lipid profile levels of experimental groups of rats

Lipid profile Control Diab Diab+ EAEx Diab+AcarT-Ch (mmolL) 068 plusmn017 172 plusmn128lowastlowastlowast 155 plusmn 058lowastlowastlowast 060plusmn017lowastlowastlowastTG (mmolL) 145plusmn026 188 plusmn046lowastlowastlowast 054 plusmn012lowastlowastlowast 138plusmn056LDL-Ch (mmolL) 050 plusmn019 099 plusmn 008lowastlowastlowast 06 plusmn023 058 plusmn 028lowastHDL-Ch (mmolL) 065 plusmn 017 025 plusmn 008lowastlowastlowast 055 plusmn 007lowastlowast 038 plusmn 084lowastValues represent mean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkgbw per day for 28 days Valuesdiffer significantly atlowastp lt005lowastlowastp lt001 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 andp lt 0001 as consideredsignificant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

0

500

1000

- A

myl

ase a

ctiv

ity (U

IL)

1500

2000

2500

Cont Diab Diab+ EAEx Diab+ Acar

lowastlowastlowast

Figure 7 120572-amylase activity in serum of different experimentalgroups of rats Values represent mean plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastlowastlowastplt 0001 as considered significant comparedto control groups plt005 p lt 0001 as considered significantcompared with diabetic ratsp lt005 as considered significantcompared with diabetic rats treated with acarbose

020406080

100120140160

Cont

Panc

reat

ic li

pase

(UI

L)

Diab Diab+ EAEx Diab+ Acar

lowastlowast

lowast

lowast

Figure 8 Lipase pancreatic level in serum of different experimentalgroups of rats Values represent means plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastp lt005lowastlowastlowastp lt 0001 as considered significantcompared to control groupsplt005

plt001 and p lt 0001

as considered significant compared with diabetic rats plt005plt001 and p lt 0001 as considered significant comparedwith diabetic rats treated with acarbose

313 Effect of Anthyllis henoniana Flowers Extract in HepaticDysfunction The results showed increases in markers ofoxidative stress as TBARS and AOPP by + 6419 and+7611 in diabetic rats as compared to control groups(Table 8)The indices of hepatic dysfunction parameters ALTand AST activities obtained in serum of diabetic rats werealso increased by +3458 +4063 respectively (Table 7)Theresults presented a fascinating decrease in the CAT SODand GSH activities in hepatic tissues of diabetic animalscompared to controls groups (Table 8) Diabetic rats givenethyl acetate flower extract of Anthyllis henoniana at doses of400mgkg bw showed an apparent protective effect in hepaticfunction by decreasing significantly (plt0001) in AST andALT by -2950 and -4768 as compared with diabetic ratsand aimed to adjust the antioxidant effectiveness of hepaticfunction also to fix the transformed values by reaching thenormal levels again

All of these findings were associated with the histologicalsections of the liver which showed a tolerable vacuolation ofhepatocytes in diabetic rats (Figure 9(b)) while the treatmentwith ethyl acetate extract (400 mgkg bw) and acarboseshowed a protective effect (Figures 9(c) and 9(d))

314 Effect of Anthyllis henoniana Flowers Extract in PancreasDysfunction In diabetic rats TBARS and AOPP levels wereelevated by +6491 and +4344 respectively However theantioxidant enzymes activities such as CAT SOD and GSHhad endured important decreases by -1036 -14661 and-23184 as compared with the control group (Table 8)

By oral administration of the ethyl acetate extract (400mgkg bw) and acarbose the antioxidant ability of pancreaswas regulated and the concerned values reached the normallevels The histological study confirmed these results anddisclosed the protective action of the pancreas 120573-cells ofdiabetic rats given by the administration of the ethyl acetateextract (400 mgkg bw) (Figure 10(c))

315 Effect of Anthyllis henoniana Flowers Extract in RenalDysfunction In diabetic rats the SOD CAT and GSHactivities in the kidney decreased by -22845 -1036 and-13611 respectively as compared to control groupMoreoveran important increase in TBARS and AOPP in kidney andalso renal dysfunction parameters observed in creatinine andurea level in plasma by +3434 and +4593 respectivelywere acquired in alloxan-treated rats (Tables 7 and 8) Therenal SOD CAT and GSH antioxidant activities increased

Evidence-Based Complementary and Alternative Medicine 9

Table 7 Plasma hepatic and renal parameters of experimental groups of rats

Hepatic Renal parameters Cont Diab Diab+EAEx Diab+AcarAST (UIL) 1166plusmn 120 17825plusmn563lowastlowastlowast 125plusmn515 15166 plusmn 317ALT (UIL) 3933plusmn578 6625plusmn 118lowastlowastlowast 3466plusmn 568 4775plusmn 600Urea (mmolL) 486 plusmn 857 899 plusmn 226lowastlowastlowast 556plusmn188 700 plusmn 526Creatinine(umolL) 1854 plusmn 324 2824 plusmn 317lowastlowastlowast 1913plusmn45 1521 plusmn 525Values representmean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllishenoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly atlowastlowastlowastp lt 0001 as considered significant compared to control groupsp lt 0001 as considered significant compared with diabetic ratsp lt0001 as considered significant compared with diabetic rats treated with acarbose

Table 8 Effect of the ethyl acetate flowers extract of Anthyllis henoniana on oxidative stress markers of different experimental groups of rats

Groups SOD (Umgprotein)

CAT (umoleH2O2minmg

protein)

GSH (ugmgprotein)

TBARS (nmolMDAmgprotein)

AOPP(nmolmgprotein)

LiverCont 7012plusmn130 170plusmn132 4012plusmn125 1100plusmn025 016plusmn125Diab 6001 plusmn120lowast 080plusmn002 123plusmn002lowastlowastlowast 2850plusmn121lowastlowastlowast 067plusmn105lowastlowastlowastDiab+ EAEx 6455plusmn739 144plusmn224 2707plusmn19 1638plusmn936 052plusmn030Diab+ Acar 5022plusmn 139 15plusmn002 3000plusmn04 1447plusmn613 032plusmn002

ReinCont 3659plusmn570 125plusmn044 1785plusmn397 2012plusmn103 011plusmn156Diab 1824plusmn170lowastlowastlowast 011plusmn082lowastlowastlowast 906plusmn119lowastlowastlowast 2400plusmn023lowastlowastlowast 030plusmn1231lowastlowastlowastDiab+ EAEx 3016plusmn003 040plusmn083 1312 plusmn506 1981plusmn538 015plusmn008Diab+ Acar 2036plusmn236 079plusmn012 1900plusmn017 1831plusmn150 017plusmn025

PancreasCont 9771plusmn158 105plusmn014 717plusmn013 1000plusmn075 024plusmn012Diab 3962plusmn223lowastlowastlowast 010plusmn102lowastlowastlowast 321plusmn002lowastlowastlowast 2581plusmn170lowastlowastlowast 087plusmn014lowastlowastlowastDiab+ EAEx 5363plusmn106 289plusmn112 850plusmn610 1037plusmn319 033plusmn013Diab+Acar 6515plusmn492 081plusmn112 253plusmn100 1271plusmn173 025plusmn023

Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12mgkg bw per day for 28 days Values differsignificantly atlowastp lt005 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and plt001p lt 0001 as considered significantcompared with diabetic rats plt005 plt001 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

after administration of ethyl acetate extract (400 mgkg bw)as well as acarbose and showed also a decrease in TBARSand AOPP amounts (Table 8) and renal dysfunction indiceslevels in plasma Observation of the histological sections ofthe kidneys of all experimental rats showed that the kidneysof the diabetic rats presented a structural alteration of thecortical zone affecting the Bowman capsules These affectedcapsules showed an increase in Bowmanrsquos capsule space(Figure 11(b)) Kidney protection was remarkable due to thetreatment with the ethyl acetate extract (400 mgkg bw) andthe drug acarbose which preserved the structure of the cortexand corrected the nephropathic damage compared to diabeticrats (Figures 11(c) and 11(d))

4 Discussion

DM is one of the most common metabolic diseases it isknown as an increase of the blood glucose level and impairedmetabolism of proteins and lipids Currently diabetes is

considered as one of the most critical issues in the worldMuch research performed more efforts to seek new naturalantioxidant molecules which are considered to be relativelysafer and with less or without side effects Recently theattention is focused on plants which contain high concentra-tions of phytochemical compounds because of their potentialhealth-promoting effects [38] This study has investigatedfor the first time the potential antidiabetic and antioxidanteffects of Anthyllis henoniana flowers extracts in metabolicdisorders in alloxan-induced diabetic rats

As compared to others extracts the ethyl acetate flowersextract of Anthyllis henoniana possessed the maximum ofphenols and flavonoids contents which can be attributed toits good solubility low toxicity medium polarity and highextraction capacity Likewise ethyl acetate tends to extract away better than ethers and hexane and is known to be morenatural and safer than other chemicals because it occurs innature by contributing to the characteristic aroma of severalfruits [39]The extraction of phytochemical constituents may

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

0

100

200

300

400

500

600

Phenols (mg AGEg ofextract)

Flavonoids (mg ofQuerceting of extract)

Tanins (mg of Cateching ofextract)

HExEAExMEx

Figure 1 Total phenols flavonoids and tannins contents of flowersextracts ofAnthyllis henoniana All the values aremeans plusmn SD (n=3)HEx hexane extract EAEx ethyl acetate extract MEx methanolextract

0

20

40

60

80

100

120

0625 125 25 5

HExEAEx

MExVIT E

IP

Concentration (mgml)

Figure 2 Antioxidant activity by DPPHmethod of flowers extractsof Anthyllis henoniana at different concentrations All the values aremeans plusmn SD (n=3) HEx hexane extract EAEx ethyl acetate extractMEx methanol extract

expressed in terms of Catechin equivalent (the standard curveequation y = 7026x ndash 00191 r2 = 0999)Anthyllis henonianaflowers extracts contained a low quantity of the condensedtannins present respectively in the ethyl acetate extract (657plusmn 056 mg of Cateching) methanol extract (629 plusmn 129mg of Cateching) and hexane extract (318 plusmn 055 mg ofCateching)

33 Antioxidant Activity by DPPHAssay As evident from theFigure 2 the radical scavenging activity of the extracts andthe standard increased with the concentration elevation atthe maximum concentration (5000 120583gmL) all the extractsgave lower inhibition percentages than the vitamin E (9832

Table 2 Antioxidant activity ofAnthyllis henoniana flowers extractsusing DPPH method and total antioxidant capacity (TAC)

Extracts DPPH IC50(mgmL)

TACmg of equivalents ofVitamin E g extract

HEx - 613 plusmn 153EAEx 234 5061 plusmn 190MEx 158 5933 plusmn 166Vitamin E 02 -Values are expressed as the means plusmn SD of three independent assays (n=3)

0

005

01

015

02

025

03

035

04

045

05

0 00125 0025 005 01

HExEAEx

MExVIT E

Abso

rban

ce 7

00 n

m

Concentration (mgml)

Figure 3 Reducing power of flowers extracts ofAnthyllis henonianaAll the values are means plusmn SD (n=3) HEx hexane extract EAExethyl acetate extract MEx methanol extract

plusmn 082)Themethanol extract had the highest DPPH radicalinhibition activity (8774plusmn 119) followed by the ethyl acetateextract (758 plusmn 115) and hexane extract (2148 plusmn 063)Otherwise the comparison of IC50 (mgmL) which is thenecessary amount of each sample to decline by 50 theabsorbance of DPPH is already presented in Table 2 in thisdecreasing order vitamin E (IC50 = 02 mgmL) gtmethanolextract (IC50 = 158 mgmL) gt ethyl acetate extract (IC50 =234 mgmL) However the hexane extract with a maximuminhibition percentage at a maximum concentration did notreach 50 of DPPH radical inhibition this result may be dueto the low antioxidant activity of this extract

34 Ferric-ReducingAntioxidant Power Assay (FRAP) FRAPis used to assess the ability of an antioxidant to give anelectron and prevent from peroxidation of linoleic acidwhich is due to the existence of reductones Figure 3 showsthe reducing powers of the three extracts At 1 mgmL con-centration the methanol extract reduced the (Fe3+) to ferrousions (Fe2+) more effectively (absorbance = 0449plusmn0016) thanethyl acetate extract (absorbance = 0427plusmn0017) and hexaneextract (absorbance = 0233plusmn0018) The reducing powerof the extracts and the standard increased with increasingconcentration Among all the three extracts only the hexane

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Correlation coefficients (r2values) of total polyphenolflavonoid and condensed tannins concentration with DPPH activ-ity expressed as IC50 values TAC expressed as mg of antioxidant gof extract

Correlation R2

DPPH versus Polyphenols 0722DPPH versus Flavonoids 0712DPPH versus Tannins 0710TAC versus Polyphenols 0679TAC versus Flavonoids 0420TAC versus Tannins 0685

extract has shown the lowest reducing power Besides theethyl acetate extract andmethanol extract have demonstratedhighermaximum absorbance than vitamin Ewhich is used asa reference

35 Total Antioxidant Capacity TAC TAC was measured bya spectrophotometric method using a phosphomolybdenummethod which was based on the reduction of Mo6+ to Mo5+by the sample analytes and the subsequent formation of greenphosphate Mo5+ compound with a maximum absorption at695 nm Sharp absorbance values proved the possession ofsignificant antioxidant activity TAC was identified using thestandard curve of vitamin E (equation y = 2046x + 0043r2= 0991) and revealed that the methanol extract (5933 mgvitamin Eg extract) is endowed with the highest antioxidantcapacity followed by ethyl acetate extract (5061 mg vitaminEg extract) and hexane extract (613mg vitamin Eg extract)respectively (Table 2)

36 Correlation between Phenolics Contents and AntioxidantActivity To prove the contribution and the role of pheno-lic flavonoid and tannins contents in Anthyllis henonianaflowers extracts on antioxidant activity the Pearson cor-relation coefficient (1199032) was demonstrated in Table 3 Theresults showed the presence of a significant linear correlationbetween antioxidant activities determined by using DPPHand TACmethods and phenols flavonoids and tannins con-tents respectively The strongest correlative value is obtainedwith DDPH and phenols contents (1199032 = 072) These resultspresented a good correlation between phenolic compoundswith antioxidant activities of Anthyllis henoniana flowersextracts

37 In Vitro Antipostprandial Hyperglycemia Studies

371 120572-Amylase and 120572-Glucosidase Inhibitory Potentials Theresults demonstrated that the screening of all three flowersextracts of Anthyllis henoniana had been performed in orderto identify which one(s) have an antiamylase activity byusing pancreatic amylase as a model Antiamylase activitywas expressed as inhibition percentage of three extracts thehexane extract has the lowest result (7) then the methanolextract (184) and the highest result was established forthe ethyl acetate extract by 7245 Thus the focus was

0

20

40

60

80

100

120

0 05 1 15 2 25 3Concentration [mgml]

Resid

ual

-am

ylas

e act

ivity

()

EAExAcarbose

Figure 4 120572-amylase activity () of the ethyl acetate flowers extract(EAEx) ofAnthyllis henoniana at different concentrations comparedwith acarbose All the values are means plusmn SD (n=3)

0

20

40

60

80

100

120

0 20 40 60 80 100 120Resid

ual

-glu

cosid

ase a

ctiv

ity (

)

Concentration [gml]

EAExAcarbose

Figure 5 120572-glucosidase activity () of the ethyl acetate flowerextract (EAEx) of Anthyllis henoniana at different concentrationscompared with acarbose All the values are means plusmn SD (n=3)

directed to the EAEx The inhibition analysis of 120572-amylaseand120572-glucosidase was performed using acarbose as a positivecontrol The120572-glucosidase inhibition assay presented that theenzyme was potently inhibited by ethyl acetate extract withthe IC50 value of 17 120583gmL in resemblance with acarbose (125120583gmL) (plt0001) Interestingly in the 120572-amylase inhibitionassay the inclusion of the Anthyllis henoniana EAEx withdifferent concentrations reduced successfully the residual 120572-amylase activity which was less effective in inhibiting 120572-glucosidase than acarbose drug EAEx showed an IC50 valueof 920 120583gmL in resemblance with acarbose (IC50 of 220120583gmL) (plt0001)The important in vitro inhibitory potentialof ethyl acetate extract compared to the other two extractsguides to its use for further in vivo study (Figures 4 and 5)

38 Acute Toxicity Studies The toxicity studies demonstratedthat ethyl acetate extract from Anthyllis henonianarsquos flowersdoes not mark any sign of toxic reactions or mortality up to a

Evidence-Based Complementary and Alternative Medicine 7

Table 4 Measurement of body weights of different experimental groups of rats

Groups Day1 Day7 Day 14 Day 21 Day 28Cont 17956plusmn 06 18123 plusmn 012 18523plusmn 012 19550 plusmn 123 20412 plusmn 223Diab 18026 plusmn 123 17812 plusmn 236lowastlowastlowast 17356 plusmn 112lowastlowastlowast 17366 plusmn 112lowastlowastlowast 15523plusmn 120lowastlowastlowastDiab+EAEx 18421 plusmn 22 18856 plusmn 074 19645 plusmn 012 20533 plusmn374 21056 plusmn004Diab+Acar 1854 plusmn 312 18923 plusmn 115 19523 plusmn 12 20045 plusmn 123 20316 plusmn 046Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The valuesaremeans plusmnSD (n=8 for each group) Values differ significantly at lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and p lt 0001as considered significant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

Table 5 OGTT levels of different experimental groups of rats

Groups 0min 30min 60 min 120 min 240 minCont 963 plusmn 251 1806 plusmn 321 130 plusmn 5 98 plusmn 26 823 plusmn 251Diab 205 plusmn 1 245 plusmn 15 267 plusmn 125 258 plusmn 76 238 plusmn 74Diab+EAEx 140 plusmn 5 170 plusmn 5 198 plusmn 264 155 plusmn 5 125 plusmn 5Diab+Acar 1063 plusmn 45 124 plusmn 321 125 plusmn 404 123 plusmn76 1113 plusmn 321Control group (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkg bw per day for 28 daysDiab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The values are means plusmnSD (n=8 for each group)

dose of 2000 mgkg bwThe observation of rats behavior wasmade at an interval of every 8 h during 72 h

39 Effect of Anthyllis henoniana Flowers Extracts on BodyWeight and Blood Glucose Levels As shown in Table 4and Figure 6 diabetic rats presented significant decreases(plt0001) in body weights by -315 as compared to controlrats However treated diabetic rats by ethyl acetate extract (atdose 400 mgkg bw) and with acarbose displayed significantincreases in body weights by + 3528 and +3850 respec-tively Moreover we had noticed significant increases (plt0001) in blood glucose in diabetic rats by +5447 comparedwith the control groups Otherwise the administration ofethyl acetate extract at doses of 400 mgkg bw demonstrateda significantly (plt0001) decrease in blood glucose level by -4008 as compared to diabetic rats

310 Effect of Anthyllis henoniana Flowers Extraction OGTTin Diabetic Rats OGTT has been performed in responsivefasted rats after oral administration of ethyl acetate extract toprove its outcome and affirm the potential inhibitory effect ofdigestive enzymes on carbohydrates digestion

The results clearly showed that acute oral administrationof EAEx (400 mgkg bw) presented an important decrease ofglucose concentration peak 60 min after glucose administra-tion as compared to diabetic rats (Table 5)

311 Effect of Anthyllis henoniana Flowers Extract on 120572-Amylase and Lipase Activity in Serum As comparing thediabetic rats with a control group in both Figures 7 and8 the activities of both 120572-amylase and lipase pancreatic inthe serum of diabetic rats presented significant elevationby +5703 and +4071 accordingly However the ethylacetate flowers extract of Anthyllis henoniana administrationto diabetic rats presented a remarkable contraction in serum

0

05

1

15

2

25

3

35

4

Day 1 Day 7 Day 14 Day 21 Day 28

ContDiab

Diab+EAExDiab+Acar

Glu

cose

leve

l (g

L)

Figure 6 Blood glucose levels of different experimental groupsof rats Control group (Cont) and diabetic control (Diab) Diab+EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days The values aremeans plusmnSD (n=8 for each group)

120572-amylase and lipase activities by -2770 and by -3833respectively

312 Effect of Anthyllis henoniana Flowers Extracts in LipidProfile This study showed an important rise in lipid profileparameters (T-CH TG LDL-c and HDL-c) in diabetic ratsas compared to control group (Table 6) The treatment withethyl acetate flowers extract of Anthyllis henoniana (400mgkg bw) revealed significant decreases in T-CHby -1243TG by -7127 and LDL-c by -3939 and an increase inHDL-c by +152 as compared to diabetic rats

8 Evidence-Based Complementary and Alternative Medicine

Table 6 Effect of ethyl acetate flowers extract of Anthyllis henoniana on lipid profile levels of experimental groups of rats

Lipid profile Control Diab Diab+ EAEx Diab+AcarT-Ch (mmolL) 068 plusmn017 172 plusmn128lowastlowastlowast 155 plusmn 058lowastlowastlowast 060plusmn017lowastlowastlowastTG (mmolL) 145plusmn026 188 plusmn046lowastlowastlowast 054 plusmn012lowastlowastlowast 138plusmn056LDL-Ch (mmolL) 050 plusmn019 099 plusmn 008lowastlowastlowast 06 plusmn023 058 plusmn 028lowastHDL-Ch (mmolL) 065 plusmn 017 025 plusmn 008lowastlowastlowast 055 plusmn 007lowastlowast 038 plusmn 084lowastValues represent mean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkgbw per day for 28 days Valuesdiffer significantly atlowastp lt005lowastlowastp lt001 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 andp lt 0001 as consideredsignificant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

0

500

1000

- A

myl

ase a

ctiv

ity (U

IL)

1500

2000

2500

Cont Diab Diab+ EAEx Diab+ Acar

lowastlowastlowast

Figure 7 120572-amylase activity in serum of different experimentalgroups of rats Values represent mean plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastlowastlowastplt 0001 as considered significant comparedto control groups plt005 p lt 0001 as considered significantcompared with diabetic ratsp lt005 as considered significantcompared with diabetic rats treated with acarbose

020406080

100120140160

Cont

Panc

reat

ic li

pase

(UI

L)

Diab Diab+ EAEx Diab+ Acar

lowastlowast

lowast

lowast

Figure 8 Lipase pancreatic level in serum of different experimentalgroups of rats Values represent means plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastp lt005lowastlowastlowastp lt 0001 as considered significantcompared to control groupsplt005

plt001 and p lt 0001

as considered significant compared with diabetic rats plt005plt001 and p lt 0001 as considered significant comparedwith diabetic rats treated with acarbose

313 Effect of Anthyllis henoniana Flowers Extract in HepaticDysfunction The results showed increases in markers ofoxidative stress as TBARS and AOPP by + 6419 and+7611 in diabetic rats as compared to control groups(Table 8)The indices of hepatic dysfunction parameters ALTand AST activities obtained in serum of diabetic rats werealso increased by +3458 +4063 respectively (Table 7)Theresults presented a fascinating decrease in the CAT SODand GSH activities in hepatic tissues of diabetic animalscompared to controls groups (Table 8) Diabetic rats givenethyl acetate flower extract of Anthyllis henoniana at doses of400mgkg bw showed an apparent protective effect in hepaticfunction by decreasing significantly (plt0001) in AST andALT by -2950 and -4768 as compared with diabetic ratsand aimed to adjust the antioxidant effectiveness of hepaticfunction also to fix the transformed values by reaching thenormal levels again

All of these findings were associated with the histologicalsections of the liver which showed a tolerable vacuolation ofhepatocytes in diabetic rats (Figure 9(b)) while the treatmentwith ethyl acetate extract (400 mgkg bw) and acarboseshowed a protective effect (Figures 9(c) and 9(d))

314 Effect of Anthyllis henoniana Flowers Extract in PancreasDysfunction In diabetic rats TBARS and AOPP levels wereelevated by +6491 and +4344 respectively However theantioxidant enzymes activities such as CAT SOD and GSHhad endured important decreases by -1036 -14661 and-23184 as compared with the control group (Table 8)

By oral administration of the ethyl acetate extract (400mgkg bw) and acarbose the antioxidant ability of pancreaswas regulated and the concerned values reached the normallevels The histological study confirmed these results anddisclosed the protective action of the pancreas 120573-cells ofdiabetic rats given by the administration of the ethyl acetateextract (400 mgkg bw) (Figure 10(c))

315 Effect of Anthyllis henoniana Flowers Extract in RenalDysfunction In diabetic rats the SOD CAT and GSHactivities in the kidney decreased by -22845 -1036 and-13611 respectively as compared to control groupMoreoveran important increase in TBARS and AOPP in kidney andalso renal dysfunction parameters observed in creatinine andurea level in plasma by +3434 and +4593 respectivelywere acquired in alloxan-treated rats (Tables 7 and 8) Therenal SOD CAT and GSH antioxidant activities increased

Evidence-Based Complementary and Alternative Medicine 9

Table 7 Plasma hepatic and renal parameters of experimental groups of rats

Hepatic Renal parameters Cont Diab Diab+EAEx Diab+AcarAST (UIL) 1166plusmn 120 17825plusmn563lowastlowastlowast 125plusmn515 15166 plusmn 317ALT (UIL) 3933plusmn578 6625plusmn 118lowastlowastlowast 3466plusmn 568 4775plusmn 600Urea (mmolL) 486 plusmn 857 899 plusmn 226lowastlowastlowast 556plusmn188 700 plusmn 526Creatinine(umolL) 1854 plusmn 324 2824 plusmn 317lowastlowastlowast 1913plusmn45 1521 plusmn 525Values representmean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllishenoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly atlowastlowastlowastp lt 0001 as considered significant compared to control groupsp lt 0001 as considered significant compared with diabetic ratsp lt0001 as considered significant compared with diabetic rats treated with acarbose

Table 8 Effect of the ethyl acetate flowers extract of Anthyllis henoniana on oxidative stress markers of different experimental groups of rats

Groups SOD (Umgprotein)

CAT (umoleH2O2minmg

protein)

GSH (ugmgprotein)

TBARS (nmolMDAmgprotein)

AOPP(nmolmgprotein)

LiverCont 7012plusmn130 170plusmn132 4012plusmn125 1100plusmn025 016plusmn125Diab 6001 plusmn120lowast 080plusmn002 123plusmn002lowastlowastlowast 2850plusmn121lowastlowastlowast 067plusmn105lowastlowastlowastDiab+ EAEx 6455plusmn739 144plusmn224 2707plusmn19 1638plusmn936 052plusmn030Diab+ Acar 5022plusmn 139 15plusmn002 3000plusmn04 1447plusmn613 032plusmn002

ReinCont 3659plusmn570 125plusmn044 1785plusmn397 2012plusmn103 011plusmn156Diab 1824plusmn170lowastlowastlowast 011plusmn082lowastlowastlowast 906plusmn119lowastlowastlowast 2400plusmn023lowastlowastlowast 030plusmn1231lowastlowastlowastDiab+ EAEx 3016plusmn003 040plusmn083 1312 plusmn506 1981plusmn538 015plusmn008Diab+ Acar 2036plusmn236 079plusmn012 1900plusmn017 1831plusmn150 017plusmn025

PancreasCont 9771plusmn158 105plusmn014 717plusmn013 1000plusmn075 024plusmn012Diab 3962plusmn223lowastlowastlowast 010plusmn102lowastlowastlowast 321plusmn002lowastlowastlowast 2581plusmn170lowastlowastlowast 087plusmn014lowastlowastlowastDiab+ EAEx 5363plusmn106 289plusmn112 850plusmn610 1037plusmn319 033plusmn013Diab+Acar 6515plusmn492 081plusmn112 253plusmn100 1271plusmn173 025plusmn023

Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12mgkg bw per day for 28 days Values differsignificantly atlowastp lt005 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and plt001p lt 0001 as considered significantcompared with diabetic rats plt005 plt001 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

after administration of ethyl acetate extract (400 mgkg bw)as well as acarbose and showed also a decrease in TBARSand AOPP amounts (Table 8) and renal dysfunction indiceslevels in plasma Observation of the histological sections ofthe kidneys of all experimental rats showed that the kidneysof the diabetic rats presented a structural alteration of thecortical zone affecting the Bowman capsules These affectedcapsules showed an increase in Bowmanrsquos capsule space(Figure 11(b)) Kidney protection was remarkable due to thetreatment with the ethyl acetate extract (400 mgkg bw) andthe drug acarbose which preserved the structure of the cortexand corrected the nephropathic damage compared to diabeticrats (Figures 11(c) and 11(d))

4 Discussion

DM is one of the most common metabolic diseases it isknown as an increase of the blood glucose level and impairedmetabolism of proteins and lipids Currently diabetes is

considered as one of the most critical issues in the worldMuch research performed more efforts to seek new naturalantioxidant molecules which are considered to be relativelysafer and with less or without side effects Recently theattention is focused on plants which contain high concentra-tions of phytochemical compounds because of their potentialhealth-promoting effects [38] This study has investigatedfor the first time the potential antidiabetic and antioxidanteffects of Anthyllis henoniana flowers extracts in metabolicdisorders in alloxan-induced diabetic rats

As compared to others extracts the ethyl acetate flowersextract of Anthyllis henoniana possessed the maximum ofphenols and flavonoids contents which can be attributed toits good solubility low toxicity medium polarity and highextraction capacity Likewise ethyl acetate tends to extract away better than ethers and hexane and is known to be morenatural and safer than other chemicals because it occurs innature by contributing to the characteristic aroma of severalfruits [39]The extraction of phytochemical constituents may

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

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Evidence-Based Complementary andAlternative Medicine

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Submit your manuscripts atwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Correlation coefficients (r2values) of total polyphenolflavonoid and condensed tannins concentration with DPPH activ-ity expressed as IC50 values TAC expressed as mg of antioxidant gof extract

Correlation R2

DPPH versus Polyphenols 0722DPPH versus Flavonoids 0712DPPH versus Tannins 0710TAC versus Polyphenols 0679TAC versus Flavonoids 0420TAC versus Tannins 0685

extract has shown the lowest reducing power Besides theethyl acetate extract andmethanol extract have demonstratedhighermaximum absorbance than vitamin Ewhich is used asa reference

35 Total Antioxidant Capacity TAC TAC was measured bya spectrophotometric method using a phosphomolybdenummethod which was based on the reduction of Mo6+ to Mo5+by the sample analytes and the subsequent formation of greenphosphate Mo5+ compound with a maximum absorption at695 nm Sharp absorbance values proved the possession ofsignificant antioxidant activity TAC was identified using thestandard curve of vitamin E (equation y = 2046x + 0043r2= 0991) and revealed that the methanol extract (5933 mgvitamin Eg extract) is endowed with the highest antioxidantcapacity followed by ethyl acetate extract (5061 mg vitaminEg extract) and hexane extract (613mg vitamin Eg extract)respectively (Table 2)

36 Correlation between Phenolics Contents and AntioxidantActivity To prove the contribution and the role of pheno-lic flavonoid and tannins contents in Anthyllis henonianaflowers extracts on antioxidant activity the Pearson cor-relation coefficient (1199032) was demonstrated in Table 3 Theresults showed the presence of a significant linear correlationbetween antioxidant activities determined by using DPPHand TACmethods and phenols flavonoids and tannins con-tents respectively The strongest correlative value is obtainedwith DDPH and phenols contents (1199032 = 072) These resultspresented a good correlation between phenolic compoundswith antioxidant activities of Anthyllis henoniana flowersextracts

37 In Vitro Antipostprandial Hyperglycemia Studies

371 120572-Amylase and 120572-Glucosidase Inhibitory Potentials Theresults demonstrated that the screening of all three flowersextracts of Anthyllis henoniana had been performed in orderto identify which one(s) have an antiamylase activity byusing pancreatic amylase as a model Antiamylase activitywas expressed as inhibition percentage of three extracts thehexane extract has the lowest result (7) then the methanolextract (184) and the highest result was established forthe ethyl acetate extract by 7245 Thus the focus was

0

20

40

60

80

100

120

0 05 1 15 2 25 3Concentration [mgml]

Resid

ual

-am

ylas

e act

ivity

()

EAExAcarbose

Figure 4 120572-amylase activity () of the ethyl acetate flowers extract(EAEx) ofAnthyllis henoniana at different concentrations comparedwith acarbose All the values are means plusmn SD (n=3)

0

20

40

60

80

100

120

0 20 40 60 80 100 120Resid

ual

-glu

cosid

ase a

ctiv

ity (

)

Concentration [gml]

EAExAcarbose

Figure 5 120572-glucosidase activity () of the ethyl acetate flowerextract (EAEx) of Anthyllis henoniana at different concentrationscompared with acarbose All the values are means plusmn SD (n=3)

directed to the EAEx The inhibition analysis of 120572-amylaseand120572-glucosidase was performed using acarbose as a positivecontrol The120572-glucosidase inhibition assay presented that theenzyme was potently inhibited by ethyl acetate extract withthe IC50 value of 17 120583gmL in resemblance with acarbose (125120583gmL) (plt0001) Interestingly in the 120572-amylase inhibitionassay the inclusion of the Anthyllis henoniana EAEx withdifferent concentrations reduced successfully the residual 120572-amylase activity which was less effective in inhibiting 120572-glucosidase than acarbose drug EAEx showed an IC50 valueof 920 120583gmL in resemblance with acarbose (IC50 of 220120583gmL) (plt0001)The important in vitro inhibitory potentialof ethyl acetate extract compared to the other two extractsguides to its use for further in vivo study (Figures 4 and 5)

38 Acute Toxicity Studies The toxicity studies demonstratedthat ethyl acetate extract from Anthyllis henonianarsquos flowersdoes not mark any sign of toxic reactions or mortality up to a

Evidence-Based Complementary and Alternative Medicine 7

Table 4 Measurement of body weights of different experimental groups of rats

Groups Day1 Day7 Day 14 Day 21 Day 28Cont 17956plusmn 06 18123 plusmn 012 18523plusmn 012 19550 plusmn 123 20412 plusmn 223Diab 18026 plusmn 123 17812 plusmn 236lowastlowastlowast 17356 plusmn 112lowastlowastlowast 17366 plusmn 112lowastlowastlowast 15523plusmn 120lowastlowastlowastDiab+EAEx 18421 plusmn 22 18856 plusmn 074 19645 plusmn 012 20533 plusmn374 21056 plusmn004Diab+Acar 1854 plusmn 312 18923 plusmn 115 19523 plusmn 12 20045 plusmn 123 20316 plusmn 046Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The valuesaremeans plusmnSD (n=8 for each group) Values differ significantly at lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and p lt 0001as considered significant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

Table 5 OGTT levels of different experimental groups of rats

Groups 0min 30min 60 min 120 min 240 minCont 963 plusmn 251 1806 plusmn 321 130 plusmn 5 98 plusmn 26 823 plusmn 251Diab 205 plusmn 1 245 plusmn 15 267 plusmn 125 258 plusmn 76 238 plusmn 74Diab+EAEx 140 plusmn 5 170 plusmn 5 198 plusmn 264 155 plusmn 5 125 plusmn 5Diab+Acar 1063 plusmn 45 124 plusmn 321 125 plusmn 404 123 plusmn76 1113 plusmn 321Control group (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkg bw per day for 28 daysDiab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The values are means plusmnSD (n=8 for each group)

dose of 2000 mgkg bwThe observation of rats behavior wasmade at an interval of every 8 h during 72 h

39 Effect of Anthyllis henoniana Flowers Extracts on BodyWeight and Blood Glucose Levels As shown in Table 4and Figure 6 diabetic rats presented significant decreases(plt0001) in body weights by -315 as compared to controlrats However treated diabetic rats by ethyl acetate extract (atdose 400 mgkg bw) and with acarbose displayed significantincreases in body weights by + 3528 and +3850 respec-tively Moreover we had noticed significant increases (plt0001) in blood glucose in diabetic rats by +5447 comparedwith the control groups Otherwise the administration ofethyl acetate extract at doses of 400 mgkg bw demonstrateda significantly (plt0001) decrease in blood glucose level by -4008 as compared to diabetic rats

310 Effect of Anthyllis henoniana Flowers Extraction OGTTin Diabetic Rats OGTT has been performed in responsivefasted rats after oral administration of ethyl acetate extract toprove its outcome and affirm the potential inhibitory effect ofdigestive enzymes on carbohydrates digestion

The results clearly showed that acute oral administrationof EAEx (400 mgkg bw) presented an important decrease ofglucose concentration peak 60 min after glucose administra-tion as compared to diabetic rats (Table 5)

311 Effect of Anthyllis henoniana Flowers Extract on 120572-Amylase and Lipase Activity in Serum As comparing thediabetic rats with a control group in both Figures 7 and8 the activities of both 120572-amylase and lipase pancreatic inthe serum of diabetic rats presented significant elevationby +5703 and +4071 accordingly However the ethylacetate flowers extract of Anthyllis henoniana administrationto diabetic rats presented a remarkable contraction in serum

0

05

1

15

2

25

3

35

4

Day 1 Day 7 Day 14 Day 21 Day 28

ContDiab

Diab+EAExDiab+Acar

Glu

cose

leve

l (g

L)

Figure 6 Blood glucose levels of different experimental groupsof rats Control group (Cont) and diabetic control (Diab) Diab+EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days The values aremeans plusmnSD (n=8 for each group)

120572-amylase and lipase activities by -2770 and by -3833respectively

312 Effect of Anthyllis henoniana Flowers Extracts in LipidProfile This study showed an important rise in lipid profileparameters (T-CH TG LDL-c and HDL-c) in diabetic ratsas compared to control group (Table 6) The treatment withethyl acetate flowers extract of Anthyllis henoniana (400mgkg bw) revealed significant decreases in T-CHby -1243TG by -7127 and LDL-c by -3939 and an increase inHDL-c by +152 as compared to diabetic rats

8 Evidence-Based Complementary and Alternative Medicine

Table 6 Effect of ethyl acetate flowers extract of Anthyllis henoniana on lipid profile levels of experimental groups of rats

Lipid profile Control Diab Diab+ EAEx Diab+AcarT-Ch (mmolL) 068 plusmn017 172 plusmn128lowastlowastlowast 155 plusmn 058lowastlowastlowast 060plusmn017lowastlowastlowastTG (mmolL) 145plusmn026 188 plusmn046lowastlowastlowast 054 plusmn012lowastlowastlowast 138plusmn056LDL-Ch (mmolL) 050 plusmn019 099 plusmn 008lowastlowastlowast 06 plusmn023 058 plusmn 028lowastHDL-Ch (mmolL) 065 plusmn 017 025 plusmn 008lowastlowastlowast 055 plusmn 007lowastlowast 038 plusmn 084lowastValues represent mean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkgbw per day for 28 days Valuesdiffer significantly atlowastp lt005lowastlowastp lt001 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 andp lt 0001 as consideredsignificant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

0

500

1000

- A

myl

ase a

ctiv

ity (U

IL)

1500

2000

2500

Cont Diab Diab+ EAEx Diab+ Acar

lowastlowastlowast

Figure 7 120572-amylase activity in serum of different experimentalgroups of rats Values represent mean plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastlowastlowastplt 0001 as considered significant comparedto control groups plt005 p lt 0001 as considered significantcompared with diabetic ratsp lt005 as considered significantcompared with diabetic rats treated with acarbose

020406080

100120140160

Cont

Panc

reat

ic li

pase

(UI

L)

Diab Diab+ EAEx Diab+ Acar

lowastlowast

lowast

lowast

Figure 8 Lipase pancreatic level in serum of different experimentalgroups of rats Values represent means plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastp lt005lowastlowastlowastp lt 0001 as considered significantcompared to control groupsplt005

plt001 and p lt 0001

as considered significant compared with diabetic rats plt005plt001 and p lt 0001 as considered significant comparedwith diabetic rats treated with acarbose

313 Effect of Anthyllis henoniana Flowers Extract in HepaticDysfunction The results showed increases in markers ofoxidative stress as TBARS and AOPP by + 6419 and+7611 in diabetic rats as compared to control groups(Table 8)The indices of hepatic dysfunction parameters ALTand AST activities obtained in serum of diabetic rats werealso increased by +3458 +4063 respectively (Table 7)Theresults presented a fascinating decrease in the CAT SODand GSH activities in hepatic tissues of diabetic animalscompared to controls groups (Table 8) Diabetic rats givenethyl acetate flower extract of Anthyllis henoniana at doses of400mgkg bw showed an apparent protective effect in hepaticfunction by decreasing significantly (plt0001) in AST andALT by -2950 and -4768 as compared with diabetic ratsand aimed to adjust the antioxidant effectiveness of hepaticfunction also to fix the transformed values by reaching thenormal levels again

All of these findings were associated with the histologicalsections of the liver which showed a tolerable vacuolation ofhepatocytes in diabetic rats (Figure 9(b)) while the treatmentwith ethyl acetate extract (400 mgkg bw) and acarboseshowed a protective effect (Figures 9(c) and 9(d))

314 Effect of Anthyllis henoniana Flowers Extract in PancreasDysfunction In diabetic rats TBARS and AOPP levels wereelevated by +6491 and +4344 respectively However theantioxidant enzymes activities such as CAT SOD and GSHhad endured important decreases by -1036 -14661 and-23184 as compared with the control group (Table 8)

By oral administration of the ethyl acetate extract (400mgkg bw) and acarbose the antioxidant ability of pancreaswas regulated and the concerned values reached the normallevels The histological study confirmed these results anddisclosed the protective action of the pancreas 120573-cells ofdiabetic rats given by the administration of the ethyl acetateextract (400 mgkg bw) (Figure 10(c))

315 Effect of Anthyllis henoniana Flowers Extract in RenalDysfunction In diabetic rats the SOD CAT and GSHactivities in the kidney decreased by -22845 -1036 and-13611 respectively as compared to control groupMoreoveran important increase in TBARS and AOPP in kidney andalso renal dysfunction parameters observed in creatinine andurea level in plasma by +3434 and +4593 respectivelywere acquired in alloxan-treated rats (Tables 7 and 8) Therenal SOD CAT and GSH antioxidant activities increased

Evidence-Based Complementary and Alternative Medicine 9

Table 7 Plasma hepatic and renal parameters of experimental groups of rats

Hepatic Renal parameters Cont Diab Diab+EAEx Diab+AcarAST (UIL) 1166plusmn 120 17825plusmn563lowastlowastlowast 125plusmn515 15166 plusmn 317ALT (UIL) 3933plusmn578 6625plusmn 118lowastlowastlowast 3466plusmn 568 4775plusmn 600Urea (mmolL) 486 plusmn 857 899 plusmn 226lowastlowastlowast 556plusmn188 700 plusmn 526Creatinine(umolL) 1854 plusmn 324 2824 plusmn 317lowastlowastlowast 1913plusmn45 1521 plusmn 525Values representmean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllishenoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly atlowastlowastlowastp lt 0001 as considered significant compared to control groupsp lt 0001 as considered significant compared with diabetic ratsp lt0001 as considered significant compared with diabetic rats treated with acarbose

Table 8 Effect of the ethyl acetate flowers extract of Anthyllis henoniana on oxidative stress markers of different experimental groups of rats

Groups SOD (Umgprotein)

CAT (umoleH2O2minmg

protein)

GSH (ugmgprotein)

TBARS (nmolMDAmgprotein)

AOPP(nmolmgprotein)

LiverCont 7012plusmn130 170plusmn132 4012plusmn125 1100plusmn025 016plusmn125Diab 6001 plusmn120lowast 080plusmn002 123plusmn002lowastlowastlowast 2850plusmn121lowastlowastlowast 067plusmn105lowastlowastlowastDiab+ EAEx 6455plusmn739 144plusmn224 2707plusmn19 1638plusmn936 052plusmn030Diab+ Acar 5022plusmn 139 15plusmn002 3000plusmn04 1447plusmn613 032plusmn002

ReinCont 3659plusmn570 125plusmn044 1785plusmn397 2012plusmn103 011plusmn156Diab 1824plusmn170lowastlowastlowast 011plusmn082lowastlowastlowast 906plusmn119lowastlowastlowast 2400plusmn023lowastlowastlowast 030plusmn1231lowastlowastlowastDiab+ EAEx 3016plusmn003 040plusmn083 1312 plusmn506 1981plusmn538 015plusmn008Diab+ Acar 2036plusmn236 079plusmn012 1900plusmn017 1831plusmn150 017plusmn025

PancreasCont 9771plusmn158 105plusmn014 717plusmn013 1000plusmn075 024plusmn012Diab 3962plusmn223lowastlowastlowast 010plusmn102lowastlowastlowast 321plusmn002lowastlowastlowast 2581plusmn170lowastlowastlowast 087plusmn014lowastlowastlowastDiab+ EAEx 5363plusmn106 289plusmn112 850plusmn610 1037plusmn319 033plusmn013Diab+Acar 6515plusmn492 081plusmn112 253plusmn100 1271plusmn173 025plusmn023

Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12mgkg bw per day for 28 days Values differsignificantly atlowastp lt005 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and plt001p lt 0001 as considered significantcompared with diabetic rats plt005 plt001 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

after administration of ethyl acetate extract (400 mgkg bw)as well as acarbose and showed also a decrease in TBARSand AOPP amounts (Table 8) and renal dysfunction indiceslevels in plasma Observation of the histological sections ofthe kidneys of all experimental rats showed that the kidneysof the diabetic rats presented a structural alteration of thecortical zone affecting the Bowman capsules These affectedcapsules showed an increase in Bowmanrsquos capsule space(Figure 11(b)) Kidney protection was remarkable due to thetreatment with the ethyl acetate extract (400 mgkg bw) andthe drug acarbose which preserved the structure of the cortexand corrected the nephropathic damage compared to diabeticrats (Figures 11(c) and 11(d))

4 Discussion

DM is one of the most common metabolic diseases it isknown as an increase of the blood glucose level and impairedmetabolism of proteins and lipids Currently diabetes is

considered as one of the most critical issues in the worldMuch research performed more efforts to seek new naturalantioxidant molecules which are considered to be relativelysafer and with less or without side effects Recently theattention is focused on plants which contain high concentra-tions of phytochemical compounds because of their potentialhealth-promoting effects [38] This study has investigatedfor the first time the potential antidiabetic and antioxidanteffects of Anthyllis henoniana flowers extracts in metabolicdisorders in alloxan-induced diabetic rats

As compared to others extracts the ethyl acetate flowersextract of Anthyllis henoniana possessed the maximum ofphenols and flavonoids contents which can be attributed toits good solubility low toxicity medium polarity and highextraction capacity Likewise ethyl acetate tends to extract away better than ethers and hexane and is known to be morenatural and safer than other chemicals because it occurs innature by contributing to the characteristic aroma of severalfruits [39]The extraction of phytochemical constituents may

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

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Disease Markers

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Immunology ResearchHindawiwwwhindawicom Volume 2018

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Table 4 Measurement of body weights of different experimental groups of rats

Groups Day1 Day7 Day 14 Day 21 Day 28Cont 17956plusmn 06 18123 plusmn 012 18523plusmn 012 19550 plusmn 123 20412 plusmn 223Diab 18026 plusmn 123 17812 plusmn 236lowastlowastlowast 17356 plusmn 112lowastlowastlowast 17366 plusmn 112lowastlowastlowast 15523plusmn 120lowastlowastlowastDiab+EAEx 18421 plusmn 22 18856 plusmn 074 19645 plusmn 012 20533 plusmn374 21056 plusmn004Diab+Acar 1854 plusmn 312 18923 plusmn 115 19523 plusmn 12 20045 plusmn 123 20316 plusmn 046Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The valuesaremeans plusmnSD (n=8 for each group) Values differ significantly at lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and p lt 0001as considered significant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

Table 5 OGTT levels of different experimental groups of rats

Groups 0min 30min 60 min 120 min 240 minCont 963 plusmn 251 1806 plusmn 321 130 plusmn 5 98 plusmn 26 823 plusmn 251Diab 205 plusmn 1 245 plusmn 15 267 plusmn 125 258 plusmn 76 238 plusmn 74Diab+EAEx 140 plusmn 5 170 plusmn 5 198 plusmn 264 155 plusmn 5 125 plusmn 5Diab+Acar 1063 plusmn 45 124 plusmn 321 125 plusmn 404 123 plusmn76 1113 plusmn 321Control group (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkg bw per day for 28 daysDiab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days The values are means plusmnSD (n=8 for each group)

dose of 2000 mgkg bwThe observation of rats behavior wasmade at an interval of every 8 h during 72 h

39 Effect of Anthyllis henoniana Flowers Extracts on BodyWeight and Blood Glucose Levels As shown in Table 4and Figure 6 diabetic rats presented significant decreases(plt0001) in body weights by -315 as compared to controlrats However treated diabetic rats by ethyl acetate extract (atdose 400 mgkg bw) and with acarbose displayed significantincreases in body weights by + 3528 and +3850 respec-tively Moreover we had noticed significant increases (plt0001) in blood glucose in diabetic rats by +5447 comparedwith the control groups Otherwise the administration ofethyl acetate extract at doses of 400 mgkg bw demonstrateda significantly (plt0001) decrease in blood glucose level by -4008 as compared to diabetic rats

310 Effect of Anthyllis henoniana Flowers Extraction OGTTin Diabetic Rats OGTT has been performed in responsivefasted rats after oral administration of ethyl acetate extract toprove its outcome and affirm the potential inhibitory effect ofdigestive enzymes on carbohydrates digestion

The results clearly showed that acute oral administrationof EAEx (400 mgkg bw) presented an important decrease ofglucose concentration peak 60 min after glucose administra-tion as compared to diabetic rats (Table 5)

311 Effect of Anthyllis henoniana Flowers Extract on 120572-Amylase and Lipase Activity in Serum As comparing thediabetic rats with a control group in both Figures 7 and8 the activities of both 120572-amylase and lipase pancreatic inthe serum of diabetic rats presented significant elevationby +5703 and +4071 accordingly However the ethylacetate flowers extract of Anthyllis henoniana administrationto diabetic rats presented a remarkable contraction in serum

0

05

1

15

2

25

3

35

4

Day 1 Day 7 Day 14 Day 21 Day 28

ContDiab

Diab+EAExDiab+Acar

Glu

cose

leve

l (g

L)

Figure 6 Blood glucose levels of different experimental groupsof rats Control group (Cont) and diabetic control (Diab) Diab+EAEx diabetic rats treated with EAEx of Anthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days The values aremeans plusmnSD (n=8 for each group)

120572-amylase and lipase activities by -2770 and by -3833respectively

312 Effect of Anthyllis henoniana Flowers Extracts in LipidProfile This study showed an important rise in lipid profileparameters (T-CH TG LDL-c and HDL-c) in diabetic ratsas compared to control group (Table 6) The treatment withethyl acetate flowers extract of Anthyllis henoniana (400mgkg bw) revealed significant decreases in T-CHby -1243TG by -7127 and LDL-c by -3939 and an increase inHDL-c by +152 as compared to diabetic rats

8 Evidence-Based Complementary and Alternative Medicine

Table 6 Effect of ethyl acetate flowers extract of Anthyllis henoniana on lipid profile levels of experimental groups of rats

Lipid profile Control Diab Diab+ EAEx Diab+AcarT-Ch (mmolL) 068 plusmn017 172 plusmn128lowastlowastlowast 155 plusmn 058lowastlowastlowast 060plusmn017lowastlowastlowastTG (mmolL) 145plusmn026 188 plusmn046lowastlowastlowast 054 plusmn012lowastlowastlowast 138plusmn056LDL-Ch (mmolL) 050 plusmn019 099 plusmn 008lowastlowastlowast 06 plusmn023 058 plusmn 028lowastHDL-Ch (mmolL) 065 plusmn 017 025 plusmn 008lowastlowastlowast 055 plusmn 007lowastlowast 038 plusmn 084lowastValues represent mean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkgbw per day for 28 days Valuesdiffer significantly atlowastp lt005lowastlowastp lt001 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 andp lt 0001 as consideredsignificant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

0

500

1000

- A

myl

ase a

ctiv

ity (U

IL)

1500

2000

2500

Cont Diab Diab+ EAEx Diab+ Acar

lowastlowastlowast

Figure 7 120572-amylase activity in serum of different experimentalgroups of rats Values represent mean plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastlowastlowastplt 0001 as considered significant comparedto control groups plt005 p lt 0001 as considered significantcompared with diabetic ratsp lt005 as considered significantcompared with diabetic rats treated with acarbose

020406080

100120140160

Cont

Panc

reat

ic li

pase

(UI

L)

Diab Diab+ EAEx Diab+ Acar

lowastlowast

lowast

lowast

Figure 8 Lipase pancreatic level in serum of different experimentalgroups of rats Values represent means plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastp lt005lowastlowastlowastp lt 0001 as considered significantcompared to control groupsplt005

plt001 and p lt 0001

as considered significant compared with diabetic rats plt005plt001 and p lt 0001 as considered significant comparedwith diabetic rats treated with acarbose

313 Effect of Anthyllis henoniana Flowers Extract in HepaticDysfunction The results showed increases in markers ofoxidative stress as TBARS and AOPP by + 6419 and+7611 in diabetic rats as compared to control groups(Table 8)The indices of hepatic dysfunction parameters ALTand AST activities obtained in serum of diabetic rats werealso increased by +3458 +4063 respectively (Table 7)Theresults presented a fascinating decrease in the CAT SODand GSH activities in hepatic tissues of diabetic animalscompared to controls groups (Table 8) Diabetic rats givenethyl acetate flower extract of Anthyllis henoniana at doses of400mgkg bw showed an apparent protective effect in hepaticfunction by decreasing significantly (plt0001) in AST andALT by -2950 and -4768 as compared with diabetic ratsand aimed to adjust the antioxidant effectiveness of hepaticfunction also to fix the transformed values by reaching thenormal levels again

All of these findings were associated with the histologicalsections of the liver which showed a tolerable vacuolation ofhepatocytes in diabetic rats (Figure 9(b)) while the treatmentwith ethyl acetate extract (400 mgkg bw) and acarboseshowed a protective effect (Figures 9(c) and 9(d))

314 Effect of Anthyllis henoniana Flowers Extract in PancreasDysfunction In diabetic rats TBARS and AOPP levels wereelevated by +6491 and +4344 respectively However theantioxidant enzymes activities such as CAT SOD and GSHhad endured important decreases by -1036 -14661 and-23184 as compared with the control group (Table 8)

By oral administration of the ethyl acetate extract (400mgkg bw) and acarbose the antioxidant ability of pancreaswas regulated and the concerned values reached the normallevels The histological study confirmed these results anddisclosed the protective action of the pancreas 120573-cells ofdiabetic rats given by the administration of the ethyl acetateextract (400 mgkg bw) (Figure 10(c))

315 Effect of Anthyllis henoniana Flowers Extract in RenalDysfunction In diabetic rats the SOD CAT and GSHactivities in the kidney decreased by -22845 -1036 and-13611 respectively as compared to control groupMoreoveran important increase in TBARS and AOPP in kidney andalso renal dysfunction parameters observed in creatinine andurea level in plasma by +3434 and +4593 respectivelywere acquired in alloxan-treated rats (Tables 7 and 8) Therenal SOD CAT and GSH antioxidant activities increased

Evidence-Based Complementary and Alternative Medicine 9

Table 7 Plasma hepatic and renal parameters of experimental groups of rats

Hepatic Renal parameters Cont Diab Diab+EAEx Diab+AcarAST (UIL) 1166plusmn 120 17825plusmn563lowastlowastlowast 125plusmn515 15166 plusmn 317ALT (UIL) 3933plusmn578 6625plusmn 118lowastlowastlowast 3466plusmn 568 4775plusmn 600Urea (mmolL) 486 plusmn 857 899 plusmn 226lowastlowastlowast 556plusmn188 700 plusmn 526Creatinine(umolL) 1854 plusmn 324 2824 plusmn 317lowastlowastlowast 1913plusmn45 1521 plusmn 525Values representmean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllishenoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly atlowastlowastlowastp lt 0001 as considered significant compared to control groupsp lt 0001 as considered significant compared with diabetic ratsp lt0001 as considered significant compared with diabetic rats treated with acarbose

Table 8 Effect of the ethyl acetate flowers extract of Anthyllis henoniana on oxidative stress markers of different experimental groups of rats

Groups SOD (Umgprotein)

CAT (umoleH2O2minmg

protein)

GSH (ugmgprotein)

TBARS (nmolMDAmgprotein)

AOPP(nmolmgprotein)

LiverCont 7012plusmn130 170plusmn132 4012plusmn125 1100plusmn025 016plusmn125Diab 6001 plusmn120lowast 080plusmn002 123plusmn002lowastlowastlowast 2850plusmn121lowastlowastlowast 067plusmn105lowastlowastlowastDiab+ EAEx 6455plusmn739 144plusmn224 2707plusmn19 1638plusmn936 052plusmn030Diab+ Acar 5022plusmn 139 15plusmn002 3000plusmn04 1447plusmn613 032plusmn002

ReinCont 3659plusmn570 125plusmn044 1785plusmn397 2012plusmn103 011plusmn156Diab 1824plusmn170lowastlowastlowast 011plusmn082lowastlowastlowast 906plusmn119lowastlowastlowast 2400plusmn023lowastlowastlowast 030plusmn1231lowastlowastlowastDiab+ EAEx 3016plusmn003 040plusmn083 1312 plusmn506 1981plusmn538 015plusmn008Diab+ Acar 2036plusmn236 079plusmn012 1900plusmn017 1831plusmn150 017plusmn025

PancreasCont 9771plusmn158 105plusmn014 717plusmn013 1000plusmn075 024plusmn012Diab 3962plusmn223lowastlowastlowast 010plusmn102lowastlowastlowast 321plusmn002lowastlowastlowast 2581plusmn170lowastlowastlowast 087plusmn014lowastlowastlowastDiab+ EAEx 5363plusmn106 289plusmn112 850plusmn610 1037plusmn319 033plusmn013Diab+Acar 6515plusmn492 081plusmn112 253plusmn100 1271plusmn173 025plusmn023

Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12mgkg bw per day for 28 days Values differsignificantly atlowastp lt005 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and plt001p lt 0001 as considered significantcompared with diabetic rats plt005 plt001 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

after administration of ethyl acetate extract (400 mgkg bw)as well as acarbose and showed also a decrease in TBARSand AOPP amounts (Table 8) and renal dysfunction indiceslevels in plasma Observation of the histological sections ofthe kidneys of all experimental rats showed that the kidneysof the diabetic rats presented a structural alteration of thecortical zone affecting the Bowman capsules These affectedcapsules showed an increase in Bowmanrsquos capsule space(Figure 11(b)) Kidney protection was remarkable due to thetreatment with the ethyl acetate extract (400 mgkg bw) andthe drug acarbose which preserved the structure of the cortexand corrected the nephropathic damage compared to diabeticrats (Figures 11(c) and 11(d))

4 Discussion

DM is one of the most common metabolic diseases it isknown as an increase of the blood glucose level and impairedmetabolism of proteins and lipids Currently diabetes is

considered as one of the most critical issues in the worldMuch research performed more efforts to seek new naturalantioxidant molecules which are considered to be relativelysafer and with less or without side effects Recently theattention is focused on plants which contain high concentra-tions of phytochemical compounds because of their potentialhealth-promoting effects [38] This study has investigatedfor the first time the potential antidiabetic and antioxidanteffects of Anthyllis henoniana flowers extracts in metabolicdisorders in alloxan-induced diabetic rats

As compared to others extracts the ethyl acetate flowersextract of Anthyllis henoniana possessed the maximum ofphenols and flavonoids contents which can be attributed toits good solubility low toxicity medium polarity and highextraction capacity Likewise ethyl acetate tends to extract away better than ethers and hexane and is known to be morenatural and safer than other chemicals because it occurs innature by contributing to the characteristic aroma of severalfruits [39]The extraction of phytochemical constituents may

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

Table 6 Effect of ethyl acetate flowers extract of Anthyllis henoniana on lipid profile levels of experimental groups of rats

Lipid profile Control Diab Diab+ EAEx Diab+AcarT-Ch (mmolL) 068 plusmn017 172 plusmn128lowastlowastlowast 155 plusmn 058lowastlowastlowast 060plusmn017lowastlowastlowastTG (mmolL) 145plusmn026 188 plusmn046lowastlowastlowast 054 plusmn012lowastlowastlowast 138plusmn056LDL-Ch (mmolL) 050 plusmn019 099 plusmn 008lowastlowastlowast 06 plusmn023 058 plusmn 028lowastHDL-Ch (mmolL) 065 plusmn 017 025 plusmn 008lowastlowastlowast 055 plusmn 007lowastlowast 038 plusmn 084lowastValues represent mean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkgbw per day for 28 days Valuesdiffer significantly atlowastp lt005lowastlowastp lt001 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 andp lt 0001 as consideredsignificant compared with diabetic ratsp lt005 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

0

500

1000

- A

myl

ase a

ctiv

ity (U

IL)

1500

2000

2500

Cont Diab Diab+ EAEx Diab+ Acar

lowastlowastlowast

Figure 7 120572-amylase activity in serum of different experimentalgroups of rats Values represent mean plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastlowastlowastplt 0001 as considered significant comparedto control groups plt005 p lt 0001 as considered significantcompared with diabetic ratsp lt005 as considered significantcompared with diabetic rats treated with acarbose

020406080

100120140160

Cont

Panc

reat

ic li

pase

(UI

L)

Diab Diab+ EAEx Diab+ Acar

lowastlowast

lowast

lowast

Figure 8 Lipase pancreatic level in serum of different experimentalgroups of rats Values represent means plusmn SD (n = 8 for each group)Control groups (Cont) and diabetic control (Diab) Diab+ EAExdiabetic rats treated with EAEx of Anthyllis henoniana at 400 mgkgbw per day for 28 days Diab+Acar diabetic rats treated withacarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly at lowastp lt005lowastlowastlowastp lt 0001 as considered significantcompared to control groupsplt005

plt001 and p lt 0001

as considered significant compared with diabetic rats plt005plt001 and p lt 0001 as considered significant comparedwith diabetic rats treated with acarbose

313 Effect of Anthyllis henoniana Flowers Extract in HepaticDysfunction The results showed increases in markers ofoxidative stress as TBARS and AOPP by + 6419 and+7611 in diabetic rats as compared to control groups(Table 8)The indices of hepatic dysfunction parameters ALTand AST activities obtained in serum of diabetic rats werealso increased by +3458 +4063 respectively (Table 7)Theresults presented a fascinating decrease in the CAT SODand GSH activities in hepatic tissues of diabetic animalscompared to controls groups (Table 8) Diabetic rats givenethyl acetate flower extract of Anthyllis henoniana at doses of400mgkg bw showed an apparent protective effect in hepaticfunction by decreasing significantly (plt0001) in AST andALT by -2950 and -4768 as compared with diabetic ratsand aimed to adjust the antioxidant effectiveness of hepaticfunction also to fix the transformed values by reaching thenormal levels again

All of these findings were associated with the histologicalsections of the liver which showed a tolerable vacuolation ofhepatocytes in diabetic rats (Figure 9(b)) while the treatmentwith ethyl acetate extract (400 mgkg bw) and acarboseshowed a protective effect (Figures 9(c) and 9(d))

314 Effect of Anthyllis henoniana Flowers Extract in PancreasDysfunction In diabetic rats TBARS and AOPP levels wereelevated by +6491 and +4344 respectively However theantioxidant enzymes activities such as CAT SOD and GSHhad endured important decreases by -1036 -14661 and-23184 as compared with the control group (Table 8)

By oral administration of the ethyl acetate extract (400mgkg bw) and acarbose the antioxidant ability of pancreaswas regulated and the concerned values reached the normallevels The histological study confirmed these results anddisclosed the protective action of the pancreas 120573-cells ofdiabetic rats given by the administration of the ethyl acetateextract (400 mgkg bw) (Figure 10(c))

315 Effect of Anthyllis henoniana Flowers Extract in RenalDysfunction In diabetic rats the SOD CAT and GSHactivities in the kidney decreased by -22845 -1036 and-13611 respectively as compared to control groupMoreoveran important increase in TBARS and AOPP in kidney andalso renal dysfunction parameters observed in creatinine andurea level in plasma by +3434 and +4593 respectivelywere acquired in alloxan-treated rats (Tables 7 and 8) Therenal SOD CAT and GSH antioxidant activities increased

Evidence-Based Complementary and Alternative Medicine 9

Table 7 Plasma hepatic and renal parameters of experimental groups of rats

Hepatic Renal parameters Cont Diab Diab+EAEx Diab+AcarAST (UIL) 1166plusmn 120 17825plusmn563lowastlowastlowast 125plusmn515 15166 plusmn 317ALT (UIL) 3933plusmn578 6625plusmn 118lowastlowastlowast 3466plusmn 568 4775plusmn 600Urea (mmolL) 486 plusmn 857 899 plusmn 226lowastlowastlowast 556plusmn188 700 plusmn 526Creatinine(umolL) 1854 plusmn 324 2824 plusmn 317lowastlowastlowast 1913plusmn45 1521 plusmn 525Values representmean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllishenoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly atlowastlowastlowastp lt 0001 as considered significant compared to control groupsp lt 0001 as considered significant compared with diabetic ratsp lt0001 as considered significant compared with diabetic rats treated with acarbose

Table 8 Effect of the ethyl acetate flowers extract of Anthyllis henoniana on oxidative stress markers of different experimental groups of rats

Groups SOD (Umgprotein)

CAT (umoleH2O2minmg

protein)

GSH (ugmgprotein)

TBARS (nmolMDAmgprotein)

AOPP(nmolmgprotein)

LiverCont 7012plusmn130 170plusmn132 4012plusmn125 1100plusmn025 016plusmn125Diab 6001 plusmn120lowast 080plusmn002 123plusmn002lowastlowastlowast 2850plusmn121lowastlowastlowast 067plusmn105lowastlowastlowastDiab+ EAEx 6455plusmn739 144plusmn224 2707plusmn19 1638plusmn936 052plusmn030Diab+ Acar 5022plusmn 139 15plusmn002 3000plusmn04 1447plusmn613 032plusmn002

ReinCont 3659plusmn570 125plusmn044 1785plusmn397 2012plusmn103 011plusmn156Diab 1824plusmn170lowastlowastlowast 011plusmn082lowastlowastlowast 906plusmn119lowastlowastlowast 2400plusmn023lowastlowastlowast 030plusmn1231lowastlowastlowastDiab+ EAEx 3016plusmn003 040plusmn083 1312 plusmn506 1981plusmn538 015plusmn008Diab+ Acar 2036plusmn236 079plusmn012 1900plusmn017 1831plusmn150 017plusmn025

PancreasCont 9771plusmn158 105plusmn014 717plusmn013 1000plusmn075 024plusmn012Diab 3962plusmn223lowastlowastlowast 010plusmn102lowastlowastlowast 321plusmn002lowastlowastlowast 2581plusmn170lowastlowastlowast 087plusmn014lowastlowastlowastDiab+ EAEx 5363plusmn106 289plusmn112 850plusmn610 1037plusmn319 033plusmn013Diab+Acar 6515plusmn492 081plusmn112 253plusmn100 1271plusmn173 025plusmn023

Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12mgkg bw per day for 28 days Values differsignificantly atlowastp lt005 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and plt001p lt 0001 as considered significantcompared with diabetic rats plt005 plt001 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

after administration of ethyl acetate extract (400 mgkg bw)as well as acarbose and showed also a decrease in TBARSand AOPP amounts (Table 8) and renal dysfunction indiceslevels in plasma Observation of the histological sections ofthe kidneys of all experimental rats showed that the kidneysof the diabetic rats presented a structural alteration of thecortical zone affecting the Bowman capsules These affectedcapsules showed an increase in Bowmanrsquos capsule space(Figure 11(b)) Kidney protection was remarkable due to thetreatment with the ethyl acetate extract (400 mgkg bw) andthe drug acarbose which preserved the structure of the cortexand corrected the nephropathic damage compared to diabeticrats (Figures 11(c) and 11(d))

4 Discussion

DM is one of the most common metabolic diseases it isknown as an increase of the blood glucose level and impairedmetabolism of proteins and lipids Currently diabetes is

considered as one of the most critical issues in the worldMuch research performed more efforts to seek new naturalantioxidant molecules which are considered to be relativelysafer and with less or without side effects Recently theattention is focused on plants which contain high concentra-tions of phytochemical compounds because of their potentialhealth-promoting effects [38] This study has investigatedfor the first time the potential antidiabetic and antioxidanteffects of Anthyllis henoniana flowers extracts in metabolicdisorders in alloxan-induced diabetic rats

As compared to others extracts the ethyl acetate flowersextract of Anthyllis henoniana possessed the maximum ofphenols and flavonoids contents which can be attributed toits good solubility low toxicity medium polarity and highextraction capacity Likewise ethyl acetate tends to extract away better than ethers and hexane and is known to be morenatural and safer than other chemicals because it occurs innature by contributing to the characteristic aroma of severalfruits [39]The extraction of phytochemical constituents may

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

Table 7 Plasma hepatic and renal parameters of experimental groups of rats

Hepatic Renal parameters Cont Diab Diab+EAEx Diab+AcarAST (UIL) 1166plusmn 120 17825plusmn563lowastlowastlowast 125plusmn515 15166 plusmn 317ALT (UIL) 3933plusmn578 6625plusmn 118lowastlowastlowast 3466plusmn 568 4775plusmn 600Urea (mmolL) 486 plusmn 857 899 plusmn 226lowastlowastlowast 556plusmn188 700 plusmn 526Creatinine(umolL) 1854 plusmn 324 2824 plusmn 317lowastlowastlowast 1913plusmn45 1521 plusmn 525Values representmean plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllishenoniana at 400 mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12 mgkg bw per day for 28 days Values differsignificantly atlowastlowastlowastp lt 0001 as considered significant compared to control groupsp lt 0001 as considered significant compared with diabetic ratsp lt0001 as considered significant compared with diabetic rats treated with acarbose

Table 8 Effect of the ethyl acetate flowers extract of Anthyllis henoniana on oxidative stress markers of different experimental groups of rats

Groups SOD (Umgprotein)

CAT (umoleH2O2minmg

protein)

GSH (ugmgprotein)

TBARS (nmolMDAmgprotein)

AOPP(nmolmgprotein)

LiverCont 7012plusmn130 170plusmn132 4012plusmn125 1100plusmn025 016plusmn125Diab 6001 plusmn120lowast 080plusmn002 123plusmn002lowastlowastlowast 2850plusmn121lowastlowastlowast 067plusmn105lowastlowastlowastDiab+ EAEx 6455plusmn739 144plusmn224 2707plusmn19 1638plusmn936 052plusmn030Diab+ Acar 5022plusmn 139 15plusmn002 3000plusmn04 1447plusmn613 032plusmn002

ReinCont 3659plusmn570 125plusmn044 1785plusmn397 2012plusmn103 011plusmn156Diab 1824plusmn170lowastlowastlowast 011plusmn082lowastlowastlowast 906plusmn119lowastlowastlowast 2400plusmn023lowastlowastlowast 030plusmn1231lowastlowastlowastDiab+ EAEx 3016plusmn003 040plusmn083 1312 plusmn506 1981plusmn538 015plusmn008Diab+ Acar 2036plusmn236 079plusmn012 1900plusmn017 1831plusmn150 017plusmn025

PancreasCont 9771plusmn158 105plusmn014 717plusmn013 1000plusmn075 024plusmn012Diab 3962plusmn223lowastlowastlowast 010plusmn102lowastlowastlowast 321plusmn002lowastlowastlowast 2581plusmn170lowastlowastlowast 087plusmn014lowastlowastlowastDiab+ EAEx 5363plusmn106 289plusmn112 850plusmn610 1037plusmn319 033plusmn013Diab+Acar 6515plusmn492 081plusmn112 253plusmn100 1271plusmn173 025plusmn023

Values represent means plusmn SD (n = 8 for each group) Control groups (Cont) and diabetic control (Diab) Diab+ EAEx diabetic rats treated with EAEx ofAnthyllis henoniana at 400mgkg bw per day for 28 days Diab+Acar diabetic rats treated with acarbose at dose of 12mgkg bw per day for 28 days Values differsignificantly atlowastp lt005 and lowastlowastlowastp lt 0001 as considered significant compared to control groupsplt005 and plt001p lt 0001 as considered significantcompared with diabetic rats plt005 plt001 and p lt 0001 as considered significant compared with diabetic rats treated with acarbose

after administration of ethyl acetate extract (400 mgkg bw)as well as acarbose and showed also a decrease in TBARSand AOPP amounts (Table 8) and renal dysfunction indiceslevels in plasma Observation of the histological sections ofthe kidneys of all experimental rats showed that the kidneysof the diabetic rats presented a structural alteration of thecortical zone affecting the Bowman capsules These affectedcapsules showed an increase in Bowmanrsquos capsule space(Figure 11(b)) Kidney protection was remarkable due to thetreatment with the ethyl acetate extract (400 mgkg bw) andthe drug acarbose which preserved the structure of the cortexand corrected the nephropathic damage compared to diabeticrats (Figures 11(c) and 11(d))

4 Discussion

DM is one of the most common metabolic diseases it isknown as an increase of the blood glucose level and impairedmetabolism of proteins and lipids Currently diabetes is

considered as one of the most critical issues in the worldMuch research performed more efforts to seek new naturalantioxidant molecules which are considered to be relativelysafer and with less or without side effects Recently theattention is focused on plants which contain high concentra-tions of phytochemical compounds because of their potentialhealth-promoting effects [38] This study has investigatedfor the first time the potential antidiabetic and antioxidanteffects of Anthyllis henoniana flowers extracts in metabolicdisorders in alloxan-induced diabetic rats

As compared to others extracts the ethyl acetate flowersextract of Anthyllis henoniana possessed the maximum ofphenols and flavonoids contents which can be attributed toits good solubility low toxicity medium polarity and highextraction capacity Likewise ethyl acetate tends to extract away better than ethers and hexane and is known to be morenatural and safer than other chemicals because it occurs innature by contributing to the characteristic aroma of severalfruits [39]The extraction of phytochemical constituents may

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

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Disease Markers

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Hindawiwwwhindawicom Volume 2013

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Oxidative Medicine and Cellular Longevity

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PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 9 Histopathological observation of liver in the control and experimental groups of rats Section of the liver from a control rat (a)showing normal architecture (b) liver of diabetic rat showing a moderate vacuolation of hepatocytes and infiltration of lymphocyte cells (c)liver of diabetic rat treated with EAEx (400mgkg bw) of Anthyllis henoniana showed a potential protective effect and (d) diabetic rat treatedwith acarbose protective action was shown (HampE times400)

(a) (b)

(c) (d)

Figure 10Histopathological observation of pancreatic islet cells in the control and experimental groups (a) Control group of rat the pancreasshows normal pancreatic islet cells (b) diabetic rats pancreas showing a distorted and atrophic islet of Langerhans displaying cells withvacuolated cytoplasm (c) in treated diabetic rats with EAEx of Anthyllis henoniana (400 mgkg bw) showing normal islet of Langerhanswith numerous 120573 cells and (d) in diabetic rats treated with acarbose showing a distorted islet of Langerhans displaying cells with vacuolatedcytoplasm (HampE times400)

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

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Oxidative Medicine and Cellular Longevity

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PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

(a) (b)

(c) (d)

Figure 11 Histopathological observation of kidney in the control and experimental groups of rats (a) Section of the kidney from a controlrat (b) diabetic rat showing Bowmanrsquos space size (c) diabetic rat treatedwith EAEx (400mgkg bw) ofAnthyllis henoniana showed a potentialprotective effect and (d) diabetic rat treated with acarbose demonstrated also a potential protective effect was shown (HampE times400)

depend on the polarity and the molecular weight of thechosen solvent these factors present a major role in naturalsubstances isolation According to Naczk et al [40] thesolubility of phenolics is affected by the polarity of usedsolvents thus it is very difficult to develop an extractionprocedure suitable for extraction of all plant phenolicsOverall the findings indicated that the ethyl acetate extractof Anthyllis henonianarsquos flowers was rich in flavonoid andphenolic contents which could be the main contributorto their antioxidative properties as many studies affirmedthat flavonoids and phenols offered the highest ability ofscavenging activity in medicinal plants [41 42]

In this study the results clearly stated that the methanoland ethyl acetate extracts which contained the highestamounts of total phenolic content had the strongest radicalscavenging activities and total antioxidant capacities Thepositive correlation between the phenolic compounds andthe antioxidant activity using DDPH and TAC confirmed thehypothesis of the effective role of these types of compoundsMoreover both of ethyl acetate andmethanol extracts showedhigh activities in the reducing power at a maximum concen-tration of 1mgmL Miceli et al [43] have reported similarresults by revealing a positive relationship between totalphenols flavonoids and the antioxidant activities (DDPHand FRAP) These results could be allotted to the presence ofphenolic compounds which play an efficient role as a hydro-gen donator reducing agents and singlet oxygen quenchers[44 45] Many plants from the Fabaceae family were usedin traditional complementary and alternative medicine [4647] So this research aims to seek the antidiabetic potential

by determination of 120572-glucosidase inhibitory and 120572-amylaseassay The inhibitory effect of ethyl acetate extract against120572-amylase showed a good concentration-dependent activityActually 120572-amylase and 120572-glucosidase inhibitory activitieswere marked to elevate in a dose-dependent approach withthe observation of the highest inhibitory effect in the ethylacetate extract

Therefore in vivo studies had been proceeding withthe ethyl acetate extract and confirmed the role of alloxaninjection in inducing hyperglycemia by +5474 as comparedto control groups However it was noted that the EAExadministration at doses (400 mgkg bw) to diabetic ratsinhibited in vivo blood 120572-amylase by involving an importantblood glucose decrease In fact the inhibitory potential of thisextract was quite similar to that established by acarbose Thepresence of phytochemical compounds such as flavonoidsphenols and tannins with important contents in the ethylacetate extract could be related to the antidiabetic effectMoreover many glycosides have been assumed as potentialantidiabetic agents due to their significant inhibitory effectagainst 120572-amylase and their role in DM prevention byincluding them in the dietary strategy [48 49] Other studieshave suggested that inhibiting activity of different flavonoidson glucose absorption is possibly due to the competitive inhi-bition of sodium-dependent glucose transporter 1 [48 50]eg especially isoquercitrin has shown in previous studiesits hypoglycemic effect [51ndash53] The current findings showedthe ability of the ethyl acetate extract to reduce blood glucoselevel due to the improvement of insulin action to improveglycemic control in peripheral tissue and to the inhibitory

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

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Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

effects of intestinal disaccharidases This extract (EAEx) pre-sented a protective effect in pancreatic 120573-cell function andordecreased 120573-cell mass and lytic changes in the pancreas isletinduced by alloxan as have been seen in the histologicalexamination DM causes a disorder in lipid metabolismwhich led more investigations to survey and understandsthe pathogenesis [54] The hyperglycemia accompanied bya dyslipidemic disturbance proven by a significant elevationof TG T-Ch and LDL-c and a decrease in HDL-c in serumpancreatic lipase Many studies had mentioned the dangercaused by LDL oxidation which contributes to the formationof atherosclerosis plaque lesions [55] After the ethyl acetateextract administration to diabetic rats the blood glucose levelhas been lowered and the lipid metabolism has enhancedwhich caused a remarkable reduction in serums TG T-Chand LDL-c and an elevation in HDL-c levels as comparedrespectively with diabetic rats Increasing HDL-C or HTRratios has been quoted among the most substantial criteriaof antiatherogenic agents There has been much researchreported on the association between the low incidence ofcardiovascular diseases and higher levels of HDL-c [56 57]Thehypolipidemic action of ethyl acetate extract might be theresult of the carbohydrate retardation and lipid absorptioncaused by the presence of biologically active compounds inthe flowers extract these results were similar to the studyof Daisy and Rajathi [58] that showed the hypoglycemicand hypolipidemic effects of aqueous extract from flowersand leaves of Clitoria ternatea Linn (Fabaceae) at doses of400 mgkg bw by decreasing in serum level of T-Ch LDL-cand serum glucose glycosylated hemoglobin and insulin indiabetic rats Besides the antidiabetic and antihyperlipidemicactions the ethyl acetate extract administration preventsliver-kidney dysfunctions AST and ALT along with otherblood plasma enzymes were used to evaluate the hepaticdysfunction [59 60] The increased liver enzyme activitiesare biomarkers for hepatic disorder and the high increaseof transaminases causes inflammation or hepatocellular dis-orders [61 62] These findings were in accordance with thecurrent study which was demonstrated by the increasedhepatic levels in diabetic rats as compared to control groupsOtherwise the ethyl acetate extract administration (400mgkgbw) marked significant decreases in AST and ALTas compared to diabetic rats The hepatoprotective effect ofethyl acetate extract was proved through histological resultsin liver tissues by reducing the liver fat levels Thus theantioxidant compounds of the ethyl acetate flowers extractof Anthyllis henoniana decreased the oxidative stress andprotected the hepatic The result exhibited that the treatmentwith ethyl acetate extract (400 mgkg bw) displayed animportant increase in antioxidants enzymatic and nonenzy-matic status (SOD CAT and GSH levels) and a decreasein TBARS and AOPP respectively as compared to diabeticrats Besides the amelioration effect of ethyl acetate extractEAEx in liver toxicity the administration of this extractand acarbose ameliorates the renal toxicity by decreasingthe serum creatinine and serum urea rates as compared todiabetic animals

Furthermore the antioxidant activity performed aremarkable increase in SOD CAT and GSH activities in

the kidney of treated diabetic rats with EAEx (400 mgkgbw) respectively The positive effect of ethyl acetate extractwas proved by histological reporting in kidney tissues byreducing the increase of glomerular condensation and thecapsular space The antihyperglycemic activity of this extracthas been associated with its flavonoids and total phenoliccontents which may contribute to protective property ofethyl acetate extract for the complication of the liver andkidney toxicity against alloxan-induced diabetic rats

5 Conclusion

The ethyl acetate extract from flowers extracts of Anthyllishenoniana (Coss) showed different levels of phytochemicalsantioxidant and antipostprandial hyperglycemia activitiesThe highest content of phenolics plays a major role in deter-mining the antioxidant and antihyperlipidemia activities ofthis plant In conclusion this study hands over a clearevidence for the ethnobotanical uses of Anthyllis henoniana(Coss) flowers extract in the cure of diabetes mellitus Theencouraging results for such first antidiabetic effect studyof Anthyllis henoniana lead us to enhance more specificinvestigations to isolate and identify the responsible bioactivemolecule (s) in the futuristic research

Data Availability

All data used to support the findings of this study are availablefrom the corresponding author upon a reasonable request

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Ministry of HigherEducation and Scientific Research in Tunisia

References

[1] P Zimmet KGMMAlberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[2] L Rossetti A Giaccari and R A DeFronzo ldquoGlucose toxicityrdquoDiabetes Care vol 13 no 6 pp 610ndash630 1990

[3] J-L Chiasson and R Rabasa-Lhoret ldquoPrevention of type 2diabetes insulin resistance and 120573-cell functionrdquo Diabetes vol53 no 3 pp S34ndashS38 2004

[4] H G Eichler A Korn S GasicW Pirson and J Businger ldquoTheeffect of a new specific 120572-amylase inhibitor on post-prandialglucose and insulin excursions in normal subjects and Type 2(non-insulin-dependent) diabetic patientsrdquo Diabetologia vol26 no 4 pp 278ndash281 1984

[5] N Landrault P Poucheret J Azay et al ldquoEffect of apolyphenols-enriched chardonnay white wine in diabetic ratsrdquoJournal of Agricultural and Food Chemistry vol 51 no 1 pp 311ndash318 2003

[6] K N Chidambara Murthy R P Singh and G K JayaprakashaldquoAntioxidant activities of grape (Vitis vinifera) pomace

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 13

extractsrdquo Journal of Agricultural and Food Chemistry vol 50no 21 pp 5909ndash5914 2002

[7] J H OrsquoKeefe and D S Bell ldquoPostprandial hyperglycemiahyperlipidemia (postprandial dysmetabolism) is a cardiovascu-lar risk factorrdquo American Journal of Cardiology vol 100 no 5pp 899ndash904 2007

[8] J KimC Kwon andKH Son ldquoInhibition of alpha-glucosidaseand amylase by luteolin a flavonoidrdquo Bioscience Biotechnologyand Biochemistry vol 64 no 11 pp 2458ndash2461 2000

[9] D-S Lee and S-H Lee ldquoGenistein a soy isoflavone is a potent120572-glucosidase inhibitorrdquo FEBS Letters vol 501 no 1 pp 84ndash862001

[10] S Passamonti U Vrhovsek A Vanzo and F Mattivi ldquoFastaccess of some grape pigments to the brainrdquo Journal of Agricul-tural and Food Chemistry vol 53 no 18 pp 7029ndash7034 2005

[11] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985

[12] GObohA T Isaac A J Akinyemi andRAAjani ldquoInhibitionof key enzymes linked to type 2 diabetes and sodium nitroprus-side induced lipid peroxidation in ratsrsquo pancreas by phenolicextracts of avocado pear leaves and fruitrdquo International Journalof Biomedical Science vol 10 no 3 pp 210ndash218 2014

[13] P S Unnikrishnan K Suthindhiran and M A JayasrildquoInhibitory potential ofTurbinaria ornata against keymetabolicenzymes linked to diabetesrdquoBioMedResearch International vol2014 Article ID 783895 pp 1ndash10 2014

[14] P Nain V Saini S Sharma and J Nain ldquoAntidiabetic andantioxidant potential of Emblica officinalis Gaertn leavesextract in streptozotocin-induced type-2 diabetes mellitus(T2DM) ratsrdquo Journal of Ethnopharmacology vol 142 no 1 pp65ndash71 2012

[15] P Saleh B Asghari M A Esmaeil H Dehghan and I Ghazildquo120572-glucosidase and 120572-amylase inhibitory effect and antioxidantactivity of ten plant extracts traditionally used in Iran fordiabetesrdquo Journal of Medicinal Plants Research vol 7 no 6 pp257ndash266 2013

[16] SDerbel andMChaieb ldquoGrowth establishment andphenologyof four woody Saharan speciesrdquo African Journal of Ecology vol51 no 2 pp 307ndash318 2013

[17] J Aronson C Floret E Le Flocrsquoh C Ovalle and R PontanierldquoRestoration and rehabilitation of degraded ecosystems in aridand semi-arid lands II Case studies in Southern TunisiaCentral Chile and Northern Cameroonrdquo Restoration Ecologyvol 1 no 3 pp 168ndash187 1993

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[19] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[20] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[21] M L Price S V Scoyoc and L G Butler ldquoA critical evaluationof the vanillin reaction as an assay for tannin in sorghum grainrdquoJournal of Agricultural and Food Chemistry vol 26 no 5 pp1214ndash1218 1978

[22] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[23] M Oyaizu ldquoStudies on products of browning reaction antiox-idative activity of products of browning reaction prepared fromglucosaminerdquo The Japanese Journal of Nutrition and Dieteticsvol 44 pp 307ndash315 1986

[24] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[25] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010

[26] T Nishioka J Kawabata and Y Aoyama ldquoBaicalein an 120572-glucosidase inhibitor from Scutellaria baicalensisrdquo Journal ofNatural Products vol 61 no 11 pp 1413ndash1415 1998

[27] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[28] K G Mustafa B A Ganai S Akbar M Y Dar and AMasoodldquo120573-Cell protective efficacy hypoglycemic and hypolipidemiceffects of extracts of Achillea millifolium in diabetic ratsrdquoChinese Journal of Natural Medicines vol 10 no 3 pp 185ndash1892012

[29] L Zanatta E de Sousa L H Cazarolli et al ldquoEffect ofcrude extract and fractions fromVitex megapotamica leaves onhyperglycemia in alloxan-diabetic ratsrdquo Journal of Ethnophar-macology vol 109 no 1 pp 151ndash155 2007

[30] R Ben Abdallah Kolsi A Ben Gara N Jardak et al ldquoInhibitoryeffects of Cymodocea nodosa sulphated polysaccharide on 120572 -amylase activity liver-kidney toxicities and lipid profile disor-ders in diabetic ratsrdquo Archives of Physiology and Biochemistryvol 121 no 5 pp 218ndash227 2015

[31] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[32] T Yoshioka K Kawada T Shimada and M Mori ldquoLipidperoxidation in maternal and cord blood and protective mech-anism against activated-oxygen toxicity in the bloodrdquoAmericanJournal of Obstetrics amp Gynecology vol 135 no 3 pp 372ndash3761979

[33] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidation inpost-mitotic tissues of ageing ratrdquoCell Biochemistry amp Functionvol 24 no 1 pp 79ndash85 2006

[34] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[35] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[36] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

14 Evidence-Based Complementary and Alternative Medicine

[37] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1985

[38] I-M Chung E-H Kim M-A Yeo S-J Kim M-C Seo andH-I Moon ldquoAntidiabetic effects of three Korean sorghum phe-nolic extracts in normal and streptozotocin-induced diabeticratsrdquoFoodResearch International vol 44 no 1 pp 127ndash132 2011

[39] M A M El Hadi F-J Zhang F-FWu C-H Zhou and J TaoldquoAdvances in fruit aroma volatile researchrdquo Molecules vol 18no 7 pp 8200ndash8229 2013

[40] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[41] D Krishnaiah A Bono R Sarbatly and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversity - Engineering Sciences vol 27 no 1 pp 63ndash67 2015

[42] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[43] N Miceli L P Buongiorno M G Celi et al ldquoRole ofthe flavonoid-rich fraction in the antioxidant and cytotoxicactivities ofrdquo Natural Product Research vol 30 no 11 pp 1229ndash1239 2015

[44] C Gardeli P Vassiliki M Athanasios T Kibouris and MKomaitis ldquoEssential oil composition of Pistacia lentiscus LandMyrtus communis L Evaluation of antioxidant capacity ofmethanolic extractsrdquo Food Chemistry vol 107 no 3 pp 1120ndash1130 2008

[45] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[46] R Kaewamatawong ldquoSecondarymetabolites and pharmacolog-ical activities of Bauhinia genus plantsrdquo Science journal UbonRatchathani University ScJ ubu vol 10 pp 116ndash130 2008

[47] T Gao H Yao J Song et al ldquoIdentification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2rdquoJournal of Ethnopharmacology vol 130 no 1 pp 116ndash121 2010

[48] Y Sireesha R B Kasetti S A Nabi S Swapna and CApparao ldquoAntihyperglycemic and hypolipidemic activities ofSetaria italica seeds in STZ diabetic ratsrdquo Pathophysiology vol18 no 2 pp 159ndash164 2011

[49] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity Potential targets forglucose homeostasisrdquo Nutrition Journal vol 27 no 11-12 pp1161ndash1167 2011

[50] L H Cazarolli L Zanatta E H Alberton et al ldquoFlavonoidscellular and molecular mechanism of action in glucose home-ostasisrdquoMini-Reviews in Medicinal Chemistry vol 8 no 10 pp1032ndash1038 2008

[51] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006

[52] S KurakaneN Yamada H Sato andK Igarashi ldquoAnti-diabeticeffects ofActinidia arguta polyphenols on rats andKK-AymicerdquoFood Science and Technology Research vol 17 no 2 pp 93ndash1022011

[53] H Y Park K ToumeM A Arai T Koyano T Kowithayakornand M Ishibashi ldquo120573-Sitosterol and flavonoids isolated from

Bauhinia malabarica found during screening for Wnt signalinginhibitory activityrdquo Journal of Natural Medicines vol 68 no 1pp 242ndash245 2014

[54] P Fumelli F Romagnoli G Carlino C Fumelli and MBoemi ldquoDiabetes mellitus and chronic heart failurerdquo Archivesof Gerontology and Geriatrics vol 23 no 3 pp 277ndash281 1996

[55] G Assmann and J-R Nofer ldquoAtheroprotective effects of high-density lipoproteinsrdquo Annual Review of Medicine vol 54 pp321ndash341 2003

[56] P K Shah S Kaul J Nilsson and B Cercek ldquoExploiting thevascular protective effects of high-density lipoprotein and itsapolipoproteins An idea whose time for testing is coming partIrdquo Circulation vol 104 no 19 pp 2376ndash2383 2001

[57] I S Young ldquoLipids for psychiatrists - An overviewrdquo Journal ofPsychopharmacology vol 19 no 6 pp 66ndash75 2005

[58] P Daisy and M Rajathi ldquoHypoglycemic effects of Clitoriaternatea Linn (Fabaceae) in alloxan-induced diabetes in ratsrdquoTropical Journal of Pharmaceutical Research vol 8 no 5 2009

[59] G S Achliya S G Wadodkar and A K Dorle ldquoEvaluationof hepatoprotective effect of Amalkadi Ghrita against car-bon tetrachloride-induced hepatic damage in ratsrdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 229ndash232 2004

[60] M IThabrew PD TM Joice andWRajatissa ldquoA comparativestudy of the efficacy of Pavetta indica and Osbeckia octandra inthe treatment of liver dysfunctionrdquo Planta Medica vol 53 no3 pp 239ndash241 1987

[61] W C Fortson F J Tedesco E C Starnes and C T ShawldquoMarked elevation of serum transaminase activity associatedwith extrahepatic biliary tract diseaserdquo Journal of ClinicalGastroenterology vol 7 no 6 pp 502ndash505 1985

[62] R Hultcrantz H Glaumann G Lindberg and L NilssonldquoLiver investigation in 149 asymptomatic patients with moder-ately elevated activities of serum Aminotransferasesrdquo Scandi-navian Journal of Gastroenterology vol 21 no 1 pp 109ndash1131986

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom