Research Article Antinociceptive and Antioxidant Activity ...downloads.hindawi.com/journals/tswj/2014/869537.pdf · Antinociceptive and Antioxidant Activity of Zanthoxylum budrunga

Research ArticleAntinociceptive and Antioxidant Activity of Zanthoxylumbudrunga Wall (Rutaceae) Seeds

Md Khirul Islam1 Nripendra Nath Biswas2 Sanjib Saha1 Hemayet Hossain3

Ismet Ara Jahan3 Tanzir Ahmed Khan3 Khalijah Awang4 and Jamil A Shilpi5

1 Pharmacy Discipline Life Science School Khulna University Khulna 9208 Bangladesh2 School of Chemistry University of New South Wales Sydney NSW 2052 Australia3 Chemical Research Division BCSIR Laboratories Bangladesh Council of Scientific and Industrial Research (BCSIR)Dhaka 1205 Bangladesh

4Department of Chemistry Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia5 Centre for Natural Products and Drug University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Jamil A Shilpi jamilshilpiyahoocom

Received 9 December 2013 Accepted 16 January 2014 Published 23 February 2014

Academic Editors M Galuppo and V Seidel

Copyright copy 2014 Md Khirul Islam 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

Different parts of the medicinal plant Zanthoxylum budrungaWall enjoy a variety of uses in ethnobotanical practice in BangladeshIn the present study a number of phytochemical and pharmacological investigations were done on the ethanol extract of Zbudrunga seeds (ZBSE) to evaluate its antinociceptive and antioxidant potential ZBSE was also subjected to HPLC analysis todetect the presence of some common antioxidants In acetic acid induced writhing test in mice ZBSE showed 6528 and 7430inhibition of writhing at the doses of 250 and 500mgkg and the results were statistically significant (119875 lt 0001) In hot-platetest ZBSE raised the pain threshold significantly (119875 lt 0001) throughout the entire observation period In DPPH scavengingassay the IC

50of ZBSE was observed at 8260 120583gmL The phenolic content was found to be 33877mg GAE100 g of dried plant

material In reducing power assay ZBSE showed a concentration dependent reducing ability HPLC analysis indicated the presenceof caffeic acid with a concentration of 7545mg100 g ZBSE Present investigation supported the use of Zanthoxylum budrunga seedin traditional medicine for pain management Constituents including caffeic acid and other phenolics might have some role in theobserved activity

1 Introduction

Zanthoxylum budrunga Wall (Syn Zanthoxylum rhetsaRoxb) (Rutaceae) is a deciduous medium-sized tree nativeto subtropical areas of the world and widely distributed inBangladesh India Sri Lanka Myanmar China ThailandMalaysia Indonesia Philippines and Papua New GuineaIt grows up to 35m with sparsely armed branches havingstraight or ascending prickles In Bangladesh it is distributedin Sylhet Chittagong Hill Tracts Coxrsquos Bazar Gazipur andTangail and enjoys a number of uses in ethnobotanicalpractice In traditional medicine the fruit of the plant is usedin the treatment of rheumatism cholera dysentery asthmainfections heart disease bronchitis and piles The essential

oil extracted from leaves is used in cholera Juice of thebark is considered beneficial in vomiting cough dysenteryand headache [1] The seed oil is antiseptic and is used inthe treatment of inflammatory dermatitis dry eczema anddandruff [2] In a previous study the methanol extract ofstem bark showed antinociceptive and antidiarrhoeal activityin experimental mice [3] In another study three terpenesisolated from the bark showed cytotoxic activity althoughno names or structures of the compounds were available inthe report [4] Several alkaloids and lignans were isolatedfrom the stem bark [5] The chemical composition of thevolatile oil of seed coat was analyzed and thirty-four com-pounds were identified including terpinen-4-ol (321) 120572-terpineol (82) sabinene (81)120573-phellandrene (74) and

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 869537 7 pageshttpdxdoiorg1011552014869537

2 The Scientific World Journal

2-undecanone (71) [6] Volatile constituents (hydroxy 120572-sanshool 120573-phellandrene 120573-pinene and pipertone mullilamdiol) and alkaloids (glycosine rutaecarpine) were isolatedfrom the fruits [7ndash9] A monoterpene triol trihydroxy-119901-menthane was reported from the roots [10] while threealkaloids namely pseudophrynamine 2-(210158404101584061015840-trimethylheptenyl)-4-quinozolone and lunacridine were isolatedfrom the leaves [11] The present study was designed toevaluate antinociceptive and antioxidant activities of theethanol extract of Z budrunga seeds

2 Materials and Methods

21 Plant Material The seeds of Z budrunga was collectedfrom Tangail Bangladesh in September 2011 and identi-fied by experts at Bangladesh National Herbarium DhakaBangladesh A voucher specimen (DACB 37522) has beensubmitted there for future referenceThe seeds were removedfrom the fruit rind and separated from other parts andextraneous materials

22 Extraction The dried seeds were grinded into coarsepowder (210 g) and macerated in ethanol (95 1 L) for threedays with occasional shaking It was filtered through a clearcotton plug to remove marc Filtrate was evaporated to getgummy reddish crude extractThe seeds yielded 145 extractof dried plant material and designated as Z budrunga seedextract (ZBSE)

23 Test Animals Randomly screened young Swiss-Albinomice aged 4-5 weeks weighing 20ndash25 g were selectedfor the pharmacological investigation The mice were pur-chased from the Animal Resources Branch of InternationalCentre for Diarrhoeal Disease and Research Bangladesh(ICCDRB) and fed with ICCDRB formulated commercialrodent food and water ad libitum The investigations werecarried out following the animal ethics guidelines providedby ICDDRB

24 Chemicals and Reagents Gallic acid (+)-catechinhydrate vanillic acid caffeic acid (minus)-epicatechin 119901-coumaric acid rutin hydrate ellagic acid quercetin and11-Diphenyl-2-picrylhydrazyl (DPPH) were purchased fromSigma-Aldrich USA Folin-Ciocalteursquos reagent ascorbicacid trichloroacetic acid potassium ferricyanide sodiumcarbonate ferric chloride acetic acid and dimethyl sulfoxide(DMSO) were purchased from Merck Germany Tween-80 was purchased from Loba Chemie India Diclofenacsodium morphine and vincristine sulphate were obtainedfrom Beximco Pharmaceuticals Ltd Bangladesh PopularPharmaceuticals Ltd Bangladesh and Cipla PharmaceuticalsIndia respectively

25 Phytochemical Analysis ZBSEwas subjected to a numberof standard tests to detect the presence of major phytochem-ical groups [12 13]

26 DPPH Radical Scavenging Activity In vitro antioxidantactivity was determined based on the ability of the seedextract to scavenge the stable free radical DPPH [14] Stocksolution of ZBSE was prepared in ethanol and seriallydiluted to obtain different concentrations with the maximumand minimum of 512 and 1 120583gmL respectively From eachconcentration 1mL was mixed with 2mL DPPH solution(0004 in ethanol) The mixture was kept for 30 minutesto complete the reaction The absorbance was measured at517 nm in a UV-visible spectrophotometer Ascorbic acid wasused as standard antioxidant The percent inhibition wascalculated according to the formula (1minus119860

11198600) times 100 where

1198600is the absorbance of control and 119860

1is the absorbance of

ZBSE or standard IC50value was determined from the graph

obtained by plotting inhibition versus concentration

27 Determination of Total Phenolic Content (TPC) Grindedplant material (05 g) was mixed with 50mL of 80 aqueousmethanol and sonicated for 20min An aliquot of 2mLwas taken from it and centrifuged for 15min at 14000 rpmTotal phenolic content was determined by Folin-Ciocalteursquosreagent [15] Standard gallic acid solutions were preparedby serial dilution to get concentrations of 500 250 125625 3125 and 1562mgL Gallic acid solutions of eachconcentration and ZBSE of 1mL were transferred to 25mLvolumetric flasks and 9mL distilled water was added Folin-Ciocalteursquos reagent of 1mLwas added to each volumetric flaskwith continuous shaking After 5min 10mL of 7 Na

2CO3

was added and adjusted with distilled water to make thefinal volume of 25mL It was kept for 30min to completeany reaction that occurs The absorbance was measured at750 nm against blank Blank was prepared by following thesame steps mentioned above without adding ZBSE or gallicacid Standard curve of gallic acid was prepared by plottingabsorbance versus concentration The total phenolic contentof ZBSE was determined using the standard calibration curveof gallic acid and the value was expressed as gallic acidequivalent (GAE)100 g of dried plant material

28 Reducing Power Assay Reducing power of the extractwas determined according to the method described byOyaizu [16] ZBSE of 1mL with the concentrations of 500250 125 625 3125 and 1562 120583gmL were mixed with25mL of 02M phosphate buffer (pH 66) and 25mLof 1 potassium ferricyanide with vigorous shaking Themixtures were incubated for 20min at 50∘C After coolingat room temperature 25mL of 10 trichloroacetic acid wasadded and centrifuged at 3000 rpm for 10min An aliquot(25mL) of the supernatant wasmixedwith 25mL of distilledwater and 05mL of ferric chloride (01) After 5min theabsorbance was measured at 700 nm Ascorbic acid was usedas standard

29 HPLC Detection of Phenolics Detection of selectedphenolic compounds in ZBSE was determined by HPLCanalysis as described by Chuanphongpanich and Phanich-phant with some modifications [17] It was carried out ona Dionex UltiMate 3000 system equipped with quaternary

The Scientific World Journal 3

rapid separation pump (LPG-3400RS) and photodiode arraydetector (DAD-3000RS) Separation was performed usingAcclaim C

18(5 120583m) Dionex column (46 times 250mm) at

30∘C with a flow rate of 1mLmin and an injection vol-ume of 20120583L The mobile phase consisted of acetonitrile(solvent A) acetic acid solution pH 30 (solvent B) andmethanol (solvent C) with the gradient elution program of5A95B (0ndash9min) 10A80B10C (10ndash19min) and20A60B20C (20ndash30min) with postrun equilibrationof the systemwith 100A (5min)TheUVdetector was set to280 nm for 180min changed to 320 nm for 6min and finallyto 380 nm and held for the rest of the analysis period whilethe diode array detector was set at an acquisition range from200 nm to 700 nm For the preparation of calibration curve astandard stock solution was prepared inmethanol containinggallic acid vanillic acid (+)-catechin (minus)-epicatechin 119901-coumaric acid rutin ellagic acid (20120583gmL each) caffeic acid(8 120583gmL) and quercetin (6 120583gmL) A solution of ZBSE wasprepared in methanol having a concentration of 5mgmLPrior to HPLC analysis all solutions (mixed standardssample and spiked solutions) were filtered through 02 120583mnylon syringe filter (Sartorius Germany) and degassed in anultrasonic bath (Hwashin Korea) for 15min Data acquisi-tion peak integration and calibrations were performed withDionex Chromeleon software (Version 680 RS 10)

210 Acetic Acid Induced Writhing Test Antinociceptiveactivity was evaluated by acetic acid induced writhing modelin mice [18] Control group received vehicle (1 tween-80 indistilledwater) at the dose of 10mLkg Positive control groupwas treated with diclofenac sodium at the dose of 25mgkgbodyweight and test groupswithZBSE at the doses of 250 and500mgkg bodyweight All the treatmentswere administeredorally For proper absorption of the extract or drug aperiod of 30minwas provided followed by the intraperitonealadministration of acetic acid (07) at the dose of 10mLkg toinduce abdominal contraction or writhing After an intervalof 5min writhing was counted for 10min Total numberof writhing of positive control group and test groups wascompared with the control group

211 Hot-Plate Test In hot-plate test experimental micewere subjected to pain stimulus when placed on hot-platemaintained at the temperature of 55plusmn05∘C [19]Mice havinglatency time of 3 to 5 sec were selected for the study Thelatency time was considered as the time between placing themice on hot plate and the time when mice licked their foreand hind paws or jumped from the plate Latency time wasrecorded at 0 30 60 90 and 120min following the oraladministration of vehicle (1 tween-80 in distilled water10mLkg) for control morphine (5mgkg) for the positivecontrol and ZBSE (250 and 500mgkg) for the test groupsA cut-off point of 15 sec was used to avoid accidental pawdamage Latency time of positive control and test groups wascompared with control group

212 Statistical Analysis All the values were expressed asmean plusmn SEM Statistical analysis was performed by Studentrsquos

Table 1 Results of phytochemical group test of Z budrunga seedextract

Phytochemical group ResultReducing sugars +Alkaloids +Glycosides +Steroids minus

Gums +Saponins minus

Flavonoids +Tannins minus

+ present minus absent

0

20

40

60

80

100

120

1 2 4 8 16 32 64 128 256 512

Inhi

bitio

n (

)

Ascorbic acid

Concentration (120583gmL)

Z budrunga

Figure 1 DPPH scavenging activity of Z budrunga seed extract

t-test Experimental results were considered statistically sig-nificant when 119875 lt 005

3 Results

31 Phytochemical Group Test Phytochemical screening ofZBSE revealed the presence of reducing sugars alkaloidsglycosides flavonoids and gumswhile saponins tannins andsteroids were absent (Table 1)

32 DPPH Scavenging Activity ZBSE showed DPPH radicalscavenging activity in a concentration dependent mannerThe IC

50value for the extractwas 8260120583gmLwhile the value

was 1258 120583gmL for ascorbic acid used as standard in thisassay (Figure 1)

33 Phenolic Content Determination Phenolic content ofZBSE was found to be 33877mg GAE100 g of dried plantmaterial Gallic acid standard calibration curve equation was119910 = 0098119909+00011198772 = 0991 where119910 is the absorbance and119909 is the phenolic content expressed in gallic acid equivalent(Figure 2)

34 Reducing Power Assay Reducing ability of ZBSEincreased in a concentration dependent manner At theconcentrations of 1562 3125 625 125 250 and 500 120583gmLZBSE showed absorbances of 0502 0528 0568 0625


Table 2 Parameters of calibration graphs for the nine phenolic standards used

Peak no Polyphenoliccompound

Linearity range(120583gmL)

Correlationcoefficients (1199032)

Detection limit(120583gmL)dagger

Quantitation limit(120583gmL)dagger

Recovery()Dagger

1 Gallic acid 125ndash20 09957 025 085 983 plusmn 279

2 (+)-Catechin 125ndash20 09966 030 112 967 plusmn 165

3 Vanillic acid 125ndash20 09958 021 101 979 plusmn 285

4 Caffeic acid 050ndash80 09975 014 047 1022 plusmn 319

5 (minus)-Epicatechin 125ndash20 09955 035 120 983 plusmn 288

6 119901-Coumaric acid 125ndash20 09992 026 102 1031 plusmn 274

7 Rutin 125ndash20 09986 028 109 1028 plusmn 320

8 Ellagic acid 125ndash20 09990 031 117 992 plusmn 202

9 Quercetin 0375ndash60 09991 011 037 1003 plusmn 395

daggerData were expressed as the mean of triplicate measurementsDaggerRecoveries expressed as mean plusmn standard deviation carried out on ZBSE

0

01

02

03

04

05

06

07

157 3125 625 125 250 500Concentration (mgL)

Gallic acidLinear (gallic acid)

Abso

rban

ce at

750

nm

y = 00985x + 00012

R2 = 09917

Figure 2 Standard calibration curve of gallic acid for determinationof total phenolic content

0

05

1

15

2

25

3

0 1562 3125 625 125 250 500

Ascorbic acid


Abso

rban

ce at

700

nm

Z budrunga

Figure 3 Reducing power of Z budrunga seed extract

0695 and 0764 while ascorbic acid showed absorbancesof 0702 0863 1132 1581 2121 and 2567 respectively(Figure 3)

Table 3 Contents of polyphenolic compounds in Z budrunga seedextract (119899 = 5)

Polyphenoliccompound

Z budrunga seedsContent (mg100 g of dry extract) RSD

Caffeic acid 7545 096

Table 4 Effect of Z budrunga seed extract on acetic acid inducedwrithing in mice

Treatment (119899 = 6) Dose (mgkg) Number of writhes inhibitionControl mdash 288 plusmn 116 mdashDiclofenac sodium 25 52 plusmn 037

lowast 8195ZBSE 250 102 plusmn 071

lowast 6528500 74 plusmn 068

lowast 7430Values are expressed as mean plusmn standard error for mean 119899 number of mice(6) Studentrsquos t-test lowast119875 lt 0001 versus control

35 HPLC Analysis Among the selected phenolic com-pounds only caffeic acid was found to be present in ZBSEwith a concentration of 7545mg100 g ZBSE Gallic acidvanillic acid (+)-catechin (minus)-epicatechin caffeic acid 119901-coumaric acid rutin quercetin and ellagic acid were absentor were present beyond detection level The chromatogramalso showed peaks in regions that represent simple polyphe-nols and catechins (Figures 4 and 5 and Tables 2 and 3)

36 Writhing Test In acetic acid induced writhing ZBSEshowed 6528 and 7430 inhibition of writhing at the dosesof 250 and 500mgkg body weight respectively whereasdiclofenac sodium (25mgkg) showed 8195 inhibition(Table 4)

37 Hot-Plate Test In hot-plate test ZBSE increased thelatency time to pain stimulusThemaximum latency time forthe extract at the doses of 250 and 500mgkg bodyweight was590plusmn009 and 661plusmn007 sec respectively whereasmorphine(3mgkg) showed a maximum latency time of 941 plusmn 016 sec(Table 5)


Table 5 Effect of Z budrunga seed extract in hot-plate test on mice

Treatment (119899 = 6) Dose (mgkg) Latency time (sec)0min 30min 60min 90min 120min

Control mdash 326 plusmn 004 326 plusmn 004 327 plusmn 008 323 plusmn 007 323 plusmn 006

Morphine 5 361 plusmn 003lowast

638 plusmn 015lowast




ZBSE 250 374 plusmn 005lowast





500 382 plusmn 004lowast





Values are expressed as mean plusmn standard error for mean 119899 number of mice (6) lowast119875 lt 0001 versus control (Studentrsquos t-test)

00 50 100 150 200 250 300

Retention time (min)

minus5000

10000

20000

30000

40000

50000

60000

70000

Abso

rban

ce (m

AU)

Mixed standard

1

2

34

5

6

78

9

WVL 280nmW

VL320

nm

WV

L380

nm

Figure 4 HPLC chromatogram of a standard mixture of polyphe-nolic compounds (1 gallic acid 2 (+)-catechin 3 vanillic acid 4caffeic acid 5 (minus)-epicatechin 6119901-coumaric acid 7 rutin 8 ellagicacid and 9 quercetin)

00

0

50 100 150 200 250 300

minus100000

100000

200000

300000

400000

500000

600000

700000

1

WVL 280nm

WV

L320

nm

WV

L380

nm

Figure 5 HPLC chromatogram of Z budrunga seed extract (1caffeic acid)

4 Discussion

Phytochemicals belonging to the class of alkaloids glyco-sides and flavonoids are often associated with various bioac-tivities including antioxidant and antinociceptive activities[20] Results of the phytochemical group tests indicated thepresence of some interesting class of secondary metabolitesincluding alkaloids and flavonoids DPPH radical scavengingactivity revealed that Z budrunga seed extract possessesmoderate antioxidant activity Phenolic compounds are oneof the major groups of natural antioxidants that neutralize

free radicals by redox reactions [21] Phenolic content deter-mination by Folin-Ciocalteursquos reagent allows the estimationof all phenolic compounds present in a sample [22] Theextract was further subjected to reducing power assay sinceantioxidant activity is based on the reducing ability [23]The extract showed a high level of phenolics as well asconcentration dependent reducing powerHPLC analysis wascarried out to detect selected phenolics that are abundant inplants Although only caffeic acid was detected in the extractsome peaks were also present in regions indicating that theextract might contain compounds belonging to the class ofsimple polyphenols and catechins [24]

Acetic acid induced writhing is an easy but reliablemethod to evaluate peripherally acting antinociceptive activ-ity [18] Acetic acid triggers the liberation of free fattyacids from the phospholipids of the cell membrane whichfurther converts into various eicosanoids including prosta-cyclin (PGI

2) by the enzyme phospholipase A

2 Synthesis

of prostaglandins including PGI2through cyclooxygenase

pathway is one of the major causes of pain sensation [25]Common NSAIDs including diclofenac sodium used aspositive control in the present study exert their analgesiceffect through the inhibition of cyclooxygenase mediatedprostaglandin synthesis Inhibition of writhing in the testgroups indicated that the extract might have antinociceptiveeffect via inhibition of synthesis and release of prostaglandinsand other endogenous pain mediators Caffeic acid which ispresent in the extract is known to exert anti-inflammatoryactivity through the inhibition of arachidonic acid synthesis[26] In addition caffeic acid also inhibits the gene expressionof COX-2 enzyme [27] Caffeic acid is also reported to haveantinociceptive activity [28] However the low concentrationof caffeic acid in the extract and extent of antinocicep-tive activity suggests that some other compounds presentin the extract also contributed to the observed activityInflammatory tissue damages liberate reactive oxygen speciesduring the process of phagocytic action at inflammatory sites[29] Antioxidants can relieve pain through the preventionof lipid peroxidation that is prostaglandin synthesis fromphospholipids by neutralising reactive oxygen species [30]Thus the antioxidants present in the extract might have someassociation in the observed activity

The extract showed pain inhibitory activity in hot-platetest which represents centrally acting algesia by inducingthermal pain stimulus through changes in spinal cord level[31] Natural products of various chemical classes includingalkaloids and flavonoids can give analgesics effect through


the inhibition of opioid and other receptors that act in themodulation of pain in CNS [32ndash35]

Present investigation supported the traditional use of Zbudrunga seeds in the management of pain and antioxidantspresent in the seeds might have some role in the observedactivity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors like to thank Beximco Pharmaceuticals LtdBangladesh for providing them with diclofenac sodiumJamil A Shilpi is a postdoctoral fellow at the Centre forNatural Products and Drug University of Malaya (ResearchGrant no UMC6251HIRMOHESC37)

References

[1] K R Kirtikar and B D Basu Indian Medicinal Plants vol 2Dehra Dun Chennai India 1993

[2] M Yusuf J Begum M N Hoque and J U ChowdhuryMedicinal Plants of Bangladesh BCSIR Chittagong ChittagongBangladesh 2009

[3] M T Rahman M Alimuzzaman S Ahmad and A AChowdhury ldquoAntinociceptive and antidiarrhoeal activity ofZanthoxylum rhetsardquo Fitoterapia vol 73 no 4 pp 340ndash3422002

[4] A I A Sayeed M A U Islam G R M A M Khan andM S A Bhuiyan ldquoBrine shrimp lethality bioassay of threeterpenes isolated from the bark of Zanthoxylum budrunga walla cytotoxic evaluation of plant bioactive principlesrdquo PakistanJournal of Pharmacology vol 20 no 1 pp 1ndash5 2003

[5] MM RahmanM A Islam P Khondkar and A I Gray ldquoAlka-loids and lignans from Zanthoxylum budrunga (Rutaceae)rdquoBiochemical Systematics and Ecology vol 33 no 1 pp 91ndash962005

[6] V S Rana andM A Blazquez ldquoVolatile constituents of the seedcoat of Zanthoxylum rhetsa (Roxb) DCrdquo Journal of Essential OilResearch vol 22 no 5 pp 430ndash432 2010

[7] H Banerjee S Pal and N Adityachaudhury ldquoOccurrence ofrutaecarpine in Zanthoxylum budrungardquo PlantaMedica vol 55no 4 p 403 1989

[8] S K Paknikar and V P Kamat ldquop-Menthan-112057221205734120573-triol-revised structure ofmullilamdiol a constituent ofZanthoxylumrhetsa DCrdquo Journal of Essential Oil Research vol 5 no 6 pp659ndash661 1993

[9] B Tirilline and A M Stoppini ldquoVolatile constituents of fruitsecretory glands of Zanthoxylum budrungardquo Journal of EssentialOil Research vol 6 no 3 pp 249ndash252 1994

[10] R K Thappa K L Dhar and C K Atal ldquoA new monoterpenetriol from Zanthoxylum budrungardquo Phytochemistry vol 15 no10 pp 1568ndash1569 1976

[11] M U Ahmad M A Rahman E Huq and R ChowdhuryldquoAlkaloids of Zanthoxylum budrungardquo Fitoterapia vol 74 no1-2 pp 191ndash193 2003

[12] J B Harborne Phytochemical Methods-A Guide to ModernTechniques of Plant Analysis Chapman and Hall London UK1998

[13] C K Kokate Practical Pharmacognosy Edition VallaphPrakashan Delhi India 4th edition 1994

[14] C NicolaM Salvador A E Gower SMoura and S Echeverri-garay ldquoChemical constituents antioxidant and anticholinestera-sic activity of Tabernaemontana catharinensisrdquo The ScientificWorld Journal vol 2013 Article ID 519858 10 pages 2013

[15] D Marinova F Ribarova and M Atanassova ldquoTotal phenolicsand total flavonoids in Bulgarian fruits and vegetablesrdquo Journalof the University of Chemical Technology andMetallurgy vol 40no 3 pp 255ndash260 2005

[16] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucosaminerdquo Japanese Journal of Nutrition vol 44 no 6pp 307ndash315 1986

[17] S Chuanphongpanich and S Phanichphant ldquoMethod develop-ment and determination of phenolic compounds in Broccoliseeds samplesrdquo Chiang Mai Journal of Science vol 33 no 1 pp103ndash107 2006

[18] R Koster M Anderson and E J Beer ldquoAcetic acid for analgesicscreeningrdquo Federation Proceedings vol 18 pp 412ndash417 1959

[19] A B Aquino L H Cavalcante-Silva C B Matta et alldquoThe antinociceptive and anti-inflammatory activities of Aspi-dosperma tomentosum (Apocynaceae)rdquo The Scientific WorldJournal vol 2013 Article ID 218627 8 pages 2013

[20] A A Adeolu O S Margaret M Viola M Busani J MPatrick and J A Anthony ldquoAnti-inflammatory and analgesicactivities of the aqueous extract of Cussiona paniculata stembarkrdquo Records of Natural Products vol 2 no 2 pp 46ndash53 2008

[21] F Shahidi and P K Wanasundara ldquoPhenolic antioxidantsrdquoCritical Reviews in Food Science and Nutrition vol 32 no 1 pp67ndash103 1992

[22] S Benvenuti F Pellati M Melegari and D Bertelli ldquoPolyphe-nols anthocyanins ascorbic acid and radical scavenging activ-ity of Rubus Ribes and Aroniardquo Journal of Food Science vol 69no 3 pp FCT164ndashFCT169 2004

[23] S Meir J Kanner B Akiri and S Philosoph-Hadas ldquoDeter-mination and involvement of aqueous reducing compounds inoxidative defense systems of various senescing leavesrdquo Journalof Agricultural and Food Chemistry vol 43 no 7 pp 1813ndash18191995

[24] H Sakakibara Y Honda S Nakagawa H Ashida and KKanazawa ldquoSimultaneous determination of all polyphenols invegetables fruits and teasrdquo Journal of Agricultural and FoodChemistry vol 51 no 3 pp 571ndash581 2003

[25] J A Shilpi M E Islam M Billah et al ldquoAntinociceptive anti-inflammatory and antipyretic activity of mangrove plants amini reviewrdquo Advances in Pharmacological Sciences vol 2012Article ID 576086 7 pages 2012

[26] K Frenkel H Wei R Bhimani et al ldquoInhibition of tumorpromoter-mediated processes in mouse skin and bovine lens bycaffeic acid phenethyl esterrdquo Cancer Research vol 53 no 6 pp1255ndash1261 1993

[27] P Michaluart J L Masferrer A M Carothers et al ldquoInhibitoryeffects of caffeic acid phenethyl ester on the activity andexpression of cyclooxygenase-2 in human oral epithelial cellsand in a rat model of inflammationrdquo Cancer Research vol 59no 10 pp 2347ndash2352 1999

[28] J S Bae and T H Kim ldquoEnzymatic transformation of caf-feic acid with enhanced cyclooxygenase-2 inhibitory activityrdquo


Bioorganic and Medicinal Chemistry Letters vol 22 no 2 pp793ndash796 2012

[29] D V Parke and A Sapota ldquoChemical toxicity and reactiveoxygen speciesrdquo International Journal of Occupational Medicineand Environmental Health vol 9 no 4 pp 331ndash340 1996

[30] M Ferdous R Rouf J A Shilpi and S J Uddin ldquoAntinoci-ceptive activity of the ethanolic extract of Ficus racemosa Lin(Moraceae)rdquo Oriental Pharmacy and Experimental Medicinevol 8 no 1 pp 93ndash96 2008

[31] H O Vongtau J Abbah O Mosugu et al ldquoAntinociceptiveprofile of the methanolic extract of Neorautanenia mitis root inrats and micerdquo Journal of Ethnopharmacology vol 92 no 2-3pp 317ndash324 2004

[32] J B Calixto A Beirith J Ferreira A R S Santos V CFilho and R A Yunes ldquoNaturally occurring antinociceptivesubstances from plantsrdquo Phytotherapy Research vol 14 no 6pp 401ndash418 2000

[33] Z H U Xing-Zu ldquoDevelopment of natural products as drugsacting on central nervous systemrdquo Memorias do InstitutoOswaldo Cruz vol 86 supplement 2 pp 173ndash175 1991

[34] P L Katavic K Lamb H Navarro and T E PrisinzanoldquoFlavonoids as opioid receptor ligands identification and pre-liminary structure-activity relationshipsrdquo Journal of NaturalProducts vol 70 no 8 pp 1278ndash1282 2007

[35] D E Webster Y He S Chen G F Pauli N R Farnsworth andZ J Wang ldquoOpioidergic mechanisms underlying the actions ofVitex agnus-castus Lrdquo Biochemical Pharmacology vol 81 no 1pp 170ndash177 2011

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MEDIATORSINFLAMMATION

of


2-undecanone (71) [6] Volatile constituents (hydroxy 120572-sanshool 120573-phellandrene 120573-pinene and pipertone mullilamdiol) and alkaloids (glycosine rutaecarpine) were isolatedfrom the fruits [7ndash9] A monoterpene triol trihydroxy-119901-menthane was reported from the roots [10] while threealkaloids namely pseudophrynamine 2-(210158404101584061015840-trimethylheptenyl)-4-quinozolone and lunacridine were isolatedfrom the leaves [11] The present study was designed toevaluate antinociceptive and antioxidant activities of theethanol extract of Z budrunga seeds

2 Materials and Methods

21 Plant Material The seeds of Z budrunga was collectedfrom Tangail Bangladesh in September 2011 and identi-fied by experts at Bangladesh National Herbarium DhakaBangladesh A voucher specimen (DACB 37522) has beensubmitted there for future referenceThe seeds were removedfrom the fruit rind and separated from other parts andextraneous materials

22 Extraction The dried seeds were grinded into coarsepowder (210 g) and macerated in ethanol (95 1 L) for threedays with occasional shaking It was filtered through a clearcotton plug to remove marc Filtrate was evaporated to getgummy reddish crude extractThe seeds yielded 145 extractof dried plant material and designated as Z budrunga seedextract (ZBSE)

23 Test Animals Randomly screened young Swiss-Albinomice aged 4-5 weeks weighing 20ndash25 g were selectedfor the pharmacological investigation The mice were pur-chased from the Animal Resources Branch of InternationalCentre for Diarrhoeal Disease and Research Bangladesh(ICCDRB) and fed with ICCDRB formulated commercialrodent food and water ad libitum The investigations werecarried out following the animal ethics guidelines providedby ICDDRB

24 Chemicals and Reagents Gallic acid (+)-catechinhydrate vanillic acid caffeic acid (minus)-epicatechin 119901-coumaric acid rutin hydrate ellagic acid quercetin and11-Diphenyl-2-picrylhydrazyl (DPPH) were purchased fromSigma-Aldrich USA Folin-Ciocalteursquos reagent ascorbicacid trichloroacetic acid potassium ferricyanide sodiumcarbonate ferric chloride acetic acid and dimethyl sulfoxide(DMSO) were purchased from Merck Germany Tween-80 was purchased from Loba Chemie India Diclofenacsodium morphine and vincristine sulphate were obtainedfrom Beximco Pharmaceuticals Ltd Bangladesh PopularPharmaceuticals Ltd Bangladesh and Cipla PharmaceuticalsIndia respectively

25 Phytochemical Analysis ZBSEwas subjected to a numberof standard tests to detect the presence of major phytochem-ical groups [12 13]

26 DPPH Radical Scavenging Activity In vitro antioxidantactivity was determined based on the ability of the seedextract to scavenge the stable free radical DPPH [14] Stocksolution of ZBSE was prepared in ethanol and seriallydiluted to obtain different concentrations with the maximumand minimum of 512 and 1 120583gmL respectively From eachconcentration 1mL was mixed with 2mL DPPH solution(0004 in ethanol) The mixture was kept for 30 minutesto complete the reaction The absorbance was measured at517 nm in a UV-visible spectrophotometer Ascorbic acid wasused as standard antioxidant The percent inhibition wascalculated according to the formula (1minus119860

11198600) times 100 where

1198600is the absorbance of control and 119860

1is the absorbance of

ZBSE or standard IC50value was determined from the graph

obtained by plotting inhibition versus concentration

27 Determination of Total Phenolic Content (TPC) Grindedplant material (05 g) was mixed with 50mL of 80 aqueousmethanol and sonicated for 20min An aliquot of 2mLwas taken from it and centrifuged for 15min at 14000 rpmTotal phenolic content was determined by Folin-Ciocalteursquosreagent [15] Standard gallic acid solutions were preparedby serial dilution to get concentrations of 500 250 125625 3125 and 1562mgL Gallic acid solutions of eachconcentration and ZBSE of 1mL were transferred to 25mLvolumetric flasks and 9mL distilled water was added Folin-Ciocalteursquos reagent of 1mLwas added to each volumetric flaskwith continuous shaking After 5min 10mL of 7 Na

2CO3

was added and adjusted with distilled water to make thefinal volume of 25mL It was kept for 30min to completeany reaction that occurs The absorbance was measured at750 nm against blank Blank was prepared by following thesame steps mentioned above without adding ZBSE or gallicacid Standard curve of gallic acid was prepared by plottingabsorbance versus concentration The total phenolic contentof ZBSE was determined using the standard calibration curveof gallic acid and the value was expressed as gallic acidequivalent (GAE)100 g of dried plant material

28 Reducing Power Assay Reducing power of the extractwas determined according to the method described byOyaizu [16] ZBSE of 1mL with the concentrations of 500250 125 625 3125 and 1562 120583gmL were mixed with25mL of 02M phosphate buffer (pH 66) and 25mLof 1 potassium ferricyanide with vigorous shaking Themixtures were incubated for 20min at 50∘C After coolingat room temperature 25mL of 10 trichloroacetic acid wasadded and centrifuged at 3000 rpm for 10min An aliquot(25mL) of the supernatant wasmixedwith 25mL of distilledwater and 05mL of ferric chloride (01) After 5min theabsorbance was measured at 700 nm Ascorbic acid was usedas standard

29 HPLC Detection of Phenolics Detection of selectedphenolic compounds in ZBSE was determined by HPLCanalysis as described by Chuanphongpanich and Phanich-phant with some modifications [17] It was carried out ona Dionex UltiMate 3000 system equipped with quaternary













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Autoimmune Diseases




Journal of


Pharmaceutics



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Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





















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4 Discussion





2 Synthesis









Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of














Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Research Article Antinociceptive and Antioxidant Activity ...downloads.hindawi.com/journals/tswj/2014/869537.pdf · Antinociceptive and Antioxidant Activity of Zanthoxylum budrunga