ORIGINAL ARTICLE

Antidiabetic effect of Artemisia absinthium extractson alloxan-induced diabetic rats

Haytham M. Daradka & Mohaned Mohamed Abas &Mukhallad A. M. Mohammad & Mohamed Mousa Jaffar

Received: 3 April 2014 /Accepted: 17 June 2014# Springer-Verlag London 2014

Abstract The aim of our study was to evaluate the effect ofArtemisia absinthium (A. absinthium) ethanol extract onalloxan-induced diabetic rats. Forty-eight albino rats (300 g)were used in this experiment and divided into six groups.Diabetes was induced in five rat groups by a single intraperito-neal injection of alloxan (150 mg/kg body weight). Afterhyperglycemia was confirmed, one group was considered asdiabetic control and one group was treated with glibenclamide(10 mg/kg body weight/ daily) where the remaining threegroups received daily treatments with three different doses ofA. absinthium extract (250, 500, and 1,000mg/kg body weight)for 10 days each dissolved in 0.2 ml distilled water was admin-istered intraperitoneally to each corresponding rat group. Bloodserum biochemical markers such as urea, creatinine, cholester-ol, and total serum protein levels were recorded after thetreatment ended. Findings indicate that treatment with mediumand high doses of A. absinthium extract (500 and 1,000 mg/kg/body weight) reduces blood sugar values to significant levels(P<0.01 and P<0.001) in rats after 7 and 10 days of treatmentwhen compared with diabetic control alloxan-induced rats in asimilar fusion as in glibenclamide treatment (P<0.001). Allelevated blood serummarkers induced by the alloxan treatmentwere reduced to significant levels in rats treated withA. absinthium at both medium and high doses (P<0.01 andP<0.001) and also after glibenclamide treatment (P<0.001).

We can conclude that A. absinthium treatment exhibits a sig-nificant antihyperglycemic effect without altering the bodyweight and can correct some biochemical markers induced bydiabetes in a similar manner to glibenclamide treatment.

Keywords Alloxan . Diabetes .A. absinthium . Bloodglucose . Antihyperglycemic effect . Biochemical markers

Introduction

Diabetes mellitus (DM) is a pattern of impaired carbohydrate,fat, and protein metabolism caused by either deficiency ininsulin secretion or reduced sensitivity of the tissues to insulin(Alberti and Zimmet 1998). From ancient times and eventoday, DM remains a major health problem that affects almost5 % of the population (Lokesh and Amit 2006). The earliestdescription of its symptoms is found in an Egyptian papyrusdating back to 500 BC (Ebbel 1937). In the second century,The first known clinical description of diabetes appears tohave been made by Aulus Cornelius Celsus; but the Greekphysician Aretaeus provided a detailed and accurate accountand introduced the name “diabetes” from the Greek word for“siphon” (Ebbel 1937). “Diabetes he said is a strange diseasewhich consists of the flesh and bones running together in tourine” (Sanders 2002). This rightly explains the basicsymptoms of abundant urine flow accompanied by se-vere weight loss and depletion of both muscle and fat.In 1674, Sir ThomasWillis observed that the urine of diabeticsis sweet to the taste, hence the descriptive term “mellitus” orhoneyed (Willis et al. 1684).

More than 180 million people worldwide are diabetic, andthe occurrence is expected to more than double by the year2030 (Torben 2002).

In current years, developed nations have witnessed anexplosive rising in the occurrence of DM mainly related to

H. M. Daradka (*)Department of Biological Sciences, Faculty of Science, TaibahUniversity, Al-Madinah Al-Munawwarah 41321, Saudi Arabiae-mail: hmdaradka@yahoo.com

M. M. Abas :M. A. M. Mohammad :M. M. JaffarDepartment of Physiology, Faculty ofMedicine, Jordan University ofScience & Technology, P.O. Box 3030, Irbid 22110, Jordan

H. M. DaradkaBiology Department, Faculty of Science, Taibah University,Al-Madinah Al-Munawwarah 41321, Saudi Arabia

Comp Clin PatholDOI 10.1007/s00580-014-1963-1

lifestyle changes and the resulting surge in obesity(Ramachandran et al. 2002). At present, India became thecapital of diabetes, for the reason that they have prevalenceof diabetic patients (Sicree et al. 2006). Also, there are manypatients in the community with undiagnosed diabetes.Decreased physical activity, increasing fatness, stress, andchanges in food have been concerned in this growing preva-lence in the past two decades. DM is being presented as theWorld’s major disabler and murderer in the next 25 years(Edwin et al. 2006).

Simply, diabetes can be defined as “too much glucose inblood.” It is not a single disease but a heterogonous group,resulting frommany environmental and some heredity factors.It is not only a metabolic disease with hyperglycemia andinsulin deficiency but also vascular disease, and theseacting jointly and during the course of the disease affectthe blood vessels particularly of the eyes, kidney, heart, andbrain (Duby et al. 2004).

Alloxan (2, 4, 5, 6-tetraoxypyrimidine; 2, 4, 5, 6-pyrimidine tetrone) is an oxygenated pyrimidine derivativewhich is a toxic glucose analogue that can selectively destroythe insulin-producing cells in the pancreas when administeredto rodents and many other animal species (Lenzen 2008). Thiswill lead to an insulin-dependent DM (called “alloxan-in-duced diabetes”) in these animals. This diabetic inductionhas characteristics similar to type I diabetes in humans(Lenzen 2008).

Artemisia absinthium, also known as commonwormwood,is a member of the Asteraceous family, which is an herbaceousperennial plant with strong sage odor (Nezhadali and Parsa2010). The plants are normally about 1–1.2-m tall at maturitybut can grow up to 2 m. Leaves are 2–5-in. lengthy, separatedtwo to three times into deeply lobed leaflets, and are bright toolive green in color. The leaves and stems of the plant areenclosed with small smooth hairs that provide the plant a graymanifestation. The stems are also woody at the base of theplant; flower tails appear into view at each superior leaf knoband make many yellow flower tops that are 1/8 in. in width.A. absinthium seed is less than 1/16-in. long, fine, plane, andbright gray brown in color. The leaves and flowers are vine-gary, with a characteristic smell similar to that of thujone(Palevitch and Yaniv 1991).

The common wormwood is native to warm Mediterraneancountries, usually developing on waysides and waste places,preferring to a nitrogen-rich stony and hence loose soil(Nezhadali and Parsa 2010).It is originating over the greatersection of Europe, Asia, and northern Africa (Nezhadali andParsa 2010). The flowering begins from early summer to earlyautumn with anemophilous pollination (Nezhadali and Parsa2010). The entire plant (leaves, leg, and tops) is used forpreparation of traditional medicine.

The wormwood (A. absinthium) contains the monoterpene(thujone) active component and absinthen (John et al. 1999;

Olsen 2000), beside azulenes, phenolic compounds, and fla-vonoids, which give antiradical and antioxidative activity(Jasna et al. 2004).

The major chemical constituents of the essential oil ofA. absinthium include the volatile oil consisting of α- and β-thujones, thujyl alcohol, cadinene, camphene, sabinene, pi-nene, and phellandrene (Williamson 2003).

Thujone, a monoterpene, which exists as two stereo iso-mers (α- and- β- thujone), is an ingredient of essential oils ofmany great different herbs, including A. absinthium. Thujoneoil has long been used in beverages, food additives, and herbalmedicine (Lopes-Lutz et al. 2008), and it is thought to be themain constituent of several medicinal herbs that have antidi-abetic properties (Al-Mustafa and Al-Thunibat 2008;AlarconA et al. 2002).

Thujone

The name “wormwood” is resulting from its antihelminticcharacters; this was identified through early people of Egypt(Padosch et al. 2006).

The medical necessity dates back to the Ancient EgyptianMedicine and the early conserved remedial files (Anon 1937).The Greek word apsinthion, meaning “undrinkable,” is likelythe ancestor of the word absinthe, which was used in Frenchfor the plant spices as well as for the alcoholic beverage(Padosch et al. 2006). The Greek Pythagoras of Samos sug-gested wine-waterlogged A. absinthium as a reliever of painsaccompanied with labor. Hippocrates used A. absinthiumin the management of menstruation pains and any dis-order of the extremities or back characterized by pain andstiffness (Baker 2001).

At the period of European history between ancient andmodern times, A. absinthium was used as purified and vermi-fuge and was described as “full medicinal description formany different disease” (Baker 2001). The soldiers takeA. absinthium as a protection from diseases caused by para-sitic worms in the wars and to reduce body temperature, and ifflooded, held to destroy germs and ward off dysentery(Padosch et al. 2006; Nathan 2008).

A. absinthium had been suggested to relieve gastric painand as a cardiac stimulant and a curative of declining mentalfunctions (Grieve 1980).

Other pharmacological properties of A. absinthium havebeen reported to exhibit antiinflammatory effect (Ikram et al.1985). Additionally, protective property of this extract has

Comp Clin Pathol

been experimentally proven on hepatic and gastric damage inanimals (Gilani and Janbaz 1995). Because of its bitter taste,A. absinthium extract has been added to beer, tea, coffee, andfood as spices and because this properties, the women put it totheir nipples for ablactating their children (Ikram et al. 1985).Thujone of A. absinthium has a chemical constitutionsimilar to that of delta9 tetrahydrocannabinol (an activeingredient of marijuana) and a high dose of this substanceinduced on the receptors common in the CNS causes halluci-nation, derangement, and convulsion in humans (Williamson2003; John et al. 1999).

The dried leaves, flowering tops, and essential oil of worm-wood have traditionally been used as an antiseptic, antispas-modic, carminative, sedative, stimulant, stomachic, and tonic(Simon et al. 1984).

Other uses of wormwood comprise the management ofdyspepsia, colds (Nezhadali and Parsa 2010), esophagealreflux, irritable bowel syndrome (Gambelunghe and Melai2007), flatulence and gastric pain (Guarrera 2005), inflamma-tion, joint pain, anorexia (Guarrera 2005), hypertension, andcardiac disease (Eddouks et al. 2007) as well as insomnia,epilepsy, and menstrual problems (Gambelunghe and Melai2007). The antimalarial activity against Plasmodiumfalciparum of A. absinthium was demonstrated (Rucker et al.1991) as well as choleretic, depurative, digestive, diuretic, andin treating leukemia and sclerosis (Canadanovic et al. 2004),antifungal (Kordali et al. 2005), and antimicrobial activity(Lopes-Lutz et al. 2008).

A. absinthium failed to show direct antitumor activities butshowed a definite antimetastatic effect which could beexploited as a correction against homeostasis disturbance(Gribel and Pashinskii 1991), absinth wormwood taints milkwhen eaten by cows (Lym et al. 1984). Wormwood is also usedto relieve pain during childbirth (Nezhadali and Parsa 2010).

This study aimed to further investigate the effects of differ-ent doses of A. absinthium on hyperglycemic rats induced byalloxan. We tested the hypothesis that daily administration ofA. absinthium for 20 days can reduce hyperglycemia in dia-betic male rats. Also, the toxicity of different extracts wasevaluated.

Materials and methods

Plant collection

A. absinthium plants were recognized and collected from theIrbid area located to the north of Jordan. The plant wasidentified by one of Jordan’s most knowledgeable professors,Ahmad Al-Olqlah (Biology Department, YarmoukUniversity). The plant was left to air dry partially for 2–3 daysat room temperature. The plant was chopped then grindedusing an electrical grinder until powder was obtained.

Plant ethanolic extraction

Each 500 g of this powder was extracted by ethanol-watermixture (70/30V/V) for 48 h. This step was repeated for threetimes then the filtrate was then refluxed in (2 l) 70% ethanol at50 °C using a rotary evaporator for 36 h in continuous extrac-tion (Soxhlet) apparatus(S.P. Verma, Popular ScienceApparatus Workshops PVT LTD, India). Pooled and concen-trated ethanol extract was filtered, and it was re-concentratedunder reduced vacuum pressure keeping a constant tempera-ture less than 50 °C. The concentrate yield from this processwas 80 g which was kept in room temperature for later use.

Experimental animals

Prior to the initiation of the experiment, an ethical clearance forperforming the experiments on animals was obtained from theInstitutional Animal Care and Use Committee (IACUC). Forty-eight (Cordoba et al. 1999) Albino rats (Wister strain) weighingapproximately 300 g were used throughout the experiments.Rats were raised in the Animal House Unit at Jordan Universityof Science and Technology, faculty of Medicine (JUST).Before the initiation of the experiment, rats were acclimatizedfor a period of 7 days with standard environmental conditionssuch as temperature (20±2 °C), relative humidity (45–55 %),and 12-h dark/light cycle. All rats were fed with rodent pelletdiet and water ad libitum under strict hygienic conditions.

Induction of diabetes in rats with alloxan treatment

The average of fasting blood sugar in normal rats is 60 mg/dl,which was determined by tail vein puncture in all rats afterdepriving them from food for 16 h with a free access to drinkingwater. Then, diabetes was induced in 40 rats by one singleintraperitoneal dose of 120 mg/kg body weight alloxanmonohydrate that was dissolved in 0.9 % normal saline, pH 7(S.d. fine-chem. Ltd., Mumbai, India) (Eman et al. 2005) toinduce type II diabetes. After 5 days of alloxan injection, hy-perglycemia was established when blood sugar levels measuredin rats were found to be above 250 mg/dl (Dunn et al. 1943).

After induction of diabetes, treatment was carried onthrough daily oral administration of A. absinthium andGlibenclamide for a period of 10 days (Sharma et al. 2010),using an animal feeding intubation needle as single daily doseat the same day when diabetes had been established (Popperand Sons, New York).

Determination of LD50 for A. absinthium extracttreatment in rats

Acute toxicity of the plant was determined by the calculationof LD50 which represents the dose that can be fatal to 50 % ofany rats group (Hruskova et al. 1961).

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Determination of LD50 in rats was conducted to determinethe proper treatment dose that should be used in this experi-ment. For this procedure, 24 albino rats were needed and used,distributed into four different groups each containing 6 albinorats (300 g). Different dosage A. absinthium root ethanol ex-tract, namely, 200, 400, 600, 1,000, 3,000, 4,000, and 5,000 mgeach dissolved in 0.2 ml normal saline (NaCl 0.9 %) wasadministered intraperitoneally to each corresponding rat group.Rats were then housed in transparent plastic cages under con-trolled temperature of 24 °C and monitored for 24 hrepresenting the time length of this experiment for any toxicsymptoms. The number of deceased rats was counted in eachgroup after 24 h, and the percentage of mortality was calculat-ed. Treated rats were compared with six controlled rats that hadreceived an intraperitoneal infusion of 1 ml distilled wateralone, in the same manner with the experimental rats.

Rat grouping and treatment

Rats were divided in six groups (A to F) each group contain-ing eight albino rats weighing approximately 300 g each. Onerat group that was not treated with alloxan is considered acontrol group representing the nondiabetic rats indicated asgroup A. All other rat groups were treated with alloxanmonohydrate after hyperglycemia was induced and con-firmed, and were then divided into five groups B–F as thefollowing:

Group A: Untreated normal rats (control) receiving 2 mlnormal saline

Group B: Diabetic induced rats with alloxan monohydratereceiving 2 ml normal saline.

Group C: Diabetic induced rats with alloxan monohydratetreated with Glibenclamide at a dose of 10 mg/kgbody weight dissolved in 2 ml normal saline

Group D: Diabetic induced rats with alloxan monohydratetreated with A. absinthium ethanol extract at adose of 250 mg/kg body weight dissolved in2 ml normal saline (low dose)

Group E: Diabetic induced rats with alloxan monohydratetreated with A. absinthium ethanol extract at adose of 500 mg/kg body weight dissolved in2 ml normal saline. (medium dose)

Group F: Diabetic induced rats with alloxan monohydratetreated with A. absinthium ethanol extract at adose of 1,000 mg/kg body weight dissolved in2 ml normal saline. (high dose).

Rats from the normal nondiabetic control (groupA) and thediabetic control groups (group B) were left in separate cageswith free access to water and food ad libitum. Treated rats inall groups had free access to standard diet and water inaddition to their daily oral treatment doses. Body weight and

blood glucose levels were estimated on the fourth, seventh,and tenth day of the treatment. By the end of the treatment,blood samples were collected by cardiac puncture from all ratsthat were left overnight fasting after anesthesia was applied forbiochemical parametric estimation.

Blood glucose estimation

Blood glucose level was estimated from collected blood sam-ple using a glucometer device (Infopia Co. Ltd 891, SouthKorea) with a special cylindrical instrument.

Procedure

Introduce the rat inside the cylindrical instrument and lock outwith rat tail outside the cylinder. And then, we cut off the tip ofthe tail to get a blood sample which will be placed on the stripof the glucometer for measuring blood glucose level.

Hematological analyses

The blood pooled from all the animals in each group wascollected into bottles containing ethylenediaminetetraaceticacid (EDTA) as anticoagulant. The hematological composi-tion of the blood was measured using all groups of rats (n=6).

The hematological parameters were measured includingthe following: red blood cells and white blood cells whichwere estimated by using the improved Neubauer countingChamber. Hemoglobin was measured by using Drabkin’ssolution as described in (Drabkin’s Cynmetheglobin methodby Fisher’s Haemo-photometer) (114). Packed cell volume(PCV) was determined.

Biochemical analyses

Serum was obtained by centrifugation of blood at 6,000 rpmfor 30 min. Serum obtained was used to examine the follow-ing biochemical tests: total cholesterol, triglycerides, serumaspartate aminotransferase (AST), serum alanine aminotrans-ferase (ALT), serum creatine kinase (CK), serum creatine,lactate dehydrogenase(LDH), total protein, creatinine, andblood urea using commercial kits from (BioSystems S.A.Costa Brava30, Bacelona-Spain). Parameter concentrationswere determined using a UV/visible spectrophotometer(Milton Roy Spectronic 601 Spectrophotometer USA).

All tests were done in the lab of physiology in theDepartment of Physiology and Biochemistry.

Statistical analyses

Data were assessed using SPSS program (version17.0, SPSS).P values <0.05 between mean values were considered statis-tically significant.

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Result

LD50 for A. absinthium extract treatment in rats

All the animals that received 0, 250, 500,1,000, 2,000, 3,000,4,000, and 5,000 mg/kg of A. absinthium extract orally weresurvived beyond the 24 h of administration. No behavioralchanges and no mortality were noted. The rats move and fednormally.

The result of the oral acute toxicity study showed thatLD50 of A. absinthium extract was not toxic even at a doseof 5,000 mg/kg body weight.

Effects of A. absinthium ethanol extract on fasting bloodglucose levels in alloxan-induced diabetic rats

A marked increase in fasting blood glucose level was recordedin alloxan-induced diabetic rats (diabetic control, groupB) whencompare with the normal control (group A). Glibenclamideinduces a significant reduction (P<0.001) in blood glucoselevels when we used in treating alloxan-induced diabetic ratsin (group C). It was noticed that A. absinthium ethanol extractexhibited a time-dependent significant hypoglycemic activity inmedium dose (500 mg/kg body weight, P<0.01) and high dose(1,000 mg/kg body weight, P<0.001) (groups E and F), whichwas clearly after day 10 treatment period when compared withdiabetic control group of rats (group B). Reduction of bloodglucose levels was observed to be insignificant in diabeticinduced rats receiving a dose of 250 mg/kg body weightA. absinthium extract (group D) throughout the entire periodof the treatment (Table 1 and Fig. 1).

This reduction in blood glucose levels observed in ratstreated with A. absinthium (in groups E and F) exhibitedsimilar time-dependent trend of significance when comparedwith rats treated with Glibenclamide (group C). However,Glibenclamide and A. absinthium treatments failed to bringthe blood glucose levels to normal values as in the nondiabeticcontrol rats (group A).

Antidiabetic effect of A. absinthium ethanol extract on bodyweight of diabetic rat

Table 2 showed that the body weight of normal control groupwas found to be stable throughout the housing period. Alloxantreatment induces great reduction in rat’s body weight duringtreatment especially after 10 days. Alloxan-mediated bodyweight reduction was reversed by A. absinthium ethanol ex-tract in a dose-dependant manner especially when rats weretreated with medium and high doses (500 mg/kg and1,000 mg/kg body weight) of A. absinthium (P<0.05 for bothtreatment doses). This body weight value correction was alsoobserved to be more pronounced only after 10 days in asimilar fashion as in Glibenclamide treatment (P<0.05) andwhen compared with alloxan-induced diabetic rats (Fig. 2).

Effects of A. absinthium ethanol extract on hematologicalparameters of diabetic rats

Table 3 shows the various hematological parameters in thedifferent groups of diabetic rats. There were no significantdifferences in hematological composition of blood parametersbetween, 250 mg/kg, 500 mg/kg, and 1,000 mg/kg ofA. absinthium-treated group and diabetic control group(Fig. 3).

Effects of A. absinthium ethanol extract on biochemicalparameters of diabetic rats

These results in Table 4 showed that serum urea, serumcholesterol levels, and serum creatinine were decreased sig-nificantly to almost normal levels with significant increase inserum protein level after 10 days in diabetic rats treated withmedium and high doses (500 and 1,000 mg/kg body weight)of A. absinthium ethanol extract after alloxan treatment(P<0.01 and P<0.001). The alterations of these parameterswere observed to be more marked after treatment with highdose 1,000 mg/kg of A. absinthium extract (P<0.001) in a

Table 1 Effect of A. absinthium on fasting blood glucose level in alloxan-induced diabetic rats

Treatment Fasting blood glucose level (mg/dl)

Basal value 4th day 7th day 10th day

Normal control 92.27±2.36 94.67±2.74 94.17±2.24 90.11±2.33

Diabetic control 299.66±4.67 292.69±4.33 297.66±5.72 295.22±4.17

Diabetic + Glibenclamide (10 mg/kg) 291.5±4.87 209.1±4.46* 186.9±4.51* 181.6±4.93*

Diabetic + Ethanolic extract (250 mg/kg) 297.6±4.47 283.3±4.15 271.5±4.89 260.4±4.79

Diabetic + Ethanolic extract (500 mg/kg) 290.1±4.73 263.4±4.08* 260.9±4.84** 257.1±4.46*

Diabetic + Ethanolic extract (1,000 mg/kg) 293.3±4.04 216.14±3.82* 202.4±4.03* 193.6±4.17*

Values are mean±SEM; n=6

*P<0.05, significant compared to diabetic control

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similar trend when Glibenclamide treatment is applied(P<0.001). It was observed that treatment of A. absinthiumethanol extract at low dose (250 mg/kg body weight) failed toreverse the altered biochemical parameters in rats induced byalloxan. The other biochemical parameters were not showingalteration in values for different groups when compared withdiabetic control group.

Discussion

In the present study, we focused on A. absinthium which is anherbaceous perennial plant with a hard, woody rhizome andusing for different therapeutic uses in the field of folk medi-cine (Maw et al. 1985). We examined the antidiabetic activityof A. absinthium in alloxan-induced diabetes in experimentalrats. Alloxan causes a huge reduction in insulin secretion bythe damaging beta-cells of the islets of Langerhans, thereby

inducing hyperglycemia (Lenzen 2008). Insulin deficiencyleads to various metabolic alterations in the animals likeincreased blood glucose, increased cholesterol, and alterationin liver and kidney function (Shanmugasundaram et al. 1983),for the reason the maintenance of normal blood glucose levelis necessary to prevent diabetes complications.

Our experimental study confirms that A. absinthium showsclear hypoglycemic activity for the treatment of alloxan-induced diabetic rats. Administrations of the A. absinthiumextract significantly decrease the blood glucose level in thediabetic rats when compared to diabetic control rats as inTable 2 and Fig. 1).

In our study, the different doses of ethanolic extrac-tion of A. absinthium produced a significant reductionin blood glucose level in a dose-dependent manner. Thedose of 250 mg/kg did not show significant reduction inblood glucose level in comparison to other doses, whereasdoses 500 mg/kg and 1,000 mg/kg were showed higher

Table 2 Effect of A. absinthium ethanol extract on body weight in alloxan-induced diabetic rats

Treatment Body weight of the animal (g)

Initial 4th day 7th day 10th day

Normal control 311.69±2.08 313.27±2.18 315.97±2.52 320.51±2.38

Diabetic control 313.79±2.92 180.7±2.32 165.91±2.48 152.33±1.37

Diabetic + Glibenclamide (10 mg/kg) 316.94±2.43 313.27±2.18* 306.43±1.78* 301.71±1.52*

Diabetic + Ethanolic extract (250 mg/kg) 316.42±2.36 287.16±2.68* 271.24±2.78* 259.69±1.12*

Diabetic + Ethanolic extract (500 mg/kg) 315.89±2.32 310.04±2.88* 301.36±1.94* 291.72±1.83*

Diabetic + Ethanolic extract (1,000 mg/kg) 316.94±2.43 310.12±2.21* 303.81±1.64* 296.64±1.22*

Values are mean±SEM; n=8

*P<0.05, significant as compared to diabetic control

Fig. 1 Effect of Artemisiaabsinthium on fasting bloodglucose level in alloxan-induceddiabetic rats

Comp Clin Pathol

significant reduction in blood glucose level (P<0.001) than the250 mg/kg dose.

The significant antihyperglycemic activity of ethanolic ex-traction of A. absinthium as in Fig. 2 may be due to thepresence of active components of A. absinthium like α- andβ-thujones, thujyl alcohol, azulenes, bisabolene, cadinene,sabinene, pinene, and phellandrene (Muto et al. 2003;Clarke and Clarke 1975; Alkhateeb and Bonen 2010;Lott and Wolf 1986; Cordoba et al. 1999); the exactmechanism of action in diabetes of most of these componentsare unknown.

This reduction in blood glucose level due to presence ofthujone, a major component of A. absinthium and severalmedicinal herbs, had an effective in insulin-sensitizing action,which set free insulin-stimulated glucose transport by activa-tion of adenosine monophosphate-activated protein kinase(AMPK) which partly normalized insulin-stimulated GLUT4translocation to the cell surface. In addition, it appears that

thujone also enhanced the GLUT4 intrinsic activity(Alkhateeb and Bonen 2010).

A. absinthium extract like Glibenclamide could inducehypoglycemia by stimulating insulin release and its ac-tion, thereby enhancing cellular uptake and utilization ofglucose in rats.

The plant extracts may also contain some biomolecules thatmay sensitize the insulin receptor to insulin or stimulate the β-cells of islets of Langerhans to secret insulin which may leadto enhancement of carbohydrate metabolizing enzymes in thedirection of the re-organization of normal blood glucose level.It is possible that A. absinthium extracts may act by undeter-mined ways to stimulating insulin production from the pan-creatic islets of Langerhans besides effect of thujone.

It is clear that A. absinthium changed the body weight ofdiabetic rats (Table 2 and Fig. 2); generally, there is a decline inthe body weight of diabetic untreated group (normal control)because of consumption of glucose and shift to catabolism of

Table 3 Hematological parameters of diabetic rats after 10 days administration of A. absinthium ethanol extract

Treatment WBC (×103/μl) RBC (×106/μl) PCV (%) Hb (g/dl) MCV (fl) MCH (pg) MCHC (g/dl)

Normal control 4.01±17.7 7.40±41. 40.8±2.3 14.0±0.8 55.2±1.9 18.9±1.0 34.3±0.7

Diabetic control 4.76±12.3 7.18±22 39.8±2.4 13.8±0.4 55.2±1.9 18.2±1.02 34.5±0.7

Diabetic + Glibenclamide (10 mg/kg) 4.85±14.3 6.89±24 39.7±2.5 13.6±0.5 55.2±1.9 18.1±1.09 34.5±0.7

Diabetic + Ethanolic extract (250 mg/kg) 4.56±14.5 7.25±28.2 41.1±1.3 14.2±0.2 54.8±1.2 18.9±0.9 34.5±0.7

Diabetic + Ethanolic extract (500 mg/kg) 4.18±10.5 7.43±38.2 40.3±1.9 13.9±0.6 54.2±1.5 18.7±0.8 34.5±0.7

Diabetic + Ethanolic extract (1,000 mg/kg) 3.68±62.7 7.45±52.9 41.1±2.6 14.0±0.5 55.2±2.2 18.9±1.1 34.1±1.5

Values: mean±SEM. n=6. No significance (P value >0.05)

Fig. 2 Effect of A. absinthiumextract on body weightin alloxan-induced diabeticrats

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fats and protein, even though the food intake is more in diabeticrat groups than normal control group (Nishikawa et al. 2000).

The final weight of untreated control group (normal con-trol) was raised than at the beginning of the experiment. Onthe contrary, there was a decrease in body weight in diabeticcontrol group. Similarly, the treated groups with A. absinthiumprevent the reduction in body weight in a significant manner.Correction of body weight loss may be due to increased usageof glucose as a source of energy instead of fats and proteinssecondary to increased serum insulin (Nishikawa et al. 2000).

The shift of metabolic pathway towards carbohydrate as asource of energy with saving proteins and fats as well as their

increased biosynthesis led to prevent body weight reduction indiabetic rats treated with A. absinthium (Peavy et al. 1985).

RBC and WBC counts, hemoglobin, hematocrit, and RBCindices in diabetic rats treated with A. absinthium did notchanged significantly and remained within normal limits(Table 3 and Fig. 3).

The prevalence of atherosclerosis and hyperlipidemiaamong diabetics is on the increase worldwide. Alterations inserum lipids profile are known in diabetes, which are likely toincrease the risk of coronary heart disease (Sharma et al. 2003).

Triglyceride and total cholesterol which are altered in theserum of diabetic patients (Orchard 1990) appeared to be a

Table 4 Biochemical parameters of alloxan induced diabetic rats after given A. absinthium ethanol extract for 10 days

Treatment Serum urea (mg/dl) Serum creatinine (mg/dl) Serum cholesterol (mg/dl) Serum protein (mg/dl)

Normal control 28.18±1.05 0.52±0.12 47.53±1.88 6.23±0.4

Diabetic control 59.13±1.07 1.41±014 143.16±1.25 4.73±0.82

Diabetic + Glibenclamide (10 mg/kg) 31.22±1.18 * 0.61±.02* 55.5±2.08* 6.11±0.23*

Diabetic + Ethanolic extract (250 mg/kg) 56.83±1.45 1.23±0.02 132.5±4.66 5.18±0.11

Diabetic + Ethanolic extract (500 mg/kg) 39.75±1.62* 0.82±0.02* 69.33±2.70* 5.83±0.15*

Diabetic + Ethanolic extract (1,000 mg/kg) 34.24±0.72* 0. 65±.01* 60.08±2.69* 6.11±0.20*

Values are mean±SEM; n=6

*P<0.05, significant as compared diabetic control

Fig. 3 Effect of A. absinthiumethanol extract on hematologicalparameters of diabetic rats

Comp Clin Pathol

significant factor in the development of premature atheroscle-rosis through increase in serum triglyceride and total choles-terol levels (Betteridge 1994).

A reduction in total cholesterol could be beneficial inpreventing diabetic complications as well as improving lipidmetabolism in diabetics. Considering A. absinthium extracteffects on lipid components, it can be assumed as a potentialhypolipidimic agent which will be a great advantage both indiabetic conditions as well as the associated atherosclerosis orhyperlipidemia conditions. The levels of total cholesterol havebeen decreased significantly in diabetic rats after the admin-istration of A. absinthium (Table 4); this effect may be due tolow activity of cholesterol biosynthesis enzymes and or lowlevel of lipolysis which are under the control of insulin. Sinceinsulin has a potent inhibitory effect on lipolysis in adipocytes,insulin deficiency is associated with excess lipolysis andincreased influx of free fatty acids to the liver (Coppacket al. 1994; Ohno et al. 2000).

Renal disease is one of the most common and severecomplications of diabetes, which alter the concentration ofamino acid product (Siperstein et al. 1973; Urger and Foster1998; Shanmugasundram et al. 1990). A significant decreasein serum protein concentration was observed in alloxan-induced diabetic rats (Table 4). Insulin promote amino aciduptake, enhance protein synthesis, and inhibit protein degra-dation (Denne et al. 1991; Travlos et al. 1996).

The blood urea and creatinine are a marker of renal func-tion (Braunlich et al. 1997; Hwang et al. 1997) and aresignificantly increased in diabetic rats. Different extracts ofA. absinthium brought about a significant increase in serumprotein and a decrease in urea and creatinine levels of alloxan-induced diabetic rats (Table 4).

Our result also showed decrease in protein and increase inserum urea and creatinine concentrations, which are consid-ered as a marker of kidney dysfunction and has been correctedby administration ofA. absinthium extracts in alloxan-induceddiabetic rats. This effect is due to the antihyperglycemicactivity of the A. absinthium, which might have increasedthe uptake of glucose by the tissue and its utilization andcorrect kidney function.

This indicates that the treatment with A. absinthium is nottoxic and that it guarantees the biosecurity of using theseextracts as an antihyperglycemic treatment.

Conclusion

From this study, we can state that the extract of A. absinthiumhas a hypoglycemic effect on alloxan-induced diabetes in rats.The extract of A. absinthium also prevents the sever reductionin body weight and improvement biochemical parametersassociated with DM such as total cholesterol, serum protein,urea, and creatinine, but did not show significant differences

in hematological parameters. Further pharmacological studiesare needed to isolate the active diabetogenic components ofA. absinthium to clarify their exact mechanism of action, andwill be supportive in projecting the A. absinthium as a thera-peutic agent in diabetes.

The study suggests that A. absinthium could be used intraditional medicine for the management of DM.

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