adaptogenic activity of trigonella foenum graecum (linn) seeds in

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Document heading

Adaptogenic activity ofTrigonella foenum graecum (Linn) seeds in

rodents exposed to anoxia and immobilization stress

Vinod S Pawar1,2

, Shivakumar Hugar3*

1Dept of Pharmacology, S.V.P.Ms College of Pharmacy, Malegaon (Bk), Baramati 413115, Maharashtra, India

2P.G. Dept of Pharmacology, S.C.S. College of Pharmacy, Harapanahalli 583131, Karnataka, India

3P.G. Dept of Pharmacology, BLDEAs. College of Pharmacy, Bijapur 586103, Karnataka, India

Asian Pacific Journal of Tropical Biomedicine (2012)S208-S211

Asian Pacific Journal of Tropical Biomedicine

journal homepage:www.elsevier.com/locate/apjtb

*Corresponding author: Dr. Shivakumar HugarProfessor & Head,PGDept. ofPharmacology,BLDEAs. College ofPharmacy, Bijapur586103, Karnataka, India Tel: +919503358251 Fax: 02112254447

E-mail: [email protected]

1. Introduction

The human society has become complex and in many ways,more demanding. However, our physiological responcesdesigned to cope with the ever-incereasing adversesituations have not evolved appreciably during the pastthousand years. The failure of successful adaptation duringstressful situations has resulted in stress related illnessthat result from or are associated with dysregulaton of thestress response[1]. Varoious attempts have beeen made to

counter the aversive effects of stres, ranging from yoga andmeditation to antistress drugs, particularly the anxiloyticbenzodiazepines (BDZ). However, despite claims to thecontrary, these nonpharmacological and pharmacologicalmethods appear to have limited utility [2]. An answer tothis perplexing problem of countering stress incucedperturbations of physiological homeostasis came the plantkingdom. A group of plant based drugs, adaptogens. Theactual word adaptogen was first used by a Soviet scientist,

Dr. Nikolai Lazarev, who was funded by grants from themilitary was researching substances which produced astate of nonspecific resistance (SNIR)[3]. The idea wasto find ways to enhance the productivity and performanceof soldier, athletes and workers without using dangerousstimulants. Much of the early research into adaptogens wasdone by Dr. I. I. Brekhman who, in the late 1950s studiedPanax ginseng.Trigonella foenum graecum (TF G ) has been used

since ancient times in Indian folklore medicine for its

many medicinal properties. TFG is reported to possessimmunomodulatory, hypoglycemic, nootropic and anxiolyticactivities [4-7]. In the present study, antistress activity isinvestigated in view of its reported anxiolytic and nootropicactivity. In the present study METFGS showed significantantistress activity in swimming and cold stress models inour laboratory (unpublished data). Hence in the presentinvestigation an attempt has been made to screen theadaptogenic property of METFGS against anoxia andimmobilisation stress models.

2. Materials and methods

ARTICLE INFO ABSTRACT

Article history:Received 15February 2012Received in revised form 2March 2012Accepted 12April 2012Available online 28 April 2012

Keywords:

METFGSAdaptogenAnoxia stressImmobilisation stress

Objective:To investigate the adaptogenic activity of methanolic extract ofTrigonella foenumgraecum seeds (METFGS) at 100, 250 and 500 mg/kg doses against anoxia stress tolerance in miceand immobilisation stress models. Methods: Withania somnifera (100 mg/kg, p.o.) was selectedas reference standard and it showed significant effect in both stress models.The parameterslike anoxia stress tolerance time was recorded in anoxia stress and estimation of biochemicalmarker levels and determination of organs weight were carried out in immobilisation stressmodel. Results: Concomitant treatment with METFGS at 500 mg/kg showed marked increase inanoxia stress tolerance time. But the lower doses (100 and 250 mg/kg) ofMETFGS produced mildincrease in anoxia tolerance time. There was dose dependant significant reduction in biochemicalparameters like serum glucose, cholesterol, triglycerides and BUN levels exhibited by test extractin immobilisation stress model as compared to stressed group. The stress induced increasein liver, adrenal gland weight and decrease in weight of spleen and testes were significantlyreversed by the test extract at higher dose. But lower doses of extract caused the protectiveeffect on weight of liver and adrenal gland only.Conclusion: The results from the present studyindicate that METFGS possessed significant antistress activity.

Contents lists available at ScienceDirect


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2.1. Plant material and preparation of extract

The seeds ofTrigonella foenum graecum (METFGS) werepurchased from the local market of Harapanahalli in themonth ofMay 2006 and authenticated by Prof. K. Prabhu,Dept. of Pharmacognosy, S.C.S. College of Pharmacy,Harapanahalli. A voucher specimen (SCSCP/PG/38/2006) hasbeen deposited at the museum of college. The dried seeds

were coarse powdered and defatted with petroleum ether(60 - 80) using Soxhlet extractor. The marc obtained wasthen subjected to extraction with methanol (64 - 65.5). Theextract was concentrated using rotary flash evaporator. Thedried extract was stored in airtight container in refrigeratorbelow 10. Percentage yield of extract was 14.39%.

2.2. Preliminary phytochemical screening

Preliminary phytochemical screening was carried out onMETFGS for detection of phytoconstituents present followingthe standard methods described in practical pharmacogonosyby Dr. C.K. Kokate [8] and K.R. Khandelwal[9]

2.3. Animals

The male albino rats of wistar strain (150 - 200g) andalbino mice of either sex (20 - 30 g) were used throughoutthe experimentation. The animals were procured fromNijalingappa Medical College, Bagalkot Karnataka.After randomization into various groups, animals wereacclimatized for period of 10 days under standardhusbandory condition; room temperature: 27 - 3, relativehumidity: 65% - 10% and 12 hours. Light/dark cycle. Allthe animals were fed with rodent pellet diet (Gold mohr,Lipton India Ltd.,) and water was allowed ad libitum understrict hygienic condition. Ethical clearance for performing

experiments on animals was obtained from InstitutionalAnimal Ethics Committee (IAEC).

2.4. Acute toxicity study

An acute toxicity of METFGS was conducted in femalealbino mice (20 - 30 g) maintained under standardconditions. The animals were fasted over night prior to theexperiment. Fixed dose (OECDGuideline No. 420) method ofCPCSEA was adopted for toxicity studies[10].

2.5. Evaluation of antistress activity

2.5.1. Anoxia stress tolerance in mice[11-13]

Albino mice of either sex weighing 20 - 30 g were selectedand divided into five groups of six each as Group I Control(Received only vehicle 1 mL/kg p.o.) , Group IIStandard(Withania somnifera, 100 mg/kg p.o. )Group IIIMETFGS(100 mg/kg p.o.)Group IVMETFGS (250 mg/kg p.o.)GroupVMETFGS (500 mg/kg p.o.).Animals were treated as shownabove for the three weeks. At the end of1st, 2nd and3rd week i.e. on 7th, 14th and 21st day one hour after thetreatment stress was induced in all mice by placing eachanimal individually in the hermetic vessel of1 lit. capacityto record anoxia tolerance time. The moment when theanimal showed the first convulsions immediately removedfrom the vessel and resuscitated if needed. The time

duration of entry of the animal into the hermetic vessel and

the appearance of the first convulsion was taken as time ofanoxia tolerance. Appearance of convulsion was very sharpend point, as delay by minute of removal of the animal fromthe vessel may lead to death of the same.

2.5.2. Immobilisation stress[11,12]

Adult male albino rats of150 - 200 g were selected and dividedinto six groups of six animals each as Group INegative control

(Unstressed, untreated), Group II Positive control (Stressed,received vehicle), Group III Standard (Withania somnifera100 mg/kgp.o.), Group IV METFGS(100 mg/kgp.o.), Group VMETFGS(250 mg/kg p.o.) , Group VI METFGS (500 mg/kg p.o.)Thetreatment was made as stated above for10 days 1 hour beforethe exposure of stress. Stress was induced by immobilizingrats with head down, supine position by fixing the forelimbsand hindlimbs to a wooden board inclined at an angle of600 daily, 2 hours (11 am to 1 pm) for a period of ten days.The animals were sacrificed at the end of specified periodand blood was collected by cardiac puncture under mildether anesthesia using disposable syringe and needlefor estimation of biochemical parameters such as, serumglucose (GOD-POD method), cholesterol (CHOD-PAPmethod), triglycerides (GPO-Trinder method), BUN (BloodUrea Nitrogen, GLDH-UREASE method). The weight oforgans, such as liver, spleen, adrenal gland and testes afterwashing with alcohol was recorded per100 g body weight ofanimal.

2.6. Statistical analysis

Results have been presented as Mean SEM and theexperimental groups were compared with control groupin anoxia stress and positive control (stressed group) inimmobilisation stress model. The statistical analysis wasdone using ANOVA followed by Turkey KarmmerMultiple

Comparison Test. P value of 0.05, 0.01 and 0.001 wereconsidered as statistically significant.

3. Results

3.1. Preliminary phytochemical screening

Preliminary phytochemical investigation indicated thepresence of flavonoids, tannins, saponins glycosides,alkaloids, steroids and carbohydrates in METFGS.

3.2. Acute toxicity study

TheMETFGSwas studied for acute toxicity at dose of2000 mg/kg. Theextract was found to be safe and no mortality of the animalsobserved. Hence 2500 mg/kg was considered as LD50 cutoff value as per fixed dose method of CPCSEA. So the dosesselected for the screening of adaptogenic activity were 100,250 and 500 mg/kg.

3.3. Adaptogenic (antistress) activity

3.3.1. Anoxia stress tolerance time Anoxia stress tolerance time was significantly (P< 0.05)enhanced on 14th and 21st day in METFGS (500 mg/kg)treated group. There was increased anoxia tolerance time

also seen after one week of treatment, but the result found


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statistically not significant. However the effect ofMETFGS atdoses of100 and 250 mg/kg on anoxia stress tolerance timein mice was not statistically significant at the end of1st, 2ndand 3rd week of treatment. The results are shown in Table 1.

3.3.2. Immobilisation stressEffect on biochemical parameters - Immobilisation stress

caused marked increase in serum glucose, cholesterol,triglycerides and BUN in rats. This stress induced elevatedlevels of biochemical parameters were significantly reversedby METFGS in dose dependant manner. The results are givenin Table 2.

Effect on organs weight - METFGS at dose of500 mg/kgoffered significant protection against change in the weightof liver, adrenal gland, spleen and testes when comparedto stress group. HoweverMETFGS at doses of100 and 250mg/kg produced significant decrease in weight of liver andadrenal gland but failed to increase spleen and testes weightsignificantly. The results are presented in Table 3.

4. Discussion

In the present investigation METFGS has been evaluatedfor the antistress activity against different types of stress

viz. Anoxia and immobilisation models. The well known

adaptogen Withania somnifera was taken as a referencestandard in the present study. In case of anoxia stress tolerance test METFGS at doseof 500 mg/kg exhibited significant antistress activity asindicated by increase in duration of anoxia stress tolerancetime. The test drug also produced mild increase in anoxiatolerance at doses of100 and 250 mg/kg which was however,not significant statistically. The mechanism by which stress rises serum cholesterolis likely to be related to the enhanced activity ofhypothalamo-hypophyseal axis (HPA) resulting in liberationof catecholamines and corticosteroids. This could lead to

increase in blood cholesterol level since epinephrine isknown to mobilise lipids from adipose tissues. The effectof stress on serum triglycerides has been shown to bevariable. The increase in release of catacholamines leads toelevated levels of glucose and BUN. In immobilisation stressmodel, the test extract reduced the elevated levels of serumbiochemical parameters in dose dependant manner[14-17]. Stress induces adreno-medullary response in man.Adrenaline in turn stimulates 2 receptors on the pituitaryglands causing greater release ofACTH, which can stimulatethe adrenal medulla as well as cortex. So adrenal glandweight increases. Cortisol increases mRNA levels in livercells. This lead to increase in weight of liver. Spleen

constricts to release more blood cells (RBC) during stress. So

Table 1Effect ofMETFGS on anoxia stress tolerance time in mice.

GroupDuration of anoxia stress tolerance (min)

7th Day 14th Day 21st DayControl ( vehicle) 145.804.32 146.804.20 148.603.86Std. (Withania somnifera)100 mg/kg 185.443.62**(21.49) 192.664.18**(31.23) 201.204.22**(35.39)METFGS100 mg/kg 149.303.21(2.40) 151.134.30(2.94) 152.914.51(2.90)METFGS250 mg/kg 152.603.18(4.66) 154.603.68(5.31) 159.004.80(6.99)

METFGS500 mg/kg 160.003.38(9.73) 164.004.54*

(11.71) 168.805.20*

(13.59)Values are mean SEM(n = 6), The values in parenthesis are the % increase in anoxia tolerance, *P< 0.05, **P < 0.01 as compared to control.

Table 2Effect ofMETFGS on immobilisation stress induced biochemical parameters in rats.

GroupBiochemical estimations (mg/dl)

Glucose Cholesterol Triglycerides BUNNegative control (unstressed) 92.133.21 54.283.51 69.643.12 23.532.46Positive control (stressed, received vehicle) 146.324.18 85.284.01 103.883.52 43.922.31Std. (Withania somnifera)100 mg/kg 98.243.80** 58.783.61** 76.104.00** 27.131.65**

METFGS100 mg/kg 125.233.52* 65.133.27* 85.314.21* 34.632.10*

METFGS250 mg/kg 113.153.26** 64.883.84* 83.444.12* 33.831.93*

METFGS500 mg/kg 106.944.41** 61.794.19** 78.563.83** 29.942.42**

Values are mean SEM(n = 6).*P < 0.05, **P < 0.01, ***P< 0.001 as compared to positive control.

Table 3Effect ofMETFGS on organs weight in immobilisation stress induced rats.

GroupOrgans weight (gm/100 gm b.w.)

Liver Adrenal gland Spleen TestesNegative control (unstressed) 3.4450.13 0.0130.001 0.3990.03 1.6480.060Positive control (stressed, received vehicle) 5.8200.15 0.0300.002 0.2320.02 1.2610.032Std. (Withania somnifera)100 mg/kg 3.8010.14** 0.0170.002** 0.3720.03** 1.5600.051**

METFGS100 mg/kg 5.2130.16* 0.0230.002* 0.2650.02 1.3870.048METFGS250 mg/kg 4.3570.13** 0.0210.002* 0.2930.01 1.4200.050METFGS500 mg/kg 4.3020.18** 0.0180.002** 0.3640.03* 1.5100.043**

Values are mean SEM(n = 6, *P < 0.05, **P < 0.01 as compared to positive control.


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its weight decreases during stress[18-20]. This stress inducedchanges of organs weight were significantly reversed by thetest extract at higher dose, whereas the lower doses (100 and250 mg/kg) of extract could able to exhibit protective effecton weight of liver and adrenal gland. But failed to producestatistically significant increase in spleen and testes weightin immobilisation stress model.

Experimental studies have confirmed the adaptogenic

properties of ginseng and the effects are apparently due topresence of saponin glycoside content in the root. Saponinglycosides are the main chemical constituents present inthe seeds of fenugreek4 and this may be responsible forsignificant adaptogenic activity in our study. Literaturesurvey indicate that flavonoids and tannins were reportedto possess variety of pharmacological activities includingantistress activity[21,22]. In the present investigation alsopreliminary phytochemical screening on METFGS gavepositive tests for flavonoids and tannins, this could be thereason for significant adaptogenic property of test extract.

Increased generation of oxidative free radicals (OFR )or impaired antioxidant defense mechanism have beenimplicated in chronic stress induced perturbed homeostasisincluding immunosuppression, inflammation, diabetesmellitus, peptic ulceration and other stress relateddisease[23]. The seeds of fenugreek have shown to exertsignificant antioxidant activity[24] induced by augmentedactivity ofOFR scavenging enzymes namely superoxidedismutase (SOD), catalase (CA) and glutathione peroxidase(GPx). Thus at least part of observed adaptogenic (antistress)effect ofMETFGS may be due to the antioxidant activity.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgement

The authors are grateful to management ofShivnagarVidyaPrasarak Mandal, Malegaon (Bk), for providing the necessaryfacilities through the Principal, College ofPharmacy.

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adaptogenic activity of trigonella foenum graecum (linn) seeds in

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