Effect of Mucuna pruriens Seed Extracts on Drug Induced Myelosuppression test using albino rats

 

S. Nayak1* and VK Dixit2

1Bansal College of Pharmacy, Kokta, Anandnagar, Bhopal. 462021

2Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar. 470003

 

ABSTRACT

Seeds of Mucuna pruriens were tested for drug induced myelosuppression test using cyclophosphamide as cytotoxic drug. Ethanolic extract (50, 100 and 200mg/kg body weight, orally) its chloroform soluble fraction and insoluble fraction (50, 80 and 100 mg/kg body weight, orally) were studied on albino rats.  Results of present finding suggests the ethanolic extract and its both chloroform soluble and insoluble fractions significantly (p<0.05) protect the effect of cyclophosphamide as observed by hematological studies. These investigations justifies the diversified use of Mucuna pruriens in the traditional system of medicine.

 

KEY WORDS              Mucuna pruriens, Myelosuppression, Cyclophosphamide

 

INTRODUCTION:

Mucuna pruriens Baker, belongs to family Leguminosae1  is a herbaceous twig found in bushes and  hedges,  damp  places  throughout  the  plains  of India, Andman and Nicobar, Ceylon and Burma. The leaves of Mucuna pruriens2  are silky, stipules lanceolate, pods are firstly brown in color and afterwards steel grey with 5-6 small seeds. The bristly

hairs covering, fresh and dry pods cause intense itching on contact with skin and sometime cause blisters and dermatitis. The plant has long been valued in medicine. It is used to treat a number of diseases3,4 like dropsy, syphllis, diseases of liver, nervous system and gall bladder. Recent investigations shows the plant has a number of pharmacological activities, including analgesic-antipyretic activity, aphrodisiac, antidiabetic activities etc5,6. It is reported7  that the drug contains stigmosterol, b-sitosterol, α amyrin acetate, acacetin, luteolin, betulinic acid, ursolic acid, b-carboline, bufotenine, L-3,4dihydroxyphenylalanine or dopa, glutathione and gallic acid. It also contains alkaloids e.g. nicotine, prurienine,      mucunine, mucunadin8. Present study was undertaken to assess the potential of seed extracts in drug-induced myelosuppression on albino rats.

 

MATERIALS AND METHODS:

Seeds of Mucuna pruriens were procured from local market  and authenticated by the Department of Botany, Dr. Hari Singh Gour Univerity, Sagar (M.P.) India by matching with specimen herbarium. The drug material was dried in oven and crushed into coarse powder; powdered drug (2.5 kg) was defatted in a soxhlet apparatus with petroleum ether (40o- 60o) for about 35-40 complete cycles.  The defeated material was  dried  and  subjected  to  ethanolic  extraction  (ethanol 95%) in a soxhlet apparatus. The solvent was removed under vacuum; so obtained ethanolic extract was weighed (yield 5.3% w/w of dried seed powder) and kept in airtight containers for further studies9. Ethanolic extract was subjected to fractionation with chloroform. Weighed quantity of ethanolic extract (50gm) was refluxed with chloroform (500 ml) in a round bottom flask  fitted  with  a  condenser  for  3  hours.    Chloroform soluble portion was separated and solvent evaporated to give chloroform soluble fraction (yield 36.0 % w/w of ethanolic extract) and Chloroform insoluble fraction (yield 61.3% w/w of ethanolic extract)

 

Reagents:

Leishman's  reagent  –  Leishmans  eosine  methylene  blue solution, (E-merk, India Ltd. Mumbai); Gower's solution – RBC diluting fluid (Quailgens fine chemicals, Mumbai); WBC diluting fluid – (Quailgens fine chemicals, Mumbai); Cyclophosphamide – Two tablets of endoxan (German Remedies, India) 50 mg., was triturated with 200 ml slurry of 2% gum acacia in distilled water.

 

Table 1: Drug Induced Mylosupression Test of Mucuna pruriens after 7 days.

S. No.	Groups	Hb% Mean± S.E.M.	RBC Mean± S.E.M. million/mm3	W.B.C. Mean   ± S.E.M. thou./mm3	Neutroph ills% Mean± S.E.M.	Lymphocyt e  %  Mean ±S.E.M.	Monocyt e          % Mean ±S.E.M.	Eosinoph il         % Mean ±S.E.M.	Platelate lacs/mm3 Mean ±S.E.M.	Body wt.   gm Mean ±S.E.M.
1	I.   Control   (1mL gum acacia 2%)	13.50± 0.1622*	4.76± 0.135*	9.80± 0.065*	54.46± 1.044*	41.33± 0.545*	2.5± 0.54*	2.3± 0.44*	4.347± 0.034*	163.25± 1.882*
2	II. Cyclophosphamide (3mg/kg body wt.)	8.52± 0.2613*	3.83± 0.188*	5.54± 0.046*	60.33± 0.822*	33.66± 0.862*	2.8± 0.24*	2.8± 0.34*	3.482± 0.024*	146.20± 0.825*
3	III.          Ethanolic extract (100 mg/kg body wt.)	13.25± 0.2622	4.50± 0.056*	9.75± 0.122	52.50± 0.788	43.83± 0.7062	2.0± 0.30	1.4± 0.32	4.376± 0.014	162.75± 0.462
4	IV. Chloroform soluble fraction (50 mg/kg body wt.)	13.60 ± 0.282	4.80± 0.042	9.72± 0.165	54.83± 1.621	40.33± 0.322	3.81± 0.85*	2.8± 0.60	4.268± 0.032	163.85± 1.256*
5	V.         Chloroform Insoluble      fraction (50 mg/kg body wt.)	13.00± 0.3482	4.50± 0.122*	9.61± 0.115	53.33± 1.044	42.66± 0.484	4.3± 0.33*	1.8± 0.42*	4.152± 0.014*	158.30± 1.882*
6	VI.          Ethanolic extract (100 mg/kg body wt.) + CP	10.25± 0.4120*	3.22± 0.055*	8.60± 0.155*	54.16± 0.676*	39.36± 0.826*	3.51± 0.11	2.3± 0.32*	4.134± 0.016*	155.82± 2.304*
7	VII.         Chloroform soluble   fraction   (50 mg/kgbody wt.) + CP	11.50± 0.2482*	3.36± 0.082*	9.48± 0.016*	58.50± 0.7628*	35.62± 0.943*	3.2± 0.20*	2.9± 0.38*	3.832± 0.022*	151.50± 1.754**
8	VIII.       Chloroform Insoluble fraction (50 mg/kg body wt.) + CP	10.25± 0.2462*	3.27± 0.128*	8.10± 0.088*	52.66± 0.641*	41.33± 1.822*	2.5± 0.28	3.6± 0.22*	4.021± 0.026*	160.25± 1.255**

 

n = 6 animals in each groups, CP = Cyclophosphamide, *P<0.05, **P<0.001, comparison between control and groups

 

Table 2: Drug Induced Mylosupression Test of Celastrus paniculatus after 14 days.

S. No.	Groups	Hb% Mean± S.E.M.	RBC Mean± S.E.M. million/mm3	W.B.C. Mean   ± S.E.M. thou./mm3	Neutroph ills% Mean± S.E.M.	Lymphocyt e  %  Mean ±S.E.M.	Monocyt e          % Mean ±S.E.M.	Eosinoph il         % Mean ±S.E.M.	Platelate lacs/mm3 Mean ±S.E.M.	Body wt.   gm Mean ±S.E.M.
1	I. Control (1ml   gum   acacia	13.50± 0.2551*	4.85± 0.125*	9.75± 0.122*	54.33± 0.424*	43.66± 0.420*	1.8± 0.33	1.8± 0.30*	4.374± 0.018	169.25± 1.25*
2	II. Cyclophasphamide (3mg/kg body wt.)	6.52± 0.2243*	3.66 ± 0.183*	5.20± 0.084*	62.16± 0.612*	34.33 ± 0.240*	2.2± 0.26*	1.6± 0.30*	2.713± 0.024*	142.25± 0.682**
3	III.          Ethanolic extract (100 mg/kg body wt.)	13.40± 0.2501	4.60± 0.082	9.72± 0.145*	52.66± 0.520	44.16± 0.620*	2.3± 0.33	1.6± 1.21	4.436± 0.012	166.75± 1.64*
4	IV.      Chloroform soluble   fraction (50   mg/kg   body wt.)	13.28 ± 0.1702	4.48± 0.558	9.75± 0.185	52.26± 1.106	44.36± 0.962	2.0± 1.25	3.0± 0.30	4.365± 0.016*	166.35± 0.625*
5	V.        Chloroform Insoluble fraction (50   mg/kg   body wt.)	13.25 ± 0.3354*	4.52± 0.042*	9.62± 0.210*	54.33± 0.482	43.33± 0.216	2.3± 0.36	2.8± 0.22	4.343± 0.013	162.25± 1.602*
6	VI.          Ethanolic extract (100 mg/kg body wt.) – CP	12.82± 0.3853*	4.18± 0.010*	9.42± 0.162*	56.00± 0.472*	40.66± 0.540*	1.5± 1.22	3.4± 0.30	4.422± 0.018*	159.25± 1.276*
7	VII.     Chloroform soluble   fraction (50   mg/kg   body wt.) – CP	13.45± 0.3246*	4.43± 0.113*	9.76± 0.154*	56.50± 0.612*	40.66± 1.330**	2.1± 1.30	1.8± 1.36	4.426± 0.018*	154.35± 0.722*
8	VIII.   Chloroform Insoluble fraction (50   mg/kg   body wt.) – CP	11.45± 0.2750**	3.82± 0.142*	8.50± 0.122*	54.82± 0.222*	43.66± 0.332*	2.2± 0.28*	2.4± 0.42*	3.541± 0.052*	164.00± 0.731*

 

n = 6 animals in each groups, CP = Cyclophosphamide, *P<0.05, **P<0.001, comparison between control and groups

 

Animals

Albino rats (Sprage Dwely) of mixed population (160- 180 gms) were used for studies.  All the animals were given a balanced diet, tap water and maintained in the uniform environmental conditions. Experimental protocols were approved by institutional animal ethical committee.

 

 

Drug induced Myelosuppression Test

To determine the effect of drug-induced myelosuppression10,11  cyclophosphamide was used to produce myelosuppression12 in albino rats.  Albino rats were divided in eight groups of six each.  Group I was kept as control and given 2% gum acacia (1 ml) suspension in water.

 

Group    II    was    treated    with    cyclophosphamide 3mg/kg/day for seven days.  Group III, IV and V were administered with ethanolic extract 100 mg/kg. Chloroform soluble fraction 50mg/kg, and chloroform insoluble fraction 50 mg/kg, group VI, VII, and VIII were  fed  with  ethanolic  extract,  chloroform soluble fraction  and  chloroform insoluble  fraction  with  100 mg/kg.,  50  mg/kg.  and  50  mg/kg  respectively with cyclophosphamide 3 mg/kg/body weight/day for seven days.

 

On seventh day blood was taken from retro-orbital pluxes and subjected to hematological studies. The cyclophosphamide was withdrawn from group VI, VII and VIII and fed only ethanolic extract, chloroform soluble  and  chloroform  insoluble  fraction  for  next seven days (recovery studies).  Blood samples of each animal collected on 15th  day and again subjected to hematological13   studies  including  hemoglobin count, RBC,  WBC  count,  differential WBC  count,  platelet count and body weight.   No mortality was observed after discontinuation of drugs till one month.

 

RESULTS AND DISCUSSION:

Results are presented in Table 1 and Table 2.  In drug induced myelosuppression test, the myelosuppression was produced by administration of cytotoxic drug, cyclophosphamide that produces significant14 myelosuppression       in       experimental       animals. Cyclophosphamide produces significant bone marrow suppression resulting in cytopenia and subsequent suppression of humoral and/or cellular as well as non- specifc immune response.     Cyclophosphamide treatment resulted in significant lowering of haemoglobin concentration, RBC count, platelet count and total WBC count, as well as lymphocyte percentage¹⁵,       The suppressive effect of cyclophosphamide was significantly protected by administration of ethanolic extract of Mucuna pruriens in  100mg/kg  body  weight  of  chloroform  soluble fraction and insoluble fraction (50mg/kg body weight, (P<0.05). Recovery studies also suggests that the myelosuppressive16 drug withdrawal can be supported by the treatment with the extracts of Mucuna pruriens which results in a complete restoration of immune response even in improved immunological parameters17.  Present finding supports the use of Mucuna pruriens to treat various diseases.

 

ACKNOWLEDGMENT:

One  of  the  authors  S.  Nayak  is  Thankful  to  University Grants Commission for financial assistance.

 

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