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Chemopreventive Activity of Withania somnifera in Experimentally Induced Fibrosarcoma Tumours in Swiss Albino Mice Jai Prakash, 1 S. K. Gupta, 1 * V. Kochupillai, 2 N. Singh, 3 Y. K. Gupta 1 and S. Joshi 1 1 Department of Pharmacology, All India Institute of Medical Sciences, New Delhi - 110029, India 2 Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi - 110029, India 3 Department of Biochemistry, All India Institute of Medical Sciences, New Delhi - 110029, India The current experimental work deals with the chemopreventive studies of a hydroalcoholic extract of With- ania somnifera roots, against 20-methylcholanthrene induced fibrosarcoma tumours in Swiss albino mice. A single subcutaneous injection of 200 mg 20-methylcholanthrene in 0.1 mL of dimethylsulphoxide into the thigh region of mice produced a high incidence (96%) of tumours. Oral treatment of animals with 400 mg/kg body weight of Withania somnifera extract (one week before injecting 20-methylcholanthrene and continued until 15 weeks thereafter) significantly reduced the tumour incidence, tumour volume and enhanced the survival of the mice, compared with 20-methylcholanthrene injected mice. The tumour inci- dence was also delayed in the treatment group when compared with 20-methylcholanthrene injected mice. Liver biochemical parameters revealed a significant modulation of reduced glutathione, lipid peroxides, glutathione-S-transferase, catalase and superoxide dismutase in extract treated mice compared with 20- methylcholanthrene injected mice. The mechanism of chemopreventive activity of Withania somnifera extract may be due to its antioxidant and detoxifying properties. Copyright # 2001 John Wiley & Sons, Ltd. Keywords: Withania somnifera Dunal; chemopreventive activity; fibrosarcoma tumours. Introduction In Indian traditional systems of medicine (Ayurvedic and Unani), Withania somnifera Dunal (common name — Ashwagandha, family — Solanaceae) is known to have high medicinal values. Its roots are reported to have health maintenance and restoration properties compar- able to that of ginseng (Tripathi et al., 1996). Previous reports demonstrate the growth inhibitory, antitumour and radiosensitizing activities of Withania somnifera on transplantable tumour sarcoma-180 in mice (Devi et al., 1992; 1993). Later, modification of stem cell survival of mouse bone marrow by withaferin-A, the steroidal lactone of Withania somnifera was also observed (Ganasoundari et al., 1997). Substantial experimental evidence so far has strongly implied that free radicals are involved in both the initiation and promotion process of carcinogenesis (Sun, 1990). The proven chemopreventive action of a few antioxidants in experimental models have further strengthened this theory (Azuine and Bhide, 1992; Elangovan et al., 1994). Despite the fact that Withania somnifera is an important medicinal plant having antioxidant properties (Panda and Kar, 1997), its efficacy as a chemopreventive agent in 20-methylcholanthrene (MCA) induced solid tumours (fibrosarcomas) in Swiss albino mice has not been studied before. Therefore in the present study, the hydroalcoholic extract of Withania somnifera roots (WSE) was chosen to assess its chemopreventive activity against MCA induced experi- mental fibrosarcoma tumours in Swiss albino mice. The study also aimed to obtain insights into whether the scavenging of free radicals was associated with the chemopreventive activity of the extract. Materials and methods Drugs and chemicals. A dried powdered hydroalcoholic extract of Withania somnifera roots (batch no. 101048) consisting of 1.97% w/w of total withanolides, was a gift from Dabur Research Foundation, Sahibabad, Ghazia- bad, U.P, India. MCA, bovine albumin, Tris(hydroxy- methyl)aminomethane and 1,1,3,3-tetraethoxypropane were purchased from Sigma Chemicals Co, USA. All other chemicals were of highest purity, AR grade and purchased from local agents. Dimethylsulphoxide (DMSO) and absolute alcohol were used as solvents to dissolve 20-methylcholanthrene and 1-chloro-2,4-dini- trobenzene (CDNB) respectively. Animals. Swiss albino mice of 7–8 weeks old weighing 18–22 g were obtained from the Experimental Animal Facility of All India Institute of Medical Sciences, New Delhi. They were maintained under standard laboratory conditions of feed and water. The animals were acclimatized for 1 week before experimentation and the initial body weight of each mouse was recorded before the commencement of the experiment. PHYTOTHERAPY RESEARCH Phytother. Res. 15, 240–244 (2001) DOI: 10.1002/ptr.779 Copyright # 2001 John Wiley & Sons, Ltd. * Correspondence to: Dr S. K. Gupta, Department of Pharmacology, All India Institute of Medical Sciences, New Delhi - 110029, India. E-mail: [email protected] Received 18 June 1999 Accepted 19 May 2000

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Chemopreventive Activity of Withania somniferain Experimentally Induced FibrosarcomaTumours in Swiss Albino Mice

Jai Prakash,1 S. K. Gupta,1* V. Kochupillai,2 N. Singh,3 Y. K. Gupta1 and S. Joshi11Department of Pharmacology, All India Institute of Medical Sciences, New Delhi - 110029, India2Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi - 110029, India3Department of Biochemistry, All India Institute of Medical Sciences, New Delhi - 110029, India

The current experimental work deals with the chemopreventive studies of a hydroalcoholic extract of With-ania somnifera roots, against 20-methylcholanthrene induced fibrosarcoma tumours in Swiss albino mice. Asingle subcutaneous injection of 200�g 20-methylcholanthrene in 0.1mL of dimethylsulphoxide into thethigh region of mice produced a high incidence (96%) of tumours. Oral treatment of animals with400mg/kg body weight of Withania somnifera extract (one week before injecting 20-methylcholanthreneand continued until 15 weeks thereafter) significantly reduced the tumour incidence, tumour volume andenhanced the survival of the mice, compared with 20-methylcholanthrene injected mice. The tumour inci-dence was also delayed in the treatment group when compared with 20-methylcholanthrene injected mice.Liver biochemical parameters revealed a significant modulation of reduced glutathione, lipid peroxides,glutathione-S-transferase, catalase and superoxide dismutase in extract treated mice compared with 20-methylcholanthrene injected mice. The mechanism of chemopreventive activity of Withania somniferaextract may be due to its antioxidant and detoxifying properties. Copyright � 2001 John Wiley & Sons,Ltd.

Keywords: Withania somnifera Dunal; chemopreventive activity; fibrosarcoma tumours.

Introduction

In Indian traditional systems of medicine (Ayurvedic andUnani), Withania somnifera Dunal (common name —Ashwagandha, family — Solanaceae) is known to havehigh medicinal values. Its roots are reported to havehealth maintenance and restoration properties compar-able to that of ginseng (Tripathi et al., 1996). Previousreports demonstrate the growth inhibitory, antitumourand radiosensitizing activities of Withania somnifera ontransplantable tumour sarcoma-180 in mice (Devi et al.,1992; 1993). Later, modification of stem cell survival ofmouse bone marrow by withaferin-A, the steroidallactone of Withania somnifera was also observed(Ganasoundari et al., 1997).

Substantial experimental evidence so far has stronglyimplied that free radicals are involved in both theinitiation and promotion process of carcinogenesis(Sun, 1990). The proven chemopreventive action of afew antioxidants in experimental models have furtherstrengthened this theory (Azuine and Bhide, 1992;Elangovan et al., 1994). Despite the fact that Withaniasomnifera is an important medicinal plant havingantioxidant properties (Panda and Kar, 1997), its efficacyas a chemopreventive agent in 20-methylcholanthrene(MCA) induced solid tumours (fibrosarcomas) in Swissalbino mice has not been studied before. Therefore in thepresent study, the hydroalcoholic extract of Withania

somnifera roots (WSE) was chosen to assess itschemopreventive activity against MCA induced experi-mental fibrosarcoma tumours in Swiss albino mice. Thestudy also aimed to obtain insights into whether thescavenging of free radicals was associated with thechemopreventive activity of the extract.

Materials and methods

Drugs and chemicals. A dried powdered hydroalcoholicextract of Withania somnifera roots (batch no. 101048)consisting of 1.97%w/w of total withanolides, was a giftfrom Dabur Research Foundation, Sahibabad, Ghazia-bad, U.P, India. MCA, bovine albumin, Tris(hydroxy-methyl)aminomethane and 1,1,3,3-tetraethoxypropanewere purchased from Sigma Chemicals Co, USA. Allother chemicals were of highest purity, AR grade andpurchased from local agents. Dimethylsulphoxide(DMSO) and absolute alcohol were used as solvents todissolve 20-methylcholanthrene and 1-chloro-2,4-dini-trobenzene (CDNB) respectively.

Animals. Swiss albino mice of 7–8 weeks old weighing18–22g were obtained from the Experimental AnimalFacility of All India Institute of Medical Sciences, NewDelhi. They were maintained under standard laboratoryconditions of feed and water. The animals wereacclimatized for 1 week before experimentation and theinitial body weight of each mouse was recorded beforethe commencement of the experiment.

PHYTOTHERAPY RESEARCHPhytother. Res. 15, 240–244 (2001)DOI: 10.1002/ptr.779

Copyright � 2001 John Wiley & Sons, Ltd.

* Correspondence to: Dr S. K. Gupta, Department of Pharmacology, All IndiaInstitute of Medical Sciences, New Delhi - 110029, India.E-mail: [email protected]

Received 18 June 1999Accepted 19 May 2000

Induction of experimental fibrosarcoma tumours inmice and drug treatment schedule. The MCA inducedtumour induction model in mice was standardized in ourlaboratory. Dose tolerance studies with WSE in micewere also carried out. A daily oral dose 400mg/kg bodyweight of WSE (suspended in water) for 4 months wasfound to be the maximal tolerated dose in mice.Significant mortality was observed beyond this doselevel. Therefore this dose was chosen for chemopreven-tive studies. A total of 60 mice were distributed into threegroups as follows: Group I (n = 10) served as a vehiclecontrol and each mouse was administered a single dose of0.1mL DMSO into the thigh region subcutaneously.Group II (n = 25). Each mouse was administered a singledose of 200�g MCA/0.1mL DMSO into the thigh regionsubcutaneously. Group III (n = 25). Each mouse received400mg/kg body weight of WSE once daily by oral route,1 week before MCA administration and continued until15 weeks thereafter.

Assessment of chemopreventive activity. The chemo-preventive activity of WSE was assessed by recording thefibrosarcoma incidence weekly, tumour volume at theend of week 15, mice body weight changes at fortnightlyintervals and quantal 15 weeks survival in drug treatedand untreated mice. The following biochemical par-ameters were estimated in the liver tissues of vehicle,MCA and MCA � WSE treated mice to explore themechanism of chemopreventive action: reduced glu-tathione (GSH), lipid peroxides (MDA), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase(CAT) and protein.

Preparation of homogenate and supernatant frac-tions. The mice were killed at the end of week 15, theirlivers excised, rinsed in ice cold normal saline followedby cold 0.15M Tris-HCl (pH � 7.4), blotted dry andweighed. A 10% w/v homogenate was prepared in 0.15MTris-HCl buffer (pH � 7.4) and processed for theestimation of lipid peroxides. A part of the homogenate,after precipitating proteins with trichloroacetic acid, wasused for the estimation of GSH. The rest of thehomogenate was centrifuged at 15000 rpm for 15 minat 4°C using an ELTEK Refrigerated Centrifuge (RC4100D). The supernatant thus obtained was utilized forthe estimation of GST, CAT, SOD activities and protein.

Biochemical assays

Estimation of reduced glutathione. The level of GSHwas estimated by the method of Moron et al. (1979). To0.1mL of protein-free supernatant, 2mL of 0.6mM, 5,5�-dithiobis-[2-nitrobenzoic acid] in 0.2 M phosphate buffer(pH 8.0) was added. The volume of the reaction mixturewas made up to 3mL with 0.2 M phosphate buffer (pH8.0). The absorbance was recorded at 412nm on aBeckman’s spectrophotometer. Reduced glutathione wasused as a standard.

Estimation of lipid peroxides. Lipid peroxide estima-tion was carried out following the method of Ohkawa etal. (1979) To 0.1 mL of the homogenate, 1.5 mL of 20%acetic acid, 0.2 mL of 0.1% sodium dodecyl sulphate and1.5 mL of 0.81% thiobarbituric acid were added. Themixture was made up to 4 mL with distilled water and

heated for 30 min at 95°C in a temperature controlledwater bath. After cooling, 4 mL of n-butanol pyridine(15:1) was added and shaken well. Then centrifugationwas done at 5000 rpm for 10 min, and the absorbance ofthe organic layer read at 532 nm. The standard used was1,1,3,3-tetraethoxypropane.

Estimation of glutathione-S-transferase. GST activitywas determined at 25°C according to the method ofHabig et al. (1974). The conjugation of GSH with CDNB,a hydrophilic substrate was observed spectrophotome-trically at 340 nm. Briefly, the reaction mixture contained1.7mL of 100 mM phosphate buffer (pH 6.5), 0.1 mL of30 mM CDNB, 0.1 mL of 30 mM GSH and 0.02 mL ofthe supernatant. The change in absorbance was recordedat 340nm at 30 s intervals for 3 min. The GST activitywas calculated using the extinction coefficient of9.6mM�1cm�1 and expressed as units/mg protein.

Estimation of superoxide dismutase. SOD was assayedaccording to the method of Misra and Fridovich (1972).The assay is based upon the ability of SOD to inhibit theauto-oxidation of adrenaline to adrenochrome at alkalinepH. Briefly, the reaction mixture contained 1.5 mL of100 mM carbonate–bicarbonate buffer (pH 10.2) contain-ing 57 mg/dL of EDTA and sufficient sample. The increasein absorbance was followed for 3 min at 480 nm, afteradding 3 mM epinephrine. One unit of SOD activity is theamount of enzyme required to inhibit the rate of adrenalineauto-oxidation by 50% under the conditions of the assay.

Estimation of catalase. CAT was estimated according tothe method of Aebi (1974) using H2O2 as substrate. Theassay was conducted at 25°C. The reaction mixture(2.25 mL) consisted of 2 mL of 50 mM phosphate buffer(pH 7.0) and 0.05 mL of the supernatant. The change inabsorbance at 240 nm was followed at 10 s intervals for1 min, after the addition of 0.2 mL of 30 mM H2O2,against the sample blank. One unit of CAT activityrepresents 1 �mol of H2O2 decomposed/min under thedefined conditions of the assay.

Estimation of protein. The protein content of eachsample was estimated by the method of Lowry et al.(1951) using bovine serum albumin as standard. Theabsorbance was read at 640nm.

Statistical analysis. The results were analysed statisti-cally using Student’s t-test and Chi-square test. p valuesof less than 0.05 were considered significant.

Results

The incidence of fibrosarcoma in the control and WSEtreated mice at different weeks is shown in Fig. 1. Theonset of fibrosarcoma was observed from week 5onwards in MCA injected mice and the incidence reachedas high as 96% within 15 weeks. It was interesting to notethat fibrosarcoma incidence started from week 7 onwardsin WSE supplemented mice and reached just 60% by theend of week 15. This difference in the incidence betweenthe two groups was significant (p � 0.01). 40% of themice in WSE supplemented group still remained normalby the end of the experiment. Fibrosarcoma was not

WITHANIA SOMNIFERA ON FIBROSARCOMAS 241

Copyright � 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 240–244 (2001)

observed in the DMSO injected group of mice. There wasa significant reduction (p � 0.001) in tumour volume(calculated using the formula Tumour volume = Majordiameter � Minor diameter2/2) in WSE supplemented

mice compared with MCA alone injected mice (Fig. 2).The effect of WSE on the survival of mice is presented inTable 1. It is evident that 100% of mice survived invehicle treated group, whereas the survival in the MCAinjected group was only 56%. WSE supplementationsignificantly increased the survival (88% vs 56%,p � 0.05). The average body weight (g � SD) in vehicletreated, MCA treated and MCA � WSE treated mice, atthe end of week 15, were found to be 36.1 � 3.5,31.6 � 3.1 and 45.8 � 3.0, respectively.

Table 2 depicts the effect of WSE on liver antioxidantdefence. Liver enzymatic and non-enzymatic anti-oxidants were significantly altered in MCA injected micecompared with the vehicle treated mice (p �0.001). GSH,SOD, CAT and GST levels were 1.25, 1.52, 1.56 and 1.67fold higher respectively, with WSE supplementationwhen compared with the MCA treated mice but less thanthe vehicle treated mice. Lipid peroxide levels (expressedin terms of malondialdehyde (MDA) formation) weresignificantly high in MCA treated mice compared withvehicle treated mice (p �0.001). WSE administrationprevented the rise in MDA levels (p �0.001) but did notbring them to the normal levels.

Discussion

In our preliminary studies WSE was found to inhibitsignificantly the viability of HeLa cells (human cervicalcarcinoma) in vitro in a dose dependent manner (JaiPrakash et al., 1999). Based on findings of our studies andthat of others (Singh et al., 1986; Devi et al., 1993), afurther attempt was therefore made to evaluate theactivity of the WSE in vivo against chemically inducedcarcinogenesis (MCA induced fibrosarcoma tumours) inmice. The results of the present study indicate asignificant reduction in fibrosarcoma incidence in micewhen supplemented with the maximum tolerated dose ofWSE compared with MCA injected mice. This protectiveeffect could be due to a delay in the initiation process with1 week of drug pretreatment and a delay in the promotionprocess of carcinogenesis with continuous drug treatment,resulting in a low incidence of fibrosarcomas. The tumourvolume was also approximately 50% of the MCA controlin WSE supplemented mice group, indicating its tumourregression potential (Fig. 3–4). Tumours did not appear inDMSO injected mice (Fig. 5). The quantal survival of themice at the end of week 15 in WSE treated group wassignificantly more compared with the untreated MCA

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Table 1. Effect of Withania somnifera root extract on 15week survival of mice

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242 J. PRAKASH ET AL.

Copyright � 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 240–244 (2001)

control. This could be related to a higher death rate andincreased tumour burden in MCA injected mice comparedwith extract supplemented mice. An appreciable increasein body weight was also seen in WSE treated mice whencompared with MCA injected mice (p �0.001). Theincrease in body weight signifies that Withania somniferais an appetite stimulant and a good healthcare supplementor alternatively this could also be due to better tumourcontrol. Previous reports also suggest Withania somniferato be a good nutrient (Tripathi et al., 1996). The averagebody weight of the mice decreased significantly in thecarcinogen control group as compared with the vehicle(DMSO) injected group (p �0.01).

The role of free radicals in the multistep process ofcarcinogenesis is well established (Fischer et al., 1998)and antioxidants are reported to offer a protective effect

against cancer (Uddin and Ahmed, 1995; Schwartz et al.,1994). Recently, Withania somnifera has been reported topossess free radical scavenging activities (Bhattacharya etal., 1997; Mohanty et al., 1999). It has also been shown toinduce SOD and CAT activities in mice livers (Panda andKar, 1997). It is also well documented that the carcino-genic potential of a chemical substance depends on its rateand extent of metabolic activation and detoxification. Theantioxidant and detoxifying mechanisms in liver areknown to play a very important role in predicting theoverall carcinogenic potential of chemical carcinogens. Ithas been demonstrated that tumour bearing animals canexperience a systemic change of enzymatic and non-enzymatic antioxidants in organs distant from tumour sites(Kampschmidt, 1965; Elangovan et al. 1994). Liver, beinga versatile organ of metabolism and detoxification, isinvolved in the metabolism of chemical carcinogens suchas methylcholanthrene, a polynuclear aromatic hydrocar-bon. Taking these facts into consideration, antioxidantbiochemical parameters were estimated in the liver tissuesof treated and untreated mice. Our results indicate amaintenance of these levels (GSH, MDA, GST, SOD andCAT) by WSE supplementation while a significantreduction was seen in the MCA injected group (Table 2).

Reduced glutathione (GSH) is one of the mostabundant tripeptide non-enzymatic biological antioxi-dants present in the liver. Its functions are concerned withthe removal of free oxygen species such as hydrogenperoxide, superoxide radicals, alkoxy radicals, mainte-nance of membrane protein thiols and as a substrate forglutathione peroxidase (GP) and GST. WSE feeding(group III) significantly protected the fall of GSH levelscompared with MCA injected mice. Depletion of GSHhas been associated with an enhanced lipid peroxidation.There are reports which suggest higher lipid peroxidelevels in fibrosarcoma bearing rats (Kamarajan et al.,1991). Our results confirm earlier reports and suggest thatmaintenance of GSH levels could partly be due toprotection against lipid peroxidation by WSE asevidenced by MDA levels.

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WITHANIA SOMNIFERA ON FIBROSARCOMAS 243

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SOD, CAT and GST comprise the cellular antioxidantdefence system. It seems probable that in tumour bearinganimals, initiated cells escape the inhibitory effect ofantioxidant enzymes and induced enzymes go below thenormal level. We have found low levels of antioxidantenzymes in the livers of tumour bearing animals. Further,WSE supplementation to mice maintained the levels ofSOD and CAT suggesting either induction of theseenzymes by WSE or a prevention of the downregulationin MCA injected mice.

GSTs are a group of detoxifying enzymes whichcatalyse the conjugation of a wide variety of hydrophobicelectrophiles to reduced GSH resulting in formation ofsuch products which are generally water soluble andeasily excretable in bile or urine (Jakoby, 1978). Inaddition, GST can covalently bind to a wide variety ofcarcinogens. So the induction of a detoxifying systemcould enhance the conjugational capacity of GST and theavailability of non-critical nucleophiles for inactivationof electrophiles (Benson et al., 1978; Sparnins et al.,1982). The drugs that enhance the activity of detoxifyingenzymes have been shown to inhibit the process ofcarcinogenesis (Wattenberg, 1983, 1985). A significant

induction of GST was observed in our studies with WSEsupplementation.

Conclusions

Our results demonstrate that WSE at the maximaltolerated dose level of 400 mg/kg possesses significantchemopreventive activity in experimental fibrosarcomatumours in Swiss albino mice. The antioxidant anddetoxifying properties of WSE may, at least in part, beresponsible for the observed chemopreventive action. Inview of the promising results, it seems probable thatWSE can be used as an adjuvant to local tumour excisionand may increase the overall survival rate, however, moreexperimental and then clinical data is required to supportthis statement.

Acknowledgement

Financial assistance to Mr Jai Prakash in the form of Senior ResearchFellowship from Council of Scientific and Industrial Research, NewDelhi is duly acknowledged.

REFERENCES

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244 J. PRAKASH ET AL.

Copyright � 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 240–244 (2001)