evaluation of the anticarcinogenic activity of swertia chirata buch.ham, an indian medicinal plant,...

ANTICARCINOGENIC ACTIVITY OF SWERTIA CHIRATA 373

Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 373–378 (2004)

Copyright © 2004 John Wiley & Sons, Ltd.

Received 11 September 2002Accepted 23 June 2003

PHYTOTHERAPY RESEARCHPhytother. Res. 18, 373–378 (2004)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1436

Evaluation of the Anticarcinogenic Activity ofSwertia chirata Buch.Ham, an Indian MedicinalPlant, on DMBA-induced Mouse SkinCarcinogenesis Model

Prosenjit Saha1, Suvra Mandal2, Ashes Das2, Prabhas C. Das2 and Sukta Das1

1Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute, Kolkata, India2Department of Chemistry, Central Research Institute (Ayurveda), Bidhannagar, Kolkata, India

Considerable attention has been focused on plants which are sources of natural anti-oxidant compounds,because most of them have a modulatory role on physiological functions and biotransformation reactionsinvolved in the detoxification process. Such compounds are likely to afford protection from cytotoxic, genotoxicand metabolic actions of environmental toxicant thereby reducing the risk for cancer. The present studyreports the anticarcinogenic activity of Swertia chirata Buch.Ham, an Indian medicinal plant. All the fourdetoxification enzymes studied viz, GST, GPx, SOD and CAT were found to be activated in different degreesfollowing treatment with infusion of Swertia chirata, its crude extract and a purified ‘Amarogentin’ richextract. The activation of the enzymes was accompanied by significant reduction in lipid peroxidation andinhibition of incidence as well as multiplicity of Dimethylbenz(a)anthracene (DMBA) induced papillomas.The effect of S.chirata on apoptosis and cell proliferation was also studied in mice skin exposed to DMBA.Both the crude and purified extracts significantly inhibited cell proliferation and induced apoptosis. This isthe first report of its kind and the observation suggests the chemopreventive potential of Swertia chirata.Copyright © 2004 John Wiley & Sons, Ltd.

Keywords: Swertia chirata; amarogentin; detoxification enzymes; cell proliferation; apoptosis; cancer chemoprevention.

* Correspondence to: Dr S. Das, Head, Department of Cancer Chemo-prevention, Chittaranjan National Cancer Institute, 37, SP MukherjeeRoad, Kolkata 7000 26, India.E-mail: [email protected] or [email protected]

INTRODUCTION

The plant Swertia chirata, Buch.Ham. (Gentianaceae),commonly known as ‘Chirata’ or ‘Kirata-tikta’ inSanskrit, is well known for its multifarious therapeuticvalue since the era of ‘Atharva Veda’ (CharakaSamhita) and is widely used in the Indian System ofMedicine as crude drug. It is used as an antimalarial,a bitter stomachic, febrifuge, antihelminthic, and asremedy for scanty urine, epilepsy, ulcer, bronchialasthma, melancholia and certain type of mental dis-order (Chatterjee and Pakrashi, 1995). The chemicalcompounds identified in S.chirata (Mandal et al., 1997)account for the varied medicinal propoerties of theplant. The chemical constituents of S.chirata includethe first isolated dimeric xanthone ‘chirantanin’ anda large number of xanthones mostly teratogenated(Mandal and Chatterjee, 1987; Mandal et al., 1992).S.chirata also contains a number of triterpenoids, alka-loids and secoiridoid bitters (Karan et al., 1999a). Thechloroform soluble fraction of the methanol extract ofS.chirata demonstrated potent anti-hepatotoxic activity(Karan et al., 1999b) and methanol extract of S.chirata

is reported to possess superoxide scavenging property(Khanom et al., 2000). It has been shown that ethanolicextract of S.chirata can reduce experimentally inducedgastric ulcers in rat and guinea pig (Rafatullah et al.,1993). A methanol extract of S.chirata, secoiridoidglycoside amarogentin, the bitterest compound, has beentraditionally used for centuries mainly as a digestiveaid as well as to protect against hepatic injury and hasalso been used in folk medicine for the treatmentof skin tumour by topical application. It was found tobe an inhibitor of topoisomerase I of Leishmaniadonovani (Ray et al., 1996) and this compound wasshown to possess antileishmanial property (Meddaet al., 1999).

The present report furnishes results of our study onthe effect of crude and purified extracts of S.chirata onphase II detoxification enzymes (GST, GPx, SOD andCAT) and lipid peroxidation in liver of mice and alsoon the incidence and multiplicity of papillomas in mouseskin induced by DMBA (Filed for Indian Patent in2002). The effect on in situ cell proliferation andapoptosis was also studied.

MATERIALS AND METHODS

Plant material. Swertia chirata (whole plant), collectedfrom a local plant supplier of Kolkata, was authenti-cated by Dr S. R. Das, Ex-Survey Officer, Central

374 P. SAHA ET AL.


Research Institute (Ayurveda), Kolkata. The plantspecimen has been preserved in the Herbarium of theCentral Research Institute (Ayurveda), Kolkata.

Preparation of the plant infusion and extract. Infusionof the dry powdered plant material was prepared bysoaking overnight in distilled water followed by decant-ing. The coarsely powered plant material (3.5 kg) wasextracted with normal hexane, the defatted materialsoaked in alcohol and concentrated under reducedpressure to yield 200 gm of a semisolid mass (crudeextract). This crude mass was further purified bychromatography using organic solvents of increasingpolarity. Ethylacetate eluates were concentrated toyield a purified extract which was further chromato-graphed and purified by repeated crystallization andfinally dried under vacuum. Yield was 1.5 gm of drypower (90–95% amarogentin).

Experimental animals. Adult (5 weeks) Swiss albinomale mice (23 ± 2 gm), bred in the animal colony ofChittaranjan National Cancer Institute, Kolkata, usedfor the study, were maintained at controlled tempera-ture under alternating light and dark conditions. Stand-ard food pellets (Lipton India Ltd., Bangalore) anddrinking water were provided ad libitum.

Carcinogenesis model. 9,10-dimethyl benz(a)anthracene(DMBA, Sigma Chemicals, USA) was applied topicallyin a shaved portion on the dorsal surface of the mice,at a dose of 1 mg/100 µl acetone/mouse twice at an in-terval of three days. This was followed by applicationof 1% croton oil on the same place twice weekly for8 weeks.

Detection of Papilloma. Animals in all experimentalgroups were continuously examined for detection ofpapillomas appearing on the skin. If left untreatedpapillomas appear on the skin which progress furtherto epithelial cell tumour in 90% of mice after a periodof 6–8 months. Morphological observation were con-firmed by histopathology of the growths. The observa-tion period for the present study was 12 weeks whenskin papilloma (precancer growth) developed in 80%of the experimental mice. Inhibition of multiplicity wascalculated by (total number of papillomas in carcino-gen control – total number of papillomas in treatedgroup) × 100/total number of papillomas in carcinogencontrol.

Toxicity testing. Evaluation of the toxicity of the crudeand purified extracts of S.chirata was done by standardprotocol (Amonkar and Adwankar, 1997). The IC50 ofcrude and purified extracts were determined on Dalton’sascitic tumour cells, treated for 24 h in vivo, and foundto be 10 mg and 0.5 mg respectively. These doses werealso found to be non-toxic to normal mice as revealedby body weight and survival.

Experimental groups. Three sets of experiments wereconducted to study the effects of aqueous infusion,crude and purified extracts of S.chirata. The test agentswere administered by interperitoneal injections (i.p.)daily for 12 weeks. 1, 2 and 5% aqueous infusion wereadministered. Three selected doses of extracts usedwere below IC50 (5, 2.5 and 1 mg/mouse of crude and

0.5, 0.2 and 0.1 mg/mouse of purified extract). Bestresponse to treatment was found at the dose of 2%infsion, 2.5 mg/mouse of crude and 0.2 mg/mouse ofpurified preparation, the results of which are presentedhere. Carcinogen control group (CC) received DMBAand croton oil and only distilled water (i.p.) in placeof treatment with the plant extracts. The treatmentgroups received carcinogen as in CC group but weretreated with the selected doses of the infusion, crudeand purified samples administered i.p. The Numberof animals in each group was 40 of which 30 weresacrificed for histological and biochemical studies andthe remaining 10 were observed for development ofskin papillomas.

Biochemical Estimations. Biochemical estimation ofthe enzymes were done after 15 days and 12 weeksof treatment. Glutathione-S-transferase (GST) activitywas measured in the liver cytosol after 15 days oftreatment following the method of Habig et al.(1974). The enzyme activity was determined fromthe increase in absorbance at 340 nm with 1-chloro-2-4-dinitrobenzene (CDNB) as the substrate and specificactivity of the enzyme expressed as formation of1-chloro-2-4-dinitrobenzene (CDNB)-GSH conjugateper minute per mg of protein. Glutathione peroxidase(GPx) activity was also determined after 15 days oftreatment in the post mitochondrial fraction by themethod of Paglia and Valentine (1967). The reactionmixture contained NADPH and glutathione reductase.The decrease in absorbance following addition ofH2O2 was recorded at 340 nm. Enzyme activity wasexpressed as µmoles of NADPH utilized per minuteper mg protein using molar extinction co-efficient at340 nm as 6200 m−1 cm−1. Activity of catalase (CAT)in liver was estimated by the method of Luck (1963).The enzyme activity was determined spectrophoto-metrically at 250 nm and expressed as unit/mg proteinwhere the unit is the amount of enzyme that liberateshalf the peroxide oxygen from H2O2 in 100 seconds at25 °C. Superoxide dismutase (SOD) activity was deter-mined by quantification of pyrogallol auto oxidationinhibition by the method of Marklund and Marklund(1974) and expressed as unit/mg protein. One unit ofenzyme activity is defined as the amount of enzymenecessary for inhibiting the reaction by 50%. Autooxidation of pyrogallol in Tris-HCL buffer (50 mM,pH 7.5) is measured by increase in absorbance at420 nm. Lipid peroxidation was estimated in livermicrosomal fraction by using the method of Okahawaet al. (1979). The level of lipid peroxides formed wasmeasured using thiobarbituric acid and expressedas thiobarbituric acid reactive substance (TBARS)formed per mg protein using extinction co-efficientof 1.56 × 105 M−1 cm−1. Protein was estimated by themethod of Lowry et al. (1951). The level of cell pro-liferation was studied in situ following incorporation,5-bromo-2′-deoxy-uridine (BrdU) using BrdU Labelingand Detection Kit II, AP (Roche Germany) as per kitprotocol. The tissue samples were then evaluated inphasecontrast microscope. The percentage of apoptosismeasured by the TUNEL method using in situ celldeath detection kit, Fluorescein (Roche Germany). Theskin tissues were fixed with 4% paraformaldeyde andsections were permeabilized with 0.1% Triton X-100in 0.1% Sodium citrate and labeled with TUNEL



Table 1. Effects of Swertia chirata on DMBA induced murine skin carcinogenesis (12 weekstreatment)

No of papilloma per Inhibition ofNo. of papilloma Incidence of papilloma bearing multiplicity

Groups bearing mice Papilloma (%) mouse ± SEM (%)

Normal 0 (out of 10) (–) (–) (–)Carcinogen Control 8 (out of 10) 80 3.6 ± 0.45 0.0Aqueous Infusion 6 (out of 10) 60 1.8 ± 0.29 63.5Crude extract 3 (out of 10) 30 2 ± 0.16 79.2Purified extract 4 (out of 10) 40 1 ± 0.25 86.1

Inhibition of multiplicity = (Total no. of papilloma in carcinogen control) – (Total no. of papillomain treated) × 100/Total no. of papilloma in carcinogen control.

reaction mixture, and observed, using fluorescencemicroscopy.

Statistical analysis. All calculations were done using MS-Excel. Results are expressed as Mean ± SEM or aspercentage. The data from biochemical determinationswere analyzed using students-t-test and a p-value of<0.05 was considered significant.

RESULTS

Incidence and multiplicity of papillomas

It may be seen from Table 1 that minimum incid-ence of papillomas was noted in the group treatedwith crude extract but maximum inhibition of multi-plicity of papillomas was produced by treatmentwith the purified extract containing the compoundamarogentin.

Effect of treatment on GST, GPx, SOD, CAT andlipid peroxidation in mouse liver

Figs 1 and 2 reveal that activities of the detoxifica-tion enzymes studied were altered following exposureto the carcinogen. But the nature of alteration variedat the two time points during carcinogenesis, selectedfor the present study, viz. 15th day and 12th week. Itmay be noted from Fig. 1 that GST activity at day 15after the first DMBA exposure was enhanced almosttwo fold as compared to the normal control group.Contrary to this GPx, SOD and CAT activities weredecreased. Treatment with the plant extracts activ-ated GST further, but a reversal of enzyme activitytowards normal was noted in case of GPx and SOD.CAT activity however continued to fall in the groupstreated with the infusion and crude extract. Observa-tion at the 12th week (Fig. 2) was somewhat different.Increased activity was noted for both GST and GPxwith no change with respect SOD and CAT in the car-cinogen control in comparison to normal. Treatmentwith S.chirata extracts resulted in reduced activitiesof GST, GPx as well as SOD, with activation of CATby crude and purified fractions only.

Lipid peroxidation, which was enhanced follow-ing carcinogen exposure, was found to be reduced

significantly by treatment with crude and purepreparations of S.chirata but not by the infusion(Fig. 3).

Apoptosis and cell proliferation

S.chirata infusion failed to produce any change in cellproliferation and apoptosis on the DMBA induced skinlesion. But crude extract and amarogentin rich purifiedfraction was found to inhibit cell proliferation andinduced apoptosis (Fig. 4) at the target site, i.e. skinepithelium.

DISCUSSION

Halliwell and Gutteridge (1989) defined free radicalsas ‘any species capable of independent existence thatcontain one or more unpaired electrons’ and in thebiological system, they can be generated throughxenobiotic metabolism, UV and ionizing radiation, andsecretion of oxidants from inflammatory leukocytes(Bland, 1986). Free radicals are able to induce cellulardamage in a variety of ways because of their very highchemical reactivity of which damage to DNA (Richteret al., 1988) is associated with and precedes carcino-genesis. Many phytochemicals are known to preventthe deleterious effects of free radicals. The presentinvestigation was undertaken to evaluate the effectof Swertia chirata extracts on the activation of thebiochemical pathways for detoxification of hazardouscompounds. GST plays an important role in initiatingdetoxification by catalyzing the conjugation of GSH tothe electrophilic foreign compounds for their elimina-tion from the system, thereby providing cellular pro-tection against a wide variety of xenobiotics (Sedlackand Lindsay 1968 and Szarka et al., 1995). GPx, SODand CAT form a part of the crucial processes involvedin cellular antioxidant defense mechanism wherebyperoxides and superoxides are inactivated (Vang et al.,1997). The present study clearly demonstrates thatextracts of Swertia chirata can induce reversal ofaltered enzyme activities, although the nature of inter-play of the four enzymes during DMBA inducedcarcinogenesis and following treatment is not quite clearat this stage. However, it is evident that modulation ofthe enzyme activities is accompanied by a reduction

376 P. SAHA ET AL.


Figure 1. Effect of Swertia chirata on the detoxification enzymes Glutathione-S-transferase (GST), Glutathione peroxidase (GPx),Superoxide dismutase (SOD), and Catalase (CAT) in liver of mice during skin carcinogenesis which received treatment for 15 days.All significance values calculated in respect to carcinogen control.* p < 0.05, ** p < 0.01, *** p < 0.001.

in lipid peroxidation, a process known to generatereactive oxygen species that is associated with tissueinjury and damage of cellular macromolecules (Sedlackand Lindsay, 1968; Szarka et al., 1995). We have alsonoted that the modulatory effects of the plant extractson the detoxification enzymes and reduction of lipidperoxidation in liver, the major site of carcinogenmetabolism, is reflected in the reduction of incidenceof skin papilloma and inhibition of multiplicity ofpapillomas.

Any mutation that involves factors which regulatecell proliferation, by conferring advantages like abilityto divide faster or ability to survive longer, can alloweven a single cell to establish itself among a popula-tion of normal cells. In mouse epidermis 5–12% of cellsin basal layer are stem cells (Potten, 1992). The cellprogeny generate a discrete column of cells from basalcells to keratinized cells, arranged in a hexagonal pat-tern and called an epidermal proliferative unit (Potten,

1981). Increases in epidermal proliferation by mutatedstem cells can lead to the establishment and develop-ment of a mutated clone, which ultimately can resultin papillomas. Programmed cell death (apoptosis) isa part of normal physiology for most metazoan speciesby virtue of which unwanted cells are removed dur-ing development, making important contributions tomorphogenesis and organogenesis (Vaux and Korsmeyr,1999). Defective apoptosis may promote cancer as anyderangement of the cell death machinery, which pre-vents the programmed turn over of cells, can increaseundesired cell accumulation, genetic instability, enhancecells longevity and permit cells to survive in a suspendedstate. Treatment with both crude and purified extractof Swertia chirata was found to result in inhibitionof cell proliferation and induction of apoptosis, at thetarget site initiated with DMBA, eventually resultingin a control on overproduction of cells at the site ofcarcinogenic exposure. This may be responsible for the



Figure 2. Effect of Swertia chirata on the detoxification enzymes Glutathione-S-transferase (GST), Glutathione peroxidase (GPx),Superoxide dismutase (SOD), and Catalase (CAT) in liver of mice during skin carcinogenesis which received treatment for 12 weeks.All significance values calculated in respect to carcinogen control.* p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3. Inhibition of Lipid peroxidation in liver of mice duringskin carcinogenesis after 12 weeks of treatment with crude andpurified extracts of Swertia chirata as compared to the carcino-gen control group (CC) and significance values calculated inrespect to carcinogen control.** p < 0.01, *** p < 0.001.

reduction in incidence of papilloma noted followingtreatment with Swertia chirata.

In view of these observations it is suggested thatSwertia chirata, the Indian medicinal plant, is a sourceof many anticarcinogenic agents which may be usefulfor prevention of cancer. Further chemical analysisof this endangerd Indian plant species and evalu-ation of the biological activities of the different frac-tions and purified compounds from this plant aretherefore required to explore their anticarcinogenicpotentials.

Acknowledgements

The authors express their profound gratitude to Dr N. Frank, DKFZ,Heidelberg, Germany for his help and support. Kind acknowledge-ment is also made to the Directors of Chittaranjan National CancerInstitute and Central Research Institute (Ayurveda), Kolkata, fortheir interest and encouragement in this work.

378 P. SAHA ET AL.


Figure 4. Induction of Apoptosis and inhibition of Proliferation in skin of mice during carcinogenesis following 12 weeks of treatmentwith crude and purified extracts of Swertia chirata.

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evaluation of the anticarcinogenic activity of swertia chirata buch.ham, an indian medicinal plant,...

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