ANTICANCER ACTIVITY OF OCIMUM SANCTUM

Pharmaceutical Biology 1388-0209/99/3704-0285$15.00 1999, Vol. 37, No. 4, pp. 285 290 Swets & Zeitlinger ANTICANCER ACTIVITY OF OCIMUM SANCTUM K. Karthikeyan 1, P. Gunasekaran 2, N. Ramamurthy 2 and S. Govindasamy 1* 1 Department of Biochemistry and Molecular Biology, University of Madras, Guindy Campus, Madras, India 2 Virology Department, King Institute of Preventive Medicine, Guindy, Madras 600 032, India ABSTRACT Ocimum sanctum L. (Labiatae), a plant with various medicinal properties, has been investigated against human fibrosarcoma cells (HFS cells) in culture. Treatment with an ethanolic extract of Ocimum sanctum induced cytotoxicity at 50 g/ml and above. Morphologically the cells showed shrunken cytoplasm and condensed nuclei. The DNA was found to be fragmented on observation in agarose gel electrophoresis. Biochemically the extract-treated HFS cells showed depleted intracellular glutathione and increased levels of lipid peroxidation products. Administration of aqueous and ethanolic extracts of Ocimum sanctum to mice bearing Sarcoma-180 solid tumors mediated a significant reduction in tumor volume and an increase in lifespan. These observations clearly indicate Ocimum sanctum extracts possess anticancer activity. INTRODUCTION Keywords: Apoptosis, cytotoxicity, glutathione, human fibrosarcoma cells, Ocimum sanctum extracts, Sarcoma-180 solid tumor. * Address correspondence to: S. Govindasamy, Department of Biochemistry and Molecular Biology, University of Madras, Guindy Campus, Madras 600 025, India. Fax: 91 44 2352494, Email: sgsamy@unimad.ernet.in. Ocimum sanctum L. (Labiatae), a plant commonly called holy basil and Tulsi in Indian languages has been widely used in the Ayurvedic system of medicine for its various medicinal properties. Previously, we studied the chemopreventive role of O. sanctum on hamster buccal pouch carcinogenesis using 7,12- dimethylbenz(a)anthracene (DMBA) (Karthikeyan et al., 1999). In the present study, experiments were designed to study the effect of O. sanctum aqueous (AE) and ethanolic extracts (EE) on human fibrosarcoma (HFS)-1080 cell line and against Sarcoma-180 cells (S-180) carried in mice. MATERIALS AND METHODS Chemicals 5,5'-Dithiobisnitrobenzene, glutathione and bovine serum albumin were obtained from Sisco Research Laboratories, India. Sarcosyl, proteinase K and ethidium bromide were obtained from Sigma Chemical Co., St. Louis, MO, USA. Fetal calf serum was obtained from Gibco laboratories, UK. All other chemicals were of analytical quality. Ocimum Sanctum Extract Preparation Aqueous Extract (AE). Fresh leaves (10 g) of O. sanctum (obtained from a vegetable market, Madras, India) were refluxed with 100 ml of distilled water and filtered. The filtrate was used for in vivo treatment assuming it contained 10 g of active principle components. The extract was vacuum dried and the powder was dissolved in phosphate buffered saline (PBS) for in vitro studies. Ethanolic Extract (EE). Dried powdered leaves were soaked for 48 h in ethanol (95%) and filtered. The filtrate was evaporated to dryness and a crude paste was suspended in olive oil for the in vivo study, and the paste was dissolved in 0.3% dimethyl sulphoxide (DMSO) in distilled water for in vitro studies. Both the extracts were freshly prepared prior to use. Cell Lines. Sarcoma-180 and HFS-1080 cell lines were obtained from the National Centre for Cell Sciences (NCCS), Pune, India. Sarcoma-180 cell line is a suspension cultured cell line and HFS cell line grows as a monolayer maintained in minimum essential medium

286 K. KARTHIKEYAN ET AL. with 10% (v/v) fetal calf serum, 100 units/ml penicillin and 100 g/ml streptomycin sulphate. Cultured cells were maintained at 37 C in a humidified atmosphere with 5% CO 2 and air. In Vitro Studies Cytotoxicity. HFS cells were grown in 96-well tissue culture plates (Nunc, Denmark). The monolayer cells (1 10 4 ) cells were treated with serial dilutions of aqueous and ethanolic extracts. After 24 h of incubation, the cells were fixed in a methanol:acetic acid mixture (3:1, v/v), stained with 1% crystal violet in 50% methanol, and read in a Microelisa reader (Microskan EX, Labsystems, USA) at 570 nm. Percent viability was defined as the relative absorbance of treated versus untreated control cells. The viable cells were also counted by the trypan blue exclusion method. Cell Morphology. HFS monolayer cells (1 10 6 cells) were treated with serial doses of ethanolic extract and incubated for 24 h. After 24 h, the cells were washed twice with PBS and fixed in methanol:acetic acid mixture (3:1, v/v) and stained in 2% Giemsa solution for 15 min and visualised using a light microscope (Nikon, Japan). The cells with condensed nuclei and shrunken cytoplasm were considered apoptotic. DNA Fragmentation Study. HFS monolayer cells (1 10 6 cells) were treated with serial doses of ethanolic extract and incubated for 24 h. After 24 h, the cells were harvested, pelleted, and washed twice with PBS. The pelleted cells were lysed and suspended in dye and electrophoresised in 1% agarose gel as reported by Zhu and Wang (1997). Biochemical Studies. HFS cells (1 10 6 ) were treated with serial doses of ethanolic extracts for 24 h. The cells were harvested by trypsinization, washed twice with PBS and pelleted by centrifugation, resuspended in phosphate buffer (0.1 M, ph 7.4), and sonicated at 100 W for 2 min (9 sec pulse followed by 9 sec no pulse). Further processes were carried out at 4 C. The lysate were centrifuged at 3,000 g for 10 min and the supernatant was used for the estimation of glutathione (GSH) (Moron et al., 1976), lipid peroxides (Ohkawa et al., 1979), and protein (Lowry et al., 1954). In Vivo Study A total of 182 (42 140) young male Swiss albino mice of Wistar strain (obtained from King Institute of Preventive Medicine, Madras, India) weighing 18 22 g were taken for the study. They were given a commercial diet (Brooke Bond Lipton India Limited, Calcutta, India) and water ad libitum. The animals were divided into control and experimental groups comprising six animals in each group. Group C 1 -control, C 2 -olive oil control (vehicle), C 3 -AE (1.2 g/kg body weight, p.o.), C 4 -EE (800 mg/kg body weight, p.o.). S-180 (1 10 6 ) cells were injected into the hind limb of the experimental group of mice and divided into three groups: Group E 1 -Sarcoma-180 control mice, E 2 -Sarcoma-180 AE (1.2 g/kg body weight, p.o.), E 3 -Sarcoma-180 EE (800 mg/kg body weight, p.o.). The extracts were administered triweekly from day zero for 4 weeks. The tumor volume was noted every week for 4 weeks and the volume was calculated by the formula v 4/3 r 1 2r 2 2, where r 1 and r 2 are the radii along two directions. For survival rate, administration of extracts were withdrawn after four weeks and the animals were observed thereafter to calculate percentage increase in lifespan (% ILS) by the formula: T C / C 100, where T represents the average number of days the treated animals survived and C represents the average number of days untreated control animals survived. All the experiments were repeated three times and the data obtained were statistically analysed using Student s t-test. RESULTS Extract Preparation Approximately 1 g of crude paste was obtained when 10 g of dried leaf powder was used for the ethanolic extract preparation. In the aqueous extract preparation, 60 70% of extract was obtained as filtrate. The crude ethanolic paste is sparingly soluble in water, PBS, but freely dissolved in DMSO and other organic solvents. In Vitro Studies Cytotoxicity and Biochemical Studies Ethanolic extract alone showed cytotoxicity at 50 g/ml and at higher doses when incubated for a 24 h period. Figure 1 depicts the percent viability of HFS cells, levels of glutathione, lipid peroxides with increase in ethanolic extract dosages. The mean lethal concentration (IC 50 ) was found to be 187.5 g/ml. HFS cells treated with ethanolic extract showed a significant decrease in the level of intracellular glutathione and

ANTICANCER ACTIVITY OF OCIMUM SANCTUM 287 Fig. 1. Percent viability, levels with glutathione and lipid peroxides in HFS cells on treatment with EE. Mean SD, six repetitions for each experiment. increase in lipid peroxides when compared to untreated cells. There was no significant changes between control and DMSO solvent control cells. Cell Morphology. Giemsa stained cells showed condensed nuclei in 50, 100, 200 and 400 g/ml EE doses (Fig. 2). DNA Fragmentation. DNA fragmentation studies with ethanolic extract at doses from 50 g/ml showed typical fragmentation patterns indicating the presence of DNA equivalent to the size of single and oligonucleosomes were seen (Fig. 3, lane D). We also included serum starved HFS cells (24 h exposure) as standard induction of DNA fragmentation (Fig. 3, lane C). DNA fragments were not detected in untreated HFS cells or in DMSO treated control cells (lanes A and B). In Vivo Studies The inoculated Sarcoma-180 cells in the hind limb of mice grew well and a significant reduction in tumor volume was noted on ethanolic extract treatment (E 3 group) (Table 1). No significant changes were observed between the control group (group C 1 ) and the control treatment group (group C 2, C 3 and C 4 ) during the experimental period. Oral administration of aqueous extract to the E 2 group of animals showed no significant changes up to the third week and a mild significant reduction in the volume of tumor was noted in the 4th week compared to untreated S-180 control animals. The S-180 control animals showed 90% survival after 4 weeks and a dramatic decrease was noted on subsequent days. At the end of 5th week, only 45% of the animals survived, and 5% survival was observed after 6 weeks. There was an increase in the lifespan of animals treated Table 1. Volume of tumor (cm 3 ) after the first, second, third and fourth weeks of groups E 1, E 2 and E 3. Group Week First Second Third Fourth E 1 0.31 ± 0.06 1.56 ± 0.14 3.22 ± 0.22 4.29 ± 0.32 E 2 0.28 ± 0.04 1.48 ± 0.12 2.88 ± 0.36 3.82 ± 0.34 a E 3 0.28 ± 0.06 1.42 ± 0.10 2.76 ± 0.28 a 3.71 ± 0.28 b Mean ± SD, six animals in each group. a: p 0.05; b: p 0.01 when compared to E 1 group.

288 K. KARTHIKEYAN ET AL. Fig. 2. Giemsa stained HFS cells after treatment with EE for 24 h. Cells with condensed nuclei were shown by ( ) and fragments by ( ). magnification 200. A B C D fourth weeks, respectively, when compared to S-180 control mice. In the third week, significance was p 0.05 compared to AE (p.o.), and p 0.01 when compared to AE and EE (p.o.) in the fourth week. DISCUSSION Fig. 3. DNA fragmentation observed after 24 h exposure to EE. Lane D 50 g/ml concentration. Lane C cells starved with serum for 24 h. Control cells and DMSO-treated cells did not display any fragmentation (lanes A and B, respectively). with aqueous extract (73%) and on ethanolic extract treated animals (118%). This showed the ethanolic extract had greater ability to reduce tumor development compared to the aqueous extract. The responses seen in S-180 mice were also reflected in tumor growth delay (data not shown). When AE was administered intraperitoneally (1.2 g/kg body weight), there was a dramatic decrease in tumor volume and an increase in lifespan (154%). The tumor volume was found to be 1.38 0.1 (p 0.05) after the second week, 2.40 0.32 (p 0.001) and 3.18 0.26 (p 0.001) after the third and Studies showed that AE did not mediate cytotoxicity at concentrations as high as 400 g/ml. The mechanism by which EE induces cytotoxicity is presently unknown but might be due to intracellular GSH depletion. The considerable cellular damage found in GSH deficiency reflects increased lipid peroxidation. It is not known whether the extract scavenges GSH directly and/or indirectly by influencing the endogenous production of free radicals. In recent years a large body of evidence has accumulated to suggest that oxidative stress may play a role as a common mediator of apoptosis (Hacker & Vaux, 1995; Slater et al., 1995). It is known apoptosis can be induced by accumulation of reactive oxygen intermediates (Papa & Skulachev, 1997). It has also been postulated that many cytotoxic drugs can generate elevated reactive oxygen intermediates through electron transport chains associated with the mitochondrial membrane (Gorman et al., 1997). Characteristic morphologic changes were evident and DNA ladder formation occurred concurrent with or slightly prior to cytotoxicity. Hence, it is likely that the damage caused by EE may trigger the cell to engage the apoptotic pathway.

ANTICANCER ACTIVITY OF OCIMUM SANCTUM 289 Administration of Ocimum sanctum extracts showed that EE had greater ability to reduce tumor development compared to AE. The observation is contrary to our previous study wherein the aqueous extract had more potency in the hamster buccal pouch carcinogenesis model than the ethanolic extract (Karthikeyan et al., 1999). We also administered (p.o.) both AE and EE to animals 14 days after injection of S-180 cells in mice. There was no significant alteration in the volume of tumor development but there was an increase in the survival rate (48 and 94% ILS in AE and EE, respectively). This shows that the extracts did not have any direct impact on tumor cells, rather they delay the development of tumor indirectly. The beneficial effect of O. sanctum may therefore be due to its direct or indirect effect on the immune system. Immuno-regulatory activity, modulatory activity of humoral immune responses in animals, and enhanced cell mediated immunity in man has been reported on O. sanctum treatment (Godhwani et al., 1988; Mediratta et al., 1988; Trivedi et al., 1995), suggesting that O. sanctum might influence the immune system to delay the neoplastic process. It is also noted that ursolic acid, a product of O. sanctum, has been reported to have antioxidant properties (Balanehru & Nagarajan, 1992). This shows that O. sanctum may have the ability to scavenge free radical mediated events in the neoplastic development. Aruna and Sivaramakrishnan (1990, 1992) reported that administration of O. sanctum to mice significantly elevated glutathione and more than 78% of glutathione S-transferase activity and prevented forestomach tumors and hepatomas. Hence, an increase in survival rate but not tumor volume on administration of O. sanctum extracts to animals after S-180 cells implantation might also be due to the antioxidant nature of the extracts. Since it is known that the crude extracts of O. sanctum contains many active principles and unknown constituents, the active principle which elicit this kind of response in HFS cells is not known at present. Since EE was found to significantly delay S-180 cell induced solid tumors in mice, and AE and EE prevent 7,12- dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis (Karthikeyan et al., 1999), AE (p.o.) may not have the ability to elicit any direct response in S-180 cell-induced solid tumor in mice, though mild significance was observed at the fourth week. This implies that AE may either lack some components or possess some additional components that inhibit the response elicited by EE. However, AE administered by the intraperitoneal route was found to have a greater effect than the oral route. Devi and Ganasoundari (1995) also reported a similar radioprotective effects of O. sanctum where intraperitoneal injection produced maximum survival in irradiated mice followed by oral, i.v., and i.m. routes. Hence, it has been suggested that some active components present in AE might be inactive on oral administration. These observations clearly indicate that O. sanctum extracts have anticancer activity and we suggest that the active principle components of AE and EE may be useful as potential anticarcinogenic and chemotherapeutic agents. ACKNOWLEDGEMENT Financial assistance provided by the Lady Tata Memorial Trust, Bombay, in the form of Senior scholarship, is gratefully acknowledged by K. Karthikeyan. REFERENCES Aruna K, Sivaramakrishnan VM (1990): Plant products protective against cancer. Indian J Exp Biol 26: 1008 1011. Aruna K, Sivaramakrishnan VM (1992): Anticarcinogenic effect of some Indian plant products. Fd Chem Toxicol 30: 953 956. Balanehru S, Nagarajan B (1992): Intervention of adriamycin-induced free radical damage by ursolic acid. Biochem Int 28: 735 744. Devi PU, Ganasoundari A (1995): Radioprotective effect of leaf extract of Indian medicinal plant Ocimum sanctum. Indian J Exp Biol 33: 205 208. Godhwani S, Godhwani JL, Vyas DS (1988): Ocimum sanctum a preliminary study evaluating its immunoregulatory profile in albino rats. J Ethanopharmacol 24: 193 198. Gorman A, McGowan A, Cotter TG (1997): Role of peroxide and superoxide anion during tumor cell apoptosis. FEBS Lett 404: 27 33. Hacker G, Vaux DL (1995): The medical significance of physiological cell death. Med Res Rev 15: 299 311. Karthikeyan K, Ravichandran P, Govindasamy S (1999): Chemopreventive effect of Ocimum sanctum on DMBAinduced hamster buccal pouch carcinogenesis. Oral Oncol 35: 112 119. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1954): Protein measurement with the folin-phenol reagent. J Biol Chem 193: 265 275. Mediratta PK, Dewan V, Bhattacharya SK, Gupta VS, Maiti PC, Sen P (1988): Effect of Ocimum sanctum Linn. on humoral immune responses. Indian J Med Res 87: 384 386. Moron MS, Pierre JN, Mannervick B (1976): Levels of glutathione, glutathione reductase, glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta 582: 67 71.

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