ORIGINAL RESEARCH ARTICLE CHEMOPREVENTIVE POTENTIAL OF METHANOL

EXTRACT OF STEM BARK OF CASSIA FISTULA L. IN MICE.

Linu Mathew a,*, Shankar Shashidharb a. School of Biosciences, M.G. University, Kottayam, Kerala, India b. Azeezia Medical College, Kollam, Kerala, India Abstract Ethnomedical survey has shown that stem bark of Cassia fistula. L is used to treat skin diseases by tribal people of forest ranges of India. Therefore, bark extracts were tested for their chemopreventive potential in two-stage murine skin chemical carcinogenesis model. The papilloma were produced on mice skin by DMBA and promoted by croton oil. The methanol extract of stem bark of Cassia fistula L. (MEC) was found to prevent the formation as well as persistence of papilloma in treated mice compared to untreated control mice. Key Words. Cassia fistula L., Chemical carcinogenesis, DMBA, croton oil, papilloma I. Introduction Cassia fistula L. (Fa. Caesalpiniaceae) is a tropical evergreen tree with seasonal flowering and is a popular avenue tree in Indian subcontinent. The
* corresponding author. Tel.: E-mail: +914812731035 linubinoi @ rediffmail. com

common name of the plant is Indian Laburnum or golden shower. Most parts of this tree have medicinal uses in the traditional Indian systems of medicine. The stem bark of the plant is considered, an astringent and antihelminthic, in the Ayurveda system of medicine. The powdered bark of the plant is used to treat several skin abnormalities by the mountain tribes of the states of West Bengal (Das and Chattopadhyay, 2003) and Madhya Pradesh (Acharya and Rai, 2005). The bark of the plant is rich in flavonoids (Siddhuraju et al., 2002). The chemical constituents of the stem bark are fully characterized (Murty et al., 1967). In the present study to verify the ethnomedical claim, the methanolic extract of C.fistula L. (MEC) was tested for its chemopreventive potential in mouse two stage skin chemical carcinogenesis model. 2. Materials and methods 2.1 Plant material Stem bark of C. fistula was collected from the premises of School of Biosciences, M G University, Kerala, India and authenticated by Dr. V. T. Antony, Taxonomist, St. Berchmans College, Changanacherry, Kerala, India and a Voucher specimen was deposited in the Regional herbarium, Botanical Survey of India (Specimen. No. 4589) in St. Berchmans College, Changanacherry, Kerala, India. The bark was dried in shade and 100g of powdered bark was extracted with different organic solvents of increasing polarity in a soxlet apparatus. The methanol extract (MEC) was found to possess maximum activity in pilot studies. The extract was dried with a rotary evaporator under reduced

pressure at 40-450c. The yield of the extract was 18%. The extract was dissolved in 0.1% DMSO. 2.2 Animals Balb/c male mice weighing 18-20g were procured from Small Animal Breeding Station, College of Veterinary Sciences, Trichur, Kerala, India. The animals were acclimatized in the animal experimentation facility in School of Biosciences. They were given standard commercial pellet diet (Lipton India Ltd), and water ad libitum and maintained at 22-280C, relative humidity 60-70% and 12h dark light cycle. All the animal experiments were performed according to the rules and regulations of the CPCSEA, Government of India. 2.3 Chemopreventive activity For the long-term carcinogenicity bioassay, random bred male Balb/C mice of 18-20g were selected. They were kept in a group of ten animals per polypropylene cage under controlled environmental condition. During the course of the experiment, hair was removed from the interscapular region over an area of 2cm 3 using electric clippers that were not lubricated with oil or grease. Animals were observed for 3 days and animals in the resting phase of hair cycle were selected for the study. Mouse skin papilloma were induced in all groups by topical application of 7, 12, dimethyl benz (a) anthracene (DMBA, Sigma chemical Co., St. Louis, USA) at the rate of 100 nmol in acetone in the 2nd week of experiment and promoted by 1% croton oil (Sigma chemical Co., St. Louis, USA) in acetone thrice weekly until 15 weeks. The animals were divided into 3 groups comprising 10 animals.

Group1: Untreated control, receiving 0.1% DMSO topically thrice weekly (on two days interval) Group2: Receiving MEC, weekly thrice @ 100mg/kg body wt by topical application on 1st and 3rd week of experiment, and 0.1% DMSO for the rest of the duration of the experiment Group3: Receiving MEC thrice weekly @) 100mg/kg body weight by

topical application throughout the study period The shaven skin of the treated mice was observed daily for the eruption of papilloma, and body weight was taken weekly. The time of first papilloma eruption (latency period), cumulative and average no. of papilloma, no. of animals bearing papilloma on 15 th week, total and average no. of papilloma per mouse at 8th, 12th and 15th week were observed. 2.4 Statistical analysis The means were expressed as Mean SEM and the results were analyzed by one-way analysis of variance (ANOVA). The means were compared against untreated control by Dunnetts test of multiple comparison. 3. Results Average and total number of papilloma appearing in different treated groups showed statistically significant difference compared to untreated control, at all time periods of observation namely 8th, 12th and 15th weeks. Untreated control animals had maximum number of papilloma followed by groups G2 and G3. Similarly, the time of initiation of first papilloma (latency period) was 8

weeks for G1 and G2, but for G3 it was 12 weeks. In the case of G1 and G2 by the end of 15th week 80% of animals developed papilloma. Nevertheless, in the case of G3 only 30% animals developed papilloma, by the end of the period of observation (15th week) (Table 1). 4. Discussion and conclusions In the mouse skin tumour promotion model, tumourigenesis is initiated by; one sub minimal dose of carcinogen and treatment with croton oil promotes the development of the visible tumour stage by clonal expansion (Berenblum and Shubik, 1949). The application of promoter to the mice skin results in rapid accumulation of inflammatory cells and an increase in the release of Reactive Oxygen Species (ROS) (Copeland, 1983, Cerrutti, 1985). ROS are suspected to be a probable reason for the pathogenesis of several chronic diseases including cancer (Klauning and Kamendulis, 2004). If not scavenged by antioxidant control mechanisms of the cell, ROS may result in the damage to cellular nucleic acids lipid and protein and unrepaired damage to DNA may result in mutation and cancer. From this study it is evident that the application of MEC prevented tumour initiation (G2) and tumour promotion (G3) in treated groups (Table 1). The ability of the MEC treatment to increase the latency period is suggestive of its ability to reduce clonal expansion and thereby, tumour promotion.

Antioxidant principles of many plants are reported to possess antitumour activity (Ruby et al, 1995). All plant parts of C. fistula L. possess in vitro antioxidant activity, and among these maximum antioxidant activity was shown by the methanol extract of stem bark (Siddhuraju et al, 2002). This property may

be attributed to the abundant polyphenolics, anthraquinones, xanthones, proanthocyanidines, and flavonols in the stem bark of the tree (Siddhuraju et al, 2002). These compounds are well known for their antioxidant, antimutagenic and chemopreventive potential (Jovanovic et al, 1994, Aitken et al, 1994). The presence of these compounds in the MEC may be the reason for its chemopreventive potential. However, further investigations are essential for the isolation of the active principle of MEC and its mechanism of action. Literature cited Acharya, D., Rai, M.K., 2005.Traditional Knowledge for Curing Various Ailments among Gonds and Bharias of Patalcot Valley, Madhya Pradesh, India. http://www.selfgrowth.com/articles/Acharya12.html/. Aitken, R. A., Bibby, M. C., Double, A., Phillips, R. M, Sharma, S. K., 1994. Synthesis and antitumour activity of 6 methyl derivatives of flavone -8-acetic acid (FAA). Bioorganic and Medicinal Chemistry Letters 4, 2313-2316 Berenblum, I., Shubik, P., 1949. An experimental study of the initiating stage of carcinogenesis, and a re- examination of the somatic cell mutation theory of cancer. British Journal of Cancer 3, 109 118. Cerrutti, P.A., 1985. Prooxidant States and Tumour Promotion. Science 227, 375 381. Copeland, E.S., 1983. Free radicals in Cancer. Cancer Research 43, 5631 5637. Das, N., Chattopadhyay, R.N., 2003. Studies on Ethnomedical Plants of Purulia Dist, West Bengal, India. Journal of Nontimber forest products 10(3/4), 218-229.

Jovanovic, S. S. V., Steen Ken, S., Tosic, M., Marjanovic, B., Simic, M.G., 1994. Flavonoids as Antioxidants. Journal of the American Chemical Society 116, 4846-4851. Klaunig, E.J., Kamendulis, M.L., 2004. The Role of Oxidative Stress in Carcinogenesis. Annual Review of Pharmacology and Toxicology 44, 239 267. Murty, V.K., Rao, T.V.P., Venketeswarlu, V., 1967. Chemical Examination of Cassia fistula. Tetrahedron 23, 515 518. Owens, D. M., Carolyn Wei, S. J., Smart, C. R., 1999. A multihit, multistage model of chemical carcinogenesis. Carcinogenesis 20(9), 1837-1844. Ruby, A.J., Kuttan, G., Babu, K.D., Rajasekaran, K.N., Kuttan, R., 1995. Antitumour and Antioxidant Activity of Natural Curcuminoids. Cancer Letters 94, 783 789. Siddhuraju, P., Mohan, P.S., Becker, K., 2002. Studies on the antioxidant activity of Indian Laburnum (Cassia fistula L): a preliminary assessment of crude extracts from stem bark, leaves, flowers, and fruit pulp. Food Chemistry 79, 61 -67.