International Journal of Research in Pharmacology & Pharmacotherapeutics

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1 314 International Journal of Research in Pharmacology & Pharmacotherapeutics Available online at Print ISSN: Online ISSN: IJRPP Volume 2 Issue Research article Anti Cancer activity of Cinnamomum Malabatrum against Dalton s ascitic lymphoma *Sorabh kumar agrawal, R.C.Chipa, K.C.Samanta Suresh. Gyan Vihar School Of Pharmacy, Jaipur, Rajasthan, India. ABSTRACT The aim of the research is to find out new anti cancer drugs from indigenous plant which are potent, nontoxic or minimal toxic and to investigate the anti cancer activity of Cinnamomum Malabatrum. The powder of Cinnamomum Malabatrum was successively Extracted with Petroleum Ether, Chloroform, Acetone, Ethyl alcohol and Aqueous.The preliminary phytochemical test were done and the LD 50 values for both alcohol and aqueous extract determined. The anti cancer activity of the alcoholic (625 mg / kg. p.o.) and aqueous Extract (500 mg / kg. P.O.) were assessed in Dalton s Ascitic Lymphoma induced cancer. KEY WORDS: Cinnamomum malabatrum, Delton, s ascitic lymphoma. INTRODUCTION Herbal medicine is the oldest form of healthcare known to Mankind herbs has been used by all cultures throughout history. It was an integral part of the development of modern civilization. Primitive men observed and appreciated the great diversity of plants available to him. The Cinnamomum Malabatrum is moderate ever green tree, bark smooth or slightly longitudinal cracked light brown, leaves are opposite or sub-opposite, elliptic to oblong, glabrous, pink when young, 3-nerved from close above the base almost to the apex. Flowers long, pale yellowish, fruits ellipsoid. Cinnamomum Malabatrum leaves contain chemical composition of cinnamic aldehyde, euganol, β-caryophyllene, benzaldehyde, camphor, cadinene, α-terpinol, limonene, geraniol, euganol acetate, ocimene, γ-terpinene, benzyl cinnamate, β-phellandrene and benzyl acetate. The oil from bark contains cinnamaldehydes (70-85%) as a major constituents. The plant also contain 3,4,5,7- * Corresponding author: Sorabh kumar agrawal address: sorabh44@gmail.com tetra hydroxyl flavones, 3,3,4,5,7-pentahydroxy flavones, kaempferol-3-o-sophoroside and quercetin 3-O- rutin. The plant has been traditionally used as stimulant, carminative, haemostatic, astringent, diaphoretic, deobstruent and galactogogue. The bark also prescribed in bowl complaints such as dyspepsia, flatulence, diarrhea and vomiting. The leaves are carminatives and are used in colic and rheumatism. They are sweetish, heating, useful in vata, scabies, disease of the anus and rectum, tridosha, piles and heart troubles. The dried buds are used with various combinations in cough and urinary disease. The plant is also used for treatment of some tumors. Hence present study was aimed to investigate the anti cancer activity of plant extract of Cinnamomum Malabatrum on DAL induced cancer in Swiss Albino Mice. MATERIAL AND METHODS The bark of Cinnamomum Malabatrum were collected from ABS Botanical Conservation,

2 315 Research and Training Center, Kaaripatti, Tamilnadu in month of July,2009. The plant was identified and authenticated by Dr.A.BALASUBRAMANIAN. The bark part of Cinnamomum Malabatrum was shaded dried at room temperature and then powdered with a mechanical grinder. The powder was passed through sieve no.40 and stored in an air tight container for further use.the solvent used Petroleum ether (60-80 c), Chloroform, Acetone, Alcohol 95% w/v and Distilled water for decoction. The Petroleum ether extract was obtained using Soxhlet apparatus. Alcoholic extract was obtained with ethyl alcohol 95% w/v for 18 hrs using Soxhlet apparatus. The aqueous extract was prepared with the remaining mass by maceration process for 7 days. The extract was dried at 55 ºC in a water bath. The Percentage yield of Petroleum ether, Chloroform, Acetone, alcoholic and aqueous extract were 1.92%,1.68%,5.16%,3.89%and 12.2% respectively.(table no.1) Phytochemical Screening The preliminary phytochemical screening of Alcoholic and aqueous extract of Cinnamomum Malabatrum were carried out as described by khandelwal. (Table no.2) Animals Male & Female Swiss albino mice weighing between gm were used for this study. The animals were obtained from animal house of Sri Venkateshwara Enterprises, Bangalore, India. On arrival the animal were placed randomly and allocated to treatment groups in propylene cages with paddy husk as bedding. Animals were housed at a temperature of 24± 2ºC and relative humidity 45-55% with 12:12 hours light/dark cycle. The animals had free assist to food and water. The animals were habituated to laboratory conditions for 48 hours prior to the experimental protocol to minimize any nonspecific stress. All the experimental process and protocols used in this study were reviewed by the institutional animal ethical committee (Pharmacology project no. 02/2010) and were in according with the guideline of the CPCSEA. Acute Toxicity Study Acute oral toxicity of alcoholic and aqueous extract were determined using adult Swiss albino mice of both sexes. The control animals received normal saline orally. The Alcoholic extract of Cinnamomum malabatrum was administered once orally at various dose levels (6000 to 6400 mg/kg body wt. to group of 6 mice of both sexes about equal in number which have been fasting overnight (about 18h.). The aqueous extract of Cinnamomum malabatrum was administered orally at various dose levels (4400 to 5200 mg/kg body wt) to other group of animals. The animals were observed continuously for 2 hours and then occasionally for further 4 hours and finally overnight mortality recorded. Experimental Protocol Test compound The Alcoholic & Aqueous extract of Cinnamomum malabatrum (625mg/kg & 500 mg/kg body weight) and standard drug 5-fluorouracil (20 mg/kg i.p) were used. The following chemicals were obtained from the indicated commercial. Experimental Setup Swiss albino Mice (20-25 gm) used in the present studies was procured from listed suppliers of Sri Venkateshwara Enterprises, Bangalore, India. The animals were fed with standard pellet diet (Hindustan lever Ltd. Bangalore) and water ab libitum. All the animals were acclimatized for a week before use. The alcoholic & aqueous extract of Cinnamomum malabatrum was dissolved in normal saline. The rats were divided into five groups of 6 animals in each Group І : Normal Control Control animal were received normal saline Group ІІ : Tumor control Animals were inoculated with 1X106 cells per mouse intraperitoneal Group III : Standard Group Animals were injected 5-fluorouracil (20mg/kg) intraperitoneal along with DAL cells (1X106 ) cells per mouse treatment. Group IV : Test group- I Animals were treated with alcoholic extract of Cinnamomum malabatrum 625mg/kg orally along with DAL cells (1X106 cells /mouse) Group V : Test group -II Animals were treated with aqueous extract of Cinnamomum malabatrum 500 mg/kg orally with DAL cells (1X106 cells/mouse) treatment.

3 316 EVALUATION PARAMETERS Effect on Solid Tumor Volume by Simultaneous Method To determine the effect of alcoholic and aqueous extract of Cinnamomum malabatrum on solid tumor, all the animals were injected with 1x10 6 cells/mouse in phosphate buffered saline into the right hind limb of all the animals simultaneously. All the treatments were started from the next day of inoculation and were continued for five alternative days. The measurement of tumor radii was taken from 11 th day of tumor induction and was repeated on every 5 th day for a period of 30 days. The volume of tumor mass was calculated from the formula V= 4/3π r 2 where r is mean of r1 and r2 which are the two independent radii of the tumor mass (Table no.3). Effect On Peritoneal Cells Counts in Mice One group of animals was treated with aqueous extract at a dose of 500mg/kg/day, p.o. of stem bark of Cinnamomum malabatrum once for a single day and the second group received the same treatment for two consecutive days. Similar treatment was given to other group with Alcoholic extract at a dose of 625 mg/kg/day/p.o. the untreated first group was used as control. Peritoneal exudates of alcoholic and aqueous extract groups were collected after 24 hr and 48 hr of treatment by repeated intraperitoneal wash with normal saline (0.9% w/v) and the cells were counted in each of the treated groups under WBC newbauer s chamber and compared with those of normal control (Table no.4). Effect On Body Weight (21-22) Four groups of six mice each were transplanted intraperitoneal with 1x10 6 Dalton s ascetic tumor cells. After 24h, the first and second group were orally treated with Alcoholic and aqueous extract of Cinnamomum malabatrum. The third group, serving as the control, received normal saline (0.9% w/v). Treatments were continued for 9 days. Body weights were recorded every 5 th day till 40 days of treatments or till the death of the animal (Table No.5). RESULTS AND DISCUSSION (23-24) Preliminary phytochemical studies show the presence of Flavonoids, Fixed oil, Amino acids, Tannins and Phytosterols according to Table no.2. The Stem bark extract of Cinnamomum malabatrum was found to be minimal toxic. Treatment of mice with DAL produces cancer. DAL is one of the most common to produce tumor then present study demonstrated as aqueous extract exhibited significantly dose depended anti cancer activity. Alcoholic and aqueous extract shows significant decrease in Solid tumor volume, Increase in Peritoneal cell count and body weight compare to tumor control group. It can be concluded that Cinnamomum malabatrum stem bark extracts posses a protective effect against DAL induced cancer in mice. Table No.1 Data Showing the Extractive Values of Cinnamomum malabatrum Plant name Part used Method of extraction Extracts Extractive Values (w/w) Petroleum Ether 1.92% Cinnamomum malabatrum Stem bark Continuous hot percolation Chloroform Acetone Alcoholic 1.68% 5.16% 3.89% Aqueous 6.67%

4 317 Table No.2 Data showing the Preliminary Phytochemical Screening for extracts of Cinnamomum malabatrum Phytochemical constituents Petroleum ether Chloroform Acetone Alcohol (95%) Aqueous ALKALOIDS FLAVANOIDS CARBOHYDRATE SAPONINS TRITERPENS STEROLS TANNINS GLYCOSIDES VE = Present -VE = Absent Table No.3 Data showing the Effect of Cinnamomum malabatrum on Solid tumor volume (Mean± SEM n=6 in each group) in rats Groups Treatment Solid Tumor Volume (ml) 15 th days 20 th days 25 th days 30 th days I Tumor Control 7.56 ± ± ± ± 0.33 II Standard 4.56 ± 0.22* 4.16 ± 0.21* 4.16 ± 0.25* 3.16 ± 0.21* III Test Group- I 6.16 ± 0.25* 6.66 ± 0.22* 7.16 ± 0.22* 7.6 ± 0.25* IV Test Group II 5.56 ± 0.22* 6.33 ± 0.21* 7.33 ± 0.21* 8.1 ± 0.34* *P<0.01 Values are represented as mean ± S.E.M (n=6) One-way ANOVA followed by Student-Newman-Keuls post test (P< 0.001) is used. a-vs group I and b-vs group II.

5 318 Table No.4 Data showing the Effect of Cinnamomum malabatrum on Peritoneal Cell Count (Mean± SEM n=6 in each group) in rats Groups Treatment Peritoneal cell count (1X10 6 ) I Normal Control 5.08 ± 0.01 II Tumor Control 3.21 ± 0.07 *** III Standard ± 0.02 *** IV Test Group I 9.48 ± 0.13** V Test Group- II ± 0.12 Values are represented as mean ± S.E.M (n=6) One-way ANOVA followed by Student-Newman-Keuls post test (P< 0.001) is used. a-vs group I and b-vs group II. Table No.5 Data showing the Effect of Cinnamomum malabatrum on Body Weight (Mean± SEM n=6 in each group) in rats S.N Group Dose 7 th Day 14 th Day 21 st Day 28 th Day 35 th Day o. I Normal ± ± ± ± ± 0.83 control II Tumor ± 0.22* ± 0.22* ± 0.65* - - control III Standard 20mg/kg/ ± ± 0.32** ± 0.6** ± ± 0.47 day IV Test Group ± ± 0.33** 32.6 ± 0.66** ± 0.32** ± 0.33 I mg/kg/day V Test Group II 500 mg/kg/day ± 0.33* ± 0.76** 30.5 ± 0.82** 31 ± ± 0.30 One-way ANOVA followed by Student-Newman-Keuls post test (P< 0.001) is used. a-vs group I and b-vs group II. ACKNOWLEDGEMENT Authors are thankful to Management and Staff for providing the necessary facilities to conduct this study and are thankful to Ms.Divya for help pertaining to complete this study. Authors are also thankful to all persons whose help in this work.

6 319 REFERENCES [1] The U.S. Food and Drug Administration, Center for food safety and Applied Nutrition. Economic of Characterization the Dietary Supplement Industry Final Report. [2] Chen etal, Selective killing of transformed cells by cyclin/cyclin dependent kinase 2 antagonists. PNAS, 1999;96(8): [3] Levakau etal, Cleavage of p21 Cip/Waf 1 and p27 kip 1 mediates apoptosis in endothelial cells through activation of CDK2; Role of caspase cascade. Mol cell, 1998;1: [4] Kirtikar & Basu. Indian Medicinal Plants, Vol.1,2 nd Edition, [5] Nadkarni, A.J. The Indian Materica Medica; Popular Prakashan Pvt., Ltd. Bombay, Vol.(1996), [6] Anonymous, The Wealth of India, (1956), Vol.IV, CSIR, New Delhi, [7] Anonymous, The Ayurvedic Pharmacopoeia of India, Vol.1, 1 st edtn [8] Chapuis, J.,Sordat, B., Hostettmann, K., Screening for cytotoxic activity of plants used in traditional medicine. J.Ethnopharmacol.23, [9] Pulok and Mukherjee, Quality Control of herbal drugs edtn.1 st published by Business Horizons, New Delhi, Page-382 [10] Khandelwal KR, Practical Pharmacognosy techniques and experiments 9 th edition, Nirali Prakashan, Pune 2000 [11] Kokate C.K, Purohit A.P, Gokhale.S.B, Pharmacognosy, 5 th edition,1997 Nirali Prakashan, Pune [12] Gothoskar, S.V., Ranadive, K.J Anticancer screening of SAN-AB; an extract of marketing nut, Semicarpus anacardium. Indian Jounal of Experimental Biology 9, [13] Ghosh, M.N. (1984) Fundamentals of experimental pharmacology, 2 nd Edn. Scientific Book Agency, Calcutta [14] Kulkarni SK, Hand book of experimental pharmacology.2004 page 168 [15] T.A. Ajith, K.K. Janardhanan, Cytotoxic and antitumor activities of a polypore macrofungus, Phellinus rimosus (Berk) Pilat., Journal Of Ethnopharmacology 84(2003) [16] Ma, Y., Mizuno, T., Ito, H., Antitumor activity of some polysaccharide isolated from a Chinese mushroom, Huangmo, the fruiting body of Hohenbuehelia serotina, Agriculture Biological Chemistry 55, [17] B. Rajkapoor etal, Antitumor activity of Elephantopus scaber Linn against Dalton s Ascitic Lymphoma, Indian Journal of Pharmaceutical Sciences, Jan-Feb.2002, PP [18] T.J. Thomas etal, Antitumor property and toxicity of Barringtonia racemosa Roxb seed extract in mice, Journal of Ethnopharmacology 82 (2002) [19] Manika Das etal, Immunomodulatory and Antineoplastic activity of common Indian Toad skin extract, Indian Journal of Pharmacology, 1998,30: [20] K.L.bairy etal, Evalution of intraperitoneal vincristine in malignant peritoneal effusion, Indian Journal of Physiol Pharmacol, 2003; 2003;47(3) [21] Kavimani, S. Manisenthil kumar, K. Effect of methanolic extract of Glinus Lotoides on Dalton s Ascitic Lymphoma Biol. Pharm Bull 22(11): [22] Brown. J.P, A review of the genetic effects of occurring flavonoids, anthraquinones and related compounds. Mutation Research, 1980, 75: *******************************