Anticancer Activity of Clitoria ternatea Linn. Against Dalton’s Lymphoma

The aim of the study was to evaluate the anticancer activity of Clitoria ternatea in Dalton’s lymphoma(DLA) bearing mice. Tumour was induced in mice by the intraperitoneal injection of DLA cells. After 24 hours of tumour inoculation, methanol extract of Clitoria ternatea(MECT) was administered at doses of 100 and 200mg/kg body weight for 14 consecutive days. The effect of MECT was assessed using in vitro cytotoxicity, survival time, peritoneal cell count, hematological studies and antioxidant parameters. Treatment with MECT led to a decrease in tumour volume, packed cell volume and viable count. It also increased the non-viable cell count and mean survival time, thereby increasing the life span of EAC bearing mice. Hematological profile reverted to more or less normal levels in the treated group. The results suggest that MECT exhibit significant antitumour effects in DLA bearing mice. key words: anticancer; antioxidant; Dalton’s lymphoma; flavonoids; clitoria ternatea. INTRODUCTION Cancer is a major public health problem worldwide. It is the second most common cause of death in the developed world and a similar trend has emerged in the developing countries too. According to the American Cancer Society, deaths arising from cancer constitute 2-3% of the annual deaths recorded worldwide.In India, it has been estimated that there is about 1.5 million cases of cancer in the country at any given point of time with about 0.5 million new cancer cases being added every year. The search of natural products for cancer therapy represents an area of great interest in which plants had been the most important source. The World Health Organization estimates that approximately 80% of the world’s inhabitants rely on traditional medicine for their primary health care and that plants have long been used in the treatment of cancer. Drugs obtained from natural sources are perceived to have fewer side effects while having same ability to cure disorders in much the same way as their synthetic counterparts. So it is anticipated that plants can provide potential bioactive compounds for the development of new leads to combat cancer. Clitoria ternatea Linn (Fabaceae), known as Aparajitha in India is a persistent, herbaceous perennial legume. It is native to south-east Asia and widely distributed through out the world, mainly in tropical countries. The roots, seeds and leaves of this plant are of medicinal importance. The plant is reputed for its folkloric uses in various diseases. The roots are bitter, ophthalmic, laxative, intellect promoting, diuretic, depurative, aphrodisiac and is used as tonic. It is used in ophthalmology, helminthiasis, leprosy, leucoderma, elephantiasis, bronchitis, asthma, ascites, ulcers and fever. The seeds are cathartic and are useful in visceralgia. Leaves are useful in otalgia, hepatopathy and eruptions. The plant has been scientifically studied for various pharmacological activities including antioxidant, anthelmintic, analgesic, anxiolytic, antidepressant, anticonvulsant, sedative, hypoglycemic, larvicidal and anticanceractivities.It has been found to enhance acetylcholine content in rat hippocampus and is also used as a local anaesthetic. Finotin, a protein isolated from the seeds of the plant possess antimicrobial properties. The present study was undertaken to evaluate antitumour activity of crude methanol extract of Clitoria ternatea against Dalton’s lymphoma. MATERIALS AND METHODS Collection and extraction of plant material: Clitoria ternatea seeds were collected from various parts of Kottayam district, Kerala. The plant was identified and authenticated by Dr.V.T Antony, Dept.of Botany, S.B college, Changanassery with the help of herbarium sheets of sample species and a voucher specimen was deposited in the lab. Preparation of plant extract The seeds were dried under shade and pulverised.100gram of the seed powder was extracted with methanol by hot continuous extraction method using a soxhlet apparatus. The solvent was evaporated under reduced pressure at 50C and dried in vacuum. The yield of solvent free methanolic extract was 4.9% (w/w). It was stored in sterile amber coloured storage vials in refrigerator until used for experiment. The dried extract obtained was dissolved in isotonic saline solution and used for all the experiments. M.S. Latha et.al./ Anticancer activity of... IJPPR, Vol-4, Issue 4, December 2012February 2013, 207-212 Pa ge 20 8 Animals: Studies were carried out using male Balb/c mice (20-25g) obtained from Veterinary College, Mannuthy, Trichur. They were housed in polypropylene cages in a controlled environment (temperature 25 ± 2C, humidity 60-70% and 12 h dark/light cycle). They were given standard pellet diet(M/s Hindustan Lever Ltd., Bombay, India) and water ad libitum. Tumour cells: Daltons lymphoma ascites tumour(DLA) were obtained from Amala Cancer Research Institute, Trichur. The cells were maintained by the intraperitoneal inoculation of 10 cells/mouse every 14 days. Determination of in vitro cytotoxicity Short term cytotoxicity studies were conducted by incubating 1X10 DLA cells in 1ml PBS containing various concentrations of the extract at 37C for 3hrs.The viable cell count was done using trypan blue exclusion method. Normal lymphocytes served as control. Antitumour activity of MECT Male albino mice were divided in to four groups of twelve animals (n=12) each. Group I Pair fed control Group II Received 1×10 DLA cells(i.p) Group III Received 1×10 DLA cells(i.p)+MECT at a dose of 100mg/kg body weight Group IV Received 1×10 DLA cells(i.p)+MECT at a dose of 200mg/kg body weight DLA cells collected from the donor mouse were suspended in sterile isotonic saline and the viable count was adjusted to 1×10 cells/ml. These were injected intraperitoneally (i.p.) on the first day of the experiment. Fourteen doses of MECT were injected intraperitoneally (i.p.) from the first day up to the 14th day at 24-hr intervals. Control animals received only vehicle (saline solution). On day 15, half of the animals (n = 6) in each cage were killed by decapitation. Ascitic fluid and blood were collected for the analysis of various parameters. The remaining animals were kept to observe for the life span of the hosts. The anti-tumor activity of the methanol extract of Clitoria ternatea (MECT) was measured in DLA animals with respect to the following parameters: Effect of MECT on ascites: The antitumour activity of the methanolic extract was measured with respect to the following parameters: Tumor volume: The volume of the ascitic fluid collected from the peritoneal cavity was measured by taking it in a graduated centrifuge tube and packed cell volume was determined by centrifuging at 1000 rpm for 5min. Tumor cell count: The ascitic fluid was taken in a WBC pipette and diluted 100 times. Then a drop of the diluted cell suspension was placed on the Neubauer counting chamber and the number of cells in the 64 small squares was counted. Viable/non-viable tumor cell count: The cells were stained with trypan blue (0.4% in normal saline) dye. The cells that did not take up the dye were viable and those that took the stain were nonviable. These viable and nonviable cells were counted. Cell count= No. of cells Χ dilution Area× Thickness of liquid film Percentage increase in life span (% ILS) The effect of MECT on tumor growth was monitored by recording the mortality daily for a period of 6 weeks and percentage increase in life span (%ILS) was calculated. Mean survival of treated group %ILS 1 100 Mean survival of control group   = − ×     st Day of 1 death + Day of last death Mean survival 2   =     Hematological Parameters: Blood collected was used for the estimation of hemoglobin (Hb) content, red blood cell count (RBC) and white blood cell count. Estimation of in vivo Antioxidants: After collecting the blood samples, the liver was excised, rinsed in ice-cold normal saline solution followed by cold 0.15 M Tris-HCl (pH 7.4), blotted dried and weighed. A 10% w/v homogenate was prepared in 0.15M Tris-HCl buffer and was used for the estimation of lipid peroxidation (LPO) and reduced glutathione(GSH).The assay of the antioxidant enzymes catalase and superoxide dismutase were also conducted. Lipid peroxidation was measured in terms of thiobarbituric acid reactive substances, TBARS. Changes in the antioxidant status were assessed by estimating the activities of GSH, catalase and superoxide dismutase. Phytochemical analysis: Preliminary phytochemical screening of the methanolic extract of Clitoria ternatea was carried out for the detection of phytochemical components using standard conventional protocols. STATISTICAL ANALYSIS The results are presented as mean±SEM.One-way analysis of variance (ANOVA) followed by Dunnet’s test was applied for statistical analysis. p values <0.05 are considered significant. RESULTS In vitro cytotoxicity assay: The results of the in vitro cytotoxicity test by the trypan blue method is given in Table 1. 53% percentage cell death occured at a concentration of 75 μg, 83% at 100 μg and 100% at a concentration of 200 μg, whereas the effect of the extract on normal lymphocytes was negligible. Table 1: In vitro cytotoxicity assay of MECT by trypan blue method Conc. of MECT 25 μg 50 μg 75 μg 100 μg 200μg DLA(1X10 cells) 14 24 53 83 100 Lymphocytes 3 8 12 M.S. Latha et.al./ Anticancer activity of... IJPPR, Vol-4, Issue 4, December 2012February 2013, 207-212 Pa ge 20 9 Antitumour activity of MECT: Antitumor activity of MECT against DLA tumor bearing mice was assessed by the parameters such as tumor volume, packed cell volume, cell count (viable and non-viable), mean survival time and percentage increase of life span. The results are shown in Table 2. The tumor volume, packed cell volume and viable cell count were found to be significantly increased and nonviable cell count was significantly low in DLA control animals when compared with normal control animals. Administration of MECT