ANTITUMOR ACTIVITY OF CAPPARIS SEPIARIA ON EHRLICH ASCITES CARCINOMA IN MICE

The methanol extract of Capparis Sepiaria (Capparaceae) bark (MECS) were evaluated for antitumor activity against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. The extract was administered at the doses of 200, and 400 mg/kg body weight per day for 14 days after 24 h of tumor inoculation. After the last dose and 18 h fasting, the mice were sacrificed. The present study deals with the effect of MECS on the growth of transplantable murine tumor, life span of EAC-bearing hosts and hematological profile MECS caused significant (P < 0.01) decrease in tumor volume, packed cell volume, and viable cell count; and it prolonged the life span of EAC-tumor bearing mice. Hematological profile converted to more or less normal levels in extract-treated mice. The results indicate that MECS exhibited significant antitumor activity in EAC-bearing mice.


INTRODUCTION
The use of medicinal plants to treat diseases is as old as human civilization. Human beings of all ages in both developing and undeveloped countries use plants in seek to treat numerous diseases and to get relief from physical ailments. Cancer is a class of diseases in which a cell or a group of cells display uncontrolled growth, invasion and sometimes metastasis. It is largest noncommunicable disease and it has a sizable contribution in the total number of deaths. The World cancer report documents that cancer rates are set to increase at an alarming rate globally. Cancer rates could increase by 50% new cases for the year 2020 1 . India is a rich source of medicinal plants and a number of plant extracts are used against diseases in various systems of medicine such as Ayurveda, Unani, and Siddha. Only a few of them have been scientifically explored. There were good number of plant products such as flavonoids, terpenes; alkaloids 2, 3, 4 and these can are used as remedies to treat various diseases and disorders. Because of their distinct pharmacological qualities including cytotoxic and cancer chemopreventive effects, it is inspired many scientists to take up independent investigations on a number of medicinal plants 5

Plant material and toxicity studies
The plant Capparis sepiaria (Family: Capparaceae) was collected in the month of August 2010 from the Talakona forest, Chittor district. The plant material was taxonomically identified by the taxonomist, S.V University, Tirupathi. The dried powder material of the whole plant of the Capparis sepiaria were extracted with methanol in a soxhlet apparatus. The methanol extract was then distilled, evaporated, and dried in vacuum. The resulting methanolic extract yield was observed to be 10.5 %. Preliminary qualitative analysis of the methanol extract showed the presence of saponins, tannins, flavonoids, alkaloids. The methanol extract of Capparis sepiaria (MECS) was used for the present study. The LD 50 value of C.sepiaria was found to be 894.43 mg/kg (i.p) according to method described by Lorke 9 .

Animals
The study was carried out after obtaining permission from Institutional Animal Ethics Committee (No. 160/SPIPS/Wgl/IAEC/2010) and CPCSEA regulations were adhered to during the study. Male swiss albino mice (20-25 g) were selected for this study. The animals were maintained under standard environmental conditions and fed with standard pellet feed and water ad libitum.

Tumor cells and Drugs
EAC cells were obtained from Centre for Cellular and Molecular Biology (CCMB) (Hyderabad, India). The EAC cells were maintained by intraperitoneal inoculation of 2×10 6 cells /mouse. Standard drug 5fluorouracil procured as a gift sample from Cadila Pharmaceuticals Ltd, Ahmedabad.
Antitumor activity 10 The activity was carried out according to the method described by 10 . Male Swiss albino mice weighing 20 ± 2 g. were randomly divided into 5 groups (n =12). All the groups were injected with EAC cells (0.2 ml of 2 × 10 6 cells/mouse) intraperitoneally (i.p) except the normal group. This was taken as day zero. On the first day, 5 ml /kg of normal saline was

Effect of MECS on Hematological studies
Blood was withdrawn from each mouse by retro orbital plexus method and the Hemoglobin content, Red blood cell (RBC), and White blood cell (WBC) counts were measured 14, 15 . Differential leukocyte count of WBC was carried out from Leishman stained blood smears [16] of normal, EAC control, and MECS treated groups, respectively.

Effect of MECS on in vitro cytotoxicity
Short-term cytotoxicity was assessed by incubating 1 X 10 6 EAC cells in 1 ml phosphate buffer saline with varying concentrations of the MECS at 37 0 C for 3 h in CO 2 atmosphere ensured using a McIntosh field jar. The viability of the cells was determined by the trypan blue exclusion method 17 .

STATISTICAL ANALYSIS
The experimental results were expressed as the mean ± S.E.M. Data were assessed by Student's t-test; P value of < 0.05 was considered as statistically significant.

RESULTS
The present investigation indicates that the MECS showed significant antitumor activity in EAC-bearing mice. The effects of MECS at the doses of 200 and 400 mg/kg on survival time, % ILS, tumor volume, packed cell volume, and tumor cell count (viable and nonviable cell) are shown in Table 1.

Effect on mean survival time
In the EAC control group, the mean survival time was 18.54 ±0.16 days, while it increased to 28.62 ± 0.16 (200 mg/kg), and 33.87 ± 0.14 (400 mg/kg) days, respectively, in the MECS-treated groups, whereas the standard drug 5fluorouracil (20 mg/kg)-treated group had a mean survival time of 37.58 ± 0.25 days.

Effect on tumor growth
Treatment with MECS at the doses of 200 and 400 mg/ kg significantly (P < 0.01) reduced the tumor volume, packed cell volume, and viable tumor cell count in a dose-dependent manner as compared to that of the EAC control group. Furthermore, nonviable tumor cell count

Effect on hematological parameters
As shown in Table 2, hemoglobin content and RBC count in the EAC control group was significantly (P < 0.001) decreased as compared to the normal group. Treatment with MECS at the dose of 200 and 400 mg/ kg significantly (P < 0.01) increased the hemoglobin content and RBC count to more or less normal levels. The total WBC counts and protein was found to be increased significantly in the EAC control group when compared with the normal group (P<0.001). Administration of MECS at the dose of 200 and 400 mg/kg in EAC-bearing mice significantly (P<0.01) reduced the WBC count and protein as compared with the EAC control. In a differential count of WBC, the presence of neutrophils increased, while the lymphocyte count decreased in the EAC control group. Treatment with MECS at different doses changed these altered parameters more or less to the normal values.

DISCUSSION
The present study was carried out to evaluate the antitumor effect of MECS in EAC-bearing mice. The MECS-treated animals at the doses of 200 and 400 mg/ kg significantly inhibited the tumor volume, packed cell volume, tumor cell Count, and brought back the hematological parameters to more or less normal levels. In EAC-bearing mice, a regular rapid increase in ascites tumor volume was noted. Ascites fluid is the direct nutritional source for tumor cells and a rapid increase in ascitis fluid with tumor growth would be a means to meet the nutritional requirement of tumor cells 18 . Treatment with MECS inhibited the tumor volume, tumor cell count, and increased the percentage of tryphan blue positive stained dead cells in tumor bearing mice. The reliable criteria for judging the value of any anticancer drug are the prolongation of the life span of animals 19 . The MECS decreased the ascites fluid volume, viable cell count, and increased the percentage of life span. It may be concluded that MECS by decreasing the nutritional fluid volume and arresting the tumor growth increases the life span of EAC-bearing mice. Usually, in cancer chemotherapy the major problems that are being encountered are of myelosuppression and anemia 20,21 . The anemia encountered in tumor bearing mice is mainly due to reduction in RBC or hemoglobin percentage, and this may occur either due to iron deficiency or due to hemolytic or myelopathic conditions 22 . In EAC control group, a differential count the presence of neutrophils increased, while the lymphocyte count decreased, the observed leucocytopenia indicates a common symptom of immunosuppression in many types of cancers 23,24 and one of the causes of neutrophilia is myeloid growth factors which are produced in malignant process as part of a paraneoplastic syndrome. In addition to this another factor granulocyte colony stimulating factor produced by the malignant cells has also been attributed to be the cause of neutrophilia because of its action on bone marrow granulocytic cells in cancer. After the repeated treatment, MECS able to reverse the changes in altered neutrophils and lymphocytes count. 25 The manual for laboratory work in mammalian physiology. 3rd ed. 11.34 ± 0.05 5.6 ± 0.07** 0.76 ± 0.04** -----Nonviable tumor cell count (× 10 7 cells/ml ) 0.32 ± 0.03 0.83 ± 0.03** 1.48 ± 0.04** -----