CHAPTER 9 ANTI-CANCER ACTIVITY OF COMPOUND-III

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1 174 CHAPTER 9 ANTI-CANCER ACTIVITY OF COMPOUND-III 9.1 IINTRODUCTION Oleanolic acid is a naturally occurring triterpenoid, widely distributed in food and medicinal plants. It is relatively non-toxic and it exhibits antitumor, hepatoprotective and antiviral properties [132]. It exists widely in medicinal herbs and natural plants in the form of free acid or aglycones. 9.2 MATERIALS AND METHODS Animals Swiss Albino mice weighing between 20-25gms were used for the study. They were maintained under standard environmental condition (temperature C and 12hrs light/dark cycle) and they were allowed with standard laboratory feed and water ad libitum. The animals were given a week s time to get acclimatized with the laboratory condition Anti-Cancer Activity After acclimatization, mature male Swiss Albino mice were divided into four groups (n=10). All the groups except group I were inoculated (i.p.) with DAL Cells of 1 x 10 6 cells/mouse weekly [120]. The day of DAL administration was considered as day 0.Group I served as control and received distilled water for 14 days, group II served as DAL control group

2 175 were inoculated (i.p.) with DAL Cells of 1 x 10 6 cells/mouse without drug treatment. Group III animals received compound III (20mcg/kg) p.o for 14 consecutive days. On day 15, five mice of each group were sacrificed 24hrs after the last dose and the rest were kept with food and water ad libitum to check the increase in the life span of the tumor hosts. The effect of methanolic extract on tumor growth and host s survival time were examined by studying the parameters like tumor volume, tumor cell count, viable tumor cell count, nonviable tumor cell count, mean survival time and increase in life span [121]. Determination of tumor volume The fluid was collected from the peritoneal cavity of the mice. The volume was measured by taking it in a graduated centrifuge tube and packed cell volume was determined by centrifuging at 1000rpm for 5 min. Determination of tumor cell count The fluid was taken in a RBC pipette and diluted to 1000 times. Then a drop of the diluted cell suspension was placed on the Neubauer s counting chamber and the number of cells in 64 small squares were counted. Estimation of viable tumor cell count The cells were then stained with Trypan blue (0.4% in normal saline) dye. The cells that did not take up the dye were considered viable and the stained as nonviable. They were counted and the viable and non viable cell counts were estimated [122].

3 176 Percentage increase in life span Percentage increase in life span was calculated by observing the mortality and the effect of extract on tumor growth. The mean survival time (MST) of each group consisting of 6 mice was noted [123]. Haematological studies Blood was drawn from each mouse from tail vein under sterilised condition and the red blood cell count, haemoglobin content and white blood cell counts were determined using cell diluting fluids and a haemocytometer. Differential cell count was carried out using Leishman stained blood smears. Serum protein concentration was estimated by Lowry s method and packed cell volume was determined by the method described by Docie et al [124] Statistical Analysis The data s were expressed as mean ± SEM, statistical analysis was performed by one way ANOVA followed by Tukey-Kramer multiple comparison test, p values <0.05 were considered as significant. Highest significant difference test performed with Graph pad instat software. 9.3 RESULTS The tumor control mice the haematological parameters on day 15 were found to be significantly altered when compared to normal group. The total WBC count, protein and PCV were found to be increased with a reduction of the hemoglobin and RBC. In a differential count of WBC, the percent of neutrophils increased while the lymphocyte count decreased. The compound treated group showed significant effect on increasing life span and retrieving all the parameters to normal level.

4 177 Effect on mean survival time and tumor growth In the DAL control group the mean survival time was 12 ± 1.5days, while it increased to 29.2±1.7 for compound treated group (p<0.001). The increase in life span of tumor bearing mice treated with compound was found to be 70%. Treatment with compound reduced the tumor volume, packed cell volume, and viable tumor cell count in a dose-dependent manner as compared to that of DAL control group. Further, nonviable tumor cell count was increased when compared with the DAL control [Table 9.1, Figures 9.1 and 9.2]. Effect on haematological parameters The haemoglobin content and the RBC count in the DAL control group were found to be decreased when compared to normal group. The haemoglobin content for DAL treated control was found to be 6.3 ± 0.5 and treatment with the compound increased the hemoglobin content to 12.8±0.9. The RBC count to was found to be increased to more or less normal level. The total WBC counts and protein were found to be increased in DAL control group 15.1±0.5 and 16.7±0.8 when compared with normal group. Administration of compound in DAL bearing mice reduced both WBC count and protein when compared with DAL control. In the differential count of WBC, increase of neutrophils and the lymphocyte count decreased in DAL control group. Treatment with compound changed these altered parameters more or less normal [Table 9.2, Figures 9.3 and 9.4].

5 Table 9.1 Effect of compound III on survival time, life span, tumor volume, viable and non-viable cell count in DAL bearing mice S.No Treatment Vehicle control (5ml/kg p.o) DAL control (x10 6 cells/ml) Compound III (20mcg/kg) Survival time (Days) Increase of life span Tumor volume (ml) Viable cell count x 10 6 cells/ml Non-viable cell count x 10 6 cells/ml ± 1.5* * ±0.25 * * 9.7 ±0.32 * * 1.7 ±0.74 * * 29.2±1.7 *** ±0.85* * * 1.4 ± ±0.43 * * * Values are mean+ SEM expressed as (n=6) p*<0.05; **<0.01; ***<0.001; as compared with group II. p< 0.05 considered significant 178

6 Vehicle Control DAL control Compound III (20mcg/kg) Survival time (Days) Increase of life span Figure 9.1 Effect of compound III on survival time and increase of life span Tumor volume (ml) Viable cell count Non viable cell count Vehicle Control DAL control Compound III (20mcg/kg) Figure 9.2 Effect of compound III on tumor volume, Viable cell count and Non viable count

7 Table 9.2 Effect of compound III on haematological parameters in DAL bearing mice S.No Treatment Hb (g/dl) RBC (106cells/mm3) WBC (103 cells/mm3 Total Protein (g/dl) PCV (mm) Differential cell count (%) Lymphocytes Neutrophils Basophils Vehicle control (5ml/kg p.o) DAL control (1x10 6 cells/ml) 14.3± ± ± ± ± ± ± ± ±0.5 ** 2.3±0.2*** 15.1±0.5** 16.7±0.8** * 27.4±0.2*** 33.4±0.7 * * * 46.9±0.2 * * * 3.7±0.7 Compound III ±0.9 *** 6.1±0.2 * * * 8.8 ±0.4 * 9.4±0.3* * 17.5±0.4*** 67.3±0.5** * 20.3±0.3** 2.4±0.5 (20mcg/kg) Values are mean+ SEM expressed as (n=6) p*<0.05; **<0.01; ***<0.001; as compared with group II. p< 0.05 considered significant 180

8 Hb(g%) RBC WBC Total Protein PCV Vehicle Control DAL control Compound III (20mcg/kg) Figure 9.3 Effect of compound III on Hb, RBC, WBC, total Protein and PCV Lymphocytes Neutrophils Basophils Vehicle Control DAL control Compound III (20mcg/kg) Figure 9.4 Effect of compound III on differential counts

9 DISCUSSION The present study was carried out to evaluate the anti-cancer activity of compound III (20 mcg/kg) on DAL bearing mice. A reliable criteria for judging the value of any anti-cancer agent is the prolongation of life span of animals[129]. A significant increase in life span of animals was observed in the compound treated animals, the tumor volume, packed cell volume, tumor cell count and also the haematological parameters were brought back to more or less normal levels. To evaluate the extract treatment indirectly inhibited the tumor cell growth, the effect of compound treatment was examined on the viable and nonviable cell counts against tumor bearing mice. The treatment with the compound was found to enhance the non-viable cell count and decrease the viable cell count. It may be due to the absorption of extract by viable cells which leads the lysis of the cells through the activation of macrophages or some cytokinnin production in the peritoneal cavity. In cancer chemotherapy the major problem encountered are myelosuppression and anaemia [124, 125]. The anaemia encountered in tumor bearing mice is mainly due to reduction in RBC or haemoglobin percentage and this may occur either due to iron deficiency or due to haemolytic or myelopathic conditions[126]. Treatment with the compound brought back the haemoglobin content, RBC and WBC cell count near to normal values. This indicates that compound III possess a significant anti-cancer activity.

THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 079 MOD: 1st Issue Page: 1 of 7

THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 079 MOD: 1st Issue Page: 1 of 7 Procedure Type: Title: General Laboratory Procedure Cell Counts