International Journal of Drug Development and Research

Key words

Hela cell line, MTT assay, DLA cells, Canthium Parviflorum, Tumor

INTRODUCTION

Tumor is a mass of tissues which proliferate rapidly, spread throughout the body and may eventually cause death of the host. The Chemotherapeutic agents are effective against various types of tumor are not totally free from side effects. This fostered our attempt to evaluate some plant products against cancer, as they are less likely to cause side effects. Many Indian species [1] and Plants[2] are quoted to be useful in different types of cancer. Canthium parviflorum Lam belonging to the family Rubiaceae. This plant is distributed in the Deccan peninsula from Gujarat and Maharashtra southwards, and in Bihar and Orissa[3]. Canthium parviflorum Lam has very limited systematically carried out investigations. The traditional systems of medicine use this plant alone or in combination with other medicinal plants for the treatment of various diseases. It was found from the tribes of south India that the plant has been used for the suppression of tumor like syndrome among their own population[4]. A vast literature collection fails to produce a scientific evidence to prove the anti tumor activity of Canthium parviflorum Lam. Hence this study was planned to evaluate Invitro and Invivo anticancer activity of ethanolic extract of leaves of Canthium parviflorum Lam.

MATERIALS AND METHODS

Plant Material

The fresh leaf of Canthium Parviflorum was obtained from Kanchipuram, Tamilnadu and the plant material was identified and authenticated by Prof Jayaraman, PARC, Chennai. Voucher specimen were deposited in the Pharmacognosy department, C.L.B.M College of Pharmacy, Chennai. The shade dried leaves was minced and extracted with 70% ethanol. The ethanolic extract of C.Parviflorum (EECP) was used to derive a series of concentration used in this paper. The Phytochemical screening proves the presence of carbohydrate, alkaloid and flavonoids[5].

Cell Culture

Hela cell line was obtained from National centre for cell sciences, Pune, India. Stock cells are routinely cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units/ ml pencillin and 100μg/ml streptomycin at 370 C an incubator containing 5% CO2 .

Experimental Animals

Colonies inbreed strains of swiss albino male mice weighing (20-25 gm),a obtained from C. L. Baid Metha College of pharmacy was used for the pharmacological studies. The animals were kept under standard conditions maintained at 23-25oC, 12 hr light/dark cycle and given standard pellet diet (Hindustan lever, Bangalore) provided ad libitum. The animals were acclimatized to the laboratory conditions for a week prior to the experimentation and randomly divided into six groups of each six animals. Principles of animal handling were strictly adhered to the guidelines and handling of animals was made under the supervision of animal ethics committee of the institute. The experimental protocol was approved by Institutional animal ethics committee (IAEC) of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals).

Acute Toxicity studies

This was performed for the extracts to ascertain safe dose by the acute oral toxic class method by the Organization of Economic Cooperation and Development (OECD). A single administration of starting dose of 2000 mg/kg body weight/po of the EECP was administered to three female mice, and the mice were observed for three days to evaluate considerable changes in body weight and other signs of toxicity.

Repeating the experiment with the same dose level of EECP for more seven days, we observed the body weight change and toxicity sign for totally fourteen days.

Invitro cytotoxic activity of EECP on DLA cells

DLA cell lines was aspirated from the peritoneal cavity and washed 3 times with phosphate buffer saline solution. One million cells were incubated with various concentrations (12.5μg/ml,25μg/ml, 50μg/ml & 100μg/ml) of the extract in a total volume of 1 ml for 3 hrs at 37° c. After incubation, the viability of the cells was determined by Trypan blue dye exclusion method

Invitro cytotoxic activity of EECP on Hela cells:

MTT ASSAY

MTT assay was performed as described (wang et al, 2001). Briefly cells were seeded at a concentration of 1.5× 104 cells/ml in a 96 well plate. After overnight incubation, serial concentration of EECP (12.5μg/ml,25μg/ml, 50μg/ml & 100μg/ml) were added. Since 1% ethanol was added to drug treatment group. Positive control (5-Flurouracil), Control group was added with 1% ethanol. Each concentration was repeated three times. These cells were incubated in a humidified atmosphere with 5% co2 for 3 days. Then 20μl MTT (3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to each well and incubated at 370 C for 4 hrs. The medium was removed and formazon was dissolved in DMSO and the absorbance is taken at 570nm. The growth inhibition was determined using Growth inhibition = (control O.D – sample O.D/ control O.D) and further IC50 value were determined.

INVIVO ANTI-CANCER ACTIVITY

Effect of EECP on survival time

Animals were inoculated with 1×106 cells per mouse on day 0 and treatment with EECP started 24 hours after inoculation at a dose of 200 mg/kg and 400 mg/kg/ day p.o. The control group was treated with the same volume of 0.9% sodium chloride solution. All the treatments were continued for 10 days and observation was carried out for 45 days. The mean survival time (MST) of each group consisting of 6 mice, and changes in body weight was noted. The antitumour efficacy of EECP was compared with that of 5-fluorouracil (20mg/ kg, i.p) . The MST of the treated group was compared with that of the control group using the following formula. MST= (T-C)/C × 100 T=Treated group C= Control group

Effect of EECP on haematological parameters:

In order to observe the effects of EECP on the haematological parameters of DLA bearing mice comparison was made amongst four groups (n = 6) of mice on the 14th day after transplantation. The four groups comprised of (1) tumour bearing mice (2) and (3) tumour bearing mice treated with EECP (200 mg/kg and 400 mg/kg bwt respectively) and (4) normal mice. Blood was drawn from each mouse from tail vein under sterilised condition and the Red Blood Cell Count (RBC) Haemoglobin content (Hb) and white blood cell count (WBC) [12] were studied using cell diluting fluids and a haemocytometer. Differential cell count was (DC) carried out from Leishman stained blood smears [13].

Statistical Analysis

All values are expressed as mean ±SEM. The data were statistically analysed by ONE WAY ANOVA followed by Tukey Kramer multiple comparison test. P values < 0.001 were considered as highly significant and <0.05 were considered significant.

Results and discussion

EECP were found to be cytotoxic towards DLA and Hela cells only at higher concentration. MTT assay is based on the reduction of MTT(3-(4,5- dimethyl thiazolyl)-2,5-diphenyl-tetrazolium bromide) by mitochondrial dehydrogenase to purple formazan product. . The IC50 value of ethanolic extract showed significant anticancer acitivity by exclusion method and MTT assay (Table 1,Figure 1). The mean survival time of tumour control group was 21 days. The EECP in both doses (200 & 400 mg/kg i.p) increased significantly the MST, 32 days in lower dose treated group (P<0.01) and 41 days in higher dose treated group (P<0.001). The results were almost comparable with the 5-flurouracil treated group. (Table 2,Figure 2) EECP at both the doses (200 & 400 mg/kg p.o.) reversed the increased values in the haemotological parameters induced by tumour to normal values. Tumour growth normally affects various haematological parameters and the anticancer activity is generally assessed by restoration of the changes in these parameters to normal and most significantly in decreased WBC and increased RBC, lymphocyte and haemoglobin content as compared to tumour control. The acceptance criteria for determining the antitumour activity of a compound is the determination of circulating WBC and the life span prolongation. [14] (Table - 3). Usually, in cancer chemotherapy, the major problems that are being encountered are of myelosuppression and anaemia 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 condition. But the results have clearly shown that EECP (400mg/ kg) has not only brought back the bone marrow nucleated cell count to normal, but also the haemoglobin content and the RBC count to normal.

Conclusion

On the basis of the above results it can be concluded that the EECP possess significant anticancer activity studied by invitro and invivo models. The study also provides a strong evidence for the use of the leaves Canthium parviflorum in folklore treatment as anticancer agent. The activity may be due to the presence of one or more phytochemical constituents present in the extract. Further studies warranted, for isolation of the constituents responsible for the activity and also to explore the exact mechanism of action of the activity.

Acknowledgement

The authors wish to thank Dr.Adhiraj from the Department of Pharmacognosy, KMCH college of Pharmacy, Coimbatore, Tamilnadu, India for allowing to use the cell culture facilities for the biological assay.

Tables at a glance

Table 1 Table 2 Table 3

Figures at a glance

Figure 1 Figure 2

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