In Vitro Cytotoxic Activity of Alcoholic Extract of Aristolochia indica

 

V.A. Kangralkar1* and A.R. Kulkarni2

1Maratha Mandal’s College of Pharmacy, Belgaum Karnataka,

1Research Scholar, Karpagam University Coimbatore.

2Department of Pharmacology, SET’s College of Pharmacy, Dharwad- 580 002, India.

*Corresponding Author E-mail: vishnuak@rediffmail.com

 

 

ABSTRACT:

Aristolochia indica root extract (alcoholic) was screened for its antitumor activity on proliferation of HT29 colon cancer cell line. The activity was determined by microculture tetrazolium viability (MTT) assay. The different concentrations (100, 50, 25, 12.5, 6.25and 3.125 μg/ml) were taken and cells were exposed with the extract. The % cytotoxicity produced in MTT assay at100μg/ml was 69.21   with IC50 of 28.56 . From these results it was observed that alcoholic extract of Aristolochia indica root has significant cytotoxic activity.

 

KEYWORDS: Aristolochia indica root, MTT assays, cytotoxicity, IC50.

 

 


INTRODUCTION:

The Cancer cases are increasing in every country of the world. It is a leading cause of death in today’s era. It is the uncontrolled cell proliferation. Carcinoma of the large bowel is equally distributed between men and women.1  Cases of colorectal cancer are increasing and it made to develop a more effective preventive measure. Natural products/various medicinal plants are rich source of medicinal chemical constituents which are having potential pharmacological activities. Several plant and dietary sources have been investigated and reported to posses chemo preventive effect in in vitro and in vivo experiments eg. Green tea2, Origanum vulgare3, Raspberry4, Curcumin longa5 etc. Similarly Aristolochia indica have been reported to have Antitumor activity, however there is paucity of information regarding their effects on colon cancer. Aristolochia indica Linn. a perennial shruby glabrous twiner with a long woody root stock; leaves simple, alternate; floweres greenish white; fruits rounded or oblong. The roots are bitter, acrid, astringent anthelmintic, cardiotonic, anti-inflammatory, diuretic and tonic.6various extracts and fractions of root showed interceptive activity7 and antiestrogenic activity in mice.8Extracts or isolates of A. indica possess anticancer activity. It stimulates phagocytic activity in guinea pigs.9

 

The present study is therefore planned to explore the the effect of Aristolochia indica Linn, on HT-29 Human colorectal cell lines.

 

MATERIALS AND METHODS:

Plant material Collection: Plant was collected from local market and are identified and authenticated in Redekar Auyrvedic college and research centre. Gadhinglaj.  Ethanolic extract is prepared using soxhlet apparatus.

Cell Culture: HT-29 human colon cancer cells were obtained from NCL, Pune, India. The cells were maintained in laboratory. MTT reagent was purchased from Sigma. The solution was preserved at 40C away from light. 

 

MTT solution preparation: 10 mg in 10 ml of Hank’s balanced solution.

 

Cell culture:

 The cell line were maintained in 96 wells micro titer plate containing Minimum Essential Medium (MEM) media supplemented with 10% heat inactivated fetal calf serum (FCS), containing 5% of mixture of Gentamycin, Penicillin (100 Units/ ml) and Streptomycin (100µg/ml) in presence of 5% CO2 at 370C for 3-4 days. After 3-4 days remove the supernatant and replace MEM media with Hank’s balanced solution supplemented with Gentamycin, Penicillin and Streptomycin. Incubate overnight. In-vitro growth inhibition effect of test compound was assessed by calorimetric or spectrophotometric determination of conversion of MTT into “Formazan blue” by living cells. Remove the supernatant from the plate and add fresh Hank’s balanced salt solution and treated with different concentrations of extract or compound appropriately diluted with DMSO. Control group contains only DMSO. After 24 hrs incubation at 370C in a humidified atmosphere of 5% CO2, the medium was replaced with MTT solution (100µl, 1mg per ml in sterile Hank’s balanced solution) for further 4 hr incubation. The supernatant carefully aspirated, the precipitated crystals of “Formazan blue’ were solubalised by adding DMSO (200µl) and optical density was measured at wavelength of 570nm. The test denotes the survival cells after toxic exposure. Percentage inhibition of the extract against all cell line was calculated using the following formula:

 

Surviving cells (%) = Mean OD of test compound×100

                                    Mean OD of control

 

% Cell inhibition = 100 - % Cell survival

 

The effects of extracts were expressed by IC50 values calculated from dose response curves.

 

Calculations and statistics:

Results were expressed by calculating percentage growth inhibition of control. IC50 values were derived from a nonlinear regression model (curve fit) based on sigmoidal dose response curve (variable) and computed using Graph pad Prism version 5.00.

 

RESULTS:

Treatment with alcoholic extract resulted in inhibition of cell growth in cell lines. The results are tabulated in table 1 and fig 1.

 

Table No: 01

Effect of extract on HT-29 Cell lines

Sl.

N0

Concentration

(μg/ml)

Cytotoxic activity (%)

IC50 (μg/ml)

Rvalue

 

1

100

69.21

 

 

28.56

 

 

0.9989

2

50

58.13

3

25

43.88

4

12.5

29.51

5

6.25

21.52

6

3.125

13.47

 

Figure: 01

DISCUSSION:

The observations showed significant cytotoxic activity against HT29 cell lines. Aristolochia indica contains many antioxidant constituents and it has already reported to have antioxidant activity.10 Antioxidants are proved to prevent many diseases. The present activity may be due to the antioxidant characters. Therefore it is suggested that the isolation of active constituent to obtain a good chemotherapeutic agent.

 

REFERENCE:

1.       Ranjit Sen. Principles and Management of Cancer A Practical Guide. B.I. Publications Pvt. Ltd. New    Delhi, 2004, pp.154

2.       Xu Donga Jia and Chi Han, chemoprevention of tea on colorectal cancer induced by dimethylhydrazine in Wistar rats. World J Gastroentrol.2000;6(5):699-703.

3.       Thummala Srihari et al, Dose-dependent effect of oregano on lipid peroxidation and antioxidant status in 1,2-dimethylhydrazine-induced rat colon carcinogenesis, J Pharm Pharmacol 2008,60:787-794.

4.       oates EM et al, Colon-available raspberry polyphenols exhibit anticancer effects on in vitro models of colon cancer, J Carcinog 2007:6:4

5.       Van Erk MJ, et al Time- and dose-dependent effects of curcumin on gene expression in human colon cancer cells. J Carcinog 2004;3:8

6.       Arya Vaidya Sala, “Indian Medicinal Plants”, Vol.1, Orient Longman Lt-Chennai, 1997; 199-201.

7.       Pakrashi A, Pakrasi P. Biological profile of p-coumaric acid isolated from aristolochia indica Linn. Indian J Exp Biol 1978;16:1285-1287.

8.       Pakrashi A, Shah C. Anti-implantation and anti-oestrogenic activity of sesquiterpine from the roots of Aristolochia indica Linn. Indian J Exp Biol 1977;15:1197-1198.

9.       The Wealth of India – ‘A Dictionary of Raw Material and Industrial Products’, Vol .I, New Delhi; Publication and Information Directorate, CSIR; 1998,423-426.

10.     Thirugnansampandan, R.G. Mahendran and V.N. Bai, Antioxidant properties of some medicinal Aristolochiaceace species. Afr. J. Biotechnol.,(7)2008,357-361.

 

 

 

 

Received on 11.10.2013       Modified on 20.10.2013

Accepted on 24.10.2013      © RJPT All right reserved

Research J. Pharm. and Tech. 6(11): November 2013; Page 1240-1241