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 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 33  |  Issue : 2  |  Page : 81-84

In vitro cytotoxic activity of menispermaceae plants against HeLa cell line


1 Department of Pharmacognosy, PSG College of Pharmacy, Peelamedu, Coimbatore, India
2 Department of Pharmacognosy,Malik Deenar College of Pharmacy, Kasargod, Kerala, India
3 Department of Phytopharmacy and Phytomedicine, JSS College of Pharmacy (JSS University, Mysore), Ooty, Tamil Nadu, India

Date of Web Publication18-Aug-2014

Correspondence Address:
B Samuel Thavamani
Associate Professor, Department of Pharmacognosy, PSG College of Pharmacy, Peelamedu,Coimbatore 641 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0257-7941.139040

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  Abstract 

Background: Menispermaceae, a family of flowering plants, is a medium-sized family of 70 genera totaling 420 extant species, mostly of climbing plants. It has various medicinal properties, which are used in the Ayurvedic system of medicine. Plants belonging to this family are rich in alkaloids, especially bisbenzylisoquinoline type. The hypothesis of this study is that the bisbenzylisoquinoline alkaloids present in the selected plants may exhibit in vitro cytotoxic property.
Aim: The present study is aimed at estimating the total alkaloidal content of methanolic extract of Cocculus hirsutus and Cissampelos pareira and evaluating the in vitro cytotoxic activity of both the extracts on the HeLa cell line.
Settings and Design: Methanolic extracts of both the plants in the concentrations of 500, 250, 125, 62.5, and 31.25 μg/ml were assessed for its cytotoxic activity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Materials and Methods: Total alkaloidal content was studied for both the plants using ultraviolet-visible spectroscopy method. Methanol extracts of both the plants were tested for its inhibitory effect on HeLa cell line. Cytotoxicity of the plant extracts was evaluated by MTT assay. Nonlinear regression graph was plotted between % cell inhibition and Log 10 concentration, and IC 50 was determined using GraphPad Prism software.
Results: Preliminary phytochemical studies confirm the presence of alkaloids in both the plants. The total alkaloids present in C. hirsutus and C. pareira were found to be 0.252%w/w and 0.1656%w/w respectively. The IC 50 values of C. hirsutus and C. pareria were found to be 111 μg/ml and 129.3 μg/ml respectively.
Conclusion: From this study, it is observed that C. hirsutus and C. pareira have in vitro cytotoxic activity against HeLa cell line.

Keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Cissampelos pareira, Cocculus hirsutus, cytotoxic


How to cite this article:
Thavamani B S, Mathew M, Dhanabal S P. In vitro cytotoxic activity of menispermaceae plants against HeLa cell line. Ancient Sci Life 2013;33:81-4

How to cite this URL:
Thavamani B S, Mathew M, Dhanabal S P. In vitro cytotoxic activity of menispermaceae plants against HeLa cell line. Ancient Sci Life [serial online] 2013 [cited 2020 Sep 28];33:81-4. Available from: http://www.ancientscienceoflife.org/text.asp?2013/33/2/81/139040


  Introduction Top


Ayurveda-a science of life has tried many herbal remedies with varying degree of success, but its main significance lies in its preventive approach. Hartwell has collected data, about 3,000 plants, which possess anti-cancer properties and subsequently been used as potent anti-cancer drugs. Ayurveda, a traditional Indian medicine of plant drugs has been successful from very early times in using these natural drugs and preventing or suppressing various tumors using various lines of treatment. [1]

In this study an attempt on in vitro anti-cancer activity of Cocculus hirsutus and Cissampelos pareira were performed in HeLa cell line. Menispermaceae, the botanical name for a family of flowering plants, has been universally recognized by taxonomists. It is a medium-sized family of 70 genera totaling 420 extant species, mostly of climbing plants. C. hirsutus and C. pareira are the climbing plants of this family used for various ailments in Ayurvedic system of medicine. C. hirsutus is used for its diuretic, laxative [2] and anti-inflammatory properties while C. pareria is used in the treatment of indolent ulcers, diarrhea, urinary tract infection and for its anti-inflammatory property. [3]

Most of the plants of this family are rich in bisbenzyl isoquinoline alkaloids. Literatures give evidence that these alkaloids have cytotoxic property. [4],[5] The hypothesis of this study is that the bisbenzyl isoquinoline alkaloids present in the selected plants may exhibit in vitro anti-cancer property.


  Materials and methods Top


Collection of plant materials

The plants were collected from Kallakad of Tirunelveli District, South India. The specimens were identified by Prof. V. Chelladurai, Research Officer-Botany, C.C.R.A.S. Government of India (Retired). A voucher specimen was prepared in our research lab and maintained with the voucher no. PSGCP/DPC/01 for C. hirsutus and PSGCP/DPC/02 for C. pareira for further reference. Immediately after collection the plants were washed thoroughly with water and shade dried at room temperature. The shade dried plants were then pulverized to form coarse powder and used for extraction.

Extraction of crude drug

Methanol extract is prepared by adding one liter of methanol to 500 g of dried powder and macerated for 7 days with occasional stirring. The mixture was then subjected to sonication for about 1 h. The powder mixer was then kept overnight in continuous shaking condition in a shaker at 1500 rpm. After that, it was filtered and the filtrate was labeled as lot 01. The Marc left after extraction was again extracted with 500 ml of methanol by it keeping overnight in shaking condition. It was then filtered and the filtrate was labeled as lot 02. Lots 01 and 02 were combined and the solvents were evaporated under vacuum using rotor vacuum. The yield obtained was 11.6% (w/w) for C. hirsutus and 12.7%w/w for C. pareira.

Phytochemical studies of the extract

Preliminary phytochemical studies and chromatographic studies (Harborne, 1998) of C. hirsutus and C. pareira shows the presence of alkaloids.

Estimation of total alkaloid

The total amount of alkaloids present in the methanolic extract was found as per the procedure carried out by Sreevidya and Mehrotra. [6]

Cancer cell line

The human cervical cancer cell line (HeLa) was obtained from National Centre for Cell Science, Pune, and grown in Eagles minimum essential medium containing 10% fetal bovine serum (FBS). All the cells were maintained at 37°C, 5% CO 2 , 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.

Cell treatment and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay

The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with the FBS medium with 5% FBS to give final density of 1 × 10 5 cells/ml. 100 μl/well of cell suspension was seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 37°C, 5% CO 2 , 95% air and 100% relative humidity. After 24 h the cells were treated with serial concentrations of the extracts and fractions. They were initially dissolved in neat dimethylsulfoxide (DMSO) and further diluted in serum free medium to produce five concentrations. 100 μl per well of each concentration was added to plates to obtain final concentrations of 500, 250, 125, 62.5 and 31.25 μg/ml. The final volume in each well was 200 μl and the plates were incubated at 37°C, 5% CO 2 , 95% air and 100% relative humidity for 48 h. The medium containing no samples were served as control. Triplicate was maintained for all concentrations.

After 48 h of incubation, 15 μl of MTT (5 mg/ml) in phosphate buffered saline was added to each well and incubated at 37°C for 4 h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100 μl of DMSO and then the absorbance was measured at 570 nm using micro plate reader. [7],[8] The % cell inhibition was determined using the following formula.

% cell inhibition = {100 -[Abs (sample)/ Abs (control)]} X 100

Nonlinear regression graph was plotted between % cell inhibition and Log 10 concentration and IC 50 was determined using GraphPad Prism 6 software (GraphPad, San Diego, CA).


  Results and discussion Top


The total alkaloid present in C. hirsutus and C. pareira was found to be 0.252%w/w and 0.1656%w/w respectively.

MTT is a yellow water soluble tetrazolium salt. Succinate-dehydrogenase, a mitochondrial enzyme in living cells, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional to the number of viable cells.

Methanol extracts of C. hirsutus and C. pareria were screened for their cytotoxicity against HeLa cell lines at different concentrations to determine the IC 50 (50% growth inhibition) by MTT assay. Results are graphically represented from [Figure 1], [Figure 2], [Figure 3], [Figure 4]. The percentage growth inhibition was found to be increasing with increasing concentration of test compounds of both plant extracts. The IC 50 values of C. hirsutus and C. pareira in HeLa cell line were found to be 111 μg/ml and 129.3 μg/ml respectively. Phytochemical studies show high content of alkaloids in both the species. Among the two plants of this family, C. hirsutus has more alkaloidal content than C. pareira. This may be the reason for C. hirsutus to have significant cytotoxic property than C. pareira. Thus from the above study, it is evident that the cytotoxic property of both the plants may be due to the alkaloidal content, which is peculiar to Menispermaceae family.
Figure 1: Graphical representation of cytotoxic activity of C. hirsutus in HeLa Cells

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Figure 2: Pictoral view of cytotoxic activity of C. hirsutus in HeLa Cells

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Figure 3: Graphical representation of cytotoxic activity of C. pareira in HeLa Cells

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Figure 4: Pictoral view of cytotoxic activity of C. pareira in HeLa Cells

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  Conclusion Top


The results of the present study demonstrate the potential cytotoxic activity of C. hirsutus and C. pareira. Alkaloids being the major phyto-constituents may be responsible for this potential cytotoxicactivity against HeLa cancer cell line. Further research has to be carried out with other cancer models to elucidate the possible mechanism of action.

 
  References Top

1.Balachandran P, Govindarajan R. Cancer-An ayurvedic perspective. Pharmacol Res 2005;51:19-30.  Back to cited text no. 1
    
2.Ganapaty S, Dash GK, Subburaju T, Suresh P. Diuretic, laxative and toxicity studies of Cocculus hirsutus aerial parts. Fitoterapia 2002;73:28-31.  Back to cited text no. 2
    
3.Amresh G, Reddy GD, Rao ChV, Singh PN. Evaluation of anti-inflammatory activity of Cissampelos pareira root in rats. J Ethnopharmacol 2007;110:526-31.  Back to cited text no. 3
    
4.Mahiou V, Roblot F, Fournet A, Hocquemiller R. Bisbenzylisoquinoline alkaloids from Guatteria boliviana (Annonaceae). Phytochemistry 2000;54:709-16.  Back to cited text no. 4
    
5.Lohombo-Ekomba ML, Okusa PN, Penge O, Kabongo C, Choudhary MI, Kasende OE. Antibacterial, antifungal, antiplasmodial, and cytotoxic activities of Albertisia villosa. J Ethnopharmacol 2004;93:331-5.  Back to cited text no. 5
    
6.Sreevidya N, Mehrotra S. Spectrophotometric method for estimation of alkaloids precipitable with Dragendorff's reagent in plant materials. J AOAC Int 2003;86:1124-7.  Back to cited text no. 6
    
7.Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.  Back to cited text no. 7
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8.Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 1991;83:757-66.  Back to cited text no. 8
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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