In vitro cytotoxic effects of Muntingia Calabura Linn. Fruits against human cancer cell lines.

Introduction

Muntingia calabura Linn is a plant that belongs to the Elaeocarpaceae family, commonly known as cherry tree. The tree bears small red fruits with enormous tiny yellow seeds. The fruits are so sweety and juicy, which attracts the people to eat. This plant has been used as the Peru traditional medicine in reducing the swell of the prostate gland and alleviating headaches and cold as well as pains associated with gastric ulcers. The fruits and leaves possess the antioxidant activity [1]. Cancer is the major cause of death worldwide, claiming over 6 million lives every year. In the recent years alternative therapies have gained importance over conventional cancer therapies for the treatment of cancer [2]. Chemotherapy is restricted by both intrinsic and acquired cell resistance to drugs. [3] This has necessitated the use of natural products for the treatment of cancer. There are compelling evidences for experimental investigations on the efficacy of plant drugs against cancer [4]. It is known that there are links between the inflammatory and nociceptive, oxidative and cancer processes. The ability to inhibit any of the process will definitely lead to the inhibition of the others [5]. Based on the fact that M. calabura fruit possess antioxidant activity, the aim of the present study was to determine the in vitro anticancer activity of methanol extracts of the fruits of M. calabura against HeLa and HEp-2 cancer cell lines using MTT assay.

Plant Material and Extraction

Collection and authentication of fruits of M. calabura Linn were collected from the surrounding areas of Erode, Tamil Nadu state, India during 2008-2009 and the plant was identified and authenticated and deposited (voucher number: BSI/SC/5/23/09-10/Tech-132) at Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu.

The fruits were collected freshly and extracted with methanol, the extract was completely dried in vacuum evaporator, stored in refrigerator at 4[degrees]C and protect from sunlight until use.

Cell line and Culture

The cell lines were obtained from National Centre for Cell Science (NCCS), Pune. The HeLa and HEp-2 cells were grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS) and NIH 3T3 fibroblasts were grown in Dulbeccos Modified Eagles Medium (DMEM) containing with 10% FBS.

In vitro cytotoxicity activity assay

The MTT assay was used to assess cytotoxicity towards the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cells (Hep-2) and mouse embryonic fibroblasts (NIH 3T3) [6]. For screening experiment, the cells were seeded into 96-well plates in 100[micro]l of respective medium containing 10% FBS, at plating density of 10,000 cells/well and incubated at 37[degrees]C, 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition of fruit extract. The fruit extract was solubilized in Dimethyl sulfoxide and diluted in respective medium containing 1% FBS. After 24 h, the medium was replaced with respective medium with 1% FBS containing the fruit extract at various concentration (eg; 0.25, 0.5, 0.75 mg/ml etc ...) and incubated at 37[degrees]C, 5% CO2, 95% air and 100% relative humidity for 48h. Triplicate was maintained and the medium containing without fruit extract was served as control. After 48h, 10[micro]l of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37[degrees]C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilised in 100[micro]l of DMSO and then measured the absorbance at 570 nm using micro plate reader.

The % cell inhibition was determined using the following formula and graph was plotted between % Cell inhibition and concentration and from this, [IC.sub.50] was calculated.

% cell Inhibition = 100- Abs (drug)/Abs (control) x 100.

Statistical analysis

The experimental data were mean [+ or -] standard deviation of three measurements (n=3). Linear regression analysis was used to calculate the efficient concentration ([IC.sub.50]) values.

Results

Effects of Muntinigia calabura fruit extract on human cancer cells

In vitro cytotoxic effect of methanol extract from M. calabura fruits were investigated at 0.25, 0.5, 0.75, 1.0, 1.25 1.5mg/ml against two human cancer cell lines and a mouse embryonic fibroblast. Fig.1 illustrates the effects of different concentrations of M. calabura fruit extract on cell proliferation of different cell lines. The inhibition of growth of the cell lines by the extract was concentration dependent. At 1.5 mg/ml, the extract showed 88 % and 90 % growth inhibition against HeLa, Hep-2, cells respectively. The findings obtained in this study show that the [IC.sub.50] Values of methanol extract from M. calabura fruits was 1.0mg/ml in HeLa cells, 800 [micro]g/ml in the Hep-2 cells. Maximum cytotoxicity by fruit extract was observed against HeLa Human cancer cell line. As illustrated in Fig 2A-2D, the extract of M. calabura fruits at 1.0 mg/ml induced about 90 % of cell death in HeLa and Hep-2 cancer cells. The adherent tumour HeLa were detached from the culture plate and become floated resulting in rounding up of cellular shape (Fig.2B). The Hep-2 cancer cells treated with fruit extract of M. calabura significantly increased the number of apoptotic cells compared to control (Fig 2C).

Discussion

The search for novel anticancer drugs from plants has been successful worldwide. Many anti-cancer components have been isolated and are nowadays used to treat human cancer. Our present study observed that the methanol extract of M calabura fruit inhibited the proliferation of human cancer cell lines such as human cervical cancer cell line (HeLa), and human laryngeal epithelial carcinoma cells (Hep-2) and a mouse embryonic fibroblast (NIN 3T3). The extract displayed dose dependent and cell specific antiproliferative cytotoxicity against the cell lines tested. From the results obtained, it is apparent that extract from M calabura fruit was active in inhibiting in vitro cell proliferation. Maximum cytotoxicity by methanolic fruit extract was observed against HeLa human cancer cell line. Significant morphological changes were observed compared to control in the fruit extract treated HeLa cells. The degenerative morphological change ultimately led the cells to death. The number of colonies decreased significantly in a concentration-dependent manner, suggesting that the M. calabura fruit extract effectively reduced the malignancy and suppressed the regeneration potential of cancer cells. The results of this study showed a remarkable activity of fruit extract against the tested cell lines though the IC50 values are not lower than the dose recommended by the protocols of the national cancer Institute of USA7 . Thus, further in vivo study is warranted to confirm its cytotoxicity.

Conclusion

It was observed that the methanol fruit extract of M. calabura was active against the tested human cancer cell lines. A wide variety of natural substances have been recognised to have the ability to induce apoptosis in various tumour cells. It is thus considered important to screen apoptotic inducers from plants, either in the form of crude extracts or as components isolated from them. Hence it is proposed for further studies on possible anticancer activity in vivo.

Each value in the table was obtained by calculating the average of three experiments [+ or -] standard deviation (n=3)

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References

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[3] Tundis R, Loizzo M R, Bonesi M, Menichini F, Dodaro D, Passalacqua N G, Statti G, Menichini F. 2009. 'In vitro cytotoxic effects of Senecio stabianus Lacaita (Asteraceae) on human cancer cell lines'. Nat Prod Res. 23(18): 1707-1718.

[4] Ferrez A, Faria D A, Benneti M N, Brondani da Rocha A, Schwartsmann G, Henriques A, Von Poser G L. 2005. 'Screening for antiproliferative activity of six southern Brazilian species of Hypericum. Phytomedicine'. 12: 115-122.

[5] Olszanecki, R., Gebska, A., Kozlovski, V.I. and Gryglewski, R.J. 2002. 'Flavonoids and nitric oxide synthase'. Journal of Physiology and Pharmacology, 53(4):571-584.

[6] Mosmann T. 1983. 'Rapid colorimetric assay for cellular growth and survival: Application to Proliferation and cytotoxicity assays'. J. Immunol Method. 65: 55-63.

[7] Geron R.L., Greenberg N.A., Mc Donald N.M., Schumacher A.M. and Abbott B.J. 1972. 'Protocols for chemical agents and natural products against tumours and other Biological system'. Cancer Chemother. Rep., 3, 17.

K. Preethi (1) *, J.M. Sasikumar, (2) and Chandramohan (3)

(1) Research and Development Centre, Bharathiar University, Coimbatore -641046, India

(2) Sasikumar, Department of Biotechnology, Karpagam Arts & Science College, Coimbatore- 641 021, India

(3) Microcore Research Laboratories India Pvt. Ltd., Erode--638115, Tamil Nadu, India

* Corresponding Author E-mail: gunpre@yahoo.com, info@microcoreresearch.com Table 1: The [IC.sub.50] for anticancer activity of M calabura fruit extract against Human cancer cell lines. Sample Cytotoxicity [IC.sub.50] (mg/ml) Methanol extract HeLa HEp-2 NIH 3T3 of M calabura fruit 1.0 0.8 0.5

Preethi, K.; Sasikumar, J.M.; Chandramohan. 2011 In vitro cytotoxic effects of Muntingia Calabura Linn. Fruits against human cancer cell lines. The Free Library (July, 1),http://www.thefreelibrary.com/In vitro cytotoxic effects of Muntingia Calabura Linn. Fruits against...-a0322903755 (accessed July 07 2015)