Anticancer activity of Morus nigra leaves extract

Plants are screened for treatment of many ailments including cancer because they possess certain potential constituents which are effective for treatment. The aim of this study was to evaluate the anticancer activity of Morus nigra leaves against human cervical cancer cell line (HeLa). n-Hexane and aqueous methanolic extract of plant’s leaves were made by maceration. Anticancer activity was estimated by methyl-thiazolyl-tetrazolium (MTT) assay and percentage inhibition of cells was calculated. Results of MTT showed that 100 µg/ml aqueous methanol extract of M. nigra inhibited 89.5-31.99% of HeLa cell line. It was concluded that M. nigra possess anticancer activity. DOI: http://dx.doi.org/10.3329/bjp.v9i4.19783


Introduction
Cancer is one of the leading death causing disease of the current era and is characterized by aberrant growth of cell mass that is uncoordinated to remain proliferating when stimulus has been removed that provoked that action.Many plants have recently be reported with anticancer activity like Casuarina equisetifolia (Shafiq et al., 2014), Aspergillus niger (Channabasavaet al., 2014) and Convolvulus arvensis (Saleem et al., 2014).
The objective of this study was to evaluate the anticancer activity of M. nigra extracts against human cervical cancer cell line ((HeLa).

Material and Method
Collection of medicinal plants: M. nigra leaves weres collected from Jinnah colony, plant nurseries located at Bilal road and Samundri road, Faisalabad.The plant material was identified from Department of Botany, University of Agriculture, Faisalabad.Prior to maceration, all plants were washed, shadow dried and grinded to coarse powder for extract formation.
Preparation of extracts: One thousand three hundred grams of powdered M. nigra (leaves) was merged in 4,000 mL of n-hexane and aqueous methanol (70% methanol and 30% distilled water) for 7 days with occasional shaking.After maceration, rotary evaporator lyophilizer was used for concentration of the extracts.
Anticancer activity: Single cell suspension was made by trypsin-ethylenediamine tetraacetic acid (EDTA) and layer of cells was separated.Final density of 1x10 5 was made using medium containing 5% FBS and diluted the cell suspension.In 96-well plates, 10,000 cells/well were seeded and incubated at 37°C, keeping 5% CO2, 95% air and 100% relative humidity.After 24 hours, different concentrations of extracts i.e., 1, 10, 25, 50 and 100 μg/mL were added and incubated under above mentioned working conditions for another 48 hours.Well without plant extracts were taken as control.Whole procedure was repeated in triplicate and viable cells were calculated using hemocytometer before and after extracts addition (Dantuet al., 2012).

Anticancer activity of Morus nigra leaves extract
MTT assay: In every well 100 μL of MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in phosphate buffered saline was added and incubated at 37°C for 4 hours.The medium with MTT was flipped off and the formed formazan crystals was solubilized in 100 μL of DMSO.Using micro plate reader the absorbance was measured at 490 nm.The %cell inhibition was determined using the following formula (Cheng et al., 2011).

Absorbance sample % Cell inhibition = 1 -x 100 Absorbance control
Graph was plotted against concentrations to calculate IC50.

Statistical analysis:
A logistic linear regression model was fit to the data using Microsoft Excel 2013 Software to calculate the IC50.The data obtained were expressed as mean ± standard deviation.A value of p<0.05 was considered as significant.

Results and Discussion
The anticancer activity of n-hexane and aqueous methanolic extract of M. nigra (leaves) against HeLa cancer cell line is shown in Table I. 100 µg/mL aqueous methanol extract of M. nigra inhibited 89.5-32.0% of HeLa cell line.Estimated IC50 of n-hexane and aqueous methanolic of M. nigra against HeLa cancer cell line at 24 hours was 185.9 ± 8.3 µg/mL and 56.0 ± 1.7 µg/mL respectively.n-Hexane and aqueous methanolic extract at 1, 10, 25, 50 and 100 µg/mL had shown dose dependent inhibition of cells.Sundararajan et al. (2006) also evaluated anticancer activity of n-hexane extract of Bidens pilosa on HeLa and KB cell lines corroborated our findings with IC50 values of 509.2 ± 6.3 µg/mL and 385.2 ± 4.7 µg/mL respecttively.A lower IC50 of Carthamus oxyacanthus (whole plant) indicates that it has phytochemical constituents that synergistically inhibit growth of cancer cells (Alesiani et al., 2010).
It can be concluded that M. nigra (leaves) possess anticancer activity against cervical (HeLa) cancer cell line.