Bangladesh Journal of Pharmacology Research Article AntiAntiAnti---cancer activity of cancer activity of cancer activity of Morus nigra Morus nigra Morus nigra leaves extractleaves extractleaves extract BJP 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 prolifer- ating 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). M. nigra (Family Moraceae), commonly as Black Mul- berry (English) and Shah-toot (Hindi/Urdu), is used as hepatoprotective (Malhi et al., 2014), anti-oxidant (Imran et al,. 2010; Ercisli and Orhan, 2008) and antimicrobial (Digrak et al., 1999). The objective of this study was to evaluate the anti- cancer 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. Anti-cancer activity Single cell suspension was made by trypsin-ethylene- diamine tetraacetic acid (EDTA) and layer of cells was separated. Final density of 1 x 105 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 A Journal of the Bangladesh Pharmacological Society (BDPS); www.bdps.info Bangladesh J Pharmacol 2014; 9: 496-97 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract 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 anti-cancer 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. Anti-cancer 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 - 32.0% of HeLa cell line. It was concluded that M. nigra possess anti-cancer activity. Article Info Received: 8 August 2014 Accepted: 31 August 2014 Available Online: 9 October 2014 DOI: 10.3329/bjp.v9i4.19783 Cite this article: Qadir MI, Ali M, Ibrahim Z. Anti- cancer activity of Morus nigra leaves extract. Bangladesh J Pharmacol. 2014; 9: 496-97. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Anti-cancer activity of Morus nigra leaves extract Muhammad Imran Qadir1,2, Muhammad Ali1 and Zubair Ibrahim2 1Institute of Molecular Biology & Biotechnology, Bahauddin Zakariya University, Multan, Pakistan; 2College of Pharmacy, GC University, Faisalabad, Pakistan. extracts addition (Dantuet al., 2012). MTT assay In every well 100 μL of MTT (3-(4,5-dimethylthiazol-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). %Cell inhibition = 1 - Absorbance sample 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 anti-cancer activity of n-hexane and aqueous metha -nolic 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 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 depen- dent inhibition of cells. Sundararajan et al. (2006) also evaluated anti-cancer 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 and 385.2 ± 4.7 µg/mL respectively. 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 anti- cancer activity against cervical (HeLa) cancer cell line. Reference Alesiani D, Canini A, D’Abrosca B, DellaGreca M, Fiorentino A, Mastellone C, Pacifico S. Anti-oxidant and anti-prolifer- ative activities of phytochemicals from Quince Cydonia vulgaris peels. Food Chem. 2010; 118: 199-207. Channabasava, Govindappa M. First report of anti-cancer agent, lapachol producing endophyte, Aspergillus niger of Tabebuia argentea and its in vitro cytotoxicity assays. Bangladesh J Pharmacol. 2014; 9: 129-39. Cheng XL, Liu Q, Peng YB, Qi LW, Li P. Steamed ginger (Zingiber officinale): Changed chemical profile and increased anticancer potential. Food Chem. 2011; 129: 1785-92. Dantu AS, Shankarguru P, Ramya DD, Vedha HB. 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Bangladesh J Pharmacol 2014; 9: 496-497 497 Table I Percentage inhibition (Mean ± SD) of HeLa cell line by Morus nigra extracts Concentration (µg/mL) n-Hexane Aqueous methanol 1 0.04 ± 0.0 0.8 ± 0.1 10 4.5 ± 0.8 7.2 ± 1.1 25 17.9 ± 0.5 20.8 ± 2.2 50 30.9 ± 0.1 48.1 ± 1.3 100 41.2 ± 0.8 89.5 ± 2.0 IC50 185.9 ± 8.3 56.0 ± 1.6 Author Info Muhammad Imran Qadir (Principal contact) e-mail: mrimranqadir@hotmail.com DatePrinted: This article was downloaded by you on: Oct 15, 2017