Plants are excellent sources of

Plants are excellent sources of food, chemicals and herbal medicines. Many important drugs have been derived from these (Khalid et al., 2011). M. nigra is one of the most important species of the genus Morus, the fruits containing substantial levels of phenolics, flavonoids, and ascorbic RG7388 (Kostic et al., 2013). It has recently been reported to exhibit biological properties, such as antidiabetic, antioxidative, anti-inflammatory and antihyperlipidemic activities. These biological activities are due to the polyphenol components, including anthocyanins, present in some varieties (Kutlu et al., 2011; Kamiloglu et al., 2013). Various studies have investigated in vitro antiproliferative and proapoptotic characteristics of different Morus species in the recent years (Fathy et al., 2013; Eo et al., 2014), but studies involving the in vitro cytotoxic effect of M. nigra extract are very limited (Qadir et al., 2014). No previous studies have investigated the cytotoxic effect of M. nigra extract on prostate cancer. Sforcin and Bankova reported that in vitro methods are useful for preliminary investigation of the possible beneficial biological effects of a natural product. If positive results are obtained from in vitro studies, then in vivo or clinical trials are recommended (Sforcin and Bankova, 2011). The PC-3 cell line is an androgen receptor negative prostate cancer cell line with high metastatic potential frequently in cancer research for in vitro prostate cancer models (Shahneh et al., 2014; Huo et al., 2015). We therefore planned this study on the prostate cancer cell line (PC-3) under in vitro conditions.
Many methods are available for extraction of active components from plant materials. Maceration is one such technique. In this method, many solvents, such as water, ethanol, methanol, ethyl acetate, dimethyl sulfoxide, hexane or acetone, are used to extract the components from plant materials directly without compromising the structure (Dai and Mumper, 2010). The DMSO extract of M. nigra was prepared using the maceration technique in this study.
Various in vitro assays can be used to determine antioxidant activity of herbal extracts, and the use of at least two different methods is recommended (Nuutila et al., 2003). The TPC and FRAP methods were employed for the determination of antioxidant activity of DEM in this study. TPC and FRAP values of M. nigra extract were 20.7±0.3mg gallic acid equivalents and 48.8±1.6mg trolox equivalents per g sample, respectively. The TPC values of various extracts of M. nigra fruit from different regions range between 1.69 and 22.37mg gallic acid equivalents/g sample (Kostic et al., 2013), while the FRAP value of 75% aqueous-methanol extract of M. nigra fruit from Turkey has been reported at 3.8mg trolox equivalents/g sample (Kamiloglu et al., 2013). Our results were broadly similar to those of other studies. Any differences may have been due to the type of extraction method and solvent, environmental factors, soil, geographic region, harvest season, post-harvesting conditions and the maturity level of the fruits.
Several reports have described the use of HPLC with a diode array detector (DAD) for the characterization and quantification of phenolic composition (Boligon and Athayde, 2014). We used the HPLC-DAD spectra system for phytochemical analysis. Seven phenolic compounds (ascorbic acid, gallic acid, 3,4-dihydroxy benzoic acid, protocatechuic acid, chlorogenic acid, p-coumaric acid and rutin hydrate) were determined in the DEM. These results agree with previous research showing that the genus Morus is rich in polyphenolic compounds such as cyanidin-3-glucoside, kaempferol-3-O-rutinoside, rutin, quercetin, catechin, quercetin-3-O-glucoside, quercetin-3-O-rutinoside, taxifolin, chlorogenic acid, p-coumaric acid, vanillic acid, gallic acid, ferulic acid, caffeic acid, and syringic acid (Hassimotto et al., 2007; Gundogdu et al., 2011; Kostic et al., 2013).
An effective and acceptable chemopreventive or anticancer agent has to meet various criteria, including having no harmful effects on normal cells (Galati and O’Brien, 2004). We therefore performed cytotoxicity experiments in PC-3 cells coupled with human normal foreskin fibroblast cells. DEM exhibited reasonable selective toxicity against PC-3 cells compared to normal fibroblast cells via MTT assay, a nonradioactive, quick, and affordable method widely used in cytotoxicity studies (Russo et al., 2004). Qadir et al. demonstrated that M. nigra leaf extract exhibited cytotoxic effect on a human cervical cancer (HeLa) cell line (Qadir et al., 2014). Shi et al. isolate numerous flavonoids with isoprenoid groups from Morus mongolica and demonstrate selective cytotoxic activity in two human oral cancer cell lines (HSC-2 and HSG) against normal human gingival fibroblasts (Shi et al., 2001). In another study, Fathy et al. reported that M. alba extract exhibits cytotoxic effect on the hepatocellular cell line (Fathy et al., 2013), while Kofujita et al. demonstrated that prenylated flavanone separated from M. alba root shows cytotoxic effect against rat hepatoma cells (Kofujita et al., 2004). Additionally, Dat et al. isolated 11 flavonoids from the leaves of the white mulberry and evaluated their cytotoxic effect on three human cancer cell lines (HeLa, MCF-7 and Hep3B). Morusin was identified as the most potent cytotoxic compound against HeLa cells (Dat et al., 2010).