Genotoxic and Genoprotective Potential of Black Mulberry (Morus nigra) Fruit

Black Mulberry (Morus nigra l.) belongs to Moraceae family. The present study evaluated the possible genotoxic and/or protective activities of black mulberry fruit juice (BMFJ), in vitro, using mitomycin C (MMC) as a positive control, by chromosomal aberrations and micronucleus assays. Human lymphocytes were treated with BMFJ concentrations alone (1/1, 1/2, 1/4, 1/8 dilutions), pretreatment (49h) (0.20 μg/ml MMC+ 1/1 BMFJ, 0.20 μg/ml MMC+1/2 diluted BMFJ, 0.20 μg/ml MMC+1/4 diluted BMFJ, 0.20 μg/ml MMC+1/8 diluted BMFJ) and simultaneous-treatment (48h) (0.20 μg/ml MMC+ 1/1 BMFJ, 0.20 μg/ml MMC+1/2 diluted BMFJ, 0.20 μg/ml MMC+1/4 diluted BMFJ, 0.20 μg/ml MMC+1/8 diluted BMFJ). The in vitro results demonstrated that BMFJ showed no genotoxicity, but it significantly decreased chromosomal aberration and micronucleus frequency induced by MMC. Our results showed that all concentrations of BMFJ revealed no genotoxicity but protective activity against genomic changes induced by anti-tumor agent MMC in human lymphocytes. Protective effects of BMFJ on MMC induced chromosomal damages most probably due to its free radical scavenging activity.


INTRODUCTION
The relation between use of plants and health has always been widely known by mankind throughout the centuries.According to the World Health Organization nearly 80% of the world's population still relies on traditional medicinal care for better health (Farnsworth et al. 1985).Vitamins, polyphenols, terpenoids and isoflavonoids are the major bioactive compounds of plants that possess anti-oxidative, anti-mutagenic, anti-carcinogenic, and immunomodulating properties (Craig et al. 1999, Santos-Cervantes et al. 2007, Karamova et al. 2011).Since many plant components possess different pharmacological properties for the prevention or treatment of diseases (Jiménez-Medina et al. 2006), interest on anti-genotoxic, anti-carcinogenic activity of plant extracts has been risen during the past few years.Plant components are generally retard oxidative degradation of lipids and other biomolecules and/or sweep up free radicals generated by mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion in cell.Oxidative stress causes macromolecular damage and is implicated in various diseases such as atherosclerosis, diabetes, cancer, neurodegeneration and aging (Barcelos et al. 2007).Black Mulberry (Morus nigra), belongs to the Moraceae family (Boschini 2002) and grow up in east, West and South-east Asia, South europe, South of North America, Northwest of South America and some areas of Africa (Calín-Sánchez et al. 2013) and also in Turkey.The cultivation for fruit production of mulberry reaches nearly 80.000 tons per year (Ercişli and Orhan 2007).Fruit, leaves and even branches of black mulberry are traditionally used as antipyretic, diuretic, lowering blood pressure in various countries (Zhishen et al. 1999).The main bioactive compounds of Black Mulberry fruit have been widely studied in the literature.It is a rich source of phenolic compounds including flavonoids, anthocyanins, phenolic acids and carotenoids (lin and Tang 2007, Du et al. 2008, Özgen et al. 2009, Kutlu et al. 2011, Fazaeli et al. 2012, Calín-Sánchez et al. 2013).Bae and Suh (2007) reported that anthocyanins are the powerful bioactive components of black mulberry.gundogdu et al. ( 2011) deeply investigated the phenolic compound contents, vitamin C (ascorbic acid) content and total antioxidant capacity (Table I ).Chlorogenic acid and rutin was to be found the highest value of phenolic content.
Recently, studies have shown that the extracts of this plant have strong antioxidant activity because of high phenolic and anthocyanin contents (gerasopoulos and Stavroulakis 1997, Zhishen et al. 1999, Kutlu et al. 2011).The extract of the black mulberry prevents cell from the lipid oxidation and bio-membranes from peroxidative damage (Naderi et al. 2004), especially flavonoids protect DNA from oxidative damage and effective on enzymes that promotes cancer development (Kris-etherton et al. 2002).Black mulberry fruit has also possess laxative, odontalgic, antihelmintic, expectorant, hypoglycemic and antimicrobial effects (Mandal and Kumar 2002, Kone et al. 2004, lin and Tang 2007, Ozgen et al. 2009).

MATERIALS AND METHODS
Black Mulberries at full mature stage were collected from the Balıkesir province in Turkey and deposited in our Department (voucher specimen number: 2015FHS38).Chromosome medium B was obtained from Biochrome.Mitomycin C (CAS No: 200-008-6), cytochalasin B (CAS No: 14930-96-2) was obtained from Sigma.Other chemicals was obtained from Sigma (USA).Before experiments ethical approval was obtained from local ethic committee .Peripheral venous blood was collected from non-smoking healthy 2 females and 2 males adults (aged 25-26 years).Whole blood (0.2 ml) was added to 2.5 ml Chromosome Medium B.
For the statistical analysis of the results, z-test for percentage of abnormal cell, CA/cell and MN were used.Dose-response relationships were determined from the correlation and regression coefficients for the percentage of abnormal cell, CA/cell and mean MN.II).

No significant difference in the induction of chromosomal aberrations and micronuclei
The results also showed that BMFJ significantly reduced the frequency of micronuclei (pre-treatment and simultaneous treatment) induced by MMC at the three concentrations (1/1, 1/2 and 1/4 dilutions) tested.Only lowest concentration in simultaneous treatment was not significant (Table III).Our results exhibited that all concentrations of BMFJ showed no genotoxicity but protective activity against genomic changes induced by antitumor agent MMC in human lymphocytes.

DISCUSSION
Mitomycin C (MMC) is an antitumor agent that has been used for a treatment of variety of tumors.However, it has bind covalently to DNA (Tomasz 1995, Paz et al. 2012), and has been shown to alkylate other cellular nucleophiles in addition to DNA, such as glutathione (gSH) (Sharma andTomasz 1994, Sharma et al. 1994  Black mulberry (Morus nigra) fruit has a high nutrient content, especially of antioxidants (Naderi et al. 2004).It has the highest acidity, reduced ascorbic acid, Fe++, total flavonoids, and total monomeric anthocyanins (especially Cyanidin) (Jiang andNie 2015, Veberic et al. 2015).extracts of mulberry fruits were found to possess free radical scavenging activity, anti-hyperlipidemia, anti-atherogenic properties and neuroprotective effects and anti-cancer effects (human lung cancer cell line A549) (Chen et al. 2005, 2006, Kang et al. 2006, Bae and Suh 2007, Jiang and Nie 2015).
Aydın et al. (2015) reported that no statistically significant difference was observed in the hepatic lipid fractions of rats containing M. nigra fruit extract.However, the level of lipid peroxidation were found to be very low in M. nigra treated group (Aydın et al. 2015).Anticancer activity of M. nigra on human breast cancer cell line (MCF-7) was investigated by Ahmed et al. (2016).Black mulberry (BM) extracts administration gave significant morphological evidence of apoptosis and increased cell death after 48 hours more than that of 24 hours-treated MCF-7 cells among fresh and dry extract groups.DNA analysis after 24h of BM extracts treated groups has no fragmentation while apoptosis is generated after 48h showing more fragmentation in fresh BM groups.The mitotic index records were significantly decreased in dose and time dependent manner showing the better anti-proliferative effect with fresh extract treatments.DNA single strand breaks were also increased among the treatment groups at dose and time dependent manner with the best results with fresh extract.
ghasemnezhad Targhi et al. ( 2017) investigated the radio protective effect of black mulberry extract on liver tissue and bone marrow cells in the rat.200   2011) reported that M. nigra leaf aqueous extract had antioxidant effect, contributing to reduce incidence of internal anomalies in offspring from diabetic rat dams.Kutlu et al. (2011) informed that acidified extract of black mulberry was higher in β-carotene prevention and DPPH radical scavenging activity than non-acidified extract.However, non-acidified extract represented a higher reducing power and metal chelating activity, and a higher content of total phenolics.As shown in the Table II no significant difference in the induction of chromosomal aberrations was observed between the groups treated with the concentrations of BMFJ and the negative control.Beyond, the treatment of the cells with BMFJ plus MMC (pre-treatment and simultaneous treatment) showed a significant reduction of chromosomal aberrations when compared to treatment with MMC only (Table II).A relationship between chromosomal damage and cancer development has been suggested since the beginning of the 20 th century (Ribbert et al. 1914, Bonassi et al. 2000), and now we clearly know that increased frequency of chromosomal aberrations cause cancer development because of genomic imbalance.
BMFJ has also significantly reduced the frequency of micronuclei (pretreatment and simultaneous treatment) induced by MMC at the three concentrations (1/1, 1/2 and 1/4 dilutions) tested.The in vitro micronucleus test detects genotoxic effects of xenobiotics in cells and predicts cancer development (Bonassi et al. 2011).Our results showed that all concentrations of BMFJ indicated no genotoxicity but protective activity against genomic changes induced by anti-tumor agent MMC in human lymphocytes.Protective effects of BMFJ on MMC induced chromosomal damages most probably due to its free radical scavenging activity.
the groups treated with the concentrations of BMFJ and the negative control.Beyond, the treatment of the cells with BMFJ plus MMC (pre-treatment and simultaneous treatment) showed a significant reduction of chromosomal aberrations when compared to treatment with MMC only (Table

TABLE III Protective role of black mulberry fruit juice (BMFJ) against MMC induced micronuclei in vitro.
Significantly different from the MMC P < 0.001.