54-58The aim of this study was to determine the cytotoxic effect of black mulberry fruit extract (Morus nigra L.) on human breast cancer (MDA-MB-231) and prostate cancer (PC3) cell lines. The extract prepared from mulberry (M. nigra) fresh fruit was applied to cancer cell lines in 5 different v/v concentrations (10%, 4%, 2%, 1.33% and 1%) for 72 h. At the end of the incubation period, cell viability was determined by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay. Results were compared with negative and positive control groups. The effect of mulberry (M. nigra) fruit extract on cell viability was determined depending on the concentration applied; extract showed a significant cytotoxic effect on PC3 cell line at the 10% concentration compared to the control group, but not detected any effect at 4%, 2%, 1.33% and 1% concentrations. In the MDA-MB-231 cell line, there was a high cytotoxic effect at a concentration of 10% compared to the control group, while a lower effect was observed at 4%, 2%, 1.33% and 1% concentrations. The data obtained from the study showed that extract prepared from black mulberry fruit has cytotoxic effect especially against breast cancer cells

Dalkiliç, L K

Dalkiliç, S

İnci, Ş

Kırbağ, S

Korkmaz, İ

NOPR

Indian Journal of Traditional Knowledge Vol 20(1), January 2021, pp 54-58 Investigation of cytotoxic effect of black mulberry (Morus nigra L.) fruit S Dalkiliç* , L K Dalkiliç, Ş İnci, İ Korkmaz & S KırbağFirat University, Faculty of Science, Department of Biology, Elazıg, Turkey E-mail: †sdalkilic@gmail.comReceived 27 October 2019; revised 10 September 2020 The aim of this study was to determine the cytotoxic effect of black mulberry fruit extract (Morus nigra L.) on human breast cancer (MDA-MB-231) and prostate cancer (PC3) cell lines. The extract prepared from mulberry (M. nigra) fresh fruit was applied to cancer cell lines in 5 different v/v concentrations (10%, 4%, 2%, 1.33% and 1%) for 72 h. At the end of the incubation period, cell viability was determined by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay. Results were compared with negative and positive control groups. The effect of mulberry (M. nigra) fruit extract on cell viability was determined depending on the concentration applied; extract showed a significant cytotoxic effect on PC3 cell line at the 10% concentration compared to the control group, but not detected any effect at 4%, 2%, 1.33% and 1% concentrations. In the MDA-MB-231 cell line, there was a high cytotoxic effect at a concentration of 10% compared to the control group, while a lower effect was observed at 4%, 2%, 1.33% and 1% concentrations. The data obtained from the study showed that extract prepared from black mulberry fruit has cytotoxic effect especially against breast cancer cells. Keywords: Breast cancer, Cytotoxicity, Morus nigra L., MTT, Prostate cancer IPC Code: Int. Cl.21: A61K 39/395, A61K 38/00, C12N 15/82,  Cancer is the most common pathology worldwide and has the highest mortality rate after cardiovascular diseases. It accounts for approximately 20% of all deaths worldwide1. According to the GLOBOCAN cancer statistics 18.1 million new cases and 9.6 million death occure every year in the world2. Population grow, lifestyle and some behaviors having main roles in this increasing3. According to data from the World Health Organization, the incidence of cancer is increasing and according to predictions 22 million new cancer cases are expected to emerge in 20304. Nowadays, thanks to the advances made especially in the field of medicine, significant success has been achieved in the diagnosis and treatment of cancer. However, despite all these developments, the success achieved in some cancer types is not at the desired level5. Since the side effects of chemotherapy agents used in cancer treatment are very high, natural and herbal treatment methods are considered as an alternative. Therefore, some natural and herbal substances have been suggested to be effective in the treatment of cancer6. Such practices are almost as old as the history of mankind and are still being practiced today. Intensive studies are carried out to identify new drug target molecules and to develop more effective drugs5,6. It is known that the leaves, bark and fruit parts of M. nigra belonging to Moraceae family are used asantipyretic, diuretic, expectorant, helping to protectoral mucosal health and complementary in thetreatment of dysentery7,10. And also; hasantimicrobial, antioxidative, antihelminthic, anti-inflammatory, antidiabetic and cytotoxic effects11,16.M. nigra is used in the treatment of certain diseasesdue to its high anthocyanin content and bioactivecompounds besides being used as a food17.Anthocyanins are a major group of molecules called flavonoids that have strong antioxidant, antiallergic, antiapoptotic properties synthesized by phenylpropanoid18,20 pathways. Flavonoids like as polyphenolic compounds which can show bioactivity in organism and its anti-cancer effect is causing controversy in the scientific world19. Various studies indicate that flavonoids can contribute to both prevention and treatment of cancer14,21. Flavonoids are promising molecules in the treatment of cancer22. The aim of this study was to determine the cytotoxic activity of M. nigra fresh fruit extract on breast cancer (MDA-MB-231) and prostate cancer (PC3) cell lines. ————— *Corresponding AuthorDALKİLİÇ et al.: ANTICANCER PROPERTIES OF BLACK MULBERRY FRUIT EXTRACT   55 Material and Methods  Obtaining the material M. nigra was collected from Arsuz district of Hatay (Turkey) in 2019 and fruits were extracted using blender. After the extraction of M. nigra, 9 mL of medium was added to 1 mL of M. nigra extract and 10% concentration was prepared as stock and serial dilutions (10%, 4%, 2%, 1.33% and 1%) were prepared from stock solution and stored at +4°C  until use.   Supply of cell lines Human breast cancer (MDA-MB-231) and prostate cancer (PC3) cell lines were obtained from Inönü University and Erzurum Technical University.  Cell culture Breast cancer (MDA-MB-231) and Prostate cancer (PC3) cells were cultured in DMEM [1% L-Glutamine, 1% Penicillin-Streptomycin and 10% FBS (Fetal Bovine Serum)] in 25 cm² flasks, at 37°C and 5% CO₂ atmospheric conditions.  Evaluation of cell viability  The MDA-MB-231 and PC3 cell lines grown in  25 cm² flasks, after 90% of confluency the medium in the flask was removed and washed with 5ml sterile PBS solution. About 1 mL Trypsin-EDTA was added to the flasks and incubated for 2 min in an incubator under conditions at 5% CO2 and 37°C. After the cells were separated from the surface, trypsin-EDTA was inactivated with 5 mL of culture medium. Cells were taken from the flask, centrifuged at 1200 rpm for  5 min, then the supernatant was removed and the cell pellet was thawed with 1 mL of fresh medium and counted in the microscope. Adjusted the cell number as 5000 cell in 100 μL and then seeded in 96-well plates. Only the medium was placed in the first row to be used as blank and incubated in an incubator at 37°C with 5% CO2 for 24 h. After incubation, 100 μL of, 10%, 4%, 2%, 1.33% and 1% of each diluted mulberry extract was added in 12 replicates and incubated under condition 5% CO₂ incubator at 37°C for 72 h. MTT test was applied after incubation. Absorbance measurements were made at 570 and  540 nm wavelength. 2.5 µg/mL Doxorubicin was used as positive control and culture medium was used as negative control23,24.  MTT assay MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay is a method used to measure cell viability, proliferation and cytotoxicity. MTT assay is based on the fact that activated mitochondria in living cells take up electrons in MTT which can pass through cell membrane and it is converted to into purple water-insoluble formazan crystals. Then, formazan crystals are dissolved with appropriate solvents and color changes are measured by spectrophotometric methods to determine the number of viable cells25-27. After the incubation period with this method, 20 μL of 5 mg/mL MTT solution was added to the wells containing the cells and incubated for 4 h at 37°C with 5% CO₂ in dark environment. After incubation, medium was removed and formazan crystals were dissolved with 100 μL DMSO (dimethyl sulfoxide). The absorbance measurements at 570 and 540 nm wavelengths were performed by ELISA plate reader28,30.  Analysis The absorbance values measured by ELISA plate reader device were recorded, then the measured absorbance values transformed to live cell percentage and calculated values were compared with the control groups and the data were transferred to the graphs.  Results In this study, cytotoxic effect of M. nigra fruit extract on human breast cancer (MDA-MB-231) and human prostate cancer (PC3) cell lines were investigated via MTT [3- (4,5-dimethylthiazol-2-yl) -2,5 -diphenyltetrazolium bromide] assay. According to our results, M. nigra fruit extract has significant cytotoxic activity on MDA-MB-231 (breast cancer) cell line at 10% concentration as shown in (Figure 1 and Figure 2). It was determined that the effect at other concentrations had a lower than 10%   Fig. 1 — Cytotoxic effect of M. nigra on MDA-MB-231 (breast cancer) cell line. Control: DMEM; (+) Control: Doxorubicin, M. nigra, 10%, 4%, 2%, 1.33% and 1%. INDIAN J TRADIT KNOW, JANUARY 2021   56 concentration. The effect on PC3 (prostate cancer) cell line showed significant cytotoxic activity at 10% concentration as shown in (Figure 3 and Figure 4), while no cytotoxic effect on cells was observed  at other concentrations. M. nigra extract was shown concentration-dependent cytotoxic activity on the MDA-MB-231 cell line compared to the PC3 cell line. Our results have shown that M. nigra extract has cytotoxic effect against to breast cancer cell line. The most effective cytotoxicity has determined in the concentration of 10%. Total live cell percentage has determined as 21% in this concentration at the 540 nm wavelength. The lowest cytotoxic effect has observed as 76.1% in the concentration of 1%. Cytotoxic effect has decreasing as a dose dependent manner. Cytotoxic effect of M. nigra against the MDA-MB-231 (breast cancer) cell line is shown in Figure 1.  The maximum cytotoxic effect has observed in concentration of 10% on MDA-MB-231 cell line in the 570 nm wavelength. This effect has better than doxorubicin effect on cancer cell in this concentration (Fig. 2). Figure 3 and Figure 4 have shown observed cytotoxicity of M. nigra extract on PC3 prostate cancer cell line. The most effective cytotoxicity has observed as 28.4% at the concentration of 10% in the 570 nm wavelength. We have not observed any significant cytotoxicity in other concentrations on PC3 cell line. Maximum cytotoxic effect has determined as 56.4% at the 10% concentration in the 540 nm wavelength. There has not observed any significant cytotoxic effect in other concentrations on PC3 cell line.  Discussion and Conclusion M. nigra has been observed to have apoptotic and antiproliferative effects on PC3 cell line in previous studies31. Leaf extract of the same species has been reported to show no significant cytotoxic activity against L-929 (fibroblast), B16F10 (melanoma), HeCat (keratinocyte) cell lines32. It has been reported that M. nigra exhibits strong antibacterial and antioxidant activity and weak cytotoxic effect33. The differences in the results obtained in the aforementioned studies and the study may be due to the different cell lines used or the difference in the experimental and extraction procedure. The fruit of M. nigra is rich in antioxidant flavonoids18,20. Flavonoids are known to inhibit carcinogenic and harmful oxidant agents such as superoxide anion radicals and nitric oxide14-16,34. In the light of these studies, it is understood that black mulberry (M. nigra) species is rich with antioxidants but it has low cytotoxic effect against some cancer cell lines.   Fig. 2 — Cytotoxic effect of M. nigra on MDA-MB-231 (breast cancer) cell line. Control: DMEM; (+) Control: Doxorubicin, M. nigra, 10%, 4%, 2%, 1.33% and 1%    Fig. 3 — Cytotoxic effect of M. nigra on PC3 (prostate cancer) cell line. Control: DMEM; (+) Control: Doxorubicin; M. nigra 10%, 4%, 2%, 1.33% and 1%    Fig. 4 — Cytotoxic effect of M. nigra on PC3 (prostate cancer) cell line. Control: DMEM; (+) Control: Doxorubicin, M. nigra 10%, 4%, 2%, 1.33% and 1%  DALKİLİÇ et al.: ANTICANCER PROPERTIES OF BLACK MULBERRY FRUIT EXTRACT   57 In the review of Lim and Choi, M. nigra particular fruit and leaf parts have various pharmacological properties such as antinociceptive, anti-inflammatory, antimicrobial, melanogenic, antidiabetic, anti-obesity, anti-hyperlipidemic and anticancer activities, as well as central nervous system, liver, and kidney protective and therapeutic effects on the gastrointestinal tract and the female reproductive system, and most of these properties are based on M. nigra antioxidant compounds such as polyphenols, flavonoids and anthocyanins35. Eo have stated that the root bark of Morus alba L. (white mulberry) has anti-inflammatory effect as well as anticancer effect and this effect is antiproliferative and apoptosis-stimulating in nature36. As pointed out in this study, the effect of M. nigra and M. alba species on cancer cells shows that not only the fruits but also parts of leaves, roots, barks, stem can have a potential effect. Fallah et al. applied antioxidants obtained from the leaves of M. alba on cancer in mice and observed a significant increase in the survival of mice37. These studies have shown that M. nigra and M. alba have anticancer effect and increase the survival of mice37. More comprehensive studies should be performed for detection of cytotoxic and antiproliferative effects of mulberry (M. nigra) and white mulberry (M. alba) fruit extract and also should test against other cancer cell lines. Further studies are needed to determine whether there is any cytotoxic effect on healthy cell lines. Likewise, more studies should perform on the mechanism of action and determination of active substances on cancer cells.  Acknowledgement We thank to Firat University Faculty of Medicine Department of Parasitology, Prof. Dr. Mustafa Kaplan which contributed to the laboratory environment in the conduct of this study.   Conflict of Interests Authors declare no conflict of interest  Author Contributions S D and L K D designed the experiments and MTT assay L K D and İ K collected and prepared fruit and extract concentration for the experiment S D, L K D, S K, Ş İ and İ K Interpreted of the results. 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Investigation of cytotoxic effect of black mulberry (Morus nigra L.) fruit

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Investigation of cytotoxic effect of black mulberry (Morus nigra L.) fruit

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