Research Article: Antioxidant Activity and ROS-Dependent Apoptotic Effect of Scurrula ferruginea (Jack) Danser Methanol Extract in Human Breast Cancer Cell MDA-MB-231

Date Published: July 13, 2016

Publisher: Public Library of Science

Author(s): Mohsen Marvibaigi, Neda Amini, Eko Supriyanto, Fadzilah Adibah Abdul Majid, Saravana Kumar Jaganathan, Shajarahtunnur Jamil, Javad Hamzehalipour Almaki, Rozita Nasiri, Irina V Lebedeva.


Scurrula ferruginea (Jack) Danser is one of the mistletoe species belonging to Loranthaceae family, which grows on the branches of many deciduous trees in tropical countries. This study evaluated the antioxidant activities of S. ferruginea extracts. The cytotoxic activity of the selected extracts, which showed potent antioxidant activities, and high phenolic and flavonoid contents, were investigated in human breast cancer cell line (MDA-MB-231) and non-cancer human skin fibroblast cells (HSF-1184). The activities and characteristics varied depending on the different parts of S. ferruginea, solvent polarity, and concentrations of extracts. The stem methanol extract showed the highest amount of both phenolic (273.51 ± 4.84 mg gallic acid/g extract) and flavonoid contents (163.41 ± 4.62 mg catechin/g extract) and strong DPPH• radical scavenging (IC50 = 27.81 μg/mL) and metal chelation activity (IC50 = 80.20 μg/mL). The stem aqueous extract showed the highest ABTS•+ scavenging ability. The stem methanol and aqueous extracts exhibited dose-dependent cytotoxic activity against MDA-MB-231 cells with IC50 of 19.27 and 50.35 μg/mL, respectively. Furthermore, the extracts inhibited the migration and colony formation of MDA-MB-231 cells in a concentration-dependent manner. Morphological observations revealed hallmark properties of apoptosis in treated cells. The methanol extract induced an increase in ROS generation and mitochondrial depolarization in MDA-MB-231 cells, suggesting its potent apoptotic activity. The present study demonstrated that the S. ferruginea methanol extract mediated MDA-MB-231 cell growth inhibition via induction of apoptosis which was confirmed by Western blot analysis. It may be a potential anticancer agent; however, its in vivo anticancer activity needs to be investigated.

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Plant-derived antioxidants protect biological systems from oxidative stress generated by free radicals or reactive oxygen species (ROS) during metabolism and other activities. Antioxidants have a preventive role in numerous disorders caused by cellular damage or oxidative injury including cancer, diabetes, cardiovascular diseases, and mutagenesis [1–3]. Secondary metabolites, such as carotenoids, phenols, ascorbic acid, and flavonoids, are potential sources of natural antioxidants with free radical scavenging capacity [4, 5]. The therapeutic potential of medicinal plants is generally attributed to the antioxidant activity of phytochemicals, particularly phenols and flavonoids [6, 7]. Herbal medicines play a key role in the development of new potential drugs. There is a large and growing body of literature on the discovery of secondary metabolites with antioxidant capacity, and new phytochemical constituents (particularly anticancer agents) from various medicinal plants [8]. Costly treatment methods and serious side effects associated with available therapies may lead to greater tendencies of using herbal medicines for health care. Mistletoe is a common semi-parasitic evergreen plant from the flowering plant family Loranthaceae, which comprises approximately 1500 species that grow on branches of many deciduous trees worldwide [9, 10]. Mistletoe is one of the most widely used herbal medicines with a long history of use in the treatment of various disorders, such as diabetes, skin infection, smallpox, and cough. Steiner [11] introduced it in the field of oncology as an alternative therapy for cancer care. For decades, natives of south Asia, Europe, and Africa have extensively used mistletoe as a complementary and alternative medicine in the treatment and management of numerous diseases including cancer. Several studies have shown that mistletoe, as an anthroposophical medicine, is one of the most important medicinal plants that are potentially efficacious against cancer [12, 13]. Numerous preclinical and in vitro studies using different commercial and standardized products of mistletoe have reported its immunomodulatory, anti-tumor, and anti-metastatic effects [14–20]. Various mistletoe extracts from different origins are capable of inducing apoptosis and cell death in numerous types of tumors and human cancer cell lines [21, 22]. The majority of the studies conducted by the European researchers, particularly investigators from Germany, employed Viscum album (European mistletoe). Extensive preclinical and clinical investigations have been carried out on European mistletoe. However, species of mistletoe from other continents have not received much attention. One such mistletoe species belonging to Loranthaceae family is Scurrula ferruginea, which is mainly distributed in tropical countries [23]. It is locally known as Dedalu in Malaysia, Singapore, and Indonesia. The plant is used traditionally to treat various diseases including gastrointestinal malfunction, high blood pressure, hypertension, and malaria [24–26]. For example, Ameer et al. [24] investigated the effect of S. ferruginea extracts on blood pressure by using in vitro and in vivo animal experimental models. They demonstrated the presence of biologically active substances in S. ferruginea and found that the methanol extract possessed the highest blood pressure lowering activity. They attributed it to the high content of phenols and flavonoids in this plant. Their results provided direct evidence of blood pressure lowering activity of S. ferruginea. Another study [27] evaluated the cytotoxic effects of S. ferruginea extract on different human cancer cell lines. The main constituents of the ethyl acetate fraction including quercetin, quercitrin, and glycoside 4-O acetylquercitrin were isolated using column chromatography. Quercetin exhibited the most potent cytotoxic activity against U251 (human glioblastoma cell line) cells with an IC50 of 35μM. S. ferruginea has been used in traditional medicine for the management of various diseases. Some studies have also reported its therapeutic potential. However, to our knowledge, the antioxidant activity of methanolic, aqueous, ethyl acetate, and hexane extracts from various parts of S. ferruginea have not been explored. Therefore, the first objective of the present study was to screen for the antioxidant activity of different extracts from stem, leaves, and flowers of S. ferruginea in vitro. In vitro assays were carried out for determination of DPPH free radical (DPPH•) scavenging capacity, total phenolic content (TPC), total flavonoid content (TFC), metal chelation capacity, and on 2,2ʹ-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid cationic radicals (ABTS•+) scavenging ability. The crude extracts exhibiting potential antioxidant activity, and high total phenolic and flavonoid contents were selected for further investigation of anticancer and apoptotic activity. Only one study, which focused exclusively on MCF-7 cells, has investigated the anticancer activity of S. ferruginea [28]. The effect of S. ferruginea extracts on claudin-low or triple-negative breast cancer cells (MDA-MB-231), which is the most aggressive subtype of breast cancer with poor prognosis, has not been previously reported. Additionally, its effect on non-cancerous cell line (HSF-1184) has not been investigated. Furthermore, the proapoptotic activity and antimetastatic potential of S. ferruginea in MDA-MB-231 cells have not been investigated. Therefore, the present study was carried out to investigate the proapoptotic activity of S. ferruginea extracts. Production of ROS and mitochondrial dysfunction were evaluated to investigate the involvement of mitochondria in cell death process after S. ferruginea treatment. To the best of our knowledge, this is the first to report that the administration of S. ferruginea methanol extract caused morphological alterations, mitochondrial disruption, ROS generation, and apoptosis induction in MDA-MB-231 cell.

The present study reports the in vitro antioxidant capacities of the aqueous, methanolic, ethyl acetate, and hexane extracts of different S. ferruginea parts, for the first time. Stems, leaves, and flowers of S. ferruginea were extracted sequentially with different solvents. The methanolic extracts exhibited the highest yield. The results of this study showed that nature and polarity of solvent affect the percentage yield of the extract. This was in agreement with the study by Osadebe et al. [45], who reported a high yield of Eastern Nigeria mistletoe (Loranthus micranthus) extracts when using polar solvents. Previous studies on other plant species also showed the yield of samples obtained in descending order of methanol > aqueous > ethyl acetate > hexane, which corroborate results of this study. The extraction yield with methanol and water was higher than that of the other solvents [46, 47]. Furthermore, results of the current study showed that different S. ferruginea extracts contained different levels of TPC and TFC. The significant TPC and TFC differences may be attributed to different parts of plant used for extraction. The stem methanolic extract had significantly higher phenolic content than other parts (P < 0.05). Results of this study were in agreement with a previous study [48], which showed that methanolic extract of European mistletoe (Viscum album) were rich in phenolic compounds. The presence of large amounts of phenolic compounds in the aqueous and methanolic extracts may contribute to the antioxidant activities and the ability to adsorb and scavenge free radicals [49, 50]. TFC obtained in the present study is in line with results of Dashora et al. [51], who stated that the stem methanolic extract of Indian mistletoe (Dendrophthoe falcata) had higher flavonoid content than the stem aqueous extract. The S. ferruginea stem methanolic extract induced cytotoxicity and apoptosis in MDA-MB-231 cells. It also induced ROS generation and disrupted the MMP. In addition, this study demonstrated that the S. ferruginea methanolic extract induced apoptosis possibly through intrinsic apoptotic signaling pathway. Antiproliferative and apoptotic effects of methanolic extract against MDA-MB-231 cells could be attributed to their antioxidant activity, high phenolic and flavonoid contents. However, specific bioactive compounds responsible for the anti-breast cancer activity of extracts require attention. Further detailed studies on the underlying mechanism responsible for the antioxidant and anticancer activities are needed. Taken together, the findings of this study showed antioxidant and anticancer activities of S. ferruginea extracts and provided the scientific rationale for using this plant in future development of chemotherapeutic drugs against breast cancer.   Source: