In vitro Anti-Angiogenic Properties of Ethanolic Crude Extract of Vernonia amygdalina

Sains Malaysiana 51(5)(2022): 1437-1447 http://doi.org/10.17576/jsm-2022-5105-14

ABSTRACT Angiogenesis is the process of generating new blood vessels that deliver tumor cells with oxygen and essential nutrients for growth and metastasis. This study examined the in vitro antiangiogenic properties of the ethanolic crude extract from Vernonia amygdalina (VA) grown in Malaysia. The direct antiangiogenic activity of VA was evaluated on EA.hy926 cells using in vitro assessments: Cell proliferation, colony formation, migration, and cell invasion assays. VA ethanolic crude extract cytotoxic activity was evident in the antiproliferative and colony formation assays. The growth inhibition (IC50) of 50% against EA.hy926 endothelial cells was achieved after 72 h treatment at a concentration of 85.43±3.57 µg/mL. Upon 48 h treatment, colony formation was inhibited completely at 100 µg/mL while 51.94% inhibition was achieved at 50 µg/mL. Moreover, the extract showed 54.72% and 31.99% inhibitory effects against migration of cells when treated for 24 h treatment at two different concentrations, 25 µg/mL and 12.5 µg/mL, respectively. The use of 100 µg/mL VA ethanolic extract inhibited cell invasion by 35.43%, which was lower than that of 57.81% inhibition achieved by the vinblastine as a positive control. All in all, the present work clearly demonstrated the antiangiogenic properties of VA ethanolic extract that may reflect a chemotherapeutic and/or chemoprevention potential for biomedical applications.

INTRODUCTION Angiogenesis is a complex and highly regulated process of generating new blood vessels. The new vessels branch off from pre-existing vessels under normal physiological homeostasis or tumorigenesis, which involves partly extracellular matrix rebuilding, endothelial migration, proliferation, and tube formation (Blood & Zetter 1990). Tumor angiogenesis delivers essential nutrients and oxygenation for tumorigenesis. Besides, it also acts as a conduit for the migration of tumor cells to adjacent and distant tissues and the formation of secondary tumors known as metastasis (Ferrara 2010). The importance of angiogenesis was first discussed upon the observation by Folkman (1971) that the tumor was unable to grow beyond 1 mm3 without angiogenesis. Hence, the ability of screening for angiogenesis inhibitors has become an attractive therapeutic choice for treating cancer patients. Medicinal plants are considered to have a rich source of active phytochemicals with pharmacological properties such as anti-inflammatory, antioxidant, and anticancer effects (Duthie et al. 2006). To date, several anticancer agents originating from plant sources have been developed as commercially available anticancer agents, such as paclitaxel, vinblastine, and vincristine (Shoeb 2006). In addition, many studies have screened medicinal plants for their antiangiogenic actives. For example, apigenin, a flavone found in medicinal plants, has anticancer characteristics that are associated with its anti-inflammatory, antioxidant, and antiangiogenic properties (Anand et al. 2008). To cite another example, abisilin, a natural terpenoid with in vitro and in vivo antiangiogenic effects, was found to inhibit neovascularization by inhibiting endothelial cell migration, proliferation, and invasion (Kudryavtseva et al. 2016).

Vernonia amygdalina (VA), which is commonly known as ‘bitter leaf’, is a tropical shrub native to Nigeria (Ogidi et al. 2019). The leaves of VA have been recognized as traditional African medicine with various health benefits. Traditionally, the leaves are used as a remedy to treat stomach discomfort, vomiting, diarrhea, and intestinal illnesses (Farombi & Owoeye 2011). As nutritional supplement rich in antioxidants and phytochemical contents, it has been deemed useful for maintaining human health and disease prevention (Yedjou et al. 2019). The crude extracts of VA have also been reported to possess immune-boosting, antioxidant, anti-bacterial, anti-inflammatory, and anticancer activities (Farombi & Owoeye 2011; Yedjou et al. 2019). These activities could be attributed to the various phytochemicals found in VA, such as vernolide, vernodalol, anthraquinones, flavonoids, lignans, terpenes, phenolic acids, and xanthones (Farombi & Owoeye 2011). The anticancer properties of VA were first reported by Kupchan et al. (1969) when a leaf chloroform extract was tested against human carcinoma of the nasopharynx. To date, studies have shown that VA decreases cell viability and induces apoptosis in numerous cancer cell lines in both concentration and time-dependent manner (Yedjou et al. 2018). The importance of this study arises from the fact that available clinically approved antiangiogenic therapies normalize the tumor vasculature by blocking the VEGF/VEGFR2 pathway (such as bevacizumab). Thus, on one side, the therapies enhance tumor perfusion and the delivery of chemotherapies, while on the other hand, resistance was developed by tumor adaptation and production of other proangiogenic factors such as fibroblast growth factor (FGF). Other clinically approved antiangiogenic therapies are small molecules targeting receptor tyrosine kinase-mediated signal transduction mechanisms such as sunitinib. Although these agents target different receptors involved in the angiogenesis process, they may have potentially life-threatening side effects.

The present study assumed that plants with antiinflammatory properties could have good antiangiogenic effects. This is because inflammation and angiogenesis are two interlinked processes that support tumor growth and progression (Albini et al. 2016). Therefore, the study aimed to evaluate in vitro antiangiogenic properties of Vernonia amygdalina shrub with previously reported anti-inflammatory effects. The direct effect of the extract was evaluated on endothelial cells EA.hy926 using antiproliferative, colony formation, migration, and cell invasion assays. Subsequently, the VA extract was further screened for its antiproliferative properties against MCF7 and HT29 human cancer cell lines. This study is valuable to introduce an extract that possesses antiangiogenic properties in addition to its previously reported anti-inflammatory effects. These bioactive properties are mediated by various active phytochemicals, which could be subsequently evaluated for in vivo prevention of tumor progress and adjuvant therapy to treat solid tumors.

Sains Malaysiana 51(5)(2022): 1437-1447 http://doi.org/10.17576/jsm-2022-5105-14