Cytotoxicity, antimicrobial and antioxidant activity of eight compounds isolated from Entada abyssinica (Fabaceae)

Oxidative stress occurs when there is excessive free radical production and/or low antioxidant defense, which leads to many pathophysiological conditions in the body [1]. To neutralize free radicals and protect the body against oxidative damage, different antioxidants which are present in normal physiological conditions are able to counteract the production of reactive oxygen species. Free radicals are known to be the main cause of various diseases such as cancer and bacterial diseases. The development of resistance to multiple drugs in microbes and tumor cells has become a major public health threat [2, 3]. Cancer is one of the leading causes of death in most well developed countries. A large body of evidence has determined that relationships exist among certain bacteria and cancers [4]. Because of the resistance that pathogenic microorganisms and malignant cells build against current antibiotics and anticancer drugs, there is great interest in the search for new therapeutic agents. Thus, in recent years there has been increased use of plants and their derivatives as an alternative modality in the treatment of various diseases, including cancer and infections caused by microorganisms [5]. Unlike synthetic drugs, bioactive natural products can have a beneficial effect on the whole organism and with less toxic effects. Therefore, natural products will continue to be extremely important as sources of discovery of new medicinal agents. Entada abyssinica A.Rich (Fabaceae) is a tree widely spread in tropical Africa. It is traditionally used to treat coughs, rheumatism, bronchitis, abdominal pains, diarrhoea and fever and to prevent miscarriage [6, 7]. Some pharmacological properties of E. abyssinica have been previously reported, including anti-inflammatory, antimicrobial and antioxidant [8–10]. Previous phytochemical screening of E. abyssinica indicated the presence of flavonoids, terpenoids and kolavic acid derivatives [11–13]. Considering the vast potential of plants as sources of antimicrobial and anticancer drugs, the objective of this study was to examine the possible antiproliferative, antimicrobial and antioxidant activity of terpenoid and flavonoid compounds isolated from E. abyssinica.

The antibacterial potential ranged from significant to weak activity. Ursolic acid is an ubiquitous compound that can be isolated from many medicinal plants and its antibacterial activities are well documented. It has been reported to be active against many bacterial species, particularly Gram-positive species, inhibiting bacterial growth of S. aureus with a MIC value of 4 µg/mL [17, 18]. It is noteworthy that the activity of compound 7 (entadanin) against S. typhimurium was comparable to the standard gentamicin. Quercitrin is a quercetin-related flavonoid and previous studies have shown that quercetin and its glycosides quercetin-3-O-α-l-arabinopyranoside and quercetin-3-O-β-d-arabinopyranoside have strong antibacterial activity against the Gram-positive S. aureus, and the Gram-negative P. aeruginosa and E. coli with MIC values ranged from 0.093 to 0.37 µg/mL [19].

The antioxidant activity of compounds can be determined in vitro by hydrogen atom transfer (HAT) method and single electron transfer (SET) method. HAT methods measure the capacity of an antioxidant to scavenge free radicals by hydrogen donation to form a stable compound. SET methods determine the ability of the antioxidant to transfer one electron to reduce compounds including metals, carbonyls and radicals [20]. The FRAP assay involves the SET method, while the DPPH and ABTS assays involve both methods, but predominantly the SET method [21]. In this study, the antioxidant activity of compounds was determined using the free radical 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the ferric reducing antioxidant power (FRAP) assays. The use of at least two different assays in evaluating antioxidant activity of plant products has been recommended by Moon and Shibamoto [22].

The antioxidant activity revealed that, the IC₅₀ values of compounds 7, 6 and 2 were significantly different from the IC₅₀ values of ascorbic acid and trolox, which are standard antioxidant agents used as positive controls. The capacity of flavonoids to act as antioxidants in vitro has been previously studied [23]. However, the antioxidant activity of entadanin, a new peltogynoid is here reported for the first time.

In order to ascertain the likely safety of compounds for their potential use, a standard cell-based toxicity assay was performed for cytotoxicity evaluation against Vero monkey kidney cells. In addition, the anti-proliferative activity was assessed on two cancerous cell lines (THP-1 and RAW 264.7). According to the in vitro cytotoxic activity criteria suggested by Syarifah et al. [24], a compound is considered as weakly active if the LC₅₀ ≥ 50 µg/mL, moderately active for 10 µg/mL < LC₅₀ < 50 µg/mL and significantly active if LC₅₀ ≤ 10 µg/mL). Considering this cut-off, the activity obtained with compound 1 (ursolic acid) against THP-1 and RAW 264.7 cells could be considered significant. Ursolic acid is a natural pentacyclic triterpenoid carboxylic acid present in a wide variety of plants, including apples, basil, bilberries, cranberries, peppermint, rosemary and oregano [25]. Several pharmacological effects of ursolic acid including anti-proliferative properties have been reported in a number of experimental systems [26]. It should be noted that this is the first report on the biological activity of compound 7, a cyclic homoflavonoid (entadanin), and compound 8 (bis-[(S)-(2,3-dihydroxypropyl)] hexacosanedioate).

Our findings suggest that among the terpenoid and flavonoid compounds studied, entadanin (compound 7), whose activities are reported here for the first time, possesses extremely interesting antibacterial activity against S. typhimurium. Therefore, this compound could be investigated further for its potential use in the treatment of bacterial diseases, especially gastrointestinal infections caused by S. typhimurium.