Chemicals and compounds
Gentamicin was obtained from Virbac, South Africa. Sodium carbonate was provided by Holpro Analytic, South Africa. Dulbecco’s Modified Eagle Medium (DMEM) and Fetal calf serum (FCS) were purchased from Highveld Biological, South Africa. Whitehead Scientific, South Africa provided trypsin and Phosphate buffered saline (PBS). p-iodonitrotetrazolium violet (INT), doxorubicin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), puromycin, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), dimethyl sulfoxide (DMSO), were provided by Sigma-Aldrich St. Louis, MO, USA, while Müller-Hinton agar and broth were from Sigma-Aldrich, India.
Naturally occurring compounds studied in this work were isolated from the leaves and stembark of Entada abyssinica. The leaves of E. abyssinica was collected in May 2012 at Balatchi (Mbouda), in the West region of Cameroon, and identified by Mr. Victor Nana (plant taxonomist) of the National Herbarium of Cameroon, Yaoundé, where a voucher specimen is deposited under reference number 32436/HNC. Compounds studied included: ursolic acid (1), quercetin-3-O-α-l-rhamnoside or quercitrin (2), quercetin-3-O-β-D-glucosyl (1→4)-α-l-rhamnoside (3), (8S)-kolavic acid 15-methyl ester (4), 13,14,15,16-tetranor-3-clerodene-12,18-dioic acid (5), methyl gallate (6), entadanin (7), bis-[(S)-(2,3-dihydroxypropyl)] hexacosanedioate (8). We previously described their isolation procedure and their structure elucidation [14]. Chemical structures are shown in Fig. 1.
Antimicrobial activity
The six bacterial strains included: Pseudomonas aeruginosa ATCC 27853, Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028 and Enterococcus faecalis ATCC 29212. The antimicrobial activity was evaluated by determining the minimal inhibitory concentration (MIC) by the rapid p-iodonitrotetrazolium violet (INT) microdilution method as previously described [15].
Antioxidant assays
ABTS radical assay
The antioxidant activity by ABTS was assessed according to the method previously described [16].
DPPH assay
The DPPH radical-scavenging activity was assessed by the method previously described [16].
Ferric reducing antioxidant power (FRAP) assay
The antioxidant activity by the ferric reducing antioxidant power (FRAP) was assessed according to the method previously described with slight modifications [16].
Cytotoxicity assay
Cell culture
Cancer cell lines including human monocytic THP-1 and murine macrophage RAW 264.7 cells and the normal mammalian Vero monkey kidney cell line were obtained from the American Type Culture Collection (Rockville, MD, USA). They were maintained in DMEM under standard cell culture conditions at 37 °C and 5% CO2 in a humidified environment.
MTT assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxicity of the compounds as previously described [15]. The selectivity index (SI) values to identify selective anti-cancer cell activity were calculated by dividing the LC50 values of normal Vero cells by the LC50 of cancer cells.
Statistical analysis
Experiments were performed three times and values were expressed as mean ± standard deviation. Differences between IC50 values were analysed for statistical significance using ANOVA and compared using the Fisher’s least significant difference (LSD) at 5% interval confidence.