Cytotoxicity, antimicrobial and antioxidant activity of eight compounds isolated from Entada abyssinica (Fabaceae)
© The Author(s) 2017
Received: 23 February 2016
Accepted: 1 March 2017
Published: 6 March 2017
Entada abyssinica is a plant traditionally used against gastrointestinal bacterial infections. Eight compounds including three flavonoids, three terpenoids, a monoglyceride and a phenolic compound isolated from E. abyssinica were investigated for their cytotoxicity, antibacterial and antioxidant activity.
Compounds 7 and 2 had remarkable activity against Salmonella typhimurium with the lowest respective minimum inhibitory concentration (MIC) values of 1.56 and 3.12 µg/mL. The antioxidant assay gave IC50 values varied from 0.48 to 2.87 μg/mL in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, from 2.53 to 17.04 μg/mL in the 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) assay and from 1.43 to 103.98 µg/mL in the FRAP assay. Compounds had relatively low cytotoxicity (LC50 values ranging from 22.42 to 80.55 µg/mL) towards Vero cells. Ursolic acid had the most potent cytotoxicity against THP-1 and RAW 264.7 cells with LC50 values of 9.62 and 4.56 μg/mL respectively, and selectivity index values of 7.32 and 15.44 respectively.
Our findings suggest that among the terpenoid and flavonoid compounds studied, entadanin (compound 7) possess tremendous antibacterial activity against S. typhimurium and could be developed for the treatment of bacterial diseases.
KeywordsCytotoxicity Antibacterial Free radical scavenging Entada abyssinica
Oxidative stress occurs when there is excessive free radical production and/or low antioxidant defense, which leads to many pathophysiological conditions in the body . 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 . 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 . 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.
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.
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 .
ABTS radical assay
The antioxidant activity by ABTS was assessed according to the method previously described .
The DPPH radical-scavenging activity was assessed by the method previously described .
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 .
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.
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 . 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.
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.
Antibacterial activity of eight compounds isolated from Entada abyssinica (MIC in µg/mL)
Antioxidant activity of eight compounds isolated from Entada abyssinica
DPPH (IC50, µg/mL)
ABTS (IC50, µg/mL)
FRAP (µmol FeSO4/g)
2.87 ± 1.19a
7.04 ± 1.29a
1.43 ± 0.80a
0.9 ± 0.06b
3.53 ± 0.39b
76.01 ± 1.10b
2.08 ± 0.19a
17.04 ± 0.26c
75.34 ± 1.06b
1.93 ± 0.14a
5.09 ± 0.40c
0.48 ± 0.02c
2.53 ± 0.49d
103.98 ± 13.70d
1.12 ± 0.10c,d
4.13 ± 0.10e
72.41 ± 2.02b,e
22.98 ± 4.29f
8.71 ± 2.03e
10.38 ± 2.4a,f
3.44 ± 1.9a,f
4.15 ± 1.21d,e
Cytotoxicity (LC50 in µg/mL) of eight compounds isolated from Entada abyssinica and their selectivity index (SI) values against normal and cancer cell lines
22.42 ± 2.48a
9.62 ± 0.59a
4.56 ± 0.020a
44.83 ± 2.83b
16.44 ± 0.20b
53.76 ± 2.05c
41.90 ± 0.43c
47.46 ± 0.63b,d
49.78 ± 3.03b
52.30 ± 1.30d
41.91 ± 1.85b,e
21.81 ± 1.11c
16.10 ± 1.00b
30.58 ± 3.09f
75.00 ± 1.68d
36.92 ± 1.27e
55.65 ± 0.30c
84.28 ± 3.30e
19.12 ± 0.25f
80.50 ± 4.83g
65.00 ± 6.88d,f
86.55 ± 4.61g
9.35 ± 0.66h
0.5 ± 0.00h
5.32 ± 0.90i
0.4 ± 0.02g
1.15 ± 0.17i
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 .
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 . The FRAP assay involves the SET method, while the DPPH and ABTS assays involve both methods, but predominantly the SET method . 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 .
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 . 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. , 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 . Several pharmacological effects of ursolic acid including anti-proliferative properties have been reported in a number of experimental systems . 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.
fetal calf serum
phosphate buffered saline
2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt
Muller Hinton broth
Dulbecco’s Modified Eagle Medium
ferric reducing antioxidant power
JPD carried out the experiments and wrote the manuscript. RM, ArTT and AlTT contributed to the compound isolation and identification. GDKWF and BTN supervised the chemical part of the study. JNE and LJM supervised the work and provided the facilities for biological activities study. All authors read and approved the final manuscript.
The University of Pretoria provided a postdoctoral fellowship to JPD.
The authors declare that they have no competing interests.
Availability of data and materials
“The chemical structures supporting the conclusions of this article are available in the http://pubchem.ncbi.nlm.nih.gov/under the CID number 77-52-1, 522-12-3, 59262-54-3, 948827-00-7, 14218259-0 and 99-24-1. Cell lines are available at: http://web.expasy.org/cellosaurus/ under references: CVCL_0493, CVCL_0006 and CVCL_0059. All other datasets supporting the conclusions of this article are included within the article.
Consent to publish
This manuscript does not contains any individual person’s data.
Ethics statement and consent
This research did not involve data collected from humans or animals. Entada abyssinica does not belong to the species under CITES, therefore no permission is required to collect and study this plant in Cameroon.
The National Research Foundation (NRF) and Medical Research Council (MRC) provided funding to support this study.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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