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Acthaside: a new chromone derivative from Acacia ataxacantha and its biological activities
© The Author(s). 2016
Received: 2 June 2016
Accepted: 29 November 2016
Published: 7 December 2016
Acacia ataxacantha (Fabaceae), used in traditional medicine grows in the South-West of Bénin. Ethyl acetate extract of the barks of this species was previously reported to display various bioactivities, including antibacterial, antifungal and antioxidant activities. In the present study, we investigate the antimicrobial and antioxidant activities of compound isolated from ethyl acetate extract of Acacia ataxacantha.
Purification, isolation and structural identification of isolated compound were done using various chromatographic and spectroscopic methods. Antimicrobial activity was investigated using a two-fold serial microdilution method. The inhibitory potency of isolated compound was evaluated by kinetic experiments. The antioxidant activity was also determined using 2, 2-diphenyl-1-picrylhydrazyl.
The isolated compound was identified as 7-hydroxy-2-methyl-6-[β-galactopyranosyl-propyl]-4H-chromen-4-one. As far as we know, this compound, named “acthaside”, reported for the first time, was active against all tested microorganisms with minimal inhibitory concentration ranging from 25 to 50 μg/ml. At 50 μl/ml, no growth was observed in almost all tested microbial after 24 h of exposure. The isolated compound had significant antioxidant activity with an IC50 value of 3.61 ± 0.12 μg/ml compared to quercetin (IC50 1.04 ± 0.01 μg/ml).
The present work demonstrates that the new chromen derivative isolated from A. ataxacantha may help treat bacterial and yeast infections. However, further studies are required to clarify the mechanism of action of this compound.
Infectious diseases are the main cause of approximately one-half of all death in tropical countries. Some bacteria and fungi extremely pathogenic, are principal causes of these human infections. For several decades until now, the antibiotherapy has been one of the most important therapies used for fighting infectious diseases and has tremendously enhanced the health aspects of human life since its introduction. The discovery of antibiotics to combat these pathogens marked a revolution in the twentieth century . However, the bacterial resistance is spreading throughout the world, inducing a steadily decreasing of relevant antibiotics . The emergence of microbial resistance and the decrease in effectiveness of currently available antimicrobial agents requires the discovery for new antimicrobial substances with novel inhibitory mechanisms. Medicinal plants represent an alternative source for new antimicrobial agent discovery . Medicinal plants have been used for centuries to treat infectious diseases and are considered as an obvious source of new antimicrobial compounds . Numbers of bioactive metabolites have been isolated from these natural sources. According to the World Health Organization (WHO), medicinal plants could be the source of a variety of drugs. Antimicrobial properties of medicinal plant are being increasingly reported from different parts of the world . The uneven availability, chemical diversity, structural complexity, lack of substantial toxic effects, and broad spectrum of antimicrobial activity of natural products, either as pure compounds or as standardized plant extracts, provide unlimited possibilities for new effective drugs and make them ideal candidates for new therapeutics [6, 7]. Members of the genus Acacia, which are widely distributed in tropical and subtropical regions, consist of about 1200 species , and are used medicinally for several purposes [9–12]. Previous phytochemical investigation on some species of the genus Acacia led to the isolation of flavonoids [13, 14], terpenoids  and polyphenols . To the best of our knowledge, no phytochemical work has yet been done on A. ataxacantha. This species is widespread in much of sub-Saharan Africa. This species has been reported in Benin, Nigeria and Kenya for the treatment of tooth decay, dysentery, bronchitis, cough and joint pain [17–19]. Antimicrobial and antioxidant activities of the extracts of A. ataxacantha have also been reported previously [20, 21]. The lack of toxicity of the bark of this species was also reported . These results suggest that this plant might contain bioactive compounds that act as antimicrobial and antioxidant agents. The present study were undertaken to evaluate antimicrobial and antioxidant properties of a new chromen derivative isolated from A. ataxacantha.
General experimental procedures
1D and 2D NMR spectra were recorded using Bruker 400 MHz spectrometer. The chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS δ = 0). The coupling constants (J) are given in Hz. Deuterated methanol (CD3OD) was used as solvent in the NMR experiments. Mass spectral data ESI-MS, were recorded on Agilent technology instrument Accurate Mass Q-ToF spectrometer. The material used for chromatographic separation was Sephadex LH-20. Pre-coated silica gel 60 F254 TLC plates (Merck, Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm). Mueller Hinton (Agar and Broth) and Sabouraud Dextrose (Agar and Broth) were used for preparation of culture media for the antibacterial and antifungal activities respectively. Gentamicin, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and quercetin were purchased from Sigma-Aldrich Co., USA. All solvents used were of analytical grade.
Collection and preparation of plant material
Fresh barks of Acacia ataxacantha were collected in September 2012 from Ouidah, Department of Atlantic, South of République du Bénin. Botanical identification was performed by a botanist from the Herbier National du Bénin at University of Abomey-Calavi and a specimen with voucher number AA 6509/HNB was deposited at the same Herbarium. The collected plant material was chopped into small pieces and air-dried in the laboratory (22 °C), under shade for two weeks. The dry plant material obtained was ground to a fine powder using an electric grinder (Excella mixer grinder).
Preparation of plant extract
Dry powdered barks of A. ataxacantha (250 g) were successively extracted with hexanes (3 × 500 ml), dichloromethane (3 × 500 ml), ethyl acetate (3 × 500 ml) and methanol (3 × 500 ml) by maceration with continuous shaking (at 24 °C for 72 h) using an orbital shaker (Ika Ks 260 basic). The extracted matter from each solvent was filtered first using a cotton plug followed by Whatman No 1 filter paper. The resulting extracts were concentrated using rotary evaporator (BUCHI Rotavapor RII, Switzerland) under reduced pressure and extracts were stored in refrigerator at 4 °C.
Isolation of bioactive compound
The best solvent system for column chromatography was selected for elution after carrying out the TLC of the ethyl acetate extract by the combinations of different solvents such as dichloromethane, ethyl acetate and methanol alternatively. Among all combination of solvents, dichloromethane: methanol combination showed the best resolution of the components of the extract on TLC plate.
The ethyl acetate extract (1 g) was dissolved in 1 ml of a mixture of CH2Cl2/CH3OH (50:50, v/v) and fractionated using Sephadex LH-20 column (diameter: 4 cm; Sephadex height: 25 cm). The elution solvent was the mixture CH2Cl2/CH3OH (50:50 and 20:80 v/v), to afford a total number of 31 fractions. A monitoring with TLC of these fractions yielded four main fractions (I-IV). Fraction III that consisted of major compounds in the extract was subjected to further purification. Purification of this fraction was done using preparative liquid chromatography (Gilson VP 250/21, Nucleodur 100-5 C18ec, Macherey-Nagel, UV detection 220 and 254 nm) by gradient elution (flow rate 20 ml/min) using water/acetonitrile (85:15 to 00:100 v/v) with 0.1% trifluoroacetic acid as mobile phase, to obtain compound 1 (9 mg).
Structural elucidation of isolated compound
Structural determination of the isolated compound was carried out by spectrophotometric methods (1D and 2D NMR, mass and UV spectrometry). 1D and 2D NMR spectrum were recorded at room temperature with a Bruker NMR spectrometer 400 and 500 MHz in CD3OD. The 2D experiments (COSY, NOESY, HSQC, HMBC) were performed using standard Bruker programs.
Acid hydrolysis of isolated compound
The isolated compound (1 mg) was dissolved in 6% HCl (1 ml) and heat at 80 °C for 2 h. The mixture was then extracted with CHCl3 (3 × 5 ml). The H2O phase was evaporated and dried to obtain the monosaccharide residue. This residue was identified as galactose by comparison with standards (galactose and glucose) on TLC in CHCl3/CH3OH/H20 (8:5:1) .
The microorganisms used in this study included Gram-positives bacteria such as Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (CIP8039), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus Methicillin Resistant (SAMR) and Gram-negative Pseudomonas aeruginosa (CIP 82118). The microorganisms were obtained from Laboratoire de Biophotonique et Pharmacologie, University of Strasbourg, France. Candida albicans (CIP 4872) strain was obtained from national laboratory of drug control in Cotonou (Bénin). Bacterial cultures were maintained on Mueller-Hinton agar (MHA) and yeast cultures were maintained in Sabouraud Dextrose Agar (SDA) at 4 °C. Sub-culturing was done weekly. The bacteria were inoculated in Mueller-Hinton broth (MHB) for 18 h at 37 °C and yeast in Sabouraud Dextrose broth (SDB) for 48 h at 30 °C, prior to the test.
Minimum inhibitory concentration
The two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) values of isolated compound against microorganisms . The stock solution (1 mg/ml) was prepared by solubilizing 1 mg of isolated compound in 50 μl of dimethyl sulfoxide (DMSO 2.5%) followed by 950 μl of MHB. Briefly, 100 μl of isolated compound (100 μg/ml), gentamicin and fluconazole (50 μg/ml) were two-fold serially diluted with Mueller-Hinton broth for antibacterial assay and Sabouraud broth for yeast assay in 96-well microplates to make eight concentrations of isolated compound (100-0.78 μg/ml) and control (50-0.39 μg/ml). 100 μl of freshly culture of bacteria (106 CFU/ml) and yeast (2 × 105 CFU/ml) was added to each well. DMSO (2.5%) was used as negative control while gentamicin and fluconazole were used as positive controls. The microplates were covered and incubated at 37 °C. After 18 h of incubation, 40 μl of 0.2 mg/ml solution of p-iodonitrotetrazolium (INT) which is an indicator solution for determination of bacterial growth were added to each well and microplates were further incubated at 37 °C. The minimal inhibitory concentration was determined 30 min after addition of INT.
Minimum bactericidal and fungicidal concentration
The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of isolated compound was determined according to the method of Escalona-Arranz et al. . To determine the MBC and MFC, aliquots of 20 μl from all dilutions not showing any growth of bacteria and yeast were inoculated on sterile MHA plates (for bacteria) and SDA (for yeast) by spreading using swab sticks. Inoculated plates were incubated at 37 °C for 24 h for all bacteria while those inoculated with fungi were incubated at 30 °C for 48 h. After incubation, the concentration at which there is no visible growth of the organisms on the agar plates was recorded as the minimal bactericidal concentration (MBC) for bacteria and minimal fungicidal concentration (MFC) for yeast. The experiment was carried out in triplicate.
Determination of MIC index
The MIC index (MBC/MIC) was calculated for isolated compounds and positive controls to determine whether a compound had bactericidal/fungicidal (MBC/MIC ≤4) or bacteriostatic/fungistatic (4 < MBC/MIC <32) effect on the growth of bacteria or fungi .
Time-kill kinetic index
The time-kill kinetic index of isolated compound was determined as described by Miyasaki et al.  with slight modifications. The objective of this test is to know the duration of bactericidal or fungicidal effect of isolated molecule. Briefly, bacterial and yeast overnight cultures were diluted to the 106 CFU/mL with MH and SD broth respectively. Equal volume of each diluted inoculum and tested compound were mixed at their respective predetermined MBC and MFC values and incubated with shaking at respective temperature of 37 °C for bacteria and 30 °C for yeast. At different time intervals viz. 0, 1, 4, 8, 12, … 36 h, 0.1 mL of the mixed suspension was spread on suitable agar petri dishes in triplicate and incubated for 36 h at suitable temperature. After 18 h incubation, viable colonies were enumerated. The results were recorded in terms of log10 CFU and plotted vs. time for each microbial tested.
In vitro antioxidant activity
Where, ABlank is the absorbance of the control reaction (containing all reagents except the test sample) and Asample is the absorbance of sample (isolated compound or quercetin).
The concentration of samples reducing 50% of free radical DPPH (IC50) was determined using the regression line representing the inhibition percentage of DPPH versus the sample concentration. The assay was replicated three times and results are expressed as mean ± standard deviation.
The results were expressed as means of triplicate determination ± standard deviation (SD). The graphical was performed using the Graph Pad Prism 6.1 software (Microsoft, USA).
Results and discussion
In our previous work, the ethyl acetate extract of bark of Acacia ataxacantha was found to have significant activity against S. aureus (ATCC 6538), S. epidermidis (CIP 8039), E. faecalis (ATCC 29212), S. aureus Methicillin Resistant (SARM) and P. aeruginosa (CIP 82118). Ethyl acetate extract was active against all bacteria with the minimum inhibitory concentrations values of 325 μg/ml against S. aureus and 625 μg/ml against all other tested bacteria . The phytochemical analysis of this active extract led to the isolation of a chromen derivative. The structural identification of isolated compound was elucidated using a combination of spectroscopic methods. Biological activity of this compound was also evaluated.
Structural elucidation of isolated compound
NMR Spectroscopic data for acthaside
Isolated compound (acthaside)
δH (J in Hz)
5.94, (d, 0.7)
6.65 (d, 2.4)
6.62 (d, 2.4)
2.97 (dd, 12.1, 7.4)
3.68 (dd, 12.1, 5.3)
1.09 (d, 6.2)
2.25 (d, 0.7)
4.45 (d, 8.0)
3.07 (dd, 8.7, 8.0)
3.75 (dd, 11.8, 1.6)
Antimicrobial activity of isolated compound
Minimum inhibitory concentration (MIC) and Minimum bactericidal and fungicidal concentrations (MBC, MFC) of acthaside (μg/ml) from A. ataxacantha against microorganisms
Minimum inhibitory concentrations (μg/ml)
Gram (+) bacteria
Gram (-) bacteria
Minimum bactericidal and fungicidal (μg/ml)
Time-kill kinetic study
The present research work through a systematic chemical and biological investigation has determined and identified for the first time the compound 7-hydroxy-2-methyl-6-[β-galacto-propyl]-4H-chromen-4-one (acthaside) as a new antimicrobial substance and antioxidant, isolated from A. ataxacantha. This study demonstrated that the isolated compound present a broad spectrum antimicrobial activities against Gram-positive and Gram-negative bacteria and also against C. albicans (yeast). The isolated compound has also showed a promising antioxidant activity when compared with standard drugs. The results obtained in the present study showed the interest of the ethnopharmacological and chemotaxonomic approaches in the search for active substances against the microbial infections. The active tested compound may, either to be a model for the synthesis of more active and less toxic analogues or to be used in association with antimicrobial commercial drug, as they may reverse the resistance of microbial to antimicrobial drugs. However whether the new compound isolated should act as an effective therapeutic agent, the study of its mechanism of action would be necessary before application.
American type culture collection
Collection Institute Pasteur
Heteronuclear multiple bond correlation
High performance liquid chromatography
Heteronuclear single quantum coherence spectroscopy
Minimum bactericidal concentration
Minimum fungicidal concentration
Minimum inhibitory concentration
Nuclear magnetic resonance
Nuclear overhauser effect spectroscopy
Sabouraud dextrose agar
Thin layer chromatography
The authors wish to thank the International Foundation for Science (IFS) for financial support (Grant F/5673-1). The authors want also to thank Wood-Whelan fellowship from International Union of Biochemistry and Molecular Biology for support to go to the Laboratoire d’Innovation Thérapeutique in Strasbourg, France for the structural elucidation of isolated compound.
International Foundation for Science (IFS) for financial support (Grant F/5673-1).
Availability of data and materials
All data generated or analysed during this study are included in the manuscript and in additional file.
LL designed the study, followed the implementation, participated to isolate compounds, wrote the manuscript, AMA isolated compounds, carried out antimicrobial, antioxidant assay and participate to write the manuscript, MB and CVS carried out structural identification of isolated compounds and participated to write the manuscript, AS coordinate the team and helped to revise the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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