Antimicrobial activity of the extracts of Albizia masikororum R.Vig., a Fabaceae from Madagascar

This study is aimed at the assessment of antimicrobial potential of Albizia masikororum, a Malagasy Fabaceae. Hexanic and methanolic extracts from fruit pods, stem bark, leaves and seeds were tested by disc diffusion and microdilution methods on 10 pathogenic microorganisms including four Gram positive bacteria, five Gram negative bacteria and one yeast. Only the leaf and seed methanolic extracts, LME and SME respectively, were active on some bacteria. LME and SME had a broad-spectrum activity, with SME more effective. The minimum inhibitory concentration (MIC) values of both extracts were <1000 μg/ml of which 30% <100 μg/ml, 20% between 100 and 500 μg/ml and 50% between 500 and 1000 μg/ml. SME MICs ranked from 6.10 μg/ml (Streptococcus pneumoniae) to 781.25 μg/ml (Yersinia enterocolitica) and those of LME from 97.65 μg/ml (Streptococcus pneumoniae) to 781.25 μg/ml (Bacillus cereus, Streptococcus pneumoniae and Yersinia enterocolitica). LME was bactericidal on all sensitive bacteria whereas SME was bactericidal on some and bacteriostatic on others. Both extracts contained different chemical groups known for their antimicrobial properties, saponins in SME and phenolic compounds in LME.


INTRODUCTION
Globally, the number of multidrug-resistant strains has increased dramatically over the past 10 years (Roca et al., 2015;Sharifi-Rad, 2016) and new more resistant strains may be expected to emerge in the coming years (McGaw et al., 2008).
This situation may exhaust the chemical defenses at our disposal.Therefore, research programs in alternative therapies including the search for new antimicrobial agents should be encouraged.Natural products still play a major role in the search for new medicines and plants represent a good source for new molecules of interest (Kuete et al., 2009;Sharifi-Rad et al., 2017a, b, c, d, e; *Corresponding author.E-mail: victor_jeannoda@yahoo.fr. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Source: The authors and Silo National des Graines Forestières (SNGF).Salehi et al., 2017).Malagasy plants are good candidates in view of the diversity of Malagasy flora whose originality could promise original molecules.
The genus Albizia is a group of plants that includes about 150 species and occurs in tropical regions.Worldwide, it has many uses including control of pests such as pathogenic microorganisms (Agyare et al., 2006;Sarkiyiayi et al., 2011;Odeyemi et al., 2014).The Malagasy species studied also showed in vitro antimicrobial properties (Randriamampianina et al., 2017).In this work, the antimicrobial potential of the endemic species A. masikororum, which has already shown appreciable biological properties on mice and chicken (Randrianarivo et al., 2014) was studied.

Plant materials
A. masikororum is a large shrub or tree up to 25 m tall (Figure 1) growing throughout the western part of Madagascar, from the North to the South.It was collected in Belo-sur-Tsiribihina/Betioky (West of Madagascar).Voucher specimens of the plant were deposited in the herbarium of Silo National des Graines Forestières (SNGF) under the reference number SNGF 873.

Microorganism strains
The 10 microorganisms used in this study included 4 Gram (+) bacteria, 5 Gram (-) bacteria and 1 yeast (Table 1).They were maintained on agar slants at 4°C and cultured on a fresh appropriate agar plates 24 h prior to any antimicrobial test.

Preparation of hexane and methanolic extracts
Ground seeds, leaves, pods and stem bark powder (50 g) were delipidated with hexane (3 X 250 ml), then extracted with methanol (3 X 250 ml).After filtration using a Whatman filter paper, extracts were evaporated to dryness under reduced pressure.

Phytochemical screening
The reactions of chemical group detection were those developed by Marini-Bettolo et al. (1981).

Antimicrobial assays
All the materials and methods used for antimicrobial assay were detailed in a previous study (Randriamampianina et al., 2017).
The results were interpreted using the scale of Ponce et al. (2003) and Celikel et al. (2008).With the disc diffusion method, bacteria are not considered sensitive for an inhibition zone diameter (IZD) less than 8 mm, sensitive for IZD of 9 to 14 mm, very  For each extract, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined on susceptible strains only.The standards used to interpret MIC results were those of Dalmarco et al. (2010).For crude extracts and fractions, a MIC lower than 100 µg/mL was considered as an excellent effect, from 100 to 500 µg/ml as moderate, from 500 to 1000 µg/mL as weak, and over 1000 µg/ml as inactive.

Extraction yields
The different crude extracts were obtained with yields ranging from 0.95% for BHE to 19.01% for SME (Table 2).

Disc diffusion
The effects of the various extracts of seeds, pods, bark and leaves at a concentration of 1 mg/disc on the microorganisms are presented in Table 4.
LME and SME only displayed activity on some bacteria.Their IZD values ranked from 11.5 to 16.5 mm for LME and from 9 to 15 mm for SME.They were less effective than neomycin which is a pure product.None of the extracts was active against Pseudomonas aeruginosa and Candida albicans.

MIC and MBC values
MIC and MBC values were determined for LME and SME only for active extracts on sensitive bacteria by the disc diffusion method (Table 5).
LME exerted a bactericidal effect against all bacteria  sensitive to it whereas SME had a bactericidal effect on 4 bacteria and bacteriostatic effect on 3 others.

DISCUSSION
Of all the 8 extracts tested, only methanolic extracts from leaves (LME) and seeds (SME) displayed antimicrobial activity.
The antibacterial compounds in the two extracts did not belong to the same chemical groups: Phenolic compounds for LME and saponins for SME.As these extracts were only semi-purified, the study results did not yet allow the conclusion on the number of active compounds in each one of them.LME was active against 4 and SME against 7 of the 10 strains tested but unlike other Malagasy Albizia species so far studied (Rakoto et al., 2011;Randriamampianina et al., 2017), both extracts were not active against Candida albicans.Generally, the strain sensitivity varied significantly with the extract.For example, Clostridium perfringens and Enterobacter cloacae were sensitive to SME but resistant to LME whereas Bacillus cereus was sensitive to LME but not to SME.This difference could lie in the fact that the two extracts contained very different chemical groups.SME was rich in saponins and LME in phenolic compounds.Both chemical groups are known for their antimicrobial properties.
LME and SME displayed an excellent effect against Streptococcus pneumoniae and Streptococcus pyogenes.Those two bacteria, because of their high prevalence in infectious diseases and their resistance to various antibiotics, have become a serious clinical and health problem in many countries (Nuermberger and Bishai, 2004;Ardanuy et al., 2014;Lu et al., 2017;Sekizuka et al., 2017;Zhao et al., 2017).
In comparison with antimicrobial activity of other malagasy Albizia assessed in similar conditions (Randriamampianina et al., 2017), LME and SME gave MIC values much lower than 1000 µg/ml, and therefore far more effective compared to the seed extracts from A. aurisparsa against B. cereus (MIC = 1980 µg/ml) and from A. mahalao (MIC = 3750 µg/ml) and A. polyphylla (MIC = 2420 µg/ml) against Staphylococcus aureus.However, their activities were more or less similar to those of A. bernieri (Randriamampianina et al., 2017) against the same sensitive bacteria.
There is no consensus on the standard scale for interpreting antimicrobial activity of natural products (Benko and Crovella, 2010).According to the scale of Dalmarco et al. (2010), used in this work, 2 bacteria were very sensitive, 3 moderately sensitive and 4 weakly sensitive.With standards used by other authors, plant extracts with MICs values higher than 500 µg/ml (Aligianis et al., 2001) and even much higher than 1000 µg/ml (Maregesi et al., 2008;Abubakar and Majinda, 2016;Touaibia and Chaouch, 2017) were classified as having strong antimicrobial activity.For comparison, LME and SME had excellent activates on all the bacteria tested.
Based on the results obtained with the strains tested, LME and SME have a relatively broad activity spectrum.The effect of SME on sensitive bacteria was bactericidal whereas SME exerted a bactericidal effect on some strains and bacteriostatic effect on others.
In conclusion, Albizia masikororum exerted antibacterial effect.LME and SME could constitute good alternatives to synthetic antibiotics, particularly against Streptococcus.Further studies should be undertaken to determine the number and the identities of active principles responsible of this property and to know whether or not they are new products.

Table 1 .
List of microorganisms used.

Table 2 .
Extraction yields of the different crude extracts.
sensitive for IZD of 15 to19 mm and extremely sensitive for IZD larger than 20 mm.

Table 3 .
Phytochemical screening of seed powder and organ methanolic extracts.

Table 4 .
Effects (IZD in mm) of A. masikororum extracts by disc diffusion method at a concentration of 1 mg/disc.