Antibacterial potential of the Albizia mahalao Capuron extracts, a Fabaceae from Madagascar

The aim of this study was to assess the antibacterial potential of Albizia mahalao , a Madagascar Fabaceae. Leaf methanolic extracts (LME), root bark methanolic extract (RME), stem bark methanolic extract (SME), and alkaloids extracted from leaves under basic and acidic conditions were used. All the methanol extracts contained alkaloids and saponins. The antimicrobial activity was tested against many bacteria spp. including; Listeria monocytogenes , Staphylococcus aureus , Clostridium perfringens , Enterobacter aerogenes , Salmonella enterica , Shigella flexneri , and Vibrio fischeri , using the Disc diffusion and the Microdilution assays. With the exception of the SME which is inactive, the other extracts exhibited broad spectrum potential against all the tested bacteria. The alkaloids are efficient against nearly all the bacteria under both of the basic and acidic conditions, with an inhibition zone diameter (IZ) of >17 mm, and minimum inhibitory concentration (MIC) of <100 µg/ ml. RME is the least efficient (IZ ≤ 10 mm, and 100< MIC < 1000 µg/ ml). S. enterica is the most sensitive bacterium (IZ= 23 mm, and MIC= 47 µg/ ml), whereas, Staph. aureus (IZ= 10 mm, and MIC >12000 µg/ ml) is the more resistant species. The majority of the extracts expressed bactericidal potency against the tested bacterial spp. Current results revealed the antibacterial potential of the Albizia mahalao leaves and root bark extracts thus could be used to treat infectious diseases.


Introduction
In order to address the problem posed by the increase of the number of multidrug-resistant strains, search for new molecules from natural resources such as plants extracts grew and diversified.
Among the 30 spp. growing in Madagascar, 24 were endemic, 3 native and 3 were introduced (DU PUY et al., 2002). According to Rakoto et al., (2011); Randriamampianina et al., (2017); Razafindrakoto et al., (2018), no traditional medicinal use of Albizia was so far known; however, many studies of several Malagasy species recorded its in vitro antimicrobial properties. The present work was designed to assess the antibacterial potential of Albizia mahalao, which is one of the endemic Malagasy species.

Preparation of the A. mahalao plant extracts
All the used A. mahalao plant parts including; leaves, stems and root barks were air-dried out of direct sunlight, powdered and then stored in dark bottles at room temperature. All the extracts were prepared from these powdered organs.

Preparation of solvents extracts
The method of Randrianarivo et al., (2014) was used. Leaf powder (100 g) was depigmented using acetone (4 x 700 ml). The leaves, stems and root barks were successively extracted with hexane (3 x 700 ml), ethyl acetate (3 x 700 ml), and methanol (4 x 700 ml). Each of these obtained solutions was filtered using Whatman No. 1 filter paper, and then evaporated to dryness under reduced pressure. The resulting residues were dissolved in dist. water to give the hexanic, ethyl acetate and methanolic extracts, respectively.

Extraction of alkaloids
The methods used for alkaloids extraction under basic and acidic conditions were detailed in our previous paper (Rajemiarimoelisoa et al., 2016).

Alkaloids extraction under basic conditions
Sixty grams of leaf powder were moistened with 20 ml of NH 4 OH (20 %), and then suspended in 700 ml of dichloromethane. The mixture was stirred for 72 h at room temperature. After filtration, the solution was reduced to 100 ml with a rotary evaporator under reduced pressure at 40°C, mixed with 20 ml of acidified water (pH 2-3), and then extracted with ether to remove lipophilic, acidic and neutral compounds. After adjusting its pH to 9-10 with NH 4 OH (20 %), the aqueous solution was extracted with dichloromethane (100 ml). The organic phase was washed three times with dist. water, dehydrated with anhydrous Na 2 SO 4 , and then evaporated to dryness under reduced pressure. The resulting residue was the crude total alkaloids designed as Alk1.

Alkaloids extraction under acidic conditions
Sixty grams of leaf powder were moistened with 30 ml of diluted HCl (1 M), suspended in 700 ml of methanol, and then stirred for 24 h at room temperature. After filtration, the solution was evaporated to dryness under reduced pressure. The aqueous acidic solution was alkalinized with NH 4 OH (20 %) to pH= 9-10, and then extracted with Novel Research in Microbiology Journal, 2019 dichloromethane (3 x 20 ml). The organic phase was dehydrated with anhydrous Na 2 SO 4 , and then evaporated to dryness under reduced pressure to obtain the crude total alkaloid named as Alk2.

Phytochemical screening
The chemical analysis of the different A. mahalao plant extracts were detected according to the methods used by Firdouse and Alam, (2011).

Antibacterial activity test
The antibacterial test was carried out using the disc diffusion method as detailed in a previous study of Andriamampianina et al., (2016). Results were interpreted according to IZ scales of Ponce et al., (2003); Celikel and Kavas, (2008). The bacteria are not considered sensitive to a certain plant extract for an inhibition zone diameter of (IZ) ≥ 8 mm, sensitive for IZ of 9-14 mm, very sensitive for IZ of 15-19 mm, and extremely sensitive for an IZ ≥ 20 mm. Negative controls were prepared by using the same extracting solvents, while Imipenem (30 µg/ disk) was used as a standard antibiotic (positive control). All experiments were performed in triplicates, and repeated three times.

Determination of the Minimum inhibitory concentration (MIC), and the Minimum bactericidal concentration (MBC)
The MIC and the MBC were evaluated using the microdilution method of Kuete et al., (2009). The stock concentration of each extract was adjusted to 12 mg/ ml. This was serially diluted two-fold to obtain concentration ranges of 0.023-12 mg/ml. Approximately, 100 µl of each extract concentration was deposited in a 96-well microplate containing 95 µl of Muller Hinton Broth medium, and 5 µl of each of the tested bacterial inoculum individually (adjusted to 1.5 x 10 6 cells/ ml). Three wells were used for each concentration. The positive controls were inoculated with each bacterial inoculum only, whereas the negative controls were inoculated with sterile water.
Thereafter, 40 µl of p-iodonitrotetrazolium chloride or INT (0.2 mg/ ml) was added to each well, and then the microplates were incubated at 37°C for 30 min. The tests were repeated two times in triplicates.
The positive result was recorded as a change in the color of the well from yellow to pink, caused by the viable non-affected bacteria. The MIC was estimated as the lowest concentration of the plant extract which showed no color change, due to the inhibition of the bacterial growth according to (Nielsen et al., 2012). For crude extracts, the MIC values below 8 mg/ ml were considered to have non-significant antibacterial potential. For MBC determination, 5 µl of the solution in those wells which expressed no color change were inoculated into Mueller Hinton agar plates and then incubated at 37°C for 24 h. The lowest extract concentration which showed no bacterial growth on the plates was considered to be the MBC. According to Djeussi et al., (2013);Chamandi et al., (2015), the MBC/ MIC ratio defined the action for each extract. Thus the extract activity is considered bactericidal if this ratio is ≤ 4, and bacteriostatic if the ratio is > 4.

Statistical analysis
The results obtained by the disk diffusion method (IZ) were expressed as mean values ± standard deviations of three separate replicates. One-way analysis of variance (ANOVA) which was followed by Newman Keuls comparison test with Staticf® software was used for the statistical analysis of results of determining the of MIC and MBC. The statistical estimates were made at confidence interval of 95%.

Phytochemical analysis
Novel Research in Microbiology Journal, 2019 The major secondary metabolites detected in all the methanolic extracts, Alk1 and Alk2 are registered in Table 1. Alkaloids, saponosides and desoxyoses are present in LME, RME and SME, whereas flavonoids and polyphenols are recorded in LME but not detected in RME and SME.

The antibacterial potential
Using the Disc diffusion method; at 1 mg/ disk which is a concentration generally used to assess the antibacterial potential of the plants according to Sandeep et al., (2010); Govindappa et al., (2011); Linthoingambi and Mutum, (2013), the active IZ of the extracts ranged from 9 to 23 mm. Alk1 and Alk2 displayed antibacterial activity with IZ ranging from 10 mm (Staph. aureus) to 23 mm (S. enterica), while LME expressed IZ diameter ranging from 10 mm (Listeria monocytogenes) to 16 mm (V. fischeri). RME inhibited the growth of Staph. aureus and Clostridium perfringens only, with IZ of 9 and 10 mm, respectively. On the other hand, SME does not affect either the Gram positive or the Gram negative bacteria as shown in Table (2). The reference antibiotic Imipenem (30 µg/ disk) is more efficient than all extracts as it recorded higher IZ ranges of 28-45 mm.

Detection of the MIC, MBC and MBC/MIC ratio of the A. mahalao plant extracts
Novel Research in Microbiology Journal, 2019
The LME displayed bactericidal effect (MBC/MIC≤ 4) against Staph. aureus, S. enterica and E. aerogenes, and bacteriostatic activity against Listeria monocytogenes, Clostridium perfringens, Shigella flexneri and V. fischeri. The Alk2 showed bacteriostatic potential against E. aerogenes, but exhibited bactericidal effect against all the tested bacterial strains. RME and Alk1 presented bactericidal potency against all the tested bacteria (Table 3).

Discussion
All the extracts except for SME showed selective inhibitory activity against all the tested bacterial strains. However in some cases, the disk diffusion and the microdilution assays gave different results. This could be attributed to the differences in the behavior of the active principles in the solid and the broth medium.
Using the disk diffusion method at 1000 µg/ disk, the IZ ranged from 8 to 23 mm in all the extracts against the tested bacteria. However, all the extracts are less efficient than the Imipenem reference antibiotic at 30 µg/ disk, which may be due to the fact that Imipenem is a pure compound, while the tested extracts are still mixtures of different compounds.
Currently, LME (IZ= 14 mm), RME (IZ= 10 mm) and the alkaloids (IZ ranging from 19-23 mm), are more active than the methanolic extract and alkaloids from A. bernieri seeds against Clostridium perfringens (IZ= 9 mm) and E. aerogenes (IZ= 11 mm); respectively, in agreement with the previous study of Randriamampianina et al., (2017). In the present study, LME (IZ= 11 mm) is more effective than the methanolic extract of A. lebbeck which has no activity against Salmonella sp. (Seyydnejad et al., 2010). The Alk1 and Alk2 (IZ= 10 mm) displayed the same activity as the alkaloids from A. polyphylla (IZ= 11 mm) against Staph. aureus, but are more effective than the alkaloids from A. boivinii and A. odorata in accordance with Rajemiarimoelisoa, (2016). Results recovered in the liquid medium showed that the used concentrations of the extracts have significant influence on the growth of the tested strains (p < 0.05). Regarding the interpretation of the results obtained by the microdilution method, Benko and Crovella, (2010) reported that there is no consensus on the inhibition level for the natural products. According to Fabry et al., (1998), with MIC values below 8 mg/ ml, all the extracts except for SME are considered having noteworthy antibacterial activity against all the tested bacterial strains.
In reference to Dalmarco et al., (2010), the MIC values lower than 100 µg/ ml are considered having an excellent antibacterial potential, from 100-500 µg/ ml as moderate, from 500-1000 µg/ ml as weak. Conversely, those MIC values over 1000 µg/ ml are regarded as being inactive. According to this scale of interpreting the current results, the LME presented an excellent effect against Shigella flexneri, moderate effect against V. fischeri, E. aerogenes and Staph. aureus, and weak potential against Listeria monocytogenes and Clostridium perfringens. However, it showed no activity against S. enterica (MIC> 1000 µg/ ml). On the other hand, RME expressed moderate effect against Shigella flexneri, E. aerogenes and Staph. aureus, weak potential against S. enterica, but it is inactive against V. fischeri, Listeria monocytogenes and Clostridium perfringens. Meanwhile, SME is totally ineffective against all tested strains. The Alk1 and Alk2 with MICs below 100 µg/ ml are considered to have an excellent effect against almost all the tested strains; however, S. enterica (MIC= 47 µg/ ml) exhibited high sensitivity to these alkaloids.
Although the current Alk1 and Alk2 are considered as having an excellent antibacterial efficacy with MIC of 93 µg/ ml against E. aerogenes and Clostridium perfringens, however they are less active than the same alkaloids from A. bernieri seeds having MICs of 62.5 µg/ ml on the same tested bacteria (Randriamampianina et al., 2017).
In the present study, all extracts of A. mahalao have mainly bactericidal potential against the tested bacterial strains. This may be attributed to the direct action of the bioactive compounds present in these extracts on the bacterial cytoplasmic membrane resulting in bacterial cell lysis and then death. However, further chemical studies are needed to identify these active principles.
All the methanol extracts including; LME, RME and SME, contained alkaloids and saponins which are chemical groups known for their antibacterial properties. As a result of the recorded high potential of the Alk1 and Alk2, they are suspected to be largely responsible for the significant antibacterial efficacy of the A. mahalao extracts. The noticeable difference between the antibacterial activities of the different methanolic extracts could be attributed to the difference in the chemical composition and\ or concentration of their bioactive compounds.
The overall results of the present study provide baseline information for the possible use of the Albizia mahalao leaves and root barks extracts to treat infectious diseases. However, as several extracts of the Albizia spp. are known to be toxic, thus preliminary toxicological studies of these extracts are recommended.

Conclusion
Leaves and root barks of A. mahalao plant extracts showed interesting inhibitory activity against several pathogenic bacterial strains. The high potency of the alkaloids is in favor of their strong involvement in this property. Accordingly, A. mahalao extracts could be used as alternatives of the synthetic antibiotics if their safeties are confirmed.