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ORIGINAL ARTICLE
Year : 2016  |  Volume : 15  |  Issue : 1  |  Page : 38-42

Inhibition of human pathogenic bacteria by Moringa oleifera cultivated in Jazan (Kingdom of Saudi Arabia) and study of synergy to amoxicillin


Department of Biology, Faculty of Science, King Khalid University, Abha, Saudi Arabia; Department of Microbiology, National Organization for Drug Control and Research (NODCAR), Cairo, Egypt

Date of Submission01-Oct-2015
Date of Acceptance15-Nov-2015
Date of Web Publication14-Jun-2016

Correspondence Address:
Mona Kilany
Department of Biology, Faculty of Science, King Khalid University, PO Box 9004, Abha, Saudi Arabia

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-4315.184029

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  Abstract 

Background The spread of multidrug-resistant strains of bacteria to most antibiotics has led to intensive searches for effective strategies to overcome bacterial infections.
Aim Moringa oleifera could be a potentially useful agent for many therapeutic applications, especially antimicrobial.
Settings and design The leaves of M. oleifera were collected in October 2014 from Jazan, located in the southern region of the Kingdom of Saudi Arabia.
Materials and methods Different organic solvents were used to extract antimicrobial substances in the plant leaves. The antibacterial activity of each leaf extract was determined in vitro and compared with some antibiotics. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of both plant extracts and amoxicillin were investigated against Bacillus spp. An evaluation of the synergistic effects of amoxicillin and plant extract was carried out.
Results The results showed that an ethanol extract is the best organic solvent for extracting the antimicrobial substance from M. oleifera. In addition, the antibacterial potential of an ethanol extract is comparable to that of some commercial antibiotics. MIC and MBC of the plant extract were 320 and 620 μg/ml, respectively, whereas MIC and MBC of amoxicillin were 25 and 50 μg/ml, respectively. The fractional inhibitory concentration of the plant extract and amoxicillin was determined to be 0.125 and 0.25, respectively. Therefore, ethanolic plant extract can be considered a good synergistic factor to amoxicillin, yielding a fractional inhibitory concentration index 0.375 ≤ 0.5 of the combination.
Conclusion M. oleifera leaves may serve as a natural alternative to antibiotics. Moreover, M. oleifera can boost the inhibitory effect of amoxicillin, leading to a reduction of administration dose as well as minimizing the side effects of antibiotics.

Keywords: antibacterial, antibiotic, ethanol, Moringa oleifera


How to cite this article:
Kilany M. Inhibition of human pathogenic bacteria by Moringa oleifera cultivated in Jazan (Kingdom of Saudi Arabia) and study of synergy to amoxicillin. Egypt Pharmaceut J 2016;15:38-42

How to cite this URL:
Kilany M. Inhibition of human pathogenic bacteria by Moringa oleifera cultivated in Jazan (Kingdom of Saudi Arabia) and study of synergy to amoxicillin. Egypt Pharmaceut J [serial online] 2016 [cited 2020 Apr 8];15:38-42. Available from: http://www.epj.eg.net/text.asp?2016/15/1/38/184029


  Introduction Top


Microbial infections are increasing in incidence rapidly worldwide; they are resulting in life-threatening diseases in developing countries [1]. Antimicrobial activities of many plants were investigated, which led to the use of these plants as a therapeutic alternative to antibiotics, particularly considering that is characterized as a natural, safer, and cheaper alternative [2],[3]. These antimicrobial agents of plant extracts were produced as secondary metabolites such as alkaloids, phenolic compounds, etc. Moringa oleifera is the most frequently cultivated species of a monogeneric family (Moringaceae) that has multiple pharmaceutical effects; therefore, it has been used as a remedy for various diseases, including bacterial and fungal infections, in the traditional medicinal system [4]. It is well known that the leaves of the plant have antimicrobial substances, which are usually found to vary depending on the type of solvent used for extraction. Notably, many antimicrobial substances of Moringa preparations have been reported such as 4-(4´-O-acetyl-a-l-rhamnopyranosyloxy)benzyl isothiocyanate, 4-(a-l-rhamnopyranosyloxy)benzyl isothiocyanate, niazimicin, pterygospermin, benzyl isothiocyanate, and 4-(a-l-rhamnopyranosyloxy)benzyl glucosinolate [5]. The results of monotherapy have improved typically with consecutive treatment for invasive infections; however, combination therapy could be a substitution for monotherapy for invasive infections that are difficult to due to multiresistant species. A combination of antimicrobial compounds might improve the effectiveness of each compound; therefore, a lower dose of each compound can be used. Moreover, some investigations have shown that antimicrobial plant extracts also promoted the effectiveness of some antibiotics. Bantar et al. [6] studied the combination of some antibiotics against carbapenem-resistant strains. Obviously, the combination of an aminoglycoside and β-lactams or a fluoroquinolone is still an optimal option for the treatment of invasive infections caused by Pseudomonas aeruginosa [7]. Palaniappan and Holley [8] studied the activity of a single natural antimicrobial substance such as carvacrol, thymol, eugenol, allylisothiocyanate, and cinnamaldehyde, as well as the activity when paired with an antibiotic using checkerboard and microdilution methods. In the former assay, the interactions between the inhibitors were characterized by calculating fractional inhibitory concentration (FIC) values. Consequently, the recent appearance of strains with reduced susceptibility to antibiotics and the recurrent spread of multidrug-resistant strains of bacteria have led to research of new infection-combat strategies [9],[10]. The current study was carried out specifically to investigate the role of different plant extracts of M. oleifera leaves cultivated in Jazan region (Kingdom of Saudi Arabia) against some human pathogenic bacteria. In addition, an attempt was made to investigate the synergy between amoxicillin as an example of a commercial antibiotic and plant extract.


  Materials and Methods Top


Collection of samples

The leaves of M. oleifera were collected in October 2014 from Jazan, located in the southern region of the Kingdom of Saudi Arabia. The fresh plant leaves were cleaned, washed, shade dried, and homogenized to a fine powder and stored in an airtight bottle.

Preparation of plant extracts

Twenty-five grams of the ground leaves were extracted separately in 500 ml conical flasks with 50 ml of methanol, ethanol, or n-hexane. The flasks were shaken at 120 rpm overnight. The extracts were filtered separately using a sterile Whatman no. 1 filter paper. The resulting filtrates were left to air dry and the residues were dissolved in 5% dimethyl sulfoxide to maintain the concentration at 1 g/ml [11]. The plant extracts were sterilized using a syringe filter (0.2 µm pore size).

Test organisms and antibiotics

Pure overnight-grown culture of bacteria was used for the evaluation of the antimicrobial potential of the leaf extracts including both Gram-negative (Proteus mirabilis, P. aeruginosa, Klebsiella spp., Shigella spp.) and Gram-positive bacteria (Bacillus spp. and Staphylococcus aureus). A standard inoculum of bacterial suspensions was performed to yield optical density 0.5 at 600 nm using a Lambda 25 UV/VIS Spectrometer. All test bacteria were maintained on nutrient agar slants at 4°C. Three antibiotics were used in this study: chloramphenicol (30 µg), streptomycin (10 µg), penicillin (10 µg), and amoxicillin.

Antimicrobial activity of different plant extracts

The well-diffusion method was used to evaluate the antimicrobial activity of M. oleifera leaf extracts[12]. Nutrient agar plates were inoculated with different strains of bacteria separately using sterile swaps. Six millimeter wells were created on the agar surface, onto which the extracts were placed. Afterwards, the plates were incubated at 37°C for 24 h. Antibacterial activity was determined by measurement of the inhibition zone.

Comparison of the efficacy of different plant extracts and antibiotics

Antimicrobial activities of the ethanolic, methanolic, and n-hexane extracts were assayed using the disc diffusion method [3]. Sterile discs (6 mm) were impregnated with each extract separately. The discs were carefully and firmly placed onto the nutrient agar plates seeded previously with standardized bacterial suspensions (optical density 0.5 at 600 nm). Paper discs of chloramphenicol (30 µg), streptomycin (10 µg), and penicillin (10 µg) were used for comparison. Filter paper discs dipped into sterile 5% dimethyl sulfoxide and allowed to dry were used as a negative control. The plates were then incubated at 37°C for 24 h. Antibacterial activity was determined by measurement of the zone of inhibition.

Determination of minimal inhibitory concentration of ethanol extract

Minimal inhibitory concentration (MIC) of ethanol extract was determined using the two-fold serial dilution method [13]. The dose levels of 20 mg/ml of plant extract were serially diluted in nutrient broth media separately to obtain 0.04, 0.08, 0.16, 0.32, 0.65, 1.25, 2.5, 5, and 10 mg/ml. Each 0.1 ml standardized suspension of Bacillus spp. was inoculated into the corresponding tubes. Nutrient broth inoculated with bacteria was used as a positive control and nutrient broth containing the plant extract was used as a negative control. MIC was measured as the lowest concentration of the samples with no visible growth.

Determination of minimal bactericidal concentration of ethanol extract

Minimal bactericidal concentration (MBC) was detected by preparing different concentrations of plant extract including MIC and three lower concentrations using nutrient agar medium on which aliquots of Bacillus spp. were inoculated and then incubated for 24 h at 37°C. The plates were examined for the growth of the challenge microorganism [3],[14].

Determination of minimal inhibitory concentration and minimal bactericidal concentration of amoxicillin

As described above, the two-fold serial dilution method was adopted [13] using different concentrations of antibiotics (0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 µg/ml). The lowest concentration of the samples with no visible growth was considered the MIC of amoxicillin.

Evaluation of synergistic effect of plant extract to amoxicillin

The synergy of the combination of plant extract and amoxicillin (in triplicate) against Bacillus spp. was investigated according to the checkerboard method [15]. The concentration gradient of both the plant extract and amoxicillin in combination ranged from 1/32 times the MIC (1/32´MIC) to 4´MIC, inoculated with the initial inoculum of Bacillus spp. (1.5 × 106 cfu/ml) and incubated at 37°C for 24 h. The concentration of the combination at which the growth was completely inhibited was considered the effective MIC for the combination. To evaluate the effect of the combinations, the FIC was calculated for each antibiotic in each combination. The following formulas were used to calculate the FIC index [16]:



FIC index = FIC of amoxicillin + FIC of plant extract.

Synergy was defined as an FIC index of less than or equal to 0.5.


  Results Top


Antimicrobial activity of different plant extracts

The results of the antimicrobial screening of different plant extracts of M. oleifera are shown in [Figure 1], which show that ethanol extract exerted the highest inhibitory effect toward all test bacteria (11–30 mm inhibition zone). Particularly, Bacillus spp., as a model of Gram-positive bacteria, is more sensitive than Gram-negative bacteria as shown in [Figure 2].
Figure 1: Antimicrobial activity of different plant extracts.

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Figure 2: Antimicrobial activity of ethanol extract.

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Comparison of the efficacy of ethanol extract and antibiotics

The results indicated that the antimicrobial activity of the plant extracts were comparable to that of antibiotics; most of the test organisms developed resistance to penicillin (10 µg), but they were all susceptible to chloramphenicol and streptomycin in various degrees ([Table 1]), whereas all of them were sensitive to the leaf extracts, except Proteus mirabilis, which was found to be resistant to hexane extract.
Table 1: Antimicrobial activity of ethanol extracts and antibiotics

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Determination of minimal inhibitory concentration and minimal bactericidal concentration of ethanol extract

The results indicated that MIC and MBC of Bacillus subtilis were found to be at a concentration 0.32 and 0.62 mg/ml, respectively.

Determination of minimal inhibitory concentration and minimal bactericidal concentration of amoxicillin

The MIC and MBC of amoxicillin against B.subtilis were determined to be 25 and 50 µg/ml, respectively.

Evaluation of the synergistic effect of amoxicillin and plant extract

The results also showed that a combination of the plant extract with amoxicillin showed a promising synergistic effect against B. subtilis; the FIC of the plant extract and amoxicillin was 0.125 and 0.25, respectively. Consequently, the FIC index of the combination was 0.375≤0.5, meaning that the plant extract is good synergistic to amoxicillin.


  Discussion Top


Different solvents have different extraction capacities and different spectra of solubility for the phytoconstituents [17]. In the current work, it was shown that an ethanol extract of M. oleifera leaves had a broad spectrum of antimicrobial activity against some human pathogenic Gram-positive bacteria more than Gram-negative bacteria at 1 g/ml to different degrees. These results are in agreement with other findings [3],18],[19],[20]. The differences in sensitivity between Gram-positive and Gram-negative bacteria may be attributed to their morphological variations; an outer phospholipidic membrane of Gram-negative bacteria carrying the structural lipopolysaccharide components led to cell wall impermeability to lipophilic solutes, whereas porinsporins represent a selective barrier to the hydrophilic solutes with an exclusion limit of about 600 Da.

However, Gram-positive bacteria are more susceptible because of the presence of an outer peptidoglycan layer, which is not an efficient permeability barrier [21]. The antimicrobial activity of the ethanol plant extract is less than that of antibiotics; this may be because antibiotics are in a purified form, whereas the plant extracts are still in a crude state. Therefore, the antimicrobial activity of the plant extract might be higher after purification. This result is in agreement with Doughari et al. [3]. However, it has been found in previous studies that the plant has an effect comparable to that of some antibiotics and is more active than the others, where M. oleifera leaf extract showed higher antimicrobial activity than that of tetracycline [22],[23]. MIC and MBC are used as the most common tools for the assessment of antimicrobial action [24]. Moreover, MIC is the lowest antibiotic concentration that inhibits growth, but does not kill the organisms. Meanwhile, a higher antibiotic concentration kills the organisms and this is the MBC. Hence, we can determine the extent of success of antibiotic in treatment. Moreover, bacteriostatic antibiotics inhibit the bacterial metabolism or ribosomal protein synthesis, whereas bactericidal antibiotics inhibit cell wall formation. The present study showed that MIC and MBC of the plant extract were higher than those of antibiotics, suggesting that the plant extract acts as a bacteriostatic at lower concentrations and as a bactericidal at higher concentrations. These findings are just in line with the observations of Aliyu et al. [25] and Rahman et al. [26]. The precise prediction of synergy between the synthetic antibiotic and a natural product relied on the results of in-vitro testing is very crucial. In the current study, the plant extract showed a synergistic effect to amoxicillin against B. subtilis. This synergistic action provides scientific bases for the use of concoctions traditionally in the treatment of diseases. This result is in agreement with Tahany et al. [27], who assessed a synergistic combination of biologically active components from wild Moringa peregrine against different pathogens, indicating the high efficacy of combination therapy over monotherapy. The FIC index of the combination of plant extract and amoxicillin is 0.375≤0.5, indicating that a plant extract is a good synergistic agent to amoxicillin. In addition, Hariharan [28] investigated the synergistic action of a combination of M. oleifera with three plant extracts, namely, Terminalia arjuna, Azadira chtaindica, and Curcuma longa against B. subtilis, reporting an FIC index of 0.291. In conclusion, M. oleifera could be a potent and natural antibiotic for human consumption.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]


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