Antibacterial effects of the methanolic extract of Glycine Max ( Soybean )

High occurrence of antibiotic resistance in pathogenic bacteria is caused by the use of natural medicinal plants to eliminate risk of infectious diseases. Glycine max is on the most popular and nutritious foods with high antimicrobial effects. The present research was done to study the antibacterial effects of the methanolic extract of G. max against Gram-negative and positive bacteria. G. max seeds of M7 and M9 varieties were purchased and their methanolic extracts were collected. Diameter of the zone of growth inhibition of extracts were measured for pathogenic bacteria using the CLSI guidelines. Antimicrobial effects of G. max extracts were also compared with several antibiotics. Diameter of the inhibition zone of tested bacteria against different treatments of G. max had a ranges of 5.93±0.18 to 22.61±1.80 mm. M7 variety had a higher antimicrobial effects on tested bacteria (P<0.05). The highest diameter of the zone of growth inhibition was seen for L. monocytogenes (22.61±1.80 mm) and S. aureus (19.33±1.56 mm). Antimicrobial effects of the methanolic extract of the G. max was also dose-dependent (P<0.05). Diameter of the inhibition zone of tested bacteria against different antibiotic agents had a ranges of 5.94±0.38 to 24.95±2.11 mm. Ciprofloxacin, penicillin G tetracycline, gentamicin and azithromycin antibiotic agents exhibited the highest diameter of zone of the growth inhibition for K. pneumoniae (15.71±1.46 mm), L. monocytogenes (23.94±2.03 mm), L. monocytogenes (20.55±1.95 mm), S. aureus (14.27±1.36 mm) and L. monocytogenes (24.95±2.11 mm), respectively. Formulation of antibiotic agent using the 100 mg/mL concentration of the M7 variety of G. max has been recommended for treatment of the cases of infectious diseases. Introduction In spite of the considerable progress of medicine, treatment of infectious diseases faced with different unfavorable problems. Occurrence of severe levels of the antibiotic resistance is the main issue facing medical practitioners.1,2 Antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria. It is an increasingly serious threat with global public health impact that requires action across all government sectors and society.3,4 Resistant bacteria caused more severe clinical diseases for longer period of time which causes abundant economic losses.1-4 Documented data revealed that pathogenic bacteria and especially Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Bacillus cereus (B. cereus), Pseudomonas aeruginosa (P. aeruginosa), Listeria monocytogenes (L. monocytogenes), Klebsiella pneumoniae (K. pneumoniae) and Salmonella typhi (S. typhi) exhibited the high levels of resistance against aminoglycosides, tetracyclines, lincosamides, macrolides, beta-lactams, quinolones, fluoroquinolone and cephems groups of antibiotics.1-8 Therefore, therapeutic and pharmacological factories tried to use from novel sources for antimicrobial agents to produce strong antibiotic drugs. Application of medicinal plants for producing of antimicrobial agents had an ancient history.9 Soybean (Glycine max (G. max)) belongs to a large botanical family Leguminosae which classically grows in tropical, subtropical and temperate climatic regions like Iran.10 Soybean is an oilseed and consists of 20% oil content. It is considered as the most significant crop for the production of edible oil. Soybean plant holds great importance in today’s world mainly because of its high protein and lipid content and other major constituents including vitamins, minerals, fatty acids and other essential nutritional factors.10-12 Besides. soybean holds much importance from medicinal perspective. Recorded data revealed the high antimicrobial and antioxidant content of the G. max.10-12 Soybean seeds are rich in proteins, isoflavones and phytoestrogens, while Genistein, a soy isoflavone, has also been reported to possess anti-cancerous, antioxidant, anti-inflammatory and anti-osteoporosis effects and is considered as potential compound for metabolic disorders’ treatment.10-12 According to the uncertain antimicrobial effects of the G. max, the present investigation was done to study the antibacterial effects of the methanolic extract of the M7 and M9 varieties of the G. max on Gramnegative and positive bacteria. Materials and Methods


Introduction
In spite of the considerable progress of medicine, treatment of infectious diseases faced with different unfavorable problems.Occurrence of severe levels of the antibiotic resistance is the main issue facing medical practitioners. 1,2Antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria.It is an increasingly serious threat with global public health impact that requires action across all government sectors and society. 3,42][3][4] Documented data revealed that pathogenic bacteria and especially Staphylococcus aureus (S. aureus), Escherichia coli (E.coli), Bacillus cereus (B.cereus), Pseudomonas aeruginosa (P.aeruginosa), Listeria monocytogenes (L.monocytogenes), Klebsiella pneumoniae (K.2][3][4][5][6][7][8] Therefore, therapeutic and pharmacological factories tried to use from novel sources for antimicrobial agents to produce strong antibiotic drugs.Application of medicinal plants for producing of antimicrobial agents had an ancient history. 9Soybean (Glycine max (G.max)) belongs to a large botanical family Leguminosae which classically grows in tropical, subtropical and temperate climatic regions like Iran. 10 Soybean is an oilseed and consists of 20% oil content.It is considered as the most significant crop for the production of edible oil.1][12] Besides.soybean holds much importance from medicinal perspective.][12] According to the uncertain antimicrobial effects of the G. max, the present investigation was done to study the antibacterial effects of the methanolic extract of the M7 and M9 varieties of the G. max on Gramnegative and positive bacteria.

Ethical issues
The

Samples
Two different seed varieties of G. max mutant of Clark (M7 and M9) were obtained from the National Agriculture Research Centre of Isfahan province, Iran.Samples were collected in sterile polyethylene bags.These two varieties of G. max were tested for their antibacterial activity.The fresh seeds were dried under shade.Weighed around 10 gram of soybeans seed and washed air, dried at room temperature and coarsely powdered in a mixer sieved.Dried seeds were disinfected with 15% H2O2 and stored at room temperature in sterile sealed bottled until its extraction.

Methanolic extract
For the preparation of methanol extracts, 50 g of each seed sample was dissolved in 500 mL of methanol solvents.After dissolution, each sample was placed in shaking incubator at moderate temperature of 37°C±2 for 7 days.After 7 days, the samples were taken out of shaker and filtered initially with muslin cloth and then with Whatman filter paper #01 (11 μm) so that transparent solution was obtained.The filtrates obtained were then placed in water bath at moderate temperature of 35°C±2 for 6-7 h daily until dried extracts were obtained.

Microorganisms used and growth conditions
Strains of bacteria selected to assess susceptibility pattern were Bacillus cereus (ATCC 10987), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 6538), Listeria monocytogenes (ATCC 19111), Klebsiella pneumoniae (ATCC 43816) and Salmonella typhi (ATCC 14028).Bacterial cultures were purchased from the Pasteur Institute of Iran.Each microorganism was reactivated prior to susceptibility testing by transferring them into a separate test tubes containing nutrient broth (NB, Merck, Germany) and incubated overnight at 37°C at shaker.

Antibacterial effects of G. max
Guidelines of the Clinical and Laboratory Standard Institute (CLSI) were used to study the antibacterial effects of G. max M7 and M9 methanolic extracts. 13imple disk diffusion method was used to study the diameter of zone of inhibition of several types of bacteria subjected to 25 mg/mL, 50 mg/mL and 100 mg/mL concentrations of G. max M7 and M9 methanolic extracts.Paper discs (Whatman No 6 mm diameter) were separately impregnated with aliquots of 10 μL of each concentration of the G. max M7 and M9 extracts and placed onto the seeded top layer of the inoculated MHA plates.Findings of the antibacterial effects of the G. max extracts were compared with several types of antibiotic agents including ciprofloxacin (5 µg/disk), penicillin G (10 u/disk), tetracycline (30 µg/disk), gentamicin (10 µg/disk) and azithromycin (15 µg/disk) (Oxoid, UK).Bacterial strains were spread on to Muller Hinton Agar (MHA, Merck, Germany) media.plates containing the discs were allowed to stand for at least 30 min before incubated at 37°C for 24 h.Interpreting of the diameter of the zone of inhibition was done according to the protocol of the Clinical Laboratory Standard Institute (CLSI). 13Distilled water was used as negative control in all reactions.

Statistical analysis
All data were presented as mean ± standard deviation (SD).SPSS.Ver.20 was used for statistical analysis.Comparison of means were studied using the one-way ANOVA and also post hoc turkey test.Pvalue ≤0.05 was determined as significant level.All tests were done 3 times.

Results
Table 1 represents the diameter of the zone of inhibition of different bacteria against methanoplic extract of M7 and M9 varieties of the G. max.Diameter of the inhibition zone of tested bacteria against different treatments of G. max had a ranges of 5.93±0.18 to 22.61±1.80mm.M7 variety of the G. max had a higher antimicrobial effects on tested bacteria than M9 variety (P<0.05).The highest diameter of the zone of growth inhibition was seen for L. monocytogenes (22.61±1.80mm) and S. aureus (19.33±1.56mm) against methanolic extract of the M7 variety of G. max at concentration of 100 mg/mL.Antimicrobial effects of the methanolic extract of the G. max was dose-dependent (P<0.05).Statistically significant differences were seen for the diameter of zone of the growth inhibition between different bacteria, different varieties and different concentrations

Discussion
Findings of the current research revealed that methanolic extract of G. max and especially its M7 variety had significant antimicrobial effects on tested bacteria and especially L. monocytogenes, B. cereus, E. coli, S. aureus and K. pneumonia.We found that inhibition zone of growth B. cereus, S. aureus, L. monocytogenes, E. coli, P. aeruginosa, K. pneumonia and S. typhi against methanolic extract of the M7 variety of G. max at concentration of 100 mg/mL were 17.72±1.48,19.33±1.56,22.61±1.80,12.99±1.27,6.70±0.42,14.33±1.17and 13.86±1.29 mm respectively.Inhibitory effects of the methanolic extract of the M7 variety of G. max at concentration of 100 mg/mL were higher than some tested antibiotic agents.
One possible explanation for the high antimicrobial effects of methanolic extract of G. max is the fact that G. max is full from antimicrobial chemical components.G. max is a rich source of phenols, saponins, micronutrients, flavonoids, and polysaccharides which may be responsible for its high antimicrobial effects. 14Phenolic compounds are well known to have a negative effect on the growth of bacteria through inhibiting their nucleic acid synthesis, enzymatic activity, cytoplasmic membrane function and energy metabolism. 15Isoflavones is one of the main chemical components of the G. max extract.Isoflavones have many biological effects including antimicrobial action.Wang et al. (2010) 16 showed that soybean isoflavones inhibited the nucleic acid synthesis of S. aureus.In addition, phenolic acids were found at significant concentrations and their antimicrobial activity has been widely investigated.Alves et al.  (2013)  17 and Saavedra et al. (2010) 18 showed that syringic, coumaric, ferullic and vanillic acids which were found in the G. max extract exhibited significant antimicrobial activity against both Gram-positive and Gram-negative bacteria.Therefore, the antibacterial activity detected in our research could be explained by the pool of phenolic compounds found in our extract and also large amount of isoflavones.
We found that Gram-positive bacteria were more susceptible against G. max than Gram-negative bacteria.This part of our research is consistent with findings of Villalobos et al. (2016). 14They showed that among tested bacteria, the highest antimicrobial activity was observed against L. monocytogenes, B. cereus and E. faecalis, with values around 50% inhibition even at the lowest concentration assayed.This is may be due to the assumption that Gram-negative bacteria are more resistant to antimicrobial compounds due to their outer lipopolysaccharide membranes.Nevertheless, this was not always true for all strains assayed, for example K. pneumoniae was inhibited by the G. max extract in our study.Ghahari et al. (2017)  19 reported that the essential oil of soybean seeds showed maximum activity against S. aureus [Minimum Inhibitory concentration (MIC) of 50 μg/L], B. subtilis and E. coli (MIC of 100 μg/mL).They showed no inhibition for P. aeruginosa with the highest concentration.The lower susceptibility of Gram-negative bacteria to extracts may be explained in terms of diffusion limitations of essential compounds, through their external membrane, owing to the presence of a hydrophilic barrier.Although this barrier is not totally impermeable, it however hinders the transport of macromolecules and hydrophobic components.Kumaran and  Citarasu (2015)  20 reported that zone of the growth inhibition of crude and pure extract of G. max against B. subtilis, E. coli, Vibrion harvey (V.harvey), Aeromonase hydrophila (A.hydrophila) and V. patahaemolyticus were 14.07±1.20 and 15.54±0.5 mm, 9.05±0.5 and 13.17±0.2mm, 8.02±0.6 and 16.07±0.8mm, 9.47±0.2and 14.32±0.2mm and 10.84±0.6 and 15.49±0.7 mm, respectively.They showed that high presence of saponin is the main factor for the high antimicrobial activities of G. max.Kumaran and Citarasu (2015) 21 reported that zone of the growth inhibition of 1 g/mL, 2 g/mL and 3 g/mL concentrations of the G. nax against E. coli, P. aeruginosa and V. harvey were 4.2, 5.9 and 2.7 mm, 3.8, 5.4 and 1.4 mm and 5.4, 6.1 and 1.8 mm, respectively.
Unauthorized and indiscriminate prescription of antibiotics and especially tetracycline, penicillin, gentamicin and ciprofloxacin in medicine and veterinary are the main reasons for the high prevalence of resistance (low zone of inhibition) in the bacterial strains of our study.In fact, the methanolic extract of G. max harbored the higher antimicrobial effects than mentioned antibiotics.

Conclusions
In conclusion, we identified a considerable antimicrobial effects of the methanolic extracts of M7 and M9 varieties of G. max against pathogenic bacteria and especially Gram-positive bacteria.M7 variety had a higher antimicrobial effects than M9.Highest in herbivory effects of the M7 variety of G. max extract was seen for L. monocytogenes, B. cereus, S. aureus and K. pneumonia.Antimicrobial effects of G. max extract were dose-depended and the highest antimicrobial effects was seen for the 100 mg/mL concentration.Judicious prescription of antibiotics can control and eliminate the occurrence of antibiotic resistance in pathogenic bacteria.We recommended the production of antibiotic agent using the methanolic extract of the M7 variety of the G. max.