Bactericidal Efficacy of Omega-3 Fatty Acids and Esters Present in Moringa...

[Full title: Bactericidal Efficacy of Omega-3 Fatty Acids and Esters Present in Moringa oleifera and Portulaca oleracea Fixed Oils Against Oral and Gastro Enteric Bacteria]

OPEN ACCESS International Journal of Pharmacology ISSN 1811-7775 DOI: 10.3923/ijp.2017.44.53 Research Article Bactericidal Efficacy of Omega-3 Fatty Acids and Esters Present in Moringa oleifera and Portulaca oleracea Fixed Oils Against Oral and Gastro Enteric Bacteria Amal Sabry Othman Department of Microbiology, Faculty of Applied Medical Sciences, October 6 th University, Egypt Abstract Background: Medicinal plants are now used as antibacterial compound that is safe for human use and overcome the multidrug resistant phenomenon. Objective: This study was done to compare the antibacterial activity of two fixed oils, purslane and MO Methodology: The antimicrobial activity was done using oil diffusion method. The GC-MS analysis was done for the effective oil and protein profile and TEM scanning was done for the highly affected bacteria. Results: Purslane oil was the most effective especially against E. coli . Protein profile and TEM scanning revealed that the effect was on the outer bacterial membrane with no change in protein profile. The GC-MS analysis revealed that the oil contained Omega-3 fatty acid " -linolenic (ALA, C 18:3 n-3); 9, 12, 15-octadecatrienoic acid methyl ester (30.83%). Conclusion: These fatty acids are bactericidal compounds that could disrupt the outer bacterial cell membrane and do not affect the internal protein content Key words: Purslane, fixed oil, bactericidal, protein profile, GC-MS analysis Received: June 17, 2016 Accepted: October 19, 2016 Published: December 15, 2016 Citation: Amal Sabry Othman, 2017. Bactericidal efficacy of Omega-3 fatty acids and esters present in Moringa oleifera and Portulaca oleracea fixed oils against oral and gastro enteric bacteria. Int. J. Pharmacol., 13: 44-53 Corresponding Author: Amal Sabry Othman, Department of Microbiology, Faculty of Applied Medical Sciences, October 6 th University, Egypt Copyright: © 2017 Amal Sabry Othman. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists Data Availability: All relevant data are within the paper and its supporting information files.

Int. J. Pharmacol., 13 (1): 44-53, 2017 INTRODUCTION Now a days medicinal plants are used for human health The antimicrobial properties of plants have been investigated by a number of studies worldwide. Many have been used as therapeutic alternatives because of their antimicrobial properties 1 . The antimicrobial properties are produced as secondary metabolites such as alkaloids, phenolic compounds, etc. The practice of using folk medicine is now increasing in developing countries in response to World Health Organization directives culminating in several pre-clinical and clinical studies 2,3 Portulaca oleracea (purslane) is reported to be rich in " -linolenic acid and s-carotene and used as a health food for patients with cardiovascular diseases 4 . It contains several types of vitamins and minerals 5 , fatty acids 6 , glutathione, glutamic acid, aspartic acid dopamine, dopa, coumarins, flavonoids, alkaloids, saponins and anthocyanin 7 . purslane seeds decoction is considered as excellent diuretic. In Africa whole plant is considered as bactericidal in bacillary dysentery, diarrhea, hemorrhoids and used as antidiabetic 8 A recent study has shown that the flavonoid apigenin isolated from ethanolic extracts of Portulaca oleracea L., has antibacterial property and could be used to develop antibacterial drugs 9 Other study was done in 2015 and revealed that the hydroalcoholic extract of Portulaca o leracea leaves and seeds showed different antimicrobial activity 10 . Londonkar and Nayaka 11 showed the antimicrobial and antifungal properties of ethanol extracts of P. oleracea on some bacteria and fungi. Bae 12 showed the antimicrobial effect of P. oleracea extracts on foodborne pathogens Antimicrobial components of Moringa Oleifera (MO) have been used against several microorganisms. Aqueous extracts of MO was found to be inhibitory against many pathogenic bacteria, including Staphylococcus aureus , Bacillus subtilis , Escherichia coli and Pseudomonas aeruginosa 13 Sayeed et al 14 revealed that the fruit extract of Moringa oleifera showed a broad-spectrum antibacterial and antifungal activity This study will compare the antibacterial activity of Moringa olifera and purslane fixed oils against some gram positive and gram negative pathogenic bacteria, detection of the active compounds by GC-MS analysis and mechanism of action using TEM and protein profile analysis. This may lead to use these healthy natural compounds as antimicrobial agent instead of using hazardous chemical compounds or antibiotics MATERIALS AND METHODS Bacterial strains: Some Gram positive and Gram negative bacterial strains were used (ATCC, US) for examination in this study, Staphylococcus aureus (ATCC: 25923), Staphylococcus epidermidis (ATCC: 12228), Streptococcus pyogenes (ATCC: 19615), Streptococcus sanguinis (ATCC: 10556), Pseudomonas aeruginosa (ATCC: 27853), klebsiella pneumoniae (ATCC: 13883), Streptococcus pneumoniae (ATCC: 6305), Salmonella typhi (ATCC: 19430), Escherichia coli (ATCC: 25922), Shigella flexneri (ATCC: 12002). Bacteria were grown on Brain Heart Infusion (BHI) broth medium and maintained on nutrient agar slants 15 at 4 E C Extraction of Moringa olifera and Purslane fixed oil Plant material: Plant materials of purslane and Moringa olifera were collected from Horbit Village, El-Sharkyea Governorate, Egypt. It was identified, authenticated taxonomically by Dr. Heba El-Gezawy, Pharmacognosy Department, Faculty of Pharmacy, October 6 th University. The seed were cleaned, dried under direct sunlight and powdered by a mechanical grinder Extraction of fixed oil: First, seeds were cleaned by hand carefully to remove the foreign materials such as other seeds, stones and small stalks, then purslane seed were dried at 50 E C for 12 h in an oven and then crushed into powder in a grinder with a size range of 0.55-1.0 mm. Purslane and Moringa olifera ground samples were mixed with hexane (1:10, m/V) at (60-80 E C) using a soxhlet apparatus. This process of extraction was repeated for 6 h, the hexane distilled out by distillation assembly, then concentrated by hot plate drying and air-drying at temperature 16 of 40±2 E C Antimicrobial susceptibility tests: Antimicrobial susceptibility testing was done using the disk diffusion method and results were interpreted using the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards (NCCLS) break point criteria 17 . Antimicrobial drugs included penicillin group (amoxil); glycopeptide group (vancocin), aminoglycosides (amikin) and cephalosporin (cephradine and imipenem) Multidrug resistant strains (MDR) were detected and defined as the non-susceptible strains to at least one agent in three or more antimicrobial categories 18 Evaluation of antibacterial activity of the fixed oils against bacterial strains: Fixed oils of Moringa oleifera and purslane 45

Int. J. Pharmacol., 13 (1): 44-53, 2017 were examined for their antimicrobial activity against the tested bacteria by agar diffusion method. Dilutions of fixed oils were carried out in DMSO (dimethyl sulfoxide) with concentrations ranging from 10-100% (v/v). Plates were incubated at 35±2 E C for 16-20 h. The mean inhibition zone diameter was measured. Each assay was carried out in triplicate Preparation of Fatty Acid Methyl Ester (FAME): Seeds (40 g) were dried overnight in hot oven at 50 E C and ground into powder with a mortar and pastle, then 0.6 mL of dichloromethane and 4.0 mL of 0.5 N sodium methoxide were added. Acidic catalyzed esterification using the boron triflouride-methanol complex (14% w/v) was added according to the method described by AOAC 19 and Rezanka and Rezankova 20 . The tube was shaken and heated for 30 min at 50 E C. The reaction was stopped by adding 5.0 mL of water containing 0.2 mL of glacial acetic acid. The esterified fatty acids were extracted with 3.0 mL petroleum ether (40-600 E C). The clear fraction was kept at -20 E C until further analysis Separation condition of fatty acids on GC/MS Instrument: The HP 6890 Series Gas Chromatograph System with an HP 5973 Mass Selective Detector. The FAME in hexane (1 µL) was injected into the column with a split ratio of 100:1. The injector and detector temperature were set at 200 and 250 E C, respectively. Helium was used as the carrier gas at a flow rate of 1.5 mL min G 1 . Separation was carried out on a TR-FAME (Thermo 260 M 142 P) (30×0.25 mm ID) with a film thickness of 0.25 µm film (70% cyanopropyl polysilphphenylene siloxane) capillary column. The column temperature was programmed from 100-160 at 2 E C min G 1 and then to 250 at 4 E C min G 1 and finally held at 250 E C for 20 min. The weights of the individual FAME were calculated on the basis of their relative peak area compared with that of internal standard and then they were corrected using the corresponding GC response factors for each fatty acid Protein profile analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 5 x sample buffer: (10% w/v SDS, 10 mM dithiothreitol or $ -mercapto-ethanol, 20% v/v glycerol, 0.2 M tris-HCl, pH 6.8 and 0.05% w/v bromophenol blue) should add up to 8 M urea for really hydrophobic proteins 1 x running buffer: (25 mM tris-HCl, 200 mM glycine and 0.1% (w/v) SDS) Preparing the sample: Mix the protein 4:1 with the sample buffer. Heat the sample by boiling for 5-10 min Running the sample on gel: Clamp in your gel and fill both buffer chambers with gel running buffer according to the instructions for your specific apparatus. Pipette the sample into the gel adjusting the volume according to the amount of protein in the sample. Be sure to include a lane with molecular weight standards. Now attach your power leads and run the gel until the blue dye front reaches the bottom with 250 V constant which in a four to twenty percent mini gel needs about 30 min total run time but adjust to the thickness of your gel, the power supply used and the resolution desired. Remove the gel for the power supply and process further-visualize the proteins using coomassie brilliant blue, silver stain or any of the other protein stains 21 Transmission electron microscope (TEM) examination: Conventional TEM microscopy is frequently selected to visualize the ultrastructural damage on both cell wall and cytoplasm membrane of entire microbes when fixed material can be used 22 At ultra structural level, a simple negative staining for TEM (JEM-1400 TEM, JEOL-Japan) of bacterial cells can report evidences on the mechanism of membrane disruption by antimicrobial proteins and peptides (AMPPs) 23 . Ultrathin sections obtained by conventional procedures, namely fixation with aldehydes, post-fixation with osmium tetraoxide, dehydration and embedding in epoxy resin, allow the observation of membrane and cytoplasmatic alterations. Treatment with AMPPs can induce several external and internal changes such as membrane bleb, ruffling or detachment, the presence of electro dense dots or fibers, hypodense cytoplasmic release and cell vacuolization 24 . The outer membrane detachment observed is generally related to the extremely high affinity of AMPPs to LPS, the main component of the Gram-negative bacteria cell wall 24 RESULTS Table 1 showed that all bacteria tested were not multidrug resistant, however 5 (45.5%) strains of them were resistant to vancomycin, 1 (9%) was resistant to amikacin, 1 (9%) for cephradines and 1 (9%) was resistant to amoxil while all of them (100%) were susceptible to Impenenems Figure 1 showed that MO fixed oil had very low effect on Gram positive bacteria tested (very small inhibition zone diameter # 1 cm) 46

Int. J. Pharmacol., 13 (1): 44-53, 2017 Fig. 1: Effect of Moringa oleifera fixed oil on Gram positive bacteria tested Fig. 2: Effect of Moringa oleifera fixed oil on Gram negative bacteria tested Table 1: Antibiogram of the tested strains to different antibiotics and detection of multi drug resistant ones Antibiotics Mean diameter of inhibition zone (mm) ----------------------------------------------------------------------------------------------------------------------------------------------------------------------- Penicillin group Cephalosporin group Aminoglycosides group Glycopeptide group ------------------------ -------------------------------------------------- ------------------------------- --------------------------- Bacterial strains Amoxil Impenem Cephradine Amikacin Vancomycin Staphylococcus aureus 32 40 15 28 18 Staphylococcus epidermidis 22 45 32 40 00 Streptococcus pyogenes 30 45 32 30 20 Streptococcus sanguinis 20 30 45 30 00 Streptococcus pneumonia 26 40 40 32 25 Pseudomonas aeruginosa 30 44 33 28 28 Klebsiella pneumoniae 22 18 35 25 18 Salmonella typhi 12 38 28 22 00 Escherichia coli 23 32 30 18 00 Shigella flexneri 28 28 00 32 00 Figure 2 also showed that MO fixed oil had very low effect on Gram negative bacteria tested (very small inhibition zone diameter # 1.2 cm) Figure 3 also represented low effect of purslane fixed oil on Gram positive bacteria tested (very small inhibition zone diameter # 1.2 cm) Figure 4 illustrated that purslane fixed oil had high effect on E. coli at concentrations between 10-70% showing the highest effect at concentration 20% The GC-MS showed that purslane contains many fatty acids. It is one of the richest plant sources of It is one of the richest plant sources of Omega-3 fatty acid alpha-linolenic (ALA, C 18:3 n-3); 9, 12, 15-octadecatrienoic acid methyl ester (30.83%) (Table 2, Fig. 5) As E. coli was the highly affected bacterial strain in this study, it was chosen to investigate its protein profile before and after treatment and to be scanned by Transmission Electron Microscope (TEM) to see the effect of purslane fixed oil on the protein content and cell membrane structure Figure 6 showed no protein profile changes before and after treatment of E. coli with purslane fixed oil 47 1.0 0.8 0.6 0.4 0.2 0.0 1 10 20 30 40 50 60 70 80 90 100 Concentration (%) I Z D ( cm) S. aureus S. epidermidis S. sanguinis S. pyogenes S. pneumonia 1.2 1.0 0.8 0.6 0.4 0.2 0.0 I Z D ( cm) 1 10 20 30 40 50 60 70 80 90 100 Concentration (%) P. aeroginosa K. pneumoniae S. typhi E. coli S. flexneri

Int. J. Pharmacol., 13 (1): 44-53, 2017 Fig. 3: Effect of purslane fixed oil on Gram positive bacteria tested Fig. 4: Effect of purslane fixed oil on Gram negative bacteria tested Table 2: GC-MS analysis of purslane fixed oil constituents Peak No Compounds Relative retention time (min) Percentage 1 Dimethyl-sulfoxide 4.955 0.84 2 2-furancarboxaldehyde, 5-methyl-2-furfural 6.086 6.11 3 Methyl heptyl ketone 7.243 9.62 4 3-(dimethyl-amino) propyl-carbamate 7.694 3.55 5 S-propyl butyl (ethyl) thiocarbamate 7.881 7.16 6 4-methoxycyclohexanone 2-aminomethyl-5-methylamino-1,3,4-oxadiazole 8.415 2.40 7 N-isobutyl-2-oxoimidazolidine-1-car-boxamide 12.297 0.85 8 p-hydroxybenzyl alcohol, 4-methylolphenol 13.236 0.99 9 4-hydroxy-benzeneacetonitrile 14.642 5.42 10 4-hydroxy-benzeneacetonitrile, 4-hydroxy-phthalimidine 14.683 9.22 11 Hexadecanoic acid, methyl ester 18.046 0.52 12 n-hexadecanoic acid 18.513 4.91 13 7-octadecatrienoic acid methyl ester, methyl ester 19.737 2.20 14 6-octadecatrienoic acid methyl ester 20.209 30.83 15 9-octadecatrienoic acid methyl Ester 20.360 7.66 16 Succinic anhydride oleic acid 21.055 1.01 17 2-ethoxy-2,3-dihydro-3,3-dimethyl-benzofuran-5-yl methanesulphonate 23.307 1.30 18 1,2,3-tris(t-butyl)-2-cyclopropene 23.691 0.54 Transmission Electron Microscope showed that the effect of purslane fixed oil on E. coli was on the outer cell membrane (Fig. 7 b). Figure 7 showed disruption in bacterial cell membrane with releasing of the internal bacterial cell content DISCUSSION Purslane is a very good source of alpha-linolenic acid (Omega-3 fatty acid) which plays an important role in human growth and development and in preventing diseases. Purslane 48 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 I Z D ( cm) 1 10 20 30 40 50 60 70 80 90 100 Concentration (%) P. aeroginosa K. pneumoniae S. typhi E. coli S. flexneri 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1 10 20 30 40 50 60 70 80 90 100 Concentration (%) I Z D ( cm) S. aureus S. epidermidis S. sanguinis S. pyogenes S. pneumonia

Int. J. Pharmacol., 13 (1): 44-53, 2017 Fig. 5: Total chromatogram for GC-MS analysis of purslane fixed oil constituents Fig. 6: Protein profile for E. coli before and after treatment with purslane fixed oil 49 M 1 2 180 kDa 1350 kDa 100 kDa 75 kDa 63 kDa 48 kDa 35 kDa Abundance 8000000 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 7500000 7000000 6500000 6000000 5500000 5000000 4500000 4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000

Int. J. Pharmacol., 13 (1): 44-53, 2017 Fig. 7(a-b): TEM for the effect of purslane fixed oil on E. coli , E. coli before treatment and (b) E. coli after treatment has been shown to contain five times higher Omega-3 fatty acids than spinach. Omega-3 fatty acids belong to a group of polyunsaturated fatty acids essential for human growth, development, prevention of numerous cardiovascular diseases and maintenance of a healthy immune system 25 This study compared the activity of two fixed oils against some gram positive and Gram negative bacteria. The study showed that purslane was more efficient than MO showing the higher effect against E. coli especially at low concentrations (20%). This result was in agreement with 50 (a) (b) 13.tif Print mag: 91100×@ 211 mm TEM mode: Imaging 7.tif Print mag: 41400×@ 211 mm TEM mode: Imaging 500 nm HV = 80.0 kV Direct mag: 40000 x 500 nm HV = 80.0 kV Direct mag: 20000 x

Int. J. Pharmacol., 13 (1): 44-53, 2017 some researches. Zhao et al 26 in their study found that different additive dosages of P. oleracea extracts in daily ration would bring different effects on cecal Lactobacillus and E. coli . Shuai Peng and his colleagues in their study also showed that purslane seed oil could affect E. coli and other Gram positive cocci 27 . The same was done on methanol extract of Portulaca oleracea and showed high activity against both Gram positive organisms Bacillus subtilis and Staphylococcus aureus and only active against one Gram-negative bacteria namely: Pseudomonas aeruginosa 28 In contradiction to the current result, it has been reported that crushed seed extract of Moringa oleifera had bactericidal activity against Staphylococcus pyogenus and Pseudomonas aerogenosa 29 . Harvey 30 also reported that pterygospermin, a bactericidal and fungicidal compound contained in an aqueous extract made from seed of Moringa oleifera was effective against Staphylococcus aureus as the antibiotic neomycin The present study also indicated that all the strains were resistant to all dilutions of Moringa olifera fixed oil this was in agreement with other study done by examining aqueous methanolic extract and fixed oil of MO against some bacteria and viruses showing varying degree of antimicrobial activity was observed ranging from sensitive for B. stearothermophilus to resistant for P. aeruginosa 31-33,9 Also another study was done comparing relative antimicrobial activity of seed extracts against bacteria ( Pasturella multocida , E. coli , B. subtilis and S. aureus ) and fungi ( Fusarium solani and Rhizopus solani ) revealed that P. multocida and B. subtilis were the most sensitive strains and their activity was influenced by cations (Na + , K + , Mg 2+ and Ca 2+ ) 13 Nayaka et al 9 showed the antibacterial activity of hydroalcoholic extract of some parts of P. oleracea by five pathogenic bacteria, including Salmonella typhimurium and P. mirabilis . The highest inhibition zones for Pseudomonas aeruginosa , Klebsiella pneumonia and Enterobacter aerogenes were 14.56±0.21 and 11.68±0.13 mm, respectively 34 . Londonkar and Nayaka 11 studied the ethanol extract of the aerial parts of P. oleracea and showed its inhibitory effect against strains of S. aureus , K. pneumonia , Bacillus cereus and Aspergillus fumigates Bae 12 showed that the ethyl acetate extract of P. oleracea had the highest antimicrobial activity against S aureus and Shigella dysenteriae In contrast to these results a relative study comparing the antibacterial andantifungal efficacy of MO steam distillate observed more inhibition for E. coli followed by S. aureus , Klebsiella pneumoniae , P. aeruginosa and B. subtilis. In case of fungi, Aspergillus niger was strongly inhibited followed by Aspergillus oryzae , Aspergillus terreus and Aspergillus nidulans 35 In the present study purslane fixed oil was analyzed by GC-MS to detect the active antibacterial ingredient that could affect E. coli . It was found that Omega 3 was the most prevalent compound (30.83%) Omega 3 and oleic acids are of polyunsaturated fatty acid which are known for their inhibitory effect on Gram negative bacteria compared to saturated fatty acids. They are incorporated to the outer cell membrane and increase membrane permeability so the concentration gradient necessary between the organism and its environment may be dissipated resulting in death of the organism 36 . This may interpret why there was no change in protein profile of E. coli before and after treatment with purslane oil while the TEM showed disruption of the outer cell membrane and releasing of all internal cell content CONCLUSION Plant oil extract has antibacterial activity. It contains unsaturated fatty acids that have bactericidal effect. This study showed that purslane fixed oil was more effective than MOʼs E. coli was the highly affected bacterial strain. Omega 3 and oleic acids are of polyunsaturated fatty acids that present in purslane oil and has bactericidal activity by disrupting the outer cell membrane. Using of natural compounds can reduce the phenomenon of antimicrobial resistance ACKNOWLEDGMENT The author thanks deeply Prof. Mohammed Abdulla Hussein, Biochemistry Department, Faculty of Pharmacy, October 6 th University, for his grateful help and support REFERENCES 1 Bugno, A., M.A. Nicoletti, A.A.B. Almodovar, T.C. Pereira and M.T. Auricchio, 2007. Antimicrobial efficacy of Curcuma zedoaria extract as assessed by linear regression compared with commercial mouthrinses. Braz. J. Microbiol., 38: 440-445 2 Vijaya, K. and S. Ananthan, 1997. Microbiological screening of Indian medicinal plants with special reference to enteropathogens. J. Altern. Complement. Med., 3: 13-20 3 Dilhuydy, J.M., 2003. [Patients' attraction to Complementary and Alternative Medicine (CAM): A reality which physicians can neither ignore nor deny]. Bull. Cancer, 90: 623-628, (In French) 51

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