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Bioactivity and application of plant seeds’ extracts to fight resistant strains of Staphylococcus aureus
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Bioactivity and application of plant seeds’ extracts to fight resistant strains of Staphylococcus aureus ⁎
Ahmed A. Tayela, , Sahar M. Shabanb, Shaaban H. Moussac, Nihal M. Elguindyd, Amany M. Diaba, Khaled E. Mazrouc, Reem A. Ghanema, Sabha M. El-Sabbaghb a
Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Egypt Faculty of Science, Minoufiya University, Egypt c Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Egypt d Faculty of Science, Alexandria University, Alexandria, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobial Mode of action Phytochemicals Plant extracts Practical uses
Staphylococcus aureus is a highly dangerous pathogen that causes lots of health problems. The resistant strains to methicillin (MRSA) are dangerously health threatens. Nine plant seeds’ extracts (Alium ampeloprasum, Allium cepa, Brassica juncea, Lycium shawii, Nigella sativa, Ocimum basilicum, Peganum harmala, Phyllanthus emblica and Portulaca oleracea) were evaluated as microbial inhibitor agents against S. aureus isolates, including MRSA strains. The crude extracts of L. shawii and P. emblica seeds proved to be the most active antimicrobials, against the entire S. aureus strains, using both quality and quantity assays for their bactericidal activity. Both L. shawii and P. emblica seeds contained remarkable amounts from active phytochemicals, alkaloids, phenolic compounds, tannins and flavonoids. P. emblica seeds were exceedingly rich sources of phenolic compound and flavonoids. The electron scanning micrographs of S. aureus cells, after exposure to plant seeds’ extracts, showed that bacterial cells were shrunk and became tiny, diminutive and dehydrated after 3 h and the entire cells were fully lysed, exploded or disrupted after 6 h of exposure to P. emblica extract; whereas exposure to L. shawii extract derived treated cells to lyse and combine with each other’s after 3 h then complete cell wall lysis was observed after 6 h of exposure. The applications of plant seeds’ extracts, for textile finishing and ointment formulation, confirmed their efficacy as potent applicable anti-MRSA agents. It may be recommended to apply plant seeds’ extract, e.g. P. emblica and L. shawii, as powerful antibacterial agents for the control of the skin and foodborne pathogenic bacteria, S. aureus, and their resistant MRSA strains.
1. Introduction Plants continued to be marvelous rich sources of medicinal compounds that recurrently helped to save human health since ancient history; plant extracts and their bioactive phytochemical constituents were reported in traditional therapies and folk medicine of 80% from the earth population (Cowan, 1999). Moreover, more than the half of all current modern clinical drugs was derived from natural plant origins (Kirbag et al., 2009). The screening of antimicrobial agents, from plant phytochemicals and derivatives, was repeatedly positioned as the starting point for the discovery of novel antimicrobial drugs (Cederlund and Mårdh, 1993; Tayel et al., 2013a). The wide variety of bioactive phytochemicals in plant derivatives promoted the researchers for investigating more and more pharmaceutical usages from their mostly safe compounds (Aiyegoro and Okoh, 2009).
The emergence of additional bacterial resistance to commonly used antibiotics urged the requisite for novel powerful antimicrobial agents, from non- conventional sources (Cowan, 1999). These complex factors have obligated researchers to explore innovative antimicrobial agents from all available sources to act as alternative antimicrobial chemotherapeutic compounds; the high production cost of synthetic drugs and their adverse effects, compared with naturally plant derived agents, persuade the direction back to nature (Cowan, 1999; Tayel et al., 2013a). Staphylococcus aureus is the Gram positive bacteria that mostly involved in food poisoning, wound infections, toxic shock and scaldedskin syndrome, osteomyelitis and endocarditis (Lowy, 1998; Winn et al., 2006), via their production of diverse dangerous toxins; enterotoxin A-E, exfoliated toxins A and B and toxic shock syndrome toxin-1 (TSST-1) (Projan and Novick, 1997). The ingestion of S. aureus
Peer review under responsibility of Faculty of Agriculture, Ain-Shams University. ⁎ Corresponding author at: Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, El−Geish St., 33516 Kafrelsheikh City, Egypt. E-mail addresses: [email protected], [email protected] (A.A. Tayel). https://doi.org/10.1016/j.aoas.2018.04.006 Received 31 January 2018; Received in revised form 28 April 2018; Accepted 29 April 2018 0570-1783/ 2018Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Tayel, A.A., Annals of Agricultural Sciences (2018), https://doi.org/10.1016/j.aoas.2018.04.006
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spread onto solidified Nutrient agar (NA) plates for bacterial isolates, allowed to dry for 5 min. then holes of 8 mm diameter were made using a sterile cork borer. Aliquots from each plant seeds’ crude extract (50 μl of diluted extracts at 10% w/v) were pipetted into each well and the plates thereafter were incubated at 37 °C for 24–48 h. The appeared zones of bacterial growth inhibition were measured for each extract. The MIC determination of plant seeds’ extracts was performed using the microdilution method described by Tayel et al. (2010), using extracts concentration range of 1–10 mg/ml. Triphenyl tetrazolium chloride (TTC) was used as indicator for confirming the bactericidal activity of plant seed extracts.
enterotoxin from contaminated food is a very dangerous source of food poisoning worldwide (Howard and Kloos, 1987). S. aureus was reported to be the most important pathogen in seafood; it was recorded to contaminate 20% of various fisheries products including whole fish, fish fillets, crab-meat, shellfish-meat and shrimp tails (Ayulo et al., 1994). S. aureus was reported to facilitate and increase the susceptibility of fish infection with Streptococcus agalactiae that causes the mortality of > 60% from infected fish (Amal et al., 2008). S. aureus is from the well-established organisms that have the ability to acquire resistance toward many chemical antibiotics; the resistant bacteria to methicillin and its derivatives (MRSA) is considered as the most threatening cause of nosocomial infections. MRSA infections are very challenging to treat because of bacterial resistance to most of clinically usable antibiotics (Brooks et al., 2007). A large number of S. aureus isolates was obtained from tilapias fish (Oreochromis niloticus); 50% from them were characterized as MRSA strains (Atyah et al., 2010). The bacterial resistance to antimicrobial is multifactorial, involving the relationship between bacterial cell and antibiotic, environmental factors, the type of antibacterial agent usage and host characteristics (Adwan and Mhanna, 2008). However, current study was designed to evaluate the effectiveness of many plant seeds’ extract as potential antibacterial agents against S. aureus and their MRSA strains, and to elucidate their potential antibacterial actions against the microbial strains.
2.4. Quantitative determination of some phytochemical constituents
2. Materials and methods
The contents of phytochemical groups in plant seed materials were quantitatively determined as follow: The total alkaloids contents and total phenolic compounds were measured according to previously described methods (Harborne, 1998); alkaloids contents were expressed as mg/g of the plant sample dry weight, whereas total phenolics were spectrophotometerically estimated using the Folin Ciocalteau’s reagent and expressed as mg equivalent of Gallic acid/g plant material. The total tannins were estimated using the Gravimetric method and their contents were calculated as illustrated by Ali (1991). Flavonoids were determined as mg/g using the described modified aluminum chloride colorimetric method (Chang et al., 2002), with rutin as a standard for comparison.
2.1. Plant seeds
2.5. Imaging with scanning electron microscopy (SEM)
The examined plant seeds, for their antibacterial activity, i.e. Alium ampeloprasum, Allium cepa, Brassica juncea, Lycium shawii, Nigella sativa, Ocimum basilicum, Peganum harmala, Phyllanthus emblica and Portulaca oleracea, were kindly obtained from the Vegetable and Medicinal Plants Research Centre, Giza, Egypt. Plant seeds were washed with distilled water, dried with hot air at 42 °C in an electrical oven, then ground using electrical grinder to have plant particle sizes of about 40 mesh. Weights of 200 g from each seed powder were extracted using 1 L of solvent, i.e. methanol (70%), for 72 h with occasional shaking at 120 x g. Extracts were then filtered, completely evaporated under reduced pressure at 40 °C, weighted and re-suspended in 20% dimethyl sulfoxide (DMSO) solution to attain concentrations of 100 mg/ml (Tayel et al., 2012). DMSO solution was used as negative control for comparison.
For the potential explanation of antimicrobial action from the most powerful plant seeds’ extracts toward S. aureus strains, the bacteria cells were treated with seed extracts and the micrographs were captured using SEM (Jeol JSM-5300), operated at 20 kV and magnification of 20 Kx, after 0, 3 and 6 h from the treatment with extracts. The fixation of bacterial cells was performed using 2% Osmium peroxide (OsO₄), then they were dehydrated at 4 °C using a graded ethanol series. Samples were critical dried using a carbon paste and coated with gold to a thickness 400A° inside a sputter – coating unit (JFC-1100 E). Micrographs capturing was depended on the variation in cells morphology after exposure period to extracts. 2.6. Application of anti-MRSA seed extracts 2.6.1. Fabrication of extracts-treated textiles Standard and scoured cotton textile (104 g/m2 plain weave, Style S/ 400, TESTEX, Germany) were used for impregnating with plant seeds’ extracts. The method of “pad-dry-cure” was performed for textile finishing. 1 × 1 cm squares from fabrics were cut and immersed in extracts solution, at their MIC levels, stirred for 2 h at 50 °C, then padded and squeezed using 2 nips and dips to 100% wet pick up. Treated fabric pieces were then dried for 3 min at 75 °C, then cured at 125 °C for 5 min, as modified after Tayel et al. (2013b) and Koh and Hong (2014). The antibacterial evaluation of extract-treated fabrics was conducted using inhibition zone assay on inoculated NA plates with S. aureus and MRSA strains.
2.2. Isolation of bacterial pathogens The examined bacterial strains were isolated from skin wound infections in patients at some universities hospital, by the aid of a physician. Samples were aseptically collected using sterile cotton swabs, spread on mannitol salt agar medium (MSA) and incubated overnight at 37 °C, the appeared colonies were then individually isolated, purified and kept in MSA slants. Isolates were then subjected to the described biochemical tests by Cowan and Steel's Manual (Barrow and Feltham, 1993), to confirm their identity. The isolates identification was further confirmed using highly automated VITEK 2 System, produced from bioMérieux Co., France. A reference strain, i.e. S. aureus (ATCC 25923), was used for comparison and result confirmation.
2.6.2. Extracts topical formulation The topical formulation of seeds extracts was conducted using an ointment base from soft white paraffin, supplemented with 1% Sodium lauryl sulfate (SD Chemicals, India), plant seeds’ extracts were blended with the liquefied ointment base (at 45 °C), to have a concentration of 10 mg/ml from each extract (Gaud and Gupta, 2006). For the antibacterial evaluation, 50 μl from liquefied extract-supplemented ointment were pipetted into 6 mm diameter wells, made in inoculated NA plates with bacterial strains using sterile cork borer, and incubated at
2.3. Assays for plants seed extracts’ antimicrobial activity The antibacterial activities of plant seed extracts were evaluated using both qualitative and quantitative assays, i.e. well diffusion method and minimal inhibitory concentration (MIC), respectively. The well diffusion assay was conducted according to Obeidat et al. (2012); 100 μl of bacterial suspension (∼2 × 106 CFU) was uniformly 2
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Fig. 1. Antibacterial activity of different plant seeds’ extracts (as zones of inhibition, mm) against examined Staphylococcus aureus strains.
25923), (Isolate 1), (Isolate 2), MRSA (Isolate3) and MRSA (Isolate 4), respectively. Whereas the recorded MIC values from P. emblica extract were 2, 2, 6, 8 and 10 mg/ml to inhibit the same strains, respectively. Thus, only L. shawii and P. emblica seed extracts were chosen to perform further investigations to elucidate their potential antibacterial actions and their phytochemical contents. The phytochemical contents, in the seeds materials of L. shawii and P. emblica, are presented in Table 1. It is observed that L. shawii seeds’ extract had higher content from alkaloids than in P. emblica seeds extract. Oppositely, the extract of P. emblica seeds had much higher contents from the other determined phytochemical groups, i.e. phenolic compounds, tannins and flavonoids, than their relevant contents in L. shawii seeds (Table 1). P. emblica seeds were proved to be very rich sources of phenolic compound and flavonoids, with values of 78.51 and 57.14 mg/g seed powder, respectively. The SEM investigation was performed to elucidate the morphological changes of treated S. aureus cells with MIC concentration from the crude extract of P. emblica and L. shawii (Fig. 2). The treatment with
37 °C for 24 h. Inhibition zones were precisely measured in triplicates and the mean zones diameter were calculated. 2.7. Statistical analysis The experiments were conducted in triplicates, the calculation of results means and standard deviations was performed using Excel software (Microsoft office 2013). The statistical software MedCalc (V. 11.6.1, Ostend, Belgium) was applied to analyze results significance at confidence interval of ≥95%. 3. Results From 20 isolated bacterial strains, from infected wounds, four strains were identified and characterized as S. aureus isolates. By examining the sensitivity of isolated strains to eight standard antibiotics (data not shown), two of them were confirmed as MRSA strains, i.e. MRSA (Isolate 3) and MRSA (Isolate 4). The qualitative antibacterial activity of examined plant seeds’ extracts (as appeared zones of growth inhibition, mm), against S. aureus strains, is illustrated in Fig. 1. The extracts of P. emblica and L. shawii exhibited their antibacterial activity toward all examined S. aureus strains, including the MRSA strains. The extracts of N. sativa, O. basilicum, P. oleracea, A. ampeloprasum and A. cepa had only antibacterial activities against normal S. aureus strains but not toward MRSA isolates. On the other hand, B. juncea did not exhibit any antibacterial activity against S. aureus strains (Fig. 1). The negative control (DMSO) did not exhibit any antimicrobial activity against examined strains, using the above assay. Regarding the antimicrobial activity assay as MIC (mg/ml), it was observed that L. shawii and P. emblica seed extracts were the only extracts, among the examined plants, that could inhibit the MRSA strains within the value of ≤10 mg/ml. The recorded MIC values for L. shawii extract were 1, 1, 5, 10 and 10 mg/ml to inhibit S. aureus (ATCC-
Table 1 Phytochemical content in the extracts of Lycium shawii and Phyllanthus emblica seeds. Plant seeds
Lycium shawii Phyllanthus emblica
Phytochemical content* Alkaloids (mg/g dry weight)
Total phenolics (mg/g dry weight)
Tannins (mg/ml)
Flavonoids (mg/g dry weight)
46.1 ± 1.3a
10.96 ± 0.42a
36.21 ± 0.18a
8.62 ± 0.72a
34.4 ± 2.4b
78.51 ± 0.63b
50.41 ± .0.27b
57.14 ± 0.65b
Different superscript letters in the same column indicate difference significance at CI ≥ 95%. * Values are means of triplicates ± standard deviation. 3
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Fig. 2. Scanning electron micrographs of the cells of Staphylococcus aureus MRSA-1 after treatment with seed extracts of Phyllanthus emblica (1) and Lycium shawii (2) after zero time (A), 3 h (B) and 6 h (C) of exposure to extracts.
The applications of plant seeds’ extracts, i.e. P. emblica and L. shawii, for textile finishing and ointment formulation proved to be successful for the control of normal S. aureus and MRSA strains. Both extracts and applications were effective for inhibiting the examined S. aureus strains. The extract treated textiles and ointments were more forceful against normal S. aureus strains than for MRSA strains. Regarding the antiMRSA activity of extract treated products, their antibacterial potentialities were very remarkable, as appeared from the growth inhibition zones, with a higher antibacterial activity of P. emblica extract than L. shawii extract toward the two examined MRSA strains (Fig. 3). The mean diameters of inhibition zones, from P. emblica extract-treated textiles, were 32.3, 31.7, 25.2 and 27.4 mm, including the textile length of 10 mm, against S. aureus strains (isolate 1, isolate 2, MRSA 1 and MRSA 2), respectively. Whereas the appeared zones, after challenging these strains with loaded textiles with L. shawii extract, were 17.5, 16.7, 14.6 and 15.3 mm, respectively.
extracts caused notable morphological alterations in comparison with the control, no micrographs for DMSO treated cells were captured in this study. It may observed, from the SEM micrographs of the zero-time treated and treated S. aureus cells, for 3 and 6 h, that zero-time treated cells (A-1, A-2) appeared with normal, round shape and smooth surfaces. The SEM micrographs seem to show homogenous and heterogeneous biofilm formation. After 3 h of exposure to P. emblica extract (B-1), the treated bacterial cells were shrunk, became tiny, diminutive and dehydrated and after 6 h of exposure (C-1), the entire bacterial cells were fully lysed, exploded or disrupted; the interior dehydrated cellular components and debris were the only observable matrix. Regarding L. shawii extract treatment, it is observable that after 3 h of exposure (B-2), the bacterial treated cells began to lyse and combine with each other’s. After 6 h of bacterial exposure to L. shawii extract (C-2); a complete cell wall lyses was observed and the only detectable materials were the residues of lysed cell walls combined with the released interior components from bacterial cells. It may be suggested that, at this stage, that the entire treated cells had lost all of their metabolic functions completely. 4
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Fig. 3. Anti-MRSA activity, as appeared inhibition zones, of seed extracts-treated textiles (T) and ointments (C) for the extracts of Phyllanthus emblica (P) and Lycium shawii (L) against the examined MRSA strains (1 and 2).
4. Discussion
possess an antibacterial activity against many bacterial strains except MRSA (Enomoto et al., 2001); thymol contents in N. sativa was suggested to be the main responsible of seeds antibacterial activity (Karapinar and Aktug, 1987). The P. oleracea extract was suggested to have a remarkable activity against both Gram + bacteria, Bacillus subtilis and S. aureus, and it was only active against one Gram negative strain, i.e. Pseudomonas aeruginosa (Bakkiyaraj and Pandiyaraj, 2001). The Allium spp. seed extracts did not show notable antibacterial activity against MRSA and other tested strains; this results could be supported with other reported results regarding A. ampeloprasum extract (Anuswedha and Rao, 2015) and for the extract of A. cepa (Grover et al., 2011). The powerful antimicrobial plant seeds in current investigation, i.e. P. emblica and L. shawii, had relatively varied amounts from bioactive phytochemicals, which could play an important role to force their bactericidal activity. The advantageous pharmaceutical properties of plant materials could be characteristically attributed to their contents from secondary products combination, e.g. phenol compounds, flavonoids, alkaloids, steroids and tannins (Cowan, 1999; Davidson, 2001). The bioactive phytochemicals in plants may have more effective mechanisms than those of chemical or synthetic antimicrobial drugs; thus they could be more significantly employed in the fighting of resistant microbes (Ali, 1991). It was suggested that P. emblica extract could be utilized as a bactericidal agent because of its containment from bioactive compounds, e.g. tannins such as emblicanin A and B, flavonoids, saponins and phenols, which were affirmed as effective chemotherapeutic (Javale and Sabnis, 2010; Jyothi and Subba, 2011; Kumar and Pandey, 2013). The major bioactive principals in P. emblica extract was indicated to contain ascorbic acid, gallic acid, flavonoids (quercetin), hydrolysed tannins (emblicanin A and B) and alkaloids (phyllantidine, phyllantine) (Patil et al., 2012). Tannins, from plant origin, were verified to possess an effectual antimicrobial activity (Min et al., 2008); the suggested action of that is to interact with cell proteins through many bioactive forces such as hydrogen bonding, formation of covalent bond or hydrophobic possessions. Accordingly, tannins could perform their antibacterial action through inactivating of microbial vital mechanisms, e.g. extracellular enzymes activity, microbial adhesions, suppression of oxidative
The multiple bacterial resistance to antibiotic is a great threat to human health worldwide; MRSA was considered from the most dangerous resistant pathogens that could endanger human life (Safdar et al., 2006). Almost all compounds derived from plant are supposed to possess high levels from biosafety to human, due to their natural, organic, biodegradable and biocompatible natures, and their recurrent usage by people for centuries (Kumar and Pandey, 2013; Tayel et al., 2013a). Many natural compounds, found in plants, spices and herbs, were proved to possess antimicrobial actions and could be served as sourced for antimicrobial drugs against microbial pathogens (Cowan, 1999). The variation in the antibacterial potentialities of examined plant seeds, in current study, could be anticipated to their dissimilar contents from microbicidal agents; this suggestion was mentioned in many previous studies that recommended that medicinal plants could be the ideal potential sources to explore novel antibacterial drugs even against antibiotic-resistant bacterial strains (Davidson, 2001; Ceylan and Fung, 2004). The total crude extract of P. emblica seeds exhibited a powerful antibacterial activity against tested S. aureus strains; these results are harmonized with other previously obtained result, which indicated the efficacy of methanolic and ethyl acetate extracts (Anbuselvi and Jha, 2015) and the aquas extract (Varghese et al., 2013) of P. emblica seeds against S. aureus isolates. Additionally, the potentiality of P. emblica extracts to act as antibacterial agents against Gram-positive and negative bacteria was indicated, exercising S. aureus and K. pneumoniae as representative pathogenic organisms (Reghu and Ravindra, 2010). The seed extract of L. shawii had a potent antibacterial activity against S. aureus, which is in disagreement with the results of Aldoweriej et al. (2016), who reported that the methanolic extract of aerial parts from L. shawii had weak activity against S. aureus. This could be indicate that the main antimicrobial active compounds in L. shawii present in its seeds and not in the aerial parts. Regarding the other examined plant seeds’ extracts, it was indicated that the extracts from P. harmala seeds have exhibited some antimicrobial potentiality toward pathogenic bacteria activity (Prashanth and John, 1999). The methanolic extract of black seeds (N. sativa) had also stated to 5
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phosphorylation or transport proteins in cell envelope (Scalbert, 1991). In addition, tannins were proposed to bind with proline-rich peptides, leading to the restriction of protein synthesis (Shimada, 2006). The applications of P. emblica and L. shawii seed extracts for fabrication of anti-S. aureus products, proved their effectiveness, in both loaded fabrics and ointment with extracts. This could indicate that plant seeds’ extracts were able to maintain their bioactivity after their loading onto cellulose fibers (textile) or into white paraffin (ointment), without executing chemical reaction with the delivery carrier materials. The decrement of antimicrobial activity in extracts-treated textiles and ointment, when compared to agar well diffusion test, could be attributed to the heat treatment in curing process, for textile, which could negatively affect the activity of antimicrobial compounds, and the barricade effect of white paraffin, in ointment, that might hinder the extract release and action into the growth media. The captured SEM micrographs, of treated S. aureus cells with P. emblica and L. shawii, clearly indicated the influence of extracts treatment on the cell morphology and could indicate the possible antimicrobial modes of action from the both extracts. Regarding the SEM micrographs of treated cells with P. emblica extract, it may be suggested that that the extract increased the osmotic pressure in the surrounding media, because of its high contents from bioactive phytochemicals, and thus derived the cells to release their interior moist contents and appeared with shrunk and diminutive shape, after 3 h of exposure to the extract. While after 6 h from the exposure to P. emblica extract, and due cells loss of their water contents, it could be expected that all of the enzymatical and biological process inside the cells, including cell walls synthesis mechanisms, was stopped and the cells tended to deform and lyse. From the SEM micrograpgh of treated S. aureus cells with L. shawii extract, it could be suggested that the extract performed its antibacterial action with different mechanism; the extract either interact with the outer membrane and cell walls of the bacteria, to increase their permeability and cause the release of their interior components, or may be penetrated inside the cells and interact with their vital components, e.g. DNA, RNA, Enzymes, etc., causing their inactivation or inhibiting their synthesis or action. The finished textiles with anti-MRSA extracts could be suggested for application in the manufacturing of intensive care and surgery coats, medical antibacterial bandages, bed covers and wound dressings, whereas the treated ointments could be applied for the topical and superficial usages on infected skins.
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Safdar, N., Marx, J., Meyer, N., Maki, D., 2006. Effectiveness of preemptive barrier precautions in controlling nosocomial colonization and infection by methicillin-resistant Staphylococcus. Am. J. Infect. Control. 34, 478–483. Scalbert, A., 1991. Antimicrobial properties of tannins. Phytochemistry 30, 3875–3883. Shimada, T., 2006. Salivary proteins as a defense against dietary tannins. J. Chem. Ecol. 32, 1149–1163. Tayel, A.A., Abdel-Monem, O.A., Moussa, S.H., Al-Turki, A.I., 2013a. Plant extracts as antimicrobials: prospects in food safety and health protection, in: Giordano, A., Costs, A. (Eds.), Plant Extracts: Role in Agriculture, Health Effects and Medical Applications, Nova Science Publishers, Inc., Hauppauge, NY, pp. 311–326. Tayel, A.A., El-Tras, W.E., Abdel-Monem, O.A., El-Sabbagh, S.M., Alsohim, A.S., El-Refai, E.M., 2013b. Production of anticandidal cotton textiles treated with oak gall extract. Rev. Argent. Microbiol. 45, 271–276. Tayel, A.A., El-Tras, W.E., Moussa, S.H., El-Sabbagh, S.M., 2012. Surface decontamination
5. Conclusion The above-mentioned results may recommend the application of plant seeds’ extract, e.g. P. emblica and L. shawii, as powerful antibacterial agents for the control of the foodborne pathogenic bacteria, S. aureus, and their resistant MRSA strains. The successfulness of plant seeds applications for production of anti-S. aureus textiles, including MRSA strains, emphasize their effectiveness and applicability for pathogens treatment. References Adwan, G., Mhanna, M., 2008. Synergistic effects of plant extracts and antibiotics on Staphylococcus aureus strains isolated from clinical specimens. Middle-East J. Sci. Res. 3, 134–139. Aiyegoro, O., Okoh, A., 2009. Use of bioactive plant products in combination with standard antibiotics: implications in antimicrobial chemotherapy. J. Med. Plants. Res. 3, 1147–1152. Aldoweriej, A.M., Alharbi, K.B., Saeed, E.M., El-Ashmawy, I.M., 2016. Antimicrobial activity of various extracts from some plants native to Alqassim Region, Saudi Arabia. J. Food. Agric. Environ. 14, 238–243. Ali, R.M., 1991. Changes in chemical composition of fruits of salinized Datura stramonium. Med. J. Islamic World Acad Sci. 4, 289–292. Amal, A.M., Zamri-Saad, M., Siti-Zahrah, A., Nur-Nazifah, M., Nurazlan, W., Shahidan, H., 2008. Bacterial isolation pattern from tilapia infected with Streptococcus agalactiae. In: Proc 1st Int Cong. Aquatic. Anim. Health Management Dis, pp. 177.
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antibacterial activity from whole plant (fruits, seeds, stem, leaves and roots) of Emblica officinalis Gaertn. Int. J. Ayurvedic. Herbal. Med. 3, 1420–1425. Winn, W.C., Allen, S., Janda, W.M., Koneman, E.W., Procop, G., Schreckenberger, P.C., Woods, G., 2006. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. Lippincott Williams & Wilkins, Philadephia.
and quality enhancement in meat steaks using plant extracts as natural biopreservatives. Food Borne Path. Dis. 9, 755–761. Tayel, A.A., Moussa, S., Opwis, K., Knittel, D., Schollmeyer, E., Nickisch- Hartfiel, A., 2010. Inhibition of microbial pathogens by fungal chitosan. Int. J. Biol. Macromol. 47, 10–14. Varghese, L.S., Alex, N., Ninan, M.A., Soman, S., Jacob, S., 2013. Evaluation of in vitro
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Bioactivity and application of plant seeds’ extracts to fight resistant strains of Staphylococcus aureus ⁎
Ahmed A. Tayela, , Sahar M. Shabanb, Shaaban H. Moussac, Nihal M. Elguindyd, Amany M. Diaba, Khaled E. Mazrouc, Reem A. Ghanema, Sabha M. El-Sabbaghb a
Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Egypt Faculty of Science, Minoufiya University, Egypt c Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Egypt d Faculty of Science, Alexandria University, Alexandria, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobial Mode of action Phytochemicals Plant extracts Practical uses
Staphylococcus aureus is a highly dangerous pathogen that causes lots of health problems. The resistant strains to methicillin (MRSA) are dangerously health threatens. Nine plant seeds’ extracts (Alium ampeloprasum, Allium cepa, Brassica juncea, Lycium shawii, Nigella sativa, Ocimum basilicum, Peganum harmala, Phyllanthus emblica and Portulaca oleracea) were evaluated as microbial inhibitor agents against S. aureus isolates, including MRSA strains. The crude extracts of L. shawii and P. emblica seeds proved to be the most active antimicrobials, against the entire S. aureus strains, using both quality and quantity assays for their bactericidal activity. Both L. shawii and P. emblica seeds contained remarkable amounts from active phytochemicals, alkaloids, phenolic compounds, tannins and flavonoids. P. emblica seeds were exceedingly rich sources of phenolic compound and flavonoids. The electron scanning micrographs of S. aureus cells, after exposure to plant seeds’ extracts, showed that bacterial cells were shrunk and became tiny, diminutive and dehydrated after 3 h and the entire cells were fully lysed, exploded or disrupted after 6 h of exposure to P. emblica extract; whereas exposure to L. shawii extract derived treated cells to lyse and combine with each other’s after 3 h then complete cell wall lysis was observed after 6 h of exposure. The applications of plant seeds’ extracts, for textile finishing and ointment formulation, confirmed their efficacy as potent applicable anti-MRSA agents. It may be recommended to apply plant seeds’ extract, e.g. P. emblica and L. shawii, as powerful antibacterial agents for the control of the skin and foodborne pathogenic bacteria, S. aureus, and their resistant MRSA strains.
1. Introduction Plants continued to be marvelous rich sources of medicinal compounds that recurrently helped to save human health since ancient history; plant extracts and their bioactive phytochemical constituents were reported in traditional therapies and folk medicine of 80% from the earth population (Cowan, 1999). Moreover, more than the half of all current modern clinical drugs was derived from natural plant origins (Kirbag et al., 2009). The screening of antimicrobial agents, from plant phytochemicals and derivatives, was repeatedly positioned as the starting point for the discovery of novel antimicrobial drugs (Cederlund and Mårdh, 1993; Tayel et al., 2013a). The wide variety of bioactive phytochemicals in plant derivatives promoted the researchers for investigating more and more pharmaceutical usages from their mostly safe compounds (Aiyegoro and Okoh, 2009).
The emergence of additional bacterial resistance to commonly used antibiotics urged the requisite for novel powerful antimicrobial agents, from non- conventional sources (Cowan, 1999). These complex factors have obligated researchers to explore innovative antimicrobial agents from all available sources to act as alternative antimicrobial chemotherapeutic compounds; the high production cost of synthetic drugs and their adverse effects, compared with naturally plant derived agents, persuade the direction back to nature (Cowan, 1999; Tayel et al., 2013a). Staphylococcus aureus is the Gram positive bacteria that mostly involved in food poisoning, wound infections, toxic shock and scaldedskin syndrome, osteomyelitis and endocarditis (Lowy, 1998; Winn et al., 2006), via their production of diverse dangerous toxins; enterotoxin A-E, exfoliated toxins A and B and toxic shock syndrome toxin-1 (TSST-1) (Projan and Novick, 1997). The ingestion of S. aureus
Peer review under responsibility of Faculty of Agriculture, Ain-Shams University. ⁎ Corresponding author at: Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, El−Geish St., 33516 Kafrelsheikh City, Egypt. E-mail addresses: [email protected], [email protected] (A.A. Tayel). https://doi.org/10.1016/j.aoas.2018.04.006 Received 31 January 2018; Received in revised form 28 April 2018; Accepted 29 April 2018 0570-1783/ 2018Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Tayel, A.A., Annals of Agricultural Sciences (2018), https://doi.org/10.1016/j.aoas.2018.04.006
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spread onto solidified Nutrient agar (NA) plates for bacterial isolates, allowed to dry for 5 min. then holes of 8 mm diameter were made using a sterile cork borer. Aliquots from each plant seeds’ crude extract (50 μl of diluted extracts at 10% w/v) were pipetted into each well and the plates thereafter were incubated at 37 °C for 24–48 h. The appeared zones of bacterial growth inhibition were measured for each extract. The MIC determination of plant seeds’ extracts was performed using the microdilution method described by Tayel et al. (2010), using extracts concentration range of 1–10 mg/ml. Triphenyl tetrazolium chloride (TTC) was used as indicator for confirming the bactericidal activity of plant seed extracts.
enterotoxin from contaminated food is a very dangerous source of food poisoning worldwide (Howard and Kloos, 1987). S. aureus was reported to be the most important pathogen in seafood; it was recorded to contaminate 20% of various fisheries products including whole fish, fish fillets, crab-meat, shellfish-meat and shrimp tails (Ayulo et al., 1994). S. aureus was reported to facilitate and increase the susceptibility of fish infection with Streptococcus agalactiae that causes the mortality of > 60% from infected fish (Amal et al., 2008). S. aureus is from the well-established organisms that have the ability to acquire resistance toward many chemical antibiotics; the resistant bacteria to methicillin and its derivatives (MRSA) is considered as the most threatening cause of nosocomial infections. MRSA infections are very challenging to treat because of bacterial resistance to most of clinically usable antibiotics (Brooks et al., 2007). A large number of S. aureus isolates was obtained from tilapias fish (Oreochromis niloticus); 50% from them were characterized as MRSA strains (Atyah et al., 2010). The bacterial resistance to antimicrobial is multifactorial, involving the relationship between bacterial cell and antibiotic, environmental factors, the type of antibacterial agent usage and host characteristics (Adwan and Mhanna, 2008). However, current study was designed to evaluate the effectiveness of many plant seeds’ extract as potential antibacterial agents against S. aureus and their MRSA strains, and to elucidate their potential antibacterial actions against the microbial strains.
2.4. Quantitative determination of some phytochemical constituents
2. Materials and methods
The contents of phytochemical groups in plant seed materials were quantitatively determined as follow: The total alkaloids contents and total phenolic compounds were measured according to previously described methods (Harborne, 1998); alkaloids contents were expressed as mg/g of the plant sample dry weight, whereas total phenolics were spectrophotometerically estimated using the Folin Ciocalteau’s reagent and expressed as mg equivalent of Gallic acid/g plant material. The total tannins were estimated using the Gravimetric method and their contents were calculated as illustrated by Ali (1991). Flavonoids were determined as mg/g using the described modified aluminum chloride colorimetric method (Chang et al., 2002), with rutin as a standard for comparison.
2.1. Plant seeds
2.5. Imaging with scanning electron microscopy (SEM)
The examined plant seeds, for their antibacterial activity, i.e. Alium ampeloprasum, Allium cepa, Brassica juncea, Lycium shawii, Nigella sativa, Ocimum basilicum, Peganum harmala, Phyllanthus emblica and Portulaca oleracea, were kindly obtained from the Vegetable and Medicinal Plants Research Centre, Giza, Egypt. Plant seeds were washed with distilled water, dried with hot air at 42 °C in an electrical oven, then ground using electrical grinder to have plant particle sizes of about 40 mesh. Weights of 200 g from each seed powder were extracted using 1 L of solvent, i.e. methanol (70%), for 72 h with occasional shaking at 120 x g. Extracts were then filtered, completely evaporated under reduced pressure at 40 °C, weighted and re-suspended in 20% dimethyl sulfoxide (DMSO) solution to attain concentrations of 100 mg/ml (Tayel et al., 2012). DMSO solution was used as negative control for comparison.
For the potential explanation of antimicrobial action from the most powerful plant seeds’ extracts toward S. aureus strains, the bacteria cells were treated with seed extracts and the micrographs were captured using SEM (Jeol JSM-5300), operated at 20 kV and magnification of 20 Kx, after 0, 3 and 6 h from the treatment with extracts. The fixation of bacterial cells was performed using 2% Osmium peroxide (OsO₄), then they were dehydrated at 4 °C using a graded ethanol series. Samples were critical dried using a carbon paste and coated with gold to a thickness 400A° inside a sputter – coating unit (JFC-1100 E). Micrographs capturing was depended on the variation in cells morphology after exposure period to extracts. 2.6. Application of anti-MRSA seed extracts 2.6.1. Fabrication of extracts-treated textiles Standard and scoured cotton textile (104 g/m2 plain weave, Style S/ 400, TESTEX, Germany) were used for impregnating with plant seeds’ extracts. The method of “pad-dry-cure” was performed for textile finishing. 1 × 1 cm squares from fabrics were cut and immersed in extracts solution, at their MIC levels, stirred for 2 h at 50 °C, then padded and squeezed using 2 nips and dips to 100% wet pick up. Treated fabric pieces were then dried for 3 min at 75 °C, then cured at 125 °C for 5 min, as modified after Tayel et al. (2013b) and Koh and Hong (2014). The antibacterial evaluation of extract-treated fabrics was conducted using inhibition zone assay on inoculated NA plates with S. aureus and MRSA strains.
2.2. Isolation of bacterial pathogens The examined bacterial strains were isolated from skin wound infections in patients at some universities hospital, by the aid of a physician. Samples were aseptically collected using sterile cotton swabs, spread on mannitol salt agar medium (MSA) and incubated overnight at 37 °C, the appeared colonies were then individually isolated, purified and kept in MSA slants. Isolates were then subjected to the described biochemical tests by Cowan and Steel's Manual (Barrow and Feltham, 1993), to confirm their identity. The isolates identification was further confirmed using highly automated VITEK 2 System, produced from bioMérieux Co., France. A reference strain, i.e. S. aureus (ATCC 25923), was used for comparison and result confirmation.
2.6.2. Extracts topical formulation The topical formulation of seeds extracts was conducted using an ointment base from soft white paraffin, supplemented with 1% Sodium lauryl sulfate (SD Chemicals, India), plant seeds’ extracts were blended with the liquefied ointment base (at 45 °C), to have a concentration of 10 mg/ml from each extract (Gaud and Gupta, 2006). For the antibacterial evaluation, 50 μl from liquefied extract-supplemented ointment were pipetted into 6 mm diameter wells, made in inoculated NA plates with bacterial strains using sterile cork borer, and incubated at
2.3. Assays for plants seed extracts’ antimicrobial activity The antibacterial activities of plant seed extracts were evaluated using both qualitative and quantitative assays, i.e. well diffusion method and minimal inhibitory concentration (MIC), respectively. The well diffusion assay was conducted according to Obeidat et al. (2012); 100 μl of bacterial suspension (∼2 × 106 CFU) was uniformly 2
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Fig. 1. Antibacterial activity of different plant seeds’ extracts (as zones of inhibition, mm) against examined Staphylococcus aureus strains.
25923), (Isolate 1), (Isolate 2), MRSA (Isolate3) and MRSA (Isolate 4), respectively. Whereas the recorded MIC values from P. emblica extract were 2, 2, 6, 8 and 10 mg/ml to inhibit the same strains, respectively. Thus, only L. shawii and P. emblica seed extracts were chosen to perform further investigations to elucidate their potential antibacterial actions and their phytochemical contents. The phytochemical contents, in the seeds materials of L. shawii and P. emblica, are presented in Table 1. It is observed that L. shawii seeds’ extract had higher content from alkaloids than in P. emblica seeds extract. Oppositely, the extract of P. emblica seeds had much higher contents from the other determined phytochemical groups, i.e. phenolic compounds, tannins and flavonoids, than their relevant contents in L. shawii seeds (Table 1). P. emblica seeds were proved to be very rich sources of phenolic compound and flavonoids, with values of 78.51 and 57.14 mg/g seed powder, respectively. The SEM investigation was performed to elucidate the morphological changes of treated S. aureus cells with MIC concentration from the crude extract of P. emblica and L. shawii (Fig. 2). The treatment with
37 °C for 24 h. Inhibition zones were precisely measured in triplicates and the mean zones diameter were calculated. 2.7. Statistical analysis The experiments were conducted in triplicates, the calculation of results means and standard deviations was performed using Excel software (Microsoft office 2013). The statistical software MedCalc (V. 11.6.1, Ostend, Belgium) was applied to analyze results significance at confidence interval of ≥95%. 3. Results From 20 isolated bacterial strains, from infected wounds, four strains were identified and characterized as S. aureus isolates. By examining the sensitivity of isolated strains to eight standard antibiotics (data not shown), two of them were confirmed as MRSA strains, i.e. MRSA (Isolate 3) and MRSA (Isolate 4). The qualitative antibacterial activity of examined plant seeds’ extracts (as appeared zones of growth inhibition, mm), against S. aureus strains, is illustrated in Fig. 1. The extracts of P. emblica and L. shawii exhibited their antibacterial activity toward all examined S. aureus strains, including the MRSA strains. The extracts of N. sativa, O. basilicum, P. oleracea, A. ampeloprasum and A. cepa had only antibacterial activities against normal S. aureus strains but not toward MRSA isolates. On the other hand, B. juncea did not exhibit any antibacterial activity against S. aureus strains (Fig. 1). The negative control (DMSO) did not exhibit any antimicrobial activity against examined strains, using the above assay. Regarding the antimicrobial activity assay as MIC (mg/ml), it was observed that L. shawii and P. emblica seed extracts were the only extracts, among the examined plants, that could inhibit the MRSA strains within the value of ≤10 mg/ml. The recorded MIC values for L. shawii extract were 1, 1, 5, 10 and 10 mg/ml to inhibit S. aureus (ATCC-
Table 1 Phytochemical content in the extracts of Lycium shawii and Phyllanthus emblica seeds. Plant seeds
Lycium shawii Phyllanthus emblica
Phytochemical content* Alkaloids (mg/g dry weight)
Total phenolics (mg/g dry weight)
Tannins (mg/ml)
Flavonoids (mg/g dry weight)
46.1 ± 1.3a
10.96 ± 0.42a
36.21 ± 0.18a
8.62 ± 0.72a
34.4 ± 2.4b
78.51 ± 0.63b
50.41 ± .0.27b
57.14 ± 0.65b
Different superscript letters in the same column indicate difference significance at CI ≥ 95%. * Values are means of triplicates ± standard deviation. 3
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Fig. 2. Scanning electron micrographs of the cells of Staphylococcus aureus MRSA-1 after treatment with seed extracts of Phyllanthus emblica (1) and Lycium shawii (2) after zero time (A), 3 h (B) and 6 h (C) of exposure to extracts.
The applications of plant seeds’ extracts, i.e. P. emblica and L. shawii, for textile finishing and ointment formulation proved to be successful for the control of normal S. aureus and MRSA strains. Both extracts and applications were effective for inhibiting the examined S. aureus strains. The extract treated textiles and ointments were more forceful against normal S. aureus strains than for MRSA strains. Regarding the antiMRSA activity of extract treated products, their antibacterial potentialities were very remarkable, as appeared from the growth inhibition zones, with a higher antibacterial activity of P. emblica extract than L. shawii extract toward the two examined MRSA strains (Fig. 3). The mean diameters of inhibition zones, from P. emblica extract-treated textiles, were 32.3, 31.7, 25.2 and 27.4 mm, including the textile length of 10 mm, against S. aureus strains (isolate 1, isolate 2, MRSA 1 and MRSA 2), respectively. Whereas the appeared zones, after challenging these strains with loaded textiles with L. shawii extract, were 17.5, 16.7, 14.6 and 15.3 mm, respectively.
extracts caused notable morphological alterations in comparison with the control, no micrographs for DMSO treated cells were captured in this study. It may observed, from the SEM micrographs of the zero-time treated and treated S. aureus cells, for 3 and 6 h, that zero-time treated cells (A-1, A-2) appeared with normal, round shape and smooth surfaces. The SEM micrographs seem to show homogenous and heterogeneous biofilm formation. After 3 h of exposure to P. emblica extract (B-1), the treated bacterial cells were shrunk, became tiny, diminutive and dehydrated and after 6 h of exposure (C-1), the entire bacterial cells were fully lysed, exploded or disrupted; the interior dehydrated cellular components and debris were the only observable matrix. Regarding L. shawii extract treatment, it is observable that after 3 h of exposure (B-2), the bacterial treated cells began to lyse and combine with each other’s. After 6 h of bacterial exposure to L. shawii extract (C-2); a complete cell wall lyses was observed and the only detectable materials were the residues of lysed cell walls combined with the released interior components from bacterial cells. It may be suggested that, at this stage, that the entire treated cells had lost all of their metabolic functions completely. 4
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Fig. 3. Anti-MRSA activity, as appeared inhibition zones, of seed extracts-treated textiles (T) and ointments (C) for the extracts of Phyllanthus emblica (P) and Lycium shawii (L) against the examined MRSA strains (1 and 2).
4. Discussion
possess an antibacterial activity against many bacterial strains except MRSA (Enomoto et al., 2001); thymol contents in N. sativa was suggested to be the main responsible of seeds antibacterial activity (Karapinar and Aktug, 1987). The P. oleracea extract was suggested to have a remarkable activity against both Gram + bacteria, Bacillus subtilis and S. aureus, and it was only active against one Gram negative strain, i.e. Pseudomonas aeruginosa (Bakkiyaraj and Pandiyaraj, 2001). The Allium spp. seed extracts did not show notable antibacterial activity against MRSA and other tested strains; this results could be supported with other reported results regarding A. ampeloprasum extract (Anuswedha and Rao, 2015) and for the extract of A. cepa (Grover et al., 2011). The powerful antimicrobial plant seeds in current investigation, i.e. P. emblica and L. shawii, had relatively varied amounts from bioactive phytochemicals, which could play an important role to force their bactericidal activity. The advantageous pharmaceutical properties of plant materials could be characteristically attributed to their contents from secondary products combination, e.g. phenol compounds, flavonoids, alkaloids, steroids and tannins (Cowan, 1999; Davidson, 2001). The bioactive phytochemicals in plants may have more effective mechanisms than those of chemical or synthetic antimicrobial drugs; thus they could be more significantly employed in the fighting of resistant microbes (Ali, 1991). It was suggested that P. emblica extract could be utilized as a bactericidal agent because of its containment from bioactive compounds, e.g. tannins such as emblicanin A and B, flavonoids, saponins and phenols, which were affirmed as effective chemotherapeutic (Javale and Sabnis, 2010; Jyothi and Subba, 2011; Kumar and Pandey, 2013). The major bioactive principals in P. emblica extract was indicated to contain ascorbic acid, gallic acid, flavonoids (quercetin), hydrolysed tannins (emblicanin A and B) and alkaloids (phyllantidine, phyllantine) (Patil et al., 2012). Tannins, from plant origin, were verified to possess an effectual antimicrobial activity (Min et al., 2008); the suggested action of that is to interact with cell proteins through many bioactive forces such as hydrogen bonding, formation of covalent bond or hydrophobic possessions. Accordingly, tannins could perform their antibacterial action through inactivating of microbial vital mechanisms, e.g. extracellular enzymes activity, microbial adhesions, suppression of oxidative
The multiple bacterial resistance to antibiotic is a great threat to human health worldwide; MRSA was considered from the most dangerous resistant pathogens that could endanger human life (Safdar et al., 2006). Almost all compounds derived from plant are supposed to possess high levels from biosafety to human, due to their natural, organic, biodegradable and biocompatible natures, and their recurrent usage by people for centuries (Kumar and Pandey, 2013; Tayel et al., 2013a). Many natural compounds, found in plants, spices and herbs, were proved to possess antimicrobial actions and could be served as sourced for antimicrobial drugs against microbial pathogens (Cowan, 1999). The variation in the antibacterial potentialities of examined plant seeds, in current study, could be anticipated to their dissimilar contents from microbicidal agents; this suggestion was mentioned in many previous studies that recommended that medicinal plants could be the ideal potential sources to explore novel antibacterial drugs even against antibiotic-resistant bacterial strains (Davidson, 2001; Ceylan and Fung, 2004). The total crude extract of P. emblica seeds exhibited a powerful antibacterial activity against tested S. aureus strains; these results are harmonized with other previously obtained result, which indicated the efficacy of methanolic and ethyl acetate extracts (Anbuselvi and Jha, 2015) and the aquas extract (Varghese et al., 2013) of P. emblica seeds against S. aureus isolates. Additionally, the potentiality of P. emblica extracts to act as antibacterial agents against Gram-positive and negative bacteria was indicated, exercising S. aureus and K. pneumoniae as representative pathogenic organisms (Reghu and Ravindra, 2010). The seed extract of L. shawii had a potent antibacterial activity against S. aureus, which is in disagreement with the results of Aldoweriej et al. (2016), who reported that the methanolic extract of aerial parts from L. shawii had weak activity against S. aureus. This could be indicate that the main antimicrobial active compounds in L. shawii present in its seeds and not in the aerial parts. Regarding the other examined plant seeds’ extracts, it was indicated that the extracts from P. harmala seeds have exhibited some antimicrobial potentiality toward pathogenic bacteria activity (Prashanth and John, 1999). The methanolic extract of black seeds (N. sativa) had also stated to 5
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phosphorylation or transport proteins in cell envelope (Scalbert, 1991). In addition, tannins were proposed to bind with proline-rich peptides, leading to the restriction of protein synthesis (Shimada, 2006). The applications of P. emblica and L. shawii seed extracts for fabrication of anti-S. aureus products, proved their effectiveness, in both loaded fabrics and ointment with extracts. This could indicate that plant seeds’ extracts were able to maintain their bioactivity after their loading onto cellulose fibers (textile) or into white paraffin (ointment), without executing chemical reaction with the delivery carrier materials. The decrement of antimicrobial activity in extracts-treated textiles and ointment, when compared to agar well diffusion test, could be attributed to the heat treatment in curing process, for textile, which could negatively affect the activity of antimicrobial compounds, and the barricade effect of white paraffin, in ointment, that might hinder the extract release and action into the growth media. The captured SEM micrographs, of treated S. aureus cells with P. emblica and L. shawii, clearly indicated the influence of extracts treatment on the cell morphology and could indicate the possible antimicrobial modes of action from the both extracts. Regarding the SEM micrographs of treated cells with P. emblica extract, it may be suggested that that the extract increased the osmotic pressure in the surrounding media, because of its high contents from bioactive phytochemicals, and thus derived the cells to release their interior moist contents and appeared with shrunk and diminutive shape, after 3 h of exposure to the extract. While after 6 h from the exposure to P. emblica extract, and due cells loss of their water contents, it could be expected that all of the enzymatical and biological process inside the cells, including cell walls synthesis mechanisms, was stopped and the cells tended to deform and lyse. From the SEM micrograpgh of treated S. aureus cells with L. shawii extract, it could be suggested that the extract performed its antibacterial action with different mechanism; the extract either interact with the outer membrane and cell walls of the bacteria, to increase their permeability and cause the release of their interior components, or may be penetrated inside the cells and interact with their vital components, e.g. DNA, RNA, Enzymes, etc., causing their inactivation or inhibiting their synthesis or action. The finished textiles with anti-MRSA extracts could be suggested for application in the manufacturing of intensive care and surgery coats, medical antibacterial bandages, bed covers and wound dressings, whereas the treated ointments could be applied for the topical and superficial usages on infected skins.
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