| کلیدواژههای انگلیسی مقاله |
Herbal medicine, Anti-bacterial agents, Wounds and injuries, Phenols, Flavonoids, What&,rsquo s Known Plants are a good natural source for the development of novel, safe, and cost-effective antibacterial agents. In southern Iran, Prunus scoparia (P. scoparia) root is a commonly used herbal medicine to treat burn wound infections. Studies on the antibacterial activity of P. scoparia root are scarce. What&,rsquo s New For the first time, we demonstrated the presence of various secondary metabolites and the antibacterial activity of the methanolic extract of P. scoparia root against the most common burn wound pathogens. Prunus scoparia root showed potent anti-Methicillin-resistant Staphylococcus aureus activity. IntroductionMajor burn wounds can be a traumatic injury, and the affected patients need immediate treatment to limit potential complications and even death. 1, Burn injuries initially damage the skin the first barrier against various pathogens. Skin damage along with diminished local and systemic immune responses creates an environment for pathogens to colonize and grow rapidly on the burn wound surface. Infections and sepsis are the leading causes of death in patients with burn injuries. 1,, 2, The burn wound surface provides a favorable environment for microbial colonization and proliferation. 3, Typically, Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes) are the first bacterial invaders to colonize the burn wound surface, followed by various bacteria from normal gut flora and infections during hospitalization. 4, The mortality rate due to sepsis is high in patients with burn injuries. This is a type of body reaction that can eventually lead to death by triggering a syndrome known as multiple organ dysfunction syndromes. 5, The discovery of multidrug-resistant organisms has encouraged researchers to seek novel drugs with higher efficiency, higher safety, and particularly lower costs. 6, Plants are a vast natural source to develop novel antibacterial agents and a cost-effective treatment option for many human diseases. 7, To date, several studies have been conducted to utilize herbs and plants to develop alternative therapeutics against bacterial pathogens. 7,- 9, Prunus scoparia (P. scoparia) is a wild almond species native to Iran, Turkey, Turkmenistan, and Afghanistan. 10, This plant has been widely used as herbal medicines to treat respiratory and cardiovascular diseases, headaches, rheumatism, and wounds. Previous studies have shown the antidiabetic, 11, antioxidant, and antifungal 12, properties of the shoot extract of P. scoparia. However, as concluded from our literature review, no studies have investigated the antibacterial activities of the root extract of P. scoparia. In southern Iran (Fars Province), P. scoparia root is a commonly used herbal medicine in the treatment of burn wound infections. Hence, The present ethnobotanical study aimed to assess the in vitro antibacterial activity of the methanolic extract of P. scoparia against the most common burn wound pathogens. Materials and MethodsThe study protocol was approved by the Ethics Committee of AJA University of Medical Sciences, Tehran, Iran. The experiments were performed at the School of Pharmacy at Shiraz University of Medical Sciences, Shiraz, Iran, during 2018-2019. ReagentsMethanol, petroleum ether, dichloromethane, ethyl acetate, and all phytochemical screening reagents were purchased from Merck, Germany. Mueller-Hinton agar and Mueller-Hinton broth were purchased from Sigma-Aldrich, USA.Plant CollectionThe plants were collected from the mountains in southern Iran during spring 2018. The exact location was Gal&,#363 Bor&,#257 q village near the city of Jahrom, Fars Province, Iran (28&,deg 23&,prime 05&,Prime N 53&,deg 54&,prime 14&,Prime E). The collected plants were authenticated by an expert at Shiraz University of Medical Sciences, Shiraz, Iran (voucher number 3014).Preparation of Plant ExtractsThe roots of the collected plants were dried and ground using a grinding apparatus (MX-110PN, Japan). The extraction process was carried using 80% methanol solvent. In brief, 200 mL of the solvent was mixed with 100 g of the powdered plant root. The mixture was properly stored for 24 hours during which it was stirred several times with a glass rod. Then, the mixture was placed in a rotary evaporator (Heidolph Co., Germany) to condense the extract until the solvent was evaporated. The extract was kept in a freezer (LG, South Korea) at -20 &,deg C until further use. 13, MicroorganismsBacterial strains included Methicillin-resistant Staphylococcus aureus (MRSA) (a clinical isolate from a patient with burn wounds at Nemazee Hospital, Shiraz, Iran), Enterococcus faecalis (ATCC 29212), Acinetobacter baumannii (NCTC 13304), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 700603), and Serratia marcescens (ATCC BAA2808). Broth Microdilution and Agar Well Diffusion AssaysBroth microdilution assay was performed to obtain minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) of the plant root extracts against the bacterial strains. 8, In brief, the methanol extract was dissolved in 1% dimethyl sulfoxide and water and transferred onto 96-well plates to obtain serial dilutions (0.39-50 mg/mL). Then, the bacterial suspension with a turbidity standard of 0.5 McFarland was added into the wells to reach a bacterial concentration of 1.5&,times 105 CFU/mL in each well with a final volume of 200 &,mu L. The lowest concentration of the extract that showed no visible bacterial growth in the broth (after 24 hours incubation at 37 &,deg C) was noted as MIC. Additionally, the lowest extract concentration killing 99.9% of bacterial inoculum was noted as MBC. MBC values were determined after sub-culturing 100 &,mu L of the bacterial suspension on the Mueller-Hinton agar. All procedures were carried out in triplicates, and the values were presented as the mean of the three datasets. In accordance with a previous study, the antibacterial activity of the plant root was evaluated using the agar well diffusion method. 8, Fractionation of the Crude ExtractsFractionation was performed using a separating funnel. In brief, the extract was suspended in 150 mL of the methanol-water mixture (2,1). The separation was carried out by applying organic solvents such as petroleum ether, dichloromethane, and ethyl acetate to enhance polarity (figure 1,).Figure 1. The fractionation procedure to obtain pure extracts is illustrated.Preliminary Phytochemical ScreeningThe total plant extract and the fractions were checked in accordance with a previously described protocol 14, for the presence of various secondary metabolites including phenols, flavonoids, alkaloids, saponins, tannins, anthocyanins, anthraquinones, and terpenoids.Folin-Ciocalteu (F-C) Assay for Total Phenolic Content (TPC)Briefly, 500 &,micro L of different concentrations of the plant extract fractions in water was mixed with 1.5 mL of prediluted Folin-Ciocalteu reagent, gallic acid, (40 &,mu L, Merck, Germany). The mixture was then incubated at room temperature for five minutes. Sodium bicarbonate (1.2 mL, 7.5% w/v) was added, and the mixture was again incubated at room temperature for 60 minutes. Then, the absorbance of the mixture was measured at 765 nm. For calibration, several concentrations of gallic acid (10-500 mg/L) were used, and the measurements were expressed in mg of gallic acid equivalents (GAE) per g of the sample.Total Flavonoid ContentThe total flavonoid content (TFC) was determined based on a colorimetric assay method. 15, In brief, after mixing 1 mL of the extract fraction with 4 mL of distilled water in a tube, 0.3 mL of sodium nitrate (5%) and 0.3 mL of aluminum chloride (10%) were added to the mixture. After incubation at room temperature for five minutes, 2 mL sodium hydroxide (1 M) was added to the tube, and distilled water was added to the mixture to increase the volume to 10 mL. The test tube was placed on a shaker for five minutes, and the absorbance was measured at 510 nm. For calibration, several concentrations of catechin (50-100 mg/L) were used, and the measurements were expressed in mg of catechin equivalents (CEQ) per g of the sample. All the reagents were purchased from Merck, Germany.ResultsThe MIC and MBC values of the total plant root extract and fractions against the tested microorganisms are presented in table 1,. The results showed effective antibacterial activity of the root extract of P. scoparia against the microorganisms. We observed low or no antibacterial activity from the aqueous and chloroformic extracts of the plant root. The methanol extract was used for fractionation and phytochemical analysis. In most cases, the ethyl acetate fraction of the methanolic extract showed a better antibacterial response against the tested bacterial strains. PlantExtractTested microorganisms and antibacterial parametersS. aureus (MRSA isolate)E. faecalis (ATCC 29212)A. baumannii (NCTC 13304)E. coli (ATCC 25922)P. aeruginosa (ATCC 27853)K. pneumoniae (ATCC 700603)S. marcescens (ATCC BAA2808)MICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBCP. scoparia (root)Chloroform 25255050252512.5012.505050505012.5012.50Aqueous--------------Methanol extract1.151.156.256.256.256.2512.5012.5012.5012.5025256.256.25Petroleum ether--------------Dichloromethane--------------Ethyl acetate0.781.153.123.123.123.126.256.253.126.253.123.126.2512.50Remaining methanol extract3.126.25----------12.5025 |
| نویسندگان مقاله |
Amir Roointan |
Department of Genetics and Advanced Medical Technology, School of Medicine, AJA University of Medical Sciences, Tehran, Iran
Reza Kamali-Kakhki |
Department of Genetics and Advanced Medical Technology, School of Medicine, AJA University of Medical Sciences, Tehran, Iran
Mohammad Fathalipour |
Department of Genetics and Advanced Medical Technology, School of Medicine, AJA University of Medical Sciences, Tehran, Iran
Zohreh Hashemi |
Department of Genetics and Advanced Medical Technology, School of Medicine, AJA University of Medical Sciences, Tehran, Iran
Mohammad Mehdi Zarshenas |
Department of Phytopharmaceuticals (Traditional Pharmacy), School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
Mohammad Soleimani |
Department of Microbiology, School of Medicine, AJA University of Medical Sciences, Tehran, Iran
Ruhola Mirjani |
Department of Genetics and Advanced Medical Technology, School of Medicine, AJA University of Medical Sciences, Tehran, Iran
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