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Antibacterial Activity and Mode of Action of Mentha Arvensis Ethanol Extract Against Multidrug-Resistant Acinetobacter Baumannii

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Antibacterial Activity and Mode of Action of Mentha Arvensis Ethanol Extract Against Multidrug-Resistant Acinetobacter Baumannii Zhang et al Tropical Journal of Pharmaceutical Research November 2015; 14 (11): 2099-2106 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v14i11.21 Original Research Article Antibacterial Activity and Mode of Action of Mentha arvensis Ethanol Extract against Multidrug-Resistant Acinetobacter baumannii Ling Zhang1, Shu-gen Xu1, Wei Liang2, Jun Mei1, Yue-ying Di1, Hui-hua Lan1, Yan Yang1, Wei-wei Wang1, Yuan-yuan Luo1 and Hou-zhao Wang1* 1Central Laboratory, The 174th Hospital of the Chinese People's Liberation Army, The Affiliated Chenggong Hospital of Xiamen University, 2Hospital Infection Management Department, The First Affiliated Hospital of Xiamen University, Xiamen 361003 China *For correspondence: Email: [email protected]; Tel/Fax: 0086-592-6335560 Received: 19 May 2015 Revised accepted: 6 October 2015 Abstract Purpose: To evaluate the antibacterial effect of ethanol extract of Mentha arvensis against multi-drug resistant Acinetobacter baumannii using liquid chromatography–mass spectrometry (LC-ESI-MS). Methods: Disc diffusion and microdilution assays were used to evaluate the antibacterial effect of the extract by measuring the zone of inhibition, minimum inhibitory concentration (MIC) and and minimum bacteriocidal concentration (MBC) of the extract against the test bacteria. Scanning electron microscopy (SEM) was employed to evaluate the morphological changes induced by the extract in cellular membrane of the bacteria. Reactive oxygen species (ROS) generation and protein leakage from the bacterial cells induced by the extract were also evaluated. Results: The extract showed dose-dependent growth inhibitory effects against A. baumannii with MIC and MBC of 23.5 and 72.1 µg/mL, respectively. The extract also induced potent ROS generation and protein leakage in A. baumannii bacterial cells. SEM findings revealed that the extract induced potential cellular damage which increased with increasing extract concentration. Conclusion: The ethanol extract of Mentha arvensis is a potent antibacterial agent against A. baumannii and acts by inducing lethal cellular damage to the bacterium. Keywords: Mentha arvensis, Acinetobacter baumannii, Reactive oxygen species, Antibacterial activity, Cellular membrane damage Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION drug resistance could lead to serious epidemic since, no drug can have any visible effect on the Infectious diseases caused by bacterial and pathogenic microbes [1]. The less accessibility fungal infections are the key causes of mortality and high price of new generation antibiotics in tropical and subtropical countries. It is now demands looking for the substances from well established that due to the indiscriminate alternative medicines with proved antimicrobial uses of various antibiotic, predominantly potential. A huge number of medicinal plants synthetic drugs, the pathogenic microbes have have been reported to show antimicrobial activity acquired multidrug resistance which have especially against drug resistant microbes. It is created severe health issues mainly in estimated that plants have provided the developing countries. Most of the time, this multi- Trop J Pharm Res, November 2015; 14(11): 2099 Zhang et al prototypes for approximately 50 % of Western column (5.6 mm ID, 60 mm length) (Merck) was drugs [2,3]. used. The mobile phase was a gradient prepared from solvent A (0.3 % aqueous formic acid) and Acinetobacter baumannii is a gram-negative solvent B (acetonitrile), and the conditions used opportunistic pathogenic bacterium responsible for gradient elution were: 0-5 min, 5 – 20 % B; 5 - for various nosocomial infections, particularly in 10 min, 25 % B; 10 - 15 min, 25 - 35 % B; 15 - 20 intensive care units (ICU) of hospitals. This min, 45 – 100 % B; 20 - 25 min, 100 % B. The pathogenic microbe causes a range of infections separation was conducted at a flow rate of 0.5 including urinary tract infections, ventilator mL/min. The injection volume was 5 μL. associated pneumonia, surgical-site infections, etc. Multidrug resistance has been recently LC–MS equipment (LC–MS QqQ-6410B Agilent reported in most A. baumannii infections [4,5]. As Technologies) consisted of a chromatographic a result, A. baumannii has emerged as one of the system (1260 Infinity Agilent Technologies) most infection-causing and challenging microbial coupled with an Agilent Triple Quad mass pathogen with limited treatment options, since spectrometer fitted with an ESI source. MS only a few currently used antibiotics can have conditions were the following: MS range 100– appreciable effects on it. 1200 Da, MSn spectra were obtained using both positive and negative modes, nebulizer gas 45 The objective of the current study was to Psi, gas temperature 325 oC, capillary voltage evaluate the bioactivities and the antibacterial 4000 V. activity of ethanol extract of Mentha arvensis against multi-drug resistant bacterial strains of Bacterial strain and culture media Acinetobacter baumannii. A. baumannii strain ATCC 10545 was used in the EXPERIMENTAL current study. The bacterial strain was procured from the State Key Laboratory of Microbial Solvents Resources (SKLMR), the institute of microbiology, Chinese academy of Sciences, HPLC-MS grade acetonitrile were purchased China. The Bacterial strain was grown on nutrient from Merck Co., (Darmstadt, GER). High–purity agar plates at 37 ºC and maintained on nutrient deionized water was obtained from a Milli-Q agar slants. Cell suspension of A. baumannii water purification system (Millipore Bedford, MA, microorganisms in 0.5 % NaCl was adjusted at 5 USA). Methanol used for plant extraction was 0.5 Mcfarland to obtain approximately 10 from ANPEL Scientific Instrument Co. (Shanghai, cfu/mL. China). Initial screening for antimicrobial Preparation of the plant extract susceptibility by disc diffusion assay The aerial parts of Mentha arvensis were The antimicrobial test was performed by disc collected during August 2014 from Xiamen, diffusion assay as per NCCLS, 1997 [6]. The China. The plant material was identified by Prof nutrient agar plates containing an inoculum size 5 Jian Zunzhao, a voucher specimen (no. 14-HSU- of 10 cfu/mL on Saboraud glucose agar plates 779-23) was deposited in the Herbarium of were used. Earlier prepared extract impregnated Southeast University, Nanjing, China. The aerial disc (6 mm in diameter) at the concentrations of parts of the plant were thoroughly washed with 200 μg/mL for bacterial strains were placed tap water, shade-dried and then chopped into aseptically on sensitivity plates with proper small pieces. Ethanol (95 %) was used for hot controls. Oxacillin and ciprofloxacin (100 μg/mL) extraction for 3 h using a Soxhlet extraction was used as standard antibacterial agents. o apparatus. The extract was then concentrated Plates were incubated at 37 C for 24 h. under reduced pressure in a rotary evaporator at Antibacterial susceptibility was recorded by 45 oC and was then kept in a refrigerator at 4 oC measuring the diameter of zones of growth before use. inhibition on agar surface around the discs. Liquid chromatography–mass spectrometry Determination of minimum inhibitory (LC–ESI-MS)/HPLC analysis concentration (MIC) and minimum bactericidal concentration (MBC) HPLC analysis was carried out by a Nexera HPLC system (Shimadzu, Japan) with a double- MIC and MBC tests were done by the broth pump (LC-30AD), column oven and Auto microdilution method [7]. Mentha arvensis extract sampler (SIL-30AC). A Chromolith RP-18e was dissolved in sterilized physiological saline Trop J Pharm Res, November 2015; 14(11): 2100 Zhang et al solution (0.8 %) supplemented with Tween-80 solution containing 0.2 % Tween-80. Different (Sigma) at final concentration of 0.5 % (v/v). concentrations of the extract (0, 10, 50 and 100 Sequential doubling dilutions of the extract were µg/mL) were prepared and added into this prepared in a 96-well microtiter plate ranging suspension and was incubated at room from 10 % to 0.125 %. Each dilution (100 μL) temperature. After 24 h, the bacterial cells were was distributed into the wells, then inoculated centrifuged at 12,000 x g for 15 min. The with (100 μL) of the bacterial suspension. The bacterial cells were then washed with 0.5 mol/L final concentration of each strain was adjusted to Tris-acetate buffer (PH 7.4), fixed in tris-acetate 105 - 106 CFU/mL. The concentration of the buffer containing 2.5 % glutaraldehyde, and then extract at which the microorganisms reveal no freeze-dried. The bacterial culture was then observable growth is defined as minimum analyzed by SEM (Hitachi, Japan) at inhibitory concentration (MIC) while the extract magnifications of 4,000 x. The bacterial cell concentration at which the bacterial pathogens suspension in saline with no extract treatment are killed is defined as minimum bactericidal served as a negative control. concentration (MBC). All the experiments were done in triplicate. Statistical analysis Effect of extract on reactive oxygen species All
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  • Wild Mint (Mentha Arvensis L.) Plant Family: Lamiaceae
    BWSR Featured Plant Name : Wild Mint (Mentha arvensis L.) Plant Family: Lamiaceae Recognized world-wide, Mentha arvensis is a Plant Family: Legume Statewide Wetland popular species that possesses an interesting Indicator Status: origin story in Greek mythology. Mentha, the name deriving from Mintho, was mistress to FACW Hades. Upon discovery of Mintho, jealous Persephone trampled her until she transformed into a lowly plant to be forever walked on. As one of the few native mint species in the United States, it is a vigorous grower and a favorite of pollinators due to its sweet smell. The sweet smell is reportedly because of Hades attempting to make poor Mintho’s fate more tolerable – the more the Whorl of tubular flowers plant is trampled, the stronger and sweeter its aroma. Identification Sprawling wild mint generally grows between 6” to 18” tall with a square angular stem covered with fine hairs. The stem is stiff with little branching and will vary in color from red to bright green. Its leaves are opposite, egg-shaped and elliptic with a pointed tip and serrated edge. One to two inches long and a half inch to one-inch-wide, these edible leaves are a major reason for the plant’s aroma which is only noticeable when crushed. The flowers are tubular whorls that emerge around the axis of the upper leaves. Usually 4 lobed but sometimes irregular, each flower grows to about 1/8” long and can be white, pink or lavender. The bloom period is from July to September in which each flower produces small nutlets containing one 1mm long seed.
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