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Antibacterial activities, phytochemical analysis and chemical composition Makhlaseh extracts against the growth of some pathogenic strain causing poisoning and infection
Accepted Manuscript Antibacterial activities, phytochemical analysis and chemical composition Makhlaseh extracts against the growth of some pathogenic strain causing poisoning and infection Behrooz Alizadeh Behbahani, Abbas Ali Imani Fooladi PII:
S0882-4010(17)31604-2
DOI:
10.1016/j.micpath.2017.12.002
Reference:
YMPAT 2638
To appear in:
Microbial Pathogenesis
Received Date: 27 November 2017 Revised Date:
1 December 2017
Accepted Date: 1 December 2017
Please cite this article as: Alizadeh Behbahani B, Imani Fooladi AA, Antibacterial activities, phytochemical analysis and chemical composition Makhlaseh extracts against the growth of some pathogenic strain causing poisoning and infection, Microbial Pathogenesis (2018), doi: 10.1016/ j.micpath.2017.12.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Antibacterial activities, phytochemical analysis and chemical composition Makhlaseh extracts against the growth of some pathogenic strain causing poisoning and infection
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Behrooz Alizadeh Behbahani1, Abbas Ali Imani Fooladi1* 1.Applied Microbiology Research Center, System Biology and Poisoning institute, Baqiyatallah University of Medical Sciences, Sheikh Bahaei Street, Molla Sadra Street, Vanak Sq., Tehran 984359-44711, Iran
Corresponding author: [email protected] or [email protected] Abstract
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The aim of this study was to investigate the antibacterial activities and phytochemical
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analysis of extracts against the growth of some pathogenic strain causing poisoning and infection
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(Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, Enterobacter
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aerogenes, Escherichia coli and Shigella flexneri). Makhlaseh components were identified via
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gas chromatography/mass spectrometry (GC/MS). Total phenolic content (TPC), alkaloids,
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tannins and saponins were determined. Antioxidant activity was determined calorimetrically for
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2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. Antimicrobial effect of extracts was
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evaluated by five methods, pour plate, well diffusion, disk diffusion, minimum inhibitory
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concentration (MIC), and minimum bactericidal concentration (MBC). Camphor was the major
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compound of Makhlaseh. The TPC of aqueous and ethanolic Makhlaseh extracts was equal to
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79.45 ± 1.15 and 115.26 ± 1.23 µg GAE/mg, respectively. The antioxidant activity (IC50) test of
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aqueous and ethanolic Makhlaseh extracts showed 315.50 ± 1.12 and 118.35 ± 1.08 µg/ml,
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respectively. MIC of the aqueous extract of Makhlaseh for Enterobacter aerogenes, Escherichia
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coli, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus
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pyogenes were 32, 32, 16, 16, 8 and 8 mg/ml, respectively, and the MIC of the ethanolic extract
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were 16, 16, 16, 8, 4, and 4 mg/ml, respectively. The MBCs of the Makhlaseh extracts varied
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from 4 mg/ml to 128 mg/ml. Increasing concentration of Makhlaseh extracts had a significant
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effect (p≤ 0.05) on inhibition zone diameter. In conclusion, using Makhlaseh extracts as a natural
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antibacterial composite in vitro have significant antibacterial ability over the studied strains.
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Keywords: Makhlaseh, Extracts, Antimicrobial effect, Phytochemical analysis.
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1- Introduction
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The increase in resistance of pathogenic bacteria due to multi-various use of number of
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common therapeutic antibiotics has encouraged scientists all over the world to search for new
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antibacterial and antifungal substances from various antecedents comprising medicinal herbal.
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Therefore, it is highly appreciated to find out ways overcoming these issues. For a long age of
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time, plants have been a worth source of natural products for maintaining human safety. The use
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of herb extracts and phytochemicals, both with known antimicrobial attributes, can be of great
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significance in therapeutic treatments. Nowadays, a new approach has emerged to the use of
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herbal medicines to treat infections, since herbal medicines are stronger antimicrobial activity
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and fewer side effects than chemical drugs (1-3). In various regions of Iran (Mazandaran, Gilan,
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Golestan, Kurdistan, Fars, Khorasan, Tehran and Semnan) Makhlaseh is known as Qaliheh and
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Noroozi plant. Makhlaseh a member of Asteraceae, is a perennial herbaceous plant with sporadic
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fluff and a short straight root (4, 5).
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Enterobacter aerogenes is a pathogenic bacterium gram negative that reason opportunistic
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infections including most types of infections. Streptococcus pyogenes is a pathogenic bacterium
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gram positive can also cause disease in the form of post infectious “nonpyogenic” syndromes.
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Septicemia and endocarditis are also diseases be associated with Staphylococcus epidermidis.
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Shigella flexneri infections can usually be treated with common therapeutic antibiotics, however
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some strains have become resistant. Escherichia coli cause urinary tract infections, bacteremia
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and other infections in humans (6-9).
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The useful effects of phenolic composition such as anticancer, antimutagenic and heart-
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protective attributed, also the synthetic antioxidants being suspected to toxicity, have led to
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numerous studies for the extraction of these composition from natural resources. Phenolic
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composition are the constituents with antioxidant activity (10,11). The aim of this study was to identify the chemical compositions and to measure
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phytochemical analysis and the antioxidant activity of aqueous and ethanolic Makhlaseh extracts
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in addition to its free radical scavenging activity. The other aim of this research was to
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investigate the antibacterial effects of aqueous and ethanolic Makhlaseh extracts on pathogenic
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strain causing infection.
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2- Materials and methods 2-1- Chemicals and reagents
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All reagents and microbial media show in Table 1.
Table 1. All microbial media and reagents
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2-2- Extract preparation
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Makhlaseh was collected from Kurdistan province, Iran. After the confirmation of the plant
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scientific name (the species of the plant was identified and confirmed by herbarium, the herbal
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systematic laboratory of Ferdowsi University of Mashhad), it was rinsed with cold water,
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shadow dried and powdered using a lab grinder. The extraction of the Makhlaseh was conducted
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according to the method of Alizadeh Behbahani et al., (2017) (11) with some modifications.
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Water and ethanol were used for the maceration method. For this purpose, 25 g of the powdered
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Makhlaseh was added to 225 ml of the solvent (water and ethanol) for 48 hours (shaking
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occasionally with a shaker). After dissolving process, materials were filtered (Whatman no. 1
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filter paper) and centrifuged in 9000 g for 10 minutes. Then extracts were evaporated using
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rotary evaporator (Rota-vapor R-205, England) and dried extracts were obtained and stored at
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4°C in air tight screwcap tube.
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2-3- Determination of the dry weight of the extracts
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The extraction of the Makhlaseh was conducted according to the method of Tabatabaei
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Yazdi et al., (2015) (12). Briefly, in order to determine the dry weight, first a test tube was
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weighed using a digital balance. one ml of the Makhlaseh extracts (aqueous and ethanolic) was
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poured into it and dried at 25°C. The test tube was weighed again after drying. The difference
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between the weighs of the test tube before and after drying was recorded.
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2-4- Phytochemical analysis
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2-4-1- Alkaloids
The alkaloids Makhlaseh was conducted according to the method of Njoku and Obi., (2009)
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(13).
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2-4-2- Tannins
The tannins Makhlaseh was conducted according to the method of Njoku and Obi., (2009)
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(13).
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2-4-3- Saponins
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The saponins Makhlaseh was conducted according to the method of Njoku and Obi., (2009) (13).
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2-4-4- Phenolics
The phenolics Makhlaseh was conducted according to the method of Alizadeh Behbahani et
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al., (2017) (11).
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2-5- Identification of chemical composition
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chromatograph and coupled to a mass spectrometer (14).
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Identification of the Makhlaseh substances was performed by injecting Makhlaseh into a gas
2-6- Estimation of total phenolic content (TPC) and Antioxidant activity
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For this purpose, the method of Folin-Ciocalteu is applied for the determination of TPC. The
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antioxidant activity of Makhlaseh was estimated according to the method previously described
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by Alizadeh Behbahani et al (2017) (15).
2-7- Preparation of the microbial strains
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The following Gram-positive and Gram-negative bacteria were used for testing antibacterial activity. Staphylococcus epidermidis ATCC 12228, Streptococcus pyogenes ATTC 19615,
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Staphylococcus aureus ATTC 25923, Enterobacter aerogenes ATTC 13048, Escherichia coli
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ATTC 25922 and Shigella flexneri ATCC12022 all American type culture collections were
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obtained from the department of food science and technology of Ferdowsi University of
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Mashhad (FUM), Mashhad, Iran.
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2-8- Suspension Preparation
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based on a standard 0.5 McFarland (16).
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For this purpose, its concentration was adjusted to 1.5×108 CFU (colony forming units)/ml
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2-9- Antimicrobial test
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from each other and the plate edge. To that end, the concentrations of 10, 20, 30 and 40 mg/ml
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were prepared in a suitable solvent. The disks of gentamycin and vancomycin were also used at a
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concentration of 10 µg/ml. after that, the culture media containing bacteria were incubated at 37
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°C for 24. Eventually, the inhibition zone diameters formed around the disks were measured
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using a ruler and recorded (17).
2-9-1- Disk diffusion agar (DDA)
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2-9-2- Well diffusion agar (WDA)
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In this method, blank paper disks were placed on the culture medium at certain distances
In this method, 0.5 McFarland suspension was poured onto MHA at three points for bacteria and spread using a L-shaped spreader. Next, 4 wells with diameters of 6 mm were created on the
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surface of the culture media using the bottom of the Pasteur pipette. After incubating the culture
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media containing bacteria at 37 °C for 24, given each well diameter, the diameter of the
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inhibition zone surrounding the well was quantified using a ruler and expressed in mm (14, 15).
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2-9-3- Pour plate method (PPM)
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PPM is a qualitative method and its results are expressed as sensitive, semi-sensitive and resistant (11).
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2-9-4- Determination of minimum inhibitory concentration (MIC) through micro dilution broth
In this method, a stock solution with concentration of 512 mg/ml was prepared from
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Makhlaseh. Then, prepare sequential concentrations of 2, 4, 8, 16, 32, 64, 128 and 256 mg/ml. In
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order to estimate MIC through micro dilution broth (15, 18).
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2-9-5- Minimum Bactericidal Concentration
Given the results obtained from the MIC test, 100 µl was removed from the wells free of the
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red color, and cultured on MHA media. Subsequently, the culture media were incubated at 37 °C
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for 24 h. The concentrations at which microbial growth did not happen, were regarded as the
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MBC of Makhlaseh for pathogenic strain causing infection (11).
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2-10- Statistical analysis
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Microsoft Windows Excel 2016 and SPSS (Version18.0, SPSS Inc., Chicago, USA) were
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used to analyze the obtained data. The data were initially assessed by analysis of variance
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(ANOVA), and the Duncan’s multiple range test was subsequently applied to detect significant
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(p < 0.05) differences. All experiments were triplicated.
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3- Results and discussion 3-1- chemical composition
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The results of Makhlaseh chemical composition analysis through GC-MS showed that
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Camphor was the major compound followed by camphene, α-pinene chrysanthenyl acetate, β-
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chrysanthenyl acetate and limonene. Polatoglu et al (2010) (19), reported that camphor (49%),
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trans-chrysanthenyl acetate (22.1%) and pcymene (5.2%) were the major constituents of
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Makhlaseh. Akpulat et al (2005), verified the chemical compound of Makhlaseh in Turkey and
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concluded that camphor (56.9%), camphene (12.7%) and pcymene (5.2%) were the major
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components of Makhlaseh (20). Our findings conformed to those of the others to a large extent.
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The chemical compositions of essential oils differ from one another considering the variety,
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climate, growth stage, collection time and growth location (21).
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3-2- Phytochemical analysis, antioxidant activity and total phenolic content (TPC)
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saponins, and phenolics in the aqueous and ethanolic Makhlaseh extracts. The results
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demonstrated that the phenolic compounds had the largest content in this ethanolic Makhlaseh
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extracts (Table 2). The TPC of aqueous and ethanolic Makhlaseh extracts was equal to 79.45 ±
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1.15 and 115.26 ± 1.23 µg GAE/mg, respectively. TPC was calculated on the standard curve
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through the Folin-Ciocalteu method. The antioxidant activity (IC50) test of aqueous and ethanolic
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Makhlaseh extracts showed 315.50 ± 1.12 and 118.35 ± 1.08 µg/ml, respectively. The
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antioxidant activity of the Makhlaseh was directly related to its phenolic composition. Many
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studies have proven the direct relationship between TPC and antioxidant activity (14, 15).
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Polatoglu et al (2010), reported that Makhlaseh had antioxidant activity (19). Cao et al (1998),
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(22) and Alizadeh Behbahani et al (2017), (14) reported that the differences in the TPC and
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antioxidant properties of different plants are influenced by a variety of factors such as climatic
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conditions (climate, soil and altitude).
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Table 2: Phytochemical constituents of aqueous and ethanolic Makhlaseh extracts
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3-3- Antimicrobial effect The results of the antimicrobial effects of aqueous and ethanolic Makhlaseh extracts, using
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the method of pour plate method show that 2 mg/ml concentration of ethanolic Makhlaseh
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extract, were quite effective on reduce of growth Staphylococcus epidermidis and Streptococcus
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pyogenes and were had prevent growth over the medium. However, 2 mg/ml concentration
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ethanolic Makhlaseh extract, have no significant antibacterial effect on Escherichia coli,
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Enterobacter aerogenes and Shigella flexneri. The results showed 2 mg/ml concentration of
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aqueous Makhlaseh extract, were effective on reduce of growth Staphylococcus epidermidis and
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Streptococcus pyogenes and were not had prevent growth over the medium. 2 mg/ml
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concentration aqueous Makhlaseh extract, have no significant antibacterial effect on Escherichia
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coli, Enterobacter aerogenes and Shigella flexneri.
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The antimicrobial effect of aqueous and ethanolic Makhlaseh extracts was investigated
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through disk diffusion agar (DDA) method. The results (Table 3) presented that aqueous and
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ethanolic Makhlaseh extracts had the most detrimental effect against Staphylococcus epidermidis
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and Streptococcus pyogenes at 40 mg/ml. The smallest inhibition zone diameter against different
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aqueous and ethanolic Makhlaseh concentrations belonged to Gram-negative bacteria. The
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results also indicated that no inhibition zone was observed for Enterobacter aerogenes and
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Escherichia coli at the concentrations of 10 and 20 mg/ml aqueous extract. The results indicated
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that no inhibition zone was observed for Shigella flexneri at the concentrations of 10 mg/ml
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aqueous extract. Inhibition zone was observed at all concentrations for all Gram-positive
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bacteria. The results demonstrated that the investigated bacteria were significantly different
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(p<0.05) in terms of susceptibility to Makhlaseh through the DDA method. The pairwise
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comparison between the effects of aqueous and ethanolic Makhlaseh extracts concentrations on
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the bacteria revealed that there were significant differences between the concentrations and as
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aqueous and ethanolic Makhlaseh extracts concentration increased, the inhibition zone diameter
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increased, too. The results of the antimicrobial effects of aqueous and ethanolic Makhlaseh extracts through
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well diffusion agar (WDA) method are summarized in Table 4. They showed that inhibition zone
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was observed for all Gram-positive bacteria at all aqueous and ethanolic Makhlaseh extracts
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concentrations. No inhibition zone was observed for Enterobacter aerogenes, Escherichia coli
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and Shigella flexneri at 10 mg/ml aqueous extract. In well diffusion agar method, due to the
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direct contact between the extracts and the bacteria on the surface of the culture medium, the
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inhibition zone diameter was larger than that of disk diffusion agar method, because in disk
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diffusion agar method, it was necessary that Makhlaseh diffuse into the surface of the culture
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medium through a paper disk (14, 15). Significant differences were observed for other
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microorganisms at all concentrations.
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The results of WDA and DDA demonstrated that Gram-positive bacteria were more sensitive
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to Makhlaseh than the Gram-negative ones. In general, the sensitivity profile of the bacteria
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studied in this research is as follows:
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Enterobacter aerogenes> Escherichia coli> Shigella flexneri > Staphylococcus aureus >
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Staphylococcus epidermidis > Streptococcus pyogenes
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The higher resistance of Gram-negative bacteria to the extract of medicinal herbs could be
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attach to the more assembled structure of the cell membrane of these bacteria contrast with the
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single-layer structure of the Gram-positive ones. Gram-positive bacteria have mucopeptide in
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their cell walls, while lipopolysaccharides constitute the bulk of the cell walls of Gram-negative
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bacteria like Enterobacter aerogenes, Escherichia coli and Shigella flexneri. On the other hand,
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it seems that the resistance of bacterial cells belongs to the solvability rate and extent of
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antimicrobial compounds (Makhlaseh) in the lipid moiety of the microorganism cell membrane.
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However, this could not be compelling sake for the higher susceptibility of Gram-positive
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bacteria than the Gram-negative ones. In addition, the diversity in the hydrophobicity of the cell
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membrane surfaces can also be said as an influential invoice (23, 24)
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Similar studies concerning the antimicrobial effects of the extracts of medicinal plants which
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have been carried out on a variety of Gram-positive and Gram-negative bacteria, confirm the
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above sentences (14, 15). According to the findings of the present study, camphor is the major
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constituent of Makhlaseh. Izadi et al (2010) (25) examined the antimicrobial effect of Makhlaseh
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via Kirby-Bauer method on Gram-negative bacteria and Gram-positive bacteria. They concluded
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that Makhlaseh had a more pronounced antimicrobial effect on the Gram-positive bacteria. Their
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results are consistent with ours.
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The results of minimum inhibitory concentration (MIC) are exhibited in Table 5. The MIC of
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Makhlaseh ranged from 4 to 32 mg/ml depending on the type of bacteria. The MIC of the
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aqueous extract of Makhlaseh for Enterobacter aerogenes, Escherichia coli, Shigella flexneri,
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Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes were 32, 32,
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16, 16, 8 and 8 mg/ml, respectively, and the MIC of the ethanolic extract were 16, 16, 16, 8, 4,
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and 4 mg/ml, respectively. As mentioned earlier, the higher resistance of Gram-negative bacteria
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than the Gram-positive ones is attributed to the differences in their cell walls.
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The results of minimum bactericidal concentration (MBC) are exhibited in Table 5. The
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MBC of Makhlaseh ranged from 4 to 128 mg/ml contingent the type of bacteria. MBC of the
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aqueous extract of Makhlaseh for Enterobacter aerogenes, Escherichia coli, Shigella flexneri,
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Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes were 128, 64,
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64, 32, 16 and 16 mg/ml, respectively, and the MBC of the ethanolic extract were 64, 32, 16, 8,
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4, and 4 mg/ml, respectively. Several studies have been done on the antimicrobial effects herbal extracts on pathogenic
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microorganisms (Alizadeh Behbahani et al. (2017) (14), Tabatabaei Yazdi et al. (2014) (18),
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Heidari-Sureshjani et al (2014) (26) and Jouki et al (2014) (27)). In the studies, had a more
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pronounced antimicrobial effect on the Gram-positive bacteria.
Table 3. Mean inhibition zone diameter (mm) of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing poisoning and infection (DDA)
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Table 4. Mean inhibition zone diameter (mm) of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing poisoning and infection (WDA) Table 5. MIC and MBC of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing
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poisoning and infection
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4. Conclusion
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The ethanolic extract of Makhlaseh “in vitro” showed a considerable antibacterial effect
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against Enterobacter aerogenes, Escherichia coli, Shigella flexneri as the gram-negative bacteria
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and Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus pyogenes as the
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gram-positive bacteria. using Makhlaseh extracts as a natural antibacterial composite “in vitro”
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have significant antibacterial ability over the studied strains. So, the ethanolic and aqueous
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extract of Makhlaseh can be used in pharmaceutical industry for medical treatments with perfect
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study.
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Acknowledgments The authors wish to express their profound gratitude sincerely to Applied Microbiology
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Research Center, System Biology and Poisoning institute, Research Deputy of Baqiyatallah
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University of Medical Sciences.
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oils of Tanacetum argyrophyllum (C. Koch) Tvzel. var. argyrophyllum and Tanacetum
371
parthenium (L.) Schultz Bip.(Asteraceae) from Turkey. Biochemical systematics and
372
ecology. 2005; 33:511-6.
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21. Daferera DJ, Ziogas BN, Polissiou MG. GC-MS analysis of essential oils from some
374
Greek aromatic plants and their fungitoxicity on Penicillium digitatum. Journal of
375
Agricultural and Food Chemistry. 2000; 48:2576-81.
377 378 379 380 381
22. Cao G, Prior RL. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clinical Chemistry. 1998; 44:1309-15. 23. Burt, S. Essential oils: their antibacterial properties and potential applications in foods—a
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review. International Journal of Food Microbiology, 2004; 94(3): 223-253. 24. Holley RA, Patel D. Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiology. 2005; 22:273-92. 25. Izadi Z, Esna-Ashari M, Piri K, Davoodi P. Chemical composition and antimicrobial
383
activity of feverfew (Tanacetum parthenium) essential oil. Int J Agric Biol. 2010; 12:759-
384
63.
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385
26. M.H. Sureshjani, F. Tabatabaei Yazdi, S.A. Mortazavi, B. Alizadeh Behbahani,
386
F.Shahidi, Antimicrobial effects of Kelussia odoratissima extracts against foodborne and
387
food spoilage bacteria in vitro, J. Paramed. Sci. 2014; 5 (2):115–120.
388
27. Jouki, M., Yazdi, F.T., Mortazavi, S.A., Koocheki, A., Khazaei, N. 2014. Effect of
389
quince seed mucilage edible films incorporated with oregano or thyme essential oil on
390
shelf life extension of refrigerated rainbow trout fillets. International Journal of Food
391
Microbiology, 174: 88-97.
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Table 1. All microbial media and reagents
Supplier
Mueller Hinton Agar (MHA)
Merck, Germany
Mueller Hinton Broth (MHB)
Merck, Germany
Peptone bacteriological
BDH Chemicals Ltd., England
Tryptone soya broth
East Anglia Chemicals, UK
Sodium chloride
East Anglia Chemicals, UK
Sulfuric acid
Merck, Germany
Sterile blank disc
Oxoid United Kingdom
Antibiotic disc
Oxoid United Kingdom
Ethanol
Merck, Germany
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Reagents and microbial media
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Table 2: Phytochemical constituents of aqueous and ethanolic Makhlaseh extracts Verification method
observations
Alkaloids
Mayer and Bosshardt
Formation of a yellow or brown
Tannins
Ferric chloride test
Green bluish
Saponins
Froth test
Formation of a stable foam
Phenolics
Ferric chloride
Green-bluish
+ = Present in small concentrations
+
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++ = Present in moderately high concentrations
Occurrences (A)
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Plant Constituents
(A): aqueous Makhlaseh extract
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(E): ethanolic Makhlaseh extract
Occurrences (E)
++
+
+
+
+
+
++
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Table 3. Mean inhibition zone diameter (mm) of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing poisoning and infection (DDA) S. epidermidis
E. aerogenes
7.90 ± 0.34
7.60 ± 0.50
-
9.10 ± 0.50
9.60 ± 0.52
9.50 ± 0.28
-
11.00 ± 0.52
11.80 ± 0.54
11.70 ± 0.34
13.20 ± 0.50
13.60 ± 0.32
13.40 ± 0.50
8.60 ± 0.50
8.00 ± 0.50
9.80 ± 0.52
10.40 ± 0.28
10.10 ± 0.50
11.60 ± 0.54
12.70 ± 0.50
12.30 ± 0.52
13.70 ± 0.50
14.30 ± 0.50
14.30 ± 0.50
-
7.80 ± 0.50
7.30 ± 0.50
7.60 ± 0.50
9.70 ± 0.32
9.20 ± 0.52
10.00 ± 0.50
11.30 ± 0.50
6.80 ± 0.50
7.00 ± 0.50
7.20 ± 0.52
8.60 ± 0.34
8.50 ± 0.28
9.00 ± 0.54
10.40 ± 0.52
9.90 ± 0.54
10.60 ± 0.28
11.90 ± 0.50
11.60 ± 0.50
12.70 ± 0.50
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8.10 ± 0.50
S. flexneri
-
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7.00 ± 0.50
E. coli
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S. pyogenes
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Aqueous (mg/ml) 10 20 30 40 Ethanolic (mg/ml) 10 20 30 40
S. aureus
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S. pyogenes
S. epidermidis
E. aerogenes
8.10 ± 0.50
8.00 ± 0.50
-
9.20 ± 0.50
10.10 ± 0.50
9.80 ± 0.52
7.00 ± 0.50
11.40 ± 0.50
12.20 ± 0.50
11.90 ± 0.50
13.60 ± 0.52
14.00 ± 0.52
13.80 ± 0.28
8.60 ± 0.50
8.50 ± 0.54
10.90 ± 0.54
10.10 ± 0.50
11.90 ± 0.54
13.10 ± 0.28
12.80 ± 0.50
14.00 ± 0.50
14.90 ± 0.52
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14.20 ± 0.28
S. flexneri
-
7.60 ± 0.50
8.50 ± 0.50
8.80 ± 0.50
10.00 ± 0.52
10.20 ± 0.50
10.90 ± 0.50
11.60 ± 0.52
7.10 ± 0.54
7.20 ± 0.50
7.50 ± 0.50
8.60 ± 0.50
9.00 ± 0.54
9.50 ± 0.54
10.50 ± 0.52
10.60 ± 0.52
11.20 ± 0.50
12.90 ± 0.28
13.30 ± 0.52
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8.20 ± 0.50 10.00 ± 0.54
E. coli
7.10 ± 0.50
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7.30 ± 0.54
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Aqueous (mg/ml) 10 20 30 40 Ethanolic (mg/ml) 10 20 30 40
S. aureus
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Table 4. Mean inhibition zone diameter (mm) of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing poisoning and infection (WDA)
12.40 ± 0.50
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Table 5. MIC and MBC of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing
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poisoning and infection MIC (aqueous extract)
MIC (ethanolic extract)
MBC (aqueous extract)
MBC (ethanolic extract)
S. pyogenes
8
4
16
4
S. epidermidis
8
4
16
4
S. aureus
16
8
32
8
E. aerogenes
32
16
128
64
E. coli
32
16
64
32
S. flexneri
16
16
64
16
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microorganisms
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Highlights
infection.
Determination of Makhlaseh chemical composition by GC-MS.
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Makhlaseh a strong antibacterial activity on some pathogenic strain causing poisoning and
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Ethanolic Makhlaseh extract showed greater inhibitory effect on Gram-positive bacteria.
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Total phenolic content, alkaloids, tannins and saponins were determined.
S0882-4010(17)31604-2
DOI:
10.1016/j.micpath.2017.12.002
Reference:
YMPAT 2638
To appear in:
Microbial Pathogenesis
Received Date: 27 November 2017 Revised Date:
1 December 2017
Accepted Date: 1 December 2017
Please cite this article as: Alizadeh Behbahani B, Imani Fooladi AA, Antibacterial activities, phytochemical analysis and chemical composition Makhlaseh extracts against the growth of some pathogenic strain causing poisoning and infection, Microbial Pathogenesis (2018), doi: 10.1016/ j.micpath.2017.12.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Antibacterial activities, phytochemical analysis and chemical composition Makhlaseh extracts against the growth of some pathogenic strain causing poisoning and infection
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Behrooz Alizadeh Behbahani1, Abbas Ali Imani Fooladi1* 1.Applied Microbiology Research Center, System Biology and Poisoning institute, Baqiyatallah University of Medical Sciences, Sheikh Bahaei Street, Molla Sadra Street, Vanak Sq., Tehran 984359-44711, Iran
Corresponding author: [email protected] or [email protected] Abstract
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The aim of this study was to investigate the antibacterial activities and phytochemical
14
analysis of extracts against the growth of some pathogenic strain causing poisoning and infection
15
(Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, Enterobacter
16
aerogenes, Escherichia coli and Shigella flexneri). Makhlaseh components were identified via
17
gas chromatography/mass spectrometry (GC/MS). Total phenolic content (TPC), alkaloids,
18
tannins and saponins were determined. Antioxidant activity was determined calorimetrically for
19
2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. Antimicrobial effect of extracts was
20
evaluated by five methods, pour plate, well diffusion, disk diffusion, minimum inhibitory
21
concentration (MIC), and minimum bactericidal concentration (MBC). Camphor was the major
22
compound of Makhlaseh. The TPC of aqueous and ethanolic Makhlaseh extracts was equal to
23
79.45 ± 1.15 and 115.26 ± 1.23 µg GAE/mg, respectively. The antioxidant activity (IC50) test of
24
aqueous and ethanolic Makhlaseh extracts showed 315.50 ± 1.12 and 118.35 ± 1.08 µg/ml,
25
respectively. MIC of the aqueous extract of Makhlaseh for Enterobacter aerogenes, Escherichia
26
coli, Shigella flexneri, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus
27
pyogenes were 32, 32, 16, 16, 8 and 8 mg/ml, respectively, and the MIC of the ethanolic extract
28
were 16, 16, 16, 8, 4, and 4 mg/ml, respectively. The MBCs of the Makhlaseh extracts varied
29
from 4 mg/ml to 128 mg/ml. Increasing concentration of Makhlaseh extracts had a significant
30
effect (p≤ 0.05) on inhibition zone diameter. In conclusion, using Makhlaseh extracts as a natural
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antibacterial composite in vitro have significant antibacterial ability over the studied strains.
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Keywords: Makhlaseh, Extracts, Antimicrobial effect, Phytochemical analysis.
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1- Introduction
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The increase in resistance of pathogenic bacteria due to multi-various use of number of
37
common therapeutic antibiotics has encouraged scientists all over the world to search for new
38
antibacterial and antifungal substances from various antecedents comprising medicinal herbal.
39
Therefore, it is highly appreciated to find out ways overcoming these issues. For a long age of
40
time, plants have been a worth source of natural products for maintaining human safety. The use
41
of herb extracts and phytochemicals, both with known antimicrobial attributes, can be of great
42
significance in therapeutic treatments. Nowadays, a new approach has emerged to the use of
43
herbal medicines to treat infections, since herbal medicines are stronger antimicrobial activity
44
and fewer side effects than chemical drugs (1-3). In various regions of Iran (Mazandaran, Gilan,
45
Golestan, Kurdistan, Fars, Khorasan, Tehran and Semnan) Makhlaseh is known as Qaliheh and
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Noroozi plant. Makhlaseh a member of Asteraceae, is a perennial herbaceous plant with sporadic
47
fluff and a short straight root (4, 5).
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Enterobacter aerogenes is a pathogenic bacterium gram negative that reason opportunistic
50
infections including most types of infections. Streptococcus pyogenes is a pathogenic bacterium
51
gram positive can also cause disease in the form of post infectious “nonpyogenic” syndromes.
52
Septicemia and endocarditis are also diseases be associated with Staphylococcus epidermidis.
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Shigella flexneri infections can usually be treated with common therapeutic antibiotics, however
54
some strains have become resistant. Escherichia coli cause urinary tract infections, bacteremia
55
and other infections in humans (6-9).
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The useful effects of phenolic composition such as anticancer, antimutagenic and heart-
57
protective attributed, also the synthetic antioxidants being suspected to toxicity, have led to
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numerous studies for the extraction of these composition from natural resources. Phenolic
59
composition are the constituents with antioxidant activity (10,11). The aim of this study was to identify the chemical compositions and to measure
61
phytochemical analysis and the antioxidant activity of aqueous and ethanolic Makhlaseh extracts
62
in addition to its free radical scavenging activity. The other aim of this research was to
63
investigate the antibacterial effects of aqueous and ethanolic Makhlaseh extracts on pathogenic
64
strain causing infection.
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66 67 68 69
2- Materials and methods 2-1- Chemicals and reagents
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All reagents and microbial media show in Table 1.
Table 1. All microbial media and reagents
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2-2- Extract preparation
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Makhlaseh was collected from Kurdistan province, Iran. After the confirmation of the plant
74
scientific name (the species of the plant was identified and confirmed by herbarium, the herbal
75
systematic laboratory of Ferdowsi University of Mashhad), it was rinsed with cold water,
76
shadow dried and powdered using a lab grinder. The extraction of the Makhlaseh was conducted
77
according to the method of Alizadeh Behbahani et al., (2017) (11) with some modifications.
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Water and ethanol were used for the maceration method. For this purpose, 25 g of the powdered
79
Makhlaseh was added to 225 ml of the solvent (water and ethanol) for 48 hours (shaking
80
occasionally with a shaker). After dissolving process, materials were filtered (Whatman no. 1
81
filter paper) and centrifuged in 9000 g for 10 minutes. Then extracts were evaporated using
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rotary evaporator (Rota-vapor R-205, England) and dried extracts were obtained and stored at
83
4°C in air tight screwcap tube.
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2-3- Determination of the dry weight of the extracts
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The extraction of the Makhlaseh was conducted according to the method of Tabatabaei
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Yazdi et al., (2015) (12). Briefly, in order to determine the dry weight, first a test tube was
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weighed using a digital balance. one ml of the Makhlaseh extracts (aqueous and ethanolic) was
89
poured into it and dried at 25°C. The test tube was weighed again after drying. The difference
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between the weighs of the test tube before and after drying was recorded.
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2-4- Phytochemical analysis
93
2-4-1- Alkaloids
The alkaloids Makhlaseh was conducted according to the method of Njoku and Obi., (2009)
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(13).
96
2-4-2- Tannins
The tannins Makhlaseh was conducted according to the method of Njoku and Obi., (2009)
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(13).
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2-4-3- Saponins
100 101
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The saponins Makhlaseh was conducted according to the method of Njoku and Obi., (2009) (13).
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2-4-4- Phenolics
The phenolics Makhlaseh was conducted according to the method of Alizadeh Behbahani et
105
al., (2017) (11).
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2-5- Identification of chemical composition
109 110 111 112
chromatograph and coupled to a mass spectrometer (14).
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Identification of the Makhlaseh substances was performed by injecting Makhlaseh into a gas
2-6- Estimation of total phenolic content (TPC) and Antioxidant activity
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For this purpose, the method of Folin-Ciocalteu is applied for the determination of TPC. The
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antioxidant activity of Makhlaseh was estimated according to the method previously described
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by Alizadeh Behbahani et al (2017) (15).
2-7- Preparation of the microbial strains
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The following Gram-positive and Gram-negative bacteria were used for testing antibacterial activity. Staphylococcus epidermidis ATCC 12228, Streptococcus pyogenes ATTC 19615,
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Staphylococcus aureus ATTC 25923, Enterobacter aerogenes ATTC 13048, Escherichia coli
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ATTC 25922 and Shigella flexneri ATCC12022 all American type culture collections were
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obtained from the department of food science and technology of Ferdowsi University of
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Mashhad (FUM), Mashhad, Iran.
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2-8- Suspension Preparation
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based on a standard 0.5 McFarland (16).
129
For this purpose, its concentration was adjusted to 1.5×108 CFU (colony forming units)/ml
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130 131 132 133 134
2-9- Antimicrobial test
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from each other and the plate edge. To that end, the concentrations of 10, 20, 30 and 40 mg/ml
136
were prepared in a suitable solvent. The disks of gentamycin and vancomycin were also used at a
137
concentration of 10 µg/ml. after that, the culture media containing bacteria were incubated at 37
138
°C for 24. Eventually, the inhibition zone diameters formed around the disks were measured
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using a ruler and recorded (17).
2-9-1- Disk diffusion agar (DDA)
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2-9-2- Well diffusion agar (WDA)
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In this method, blank paper disks were placed on the culture medium at certain distances
In this method, 0.5 McFarland suspension was poured onto MHA at three points for bacteria and spread using a L-shaped spreader. Next, 4 wells with diameters of 6 mm were created on the
146
surface of the culture media using the bottom of the Pasteur pipette. After incubating the culture
147
media containing bacteria at 37 °C for 24, given each well diameter, the diameter of the
148
inhibition zone surrounding the well was quantified using a ruler and expressed in mm (14, 15).
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155 156 157 158
2-9-3- Pour plate method (PPM)
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PPM is a qualitative method and its results are expressed as sensitive, semi-sensitive and resistant (11).
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2-9-4- Determination of minimum inhibitory concentration (MIC) through micro dilution broth
In this method, a stock solution with concentration of 512 mg/ml was prepared from
161
Makhlaseh. Then, prepare sequential concentrations of 2, 4, 8, 16, 32, 64, 128 and 256 mg/ml. In
162
order to estimate MIC through micro dilution broth (15, 18).
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2-9-5- Minimum Bactericidal Concentration
Given the results obtained from the MIC test, 100 µl was removed from the wells free of the
166
red color, and cultured on MHA media. Subsequently, the culture media were incubated at 37 °C
167
for 24 h. The concentrations at which microbial growth did not happen, were regarded as the
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MBC of Makhlaseh for pathogenic strain causing infection (11).
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2-10- Statistical analysis
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Microsoft Windows Excel 2016 and SPSS (Version18.0, SPSS Inc., Chicago, USA) were
172
used to analyze the obtained data. The data were initially assessed by analysis of variance
173
(ANOVA), and the Duncan’s multiple range test was subsequently applied to detect significant
174
(p < 0.05) differences. All experiments were triplicated.
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3- Results and discussion 3-1- chemical composition
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The results of Makhlaseh chemical composition analysis through GC-MS showed that
179
Camphor was the major compound followed by camphene, α-pinene chrysanthenyl acetate, β-
180
chrysanthenyl acetate and limonene. Polatoglu et al (2010) (19), reported that camphor (49%),
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trans-chrysanthenyl acetate (22.1%) and pcymene (5.2%) were the major constituents of
182
Makhlaseh. Akpulat et al (2005), verified the chemical compound of Makhlaseh in Turkey and
183
concluded that camphor (56.9%), camphene (12.7%) and pcymene (5.2%) were the major
184
components of Makhlaseh (20). Our findings conformed to those of the others to a large extent.
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The chemical compositions of essential oils differ from one another considering the variety,
186
climate, growth stage, collection time and growth location (21).
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3-2- Phytochemical analysis, antioxidant activity and total phenolic content (TPC)
191
saponins, and phenolics in the aqueous and ethanolic Makhlaseh extracts. The results
192
demonstrated that the phenolic compounds had the largest content in this ethanolic Makhlaseh
193
extracts (Table 2). The TPC of aqueous and ethanolic Makhlaseh extracts was equal to 79.45 ±
194
1.15 and 115.26 ± 1.23 µg GAE/mg, respectively. TPC was calculated on the standard curve
195
through the Folin-Ciocalteu method. The antioxidant activity (IC50) test of aqueous and ethanolic
196
Makhlaseh extracts showed 315.50 ± 1.12 and 118.35 ± 1.08 µg/ml, respectively. The
197
antioxidant activity of the Makhlaseh was directly related to its phenolic composition. Many
198
studies have proven the direct relationship between TPC and antioxidant activity (14, 15).
199
Polatoglu et al (2010), reported that Makhlaseh had antioxidant activity (19). Cao et al (1998),
200
(22) and Alizadeh Behbahani et al (2017), (14) reported that the differences in the TPC and
201
antioxidant properties of different plants are influenced by a variety of factors such as climatic
202
conditions (climate, soil and altitude).
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Table 2: Phytochemical constituents of aqueous and ethanolic Makhlaseh extracts
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3-3- Antimicrobial effect The results of the antimicrobial effects of aqueous and ethanolic Makhlaseh extracts, using
208
the method of pour plate method show that 2 mg/ml concentration of ethanolic Makhlaseh
209
extract, were quite effective on reduce of growth Staphylococcus epidermidis and Streptococcus
210
pyogenes and were had prevent growth over the medium. However, 2 mg/ml concentration
211
ethanolic Makhlaseh extract, have no significant antibacterial effect on Escherichia coli,
212
Enterobacter aerogenes and Shigella flexneri. The results showed 2 mg/ml concentration of
213
aqueous Makhlaseh extract, were effective on reduce of growth Staphylococcus epidermidis and
214
Streptococcus pyogenes and were not had prevent growth over the medium. 2 mg/ml
215
concentration aqueous Makhlaseh extract, have no significant antibacterial effect on Escherichia
216
coli, Enterobacter aerogenes and Shigella flexneri.
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The antimicrobial effect of aqueous and ethanolic Makhlaseh extracts was investigated
218
through disk diffusion agar (DDA) method. The results (Table 3) presented that aqueous and
219
ethanolic Makhlaseh extracts had the most detrimental effect against Staphylococcus epidermidis
220
and Streptococcus pyogenes at 40 mg/ml. The smallest inhibition zone diameter against different
221
aqueous and ethanolic Makhlaseh concentrations belonged to Gram-negative bacteria. The
222
results also indicated that no inhibition zone was observed for Enterobacter aerogenes and
223
Escherichia coli at the concentrations of 10 and 20 mg/ml aqueous extract. The results indicated
224
that no inhibition zone was observed for Shigella flexneri at the concentrations of 10 mg/ml
225
aqueous extract. Inhibition zone was observed at all concentrations for all Gram-positive
226
bacteria. The results demonstrated that the investigated bacteria were significantly different
227
(p<0.05) in terms of susceptibility to Makhlaseh through the DDA method. The pairwise
228
comparison between the effects of aqueous and ethanolic Makhlaseh extracts concentrations on
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the bacteria revealed that there were significant differences between the concentrations and as
230
aqueous and ethanolic Makhlaseh extracts concentration increased, the inhibition zone diameter
231
increased, too. The results of the antimicrobial effects of aqueous and ethanolic Makhlaseh extracts through
233
well diffusion agar (WDA) method are summarized in Table 4. They showed that inhibition zone
234
was observed for all Gram-positive bacteria at all aqueous and ethanolic Makhlaseh extracts
235
concentrations. No inhibition zone was observed for Enterobacter aerogenes, Escherichia coli
236
and Shigella flexneri at 10 mg/ml aqueous extract. In well diffusion agar method, due to the
237
direct contact between the extracts and the bacteria on the surface of the culture medium, the
238
inhibition zone diameter was larger than that of disk diffusion agar method, because in disk
239
diffusion agar method, it was necessary that Makhlaseh diffuse into the surface of the culture
240
medium through a paper disk (14, 15). Significant differences were observed for other
241
microorganisms at all concentrations.
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The results of WDA and DDA demonstrated that Gram-positive bacteria were more sensitive
243
to Makhlaseh than the Gram-negative ones. In general, the sensitivity profile of the bacteria
244
studied in this research is as follows:
245
Enterobacter aerogenes> Escherichia coli> Shigella flexneri > Staphylococcus aureus >
246
Staphylococcus epidermidis > Streptococcus pyogenes
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The higher resistance of Gram-negative bacteria to the extract of medicinal herbs could be
248
attach to the more assembled structure of the cell membrane of these bacteria contrast with the
249
single-layer structure of the Gram-positive ones. Gram-positive bacteria have mucopeptide in
250
their cell walls, while lipopolysaccharides constitute the bulk of the cell walls of Gram-negative
251
bacteria like Enterobacter aerogenes, Escherichia coli and Shigella flexneri. On the other hand,
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it seems that the resistance of bacterial cells belongs to the solvability rate and extent of
253
antimicrobial compounds (Makhlaseh) in the lipid moiety of the microorganism cell membrane.
254
However, this could not be compelling sake for the higher susceptibility of Gram-positive
255
bacteria than the Gram-negative ones. In addition, the diversity in the hydrophobicity of the cell
256
membrane surfaces can also be said as an influential invoice (23, 24)
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Similar studies concerning the antimicrobial effects of the extracts of medicinal plants which
258
have been carried out on a variety of Gram-positive and Gram-negative bacteria, confirm the
259
above sentences (14, 15). According to the findings of the present study, camphor is the major
260
constituent of Makhlaseh. Izadi et al (2010) (25) examined the antimicrobial effect of Makhlaseh
261
via Kirby-Bauer method on Gram-negative bacteria and Gram-positive bacteria. They concluded
262
that Makhlaseh had a more pronounced antimicrobial effect on the Gram-positive bacteria. Their
263
results are consistent with ours.
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The results of minimum inhibitory concentration (MIC) are exhibited in Table 5. The MIC of
265
Makhlaseh ranged from 4 to 32 mg/ml depending on the type of bacteria. The MIC of the
266
aqueous extract of Makhlaseh for Enterobacter aerogenes, Escherichia coli, Shigella flexneri,
267
Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes were 32, 32,
268
16, 16, 8 and 8 mg/ml, respectively, and the MIC of the ethanolic extract were 16, 16, 16, 8, 4,
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and 4 mg/ml, respectively. As mentioned earlier, the higher resistance of Gram-negative bacteria
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than the Gram-positive ones is attributed to the differences in their cell walls.
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The results of minimum bactericidal concentration (MBC) are exhibited in Table 5. The
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MBC of Makhlaseh ranged from 4 to 128 mg/ml contingent the type of bacteria. MBC of the
273
aqueous extract of Makhlaseh for Enterobacter aerogenes, Escherichia coli, Shigella flexneri,
274
Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes were 128, 64,
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64, 32, 16 and 16 mg/ml, respectively, and the MBC of the ethanolic extract were 64, 32, 16, 8,
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4, and 4 mg/ml, respectively. Several studies have been done on the antimicrobial effects herbal extracts on pathogenic
278
microorganisms (Alizadeh Behbahani et al. (2017) (14), Tabatabaei Yazdi et al. (2014) (18),
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Heidari-Sureshjani et al (2014) (26) and Jouki et al (2014) (27)). In the studies, had a more
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pronounced antimicrobial effect on the Gram-positive bacteria.
Table 3. Mean inhibition zone diameter (mm) of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing poisoning and infection (DDA)
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282 283 284 285 286 287 288 289
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Table 4. Mean inhibition zone diameter (mm) of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing poisoning and infection (WDA) Table 5. MIC and MBC of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing
291
poisoning and infection
292 293
4. Conclusion
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The ethanolic extract of Makhlaseh “in vitro” showed a considerable antibacterial effect
295
against Enterobacter aerogenes, Escherichia coli, Shigella flexneri as the gram-negative bacteria
296
and Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus pyogenes as the
297
gram-positive bacteria. using Makhlaseh extracts as a natural antibacterial composite “in vitro”
298
have significant antibacterial ability over the studied strains. So, the ethanolic and aqueous
299
extract of Makhlaseh can be used in pharmaceutical industry for medical treatments with perfect
300
study.
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Acknowledgments The authors wish to express their profound gratitude sincerely to Applied Microbiology
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Research Center, System Biology and Poisoning institute, Research Deputy of Baqiyatallah
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University of Medical Sciences.
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312 313 314
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Table 1. All microbial media and reagents
Supplier
Mueller Hinton Agar (MHA)
Merck, Germany
Mueller Hinton Broth (MHB)
Merck, Germany
Peptone bacteriological
BDH Chemicals Ltd., England
Tryptone soya broth
East Anglia Chemicals, UK
Sodium chloride
East Anglia Chemicals, UK
Sulfuric acid
Merck, Germany
Sterile blank disc
Oxoid United Kingdom
Antibiotic disc
Oxoid United Kingdom
Ethanol
Merck, Germany
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Reagents and microbial media
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Table 2: Phytochemical constituents of aqueous and ethanolic Makhlaseh extracts Verification method
observations
Alkaloids
Mayer and Bosshardt
Formation of a yellow or brown
Tannins
Ferric chloride test
Green bluish
Saponins
Froth test
Formation of a stable foam
Phenolics
Ferric chloride
Green-bluish
+ = Present in small concentrations
+
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++ = Present in moderately high concentrations
Occurrences (A)
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Plant Constituents
(A): aqueous Makhlaseh extract
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(E): ethanolic Makhlaseh extract
Occurrences (E)
++
+
+
+
+
+
++
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Table 3. Mean inhibition zone diameter (mm) of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing poisoning and infection (DDA) S. epidermidis
E. aerogenes
7.90 ± 0.34
7.60 ± 0.50
-
9.10 ± 0.50
9.60 ± 0.52
9.50 ± 0.28
-
11.00 ± 0.52
11.80 ± 0.54
11.70 ± 0.34
13.20 ± 0.50
13.60 ± 0.32
13.40 ± 0.50
8.60 ± 0.50
8.00 ± 0.50
9.80 ± 0.52
10.40 ± 0.28
10.10 ± 0.50
11.60 ± 0.54
12.70 ± 0.50
12.30 ± 0.52
13.70 ± 0.50
14.30 ± 0.50
14.30 ± 0.50
-
7.80 ± 0.50
7.30 ± 0.50
7.60 ± 0.50
9.70 ± 0.32
9.20 ± 0.52
10.00 ± 0.50
11.30 ± 0.50
6.80 ± 0.50
7.00 ± 0.50
7.20 ± 0.52
8.60 ± 0.34
8.50 ± 0.28
9.00 ± 0.54
10.40 ± 0.52
9.90 ± 0.54
10.60 ± 0.28
11.90 ± 0.50
11.60 ± 0.50
12.70 ± 0.50
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8.10 ± 0.50
S. flexneri
-
SC
7.00 ± 0.50
E. coli
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S. pyogenes
EP
Aqueous (mg/ml) 10 20 30 40 Ethanolic (mg/ml) 10 20 30 40
S. aureus
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S. pyogenes
S. epidermidis
E. aerogenes
8.10 ± 0.50
8.00 ± 0.50
-
9.20 ± 0.50
10.10 ± 0.50
9.80 ± 0.52
7.00 ± 0.50
11.40 ± 0.50
12.20 ± 0.50
11.90 ± 0.50
13.60 ± 0.52
14.00 ± 0.52
13.80 ± 0.28
8.60 ± 0.50
8.50 ± 0.54
10.90 ± 0.54
10.10 ± 0.50
11.90 ± 0.54
13.10 ± 0.28
12.80 ± 0.50
14.00 ± 0.50
14.90 ± 0.52
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14.20 ± 0.28
S. flexneri
-
7.60 ± 0.50
8.50 ± 0.50
8.80 ± 0.50
10.00 ± 0.52
10.20 ± 0.50
10.90 ± 0.50
11.60 ± 0.52
7.10 ± 0.54
7.20 ± 0.50
7.50 ± 0.50
8.60 ± 0.50
9.00 ± 0.54
9.50 ± 0.54
10.50 ± 0.52
10.60 ± 0.52
11.20 ± 0.50
12.90 ± 0.28
13.30 ± 0.52
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8.20 ± 0.50 10.00 ± 0.54
E. coli
7.10 ± 0.50
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7.30 ± 0.54
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Aqueous (mg/ml) 10 20 30 40 Ethanolic (mg/ml) 10 20 30 40
S. aureus
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Table 4. Mean inhibition zone diameter (mm) of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing poisoning and infection (WDA)
12.40 ± 0.50
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Table 5. MIC and MBC of aqueous and ethanolic Makhlaseh extracts on some pathogenic strain causing
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poisoning and infection MIC (aqueous extract)
MIC (ethanolic extract)
MBC (aqueous extract)
MBC (ethanolic extract)
S. pyogenes
8
4
16
4
S. epidermidis
8
4
16
4
S. aureus
16
8
32
8
E. aerogenes
32
16
128
64
E. coli
32
16
64
32
S. flexneri
16
16
64
16
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microorganisms
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Highlights
infection.
Determination of Makhlaseh chemical composition by GC-MS.
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Makhlaseh a strong antibacterial activity on some pathogenic strain causing poisoning and
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Ethanolic Makhlaseh extract showed greater inhibitory effect on Gram-positive bacteria.
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Total phenolic content, alkaloids, tannins and saponins were determined.