Oliveria decumbens essential oil: Chemical compositions and antimicrobial activity against the growth of some clinical and standard strains causing infection

Accepted Manuscript Oliveria decumbens essential oil: Chemical compositions and antimicrobial activity against the growth of some clinical and standard strains causing infection Behrooz Alizadeh Behbahani, Farideh Tabatabaei Yazdi, Alireza Vasiee, Seyed Ali Mortazavi PII:

S0882-4010(17)31673-X

DOI:

10.1016/j.micpath.2017.12.033

Reference:

YMPAT 2669

To appear in:

Microbial Pathogenesis

Received Date: 7 December 2017 Revised Date:

10 December 2017

Accepted Date: 10 December 2017

Please cite this article as: Behbahani BA, Tabatabaei Yazdi F, Vasiee A, Mortazavi SA, Oliveria decumbens essential oil: Chemical compositions and antimicrobial activity against the growth of some clinical and standard strains causing infection, Microbial Pathogenesis (2018), doi: 10.1016/ j.micpath.2017.12.033. 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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Oliveria decumbens essential oil: chemical compositions and antimicrobial activity against the growth of some clinical and standard strains causing infection Behrooz Alizadeh Behbahani, Farideh Tabatabaei Yazdi, Alireza Vasiee, Seyed Ali Mortazavi Department of Food Science and Technology, Ferdowsi University of Mashhad, P.O. Box: 91775-1163, Mashhad, Iran

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Corresponding author: [email protected] Abstract

Oliveria decumbens as a valuable medicinal plant is extensively used in traditional medicine.

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clinical and standard strains causing infection resistance to antimicrobial agents, is one of the

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important problems in medicine. The aim of this study was to investigate the antibacterial

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activities and phytochemical analysis of Oliveria decumbens essential oil on the growth of some

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clinical and standard strains causing infection (Pseudomonas aerogenes, Escherichia coli,

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Streptococcus pyogenes and Staphylococcus epidermidis). Oliveria decumbens essential oil

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composition was identified by gas chromatography/mass spectrometry. Phytochemical analysis

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(alkaloids, saponins, flavone and phenolic) essential oil of the Oliveria decumbens were

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appraised based on qualitative methods. Several methods (disk diffusion, minimum inhibitory

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concentration (MIC) and minimum bactericidal concentration (MBC)) were used to appraise the

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antibacterial activity of the Oliveria decumbens essential oil. Thymol (28.45%) was the major

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compound of Oliveria decumbens essential oil. The total phenolics content (TPC) of the essential

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oil positively correlated with antioxidant activity (AA). The TPC and AA of Oliveria decumbens

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essential oil was equal to 92.45 ± 0.70 µg GAE/mg and 164.45 ± 1.20 µg/ml, respectively. The

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MIC of Oliveria decumbens essential oil ranged from 1 to 8 mg/ml depending on the type of

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bacteria (clinical and standard strains). The MBC of Oliveria decumbens essential oil varied

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from 1 mg/ml to 16 mg/ml. The smallest inhibition zone diameter (IZD) on different Oliveria

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decumbens essential oil concentrations on P. aeruginosa. Results indicate that Oliveria

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decumbens essential oil can prove to be an important source of AA and antibacterial and may be

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used for the treatment of infection diseases.

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Keywords: Oliveria decumbens, Chemical compounds, Antioxidant activity, Antibacterial effect, Microbial pathogens.

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1- Introduction

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Resistance to common antimicrobials is an important issue that physicians encounter. This

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necessitates the steady progression of novel agents, which would be able to prevent resistant

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organisms from growing. Application of medicinal plants has been taken into account for many

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years, and the therapeutic efficiency of a large number of herbal species has been vastly reported

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[1, 2].

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Aromatic herbs have been long recognized. Due to their aromatic and antiseptic

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characteristics, they are employed as spices, natural food preservatives, and for aromatherapy

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and various medical aims in the perfume industry. Pleasant species produce antibacterial

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secondary metabolites, essential oils, either as a part of their normal growth or as a response to

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pathogens’ attack or stress [3, 4]. It is needed to develop alternative antibacterial drugs for curing

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infectious illnesses of different origins such as medicinal plants. Certainly, medicinal plants are

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the primary sources of drugs in both developing and developed countries for different chemo-

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therapeutic purposes [5].

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Umbelliferae family is one of the greatest vegetable families having global distribution. Most

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of the plants which belong to this family, produce terpenes and other kinds of volatile

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compounds. Several researchers have been carried out about the antibacterial and preservative

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effects of herbal essential oils such as the ones extracted from the members of umbelliferae

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family [6, 7]. Oliveria decumbens, an endemic plant of Flora Iranian, belongs to umbelliferae

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family. It grows in warm areas of south and west of Iran. It is used for indigestion, diarrhea,

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abdominal pain and fever in traditional medicine [8].

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The purpose of this research was to identify the chemical composition of Oliveria decumbens

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essential oil. The other goal of this study was to examine the antibacterial effects of Oliveria

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decumbens essential oil against the growth of some clinical and standard strains causing

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infection.

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2- Materials and methods

2-1- Collection of plant material and preparation of the essential oil

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Oliveria decumbens was collected. Then the verification of the Oliveria decumbens

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scientific name, the plant powdered apply a lab grinder. The extraction essential oil of the

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Oliveria decumbens using the glass clevenger apparatus and the study that was described by

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Alizadeh Behbahani et al., (2017) [9].

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2-2- Determination of the extraction yield and identification of chemical composition The extraction yield essential oil of the Oliveria decumbens was conducted according to the

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method of Alizadeh Behbahani et al., (2017) [10]. Identification of the essential oil chemical

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composition done by gas chromatography/mass spectrometry (GC/MS). The carrier gas (helium)

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rate and an ionization energy were 1.1 ml/min and 70 eV, respectively. The injection and

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identification chemical compounds essential oil of the Oliveria decumbens according to the study

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that was described by Alizadeh Behbahani et al., (2017) [10].

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2-3- Phytochemical analysis, total phenolic content (TPC) and antioxidant activity (AA) Phytochemical analysis (alkaloids, saponins, flavone and phenolic) essential oil of the

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Oliveria decumbens were appraised based on qualitative methods and according to the study that

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was described by Njoku and Obi., (2009) [11] and Alizadeh Behbahani et al., (2017) [12]. To

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evaluation the TPC essential oil of the Oliveria decumbens we used Folin–Ciocalteu reagent and

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the study that was described by Yin et al., (2018) [13]. 2,2-diphenyl-1-picrylhydrazyl (DPPH)

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AA test were carried out by standard protocols.

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2-4- Preparation of the microbial strains

Clinical strains (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis and Streptococcus pyogenes) and standard strains causing infection (Pseudomonas aeruginosa

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ATCC 27853, Escherichia coli ATTC 25922, Staphylococcus epidermidis ATCC 12228 and

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Streptococcus pyogenes ATTC 19615) were obtained from laboratory of Industrial

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Microbiology, Ferdowsi University of Mashhad (FUM), Mashhad, Iran. The genus and species

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clinical strains causing infection identified by standard biochemical reaction.

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2-5- Suspension preparation clinical and standard strains causing infection

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(MHB) medium at 37 °C for 24 h. The microbial suspensions turbidity was made based on 0.5

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McFarland (1.5×108 CFU/ml) [14].

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For this purpose, the isolates (clinical and standard) were grown in mueller hinton broth

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2-6- Antibacterial activity Oliveria decumbens essential oil “in vitro”

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inhibitory concentration and minimum bactericidal concentration) were used to appraise the

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antibacterial activity of the Oliveria decumbens essential oil. These methods are summarized as

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follows:

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2-6-1- Disk diffusion agar (DDA)

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solvent (dimethyl sulfoxide). The different concentrations Oliveria decumbens essential oil were

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sterilized by a 0.22 µm syringe microfilter. Then, the blank discs were plunged in the Oliveria

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decumbens essential oil for 20 minutes. Next step, microbial suspension equivalent to 1.5 × 108

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CFU/ml (0.5 McFarland standard) were prepared. The blank discs which had been previously

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plunged in the Oliveria decumbens essential oil were fixed on the mueller hinton agar (MHA)

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medium. after that, the culture media containing clinical and standard strains causing infection

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were incubated at 37 °C for 24. Finally, the inhibition zone diameters (IZD) formed around the

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disks were measured by a ruler [10,15].

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In order to do this, the concentrations of 1, 2, 4 and 8 mg/ml were prepared in an appropriate

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For this purpose, several qualitative and quantitative methods (disk diffusion, minimum

2-6-2- Determination of minimum inhibitory concentration (MIC)

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The MIC was determined according to microdilution broth method (96-well plate and

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Triphenyl tetrazolium chloride indicator). For this purpose, the final concentration of Oliveria

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decumbens essential oil in the 96-well plate ranging was from 1 mg/ml to 128 mg/ml. The 96-

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well plate were incubated at 37°C for 24 hours. In order to estimate, the first concentration in

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which microbial growth did not fall out and red color was not seen, recorded for MIC [9,16]. 5

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2-6-3- Minimum bactericidal concentration (MBC)

The MBC was determined according to pour plate method. All the well plate which showed

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no visible growth were on MHA media, then, incubated at 37°C for 24 hours. The MBC was

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taken as the concentration that kills all bacterial cells cultured and the growth of any bacteria

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colony on MHA media [9,17].

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2-7- Statistical analysis

All experiments were triplicated. Average results were reported as the mean and standard

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error and SPSS ((Version18.0, SPSS Inc., Chicago, USA) statistical software, one-way ANOVA

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followed by Duncan’s multiple range test were used to do intergroup comparison, while

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considering p < 0.05 as the significance level.

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3- Results and discussion 3-1- chemical composition

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The results of Oliveria decumbens essential oil chemical compounds analysis via GC-MS

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indicated that a total of 12 compositions were identified. Thymol (28.45%) was the major

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compound. Other Oliveria decumbens essential oil compounds included γ-terpinene (22.2%), ρ-

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cymene (17.90%), myristicin (13.55%), carvacrol (8.50%), and limonene (2.60%). According to

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our knowledge and collected data, very little information exists about the Oliveria decumbens

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essential oil chemical compounds. Hajimehdipoor et al., (2010) [8], reported that 10

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compositions were identified in the essential oil of Oliveria decumbens. Their maintained that

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the main components were γ-terpinene, myristicin, thymol, ρ-cymene and carvacrol. Amin et al.,

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(2005) [18], reported that thymol (47.06%) was the major compound in the essential oil of

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Oliveria decumbens. Other Oliveria decumbens essential oil compounds included carvacrol

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(23.31%) terpinene (18.94%) ρ-cymene (8.71%), limonene (0.76%), and myristicine (0.63%).

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Another study which has been done on Oliveria decumbens essential oil by Mahboubi et al.,

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(2008) [19]. Their maintained that the main components of essential oil are thymol (26.9%), p-

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cymene (13.3%) γ-terpinene (11%) and carvacrol (0.25%). The results of present study

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conformed to those of the other researchers to some extent. The chemical compounds of the plant

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essential oil relevant many factors such as climate, season, growth stage, herb collection time

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and climatic conditions [12].

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3-2- Phytochemical analysis, total phenolic content (TPC) and antioxidant activity (AA)

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The results of phytochemical analysis of Oliveria decumbens essential oil showed the

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existence of alkaloids (yellow or brown), saponins (formation of a stable foam), flavone (Orange

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solution) and phenolic (green-bluish). The TPC of Oliveria decumbens essential oil was equal to

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92.45 ± 0.70 µg GAE/mg. The AA test of Oliveria decumbens essential oil showed 164.45 ±

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1.20 µg/ml. The AA of Oliveria decumbens essential oil was directly related to its TPC. Several

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studies have proven the direct relationship between TPC and AA [12, 14].

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3-3- Antibacterial activity

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The results of the antibacterial activity of Oliveria decumbens essential oil using the method

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of DDA method are indicated in Table 1. The results show that Oliveria decumbens essential oil

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had the most detrimental effect on S. pyogenes at 8 mg/ml. The smallest IZD on different

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Oliveria decumbens essential oil concentrations on P. aeruginosa. The results also show that no

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IZD was observed for P. aerogenes and E. coli (clinical strains) at the concentrations of 1 and 2

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mg/ml. The results show that no IZD was observed for P. aerogenes (standard strain) at the

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concentrations of 1 mg/ml. IZD was observed at all concentrations for all gram-positive bacteria

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(clinical and standard strains). Duncan’s test range tests showed significant difference among the

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mean IZD for the Oliveria decumbens essential oil.

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The IZD in the Oliveria decumbens essential oil varied from 8.90 mm at 8 mg/ml for P.

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aeruginosa (clinical) to 13.90 mm at 8 mg/ml for S. pyogenes (clinical) and from 10.40 mm for

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P. aeruginosa (standard) to 15.10 mm for S. pyogenes (standard), respectively.

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In general, the sensitivity profile of the clinical and standard strains causing infection is as

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follows:

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P. aerogenes > E. coli > S. epidermidis > S. pyogenes

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The results of minimum inhibitory concentration (MIC) are displayed in Table 2. The MIC of

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Oliveria decumbens essential oil ranged from 1 to 8 mg/ml depending on the type of bacteria

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(clinical and standard strains). The MIC of Oliveria decumbens essential oil for clinical strains P.

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aerogenes, E. coli, S. epidermidis and S. pyogenes were 8, 4, 2 and 1 mg/ml, respectively, and

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the MIC for standard strains were 4, 4, 1 and 1 mg/ml, respectively.

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The results of minimum bactericidal concentration (MBC) are displayed in Table 2. The

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MBC of Oliveria decumbens essential oil for clinical strains P. aerogenes, E. coli, S. epidermidis

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and S. pyogenes were 16, 8, 4 and 2 mg/ml, respectively, and the MBC for standard strains were

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8, 8, 2 and 1 mg/ml, respectively.

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The medical plants are original of biologically active material. Essential oils can be a

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considerable source of a great variety of chemical compositions armed with antibacterial activity,

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the Oliveria decumbens can be practical in treatment of the infection diseases as antibiotic.

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Oliveria decumbens Essential oil can as well as have use in food industries not only as flavor but

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also as preservative of foodstuffs [4]. Hajimehdipoor et al (2010) [8], reported that Oliveria decumbens essential oil showed high

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antimicrobial activity on Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Aspergilus

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niger and Candida albicans but low antibacterial effect on Pseudomonas aeroginosa. The results

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of the present study are consistent with theirs. Motamedi et al., (2010) [20] reported that Oliveria

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decumbens extracts have antimicrobial effect some microorganisms. Mahboubi et al., (2008) [19]

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reported that antimicrobial effect Oliveria decumbens essential oil on gram positive bacteria

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more than gram negative bacteria. Thymol and carvacrol play their antibacterial pattern by

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inhibiting the ergosterol biosynthesis and destruction of the membrane integrity [21].

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The higher resistance of gram-negative bacteria (P. aerogenes, E. coli) to the essential oil medicinal herbs due to the external membranes surrounding the cell wall in the latter ones [21]. various studies have been done on the antibacterial effects essential oil on pathogenic

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microorganisms (Alizadeh Behbahani and Imani Fooladi (2017) [22], Tabatabaei Yazdi et al

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(2015) [23] and Alizadeh Behbahni et al. (2013) [24]). In the studies, had a more pronounced

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antibacterial effect on the gram-positive bacteria.

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Table 1. Mean inhibition zone diameter (mm) of Oliveria decumbens essential oil on some clinical and standard strains causing infection (DDA)

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Table 2. MIC and MBC of Oliveria decumbens essential oil on some clinical and standard strains causing

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infection

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4. Conclusion The total phenolics content of the essential oil positively correlated with antioxidant activity.

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The Oliveria decumbens essential oil showed a considerable antimicrobial effect on some clinical

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and standard strains causing infection (Pseudomonas aerogenes, Escherichia coli, Streptococcus

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pyogenes and Staphylococcus epidermidis). Results indicate that Oliveria decumbens essential

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oil can prove to be an important source of antioxidant activity and antibacterial and may be used

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for the treatment of infection diseases.

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Acknowledgments

The authors wish to express their profound gratitude sincerely to the Research Deputy of Ferdowsi University of Mashhad for funding this project with the code of 2/44177.

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5- Heidari-Sureshjani M, Tabatabaei-Yazdi F, Alizadeh-Behbahani B, Mortazavi A. Antimicrobial Effect of Aqueous, Ethanol, Methanol and Glycerin Extracts of Satureja bachtiarica on Streptococcus pyogenes, Pseudomonas aeruginosa and Staphylococcus epidermidis. Zahedan Journal of Research in Medical Sciences. 2015; 17(7): 1-5. 6- Rahman M, Shereen G. Antibacterial activity of hydrodistilled essential oil of Psammogeton canescens N.O. Umbelliferae. Biotechnol. 2002; 1 (1): 55 - 60.

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causing poisoning and infection. 10.1016/j.micpath.2017.12.002.

Microbial

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DOI:

24- Alizadeh Behbahani B, Tabatabaei Yazdi F, Mortazavi A, Zendeboodi F, Gholian MM. Effect of aqueous and ethanolic extract of Eucalyptus camaldulensis L. Journal of Paramedical Sciences. 2013;4(3): 89-99.

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Pathogenesis.

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clinical strains P. aeruginosa

-

-

E. coli

-

7.40 ± 0.50

S. epidermidis

7.30 ± 0.50

9.00 ± 0.35

8.20 ± 0.50

10.00 ± 0.35

P. aeruginosa

-

7.30 ± 0.35

E. coli

-

8.00 ± 0.28

S. epidermidis

8.10 ± 0.55

S. pyogenes

8.60 ± 0.28

S. pyogenes

standard strains

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382 383 384 385 386 387

12.70 ± 0.35

12.10 ± 0.28

13.90 ± 0.50

10.40 ± 0.50

8.60 ± 0.55

11.70 ± 0.50

11.90 ± 0.50

13.80 ± 0.55

10.90 ± 0.50

13.20 ± 0.55

15.10 ± 0.28

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10.80 ± 0.28

10.00 ± 0.55

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380

10.90 ± 0.52

9.90 ± 0.50

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377

379

8.90 ± 0.35

9.20 ± 0.35

clinical and standard strains causing infection (DDA)

375

378

7.00 ± 0.50

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Table 1. Mean inhibition zone diameter (mm) of Oliveria decumbens essential oil on some 1 mg/ml 2 mg/ml 4 mg/ml 8 mg/ml Microorganisms

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388 389 390

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391 392 393

causing infection microorganisms

MIC

clinical strains

398

P. aeruginosa

8

E. coli

4

S. epidermidis

2

S. pyogenes

1

399 400 401 402

standard strains

403 P. aeruginosa

404 E. coli

405 S. epidermidis

406 S. pyogenes

410 411 412 413 414 415

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409

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408

TE D

407

MBC

16 8

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397

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Table 2. MIC and MBC of Oliveria decumbens essential oil on some clinical and standard strains

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394 395 396

15

4 2

4

8

4

8

1

2

1

1

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Highlights

Oliveria decumbens essential oil a strong antibacterial activity on some clinical and standard

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strains causing infection. Phytochemical analysis (alkaloids, saponins, flavone and phenolic) were appraised based on qualitative methods.

Oliveria decumbens essential oil composition were identified by gas chromatography/mass

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spectrometry.

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The total phenolics content of the essential oil positively correlated with antioxidant activity.