Annals of Tropical Medicine and Public Health

: 2017  |  Volume : 10  |  Issue : 5  |  Page : 1159--1164

Chemical composition and screening of antibacterial activity of essential oil of Pistacia khinjuk against two selected pathogenic bacteria

Reza Tahvilian1, Rohallah Moradi2, Hossein Zhaleh3, Mohammad Mahdi Zangeneh4, Akram Zangeneh5, Hossein Yazdani1, Majid Hajialiani2,  
1 Research Pharmaceutical Center, School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Research Pharmaceutical Center, School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah; Department of Chemistry, Tehran Payame Noor University, Tehran, Iran
3 Substance Abuse Prevention Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
4 Research Pharmaceutical Center, School of Pharmacy, Kermanshah University of Medical Sciences; Department of Clinical Sciences, Faculty of Veterinary, Razi University, Kermanshah, Iran
5 Research Pharmaceutical Center, School of Pharmacy, Kermanshah University of Medical Sciences; Department of Basic Sciences, Faculty of Veterinary, Razi University, Kermanshah, Iran

Correspondence Address:
Mohammad Mahdi Zangeneh
Kaj Street, Kermanshah


Background: Medicinal plants are considered as modern resources for producing agents that could act as alternatives to antibiotics in demeanor of antibiotic-resistant bacteria. The aim of the present study was to evaluate the chemical composition and antibacterial activity of the essential oil of Pistacia khinjuk (combined with the dominance γ-terpinene) against P. aeruginosa and B. subtilis. Materials and Methods: The chemical composition of the essential oil was identified using gas chromatography coupled with mass spectrometer detector (GC-MS). As a screen test to detect antibacterial property of the essential oil, agar disk diffusion and agar well diffusion methods were employed. Macrobroth tube test was performed to determinate minimum inhibitory concentration (MIC). Results: According to results of the GC-MS analysis, γ-terpinene (81.14%) (w/w), β-pinene (3.93%) (w/w), and α-terpinolene (2.38%) (w/w) were the abundant components of the essential oil. The MIC and MBC values were 0.015/0.031 g/ml for essential oil of P. khinjuk in case of P. aeruginosa and B. subtilis, respectively. Conclusion: We believe that the article provides support to the antibacterial property of the essential oil. In fact, the results indicate that the essential oil of P. khinjuk can be useful as medicinal or preservative composition. Fractionation and characterization of active molecules will be the future work to investigate.

How to cite this article:
Tahvilian R, Moradi R, Zhaleh H, Zangeneh MM, Zangeneh A, Yazdani H, Hajialiani M. Chemical composition and screening of antibacterial activity of essential oil of Pistacia khinjuk against two selected pathogenic bacteria.Ann Trop Med Public Health 2017;10:1159-1164

How to cite this URL:
Tahvilian R, Moradi R, Zhaleh H, Zangeneh MM, Zangeneh A, Yazdani H, Hajialiani M. Chemical composition and screening of antibacterial activity of essential oil of Pistacia khinjuk against two selected pathogenic bacteria. Ann Trop Med Public Health [serial online] 2017 [cited 2020 Jan 29 ];10:1159-1164
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Infections due to bacterial species also stay a serious clinical difficulty. Emerging resistance of bacterial species is seriously reducing the number of efficient antimicrobials. Due to increasing pressure of consumers and legal authorities, the food industry has tended to decrease the use of chemical preservatives in their products to either entire nil or to adopt more indigenous alternatives for the maintenance or extension of product shelf life.[1]

Essential oils are made from a very intricate mixture of volatile molecules that are produced by the secondary metabolism of aromatic and medicinal plants and can be obtained by various methods, including the use of low- or high-pressure distillation of various parts of plants or the employment of liquid carbon dioxide or microwaves. The span of the essential oils action versus bacteria may achieve values that only prevent the bacterial growth (bacteriostatic) or may be used at either high concentrations or are inherently more aggressive and their action results in a reduction in the number of bacterial cells (bactericide). The bacteriostatic action has a reversible character since, after frustration of the agent, the microbial cells will meliorate their reproductive capacity. In contrast, the bactericidal effect has a constant effect; as even after the neutralization of the agent, the microbial cells are not capable of growth and reproduction.[2] The genus Pistacia (family Anacardiaceae), is widely distributed in the Mediterranean and Middle East areas.[3] Among the 15 known species of pistachios, only 3 species flourish in Iran, including Pistacia vera, Pistacia khinjuk and Pistacia atlantica.[4] These are shrubs and small trees growing to 5–15 m tall. The leaves are alternate, innately compounds and can be either evergreen or deciduous depending on species. P. khinjuk is circumfuse in places where the attitude is 700–2000 m above sea level.[5]P. khinjuk is an indigenous plant in Iran, which the plant has been used as indigestion, toothache, and astringent in Bakhtiari folk medicine. The plant is known as Khenjuk or Kelkhong in Persian.[6] Ancient Greek physicians, such as Hippocrates, Dioscorides, Theophrastos, and Galenos have recommended the use of mastic gum obtained from genus Pistacia for gastrointestinal derangements such as gastralgia, dyspepsia, and peptic ulcer.[7],[8] Some species of Pistacia have been used in folk medicine in eczema treatment, throat infections, renal stones, asthma and stomach ache, and as a astringent, anti-inflammatory, antipyretic, antibacterial, antiviral, pectoral, and stimulant.[9],[10],[11] Essential oils of some Pistacia species includes components such as γ-terpinene, cymene, linalool, β-caryophyllene, α-thujene, fenchene, sabinene, α-phellandrene, cineol, α-fenchone, borneol, and α-terpineil. The terpinenes are a group of isomeric hydrocarbons that are classified as terpenes. They have the same molecular formula and carbon fuselage, but they vary in the position of carbon-carbon double bonds. γ-terpinene is a monoterpene and a major component of essential oils made from plants fruit and shows strong antioxidant activity in several assay systems and have been isolated from a versatility of plant sources, such as coriander oil, lemon oil, cumin oil, and fragrant celery oil also present in the grapes, celery, cinnamon, cloves, cumin seeds, ginger, pepper, and tea.[12],[13]

Based on knowledge of author, in comparison to many other pharmaceutical-industrial plants, there are a very little data about chemical composition and antibacterial activity of the essential oil collected from Kermanshah province, west of Iran, and there is no study on antibacterial effect of essential oil of P. khinjuk (Combined with the Dominance γ-terpinene) in all over the world. Hence, the aim of the current study was (1): Determination of chemical composition of its hydro-distilled essential oil obtained from Kermanshah city, west of Iran by gas chromatography mass spectrometer (GC–MS), (2): Evaluation of antibacterial activity of the essential oil against common pathogens (P. aeruginosa and B. subtilis) with broth macro-dilution and agar well and disk diffusion methods.

 Materials And Methods

Plant sample collection

In the empirical-experimental study, medicine plant collected from Kermanshah. The sample was cleaned from any strange, plants, dust, or any other contaminants.

Essential oil extraction

Essential oil from fresh, clean, weighed aerial part P. khinjuk extracted by hydro-steam distillation using the Clevenger apparatus were collected and stored in sterile vials. In brief, 100–150 g of the plant was introduced in the distillation flask (1 L), which was conjuncted to a steam generator through a glass tube and to a condenser to retrieve the oil. This was recovered in a funnel tube. Aromatic molecules of the essential oil were liberated from the plant material and vaporized into hot steam. The hot steam forced the plant material to release the essential oil without burning the plant material itself. Then, steam containing the essential oil was passed through a cooling system to compress the steam. The steam was applied for 3 h. After settling the recovered mixture, essential oil was withdrawn. The supernatant essential oil was purged through anhydrous Na2 SO4 to dry the yielded essential oil. Then, the essential oil was collected in tightened vials and stored in a refrigerator. For the antimicrobial activity test, several dilutions of the oil were performed using dimethyl sulfoxide (DMSO).

Gas chromatography-mass spectrometry

Essential oil of P. khinjuk was analyzed using GC/MS (GC 7890N, AGILENT and MS 5975C, MODE EI) with two fused silica capillary column HP-5MS (30 m, 5 mm l. d, film thickness 0.25 μm) and a flame ionization detector which was operated in EI mode at 70 eV. Injector and detector temperatures were set at 220°C and 250°C, respectively. One microliter of each solution in hexane was perfused and analyzed with the column held initially at 60°C for 2 min and then increased by 3°C/min up to 300°C. Helium was used as carrier gas (1 ml/min). The relative amount of individual components of the total essential oil is expressed as percentage peak area relative to the total peak area. Qualitative reconnaissance of the several constituents was accomplished by comparison of their relative retention times and mass spectra with those of authentic reference compounds and mass spectra.

Source of microorganisms

Two bacterial species namely P. aeruginosa (ATCC No. 1707) and B. subtilis (ATCC No. 21332) were procured from the Veterinary school of Tehran University as lyophilized. Each bacterial strain was activated on Tryptic Soy broth, constant at 37°C for 18 h. Then, 60 μl of the broth was transferred to nutrient agar and incubated at 37°C for another 24 h; cell concentration was then adjusted to obtain final concentration of 108 CFU/ml using Muller-Hinton broth.

Culture media

Mueller-Hinton Agar (Muller-Hinton agar is a microbiological growth medium that is commonly used for antibiotic susceptibility testing) was prepared according to the manufacturer's instruction (Oxoid, UK), autoclaved and distributed at 20 ml per plate in 12 cm × 12 cm Petri dishes. Set plates were incubated overnight to ensure sterility before use. Then, Mueller-Hinton broth containing different concentrations of the essential oil and of the final bacteria inoculums (1 × 108 CFU/ml) were added into each well.

Evaluation of antimicrobial activity

Agar disk diffusion and agar well diffusion were used as screen tests to evaluate the antibacterial property of essential oil of P. khinjuk based on standard protocol. The solution of the compound was yielded in 1 g/ml from which six-fold serial dilutions (v/v) were prepared. A volume of 60 μl of each dilution was poured on each disk in order. After a period of 24 h incubation, the diameters of growth inhibition zones around the disks were measured. DMSO was used as negative control whereas gentamicin and cephalothin were used as positive controls in case of P. aeruginosa and B. subtilis, respectively. Minimum inhibitory concentration (MIC) means the lowest concentration of the probable antimicrobial agent which prevents growing of bacteria (regardless of killing the bacteria or stopping the growth of them). The lowest dilution which no gross microbial growth has been seen indicates MIC. Minimum bactericidal concentration (MBC) means the lowest concentration of the agent which causes death to test bacteria. The last can be revealed by pouring 60 μl of MIC tube and three dilutions before contents on an agar plate. In this case, after the incubation period, the lowest concentration which makes no growth indicates MBC. For determination of MIC value, macrobroth dilution method was applied. Interpretation of the results was done due to national accepted letter.[14]

Statistical analysis

Antibacterial effect was determined using One-way variance analysis, using the SPSS 18 software (IBM SPSS Statistics) package. Data were considered as statistically significant at P ≤ 0.05.


Chemical composition

The most substance found in essential oil of P. khinjuk was γ-terpinene. In contrast, 1-Phellandrene was the least constituents discovered in the essential oil. Composition of the plant using GC MS method can be perceived in [Table 1].{Table 1}

Agar well diffusion test

In regard to, the widest zone was seen in 0.062 g/ml, due to B. subtilis (12 mm). It was no growth inhibition in negative control and less for both bacteria. The data are discoverable in [Table 2].{Table 2}

Agar disk diffusion test

The widest zone was formed due to positive controls (34 mm for gentamicin and 32 mm for cephalothin) and after it, the widest zone was formed due to 0.062 g/ml of the essential oil in B. subtilis culture (18 mm) and it was no halo in negative control and less for both bacteria. The data are discoverable in [Table 3].{Table 3}

Minimum inhibitory concentration determination

In the essential oil, MIC was 0.015 g/ml for P. aeruginosa, whereas MIC was 0.031 g/ml for B. subtilis [Table 4].{Table 4}

Minimum bactericidal concentration ascertaining

MBC was 0.015 g/ml for P. aeruginosa, Whereas MBC was 0.031 g/ml for B. subtilis [Table 4].

As the table showed, essential oil of P. khinjuk has excluded the growth of P. aeruginosa and B. subtilis. Furthermore, by increasing the concentration of the essential oil, the inhibition zone augmented. The results defined that in tested bacteria, there was a considerable discrepancy in terms of sensitivity to the essential oil of P. khinjuk. The most sensitivity was apperceived in B. subtilis.


The use of plant compounds to remedy infections is an old practice in large part of the world, especially in developing countries where there is dependence on traditional medicine for a versatility of diseases. Interest in plants with antimicrobial properties has revived as a result of new obstacles associated with the use of antibiotics.[15]P. khinjuk is an endemic and resistance species in dry and sub-dry forests in mountainous regions of western Iran. The plant has played important roles in folk medicine and are used as anti-inflammatory, antipyretic, antibacterial, antiviral, in treatment diarrhea, and throat infection.[16]

Yield and resolution of essential oil of Pistacia khinjuk

The chemical constituents recognized by GC and GC/MS, the results concerning the qualitative and quantitative analysis of the essential oil are presented in [Table 1]. In the essential oil of P. khinjuk, 22 compounds were identified. The main constituents were found to be γ-terpinene (81.14%) (w/w), β-pinene (3.93%) (w/w), α-terpinene (2.38%) (w/w), Camphene (1.6%) (w/w), dl-Limonene (1.45%) (w/w), 3-Cyclohexene-1-carboxaldehyde (1.25%) (w/w), β-Myrcene (1.1%) (w/w), and sabinene (1.09%) (w/w). Other components (14 compounds) were present in amounts <1%. Studies in related species have identified triterpenes in the galls of P. terebinthus L. and P. Lentiscus and in the bled resin of P. uera L. were reported to contain a-pinene, P-pinene, limonene, and myrtenol (or pinocarveol).[17],[18],[19],[20] In a previous study,[21] the main components of the green external skin of fruits of P. khinjuk were reported to be 1, 8– Cineole (11.09%) (w/w), 1, 5-Heptadien-4-one, 3, 3, 6 trimethyl (35.76%) (w/w), Camphor (26.34%) (w/w), and β-Selinene (10.15%) (w/w). In the other study reported that the essential oils of leaves of P. khinjuk Stocks, P. chinensis Bunge and P. lentiscus L, prepared by hydrodistillation, and studied by GC and GC-MS, showed qualitative and quantitative differences. All three were found to be rich in monoterpene hydrocarbons. In P. lentiscus 4% sesquiterpene alcohols were found, and no monoterpene alcohols, whereas in P. khinjuk and P. chinensis 16% and 8% monoterpene alcohols respectively were found, and no sesquiterpene alcohols. Some major constituents of essential oil from the aerial parts of P. khinjuk are α-pinene, β-pinene, Myrcene, beta-caryophyllene, Germacrene B and Spathulenol.[22] Results of a recent study showed that some of the main constituents of essential oil from the aerial parts of P. khinjuk (Kermanshah, the western part of Iran) are α-pinene, β-pinene, myrcene, beta-caryophyllene, germacrene B, and spathulenol.[23] It is possible that our result on the composition of this essential oil related to method of essential oil extraction.

Antibacterial activity

The antibacterial results showed that the essential oil of P. khinjuk inhibited the two bacteria and the activity were considerably dependent upon concentration. In fact, the results indicated that P. khinjuk essential oil with 0.015/0.031 g/ml concentration has prevented from the growth P. aeruginosa and B. subtilis, respectively, also in 0.015/0.031 g/ml concentration has destroyed P. aeruginosa and B. subtilis, respectively, actually, MIC and MBC are equal for these bacteria. Thus, the research represents the antibacterial effect of the medical plant on P. aeruginosa and B. subtilis. Concerning the method of essential oil, extraction and preventing from using high temperature to decrease the rate of destruction of impressive herbal compound. Its bioactive components may be γ-terpinene and other components that we do not know. The study results agree with the past antibacterial studies related to these species.[16],[24],[25] In the essential oil, the main constituent was found to be γ-terpinene. γ-terpinene was assessed for its ability to induce cellular protein leakage in P. vulgaris and E. coli (Gram negatives) as well as L. monocytogenes and S. pyogenes (Gram positives). Both the Gram-negative and Gram-positive test bacteria showed a similar trend of protein permeation when treated with γ-terpinene. Protein permeation could be used as an index of the membrane detriment caused by chemical and physical agents. It has been offered that the cytoplasmic membrane is also a target for γ-terpinene action and the results evidencing the protein leakage corroborated this hypothesis. γ-terpinene was assessed for its capability to induce cellular lipid permeation in P. vulgaris and E. coli as well as L. monocytogenes and S. pyogenes.[26] The effect of γ-terpinene might be the result of its phenolic structure which interferes with the lipid bilayer of the outer membranes.[27] The essential oil of P. khinjuk content flavonoids and flavonoid. Furthermore, several members of the genus Pistacia have been chemically investigated. They are characterized mainly by the occurrence of flavonoids and flavonoid glycosides.[28] Flavonoids are hydroxylated phenolic substances, and they have been found in vitro to be efficient antimicrobial substances against an extensive array of microorganisms. Their activity is probably due to their ability to complex with extracellular and solvable proteins and to complex with bacterial cell walls.[29] These plants (such as P. khinjuk) have also been reported to contain phenolic compounds and triterpenoids.[30],[31] Terpenenes or terpenoids are active against bacteria.[29] It should be noted that the two main volatile constituents, α-pinene (0.3% in the essential oil) and terpinolene (2.38% in the essential oil), are compounds with interesting antibacterial properties.[32],[33] In addition, terpinolen has been identified as an antioxidant agent.[34]

From this study, it can be concluded that the essential oil of P. khinjuk (Combined with the dominance γ-terpinene) possess antibacterial effect, and the antibacterial activity of the essential oil was due to the presence of various active compounds. Hence, the phytochemical compounds responsible for the antibacterial effect of bacteria can be subjected to isolation of the therapeutic antimicrobials. Our results defend the use of the plant in traditional medicine and offer that essential oil of P. khinjuk possess compounds with good antibacterial properties. They can be used as antibacterial supplements in the developing countries toward the development of new remedial agent. Additional in vivo studies and clinical trials would be needed to justify and further evaluate the potential of the plant as an antibacterial agent in topical or oral applications.


The authors would like to thank the Medical Sciences University of Kermanshah, Iran for the financial support of this work.

Financial support and sponsorship

We, the authors wish to thank Medical Sciences University of Kermanshah, Iran for the financial support of this work.

Conflicts of interest

There are no conflicts of interest.


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