ARTICLE INFO

Article Type

Original Research

Authors

Zare Bidaki   M. (1)
Arab   M. (*)
Khazaei   M. (2)
Afkar   E. (3)
Zardast   M. (4)






(*) Clinical Biochemistry Department, Medicine Faculty, Birjand University of Medical Sciences, Birjand, Iran
(1) “Hepatitis Research Center” and “Medical Microbiology Department, Paramedics Faculty, Birjand University of Medical Sciences, Birjand, Iran
(2) Medical Microbiology Department, Paramedics Faculty, Birjand University of Medical Sciences, Birjand, Iran
(3) Deputy of Research & Technology, Birjand University of Medical Sciences, Birjand, Iran
(4) Pathology Department, Medicine Faculty, Birjand University of Medical Sciences, Birjand, Iran

Correspondence

Address: South Khorasan, Birjand, Ghaffari Street, Birjand University of Medical Sciences, Medicine Faculty, Clinical Biochemistry Department
Phone: +985632395507
Fax: +985632440388
minaarab70@yahoo.com

Article History

Received:  January  5, 2015
Accepted:  January 25, 2015
ePublished:  September 20, 2015

BRIEF TEXT


… [1] The anti-bacterial effect of essences in complete form is more than the effect of each individual component [2]. … [3-9] Thyme (Zataria multiflora Boiss) is of Lamiaceae family and is native to Iran [10]. The medical bodies of this plant are its leaves and flowers and the main compounds in the essence of this plant are carvacrol, thymol, linalool and paracymene, of which the antibacterial effect has been proved previously [11]. The essence of this plant, due to phenolic monoterpene compounds, is one of the most effective essential oil with antioxidant and antimicrobial properties [12]. … [13-16]

Several studies have been conducted to determine the antibacterial effect of thyme essential oil; and almost in all cases the strong antibacterial effect of thyme has been confirmed [10, 17-19].

This research aimed to study the antibacterial effects of Zataria multiflora Boiss essential oil against 8 species of gastrointestinal pathogenic bacteria.

This is an experimental-laboratory study.

This study was done in 2014 at the Birjand University of Medical Sciences.

Non-declared

In the stage of preparation of essence, the essential oil was extracted by steam distillation using Clevenger apparatus. Dimethyl Sulfoxide (DMSO) was used as a solvent with the concentration of 5%. The primary stock was used for next stages [19]. In the stage of preparing bacterial suspension, the understudied microorganisms were Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 11778), Klebsiella pneumonia (ATCC 700603), Psudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213), Shigella flexneri (ATCC 12022), salmonella enterica enteritidis (PTCC 1709), and Salmonella typhimurium (PTCC 1639). These bacteria were prepared separately and in lyophilized form from Pasteur Institute of Iran, and Iran Scientific and Industrial Research Center. In order to obtain pure colonies from nutrient broth medium containing pathogenic bacteria, isolation was carried out on blood agar medium in order to prepare a solution with turbidity of 0.5 McFarland standard (1.5 x 108cfu/ml) from the appeared colonies on the medium. In broth micro-dilution testing for determining Minimum Inhibitory Concentration (MIC), MIC testing was carried out in sterile micro-plates with 96 micro wells through broth micro-dilution method. These micro-plates have 8 rows of 12 micro wells with the volume of 25 µl. Then 100µl of the first micro well was removed and was poured in the second micro well and after few removing and pouring with sampler, 100 µl was removed from the second micro well and was poured into the third micro well. This process was continued until the eleventh micro well. Then 10 µl of culture medium containing each bacteria equivalent to turbidity of 0.5 McFarland was added to all the micro wells except eleventh micro well of each row. The micro wells no. 11 of each row as control essence (negative control) contained only medium and essential oils. Micro well no. 12 of each row as the bacteria control (positive control) was for determining the turbidity of bacteria in medium, DMSO and the bacteria. Then absorbance was read using an ELISA reader at zero hour and a wavelength of 620 nm. Finally, micro plate was placed in an incubator at 37°C for 24 hours and after completion of incubation, turbidity or cloudiness of the micro wells was observed by eyes and the absorbance was read by ELISA reader. To determine minimum inhibitory concentration, the lowest concentration which did not have any turbidity, i.e. without any observed bacterial growth in it, was considered as MIC number. In stage of determining the minimum bacterial concentration (MBC), for measuring the lowest concentration, 10 µl was removed from micro wells without turbidity (MIC concentrations and over that) under sterile conditions and near the flame, and it was incubated and cultivated on the agar blood medium. After 24-hour incubation at 37 ° C, the lowest dilution that could kill 99.9% of the bacteria was considered as MBC dilution. All procedures were performed in three times, and it was presented as the mean. Data were analyzed using SPSS 16 statistical software. To evaluate the significant difference between the results, One-way ANOVA and Tukey test were used (for paired comparison of the results).

The results showed significant difference in Zataria Multiflora Boiss essential oil MIC between the tested bacteria. Concentration 0.025% of the Zataria Multiflora Boiss essential oil had the most inhibitive effect against Bacillus cereus, and the concentration 0.156% of this essential oil had the least inhibitive effect against Pseudomonas aeruginosa. Also, the essence of this plant at concentrations 0.032%, 0.039%, and 0.071% inhibited the growth of other tested bacteria. The sensitivity of both species of Salmonella to this essence was at concentration 0.032% but the MBC for Zataria multiflora Boiss essential oil did not have any significant difference in the studied bacteria. However, the concentrations of this essence which inhibited the growth of Escherichia coli, Bacillus cereus, Klebsiella pneumonia, and Salmonella enteritidis, had lethal effect on them (table 1). MIC of Zataria Multiflora Boiss essential oil was compared between the studied bacteria in pairs (table 2).

The MIC and MBC range of thyme essential oil was between 0.025-0.156% and 0.025-0.091% respectively. The antibacterial effect of Zataria multiflora Boiss essential oil was remarkable. This finding is similar to results of conducted studies in this field. The MIC of thyme oil in two methods of macro-dilution and micro-dilution has been obtained 0.04% [20]. The MIC against E.coli and Candida albicans has been determined 0.03% according to broth micro-dilution testing [21] which is in accordance with the results of the current study. In investigating the effect of Zataria Multiflora Boiss essential oil on the growth and production of Staphylococcus aureus enterotoxin E, MIC, and MBC of thyme essence has been reported 0.03 and 0.04 based on micro dilution method [22], but in the current study the amount of MIC and MBC of thyme essence against staphylococcus aureus was obtained 0.071 and 0.091% respectively. The Zataria Multiflora Boiss essential oil alone in doses of 30 and 45µl/ml can significantly decrease the growth of bacillus cereus at 20 °C in 21 days in the sample of commercial soup [23]. The antibacterial effect of thyme has been greater than antibacterial effects of myrtus and peganum harmalo, in such way that 37 isolates at a concentration of 0.025 mg/ml of the plant extract has been sensitive [24]. The thyme essence, in comparison with methanol extract, has greater antibacterial effect in disk diffusion method, and the inhibitory effect of this oil essence on gram negative bacteria has been stronger [25]. In investigating the effect of thyme essential oil on the growth rate of Salmonella typhimurium in the commercial soup, the least bacterial growth has been observed in the essence with the concentration 0.03% [26] which is consistent with the results of this study. The essence of Zataria Multiflora Boiss is effective on destroying enterococcus faecalis at the concentrations 1% and 2% [27]. This essence has an appropriate antibacterial effect on Streptococcus agalactiae and Staphylococcus aureus [28]. In investigating the effect of Zataria Multiflora Boiss essential oil on Aspergillus flavus, the amounts of MIC and MFC have been 400 and 1000ppm respectively, and the effect of essence on sporulation has been higher than the effect on the mycelium growth [29]. The antifungal activity of the thyme essential oil and extract against clinical isolates of Candida albican which are resistant and susceptible to fluconazole has been confirmed [30]. The most appropriate formulation of an anti-dermatophyte cream is from hydroalcoholic extract of Zataria multiflora Boiss contains 2% of extract [31]. … [32] Total concentration of thyme essence with lethal mean 91.1% has the most lethal effect on Giardia cysts compared with metronidazole, boiled thyme extract, and thyme soxhlet extraction after 10 minutes. Remarkable effect of Zataria Multiflora Boiss essential oil on Trichomonas vaginalis is known [33]. Antioxidant, antimicrobial and anti-inflammatory effects of Zataria Multiflora Boiss have protected the mice against this disease [34]. Anti-inflammatory and analgesic effects of thyme have been confirmed [35]. … [36]

Complementary studies should be done on other bacterial and fungal pathogens. The essence of this plant should be used in food industry (as a preservative) as well as pharmaceutical industry for treatment of diseases such as food poisoning and ocular infections caused by Bacillus cereus. The effect of this plant should be studied on other types of microorganisms.

The limited number of investigated bacteria was the limitation of this study.

Zataria Multiflora Boiss essential oil has significant anti-bacterial effects.

Department of Research and Technology of Birjand University of Medical Sciences is appreciated.

Non-declared

Due to work on the plants and bacterial population, the present research has no moral consideration.

This research was sponsored by Department of Research and Technology of Birjand University of Medical Sciences.

TABLES and CHARTS

Show attach file


CITIATION LINKS

[1]Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999;12(4):564-82.
[2]Ebrahimi A, Khayami M, Nejati V. Comparison of antimicrobial effect of different parts of quercus persica against escherichia coli O157:H7. Q Horizon Med Sci. 2012;17(4):11-7. [Persian]
[3]Ates DA, Erdogrul OT. Antimicrobial activities of various medicinal and commercial plant extracts. Turk J Biol. 2003;27:157-62.
[4]Sedighi J, Maftoun F, Ziaei G. Herbal medicine: Knowledge, attitude and practice in Tehran. J Med Plant. 2005;4(13):11-8.
[5]Calixto JB. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz J Med Biol Res. 2000;33(2):179-89.
[6]Abdollahi B, Ziaei F, Shabankhani B, Azadbakht M. Effect of essential oils of Artemisia aucheri Boiss, Zataria multiflora Boiss, and Myrtus communis L. on Trichomonas vaginalis. Iran J Pharm Res. 2004;3 suppl 2:35-40.
[7]Azadbakht M, Azadbakht M. Five common herbal remedy anti-protozoan. J Mazandaran Uni Med Sci. 2008;18(67):118-32. [Persian]
[8]Bagheri A, Naghdi Badi H, Maki Zadeh M, Hemati AR, Movahedian F. Evaluation of using herbal medicine in Isfahan women population. J Med Plant. 2005;4(15):81-92.
[9]Smith Palmer A, Stewart J, Fyfe L. The potential application of plant essential oils as natural food preservatives in soft cheese. Food Microbiol. 2001;18(4):463-70.
[10]Sharififar F, Moshafi MH, Mansouri SH, Khodashenas M, Khoshnoodi M. In vitro evaluation of antibacterial and antioxidant activities of the essential oil and methanol extract of endemic Zataria multiflora Boiss. Food Control. 2007;18(7):800-5.
[11]Eftekhar F, Zamani S, Yusefzadi M, Hadian J, Nejad Ebrahimi S. Antibacterial activity of Zataria multiflora Boiss essential oil against extended spectrum β lactamase produced by urinary isolates of Klebsiella pneumonia. Jundishapur J Microbiol. 2011;4(5):S43-9.
[12]Saei Dehkordi SS, Tajik H, Moradi M, Khalighi Sigaroodi F. Chemical composition of essential oils in Zataria multiflora Boiss. From different parts of Iran and their radical scavenging and antimicrobial activity. Food Chem Toxicol. 2010;48(6):1562-7.
[13]Tiwari BK, Valdramidis VP, O’Donnell CP, Muthukumarappan K, Bourke P, Cullen PJ. Application of natural antimicrobials for food preservation. J Agric Food Chem. 2009;57:5987-6000.
[14]Mansoura A, Enayatb K, Neda MS, Behzad A. Antibacterial effect and physicochemical properties of essential oil of Zataria multiflora Boiss. Asian Pac J Trop Med. 2010;6(3):439-42.
[15]Ramezani M, Hosseinzadeh H, Samizadeh S. Antinociceptive effects of Zataria multiflora Boiss fractions in mice. J Ethnopharmacol. 2004;91(1):167-70.
[16]Sefidkon F, Rahimi Bidgoli A. Quantitative and qualitative variation of essential oil of Thymus kotschyanus by different methods of distillation and stage of plant growth. Iran J Med Aroma Plant Res. 2003;15:1-22. [Persian]
[17]Basti AA, Misaghi A, Ebrahimzade E, Abbasifar R, Radmehr SH, Akhondzade S. Effect of Zataria multiflora Boiss. essential oil on lag phase of growth of Staphylococcus aureus in a brain heart infusion broth. J Med Plant. 2004;3(11):42-7. [Persian]
[18]Rahnama M, Razavi Rohani SM, Tajik H, Khaleghi Sigarudi F, Rezazad Bari M. Effects of Zataria multiflora Boiss. essential oil and nisin, alone and in combination against listeria monocytogen in BHI broth. J Med Plant. 2009;4(32):120-31. [Persian]
[19]Mahboubi M, Bidgoli FG. Antistaphylococcal activity of Zataria multiflora essential oil and its synergy with vancomycin. Phytomedicine. 2010;17(7):548-50.
[20]Hosseinzadeh A, Mohajerfar T, Akhondzadeh Basti A, Khanjari A, Gandomi Nasrabadi H, Misaghi A, et al. Determination of minimum inhibitory concentration (MIC) of Zataria multiflora Boiss. essential oil and lysozyme on E. coli O157: H7. J Med Plant. 2011;1(8):208-17. [Persian]
[21]Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol. 1999;86(6):985-90.
[22]Azizkhani M, Misaghi A, Akhondzadeh Basti A, Gandomi Nasrabadi H, Hosseini H. Effect of Zataria multiflora Boiss. essential oil on growth and enterotoxin e production of Staphylococcus aurous ATCC 29213. J Med Plant. 2012;4(44):185-93. [Persian]
[23]Mashak Z, Moradi B, Moradi B. The Combined Effect of Zataria multiflora Boiss. and Cinnamomum zeylanicum Nees. essential Oil on the Growth of Bacillus cereus in a Food Model System. J Med Plant. 2012;2(42):62-73. [Persian]
[24]Hashemi A, Shams S. Barati M, Samedani A. Antibacterial effects of methanolic extracts of Zataria multiflora, Myrtus communis and Peganum harmala on Pseudomonas aeruginosa producing ESBL. Arak Med Univ J. 2011;14(4):104-12. [Persian]
[25]Moshafi MH, Mansuri S, Sharififar F, Khoshnoodi M. Antibacterial and antioxidant effects of the essential oil and extract of Zataria Multiflora Boiss. J Kerman Univ Med Sci. 2007;14(1):33-43. [Persian]
[26]Moosavy MH, Basti AA, Misaghi A, Jabari Khameneh H, Karim G, Zahraei Salehi T. The survey of effect of Zataria multiflora Boiss. essential oil on the growth of salmonella typhimurium in a commercial barley soup. J Med Plants. 2010;2(34):109-16. [Persian]
[27]Ravanshad S, Basiri E, Dastgheib B. Antimicrobial activity of different concentrations of essential oil of Zataria multiflora on enterococcus faecalis. Shiraz Univ Dent J. 2007;8(1):28-36. [Persian]
[28]Zahraei Salehi T, Vojgani M, Bayat M, Torshizi H, Akhondzadeh A. Determination of Minimum Inhibitory Concentration (MIC) of Extract of Zataria multiflora, against the clinical isolates of Streptococcus agalactiae Staphylococcus, aureus and E. coli. J Fac Vet Med Univ Tehran. 2005;60(2):107-10. [Persian]
[29]Gandomi Nasrabadi H, Misaghi A, Akhondzadeh Basti A, Khosravi A, Bokaei S, Abbasifar R. Effects of Zataria multiflora Boiss. essential oil on aspergillus flavus. J Med Plant. 2008;3(27):45-51. [Persian]
[30]Akbari S. Antifungal activity of Thymus vulgaris L. and Origanum vulgare L. Against Fluconazol-Resistant and Susceptible Candida albicans Isolates. J Med Plant. 2007;1(Suppl 3):53-62. [Persian]
[31]Moghimipour E, Aghel N, Ameri A, Saadatzadeh A. Anti-dermatophyte cream formulation of the hydroalcolic extract of Zataria multifora Boiss. Iran J Basic Med Sci. 2007;10(1):36-45. [Persian]
[32]Sahebani NA, Movahhed A, Farsangi MH. Cytotoxic effects of thyme on giardia cyst in vitro. South Med J. 2002;4(2):88-95. [Persian]
[33]Azadbakht M, Ziai H, Abdullahi F, Shabankhani B. Effect of essential oils of Artemisia. Zataria and Myrtus on Trichomonas vaginalis. J Med Plant. 2003;4(8):35-40. [Persian]
[34]Ashtaral Nakhai L, Mohammadirad A, Yasa N, Minaie B, Nikfar SH, Ghazanfari G, et al. Benefits of Zataria multiflora Boiss in experimental model of mouse inflammatory bowel disease. Evid Based Complement Alternat Med. 2007;4(1):43-50.
[35]Hosseinzadeh H, Ramezani M, Salmani G. Antinociceptive, anti-inflammatory and acute toxicity effects of Zataria multiflora Boiss extracts in mice and rats. J Ethnopharmacol. 2000;73(3):379-85.
[36]Sabetsarvestani MM, Sharafzadeh Sh, Alizadeh A, Rezaeian AA. Total phenolic content, antioxidant activity and antifungal property in two parts of garden thyme shoot. Int J Farming Allied Sci. 2013;2(22):1017-22.