Microbiological

Communication

Biosci. Biotech. Res. Comm. 10(1): 137-142 (2017)

Chemical composition and antimicrobial effects of

Thymus daenensis

Helicobacter pylori

Parisa Moradi

, Tahereh Falsa

, Neda Saffari

, Ebrahim Rahimi

Hassan Momtaz

and Behzad Hanedi

Department of Biology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

ABSTRACT

High occurrence of resistance in the Helicobacter pylori strains of human clinical samples caused medical practition-

ers to found a good alternative therapeutic choice. High phenolic contents of the Thymus daenensis essential oil cov-

ered its high antimicrobial effects. The present investigation was done to study the chemical composition and anti-H.

pylori effects of T. daenensis essential oil. Aerial parts of the T. daenensis were collected from the Yasuj city and

transferred to the laboratory. Essential oil was extracted using the Clevenger apparatus. Chemical components of T.

daenensis was identi ed using the GC-mass analyzer. Anti-H. pylori effects of T. daenensis was determined using the

disk diffusion method. Twelve (99.24%) chemical components were totally identi ed in the essential oil of the T. dae-

nensis. Thymol (42.81%), gamma-Terpinene (20.39%) and para-Cymene (9.72%) were the most commonly identi ed

chemical components. Distribution of beta-Myrcene, D-Limonene, beta-Pinene and Terpinen-4-ol were moderate. H.

pylori strains harbored the highest levels of sensitivity against tetracycline and ampicillin antibiotics, while those of

resistance were seen for the erythromycin, clarithromycin and metronidazole antibiotics. Inhibition zone diameter

increased from 0 to 18.6±0.5 mm which represented dose depended anti-H. pylori effects of T. daenensis essential oil.

Extensive production of T. daenensis full from thymol, gamma-Terpinene and para-Cymene will help researchers to

formulate an effective antibiotic agent for treatment of the cases of H. pylori-infection.

KEY WORDS:

THYMUS DAENENSIS

, CHEMICAL COMPONENTS, ANTIMICROBIAL EFFECTS,

HELICOBACTER PYLORI

137

ARTICLE INFORMATION:

*Corresponding Author:

Received 21

Dec, 2016

Accepted after revision 25

March, 2017

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138 ANTI-HELICOBACTER PYLORI EFFECTS OF

THYMUS DAENENSIS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Parisa Moradi and Neda Saffari

INTRODUCTION

Helicobacter pylori (H. pylori) is an extracellular gram-

negative, microaerophilic and spiral bacterium which

is known as the causative agent of various types of

gastrointestinal diseases and disorders such as gastric

adenocarcinoma, gastric ulcer, gastritis, and lymphoma

(Shrestha et al., 2012). It has been estimated that 30 to 90

percent of hospitalized patients with such gastrointesti-

nal diseases and disorders were infected with H. pylori

(Shrestha et al., 2012; Mastromarino et al., 2005; Vu and

Ng, 2000). Prevalence of H. pylori in in developed and

developing countries is about 40% and 90%, respec-

tively (Suerbaum and Michetti, 2002). Gastrointestinal

diseases caused by H. pylori often treated with antimi-

crobial agents. Prescription of ampicillin, amoxicillin,

clarithromycin, metronidazole and tetracycline antibiot-

ics is the most common way for treatment of the cases of

H. pylori (Bytzer et al., 2011; Rimbara et al., 2011; Yang

et al., 2014). Nevertheless, occurrence of extreme levels

of antibiotic resistance in the H. pylori strains caused

several concerns regarding their treatment (Bytzer et al.,

2011; Rimbara et al., 2011; Yang et al., 2014; Ghotaslou

et al., 2015).

Therefore, medical practitioners and also pharmaco-

logical companies have a tendency to found a suitable

alternative treatment for H. pylori. Medicinal plants may

be a powerful sources of antimicrobial agents which can

be used for treatment of infectious diseases. The genus

Thymus L. goes to the major family (Lamiaceae), and

included approximately 215 species in the world. Totally,

14 species of Thymus have been described in the zone

of Iran (Stahl-Biskup and Saez, 2002; Rechinger, 1982;

Mojab et al., 2008; Fachini-Queiro et al., 2012). T. daen-

ensis, T. carmanicus, T. daenensis, T. persicus, T. vulga-

ricus and T. trautvetteri are the most commonly endemic

species in Iran (Stahl-Biskup and Saez, 2002; Rechinger,

1982; Mojab et al., 2008; Fachini-Queiro et al., 2012). T.

daenensis subsp. daenensis is an endemic subspecies of

Iran and especially high altitudes of Zagros mountains

(Stahl-Biskup and Saez, 2002; Rechinger, 1982; Mojab

et al., 2008; Fachini-Queiro et al., 2012). Areal parts and

volatile constituents of T. daenensis are used in Iranian

traditional medicine.

Thymus species and especially T. daenensis are usu-

ally applied as tasty and  avoring agents in tea and

also spices and medicinal drives including antimicro-

bial, anti-fungal, antispasmodic, and anti-in amma-

tory agent (Stahl-Biskup and Saez, 2002; Rechinger,

1982; Mojab et al., 2008; Fachini-Queiro et al., 2012;

Gautam et al., 2014). Previous investigations showed

high anti-parasites, antibacterial, antiviral, antifungal,

spasmolytic and antioxidant effects of Thymus species

(Stahl-Biskup and Saez, 2002; Rechinger, 1982; Mojab

et al., 2008; Fachini-Queiro et al., 2012; Gautam et al.,

2014).

There were no previously published data on the anti-

microbial effects of Thymus daenensis on the H. pylori.

Therefore, the present investigation was carried out

to study the chemical composition and antimicrobial

effects of the essential oil extracted from the Thymus

daenensis on ten clinical isolates of H. pylori.

MATERIAL AND METHODS

PLANT AND ESSENTIAL OIL EXTRACTION

From July to August 2013, aerial parts of T. daenensis

at the  owering stage were collected from the plains of

the Dena mountain, Yasuj, South-west of Iran. The sam-

ples of the plant were identi ed and a voucher specimen

was deposited at the Herbarium of Research Center of

Agricultural of Shahrekord city, Iran. Essential oil of the

collected

T. daenensis was extracted by water distilla-

tion using the Clevenger apparatus. The essential oil was

dried over anhydrous sodium sulfate and stored at 17°C

until further analysis.

IDENTIFICATION OF THE CHEMICAL

COMPONENTS

Samples were studied by the Gas Chromatography (GC,

Agilent 7890 A) and Mass Spectrometry (MS, Agilent

5975 C). Identi cation of the chemical components was

done according to the method described previously by

Borug˘

et al. (2014) (Borug˘

et al., 2014). Conditions of

the GC stage were as follow: temperature range of 50 to

250°C at 40°C/min with a solvent delay of 5 min. Injec-

tor of the device was kept at 250°C. Helium at a  ow of

1.0 mL/min was the inert gas, and the injected volume in

the splitless mode was 1 L. The MS conditions were as

follow: ionization energy of 70 eV, quadrupole tempera-

ture of 100°C, scanning velocity of 1.6 scan/s and weight

range of 40-500 amu.

BACTERIAL STRAINS

Ten rough H. pylori strains were obtained from the

clinical cases of gastrointestinal disorders of Alzahra

Hospital, Tehran, Iran. H. pylori strains were regularly

cultured on Brucella agar (Merck, Germany) supple-

mented with 5–7% sheep blood, amphotericin (2mg/l),

polymixin-B (8mg/l), and vancomycin (6mg/l) and were

incubated at 37°C for 3–7 days on the microaerophilic

conditions (10% CO2 and 95% humidity). Then, H.

pylori strains were approved using the morphological,

Gram staining and biochemical tests (rapid urease, oxi-

dase and nitrate) and PCR ampli cation of 16S rRNA

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTI-HELICOBACTER PYLORI EFFECTS OF

THYMUS DAENENSIS

139

Parisa Moradi and Neda Saffari

gene (Forward: 5’-CTGGAGAGACTAAGCCCTCC-3’ and

Reverse: 5’-ATTACTGACGCTGATTGTGC-3’) (110 bp) (Ho

et al., 1991).

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Suspensions of the fresh cultures were made in saline

and turbidity was adjusted to 1×10

bacteria/ml (corre-

sponding to turbidity with OD 0.8 at 600 nm). Two-hun-

dred microliters of microbial suspension were placed on

50-ml Mueller Hinton agar plates containing 10% fetal

calf serum (Sigma, UK) and incubated for 2–5 days at

37°C under microaerophilic conditions. H. pylori ATCC

26695 were used as quality control. Tests were done

three times. Table 1 shows the standard values for sus-

ceptibility determination (CLSI, 2012). The antimicrobial

effects of T. daenensis essential oil were performed by

the disc diffusion method. For this purpose, sterile blank

disks (6 mm) were inoculated with concentrations of 0.6,

1.25, 2.5, 5 and 10 (%v/v) essential oil and were placed

on dense cultures of bacteria and incubated for 2–5 days

at 37°C under microaerophilic conditions. Susceptibility

of H. pylori strains were also determined against ampi-

cillin (10 µg), metronidazole (5 µg), erythromycin (5 µg),

clarithromycin (2 µg) and tetracycline (30 µg) antibiotic

agents (Oxoid, UK) (CLSI, 2012).

STATISTICAL ANALYSIS

SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) and

one-way analysis of variance (ANOVA) test were used

for statistical analysis. P <0.05 was considered as sig-

ni cant difference.

RESULTS AND DISCUSSION

Table 2 represents the chemical components of the

essential oil of the T. daenensis. Totally, 12 chemical

components were identi ed in the essential oil of the

T. daenensis (99.24%). Thymol (42.81%), gamma-Ter-

pinene (20.39%) and para-Cymene (9.72%) were the

most commonly identi ed chemical components in the

essential oil of the T. daenensis. Frequency of beta-

Myrcene, D-Limonene, beta-Pinene and Terpinen-4-ol

were 3.71%, 3.56%, 3.82% and 3.45%, respectively.

Table 3 represents the inhibition zone diameter for

each of the ten clinical isolates of H. pylori against anti-

biotic agents. Results showed that H. pylori strains had

the highest levels of sensitivity against tetracycline and

ampicillin antibiotics. Most resistance was seen for the

erythromycin, clarithromycin and metronidazole anti-

biotics. Statistically signi cant differences (P <0.05)

were seen between the zone of inhibition and types of

antibiotics.

Table 1. Standard values for determination of sensitivity and

resistance of bacteria against antibiotic agents.

Antibiotic agents

Diameter of inhibition (mm)

Sensitive Intermediate Resistant

Ampicillin (10 µg) ≥17 14-17 ≤13

Metronidazole (5 µg) ≥15 12-15 <12

Erythromycin (5 µg) ≥18 14-17 ≤12

Clarithromycin (2 µg) ≥18 14-17 ≤13

Tetracycline (30 µg) ≥19 15-18 ≤14

Table 2. Chemical components of the essential oil

of the T. daenensis.

Chemical

components

Retention Time

(RT) (min)

Frequency

(%)

beta-Phellandrene 8.26 1.92

beta-Myrcene 6.97 3.71

alpha-Thujene 5.39 2.94

alpha-Phellandrene 7.53 2.07

D-Limonene 8.04 3.56

alpha-Pinene 5.63 2.12

beta-Pinene 6.89 3.82

Terpinen-4-ol 12.55 3.45

Caryophyllene 17.32 2.71

para-Cymene 8.46 9.74

gamma-Terpinene 8.96 20.39

Thymol 16.17 42.81

Total 99.24

Parisa Moradi and Neda Saffari

140 ANTI-HELICOBACTER PYLORI EFFECTS OF

THYMUS DAENENSIS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Table 3. Inhibition zone diameter for each of the ten clinical isolates of H. pylori against antibiotic agents.

Antibiotic agents

Diameter of the zone of inhibition for H. pylori strains (mm)

HP1 HP2 HP3 HP4 HP5 HP6 HP7 HP8 HP9 HP10

Metronidazole (5

µg)

0 0 0 0 0 0 16±0.4 15.4±0.2 0 0

Tetracycline (30 µg) 15.7±0.5 17.5±0.1 17±0.6 17.9±0.5 16±0.6 16.8±0.7 16.5±0.3 14.2±0.1 0 0

Ampicillin (10 µg) 12.1±0.6 0 11.3±0.8 9.1±0.7 0 19.8±0.5 16.2±0.8 0 34.8±0.8 11.7±0.6

Erythromycin (5 µg) 0 0 0 0 0 0 18.3±0.5 0 0 0

Clarithromycin (2

µg)

0 0 0 0 0 0 23.2±0.4 0 0 0

Table 4. Inhibition zone diameter of the various concentrations of the T. daenensis essential oil against H. pylori.

Essential oil

concentrations

Diameter of the zone of inhibition for H. pylori strains (mm)

HP1 HP2 HP3 HP4 HP5 HP6 HP7 HP8 HP9 HP10

0.6% 0 5.8±0.4 6±0.5 0 5.3±0.8 0 0 4.1±0.2 11.2±0.6 5.5±0.2

1.25% 0 5.3±0.9 7.8±0.1 7±0.0 6.7±0.3 0 0 5.2±0.8 14.3±0.4 5.2±0.5

2.5% 4.6±0.3 8.3±0.0 9.7±0.4 10.2±0.5 9.1±0.1 11.4±0.5 8.8±0.2 7.5±0.5 15.5±0.1 8.4±0.3

5% 5.3±0.5 13.1±0.5 13.2±0.1 12.8±0.6 11.6±0.2 12±0.9 14.1±0.1 10.2±0.4 16.7±0.2 11.6±0.4

10% 6.2±0.7 15.7±0.2 13.6±0.2 14.3±0.4 14±0.5 17.1±0.2 16±0.3 12.3±0.4 18.6±0.5 13.2±0.8

Table 4 represents the inhibition zone diameter of

the various concentrations of the T. daenensis essential

oil against H. pylori strains. We found that increase in

the concentration of essential oil cause its higher anti-

microbial effects on the H. pylori strains isolated from

the clinical cases of gastrointestinal disorders (P <0.05).

Inhibition zone diameter increased from 0 to 18.6±0.5

mm which showed that the anti-H. pylori effects of T.

daenensis essential oil is dose depended.

Failure of majority of therapeutic options and espe-

cially ampicillin, amoxicillin, clarithromycin, metroni-

dazole and tetracycline antibiotics caused us to study

the antimicrobial effects of H. pylori strains against T.

daenensis essential oil. The results of the present inves-

tigation showed that the T. daenensis essential oil had

a high antimicrobial effects on the H. pylori strains iso-

lated from clinical cases of gastrointestinal disorders.

Several studies have been approved the anti-H. pylori

effects of some medicinal plants. Falsa et al. (2014)

(Falsa et al., 2014) reported that Satureja bachtiarica

essential oil showed strong antibacterial activity against

clinical isolates of H. pylori (17.6 ± 1.1 mm and 0.035 ±

0.13 l/ml).They showed that anti-H. pylori effects of S.

bachtiarica is due to its high carvacrol content. Esmaeili

et al. (2012) (Esmaeili et al., 2012) reported that Shoya

powder and essential oils of Thymus vulgaris and Euca-

lyptus globulus had signi cant effects on the H. pylori.

They showed that Minimum Inhibitory Concentration

(MIC) of the T. vulgaris and E. globulus were 10.8 and

46.4 (µg/ml) respectively.

The antimicrobial effects of the T. daenensis essen-

tial oil is due to the presence of some active constitu-

ents with their hydrophobicity which enables them for

rupturing cell membranes and intrastructures. We found

that Thymol (42.81%), gamma-Terpinene (20.39%) and

para-Cymene (9.72%) were the most commonly iden-

ti ed chemical components in the essential oil of the

T. daenensis. Previous study in Romania (Grigore et al.,

2010) showed that the most commonly detected compo-

nents in the essential oil of the Thymus were p-cymene,

-terpinene and thymol which was similar to our  nd-

ings, while those of Morocco (Imelouane et al., 2009)

and Spain (Ballester-Costa et al., 2013) are entirely dif-

ferent with us. P-cymene, -terpinene and thymol were

the most commonly detected components in previ-

ous studies conducted on Iran (Pirbalouti et al., 2013),

Poland (Kowalski and Wawrzykowski, 2009), Italy (De

Lisi et al., 2011) and Spain (Rota et al., 2008). Probably

types of samples, method of sampling, genus of plants,

and differences in the climate and weather of various

Parisa Moradi and Neda Saffari

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTI-HELICOBACTER PYLORI EFFECTS OF

THYMUS DAENENSIS

141

geographical regions are the main factors which may

affected on the chemical components of Thymus plants

of different studies.

Antimicrobial effects of T. daenensis essential oil is

depends on its chemical compositions. It seems that high

content of the phenolic compounds (thymol) and ter-

pene hydrocarbons (-terpinene) is the main factor for

the high anti-H. pylori effects of T. daenensis. Previ-

ous investigations showed that para-cymene does not

display any antimicrobial activities alone (Dorman and

Deans, 2000), while it has a strong antibacterial effects

in relation with thymol and -terpinene (Delgado et al.,

2004; Gallucci et al., 2009).

CONCLUSION

In conclusion, we found that T. daenensis essential oil

has a high antimicrobial effects on the H. pylori strains

of human clinical infections. As it showed, Thymol,

gamma-Terpinene and para-Cymene were the most

commonly identi ed chemical components in the essen-

tial oil of the T. daenensis. In compare with the  ndings

of other researchers, Preparation of good conditions for

growth of T. daenensis full from these chemical compo-

nents will help us to present an ef cient antibiotic agent

for treatment of the cases of H. pylori-infection.

ACKNOWLEDGEMENTS

Authors would like to thanks from Dr. Tahereh Falsa ,

Dr. Mohaddase Mahboubi, Dr. Behzad Hamedi, Prof.

Ebrahim Rahimi and Prof. Hassan Momtaz for their

important clinically support. This work was supported

by the Alzahra University, Tehran, Iran.

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