Microbiological

Communication

Biosci. Biotech. Res. Comm. 10(2): 160-164 (2017)

Antibacterial and antioxidant effects of

Thymus

daenensis

essential oil

Seyed Heibatollah Hosseini

MSc Medicinal Plants, Faculty of Agriculture and Food Industry, Islamic Azad University, Shahrekord

Branch, Shahrekord, Iran

ABSTRACT

Thymusdaenensis is an Iranian medicinal plant with high antibacterial and antioxidant effects. The present study

was done to investigate the antibacterial and antioxidant effects of T. daenensis essential oil collected from the Dena

mountain, Iran. T. daenensis was collected and immediately transferred to the laboratory. After its approval by an

expert person, its essential oil was extracted using the Clevenger apparatus. DPPH free radical scavenging activity

and antibacterial effects of various concentration of T. daenensis essential oil were studied. Higher concentrations of

the T. daenensis essential oil had higher antibacterial and antioxidant effects. T. daenensis essential oil in a concen-

tration of 1 mg/ml had the highest inhibitory effects on the DPPH radicals (96.27±8.60%). The mean dimeter of the

zone of inhibition for tested bacteria had a range of 6.03±0.28 to 19.29±1.57 millimeter. The highest antibacterial

effects were seen for the 1mg/ml concentration of T. daenensis essential oil on all tested bacteria and especially on

E. coli (19.29±1.57 mm) and K. pneumonia (18.0±1.69 mm). Bacteria were resistant against some of tested antibiotic

agents and especially tetracycline, gentamicin and penicillin G. Essential oil of T. daenensis had a higher antibacterial

effects on its high concentrations than several types of antibiotic agents. We found that T. daenensis essential oil is

a good source for production of natural antioxidant and antibacterial agents.

KEY WORDS: ANTIBACTERIAL, PATHOGENIC BACTERIA, ANTIOXIDANT, DPPH RADICALS,

THYMUS DAENENSIS

, ESSENTIAL OIL

160

ARTICLE INFORMATION:

*Corresponding Author: Heybat_s@yahoo.com.

Received 18

Feb, 2017

Accepted after revision 29

May, 2017

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Online ISSN: 2321-4007 CODEN: USA BBRCBA

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Online Contents Available at: http//www.bbrc.in/

INTRODUCTION

Pathogenic agents and especially bacteria are the most

important causes of infections in hospitals. Staphylococ-

cus aureus (S. aureus), Escherichia coli (E. coli), Liste-

ria monocytogenes (L. monocytogenes), Bacillus cereus

(B. cereus), Pseudomonas aeruginosa (P. aeruginosa)

and Klebsiella pneumoniae (K. pneumoniae) are the

most important causes of gastrointestinal, respiratory,

burn, wound, soft-tissue, and urinary tract infections

Seyed Heibatollah Hosseini

al-around the world.They are also important causes of

food-borne diseases globally (Ruhnke-Trautmann et

al. 1989; Woodford and Livermore, 2009 andKaye and

Pogue, 2015).

Bacterial strains of clinical infections harbored the

high levels of resistance against the extensive ranges

of antibiotics including beta-lactams, macrolides, tetra-

cyclines, cephalosporins, sulfonamides and aminoglyco-

sidesantibiotics (Zhang et al., 2006; Huttner et al. 2013).

Therefore, much attentions have been done to the pre-

scription of medicinal plants for treatment of various

types of infectious diseases (Zhang et al., 2006; Huttner

et al. 2013). The genus Thymus, member of the Lam-

iaceae family, contains about 400 species of peren-

nial aromatic, evergreen or semi-evergreen herbaceous

plants with many subspecies, varieties, subvarieties and

forms (Vardar-Unlü et al. 2003; Ulukanli et al. 2011; Jia

et al. 2010; Zarshenas and Krenn, et al. 2015).

They are known as “Avishan” in Persian and are

well-known aromatic perennial herbs originated from

Mediterranean region. Among 215 species of this genus

grown in the world, 14 species are distributed in Iranian

 ora (Vardar-Unlü et al. 2003; Ulukanli et al. 2011; Jia

et al. 2010; Zarshenas and Krenn, et al. 2015). Thymus

species are well known as medicinal plants because of

their biological and pharmacological properties. In tra-

ditionalmedicine, leaves and  owering parts of Thymus

species are widely used as tonic and herbal tea, anti-

septic, antitussive, antibacterial, antifungal, antioxidant

and carminative as well as treating colds (Vardar-Unlü

et al. 2003; Ulukanli et al. 2011; Jia et al. 2010; Zarsh-

enasand Krenn, et al. 2015).

Thymus oils and extracts are widely used in pharma-

ceutical, cosmetic, and perfume industry, also for  avor-

ing and preservation of several food products (Vardar-

Unlü et al. 2003; Ulukanli et al. 2011; Jia et al. 2010;

Zarshenas and Krenn, et al. 2015). Thymus daenensis

(T.daenensis) is one of those endemic species in Iran and

is widely used as medicine and food additive. It a source

of antibacterial and antioxidant components (Vardar-

Unlü et al. 2003; Ulukanli et al. 2011; Jia et al. 2010;

Zarshenas and Krenn, et al. 2015). Data on the antibac-

terial and antioxidant effects of T.daenensis are scarce

in the world. Therefore, the present study was done to

investigate the antioxidant effects of the T. daenensis

essential oil as well as its antibacterial effects on several

types of pathogenic bacteria.

MATERIALS AND METHODS

From May to August 2016, aerial parts of T. daenen-

sis at the  owering stage were collected from the Dena

mountain slopes, Yasuj, Iran. Plants were identi ed by

an expert person in the  eld of medicinal plants at the-

Herbarium of Research Center of Agricultural of Shahre-

kord city, Iran. T. daenensis samples were dried at ambi-

ent conditions for 10 days. The dried plant was used to

extract the essential oil. Essential oil of the collected T.

daenensis was extracted by water distillation using the

Clevenger apparatus. T. daenensis essential oil was pre-

pared at a concentration of 0.25, 0.5, 0.75 and 1 mg/ml.

Radical-scavenging activities of T. daenensis essential

oilwas determined against 2,2-diphenyl-1-picrylhydra-

zyl (DPPH) free radicals. Brie y, 3 mL of T. daenensis

essential oil was mixed with 1mL of 1 mMmethanolic

solution of DPPH (Merck, Darmstadt, Germany). The

mixture was vortexed and incubated in the dark at ambi-

ent temperature for 30 min. When the DPPH solution

was mixed with the sample mixture acting as a hydro-

gen atom donor, a stable non radical form of DPPH is

obtained with simultaneous change of the violet color

to pale yellow. The absorbance was then measured at

517 nm. The percentage of DPPH free radical quenching

activity was determined using the following equation:

Percent of the DPPH scavenging effect= ×100

Where Abs DPPH is the absorbance value at 517 nm

of the methanolic solution of DPPH and Abs extract is

the absorbance value at 517 nm for the sample extracts.

DPPH radical scavenging of T. daenensis essential oil-

was also compared with the BHT synthetic antioxidant.

Simple disk diffusion method was used to study the

diameter of zone of inhibition of several types of bac-

teria subjected to T. daenensis essential oil. Susceptibil-

ity of Staphylococcus aureus (S. aureus) ATCC 25923,

Escherichia coli (E. coli) ATCC 25922, Listeria monocy-

togenes (L. monocytogenes) ATCC 19111, Bacillus cereus

(B. cereus) ATCC 14579, Pseudomonas aeruginosa (P.

aeruginosa) ATCC 27853 and Klebsiella pneumoniae (K.

pneumonie) ATCC 43816 (Pasteur Institute, Iran) were

determined against the T. daenensis essential oil. Guide-

lines of the Clinical and Laboratory Standard Institute

(CLSI) were used to study the antibacterial effects of T.

daenensis essential oil. Susceptibility of bacterial strains

were also studied against tetracycline (30 µg/disk),

azithromycin (15 µg/disk), gentamicin (10 µg/disk), pen-

icillin G (10 u/disk), and cipro oxacin (5 µg/disk) anti-

biotic disks (Oxoid, UK). Base plates were prepared by

pouring Muller-Hinton (MH) agar into sterile Petri dishes

and allowed to set. Plant essential oil aliquots of 10 l

were applied per  lter paper disc (Whatman No 6 mm

diameter). The disc was air-dried and placed onto the

seeded top layer of the agar plates. The plates contain-

ing the discs were allowed to stand for at least 30 min

before incubated at 37°C for 24 h. The diameter of the

zone of inhibition produced by each antibiotic disc was

Abs of DPPH-Abs of extract

Abs of DPPH

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOLOGICAL EFFECTS OF

THYMUS DAENENSIS

161

Seyed Heibatollah Hosseini

measured and interpreted using the CLSI zone diameter

interpretative standards (CLSI, 2015). All data were pre-

sented as mean ± standard deviation (SD). SPSS. Ver. 20

was used for statistical analysis. Comparison of means

were studied using the one-way ANOVA and also post

hoc turkey test. P value ≤0.05 was determined as signi -

cant level. All tests were done 3 times.

RESULTS AND DISCUSSION

We found that increase in the concentration of the T.

daenensis essential oil caused increase in its antioxidant

and also antibacterial effects (P<0.05). Table 1 shows the

antioxidant effect of T. daenensis essential oil against

DPPH free radicals. We found that T. daenensis essential

oil in a concentration of 1 mg/ml had the highest inhibi-

tory effects on the DPPH free radicals (96.27±8.60%).

BHT synthetic antioxidant had the higher inhibitory

effects on the DPPH free radicals than 0.25 and 0.5 mg/

ml concentration of T. daenensis essential oil.

Table 2 represents the antibacterial effects of the T.

daenensis essential oil on several types of bacteria. The

mean dimeter of the zone of inhibition for tested bacte-

ria had a range of 6.03±0.28 to 19.29±1.57 millimeter.

We found that the highest antibacterial effects were seen

for the 1mg/ml concentration of the T. daenensis essen-

tial oil on all tested bacteria and especially on E. coli

(19.29±1.57 mm) and K. pneumonia (18.0±1.69 mm).

Antibacterial effect of the T. daenensis essential oil was

not considerable against S. aureus and B. cereus.

Table 3 represents the antibacterial effects of several

types of antibiotic agents on tested bacteria. The mean

dimeter of the zone of inhibition for tested bacteria had

a range of 6.91±0.46 to 14.41±1.38 millimeter. We found

that the highest diameter of the zone of inhibition of

S. aureus, K. pneumonia, P. aeruginosa, L. monocy-

togenes, E. coli and B. cereus were seen for azithromycin

(15.28±1.22 mm), cipro oxacin (12.27±1.15 mm), cip-

ro oxacin (14.41±1.38 mm), azithromycin (13.92±1.26

mm), cipro oxacin (10.71±0.74 mm) and azithromycin

(11.79±1.06 mm).We also found that the essential oil of

the T. daenensis had a higher antibacterial effects on

its high concentrations than several types of antibiotic

agents.

The present investigation emerged the high antibacte-

rial and antioxidant effects of the T. daenensis essential

oil. Results of the present study showed that T. daenensis

essential oil in concentrations of 0.75 and 1 mg/ml had

higher inhibitory effects on DPPH free radicals than BHT

synthetic antioxidant. In addition, T. daenensis essential

oil at mentioned concentration had a higher antibacte-

rial effects on all tested bacteria than tetracycline, gen-

tamicin and penicillin G antibiotic agents. High presence

Table 1. Percent of the DPPH radical

scavenging of the T. daenensis essential oil.

Treatments

Percent of the DPPH

radical scavenging

BHT 85.12±7.34b**

T. daenensis

0.25* 55.28±5.01d

0.5 76.77±7.16c

0.75 89.92±7.49b

1 96.27±8.60a

*mg/ml

**Dissimilar capital letters in this column shows

signi cant differences less than 0.05.

Table 2. Antibacterial effects of the T. daenensis essential oil on several types of bacteria.

Bacteria

Diameter of the zone of growth inhibition (mm)

0.25* 0.5 0.75 1

S. aureus 7.33±0.58C**c*** 10.31±0.92Cb 12.79±1.23Ba 13.04±1.19Ba

K. pneumoniae 9.25±0.75Bd 12.23±1.04Bc 15.71±1.33Ab 18.0±1.69Aa

P. aeruginosa 9.09 ±0.84Bc 11.89 ±1.09Bb 13.33 ±1.25Bb 16.92 ±1.58Ba

L. monocytogenes 8.85±0.67Bc 11.26±0.72Bb 13.72±1.10Bb 14.85±1.13Ba

E. coli 11.73±1.55Ad 14.25±1.31Ac 16.82±1.44Ab 19.29±1.57Aa

B. cereus 6.03±0.28Cc 8.94±0.75Db 9.21±0.53Cb 11.42±1.16Ca

mg/ml

Dissimilar capital letters in each column shows signi cant differences less than 0.05.

***

Dissimilar small letters in each row shows signi cant differences less than 0.05.

162 BIOLOGICAL EFFECTS OF

THYMUS DAENENSIS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Seyed Heibatollah Hosseini

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOLOGICAL EFFECTS OF

THYMUS DAENENSIS

163

of antioxidant,  avonoid, phenol, carotenoid and vola-

tile components are the main factor which cause high

antioxidant effects of T. daenensis essential oil. In fact,

essential oil of the T. daenensis is a source of 1,8-cin-

eole, lanolool, -terpineol, geraniol, trans-thujan-4-ol,

terpinen-4-ol, thymol and carvacrol which have poten-

tial antibacterial and antioxidant effects (Diaz-Maroto

et al. 2006).

Several investigations focused on the antibacterial

and antioxidant effects of T. daenensis essential oil

(Sabahi et al. 2013; Damjanovi

´-Vratnica et al. 2015;

Amiri 2012).Sabahi et al. (2013) showed that the antioxi-

dant potential of crude extract of T. daenensis by DPPH

free radical scavenging (IC50 = 194 ± 0.02 g/mL), nitric

oxide scavenging (74±0.0 %) and hydroxyl radical scav-

enging (60.2 ± 0.27 %) was more active than its essential

oil. In nitric oxide scavenging crude extract was more

active than the antioxidant standard BHT, 42±0.79. They

showed that high presence of Geraniol (66.8%), Geranyl

acetate (13.9%) and Beta caryophyllene (9.6%) in the

T. daenensis essential oil warrant its high antioxidant

effects.

Damjanovi

´-Vratnica et al. (2015) showed that

geraniol (25.66%), geranyl-acetate (20.34%), linalool

(10.89%) and caryophyllene oxide (9.89%) were the most

commonly detected chemical components in the T. vul-

garis essential oil. They showed that the essential oil of

the T. vulgaris had considerable antimicrobial effects on

S. aureus, E. coli, Candida albicansand K. pneumoniae.

Amiri (2012) reported that antioxidant activity of polar

subfraction of T. daenensis subsplancifolius (Celak) Jalas

was found to be higher than other thymus species in

DPPH assay. He showed that the inhibitory effects of

theT. daenensis subsplancifolius, T. daenensis subsplan-

cifoliuspolar sub-fraction and T. daenensis subsplanci-

folius non-polar sub-fraction essential oils on DPPH free

radical were 99.6±0.5, 19.1±0.1 and 248.7±1.6μg/ml,

respectively. T. tosevii var. tosevii, T. tosevii var. dege-

nii, T. tosevii var. longifrons, T. tosevii ssp. substriatus,

T. longidens var. lanicaulis and T. caramanicus are other

some Thymus species with high antioxidant and anti-

bacterial effects (Vardar-Unlü et al. 2003; Ulukanli et al.

2011; Jia et al. 2010; Zarshenas and Krenn, et al. 2015).

Nikoli

´ et al. (2013) revealed the high antimicrobial,

antioxidant and antitumoractivity of T. serpyllum L., T.

algeriensis Boiss. and Reut and T. vulgaris L. essential

oils. They showed that Thymol was a major chemical

component in all tested thymus species. Difference in

the species of plant, method of sampling, part of plant

used for essence extraction, method of extraction and

geographical area caused difference in the chemical

composition of thymus species and therefore variation

in their antibacterial and antioxidant effects.

Bacterial strains of our investigation harbored the high

levels of resistance against tetracycline, azithromycin,

gentamicin, penicillin G, and cipro oxacin antibiotic

agents. It may be due to the irregular and unauthor-

ized prescription of these antibiotics in veterinary and

also medical  elds in Iran. High prevalence of resist-

ance against these antibiotics have also been reported

by other researchers (Momtaz et al. 2013; Dehkordi et al.

2014; Shahrani et al. 2014; Ranjbar et al. 2017) which

showed an emerging demand to use from natural anti-

bacterial agent’s like T. daenensis.

Table 3. Antibacterial effects of the antibiotic agents on several types of bacteria.

Bacteria

Diameter of the zone of growth inhibition (mm)

Tetracycline Azithromycin Gentamicin Penicillin G Cipro oxacin

S. aureus 5.21±0.42C*d** 13.28±1.22Aa 6.37±0.54Cd 12.88±1.07Ab 9.15±0.39Dc

K. pneumoniae 7.38±0.67Bc 12. 13±1.15Ca 9.64±0.71Bb 10.13±1.07Bb 12.27±1.15Ba

P. aeruginosa 7.52 ±0.65Bc 11.33 ±1.05Cb 12.27 ±1.21Ab 13.14 ±1.26Aa 14.41±1.38Aa

L. monocytogenes 7.20±0.62Bc 13.92±1.26Ba 7.14±0.45Cc 11.85±1.08Ab 10.52±0.84Cb

E. coli 8.31±0.65Ab 10.71±1.00Da 9.24±0.55Bb 8.33±0.36Cb 10.79±0.74Ca

B. cereus 7.25±0.31Bc 11.79±1.06Ca 6.91±0.46Cc 10.21±1.05Ba 8.22±0.67Db

*Dissimilar capital letters in each column shows signi cant differences less than 0.05.

**Dissimilar small letters in each row shows signi cant differences less than 0.05.

Seyed Heibatollah Hosseini

164 BIOLOGICAL EFFECTS OF

THYMUS DAENENSIS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

CONCLUSION

In conclusion, we identi ed a considerable inhibitory

effects of the T. daenensis essential oil on DPPH free

radicals and also high antibacterial effects of this plant

against Gram-negative and Gram-positive bacteria.

Results showed that T. daenensis essential oil in high

concentrations had higher antibacterial and antioxidant

effects. According to results, production of drug from

the essential oil of the T. daenensis at 0.75 and 1 mg/

ml concentration is a good approach for treatment of

the cases of E. coli, K. pneumonia, and P. aeruginosa

bacteria. Judicious prescription of antibiotic according

to the results of the disk diffusion method can eliminate

the risk of occurrence of antibiotic resistance.

ACKNOWLEDGEMENTS

Author would like to thank the staff of the Food Science

and Technology and also Medicinal Plants Research

Centers of the Islamic Azad University of Shahrekord,

Iran for their important technical help.

REFERENCES

Amiri, H. (2012): Essential Oils Composition and Antioxidant

Properties of Three Thymus Species. Evid-Based Comp Alt

Med. 2012, 1-8.

Clinical and Laboratory Standards Institute (CLSI). (2015):

Performance Standards for Antimicrobial Susceptibility Test-

ing; Twenty-Fifth Informational Supplement M100-S25.

Wayne Pa.

Damjanovi

-Vratnica, B., Cakovi

, D. andPerovi

, S. (2015):

Composition and antimicrobial studies of essential oil of Thy-

mus vulgaris from Montenegro. BiolNyssana. 6, 67-73.

Dehkordi, F. S., Yazdani, F., Mozafari, J. and Valizadeh, Y.

(2014): Virulence factors, serogroups and antimicrobial resist-

ance properties of Escherichia coli strains in fermented dairy

products. BMC Res Notes. 7, 217.

Diaz-Maroto, M.S., Perez-Coello, S., Esteban, J. andSanz, J.

(2006): Comparison of the volatile composition of wild fennel

samples (Foeniculum vulgare Mill.) from Central Spain. J Agric

Food Chem. 54, 6814-6818.

Huttner, A., Harbarth, S., Carlet, J., Cosgrove, S., Goossens, H.,

Holmes, A., Jarlier, V., Voss, A. and Pittet, D. (2013): Antimi-

crobial resistance: a global view from the 2013 World Health-

care-Associated Infections Forum. Antimicrob Resist Infect

Control. 2, 31.

Jia, H. L., Ji, Q. L., Xing, S. L., Zhang, P. H., Zhu, G. L. and Wang,

X. H.(2010): Chemical composition and antioxidant, antimi-

crobial activities of the essential oils of Thymus marschalli-

anus Will. and Thymus proximus Serg. J Food Sci. 75, E59-65.

Kaye, K. S. and Pogue, J. M. (2015): Infections Caused by

Resistant Gram-Negative Bacteria: Epidemiology and Manage-

ment. Pharmacotherapy. 35, 949-62.

Momtaz, H., Dehkordi, F. S., Hosseini, M. J., Sarshar, M. and

Heidari, M. (2013): Serogroups, virulence genes and antibiotic

resistance in Shiga toxin-producing Escherichia coli isolated

from diarrheic and non-diarrheic pediatric patients in Iran.Gut

Pathog. 5, 39.

Nikoli

´, M., Glamo

lija, J., Ferreira, I., Calhelha, R. C.,

Fernandes, A., Markovi

´, T., Markovi

´, D., Giweli, A. and Sok-

ovi

´, M. (2014): Chemical composition, antimicrobial, antioxi-

dant and antitumor activity of Thymus serpyllum L., Thymus

algeriensis Boiss. and Reut and Thymus vulgaris L. essential

oils. Industrial Crops Prod. 52,183–190.

Ranjbar, R., Masoudimanesh, M., Dehkordi, F. S., Jonaidi-

Jafari, N. and Rahimi, E. (2017): Shiga (Vero)-toxin produc-

ing Escherichia coli isolated from the hospital foods; virulence

factors, o-serogroups and antimicrobial resistance properties.

Antimicrob Resist Infect Control. 6, 4.

Ruhnke-Trautmann, M., Trautmann, M., Brückner, O., Weinke,

T. and Wagner, J. (1989): Infection caused by gram-positive

and gram-negative bacteria. A comparative study. Fortschr

Med. 107, 477-80.

Shahrani, M., Dehkordi, F. S. and Momtaz, H. (2014): Charac-

terization of Escherichia coli virulence genes, pathotypes and

antibiotic resistance properties in diarrheic calves in Iran.Biol

Res. 47, 28.

Ulukanli, Z., Cigremis, Y. and Ilcim, A. (2011): In vitro anti-

microbial and antioxidant activity of acetone and methanol

extracts from Thymus leucotrichius (Lamiaceae). Eur Rev Med

Pharmacol Sci. 15, 649-57.

Vardar-Unlü, G., Candan, F., Sökmen, A., Daferera, D., Polis-

siou, M., Sökmen, M., Dönmez, E. and Tepe, B. (2003): Anti-

microbial and antioxidant activity of the essential oil and

methanol extracts of Thymus pectinatus Fisch. etMey. Var.

pectinatus (Lamiaceae). J Agric Food Chem. 51, 63-7.

Woodford, N.and Livermore, D. M.(2009): Infections caused

by Gram-positive bacteria: a review of the global challenge.

J Infect. 59, S4-16.

Zarshenas, M. M.and Krenn, L.(2015): A critical overview on

Thymus daenensis Celak.: phytochemical and pharmacological

investigations. J Integr Med. 13, 91-8.

Zhang R, Eggleston K, Rotimi V, Zeckhauser RJ. Antibiotic

resistance as a global threat: evidence from China, Kuwait and

the United States. Global Health. 2, 6.