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Antimicrobial activity of Thyme (Thymus vulgaris) essential oil cultivated in Quetta

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Abstract

The aim of the present study was to assess the antimicrobial efficiency of thyme (Thymus vulgaris) essential oil against different microbial strains viz. E. coli, Salmonella, staphylococci, Proteus, Pseudomonas, and Streptococcus strain; verified strains were collected from Bolan Medical Complex Hospital, Quetta. Thyme essential oil was extracted by using Clevenger type apparatus at BARDC Quetta and antimicrobial effect was measured by dissolving with Dimethyl sulphoxide (DMSO), using by disc diffusion technique. Results shows that the best inhibitory zones were observed by dissolving thyme oil with DMSO and the best zone was observed against E. coli (22 mm) while reduced zone was observed against Streptococcus (08 mm). This study explores its highly valuable contribution in medicinal usage. It will also improve the socio economic status of the farmers while used as replacement for some non-profitable crops by local farmers
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Inayatullah et al.
Int. J. Biosci.
2017
RESEARCH PAPER OPEN ACCESS
Antimicrobial activity of Thyme (
Thymus vulgaris
) essential oil
cultivated in Quetta, Balochistan, Pakistan
Syed Inayatullah*1, Muhammad Shafee2, Muhammad Shafiq3, Muhammad Asif1,
Shafiya Parveen4, Khadija Kakar1, Yasir Hayat Khan5, Muhammad Amir1,
Abdul Qadir6, Muhammad Yakoob7, Abdullah Baloch3
1Department of Biotechnology, BUITEMS, Pakistan
2CASVAB, University of Balochistan, Quetta, Pakistan
3Balochistan Agriculture Research and Development Centre, Quetta, Pakistan
4Department of Biochemistry, University of Balochistan, Quetta, Pakistan
5Faculty of Life Sciences, Xiaman University, China
6Departments of Plant and Environmental Protection, CDRI, NARC, Islamabad, Pakistan
7Hoticulture Research Institute, Kuzdar, Balochistan, Pakistan
Key words: Thyme dimethyl sulphoxide, Essential oil
Article published on February 28, 2017
Abstract
The aim of the present study was to assess the antimicrobial efficiency of thyme (Thymus vulgaris) essential oil
against different microbial strains viz. E. coli, Salmonella, staphylococci, Proteus, Pseudomonas, and
Streptococcus strain; verified strains were collected from Bolan Medical Complex Hospital, Quetta. Thyme
essential oil was extracted by using Clevenger type apparatus at BARDC Quetta and antimicrobial effect was
measured by dissolving with Dimethyl sulphoxide (DMSO), using by disc diffusion technique. Results shows that
the best inhibitory zones were observed by dissolving thyme oil with DMSO and the best zone was observed
against E. coli (22 mm) while reduced zone was observed against Streptococcus (08 mm). This study explores its
highly valuable contribution in medicinal usage. It will also improve the socio economic status of the farmers
while used as replacement for some non-profitable crops by local farmers
* Corresponding Author: Syed Inayatullah aghainayat@gmail.com
International Journal of Biosciences | IJB |
ISSN: 2220-6655 (Print) 2222-5234 (Online)
http://www.innspub.net
Vol. 10, No. 2, p. 105-110, 2017
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Inayatullah et al.
Int. J. Biosci.
2017
Introduction
Medicinal plants have great contribution to health
care all over the world, in Asian region particularly
identification and recognition of traditional medical
value of medicinal plants which have significant
power of healing. Due to antibiotic resistance and
failure of chemotherapy by pathogenic microbial
agents, search for plant products has increased for
their potential antimicrobial activity because of plants
safety and cost effectiveness (Hammer et al., 1999).
Plants as a source of medicine have been used by all
cultures from ancient times to the present day.
Traditional medicines as a primary health care, about
80% of the world’s people are depended. Plants
extracts are the active principle of the traditional
therapy (Murray and Shaw, 2000).
Thyme plants are perennials, belonging to the mint
family Lamiaceae, and exist in various shapes and
colors. It produces valuable essential oils, depending
on the nature of land where it has been grown. Thyme
essential oil is water distilled from the leave, flower,
dried or fresh plant. It is shown that Antibacterial,
antiviral, antioxidant; antifungal and insecticidal
properties have been possess by the Essential oils
(Burt, 2004; Kordali et al., 2005). Essential oils are
good source of biologically active compounds, has
been used in food preservation agents (Faid et al.,
1995) aromatherapy (Buttner et al., 1997) and
fragrance industries (Van de Braak and Leijten,1999).
Thyme has bactericidal and fungicidal effects and its
alcoholic extracts are expectorant (Hornok, 1992).
Thymol the active ingredient of thyme oil has
antibacterial/antimicrobial activity against Aspergillus,
Cryptococcus neoformans, Saprolegnia, Escherichia
coli, Staphylococcus aureus and Salmonella
typhimurium (WHO). The active ingridient Thymol
has successful result against virus (warts, epithelial
tumors) and worms like tapeworm and round worm
(Trattler, 1985).
According to Food and agriculture Organization of
UN about 32% world’s food supply was lost to
spoilage or waste in 2009.
Essential oils and extracts from several plant species
are able to control microorganisms related to skin and
food spoilage, including Gram-negative and Gram-
positive bacteria. As our environment is badly
contaminated by different microorganisms, it spoils
the vegetables and fruits, deteriorates the quality of
milk and complicates the wound or infections and
mainly Gram Positive and Gram Negative bacteria are
responsible for food spoilage. Folk medicine describes
thyme-vulgaris as antiseptic, sedative, antipyretic,
cramps and dermatitis treatment. Chemically the
essential oils (mainly contain thymol and caracole),
flavonoids, tannins and triterpenes (Adam et al.,
1998; Sartoratto et al., 2004).
Escrecia coli, gram-ve rod shape with high mortality
rates worldwide and the most common bacterial enter
pathogens importantly causes of neonatal meningitis
and the agent frequently associated with bloody,
watery and Traveler’s diarrhea, can also contaminant
fruits and vegetables that can easily cause an
outbreak (Chen and Frankel, 2005). Similarly species
of Salmonella cause enteric fever. Typhoid fever,
septicemia such as osteomyelitis and enter colitis.
Enterobacter is opportunistic pathogen that causes
particularly pneumonia and UTI. Streptococcus cause
a variety of infections, S. pyogene is leading bacterial
cause of pharyngitis, cellutitis, impetigo, necrotizing
fascilitis, streptococcal shock syndrome, rheumatic
fever and acute glomerulonephritis. Staphylococcus
aureus cause abscesses, various pyogenic infections,
toxic shock syndrome, found in human respiratory
tract and on skin and is the common pathogen
associated with staphylococcal food poisoning
(salads) ready to eat products (Le Loir et al., 2003).
The aim of this study is to investigate the in vitro
antibacterial properties of thyme essential oil
(Thymus vulgaris) against E. coli, Salmonella,
staphylococci, Proteus, Pseudomonas, and
Streptococcus strains.
Materials and methods
Study area
This study was conducted at Balochistan Agriculture
Research and Development Centre (BARDC) Brewery
Road Quetta and at the Center for Advanced Studies
in Vaccinology & Biotechnology (CASVAB),
University of Balochistan (UOB) Quetta, Pakistan.
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2017
Extraction of essential oil
About 70gm of Thyme vulgaris, aerial parts were
carried, dried at room temperature and were
subjected for 3hr to Neo-Clevenger type apparatus for
water distillation (Werl Lab-Germany). In a sterile
vial the essential oil was collected and with anhydrous
sodium sulphate the EO was dried for 24 hr and
stored at 4oC after filtration.
Test microorganisms
A total of six (06) numbers of different verified microbial
strains Escherichia coli, Proteus, Pseudomonas,
Salmonella, Staph aureus and Streptococcus spp were
taken from Microbiology Laboratory. Bolan Medical
Complex Hospital (BMCH),
Culture media, apparatus sterilization
At 180°C for 2 hours, the glass wares were washed
with detergent, rinsed with water, were air dried, then
wrapped in aluminum foil and sterilized in hot air
oven. Similarly all the culture media were autoclaving
at 121°C for 15 minutes after preparation with
manufacturer’s specification.
Preparation of culture medium
Eosin Methylene Blue agar (wright), Brain Heart
infusion (BHI) Agar (Oxoid) and Mueller Hinton Agar
(Oxoid), As per manufacturer’s specification, all the
said media’s were mixed with distilled water in 500
ml quantity and were autoclaved at 15 lb/in2 pressure
per square inch (PSI) for 15 minutes at 121°C and
allowed to cool down to 45°C.
The solid media were aseptically and allowed to
solidify in Petri plates. To confirm sterility, the plates
were incubated for 24 hours at 37°C Sterile media
were stored at 4°C.
McFarland standard
Specified amounts of sulfuric acid and barium
chloride were mixed together for making McFarland
standard. Barium sulfate precipitates were form by
mixing the two compounds as a result turbidity were
caused.
By mixing 9.95 ml of sulfuric acid (H2SO4)
concentration of 1% with 0.05 mL of 1.175% barium
chloride dihydrate (BaCl22H2O) a 0.5 McFarland
standard was achieved.
Preparation of normal saline
Normal saline were prepared by mixing distilled
water (100ml) with sodium chloride (0.87g) in conical
flask of 500ml the solution were autoclaved at 121°C
for 15 and stored the solution at 4°C.
Preparation of filter paper Disc
All the filter paper discs were prepared from
Whatman #6 each of 06 mm in diameter and at 121°C
for 15 minutes the paper discs were autoclaved.
Screening of antibacterial activity
Few colonies of tested organisms were picked up by
sterile loop and inoculated in 05 ml tubes containing
Normal saline and matched with 0.5 McFarland
nephelometer turbidity standards as described by (Saeed
et al., 2006). A sterile cotton swab was dipped into the
standardized bacterial test suspension to inoculate entire
surface of Muller Hinton Agar (MHA) plate.
The essential oils were mixed in dimethylsulfoxide
(DMSO). Under aseptic conditions using Laminar flow
cabinet with 0.2 micron Hepa Filters, sterilized discs
(Whatmann # 6) were impregnated with 15 µl of
different concentrations (1:1, 1:2, 1:4) of the respective
essential oils and DMSO were placed on the agar
surface as recommended by National Committee for
Clinical Laboratory Standard, (2002).
The inoculated plates were incubated at 37°C for 24
hours. Paper discs moistened with aqueous DMSO
were placed on the seeded petriplate as a vehicle
control. Studies were performed in duplicate, and the
mean values were calculated.
Results and discussion
Thyme oil showed great effect against selected
microbial strains. The findings of the study are
summarized in Table 1.
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2017
Table 1. Antimicrobial activity of thyme essential oil diluted with DMSO against different clinical microbial isolates.
Bacterial Spp.
Mean Zone of Inhibition (mm)
Oil
01:00
15µl
Concentration
DMSO*
00:01
15µl
01:01
01:02
01:04
E coli
31
22
19
16
0
Salmonella
25
17
13
10
0
Staphylococcus
25
21
15
13
0
Proteus
17
13
10
8
0
Streptococcus
10
8
7
7
0
Pseudomonas
11
10
8
7
0
*DMSO= Dimethyl sulphoxide
Best inhibitory zone were observed against E. coli, the
pure oil (15ul) zone was 31mm while diluted with
DMSO were 22mm, 19mm and 16mm, respectively.
The second best zones were against Salmonella and
Staphylococcus and were observed as 25mm for each
with pure 15ul after 24 hrs., while diluted with DMSO
the observation for Salmonella were 17mm, 13 mm
and 10mm and for Staphylococcus the observed
zones were 21mm, 15mm and 13mm, respectively.
Moderates zones of pure thyme oil were observed
against Proteus (17mm) and Pseudomonas (11mm)
strains and diluted oil with DMSO the zones were
13mm, 10mm and 08mm, respectively for Proteus.
Diluted zones for Pseudomonas were 10mm, 08 mm
and 07mm respectively. Reduced zones were
observed for Streptococcus, against pure oil were
recorded 10mm and diluted with DMSO the zones
were recorded 08mm and 07mm consequently.
Antimicrobial effects of the essential oils, mainly the
constituents of the essential oil (EO) were assessed
from variety of medicinal plants (Soylu et al., 2007).
Essential oil of the Thymus vulgaris is known for
antimicrobial effects against fungi and vides range of
microorganisms (Arras and Usai, 2001). The higher
antimicrobial effects were found against fungi
(Hammer et al., 1999) against pathogenic microbes
(Bouhdid et al., 2010; Piskernik et al., 2011) against
spoilage bacterial microbes against mold and yeast
(Tserennadmid et al., 2011) (Tyagi and Malik, 2012).
In the present study the inhibition zone diameter
(IZD) of 10mm to 31mm were observed with 15ul
essential oil and 12mm inhibition zone diameter was
consider be the effective.
Against E. coli the inhibition zone was observed as 21
mm and 31 mm using pure thyme oil with constant
quantity (15µl), indicated higher activity against E.Coli
bacteria the mean zone of inhibition against
Pseudomonas were recorded as 10 mm, showing
moderate effect against the said bacteria because no
effect was reported by Abu-Darwish et al. (2012) against
Pseudomonas aeruginosa. The mean Inhibitory zones of
Streptococcus were observed 10mm, indicating low
effect against the said tested microorganism. Against
Salmonella, the mean inhibition diameter zones were
observed about 25mm, indicating the strong
inhibitory effect. The mean inhibition zones against
Pseudomonas were 11mm, showing also a low effect
respectively. The mean inhibition diameter zones
against Proteus were observed 17mm, which indicate
an effective efficiency of the thyme oil.
The efficiency of thyme was observed as effective
against all the tested microorganism and our these
findings corroborate with the finding of (Imelouane et
al. 2009; Klaus et al., 2008) reported the strongest
effects of thyme oil about >45mm and (0.33mg mL-
1,aginst E. coli bacteria. The zones of inhibition
against Pseudomonas are in line with the finding of
Al-Fatimi et al. (2010) who also reported that 15 mm
zone of Yemen thyme species. The efficiency of
Thymu Vulgariss observed in our study against
Streptococcus, Salmonella, Pseudomonas and
Proteus were inline with the finding of (De Martino et
al. 2009) and (Azaz et al., 2004).
The findings of our study proved that the
antimicrobial effects of EOs extracted from Quetta,
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Int. J. Biosci.
2017
Pakistan species of Thymu vulgaris is as effective and
comparable to the one observed in other species of
Thyme essential oils like the findings of (Hyun et al.,
2015, Akgul and Kivanc, 1988; Nelson, 1997).
Conclusion
The findings of the study suggesting that thyme
essential oils represents antibacterial effects against
pathogenic microorganisms and can be as effective as
modern medicine and safe alternative to treat
infectious diseases. Some more research is needed to
explore its active compounds. Further investigation
needs to create awareness among farmers to cultivate
such valuable, natural medicinal herbs to improve
their socio-economic status.
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Nowadays it is essential to test new preservation and decontamination procedures using naturally occurring chemicals against important pathogenic bacteria in meat. We tested the antimicrobial effect of rosemary extracts and the bacteriocin nisin against Campylobacter jejuni at a low storage temperature (8 °C) with or without short-term pre-freezing. The antimicrobial effect of rosemary extract was four times greater in laboratory media than in chicken meat juice. Furthermore, low temperature storage conditions prolonged the survival of C. jejuni in chicken meat juice. Nisin, with an approximately 1.0 log reduction was neither effective alone nor in combination with the extract. Pre-freezing with plant extract addition proved to be effective treatment by more than 3.0 log reduction in 48 h. The results in chicken meat food model again showed the synergistic effect of freezing and plant extract antimicrobial activity. As the combination of pre-freezing and plant extract treatment reduced the cell number by more than 2.0 log reduction, studies should be conducted to further evaluate this promising treatment for Campylobacter reduction in the poultry meat supply.