National Journal of Chemistry, 2005, Volume 18, 289-298
‫المجلد الثامن عشر‬-5002-‫المجلة القطرية للكيمياء‬
Isolation and Identification of Volatile oils from Iraqi Thyme
( Thymbra Spicata ) and study the antimicrobial activity
Ikbal S. Al-Sheibany
Chemistry Dep., College of Education, Baghdad University
Kasim H. Kadeem and Amal S. Abdullah
Chemistry Dep., College of Science, Babylon University
(NJC)
(Received on 19/12 /2004)
(Accepted for publication on 22/ 6/2005)
Abstract
Thyme, is commonly used as a cough-medicine. Especially liquid thyme extracts
are in some countries a fundamental part of many galenicals with antitussive action.
Essential oils of thyme with different methods extract were compared by using
HPLC. The major constituent of the oils was carvacrol and thymol. The biological
activity against (Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae)
was studied. All essential oils inhibited all bacteria at concentrations of ( 1/50 v/v ),
the Glavenger method was found to be the most effective one than the other steam
distillation extract.
‫الخالصة‬
‫الزعتررر ن اترراس االررخ امالررتخداا مم ررال الجيرراز التا)الررص الكتررة ا خاصررة المالررتخلر المررا ص لاترراس الزعتررر‬
‫ تررا االررتخالر الزيررس‬.‫الر ي يالررتخدا عررص تلررل الالرردان ةمررادي ا ليررة لتتاررسر اللدسررد مررن المالتتارراس الاتاتيررة‬
‫ تسر‬HPLC ‫الطرريقتسن تاالرتخداا تقايرة‬
‫الطيار من اتاس الزعتر تطرريقتسن مختل)ترسن تمرس المقارارة ارسن اترا‬
‫ تررا د ارالررة ال)لاليررة التاس ل جيررة للزيررس اللطررري تجررا ثررال‬. ‫ةااررس المرةترراس امالاالررية اررص الكررارعةر ل الثررايم ل‬
289
National Journal of Chemistry, 2005, Volume 18, 289-298
‫المجلد الثامن عشر‬-5002-‫المجلة القطرية للكيمياء‬
‫ الزيررس اللطررري المالررتخلر مررن التقطسررر الاخرراري تاالررتخداا جيرراز‬. (1/50 v/v) ‫اا ر ان مررن التةتريررا ترةسررز‬
.‫ةالعساجر ةان امكثر علالية من الزيس اللطري المالتخلر من التقطسر الاخاري اللادي‬
Thymus
Introduction
Vulgaris
the
chief
Thyme, belongs to the family
components of volatile oil are
Labiatae is commonly used as a
thymol (20-55%), carvacrol (1-
cough-medicine. Especially liquid
10%) (9).
thyme
Volatile oils or essential oils are
extracts
are
in
some
countries a fundamental part of
colorless
liquids
consisting
many galenicals with antitussive
mixtures
of
action(1).
unsaturated cyclic hydrocarbons
saturated
of
or
(10)
.
Thyme (Fig.1) has several
names :- common thyme, herba
The antimicrobial activity of plant
thymi, red thyme(2), in Arabic it is
oils
called "Za`atar" and in Kurdesh is
recognized for many years(11). In
called "Jatra"(3).
1977, it was reported that 60% of
The medicinal parts are the oil
the
extracted from the fresh, flowering
examined to date were inhibitory to
herb, the dried leaves, and the fresh
fungi,
aerial part of the flowering plant(4)
bacteria(12).
.
The aims of this work were:-
The leaves of most kinds of thyme
(i)
and
extracts
essential
has
oil
while
been
derivatives
30%
inhibited
to extract the volatile oils
can be used to good effect with
from Thymbra Spicata that
almost any savoury dish: vegetable,
grown in north of Iraq by
fish, poultry, meat. The essential
two ways.
(ii)
oils produced from any of several
Study
the
species have many used in the food
activities
industry(5) .
extracts.
antimicrobial
of
different
Herba thymi contains (1.0-2.5%)
Material and Methods
volatile oil(2,4) , and about (55%)
The study was carried out on two
phenolic material(6,7) but not less
essential oils samples obtained from
than (0.5%) phenols(2), the major
different
constituent of Thymbra Spicata
Spicata.
was carvacrol (75.5%)(8), while in
290
ways
of local
Thymbra
‫المجلد الثامن عشر‬-5002-‫المجلة القطرية للكيمياء‬
National Journal of Chemistry, 2005, Volume 18, 289-298
The dry plant Thyme was obtained
will separated from the aqueous phase
from the Ministry of health/center of
with diethylether and then evaporate in
herbal
water path at 40°C and dried over
medicine.
The
plant
was
identified by " herbelizem college of
anhydras sodium sulphate.
science" Baghdad University. It was
High
air-dried,
Chromatography ( HPLC )
and
packed
in
plastic
containers until used.
Thymol and carvacrol content in
distillation and the other way by using
thyme
Glavenger apparatus (Fig. 2 ). The test
obtained
Liquid
analysis of Volatile oils.
Volatile oils were extracted by steam
organisum
Performance
from
oil
was
quantitatively
determined by HPLC model Waters
the
2795 outo injector: column 250X4.6
Biotechnology department / Baghdad
mm I.D., packed with µBondapak C18 (
University.
water Assoc.) at wavelength 283 nm,
Steam distillation
an acetonitrile-water ( 40 : 60 ) mobile
one handerd grams of the leaves
phase and a flow-rate of 1.5 ml / min
and flowers are placed in a 1000 ml
were
round-bottomed flask and add 300 ml
used(14).
components
of distilled water, the distillation
The
were
volatile
oil
identified
by
comparing their retention times with
process was carried out for seven
those of authentic samples.
hours; and then transfer the yield to
Determination of antimicrobial
the separatory funnel, the oils will
activity:-
separated from the aqueous phase with
Antimicrobial activity of essential
diethylether and then evaporate in
oils obtained by steam distillation,
water path at 40°C and dried over
were
anhydras sodium sulphate.
determined by dilution with
dimethyl sulphoxide ( DMSO ) ( 1/50
Glavenger apparatus
v/v ) by using the well method(15).
One handerd grams of plant were
1. Staphylococcus aureus.
placed in a 1000 ml round-bottomed
2. Escherichia coli.
flask, and distilled water was added in
3. Klebsiella pneumoniae.
a ratio of (5:1) (water : plant). The
distillation process was carried out for
three hours, the yield of volatile oils
291
‫المجلة القطرية للكيمياء‪-5002-‬المجلد الثامن عشر‬
‫‪National Journal of Chemistry, 2005, Volume 18, 289-298‬‬
‫‪292‬‬
National Journal of Chemistry, 2005, Volume 18, 289-298
‫المجلد الثامن عشر‬-5002-‫المجلة القطرية للكيمياء‬
It has been reported that the volatile
The two isomers carvacrol and thymol
oil that extracted from thyme plant at
were identified by using HPLC, the
concentration of essential oils at (1/10
result are shown at ( Table 1 ).The
v/v ), ( 1/20 v/v ) has no antimicrobial
HPLC
activities(16) . 50 µL of essential oils
carvacrol and thymol was shown in
concentration ( 1/50 v/v ) were placed
(Fig.3). By using 100 gm of dried
in the hole of nutrient agar pleas cover
plant, the yield obtained by Glavenger
by the above bacteria, the plate were
is 3 ml , while only 1.2ml oils obtained
left at room temperature for 1 hour
from steam distillation method.
results
of
compound
of
then been moved to incubator 37°C
Antimicrobial activity of volatile oils
were left then for 24 h. The result
extracted by two methods in present
taken for every kind of bacteria after
research as shown in (Table 2). The
been
exhibiting of oils extract
repeated
three
times.
The
diameters of the inhibitory zones were
against
bacteria as shown in (Fig.4).
measured.
Results
The major constituents of volatile
oils of Thymbra Spicata are carvacrol (
2-methyl-5-isopropylphenol)
and
thymol (5-methyl-2-isopropylphenol).
Table (1) Retention time (tR), of carvacrol and thymol compound,
using water-acetonitrile as eluent.
compound
tR ( min )
Height (µv)
Glavenger
Steam distillation
Glavenger
Steam distillation
Carvacrol
13.769
13.770
102530
27596
Thymol
15.77
15.678
910
87130
Table (2) Illustrated a biological activity against different
type of bacteria
Microorganisms
Diameter of inhibitory zone (mm)
Glavenger method
Steam distillation
Staph. aureus
54
35
E.coli
69
28
Klebsiella
81
31
293
‫المجلة القطرية للكيمياء‪-5002-‬المجلد الثامن عشر‬
‫‪National Journal of Chemistry, 2005, Volume 18, 289-298‬‬
‫‪294‬‬
‫المجلة القطرية للكيمياء‪-5002-‬المجلد الثامن عشر‬
‫‪National Journal of Chemistry, 2005, Volume 18, 289-298‬‬
‫‪295‬‬
National Journal of Chemistry, 2005, Volume 18, 289-298
‫المجلد الثامن عشر‬-5002-‫المجلة القطرية للكيمياء‬
concentration than thymol, this is in
Discussion
agreement with the finding of other
From the work described above
authors(16,17).
the conclusion which are to be drown
From the above data
are as follow:-
and
Glavenger method is the best
(Fig.4) , the antimicrobial activity has
method for extraction the essential oils,
been concluded, the Gram negative
which given yield 3 ml , while only 1.2
( E.coli ) gave inhibition zone (69mm),
ml oils from the other way. Another
while the Gram positive bacteria
study which was carried out is HPLC
(Staph.) gave inhibition zone (54mm),
technique which shown from ( Fig.3 )
and
that the major compound is carvacrol
(Klebsiella ) shown inhibition zone
and the minor is thymol in Glavenger
(81mm) are the most inhibited bacteria
method. While the major compound is
by using the oils obtained from the
thymol and the minor is carvacrol from
Glavenger method, at concentration
the steam distillation method, that
( 1/50 v/v ), this result agree with the
which possibly due to that thymol is
other study(18).
the
The
very sparingly soluble in water.
Gram
results
negative
of
this
bacteria
study
confirmed the possibility of using the
Antimicrobial activity has been
essential oils in food systems to
shown in (Table 2), the exhibited
prevent the growth of foodborne
similarly
bacteria.
levels
of
antimicrobial
activity, but the oils extracted from
Glavenger method appeared to be more
efficient, which due to the percent of
carvacrol
compound
at
a
high
296
National Journal of Chemistry, 2005, Volume 18, 289-298
9.Evans, W.C., "Pharmacognosy",
References
15thed., W.B.Saunders, (2002).
1.Van Den Brouck, C.O., and
10. Robert
Lemli, J.A., J.Planta Medica,
2.World
Health
monographs
Prevention,
Organization
on
"Medicinal
Plants
V.,
12. Cowan,
and
M.M.,J.Clinical
No.4,564.
13. Indian
Oxford University Press, (1999).
Herbal
Pharmacopoeia,
6.Al-Shahatt, N., "Organo plant
Ajoint
Puplication of National Research
agricultural
Aldar
,
Microbiology Reviews, 1999, 12,
Companion To Food",1st ed. ,
Laboratory, V.II, (1999).
Al-arabia,
14. Solinas,V.,
(1988).
7.Hussain,
Arzedi,E.
Microbiology, 1999, 29,130.
economics
5.Alan Davidson, "The Oxford
products",
Tuberoso,
Palmas,F.,J.Applied
company,(1968).
and
and
G.I.,Pisano,B., Satta,M., Mascia,
of
4.PDR For Herbal Medicines,
Medical
Diagnosis
11. Cosentino,S.
Iraq",2th ed.Baghdad, (1988).
Medical
Robertson
Edition, (1987).
3.Al-Rawi A., and Chakravarty,
,
and
Treatment",12thed., Middle East
selected
medicinal plants, (1996) Geneva.
1sted.
H.,
O.W.,"Handbook of Poisoning :
1981, 41, 129.
H.L.,
‫المجلد الثامن عشر‬-5002-‫المجلة القطرية للكيمياء‬
and
Gessa,
G.,
J.Chromatography, 1981, 219,
F.T.,"Medicinal
332.
plants",Aldar A-arabia, (1979).
8.Hasan,B.,Osman S., Gulcan O.,
15. Vignolo, G.M., Suriani, F.,
and Tahsin, K., J.Food Control,
Holgado, A.P. and Oliver, G.,
2004, 15, 169.
J.App. Bac., 1993, 75, 344.
297
National Journal of Chemistry, 2005, Volume 18, 289-298
16. Kandil,O.,
Radwan,N.,
Hassan,A., Amer A.M ., ElBanna
H.,
J.
Ethnopharmacology, 1994, 44,
19.
17. Kunle,O.,
Okogun,J.and
Shok,M.J.Phytomedicine, 2003,
10, 59.
18. Jenan,K.,Ph.D.,Thesis,
Baghdad University, (2003).
298
‫المجلد الثامن عشر‬-5002-‫المجلة القطرية للكيمياء‬