Influence of some citrus essential oils on cell viability, glutathione –S-transferase and lipid peroxidation in Ehrlich ascites carcinoma cells

Abstract

Essential oils are the volatile fraction of aromatic and medicinal plants after extraction by steam or water distillation. They have been used for their pharmaceutical potential since early times, and even now are still subject to a great deal of attention. In this study citrus essential oils isolated from mandarin (C. reticulate), orange (C. aurantium), lemon (C. limon), and tangerine (C. aurantium) species were analyzed by gas chromatography-mass spectrometry (GC-MS). Main constituents separated in mandarin oil were dl-limonene (20.88%), neo-dihydrocaveol (4.96%), and allo-ocimene (4.78%). In orange oil, the principal compounds were linalool (10.5%), α-terpinolene (7.06%), and nonyl-aldehyde (4.79%). In lemon oil, camphene (19.31%), α-citral (17.13%), citronellal (13.64%), and limonene (6.55%) were among the principal components. Major constituents presented in tangerine oil were limonene (14.08%), citronellal (9.56%), and α-terpinene (4.68%). The chemical compositions of citrus essential oils were highly different which may be due to the difference in their genetic make up. The effect of different concentrations (25-150µl/ml) of citrus essential oils on the viability of Ehrlich ascites carcinoma cells (EACC) was tested in vitro. Generally, it was found that incubation of tumor cells with different concentrations of essential oils reduced the viability of these cells. The activity of glutathione-S-transferase (GST), glutathione content (GSH), and lipid peroxidation (LPO) were studied in EACC tumor cells treated by essential oils. The essential oils treatments increased the activities of GST, increased the cellular GSH level and inhibited lipid peroxidation. These findings support the hypothesis that citrus essential oils may possess significant antitumor and antioxidant effects on EACC cell lines.

Full text

Journal of American Science 2010;6(10)
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820
Influence of Some Citrus Essential Oils on Cell Viability,
Glutathione-S-Transferase and Lipid Peroxidation in Ehrlich ascites
Carcinoma Cells
Amal A. Mohamed *
1
, Gehan A. El-Emary
2
, Hanaa F. Ali
3
1
Plant Biochemistry Department, National Research Centre, Dokki, Cairo- Egypt
2
Institute of Productive Efficiency, Zagazig University, Egypt
3
Biochemistry Department, Faculty of Agriculture, Cairo University, Egypt
*Corresponding author:
amin_amal@yahoo.com
Abstract: Essential oils are the volatile fraction of aromatic and medicinal plants after extraction by steam or water
distillation. They have been used for their pharmaceutical potential since early times, and even now are still subject
to a great deal of attention. In this study citrus essential oils isolated from mandarin (C. reticulate), orange (C.
aurantium), lemon (C. limon), and tangerine (C. aurantium) species were analyzed by gas chromatography-mass
spectrometry (GC-MS). Main constituents separated in mandarin oil were dl-limonene (20.88%), neo-dihydrocaveol
(4.96%), and allo-ocimene (4.78%). In orange oil, the principal compounds were linalool (10.5%), α-terpinolene
(7.06%), and nonyl-aldehyde (4.79%). In lemon oil, camphene (19.31%), α-citral (17.13%), citronellal (13.64%),
and limonene (6.55%) were among the principal components. Major constituents presented in tangerine oil were
limonene (14.08%), citronellal (9.56%), and α-terpinene (4.68%). The chemical compositions of citrus essential oils
were highly different which may be due to the difference in their genetic make up. The effect of different
concentrations (25-150µl/ml) of citrus essential oils on the viability of Ehrlich ascites carcinoma cells (EACC) was
tested in vitro. Generally, it was found that incubation of tumor cells with different concentrations of essential oils
reduced the viability of these cells. The activity of glutathione-S- transferase (GST), glutathione content (GSH), and
lipid peroxidation (LPO) were studied in EACC tumor cells treated by essential oils. The essential oils treatments
increased the activities of GST, increased the cellular GSH level and inhibited lipid peroxidation. These findings
support the hypothesis that citrus essential oils may possess significant antitumor and antioxidant effects on EACC
cell lines. [Journal of American Science 2010;6(10):820-826]. (ISSN: 1545-1003).
Keywords: Essential oil; glutathione; GC/MS; limonene; lipid peroxidation
1. Introduction
Essential oils (EOs), also called volatile oils
are aromatic oily liquids obtained from different plant
parts and widely used as food flavors. The
constituents of the essential oils are mainly
monoterpenes and sesquiterpines which are
hydrocarbons with the general formula (C
5
H
8
)
n
.
Oxygenated compounds derived from these
hydrocarbons include alcohols, aldehydes, esters,
ethers, ketones, phenols and oxides. Volatile oils
have been reported to exhibit various antibacterial,
antifungal, antiviral and antioxidant properties
(Prabuseenivasan et al., 2006). Citrus is one of the
most important commercial fruit crops grown in the
world. The genus citrus includes various species of
oranges, mandarins, limes, tangerines, lemons and
grapefruit. In current citrus industry, citrus fruits are
marketed fresh or as processed juices which provide
multitude health benefits not only from vitamin-C but
also from other compounds. These compounds
include the bitter limonods, carotenoids (especially β-
carotene), flavonoids, folic acid and dietary fiber and
have been shown to prevent a variety of cancers and
cardiovascular diseases. Nevertheless, citrus essential
oils contain large amounts of terpens, aliphatic
sesquiterpene, oxygenated derivatives and aromatic
hydrocarbons (Merle et al., 2004). The composition
of the terpenic mix varies depending on the examined
citrus species to which it owns. The mix of each
species is in different proportion made of: limonene,
α–pinene, β-pinene, myrcene, linalool and terpinen.
Monoterpenes are important constituents of citrus
essential oil and other plants. A number of these
monoterpenes have an antitumor activity. For
example, d-limonene which comprises > 90% of the
orange peel oil has chemopreventive activity against
skin, liver, lung and forestomach cancers (Crowel,
1999) and has been reported to induce apoptosis on
tumor cells (Hata et al., 2003). Similarly, perillyl
alcohol, a hydroxylated limonene analog, exhibits
chemopreventive activity against liver, mammary
gland, pancreas and colon cancer in rodent (Reddy,
1997). It is presumed that a number of volatile oils
could act as potential novel antiproliferative agents

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821
(Dorman et al., 1995). A rapid enzyme assay for the
screening of potential inhibitors of chemical
carcinogenesis has been developed on the basis of
induction of the detoxifying enzymes such as
glutathione -S-transferase (GST) and NAD(P)H
quinone oxidoreductase (NQO1). Moreover; GST
catalyzies a wide range of reactions involving the
conjugation of glutathione (GSH) to electrophilic
carcinogenic compounds to form less toxic
conjugates for excretion. Cellular glutathione and
related enzymes such as glutathione peroxidase and
glutathione reductase are among the principal
protective mechanisms against endogenous and
exogenous toxic substances and free radicals-
mediated damage in liver tissue (Hayes and Mclellan,
1999). Thus any compound that can induce an
increase in the activity of these detoxifying enzymes
may be considered as potential inhibitors or
anticarcinogene (Lam et al., 1994). However, there is
strong evidence that dietary supplementation with the
citrus limonoids and limonin activates glutathione-S-
transferase in the liver and small intestine of the rat
(Lam and Hasegawa, 1989). From this viewpoint the
present study was carried out to investigate the
chemical composition of four citrus essential oils
(mandarin, orange, lemon and tangerine) as well as
the bioactivity of these oils as antitumor and
antioxidant agents ( GST activity, GSH level and the
level of MDA) in order to evaluate their medicinal
potential.
2. Material and Methods
2.1 Materials
Four citrus essential oils of mandarin (C.
reticulate), orange (C. aurantium) lemon (C. limon),
and tangerine (C. aurantium) was purchased from
Katto Aromatic Co., Giza, Egypt (Producers of plant
essential oils and aromatic substances); quality of the
oils was ascertained to be more than 98% pure. These
oils were stored at -20
0
C for subsequent analysis.
2.2 Essential oil analysis
Samples of the essential oils (0.5 mg) were
suspended in 1 ml of ethyl acetate (P.A., Merck,
Germany) and 5 µl of this solution was analyzed by
gas chromatography coupled with mass spectrometer
(GC/MS, Hewlett- Packard GC-MS Model 5890
series II) equipped with a fused silica capillary
column DB-5 (30 m x 0.25 mm x 0.25 Am). The
electron impact technique (70 eV) was used and
injector temperature was 240
0
C and that of the
detector was 230
0
C. The carrier gas was helium at
the working rate of 1.7 ml/min. The column
temperature was initially 50
0
C and then was
gradually increased at the rate of 5
0
C /min up to 180
0
C and after that, the temperature was increased up to
240
0
C at the rate of 8
0
C. For detection of the oil
components a flame ionization detector was used, set
up at 230
0
C. Components of the oils were identified
by comparison of their mass spectra and retention
indices with those published in the literature (Adams,
1995).
2.3 Viability of Ehrlich ascites carcinoma cells
Female Swiss albino mice, weighing 18-22
g, 8-10 week old were used. Animals were kept under
environmental and nutritional conditions for 2 weeks
then injected intraperitoneal by Ehrlich ascites
carcinoma cells (EACC). The animals were used for
tumor cell preparation (cell line).
2.4 Tumor cells (cell line)
A line of Ehrlich ascites carcinoma resistant
to Endoxan has been used (El-Merzabani and Tawfik,
1976). The parent line was first supplied through the
courtesy of Dr. G. Klein, Amsterdam, Holland. The
tumor line is maintained in the National Cancer
Institute, Cairo-Egypt, in female Swiss albino mice
by weekly transplantation of 2 x 10
6
cells. The cells
were taken from tumor transplanted animals after 7
days of transplantation. The cells were centrifuged at
1000 rpm for 5 min, washed with saline then the
needed number of cells was prepared by suspending
the cells in the a appropriate volume of saline.
2.5 In vitro cytotoxicity
The viability percentage of tumor cells was
measured by the modified cytotoxic trypan blue-
exclusion method as described by Bennett et al.,
(1991). The culture medium used was prepared using
RPMI 1640 media, 10% fetal bovine serum and L-
glutamine (Gibco). Two ml of cells (containing 2x10
6
cells) were incubated over night, with the examined
essential oils (essential oils as well as sterile saline
solution as control) at five concentrations; 25, 50, 75,
100 and 150 µl/ml which transferred into a set of
tubes. The tubes were incubated at 37
0
C under 5%
CO
2
. Thereafter, the tubes were centrifuged at 1000
rpm for 5 min and the separated cells were suspended
in saline. For each examined extract and control, a
new small test tube was used and 10 µl of cell
suspension, 80 µl saline and 10 µl trypan blue were
added and mixed. The number of living cells was
calculated using a hemocytometer slide. Cells were
counted in duplicates under light microscope at 100
x. Cells that showed signs of staining were
considered to be dead, whereas those that excluded
trypan blue were considered as viable.
Cell viability % = No of viable cells (unstained cells)
/ Total No of cells (stained and unstained) x l00

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822
2.6 Determination of GST activity
GST activity was determined in treated (at
100 and 150 µl/ml essential oils) and untreated tumor
cells solution (2 ml containing 2x10
6
cells) as
described by Habig et al., (1974). Reaction mixture
containing 50 mM phosphate buffer, pH 7.5, 1 mM
of 1-chloro- 2, 4 dinitrobenzene (CDNB) and a
appropriate volume of cell lines. The reaction was
initiated by the addition of reduced glutathione
(GSH) and formation of S-(2, 4-dinitro phenyl)
glutathione (DNP-GS) was monitored as an increase
in absorbance at 334 nm. The result was expressed as
µmol of CDNB conjugation formed /mg protein /min.
2.7 Determination of GSH level
The level of total SH compound (GSH) was
determined with Ellman’s reagent according to
Tukendorf and Rauser (1990). GSH was assayed by
adding 2 ml of 0.5 Mm 5, 5’ –dithio-bis -2-nitro
benzoic acid, (DTNB) prepared in 0.2 M phosphate
buffer, pH 8.0 to appropriate volume of treated cell
lines solution. The GSH reacts with DTNB and forms
a yellow colored complex with DTNB. The
absorbance at 412 mm was read after 2 min (Σ=13.6
mM
-1
cm
-1
).
2.8 Lipid peroxidation (TBARS test)
The level of lipid peroxidation products was
assayed through the formation of 2- thiobarbituric
acid reactive substance (TBARS) according to Buege
and Aust (1990), based on the reaction of
malondialdehyde (MDA ) with thiobarbituric acid
(TBA) at 95
o
C. The absorbance of the end product of
lipid peroxidation (mainly malondialdehyde, MDA)
was measured at 535 nm and corrected for non-
specific turbidity by subtracting the absorbance at
600 nm. The results were expressed as n mol MDA /
mg protein.
2.9 Determination of total protein
Protein levels were determined spectrophoto-
metrically at 595 nm, using comassie blue G 250 as a
protein binding dye (Bradford, 1976). Bovine serum
albumin (BSA) was used as a protein standard.
Statistical analysis:
The Software COSTAT for Windows
(version 10.0) was used for the statistical evaluation
of the results. Statistical significance was determined
by analysis of variance (ANOVA) and by the least
significant differences (LSD) tests corrected for the
number of comparisons. The probability level of 0.05
was used
as the criterion for significance in all
procedures according to Little and Hills (1992). All
results were expressed as mean and standard
deviation of the mean (SD).
3. Results and Discussions
3.1 Chemical composition of essential oils
The analytical results of mandarin and
orange essential oils are shown in table (1), the
principal components of the mandarin oil were dl-
limonene (20.88%), neo-dihydrocaveol (4.96%), allo-
ocimene (4.78%), camphene (4.47%), and linalool
(3.52 %), which represented (38.61 %) of the total
mandarin oil. For the orange oil, the linalool was
detected at a level of (10.5%) as a major compound.
Other important compounds were α-terpinolene
(7.06%) and nonyl-aldehyde (4.79 %), carvone
(4.52%) and citronellol (3.97 %) which was found as
a major compound in orange oil. On the other hand,
camphene (19.31 %) was the major percentage of
the lemon oil followed by α-citral (17.13%) as shown
in table (2). The major compounds identified in
tangerine oil, were limonene (14.08%), citronellal
(9.56%), α-terpinene (4.68%) and trace levels (below
0.1%) of α-pinene (0.07%) and globulol (0.08%)
respectively.
Table 1. Major components (in %) of mandarin and
orange oils separated by Gas Chromato-graphy-Mass
spectroscopy.
Mandarin oil Orange oil
Compound
name Rt % Compound
name Rt %
Alpha-Pinene 3.38 3.27 Beta-
Phellandrene 3.24 2.82
dl-Limonene 5.03 20.8
8 Myrcene 3.58 1.03
Allo-ocimene 6.34 4.78 Alpha-
terpinolene 5.2 7.06
Neo-
dihydrocaveol 6.5 4.96 Nonyl-
aldehyde 7.12 4.79
Cis-Limonene
oxide 9.59 2.19 Cis-
Limonene
oxide
7.73 0.79
Linalool 11.99 3.52 Decanal 8.73 0.76
Camphene 12.04 4.47 Linalool 9.01 10.5
Linalyl acetate 12.12 0.08 Verbenol 10.3 2.87
Farnesene 13.87 0.7 Carvone 12.2 4.52
Sabinene 22.16 1.06 Mentha-
triene 12.9 0.11
Alpha-
Farnesene 22.82 0.07 Perilladehyde 13.1 1.28
Borneol 25.05 3.03 Cis-Carveol 14.1 0.46
Limonene
glycol 25.84 2.29 Caprylic acid 17.0 0.15
Undecanoic
acid 27.3 2.01 Cinnamic-
aldehyde 17.1 3.81
Methly-
anthranilate 29.69 0.09 Farnesene 17.9 1.04
Benzaldehyde 30.18 0.49 Citronellol 19.8 3.97
Carvone 31.9 1.5 Heptadecanol 20.4 0.08
Unknowns 21.7 4.5 Unknowns 16.8 9.8
Rt; Retention time (min)

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823
These results are in accordance with the
previous findings regarding GC separation of
essential oils from sweet orange, tangerine,
bergamote, and grapefruit peel. (Gancel et al., 2002;
Hognadottir and Russell, 2003; Khanum et al., 2004).
They found terpenes and oxygenated compounds
such as limonene, γ-terpinene, β-pinene, α-pinene,
myrcene, valencene, linalool, octanal, decanal, and
butylebutyrate as the major constituents through GC
separation Steam-distilled volatile peel oil of Indian
orange when analyzed through GC and GC-MS,
limonene was found more dominant followed by
myrcene, α-terpinolene and β-pinene (Kirbaslar and
Kirbaslar, 2003). Almost similar results were
reported by Feger et al., (2003); Tu et al., (2003)
while working on orange and tangerine essential oils.
Table 2. Major components (in %) of lemon and
tangerine oils (%) separated by Gas Chromato-
graphy-Mass spectroscopy.
Mandarin oil Orange oil
Compound
name Rt % Compound
name Rt %
Alpha-Pinene 3.01 2.31 Alpha-Pinene 3.17 0.07
Alpha-
Fenchene 3.27 1.17 Limonene 4.77 14.08
Cyclohexane 4.77 4.9 Citronellal 7.66 9.56
Limonene 6.14 6.55 Aloxiprin 8.31 0.11
Citronellal 9.41 13.64 Alpha-
Terpinene 9.65 4.68
Cis-Carveol 9.9 0.41 Linalool 10.63 0.18
Alpha-
Fenchene 12.13 4.87 Heptadiene 10.98 0.25
Camphene 13.62 19.31 Trans-
menthadiene 12.05 2.76
Alpha-Citral 14.51 17.13 Tarns-
Ocimene 13.32 1.72
Carvacol 16.88 0.95 Cis-Limonene
oxide 14.23 1.95
Terpniol 15.22 1.2 Trans-
Carveol 15.07 2.19
Thymol 20.83 1.54 Methyl-
heptadiene 16.05 5.0
Carvacrol 21.21 0.23 Limonene
dioxide 17.91 0.74
Heptanal 22.19 0.32 Perillyl
alcohol 18.05 0.53
Citral 29.07 0.53 Cyclo-
octanone 19.92 0.43
Dihydroiso-
pimaric 30.88 0.65 Ledol 21.12 0.15
Dihydro-
abitec 35.28 0.1 Trans-
Decalone 22.26 1.8
Unknowns 19.9 13.13 Globulol 22.48 0.08
Benzyl-
dicarboxylic 22.93 0.94
Unknowns 19.1 9.88
Rt; Retention time (min)
3.2 Antitumor activity of citrus oils
Antitumor activity of citrus essential oils
was evaluated on the basis of “Trypan blue exclusion
assay”. The viability of Ehrlich ascites carcinoma
cells (EACC) after incubation for 2 hr with different
concentration of essential oils was evaluated and the
obtained data are presented in table (3). Data showed
that, the incubation of tumor cells with different
concentration of essential oils affected the percent of
cell viability. All concentration ranges showed
decrease in cell viability as compared to that of
control treatment. Among all tested oils, by mandarin
and orange oils showed the best activity on the tumor
cells at the concentration of 150 µl/ml (the value of
dead cells was 81 and 85% respectively). These both
essential oils contained much dl-limonene (20.88%)
and linalool (10.5%) respectively, and the same trend
was found in case of lemon oil. However, samples
treated by tangerine oil showed its best activity at the
concentration of 100 µl/ml (the value of dead cells
was 62 %), this oil contained much limonene
(14.08%) and citronellal (9.56%). In case of
mandarin oil, the survival rate of the normal cells
(control) was 3% of viable cells. The killing effect of
citrus essential oils may be due to the role of
camphore and limonene which were able to increase
the cytoplasm membrane permeability. Also,
probably because their capability of dissolving into
the phospholipids bilayer aligning between the fatty
acid chains and causing a distortion of the membrane
physical structure in various tumor cell lines
(Monajemi et al., 2005). Based on these finding it has
been expected that essential oils with a higher
percentage of limonene, show greater cytotoxicity.
However, other mechanism for limonene action have
been suggested, including the induction of carcinogen
metabolizing enzymes, growth factor receptor
expression, and inhibition of 3-hydroxy-3-methyl
glutraryl CoA reductase (Poon et al., 1996).
Additionally, d-limonene oxygenated derivates, e.g.
perillyl alcohol, carveol, carvone, geraniol and
menthol, have shown biological activity in vivo
against certain types of malignant tumors (Crowell,
1999). Hence, it can be concluded that these
components may have great cytotoxic effects.
3.3 Effect of citrus oils on antioxidant compounds
The effect of essential oils at the selected
concentration (100 and 150 µl/ml) on the glutathione
S- transferase (GST), glutathione content (GSH), and
lipid peroxidation (LPO) in Ehrlisch ascites
carcinoma cells was given in table (4). Generally,
activity of the anti-oxidative enzyme GST was
enhanced by essential oils treatment compared to
control group. Mandarin oil treatment at 150 µl/ml

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824
had a higher GST activity (the value was 3.2 µmol
/mg protein /min) than in orange, lemon and
tangerine oils (2.4, 1.99, and 2.1 µmol /mg protein
/min) respectively. The increasing % of GST in
EACC cells treated by 150 µl/ml mandarin, orange,
lemon, and tangerine oils were 336.8, 252.6, 209.5
and 221.1 % respectively, (control values 100%).
3.2 Antitumor activity of citrus oils
Table 3. Effect of different concentrations of essential oils on the viability of Ehrlich ascites
Control: Tumor cells + saline solution ±SE refers to standard error (n=6)
Glutathione (GSH) content was higher in
treated cells compared to control treatment. GSH
content was gradually increased by increasing of oil
concentration. At 150 µl/ml, the level of GSH was
increased by 242.8, 142.8, 133.3 and 152.4% of the
control 100% in mandarin, orange, lemon, and
tangerine respectively. However, GSH may play a
protective role in scavenging of single oxygen,
peroxides and hydroxyl radicals (Sander, 2003). The
active ingredients of essential oils may disturb the
metabolic behavior of tumor cells in special
GSH/GSSG Redox System. This phenomenon was
previously illustrated by Gupta et al., (2004) who
studied the relationship between antitumor activity
and antioxidant role in Ehrlich ascites. The increase
in the GST activity in general, used as indication for
the antitumor activity of the tested materials in both
normal and tumor transplanted animals. Therefore,
this enzyme has been used as antitumor factor (Oude-
Ophuis et al., 1998). In the tumor cells, the increase
of cellular enzymes that regulate the cell oxidative
stress such as SOD and GST and antioxidants such as
GSH induced cancer regression and stimulated large
number of tumor necrosis factor-alpha (TNF). Tumor
necrosis factor (TNF) is one of the most important
growth modulatory cytokines produced by almost all
cell types of the immune system. This factor is
related to GSH level in cancer cells and the
sensitivity of these cells to TNF depends on GSH
content and their rate of proliferation (Obrador et al.,
1997). ROS formed in cells tissues results in lipid
peroxidation and subsequently to increase in
malondialdehyde (MDA) level. Table (4) depicts the
levels of MDA in carcinoma cells; the levels were
lower in treated cells when compared with untreated
cells. After treatment by 150 µl/ml of essential oils,
the level of lipid peroxidation in mandarin, orange,
lemon, and tangerine oils were reduced by 59.2, 42.8,
38.8 and 75.5 %, respectively in comparison to
control treatment (100%). Several researchers have
tried to correlate GSH levels with tumors. It has been
reported, for instance, that many tumors can be
considered GSH-dependent and it is suspected that
cancer cells use GSH for protection against oxidative
damage. Also, increased levels of GSH in tumors are
related to anti-chemotherapeutic effects and
multidrug resistance (Shimura et al., 2000). Lipid
peroxide formed in the primary site would be
transferred through the circulation and provoke
damage by propagating the process of lipid
peroxidation MDA, the end product of lipid
peroxidation was reported to be higher in tumor
tissue than in non diseased organs (Yagi , 1991).
4 Conclusions
In conclusion, major aroma compounds
found in essential oils of citrus; in particular
limonene, linalool, α-terpinolene, carvone, citronellal
and camphene; exhibit potent antitumor and
antioxidant activities. Furthermore, ingestion of
these aroma compounds may help to prevent in vivo
oxidation damage such as lipid peroxidation, which is
associated with cancer, premature aging and diabetes.
To confirm this conclusion investigation of cytotoxic
effects of highly pure compounds of citrus oils is
suggested.
Essential oils
Mandarin oil Orange oil Lemon oil Tangerine oil
Tumor cells +
Essential oils
(µl/ml)
% of
viable
cells
% of
dead
cells
% of
viable
cells
% of
dead
cells
% of
viable
cells
% of
dead
cells
% of
viable
cells
% of
dead
cells
25 71±0.2 29±0.1 75±0.5 25±0.3 82±0.2 18±0.1 80±1.1 20±0.5
50 55±0.4 45±0.7 66±0.2 34±0.5 78±0.5 22±1.2 73±0.6 27±1.2
75 38±0.2 62±0.4 52±0.7 48±0.7 71±0.6 29±0.6 59±1.1 41±0.6
100 30±0.5 70±1.2 34±1.5 66±0.1 62±1.2 38±0.6 38±0.9 62±0.4
150 19±0.1 81±1.0 15±1.1 85±1.7 53±0.8 47±0.2 45±0.4 55±0.3
Control 97±0.8 3±0.5 98±0.9 2±0.5 98±0.9 2±0.3 99±1.0 1±0.1

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825
Table 4. The activity of glutathione S-trausferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) in
Ehrlisch ascites carcinoma cells treated with volatile oils.
GST* GSH** LPO*** Essential
oils Treatments
(µl/ml) Sp. activity
Rel.
content Level Rel.
content MDA Rel.
content
Mandarin 100
150 2.2
d
±0.1
3.2
e
±1.09 231.5
336.8 4.4
c
±0.05
5.1
d
±0d.19 209.5
242.8 3.4
d
±0.02
2.9
c
±0.03 69.4
59.2
Orange 100
150 2.0
d
±0.08
2.4
d
±018 210.5
252.6 2.4
a
±0.07
3.0
b
±0.17 114.3
142.8 2.8
bc
±0.04
2.1
a
±0.08 57.1
42.8
Lemon 100
150 1.2
b
±0.04
1.99
a
±0.24 126.3
209.5 2.3
a
±0.11
2.8
b
±0.41 109.5
133.3 2.6
b
±0.02
1.9
a
±0.06 59.2
38.8
Tangerine 100
150 1.6
c
±0.29
2.1
a
±0.29 168.4
221.1 2.9
b
±0.01
3.2
b
±0.21 138.1
152.4 4.1
f
±0.09
3.7
e
±0.07 83.7
75.5
Control 0.95
a
±0.14 100 2.1a±0.3 100 4.9
g
±0.02 100
Tumor cells + different con. of
essential oils
LSD 0.05 0.245 0.3437 0.217
*Specific activity of GST: µmol /mg protein /min.
**GSH level: µmol /mg protein
***LPO: n mol MDA /mg protein
Rel. content: relative content
±SE refers to standard error (n=6)
MDA: Malondialdehyde
Control: Tumor cells + saline solution
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