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Oxidative Medicine and Cellular Longevity 4.The antibacterial properties of the ethanolic oregano extract were measured using an agar dilution method.p-cymene and although cytotoxic at high concentrations, did both isomers at equimolar concentrations (12 uM carvacrol -octacosano and 5 HM thymol) to those present in the oregano extract (at not contribute to the cytotoxicity of the mixture, most likely the 2.93 ug/mL concentration) resulted in a less protective attributed to their low potency and very low availability in effect than that produced by the extract (% viability of the mixture (nM range).Discussion Results from the GC-MS analysis of the ethanol extract of wild-growing herb Origanum vulgare obtained from Southern Greece revealed an abundance of monoterpene hydrocarbons and phenolic compounds with the main con stituents being carvacrol and thymol (Table 1).the control group trea l)-benzene) pinene (1-methyl-4-0 rom WI methylethyl)-1,4-cyclohexadiene) are the precursors of car- vac in species of Origanum 19] Studies examining the effect of oregano extract on cancer prevention and cytotoxicity are limited [20].In the present and thymol did not have any effect on the uptake of carvacrol study, challenge of A549 human lung adenocarcinoma (Figure 3(a)); a small but significant increase in the uptake of epithelial cells with oregano ethanolic extracts (0-250 ug/mL final concentration) resulted in a concentration-dependent decrease in cell viability with a calculated LCs0 14ug/mL thymol was observed (Figure 3 3(b)) 3.4.Inclusion of p-cyme and/or l-octacosanol to the carvacrol and thymol mixture did not contribute to the antioxidant effects of the mixture determined in this study might possess higher potencies and The mechanisms by which thymol and carvacrol cause cell death in mammalian cell lines have not been thoroughly tent inhibitor of cell growth in A549 cell and a ne play a role in the overall effects of the oregano extracts.This finding rted by other investigators who found that in spite of the high variability of individual compounds in the essential oil of O. vulgare from 23 localities scattered all over Greece, the sum of carvacrol, thymol, p- cymene, and y-terpinene was consistent, amounting to >80% [10, 17, 181.As showen in Figure 4, pretreatment of A549 cells with the oregano extract challenge of A549 cells with thymol, carvacrol, p-cymene or l-octacosanol alone resulted in a concentration-dependent at concentrations ranging from 0 to 2.93 ug/mL resulted in decrease in cell viability, with thymol being more cytotoxic a concentration-dependent protective effect against H,0,.The oregano extract inhibited the growth of reference ATCC Gram-negative and protein amount [21].

Oxidative Medicine and Cellular Longevity
4. Discussion
Results from the GC-MS analysis of the ethanol extract
of wild-growing herb Origanum vulgare obtained from
Southern Greece revealed an abundance of monoterpene
hydrocarbons and phenolic compounds with the main con
stituents being carvacrol and thymol (Table 1). This finding
rted by other investigators who
found that in spite of the high variability of individual
compounds in the essential oil of O. vulgare from 23 localities
scattered all over Greece, the sum of carvacrol, thymol, p-
cymene, and y-terpinene was consistent, amounting to >80%
[10, 17, 181. Plants collected from the northern region of
Greece were rich in thymol (30.3-42.8% of total oil), whereas
those from the southern part of the country were rich in
carvacrol (574-696% of total oil) [17]. p-Cymene (1-methyl-
75
50
25
is consistent with that
repo
0.18 0.37 0.73 1.46 2.93
24 h oregano
Pretreatment (ug/mL)
Posttreatment (500pM)+
FIGURE 4: The antioxidant effect of the ethanolic O vulgare extract
against H20-induced cytotoxicity in A549 cells. All experiments
were repeated three times in two replicates as outlined in Section 2.
the control group trea
l)-benzene)
pinene (1-methyl-4-0
rom
WI
methylethyl)-1,4-cyclohexadiene) are the precursors of car-
vac
in species of Origanum 19]
Studies examining the effect of oregano extract on cancer
prevention and cytotoxicity are limited [20]. In the present
and thymol did not have any effect on the uptake of carvacrol study, challenge of A549 human lung adenocarcinoma
(Figure 3(a)); a small but significant increase in the uptake of epithelial cells with oregano ethanolic extracts (0-250 ug/mL
final concentration) resulted in a concentration-dependent
decrease in cell viability with a calculated LCs0 14ug/mL
thymol was observed (Figure 3
3(b))
3.4. Antioxidant Protective Effects of the Oregano Extract.(Figure1). It is not known whether the cytotoxicity of oregano
extract is attributed to a specific component or combination
of components. Results shown in Figure 2 demonstrated that
To examine the antioxidant effects of the oregano extract,
noncvtotoxic concentrations of extract that corresponded
to greater than 95% cell viability were used. As showen in
Figure 4, pretreatment of A549 cells with the oregano extract
challenge of A549 cells with thymol, carvacrol, p-cymene
or l-octacosanol alone resulted in a concentration-dependent
at concentrations ranging from 0 to 2.93 ug/mL resulted in decrease in cell viability, with thymol being more cytotoxic
a concentration-dependent protective effect against H,0,.
Preincubation of A549 cells with nontoxic concentrations of
than the other three compounds. Challenge of A549 cells
with a combination of the four compounds at equimolar
carvacrol or thymol showed that both phenolic isomers pro- concentrations (56 MM carvacrol, 23.3MM thymol) to those
tected against hydrogen-peroxide induced cytotoxicity, with present in the oregano extract was less cytotoxic than the
tself. The cytotoxicity of the oregano extract is mostly
carvacrol producing a better protective effect than thymol excti
(Figure 5(a)). Preincubation of cells with a combination of attributed to presence of carvacrol and thymol. p-cymene and
although cytotoxic at high concentrations, did
both isomers at equimolar concentrations (12 uM carvacrol -octacosano
and 5 HM thymol) to those present in the oregano extract (at not contribute to the cytotoxicity of the mixture, most likely
the 2.93 ug/mL concentration) resulted in a less protective attributed to their low potency and very low availability in
effect than that produced by the extract (% viability of the mixture (nM range). It appears that other unmeasured
carvacrol thymol mixture versus oregano extract: 37.14+ ethanolic constituents at lower concentrations than t
0.77 versus 69.60 5.00) (Figure 5). Inclusion of p-cyme
and/or l-octacosanol to the carvacrol and thymol mixture
did not contribute to the antioxidant effects of the mixture
determined in this study might possess higher potencies and
The mechanisms by which thymol and carvacrol cause
cell death in mammalian cell lines have not been thoroughly
tent inhibitor of cell growth in A549 cell
and a
ne
play a role in the overall effects of the oregano extracts.
(data not shown); however, the inclusion of the other oregano
extracts to the carvacrol/thymol mixture was not examined investigated. Results from an in vitro study
carvacrol is ve
line as evidenced by the concentration
in cell number, degeneration of cell morphology,
decrease in total
-dependent decre
3.5. Antimicrobial Properties of the Oregano Extract. The
antibacterial properties of the ethanolic oregano extract were
measured using an agar dilution method. The oregano extract
inhibited the growth of reference ATCC Gram-negative and
protein amount [21]. Thymol induces cell
death in human osteosarcoma and astrocytes and may involve
apoptosis via mitochondrial pathways [22, 23. Whether
Gram-positive bacterial strains with varying degree, but no he synergistic effects of carvacrol and thymol regardin
trends were realised (Table 2). The inhibitory concentrations
cell viability are related to a combination of the purported
were 2- to 4-fold lower against nonmucoid than mucoid mechanisms is under investigation. It is evident from the
clinical isolates of P aeruginosa and exhibited greater effects results of this study that coincubation of carvacrol and
against clinical isolates of B. cenocepacia compared to the thymol increased the uptake of the more cytotoxic thymol
and enhanced the cytotoxicity of the mixture.