Food Chemistry 134 (2012) 262268

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Study of the chemical composition, antioxidant activity and anti-inammatory

activity of essential oil from Vetiveria zizanioides
Su-Tze Chou a, Chia-Pei Lai a, Chih-Chien Lin b,, Ying Shih b,
a
Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung 43301, Taiwan, ROC
b
Department of Cosmetic Science, Providence University, 200 Chung-Chi Road, Shalu, Taichung 43301, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: Vetiveria zizanioides (vetiver grass) is well known as an eco-friendly plant that prevents soil erosion and
Received 18 July 2011 rehabilitates metalliferous polluted land. V. zizanioides is also the major source of vetiver oil for medicine
Received in revised form 22 November 2011 and perfumery. Our study identied 25 compounds in V. zizanioides essential oil (VZ-EO). The major com-
Accepted 21 February 2012
ponents were cedr-8-en-13-ol (12.4%), a-amorphene (7.80%), b-vatirenene (5.94%) and a-gurjunene
Available online 1 March 2012
(5.91%). VZ-EO may suppress the inammatory responses of LPS-stimulated RAW 264.7 macrophages,
including nitric oxide production and cell apoptosis, by regulating the expression of the inammation-
Keywords:
related enzymes heme oxygenase-1, inducible nitric oxide synthase and cyclooxygenase-2 (inducible
Antimicrobial
Antioxidant
cyclooxygenase) and the inammatory cytokines tumour necrosis factor-a, interleukin-1b and inter-
Anti-inammatory feron-b. Additionally, the anti-inammatory activity of VZ-EO correlated with its antioxidant activity
Chemical composition of decreasing LPS-induced superoxide anion production and malondialdehyde levels.
Essential oil 2012 Elsevier Ltd. All rights reserved.
Vetiveria zizanioides

1. Introduction
Previous research suggests that free radicals, reactive oxygen
species (ROS) and nitric oxide (NO) play key roles in both normal
biological function and in the pathogenesis of certain human dis-
eases (Finkel & Holbrook, 2000). For instance, generation of reacti-
vated species by inammatory cells is a major microbicidal
mechanism, and long-term generation of free radicals may also
cause diseases including arthritis, diabetes, atherosclerosis and
some types of cancer (Martinez-Cayuela, 1995). Therefore, the reg-
ulation of inammation-related enzymes such as heme oxygenase-
1 (HO-1), inducible nitric oxide synthase (iNOS) and cyclooxygen-
ase-2 (COX-2; inducible cyclooxygenase) plays an important role
in inammation (Zhang et al., 2008). Similarly, several pro-inam-
matory mediators and cytokines, such as tumour necrosis factor-a
(TNF-a), interleukin-1b (IL-1b), and interferon-b (IFN-b), are pro-
duced at high levels in pathological and hypersensitivity condi-
tions, which in turn causes an inammatory response and may
damage the neighbouring tissues and the macrophages themselves
(Tiwari, Dwivedi, & Kakkar, 2010). Thus, properly regulated inam-
matory responses are necessary for a healthy immune function.
The use of plant extracts and components has become increas-
ingly important for scientic research and industrial applications
Corresponding authors. Tel.: +886 4 26328001x15410 (Y. Shih), +886 4
26328001x15409 (C.-C. Lin).
E-mail addresses: chchlin@pu.edu.tw (C.-C. Lin), yingshih@pu.edu.tw (Y. Shih).
in recent years. Extracted oils are used in medicine, food and cos-
metics primarily because of their potency and various biological
activities (Bubonja-Sonje, Giacometti, & Abram, 2011; Song et al.,
2010). In addition, plants act as antioxidant and anti-inammatory
agents by quenching free radicals, increasing antioxidant defenses
or inhibiting the release of pro-inammatory mediators (Tiwari
et al., 2010). Therefore, the use of essential oils or plant extracts
as natural additives is a good approach for reducing oxidation
and preventing abnormal inammation.
Vetiveria zizanioides (vetiver grass), a fast-growing perennial
tussock grass of the Gramineae family, is well known as an eco-
friendly plant that prevents soil erosion and is useful in the reha-
bilitation of metalliferous polluted land because of its tolerance
to elevated levels of heavy metals (Pripdeevech, Wongpornchai,
& Promsiri, 2006). V. zizanioides is also the major source of vetiver
oil, which is used in medicine and perfumery. Vetiver oil has been
identied as a national permissible natural food additive in China
(China Number System for food, N102) and which is an expensive
edible oil in the market of China (Wang, Wen, & Zhao, 2009). V.
zizanioides is a commonly used traditional medicine in Thailand
(Issaravanich et al., 2008; Manosroi, Dhumtanom, & Manosroi,
2006). Vetiver oil has been also used in the treatment of several
diseases, including mouth ulcers, fever, headache, inammation
and gastritis (Aibibu et al., 2010; Luqman et al., 2009). Moreover,
essential oil extracted from V. zizanioides has been frequently used
as a functional ingredient and fragrance in foods, aromatic prod-
ucts and cosmetics. Therefore, the interest in this grass has

0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2012.02.131
 S.-T. Chou et al. / Food Chemistry 134 (2012) 262268 263

increased in recent years; however, few articles have described the Table 1
biological activities of this plant (Kim, Chen, Wang, Chung, & Jin, Primer sequences used in RT-PCR in this study.

2005; Luqman et al., 2009), and there have been no reports on its Target Type Sequences
anti-inammatory activity to date. Thus, research on the biological b-actin Sense 50 -TGGAATCCTGTGGCATCCATGAAAC -30
properties of V. zizanioides holds signicance for many Anti-sense 50 -TAAAACGCAGCTCAGTAACAGTCCG-30
applications. iNOS Sense 50 -AGACTGGATTTGGCTGGTCCCTCC-30
The main objective of this work was to evaluate the chemical Anti-sense 50 -AGAACTGAGGGTACATGCTGGAGCC-30
composition, antioxidant activity and anti-inammatory activity COX-2 Sense 50 -GGAGAGACTATCAAGATAGT-30
of V. zizanioides essential oil (VZ-EO). The anti-inammatory mech- Anti-sense 50 -ATGGTCAGTAGACTTTTACA-30
anism of VZ-EO in lipopolysaccharide (LPS)-induced murine mac- HO-1 Sense 50 -TGAAGGAGGCCACCAAGGAGG-30
rophage cells was also investigated in this study. Anti-sense 50 -AGAGGTCACCCAGGTAGCGGG-30
TNF-a Sense 50 -GGCAGGTCTACTTTGGAGTCATTGC -30
2. Materials and methods Anti-sense 50 -ACATTCGAGGCTCCAGTGAATTCGG-30
IL-1b Sense 50 -TGCAGAGTTCCCCAACTGGTACATC -30
2.1. Essential oil and cell line Anti-sense 50 -GTGCTGCCTAATGTCCCCTTGAATC -30
IFN-b Sense 50 -GAGTTACACTGCCTTTGCC-30
Steam-distilled essential oil of Vetiveria zizanioides (VZ-EO) was Anti-sense 50 -ATTCACTACCAGTCCCAGA-30

purchased from Lorien Vana Biotech, Inc. (Taiwan, ROC). The mur-
ine macrophage cell line RAW 264.7 (BCRC 60001) was obtained
from the Bioresource Collection and Research Center (BCRC, Hsin-
applied. The cells were treated with different concentrations of the
chu, Taiwan, ROC) and was used in anti-inammatory activity
essential oil (0, 5, 7.5, 10 and 12.5 lg/ml) and 1 lg/ml LPS for 20 h.
assays.

2.2. Materials 2.5. Cell viability

Fetal bovine serum (FBS), L-glutamine, penicillinstreptomycin, The RAW 264.7 cells were plated in 12-well plates at a density
trypsinethylenediaminetetraacetic acid (trypsinEDTA), deoxy- of 3  105 cells/ml. The cells were treated with different concentra-
nucleotide triphosphate (dNTP), oligo(dT), Taq DNA polymerase tions (0, 5, 7.5, 10 and 12.5 lg/ll) of essential oil and LPS (l lg/ml)
and Dulbeccos Modied Eagle Medium (DMEM) medium were and grown at 37 C in 5% CO2 and 95% air for 20 h. An MTT assay
purchased from Gibco BRL/Invitrogen (Carlsbad, CA, USA). Lipo- was used to determine cell viability (Sladowski, Steer, Clothier, &
polysaccharide (LPS; from Escherichia coli, serotype O111: B4), 3- Balls, 1993).
(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT), Griess reagent, sodium nitrite, pyrogallol, nitro blue tetra-
zolium (NBT), bovine serum albumin (BSA), ethidium bromide, Table 2
dithiothreitol (DTT), agarose and other chemicals were purchased Constituents of essential oil from Vetiveria zizanioides determined by GCMS.
from SigmaAldrich (St. Louis, MO, USA). Deionised distilled water No Constituents Rta KIb Area
(ddH2O) for solutions and buffers was puried using a Milli-Q sys- (%)
tem (Millipore, Bedford, MA, USA). 1 2,3,5,5,8,8-Hexamethyl-cycloocta-1,3,6-triene 23.19 1314 2.28
2 1,5,9,9-Tetramethyl-2-methylene- 24.08 1328 3.96
spiro[3.5]non-5-ene
2.3. Gas chromatography and mass spectrometry analysis
3 (+)-Sativen 25.51 1339 2.83
4 4,8,8-Trimethyl-2-methylene-4-vinylbicyclo 25.62 1407 4.53
Essential oil was analysed by gas chromatography-mass spec- [5.2.0]nonane
trometry (GCMS) to identify its constituents. The GCMS 5 a-Amorphene 26.05 1440 7.80
(GCMS-QP2010 Plus, Shimadzu, Japan) was equipped with a Forte 6 2-Isopropenyl-1,3,5-trimethylbenzene 26.14 1465 2.43
7 a-Gurjunene 26.22 1479 5.91
ID-BPX5 column (30.0 m  0.25 mm i.d., 0.25 lm lm thickness, 8 b-Vatirenene 26.30 1489 5.94
SGE, AU), and the injector temperature was maintained at 250 C. 9 d-Cadinene 26.55 1499 2.57
Helium (He) was used as the carrier gas at a ow rate of 1 ml/ 10 b-Guaiene 26.93 1523 4.28
min, and the split ratio was set at 1:100. The initial oven temper- 11 Dehydro-aromadendrene 27.05 1545 5.45
12 Cubenol 28.82 1580 2.09
ature was held at 50 C for 5 min and then programmed to increase
13 (+)-Ledene 29.28 1605 4.77
by 5 C/min to 150 C; it was nally raised to 300 C at a rate of 14 Epiglobulol 29.42 1632 2.21
10 C/min. The mass spectrometry conditions were as follows: scan 15 Widdrol 29.90 1651 2.13
range, 40350 amu; ion source temperature, 230 C; and interface 16 6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a- 30.04 1690 1.97
temperature, 250 C. The essential oil constituents were identied Octahydro-naphthalen-2-ol
17 3-(2-Isopropyl-5-methylphenyl)-2- 30.17 1745 3.17
by computer matching the mass spectra with a standard library
methylpropionic acid
and comparing the measured Kovats index (KI) to a homologous 18 Cedr-8-en-13-ol 30.67 1769 12.36
series of n-alkanes (C5C26). 19 Ethyl 4-(4-methylphenyl)-4-pentenoate 31.06 1804 2.12
20 Isovellerdiol 31.11 1842 2.38
21 a-Curcumene 31.16 1867 2.44
2.4. Cell culture 22 3,3,8,8-Tetramethyl-tricyclo[5.1.0.0(2,4)] oct- 31.38 1890 4.82
5-ene-5-propanoic acid
The RAW 264.7 cells were cultured in DMEM supplemented 23 Solavetivone 31.46 1906 4.20
with 10% FBS, 2 mM L-glutamine and 1% penicillinstreptomycin 24 3,8-Dimethyl-4-(1-methylethylidene)- 31.82 1925 4.89
2,4,6,7,8,8a-Hexahydro-5(1H)-azulenone
(100 U/ml penicillin and 100 lg/ml streptomycin). The cells were 25 ()-Spathulenol 31.88 1938 2.47
maintained in a humidied incubator at 37 C with 5% CO2. The
a
cells were sub-cultured every 34 days to maintain logarithmic Retention time (min).
b
Kovats index.
growth and were allowed to grow for 24 h before treatments were
264 S.-T. Chou et al. / Food Chemistry 134 (2012) 262268

2.6. Nitrite production
Nitrite was measured as an indicator of NO production after
20 h of essential oil treatment and LPS induction. A 100 ll aliquot
of the culture supernatant was plated in a 96-well plate, and an
equal amount of Griess reagent (1% sulphanilamide and 0.1% N-
1-(naphthyl) ethylenediamine dihydrochloride in 2.5% H3PO4)
was added. The plate was then incubated for 5 min, and the absor-
bance was measured at 540 nm. The amount of NO was calculated
using a sodium nitrite standard curve (Coker & Laurent, 1998).
2.7. Assay for messenger RNA (mRNA) levels of iNOS, HO-1, COX-2, IL-
1b, TNF-a, and IFN-b
ethidium bromide and photographed under UV light according to
the method of Lu et al., 2010).
2.11. Statistical analysis
All assays were conducted at least three times with three differ-
ent sample preparations. All data are expressed as the mean stan-
dard deviation (SD). Analysis of variance was performed in SPSS
(SPSS Inc., USA). A one-way ANOVA and Scheffe test were used
for these analyses, and p < 0.05 was considered to be statistically
signicant.
3. Results and discussion

The RAW 264.7 cells were incubated with various concentra-

3.1. Chemical composition of VZ-EO
tions of essential oil (0, 5, 7.5, 10 and 12.5 lg/ml) and LPS (l lg/
ml) for 20 h, and the total RNA was extracted using the Qiagen
Steam-distilled essential oil of V. zizanioides was purchased
RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA). Briey, the re-
from Lorien Vana Biotech, Inc. (Taiwan) and then analysed by
verse transcription reaction was performed using 5 lg of total
GCMS. The results are listed in Table 2. For VZ-EO, 25 constituent
RNA, 1 ll of oligo(dT), 1 ll of dNTP mix (10 mM) and up to 12 ll
compounds were identied and are listed in Table 2, along with the
of ddH2O. The mixture was heated for 5 min at 65 C and quickly
retention times and Kovats indices. Our results show that the most
chilled on ice. Then, 4 ll of rst strand buffer, 2 ll of 0.1 M DTT
plentiful constituent of VZ-EO is cedr-8-en-13-ol (12.4%), and the
and 1 ll of RNAseOUT were added to the mixture. The mixture
other major compounds are a-amorphene (7.80%), b-vatirenene
was incubated at 37 C for 2 min, and 1 ll of M-MLV reverse trans-
criptase was added. The reaction was stopped by heating the solu-
tion to 70 C for 15 min. A 1 ll aliquot of cDNA mixture was used
for the enzymatic amplication. Polymerase chain reaction (PCR)
was performed using 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 U of Taq
DNA polymerase, and 0.1 lM each of the primers for iNOS, HO-1,
COX-2, IL-1b, TNF-a and IFN-b (Table 1). The amplied products
were separated in 2% agarose gel in TrisBorateEDTA (TBE) buffer
and stained with ethidium bromide (Lee et al., 2003).

2.8. Measurement of superoxide anion production

The RAW 264.7 cells were incubated with various concentra-

tions of essential oil (0, 5, 7.5, 10 and 12.5 lg/ml) and LPS (l lg/
ml) for 20 h prior to testing the levels of superoxide anions. The
superoxide anion measurement, based on the NBT assay, was per-
formed according to the method of Freire et al. (2003).

2.9. Measurement of lipid peroxide levels and superoxide dismutase

(SOD) activity

The RAW 264.7 cells were incubated with various concentra-

tions of essential oil (0, 5, 7.5, 10 and 12.5 lg/ml) and LPS (l lg/
ml) for 20 h. Cells were harvested and sonicated with 1 ml of
1 mM PMSF buffer in order to obtain the cell homogenate. The
thiobarbituric acid reactive substances (TBARS) method was used
to estimate cellular malondialdehyde (MDA) levels with a spectro-
photometer at 535 nm (Botsoglou et al., 1994). SOD activity was
determined by spectrophotometry at 325 nm based on the SOD-
mediated decrease in the rate of pyrogallol autoxidation under
alkaline conditions (Marklund & Marklund, 1974). The protein con-
tent of the cell homogenate was determined based on the Biuret
reaction (Smith et al., 1985), using a BCA kit (Pierce, Rockford, IL,
USA) with BSA standards. The MDA levels in cells are expressed
as nmole/mg protein, and the specic activity of SOD is expressed
as unit/mg protein.

2.10. DNA fragmentation assay

The RAW 264.7 cells were incubated with various concentra-

Fig. 1. Effects of V. zizanioides essential oil (VZ-EO) on cell viability (A) and NO
tions of essential oil (0, 5, 7.5, 10 and 12.5 lg/ml) and LPS (l lg/ production (B) by LPS-stimulated RAW 264.7 macrophages. Data are means SD of
ml) for 20 h. The cells were then harvested by centrifugation. The three independent experiments. Indicates signicant differences (p < 0.05) com-
DNA was isolated, separated by gel electrophoresis, stained with pared to the LPS-treated group.
 S.-T. Chou et al. / Food Chemistry 134 (2012) 262268
(5.94%) and a-gurjunene (5.91%). For comparison, a previous study
reported that the essential oil of Peucedanum longifolium was abun-
dant in cedr-8-en-13-ol (33.74%) and exhibited potent scavenging
of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and inhibi-
tion of lipid peroxidation (Tepe, Akpulat, & Sokmen, 2011). More-
over, cedr-8-en-13-ol has some insecticidal activity against
mosquitoes (Adams, McDaniel, & Carter, 1988). In addition, a-
amorphene is one of the key constituents of Alpinia oxyphylla Miq
essential oil, which can inhibit the growth of Staphylococcus aureus
(Xin, Xiaojing, Jiao, Haofu, & Wenquan, 2010). The presence of b-
vatirenene also reveals an important role for vetiver oil in scaveng-
ing DPPH free radicals and chelating ferrous ions (Kim et al., 2005).
Accordingly, although the VZ-EO used in this study is thought to
have some signicant biological functions, including antioxidant
activity, its anti-inammatory activity should be studied further.
3.2. Effects of VZ-EO on cell viability and NO production
To evaluate the protective activity of VZ-EO on LPS-stimulated
(l lg/ml) RAW 264.7 macrophages and to determine the appropri-
ate concentrations for the subsequent analyses, a standard MTT as-
say was used to test the effect of VZ-EO on cell viability, and the
result is shown in Fig. 1A. All tested concentrations (from 5 to
12.5 lg/ml) of VZ-EO clearly did not decrease the cell viability of
LPS-stimulated RAW 264.7 macrophages. Moreover, all tested con-
centrations of VZ-EO increased the number of LPS-stimulated RAW
264.7 macrophages (Fig. 1A). This result indicates that VZ-EO has
an anti-inammatory effect that protects LPS-stimulated RAW
Fig. 2. (A) Effects of V. zizanioides essential oil (VZ-EO) on the expression of HO-1, iNOS and COX-2 mRNA by LPS-stimulated RAW 264.7 macrophages. (BD) Quantisation of
the results. The mRNA expression levels of target genes were determined by RT-PCR. Data are means SD of three independent experiments. Indicates signicant differences
(p < 0.05) compared to the LPS-treated group.
265
264.7 macrophages from the damage caused by the inammatory
response.
NO, a potentially toxic molecule, is released by the innate im-
mune cells during pathogenesis; therefore, NO production is also
the expected response for LPS-stimulated RAW 264.7 macrophages
(Fig. 1B). In this study, VZ-EO decreased the NO production of LPS-
stimulated RAW 264.7 macrophages at concentrations of 7.5, 10
and 12.5 lg/ml. The 12.5 lg/ml concentration of VZ-EO reduced
the NO production to 26.5% of that seen in the unstimulated
RAW 264.7 macrophages (Fig. 1B). Moreover, this inhibition by
VZ-EO was dose-dependent. These results indicate that VZ-EO
has no cytotoxicity at any of the tested concentrations and may de-
crease the NO production of LPS-stimulated RAW 264.7
macrophages.
3.3. Effects of VZ-EO on the expression of HO-1, iNOS and COX-2 mRNA
In pathogen- or cytokine-activated macrophages, HO-1, iNOS
and COX-2 are the key enzymes that participate in the inamma-
tory process. The induction of HO-1 may decrease the level of
ROS and suppress the inammatory response by reducing the func-
tion of NO. Additionally, the activation of iNOS may cause NO accu-
mulation in the cell supernatant. The activation of COX-2
(inducible COX) converts arachidonic acid (AA) to prostaglandins
and then increases the inammatory response. Therefore, exami-
nation of the gene expression of these inammation-related en-
zymes in VZ-EO-treated macrophages may help us to understand
the relationship between the anti-inammatory activity of VZ-EO
and the regulatory mechanism. The specic primers for reverse
266 S.-T. Chou et al. / Food Chemistry 134 (2012) 262268
transcription polymerase chain reaction (RT-PCR) are listed in Ta-
ble 1, and the results are shown in Fig. 2. For HO-1, VZ-EO may in-
crease the mRNA levels in the LPS-stimulated RAW 264.7
macrophages at each tested concentration (Fig. 2A). The quantisa-
tion result is shown in Fig. 2B. The increase in the levels of HO-1
mRNA expression ranged from approximately 10% to 25%. These
results indicate that the protective effect of VZ-EO on LPS-stimu-
lated RAW 264.7 macrophages may be related to HO-1 induction
(Kunwar, Sandur, Krishna, & Priyadarsini, 2009). Additionally,
Fig. 2 shows similar dose-dependent decreases in the mRNA levels
of iNOS (Fig. 2A and C) and COX-2 (Fig. 2A and D) in treated cells at
all tested concentrations of VZ-EO. The iNOS and COX-2 mRNA lev-
els in cells treated with 12.5 lg/ml VZ-EO decreased to approxi-
mately 67% and 75%, respectively, of the levels in LPS-stimulated
cells. This result shows that VZ-EO may protect macrophages from
the inammatory response through the inhibition of iNOS and
COX-2 expression, which is also reected in the anti-inammatory
activity of VZ-EO (Kim et al., 2008).
3.4. Effects of VZ-EO on the expression of TNF-a, IL-1b and IFN-b mRNA
Numerous pro-inammatory cytokines are highly expressed
during inammation; for example, TNF-a, IL-1b and IFN-b. Thus,
the mRNA expression levels for TNF-a, IL-1b and IFN-b are also
important indicators for the analysis of anti-inammatory activity.
The expression levels of TNF-a, IL-1b and IFN-b mRNA in VZ-EO-
treated cells are shown in Fig. 3. For TNF-a, the mRNA level clearly
decreased when the VZ-EO concentration was higher than 7.5 lg/
ml (Fig. 3A and B). Moreover, the suppression of mRNA expression
Fig. 3. (A) Effects of V. zizanioides essential oil (VZ-EO) on the expressions of TNF-a, IL-1b and IFN-b mRNA by LPS-stimulated RAW 264.7 macrophages. (BD) Quantisation of
the results. The mRNA expression of target genes was determined by RT-PCR. Data are means SD of three independent experiments. Indicates signicant differences
(p < 0.05) compared to the LPS-treated group.
was not correlated with the VZ-EO concentration, even if the sup-
pression effects have at least 23% (Fig. 3B). Unlike TNF-a mRNA
expression, IL-1b and IFN-b mRNA expression in VZ-EO-treated
cells exhibited a signicant decrease at all tested concentrations
of VZ-EO and was also dose-dependent (Fig. 3A, C and D). The
greatest mRNA expression inhibition, a suppression of up to 81%,
was observed for IL-1b in macrophages treated with 12.5 lg/ml
VZ-EO (Fig. 3C). In the LPS-stimulated macrophages, the NF-jB-
mediated transcriptional induction of inammation-associated
genes, such as that for TNF-a, IL-1b, iNOS and COX-2, is the most
signicant response to inammation (Ansari, Khodagholi, Amini,
& Shaerzadeh, 2011; Kim et al., 2008). Therefore, our results indi-
cate that VZ-EO may suppress the LPS-induced inammatory re-
sponse of RAW 264.7 macrophages. This suppression was
mediated by the up-regulation of HO-1 expression and down-reg-
ulation of iNOS, COX-2, TNF-a, IL-1b and IFN-b expression.
3.5. Effects of VZ-EO on superoxide anion production, MDA levels and
SOD activity
To verify that the anti-inammatory activity of VZ-EO on LPS-
stimulated RAW 264.7 macrophages was due to its antioxidant
function, the oxidative indicators of the VZ-EO-treated cells were
analysed. First, we examined the superoxide anion production of
the VZ-EO-treated cells; the result is shown in Fig. 4A. Compared
with the LPS-treated cells, the superoxide anion production of
the VZ-EO-treated cells decreased by 12% and 20% at concentra-
tions of 7.5 and 12.5 lg/ml, respectively. Thus, VZ-EO can decrease
the superoxide anion production of LPS-stimulated RAW 264.7
 S.-T. Chou et al. / Food Chemistry 134 (2012) 262268 267

Fig. 4. Effects of V. zizanioides essential oil (VZ-EO) on superoxide anion production (A), MDA (TBARS) level (B), SOD activity (C) and DNA damage (D) in LPS-stimulated RAW
264.7 macrophages. Data are means SD of three independent experiments. #Indicates signicant differences (p < 0.05) compared to the control group; indicates signicant
differences (p < 0.05) compared to the LPS-treated group. For DNA fragmentation, cellular DNA was extracted and analysed by agarose gel electrophoresis.

macrophages. The lipid peroxidation was also tested through the
production of MDA by LPS-stimulated macrophages and analysed
the cellular SOD activity to verify how VZ-EO affects the cells.
The results are shown in Fig. 4B and D. The MDA production of
VZ-EO-treated cells clearly decreased in a dose-dependent manner
when compared with LPS-treated cells, and the MDA levels for cells
treated with 10 and 12.5 lg/ml VZ-EO were lower than that of nor-
mal RAW 264.7 macrophages (Fig. 4B). Our results indicate that
VZ-EO may effectively suppress the cellular lipid peroxidation of
LPS-stimulated macrophages. The SOD activity of VZ-EO-treated
cells also decreased at concentrations higher than 10 lg/ml,
although the suppressive effect was not clearly visible at low con-
centrations of VZ-EO (Fig. 4C). This result revealed that the anti-
antioxidant activity of VZ-EO was not affected by increasing SOD
activity in the LPS-stimulated macrophages. Some compounds
demonstrate antioxidant activity when levels of antioxidant-re-
lated enzymes increase (Choi, Cho, Park, Cho, & Song, 2003; Park,
Park, Noh, Shin, & Song, 2011; Tiwari et al., 2010). However, the
activities of antioxidant-related enzymes are reduced in some anti-
oxidant-treated cells, as with the natural antioxidants apocynin
and NAO (Ben-Shaul et al., 2001). This phenomenon results when
the antioxidant activity of these antioxidants, and it is not exe-
cuted through an increase in antioxidant-related enzymes. Hence,
these results indicate that VZ-EO can suppress the oxidative stress
and lipid peroxidation of LPS-stimulated macrophages, and this
antioxidant activity is not executed through increased SOD
activity.
3.6. Effects of VZ-EO on LPS-induced DNA damage
Since the inammatory response of LPS-stimulated macro-
phages may damage neighbouring tissues and the macrophages
themselves, the LPS-induced DNA damage of VZ-EO-treated cells
were analysed by a DNA fragmentation assay to examine whether
VZ-EO can protect cells from the injury of inammation. Fig. 4D
shows that cells treated with all tested concentrations of VZ-EO ap-
pear lower on the DNA ladder than the LPS-treated cells. This result
also indicates that VZ-EO may suppress LPS-induced apoptosis in
RAW 264.7 macrophages. Induction of iNOS in RAW 264.7 macro-
phages by LPS produced the typical morphological and biochemical
changes of apoptosis, such as chromatin condensation and DNA
fragmentation. These alterations were prevented by an inhibitor
of NO synthase, thereby suggesting a link between NO generation
and induction of apoptosis (Brune, von Knethen, & Sandau, 1998).
Therefore, suppression of apoptosis in LPS-induced RAW 264.7
macrophages by VZ-EO is due to its anti-inammatory activity,
which correlates with its antioxidant function.
268 S.-T. Chou et al. / Food Chemistry 134 (2012) 262268
4. Conclusion
In conclusion, 25 compounds of VZ-EO have been identied, and
the major components are cedr-8-en-13-ol (12.4%), a-amorphene
(7.80%), b-vatirenene (5.94%) and a-gurjunene (5.91%). VZ-EO
may suppress the inammatory responses of LPS-stimulated
RAW 264.7 macrophages, including NO production and cell apop-
tosis, through the regulation of the expression of the inamma-
tion-related enzymes HO-1, iNOS and COX-2 and the
inammatory cytokines TNF-a, IL-1b and IFN-b. Additionally, the
anti-inammatory activity of VZ-EO correlates with its antioxidant
activity, which decreasing LPS-induced superoxide anion produc-
tion and MDA levels. Because of these antioxidant and anti-inam-
matory activities, the essential oil of V. zizanioides may be used in
many applications in the future.
Acknowledgements
The authors are grateful for nancial support from the National
Science Council of the Republic of China awarded to Dr. S.-T. Chou
(NSC98-2313-B-126-004-MY3), Dr. Y. Shin (NSC-98-2113-M-126-
005-MY3) and Dr. C.-C. Lin (NSC99-2313-B-126-002-MY3).
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