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Antioxidante , Anticarcinogenico e atividade Cupinicida do óleo de Vetiver
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Poster Papers
Antioxidant, Anticarcinogenic and Termiticidal Activities of Vetiver Oil
Feng Chen1, Xi Wang2, and Hyun-jin Kim1
1Department of Food Science and Human Nutrition, Clemson University, SC 29634
2Department of Biochemistry and Genetics, Clemson University, SC 29634
Abstract: Vetiver oil (VO) was assayed for its antioxidant, anticarcinogenic and termiticidal properties.
At the concentration of 10_l/ml, the DPPH radical scavenging effect of vetiver oil (93%) in antioxidant
activity test was higher than that of 1mM BHT (73%) and equivalent to 1mM _-tocopherol (93%). At
100ppm in cancer cell lines, vetiver oil inhibited the growth up to 89% of SiHa cervical cells, 88% of
CaSki cervical cells and 89% of MCF-7 breast cancer cells. Besides, vetiver oil was found to possess
strong termiticidal activity against the Formosan subterranean termite Coptotermes formosanus. Vetiver
oil decreased termite tunneling at concentration as low as 5_g/g sand, and entirely inhibited termites’
tunneling and paper consumption at concentrations higher than 25_g/g sand.
Contact: Feng Chen
1 INTRODUCTION
Vetiver oil is an important plant-derived product that contains hundreds of terpenes, terpenoids,
phenols, etc. It currently has three primary commercial applications: as aromas in perfumes, as food
flavoring additives, and as natural insecticide.
In recent years, biological activities, e.g. antioxidant, antimicrobial, insecticidal, anticancer effects, etc.,
of various essential oils and natural plant extracts have been widely and deeply studied. However, except
of few reports of the chemical composition of vetiver oil, few studies have been conducted on the
biological activities of vetiver oil (Zhu et al., 2001). Therefore, the objective of this study was to evaluate
the antioxidant, anticarcinogenic and termiticidal activities of the VO to explore its more practical
applications.
2 METHODS AND MATERIALS
Vetiver (Vetiveria zizanoides, Haiti) oil was purchased from the Good Scents Company (TGSC,
Oak Creek, WI). The authentic standard of nootkatone (crystalline, 97%) was purchased from the
Lancaster Synthesis Inc. (Windham, New Hampshire). Other standards were purchased from Sigma-
Aldrich, Inc. USA.
2.1 Silica Gel Column Chromatography
The column (3 x 2.5 cm) packed with silica gel (70-230 mesh, 60Å) was equilibrated with
hexane and eluted with the mixture solvents of hexane, methylene chloride (DCM) and methanol. Five ml
of VO was loaded and fractionated in size of 1ml.The first two fractions were isolated with pure hexane,
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other fractions were separated with DCM and methanol (70:30,V/V).
2.2 HPLC Separation
A Pinnacle II C18 column (150 X 4.6mm, 5_m; Restek, PA, USA) was connected with Shimadzu
LC-10AT HPLC system (Kyoto, Japan). 50 _l of vetiver oil partially purified by silica gel was injected
and eluted with methanol for further antioxidant test and chemical identification by GC-MS. The flow
rate was 1ml/min and the eluant was detected by Shimadzu SPD-M10V PAD.
2.3 GC-MS Identification
Chemical profile of VO was performed on Shimadzu GC-17A coupled with QP5050 MSD.
Chemicals were separated by DB-5 capillary column (60m X 0.25mm X 0.25_m; J&W Scientific,
Folsom, CA). Oven temperature was set from 60C to 240C at the increase rate of 3C/min, and held at
240C for 10 min. Identification of compounds were based on comparison of mass spectra and retention
indices with authentic standards or references.
3 BIOASSAY OF VO
Formosan subterranean termites (Figures 3 and 4) were collected in a fallen oak tree from New
Orleans, Louisiana, USA
3.1 Repellency Assay
The testing procedure was modified from Lewis et al. (1978). A Petri dish (5x1 cm) was used for
testing the repellency and toxicity of the VO against workers of the termite. The VO was dissolved in
ethyl alcohol and prepared in a series of dilutions for testing. One ml of hot agar solution (1.5%) was
spread evenly in the bottom of each dish, and covered with blasting sand (fine, #4). In each dish, one half
of the sand was chemically treated and covered with a piece of treated filter paper (Whatman #1,
0.5x1cm). The other half part was covered with sand and paper that were not chemically treated. Four
concentrations (5, 10, 25, 50_g/g sand) of the VO was prepared, and three replicates at each
concentration and control were tested. This experiment was repeated twice. Ten worker termites were
added into each Petri dish. Termite activities were recorded every 24 hours for 4 weeks. If more than 23
out of 30 termites were observed on the untreated sand, then the oil was considered repellent to termite. If
the termites behaved sluggishly or was found moribund and dead, the oil was considered toxic.
3.2 Tunneling, Paper Consumption and Mortality Assay
The experimental method of this bioassay is the same as that published (Zhu et al., 2001).
Tunneling response of termite to chemicals were recorded by a scanner for measurement for total tunnel
length. Paper consumption was calculated as the difference between the weight of filter paper before and
after the test. ANOVA test was used for the analysis of tunneling response, paper consumption and
mortality rate.
3.3 DPPH Assay for the Determination of Antioxidative Capacity
DPPH method of Yamahuchi et al. (2002) was modified to determine the radical scavenging
activity of VO. Butylated hydroxytoluene (BHT) and _-tocopherol was used as comparable standard
antioxidants. The reactant mixed with 0.4ml of 0.5mM DPPH solution, 0.2ml of ethanol, and 0.2ml of
sample was incubated in the darkness at room temperature for 1 hr. Antioxidative activity was measured
spectrophotometrically at 517nm and calculated.
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3.4 Anticancer Bioassays
VO was tested against cultures of two aggressive cervical cancer cell lines (CaSki & SiHa) and
one breast cancer cell line (MCF-7). The anticancer activity was assessed with the MTS viability assay
(CellTiter 96 AQueous One Solution kit, Promega). 15,000 cells of each cancer cell line per well were
added into 96-microtiter plate, mixed with appropriate concentrations of the vetiver oil. The cells then
were incubated for 24 hrs at 37C. After that 20 _l of MTS solution was added and incubated for 4 hours,
then the absorbance was read at 490nm in an ELISA plate reader. Dead cells%= (1 _ Abs.@490nm of
sample/Abs.@490nm of control) x 100%.
4 RESULTS AND DISCUSSION
Fig. 1 shows the DPPH radical scavenging effect of vetiver oil at different concentrations
compared to 1mM BHT and _-tocopherol. VO has the values at 44%, 66%, and 93% at the concentration
of 0.1 _l/ml, 1 _l/ml, and 10 _l/ml, respectively, compared to that of 73% for 1mM BHT and 93% 1 mM
_-tocopherol. This result indicated that VO is an excellent alternative natural antioxidant. However,
because of the complicated chemical composition of the VO, further instigation is underway to identify
the components responsible for the strong antioxidant effect.
Fig. 1 DPPH radical scavenging effect of Fig. 2 Inhibition of cell reproduction by
VO compared with BHT and tocopherol vetiver oil
Fig. 3 Termite Damage Fig. 4 Coptotermes formosanus
0
20
40
60
80
100
Relative antioxidant activity (%)
0.1 1 10 BHT Tocopherol
Vetiver oil Concent. (uL/mL)
Antioxidant activity of crude vetiver oil
0
20
40
60
80
100
Dead cells (%)
SiHa CasKi MCF-7
1ppm 10ppm 100ppm
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Fig. 2 shows the values representing the anticancer activity against three cancer cell lines at
concentrations of VO at 1ppm, 10ppm, and 100ppm. VO strongly inhibited the growth of all three cancer
cell lines approximately up to 89% at the 100ppm level. Even at the 1ppm level, the cervical cancer cells,
i.e. SiHa and Caski cells, were kill up to 40% and 50%, respectively, and the dead percentage MCF-7
breast cancer cells was in 20%.
VO also demonstrated strong repellency and antifeedant activity against the Formosan
subterranean termites, Coptotermes formosanus (Table 1). At the concentration of 5_g/g sand, VO
significantly decreased the termite tunneling activity and paper consumption at concentration as low as
10_g/g sand (F=39, 40, df=4, 15; P<0.0001). When the concentration of VO increased to 25ug/g sand and
above, the tunneling activity and paper consumption of termites entirely stopped. Further investigation
found that nootkatone was one of the major chemicals in VO responsible for the termiticidal activity, for
which patents have been filed.
Table 1 Mean (±SD) of paper consumption, percent mortality and tunneling length of Formosan
subterranean termite after a 14-day exposure
From the above results, it can be concluded that vetiver oil possesses multi-functional biological
activities. VO is not only an excellent natural antioxidant, also a potential alternative termiticide.
Considering its anticarcinogenic ability, VO also might be used in aromatherapy and provide clues for
novel drugs against some specific cancers.
References
Lewis DL, Michaels GE, Hays DB, et al. 1978. Evaluation of the antitermitic activity of
hydroxyquinoline and naphthol derivative formulations using Reticulitermes in laboratory and field
experiments. J. Econ. Entomol., 71: 818-821
Wedge DE, Meepagala KM, Magee JB, et al. 2001. Short communication. Anticarcinogenic activity of
strawberry, blueberry, and raspberry extracts to breast and cervical cancer cells. Journal of Medicinal
Food., 4(1): 49-51
Yamaguchi T, Takamura H, Matoba T, et al. 1998. HPLC method for evaluation of the free radicalscavenging
activity of foods by using 1,1-diphenyl-2-picrylhydrazyl. Biosci. Biotechnol. Biochem., 6:
1200-1204
Zhu BCR, Herderson G, Chen et al. 2001. Nootkatone is a repellent for Formosan subterranean termite
(Coptotermers formosanus). J. Chem. Ecol., 27: 523-531
50 0.00 ± 0.00 C 15.50 ± 9.32 A 0.00 ± 0.00 C
25 0.00 ± 0.00 C 13.20 ± 3.78 A 0.00 ± 0.00 C
10 8.65 ± 5.05 BC 13.00 ± 4.66 A 19.87 ± 4.52 B
5 15.80 ± 8.35 B 21.33 ± 2.08 A 27.87 ± 2.86 AB
0 64.05 ± 8.85 A 13.50 ± 4.03 A 39.75 ± 3.18 A
Tunneling
length
(cm)
% Termite
mortality
Weight loss of filter
paper (mg)
Concentration of
vetiver oil
(mg/g sand)
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