Identification of bioactive compounds in oil of tea seed (Camellia oleifera Abel)
Chia-Pu Lee and Gow-Chin Yen
Department of Food Science, National Chung Hsing University, 250 Kuokuang Road,
Taichung 40227, Taiwan
The oil of tea seed (
Camellia oleifera
Abel.) is used extensively in China for cooking oil and cosmetics. Due to its unique components, such as saponins, tea seed oil displays a number of remarkable antiseptic, fungicide, bactericide and lipid-lowering effects. Two subfractions separated from methanol extract of tea seed oil (METSO) contributed the most significant activity. METSO was isolated from UV absorption and characterized by MS, IR, 1 H NMR and 13 C NMR techniques. Two compounds were isolated from METSO and identified as sesamin and a novel compound: 2,5-Bis-benzo[1,3]dioxol-5-yl-tetrahydro-furo[3,4-d][1,3]dioxine
(compound B). Sesamin and compound B decreased H
2
O
2
-mediated ROS production in RBCs, inhibited RBCs hemolysis induced by AAPH, and increased the lag time of conjugated diene formation in human LDL. The results indicated that both compounds exhibited remarkable antioxidant activity. Antioxidant properties of METSO might be due to both compounds contained in it. Apart from the traditional pharmacological effects of
Camellia oleifera
, the oil of tea seed may also act as a prophylactic agent to prevent free radical related diseases.
1

Introduction
The oil seeds of cotton, mustard, linseed, borage, Evening Primose, etc are usually extracted as various antioxidant factors in food. The natural chemically active components are different from kinds of seeds. Lots of evidence from the epidemiological and clinical research indicated that uptake of the vegetative foods can lower down the probability causing cardiovascular disease. Those non-nutritive constituents from plants play roles in physiological activity are generally called phytochemicals.
Tea seeds oil (
Camellia oleifera
Abel.) are main edible oil from woody plants in
China (Yu et al., 1999). It was traditionally applied as a medicine of the stomachache and burning injury in China. Huang et al. (2002) had stated that it contains five-ring triterpenes that can suppress the microbial growth. Chen et al. (1998) also stated that its saponin could lower down the cholesterols, triglycerides and low density-lipoprotein in the blood of the rats. Zhang and Zhou (1995) found that it have the antioxidation ability to reduce liver ROS in the mature rats. The fatty acids was mainly composed of oleic and linoleic acid, which are very similar to those in the olive oil (Liu et al., 1979, Shyu et al., 1990). Due to its unique components, such as saponins, tea seed oil displays a number of remarkable antiseptic, fungicide, bactericide and lipid-lowering effects. Our previous study showed that methanol extract of tea seed oil (METSO) exhibited antioxidant activity. Two subfractions separated from METSO contributed the most significant activity. However, very little is known about the antioxidant compounds in METSO and their antioxidant properties. This study is mainly aimed on the antioxidation of the non-nutritive constituents in tea seed oil. To isolate and identify the bioactive compounds of
METSO and to characterize the antioxidant properties of these compounds and their effect on health.
Materials and Methods
2.1. Materials and chemicals
The sample of tea seed (
Camellia oleifera
Able.) was supplied from the Hsin-I country farmer’s association (Nantou, Taiwan).
The tea seed was sealed in a plastic bag, and stored at -20 o C until used. Methanol (HPLC grade) was obtained from Tedia
Co. (Fairfield, OH, USA). Copper sulfate pentahydrate (CuSO
4
-5H
2
O), Sodium dihydrogen-phosphate, disodium hydrogen phosphate, 2,
2’-azobis-(2-amidinopropane)-dihydropropane (AAPH), potassium bromide and
2

ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) were obtained from
Wako Pure Chemical Co. (Osaka, Japan). Sodium bromide and sodium chloride were obtained from Merck Co. (Darmstadt, Germany). 2’-7’-Dichlorofluorescin diacetate
(DCFH-DA) was obtained from Sigma Co (Missouri, USA).
2.2. Extraction
Tea seeds was roasted at 120 o C for 20 minute and then pressed by twin screw extruder to obtain the tea seed oil. Tea seed oil (100g) was extracted with 200 mL of methanol for 1 hour in a shaking incubator at room temperature. The extracts were filtered, and the residue was re-extracted under the same conditions. The combined filtrates were evaporated under vacuum below 40 o C using a rotary evaporator to a final volume of 5 mL.
2.3. Isolation of antioxidative substances
The residue was submitted separately to Kanto Chemical RP-18 column (250 × 20,
5 µ m), eluted with water-methanol gradient. For the gradient elution, the following programme was used: (A) H
2
O (B) MeOH, isocratic at 75% A for 20 min, then 50% A in 40 min; then 10% A in 60 min; then 0% A in 65 min, and isocratic for 20 min, followed by 5 min isocratic wash at 75% A.
2.4. Spectrometry
The UV-vis absorption spectra of the active components in methanol were recorded on a Hitachi U-3000 spectrophotometer (Tokyo, Japan). The mass spectra of the components were obtained using the EI-MS mode on a JEOLDMX 300 mass spectrometer (JEOL Co.,Tokyo, Japan). The nuclear magnetic resonance (NMR) spectra were measured in CDCl
3
and methanol-d4 with a Brücker AC-250 NMR spectrometer (Varian Inc., Walnut Creek, CA, USA) operating at 600 MHz for 1 H
NMRand 75 MHz for 13 C NMR with complete proton decoupling. The sweep width, pulse angle, repetition delay and acquisition time for 1 H NMR were 4500.0 Hz, 7.0 ms, 0 and 2.0, respectively, and for 13 C NMR were 25,000.0 Hz, 7.0 ms, 2.0 and 1.0 s, respectively.
The chemical shifts are reported in parts per million (ppm) from tetramethylsilane.
The Infrared spectra were measured with Bruker Equinox 55 s. Melting points were determined on a Büchi 535 apparatus.
3

2.5. Lipoproteins perparation
Fasting plasma, for LDL isolation, was collected from normal human volunteers in tubes containing ethylene diamine tetraacetic acid (EDTA; 1 mg/mL). LDL (d
1.019
– 1.063 g/mL) was isolated by sequential ultracentrifugation using a Hitachi ultracentrifuge (Himac CS 120GX, Hitachi, Tokyo, Japan) as described by Yamanaka et al. (1997) with a minor modification. LDL solution was freshed with N
2
, stored at 4 o C, and used within 1 week after preparation. Protein was measured using a Bio-Red kit, with bovine serum albumin as a standard. For oxidation experiments, LDL was dialyzed three times against 1L (1000-fold volume) of phosphate buffered saline (PBS, containing 0.01 M phosphate-buffer and 0.15 M NaCl, pH 7.4) in the dark at 4 o C for
24 h.
2.6. Oxidation of LDL
Dialyzed LDL (100 mg protein/mL) was diluted in 10 mM PBS and incubated at
37 o C in the presence or absence of 10 mM CuSO
4
. Oxidation was performed with or without the compound of tea seed oil. After incubation, lipid peroxidation of the LDL was measured as described below.
2.7. Detection of conjugated dienes
Conjugated diene formation was measured by determining the absorbance increase at 232 nm of the solution of LDL (100 mg protein/mL) in PBS incubated with 10 mM CuSO
4
in the absence or presence of compounds of tea seed oil. The absorbance was measured every 5 min for 250 min using a Hitachi U-3000 UV-VIS spectrophotometer, and the results were expressed as relative absorbance at 232 nm.
The duration of the lag phase was calculated by extrapolating from the propagation phase.
2.8. Erythrocyte Hemolysis
The method described by Zhu et al. (2002). Blood was obtained from healthy volunteers and collected into heparinized tubes. Erythrocytes were separated from plasma and the buffy coat and washed three times with 5 volumes of phosphate buffered saline, pH 7.4. During each wash, the erythrocytes were centrifuged at 3000 g for 10 min to obtain a packed cell preparation. After the last wash, the packed erythrocytes were suspended in 4 volumes of PBS solution. Erythrocyte oxidative hemolysis was induced by 2, 2’-azobis-(2-amidinopropane)-dihydropropane (AAPH), a peroxyl radical initiator. Addition of AAPH to the suspension of washed erythrocytes induces the oxidation of membrane lipids and proteins, resulting in hemolysis. Two mL of the erythrocyte suspension was mixed with 2 mL of PBS solution containing different concentrations of compounds in tea seed oil. Two mL of
200 mM AAPH in PBS was then added to the mixture. The reaction mixture was shaken gently while being incubated at 37 ° C for 3 h. After incubation, the reaction mixture was removed, diluted with 8 volumes of PBS and centrifuged at 3000 g for 5
4

min. The absorbance (A) of the supernatant fraction at 540 nm was recorded in a
Hitachi U-3000 spectrophotometer. Percent inhibition was calculated by equation
(Zou et al., 2001).
2.9. Detection of intracellular ROS
ROS was measured by a method previously described rewire modification (Bass et al., 1983). RBCs were incubated in culture medium containing DCFH-DA
(2’,7’-dichlorofluorescin diacetate) of 30 mM for 1 hour to form a stable intracellular probe (Royall and Ischiropoulos, 1993). The same concentration of DCFH-DA was continuously present during experiments. After that, the cells were washed with PBS, then removed for the 2’,7’-dichlorofluoresincein (DCF) fluorescence intensity.. The
DCF fluorescence intensity of the cells is an index of intracellular levels of ROS and determined by flow cytometry.
2.10. Flow cytometry
RBC were analyzed by a Fluorescence Activated Cell Sorter (FACS-calibur,
Becton- Dickinson, Immunofluorometry systems, Mountain View, CA). RBC were passed at a rate of about 1,000 per second, using saline as the sheath fluid. A 488 nm argon laser beam was used for excitation. A two-parameter dot-plot of the side light scatter (SSC) and forward light scatter (FSC) of the population was first analysed.
Green fluorescence of 10,000 gated RBC was then measured using linear amplification. The arithmetic Mean Fluorescence Channel (MFC) was derived by the
CellQuestR software.
2.11. Statistical analysis
Data in the figures are given as mean ± SEM. Statistical significance was examined through one-way analysis of variance and Dunnett's multiple range test.
Significant differences were accepted at p<0.05.
Results and Discussion
I.
The Extraction and Purification of Antioxidant Compounds in Tea Seed Oils
Two purified crystal phytochemicals in lignans were found by using reverse-phase column chromatography in methanol extracts of tea seed oils. Firstly the HPLC chart was shown in Fig. 2. Their peaks appeared at retention time 66 and
69 min. They seemed to be two pure compounds by using photo diode array
5

scanning during 200 to 610 nm. Therefore, they were separated and collected by preparative HPLC to obtain 2 colorless crystals. The first fraction was identified by comparison of its NMR and mass spectra; Colorless needles, mp 128-130 o C. 1 H
NMR analysis of compound A, δ 6.78 (1Η, d, J=7.8Hz
), 6.84 (1Η, dd, J=1.8 &
7.8Hz
) and 6.88 (1Η, d, J=1.8Hz
); δ 3.09(m), 3.84(dd, J=4.0 & 9.0Hz), 4.22(m) and
4.07(d, J=4.0Hz) 。
13 C NMR: 102.4, 72.6. NMR data: see the literature (Pelter et al.
,
1982). IR (CHCl
3
) ν : 1493.9, 1243.9, 1038.9 and 927.8 cm -1 ; EI-MS
m/z
: 354.4. The secondary fraction was identified by comparison of its NMR and mass spectra;
Colorless needles, mp 128-130 o C. 1 H NMR analysis of compound B, δ 6.51 (1Η, dd,
J=2.4 & 8.4Hz
), 6.62(1Η, dd, J=2.4Hz
), 6.70(1Η, d, J=8.4Hz
); δ 6.79(1Η, d,
J=7.8Hz
), 6.86(1Η, dd, J=1.2 & 7.8Hz
), 6.90(1Η, d,
J=1.2Hz
); δ 2.95(1Η, m ), 3.62(1Η, dd, J=7.8 & 9.0Hz
), 4.408(1Η, d,
J=9.0Hz
); δ 3.30(1Η, m ) 、 3.98(1Η, d, J=9.0Hz
) 及 4.10(1Η, dd, J=6.6 & 9.0Hz
). 13 C
NMR: 70.8, 72.25. IR (CHCl
3
) ν : 1486.0, 1245.6, 1038.1, 927.6 cm -1
370.3, molecular formula was C
20
H
18
O
7
.
; EI-MS m/z :
Through mass, IR and NMR chromatogram identification, they were sesamin and another newly found pure compound, B. The compound B is similar to sesamin in structure, only different in 2 unsymmetrical penta-rings at central skeleton.
The sesamin, and sesamolin, are 2 major components of the sesame oil lignans.
Nakai et al. (2003) noted that the lignans were highly correlated to the antioxidation of sesame. Both in vitro and in vivo tests showed sesamin could protect oxidation, lower cholesterlos and improve lipid metabolism (Hirose et al., 1991 and Hirata et al.,
1996). On the other hand, Miyahara et al. (2000) found that the sesamin in the unbaked sesame could suppress the growth of lymphoid leukemia Molt 4B cell and induce the apoptosis.
Whether dietary supplementation with sesamin augments , these changes were associated with significant reductions in plasma prostaglandin E2 concentrations in animals fed safflower oil with sesamin compared with those fed safflower oil
(Utsunomiya et al. 2000). High amounts of both sesamin and sesamolin have been identified in sesame (Sirato-Yasumoto et al. 2001), and them have antioxidant and health promoting activities. Dietary sesame seed elevated α -tocopherol concentrations in liver, kidney, brain and serum, and decreased urinary excretion of tocopherol metabolism product (Ikeda et al. 2002).
According to the chemical structure, and the phytochemicals, the new compound B is one of the lignans. The sesamin, sesamol and sesaminol are all of the antioxidants. Among them, sesamin is the least effective antioxidant, but can be turned into more antioxidative derivatives. It is well known that liver play a very important role in metabolism, related to all kinds saccharides, lipids and proteins.
The acetic acid and ethaldehyde injury through the liver metabolism of the ethanol can be avoided by providing the sesamin to the small rat, and sesamin can keep the
GOT and GPT constant (Nakai et al., 2003).
6

II The Inhibition of the Hemolysis of the Sesamin and Compound B
The red blood cells (RBCs) act as an oxygen carrier. They are always exposed in high oxygen tension, therefore are easily causing oxidation injury by such as superoxides, H
2
O
2
, and hydroxyl free radicals in metabolites. Many enzymic and non-enzymic intracellular antioxidation would happen. The non-enzymic protection might be due to vitamin E and the other anioxidants in cell membrane (Kitagawa et al.,2004). Mammalian RBCs are often used as a model research to test the effect of antioxidant. The human hemolysis was also used in this study to check the antioxidation effect of tea seed oils. Fig. 2, showed the hemolysis effect of sesamin and compound B induced by AAPH under 3 different concentrations. From Fig. 3, inhibition effect seemed not in accordance with the increase of the sesamin concentration in the testing range. On the other hand, inhibition of compound B would significantly increase (p<0.05) with the increases of concentration from 135 to
270 µ M. The biomembrance was most vulnerable to free-radical attack due to its content of polyunsaturated fatty acid. Heomlysis of RBC incubated by AAPH was associated with fatty acid that EPA and DHA decrease (Zhang et al. 1997). Results also indicated that sesamin and compound B were able to suppress the RBCs’ oxidation injury by free radicals, up to 49 and 39-48%, respectively. Manna et al
(1999) reported that in olive oil, both in vivo and in vitro, not only the triglycerides but also the phenolic compound could suppress the hemolysis caused by the oxidative stress. When the cell was attacked by free radicals, the polyunsaturated fatty acids in the cell membrane would be oxidizied (Kitagawa et al., 2004). The membrane composed of the lower saturated fatty acids would result in better fluidity, but the peroxidative metabolites MDA would lead to the cross linking of protein and reduce the fluid function (Patel and Block, 1988). On the other hand, this membrane would cause the malfunction of protein, change the ion permeability, induce the cell toxic substance, reduce the enzymic antioxidativity, and inhibit the regeneration of GSH
(Tesoriere et al., 2002).Free radical could induce polyunstat membrance their inhibitory ability on membrane lipid peroxidation and free radical formation or due to their free radical scavenging ability (Liu et al., 2004).Lipophilic polyphenols which easily penetrate the cytoplasm of erythrocytes, seem to react with hemoglobin
(Kitagawa et al. 2004), and sesame seeds could increase plasma γ -tocopherol and alter plasma tocopherol ratios in humans and enhanced vitamin E bioactivity (Cooney et al.,
2001). Sesamin and compound B was isolation from oil, supposed could bind at cell membrane and protect damage from free radical attack.
7

III The Inhibition of the Active Oxygen Production in Intracellular RBCs by
Sesamin and Compound B
Many diseases are highly correlated to free radicals of active oxygen that cause oxidation of lipids. There are many, mechanisms to avoid the injury of active oxygen. Since the antioxidative enzymes on health are concerned, therefore, the antioxidative foods are accounted as important roles in the body. Chang et al. (1995) found that by feeding the tea seed oil to Wister rat, the probability of arteriosclerosis decreased, because the superoxide dimutase (SOD) activity of antioxidative enzymes increased, the malondialdehyde (MDA) of serum and liver decreased. The multi-unsaturated fatty acids will inhibit the activity of glucose 6 phosphorate dehydrogenase (G-6-PDH), affected the content of GSH in the rat. The coenzyme
NADPH of GRd would not regenerate when enzyme activity decreased. Then the rat can not proceed the normal antioxidation function. Tesoriere et al. (2002) stated that MDA would suppress the G-6-PDH in the RBCs to increase the met-haemoglobin which could not carry oxygen. The MDA also decreased catalase activity, induce cell membrane lipid to be oxidized into conjugated diene, and consumed the vitamin
E in membrane, eventually, affected the RBCs’ physiology. oxidation of lipids
The fluorescence of dichlorofluorescein (DEC) was widely used to detect the reactive oxygen species (ROS) in cells. By using DCFH-DA as fluorescent dye to detect inhibition the H
2
O
2
-induced ROS in cell after applying the tea seed oil was shown in the Fig. 4. Among the testing range 0.23 to 0.54
µ m, the inhibition of sesamin decreased with the increase of the concentration, but not significantly. On the contrast, effect of compound B increase with the increase of concentration, from
14 to 86%. Apparently, the effect of compound B is much higher than that of sesamin, even up to 16 times at 0.54 µ m.
The sesamin was proved that could reduce the injuries of the PC12 cell. The reason might be the ROS production decrease due to the inhibition of mitochondrion activated protein kinases (Hou et al., 2003). Lack of the antioxidants will result in the concentration increase of free radical, such as ROS. Concurrently, the cell will be dead because of the damage of DNA, protein and membrane, etc. The cell injury caused by H
2
O
2
is highly correlated to ROS. The compound B can suppress the
ROS production more effectively than that of the sesamin. It is thought that compound B could reduce the decrease potential of the oxidation injury in the aerobic metabolism.
8

IV The Suppression Effect on LDL Oxidation by the Sesamin and Compound B
It is generally accepted that higher intake of antioxidants can protect against oxidative stress. A vegetarian diet results in higher intake of vitamins and micronutrients which, although providing antioxidant defence, might lead to deficiency of other micronutrients involved in DNA metabolism and stability (Kažimírová et al. 2004).
Oxidative stress such as DNA damage, oxidation of lipids, proteins and sugars could bring the cellular and sub-cellular changes which may be associated with chronic degenerative diseases such as cancer, cardiovascular diseases, cataracts and others, either as cause or consequence.
Studies on the antioxidants for inhibiting LDL oxidation have received more attention recently. Many evidences indicated that the oxidized LDL would cause arteriosclerosis (Esterbauer et al.; 1991, Rota et al.; 1998; Young and McEneny, 2001).
The Fig. 5 showed that the functional groups of tea seed oil slowed down the formation of LDL conjugated diene under the induction of copper ion. The experiments were performed under 13 and 26 µ m compared with the blank, respectively. Results indicated that addition of either sesamin or compound B could retard the LDL oxidation. The oxidation would be delayed from 38 to 51-89 min.
On the other hand, no conjugated diene increase was found in the trials in case no copper ion added. In this test, the compound B was obviously more effective than that of the sesamin. The time delay compound B trials were 9 and 27 min more than those of the sesamin at concentration 13 and 26 µ m, respectively.
It is well known that fatty acids and antioxidant can retard the LDL oxidation.
Epidemiological and clinical research indicated that less arteriosclerosis and coronary artery disease happened around Mediterranean was due to the antioxidant rich vegetables, beans, cereals, and the mono unsaturated fatty acid rich olive oil
(Serra-Majem et al., 1995). The sesame that contains sesamin is accounted as healthy food in the oriental countries, especially in protection of arteriosclerosis, hypertension and aging. Besides, parts of lignans can be metabolized as enzyme skeletons of bacterium p450 in mammalian intestine. Those enzymes can suppress the female hormones, preventing the woman’s breast cancer and man’s prostate cancer
(Niemeyer and Metzler, 2002; Wang, 2002).
Recently, epidemiological and biochemical survey indicated that antioxidants of vitamins, as well as non-vitamins such as polyphenols are playing roles at human health. Gimeno et al. (2002) declared that in short-term test, the olive oil can increase the contents of oleic acids, vitamin E and phenolic compounds, and enhance the antioxidation ability of a healthy person. Apparently the blood lipid ratio and
LDL fatty acid composition were quickly affected by the food. Yamashita et al.
9

(2002) found that in the rat test, the sesame lignans could increase the tocotrienol and tocopherol in adult rat liver, kidney and blood plasma. Tocopherol has been proved highly correlated to arteriosclerosis. The LDL fatty acid compositions in blood would be changed if the multi-unsaturated fatty acids were substituted by the mono-ones, and would protect the oxidation (Hargrove et at. 2001). The tea seed oil also could reduce the total cholesterols, especially the low density ones, in the rat blood because its fatty acids are mostly monounsaturated (Chen et al., 1996). In comparison with the highly saturated lipid, the fatty acid composition apparently would affect the rat lipid pattern and distribution. In the rat test, tea seed oil could significantly lower the contents of the total cholesterols triglycerides and the LDL cholesterols. Meantime, it could reduce both the mobile of membranes and deformation of RBCs, concurrently, the possibility of arteriosclerosis. It also could enhance the cell membrane fluidity and retard the peroxidation of blood lipids (Sun and Zhang, 1994). The animal test indicated that fatty acid composition definitely could affect the physiological mode of action, but whether only due to the chemical structure was not identified yet. Owen et al. (2000) also have proved that the mono-unsaturated fatty acids in olive oil could stabilize the liver and kidney lipids, free from oxygen attack and reduce the artery plaques. But on the other hand, they also found that the non-nutritive constituents in olive oil contributed the effect of disease protection in animal, also in human body. Sesamin is highly related to the
LDL tocopherol and oxidation under copper ion (Hirata et al., 1996), but whether the key components can enter the blood after digestion is remained unelucidated.
V. The Suppression of Genotoxicity Injury in Human Lymphocyte Cell by Sesamin and Compound B
Chromosome aberrations are a marker validated in studies as predictive of cancer risk (Griffths et al. 2002). The comet analysis, single cell gel electrophoresis, is used to detect and analyze the mammalian single cell DNA break, in order to assess the toxic effect on gene by the chemicals, or the effect of detoxication by phytochemicals. The DNA chain will become comet-like after electrophoresis if harmed. The genotoxicity was judged by measuring the length of the comet tail.
Researchers have found that H
2
O
2
induces oxidative DNA damage in mammalian cells ( Collin et al. 1997 ), and H
2
O
2
easily goes through the cell membrane into a cell.
If phytichemicals can retard the H
2
O
2
-induced DNA injury, indicate that they can protect the free radical attack on DNA. Fig. 6 showed both sesamin and compound B were not toxic to cell even under highest dosage (1000 µ M). The oxidative DNA damage of lymphocytes induced by H
2
O
2
was effectively suppressed when
10

compounds from tea seed oil were added. No inhibitory effect was found in sesamin trials but the effect increased with the dosage increase. The inhibitory effect at 1000
µ M is significantly higher than that at 500 µ M (p<0.05). Through the clinical
14-week survey of kidney dialysis, Kan et al. (2002) found that the vitamin E could reduce the damage of lymphocyte DNA at comet analysis. Moller and Loft (2002) also found vitamin C, E and carotenoid could reduce the oxidation injury during several hours in human body. Sesamin can form 4 different compounds after oral administration and through rat metabolism. The metabolites in liver owned the antioxidation ability. The main physiological mechanism is the hydroxyphenyl beta-glucuronidation . But sesamin itself has no antioxidative property because of no OH-group in structure, and it was used as an antioxidant prodrug possibly due to the function of its metabolites in the body (Nakai et al., 2003). Besides, sesamin could elevate the gamma-tocopherol concentration due to the inhibition of cytochrome P450 (CYP3A)-dependent metabolism of gamma-tocopherol in rat test
(Ikeda et al., 2002). A serum if in vivo test will be performed to reveal whether sesamin can have antioxidation effect by its derivatives through animal metabolism.
The lower level of oxidative DNA damage in compound B suggests that may have the greatest degree of protection against oxidative stress, although this is not reflected in their sensitivity to H
2
O
2
- induced damage.
H
2
O
2
The inhibitory effect of compound B on oxidative DNA damage induced by
in lymphocytes was more significant (P<0.05) than that of sesamin. The lignans compounds of tea seed oil have been found to offer effective protection.
Epidemiological study show that vegetables and fruits can effectively inhibit epithelial carcinoma (Tavani and Vecchia, 1995). Some antioxidants such as quercetin, caffeic acid in dietary antioxidants using comet assay for DNA damage showed efficacious protection (Szeto and Benzie, 2002). A significant positive correlation between age and oxidative DNA damage was found in non-vegetarians, vegetarian diet can lead to a slight decrease in oxidative DNA damage in lymphocytes
(Kažimírová et al. 2004).
Conclusion
There are two major constituents of lignans, sesamin and compound B, in tea seed oil. By using in vitro
experiment, this study proved that they could reduce the oxidation injury of DNA caused by the elevation of environmental oxidation stress.
Instead of the former elucidated investigations concerning about fatty acids, lignans were proved able to protect the coronary heart disease in this study. The antioxidation ability in the tea seed oil was known not only due to the unsaturated fatty acid but also the lignans. Apart from the traditional pharmacological effects of
Camellia oleifera
, the oil of tea seed may also act as a prophylactic agent to prevent
11

free radical related diseases.
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Figure 1. HPLC chromatogram of methanol extract from tea seed oils.
17

60
50
40
30
20
10
0
0.13 0.27 0.54
Fig 2
. Inhibitory Effect (%) of sesamin and compound B from tea seed oil on
AAPH-Induced Hemolysis of human Erythrocytes in Vitro.
used as a positive control. Data are expressed as mean ± SD of n
=3 samples.
18

100
80
60
40
20 sesamin compound B
0
0.13 0.27 0.54
Fig 3 . Inhibitory Effect (%) of sesamin and compound B from tea seed oil on
H
2
O
2
-stimulated ROS of human Erythrocytes. Data are expressed as mean of n
= 10000 samples.
19

1.0
0.8
0.6
LDL control
Cu
2+ sesamin(13.3
µ
M) sesamin (26.6
µ
M)
B (13.3
µ
M)
B (26.6
µ
M)
0.4
0.2
0.0
0 50 100 150 200
Fig 4
. Effect of compounds from tea seed oils on Cu 2+ mediated conjugated diene formation in LDL. LDL (100 mg protein/ml) was incubated with 10 µ M CuSO at 37 ℃ in the absence or presence of compounds of tea seed oil. Conjugated
4 diene formation was measured by determining the absorbance at 234 nm every
5 min for 250 min and the results are expressed as relative absorbance at 234 nm.
20

60
50
40
30
20
10 sesamin compound B
0
10 500 1000
Fig 5.
Inhibitory effect of compounds from tea seed oils on the genotoxicity of H
2
O
2 toward lymphocyte cells. Results are mean ± SD for n = 3.
21