Oriental Foods and Herbs - American Chemical Society

Chapter 14

Antioxidant Properties of Hsian-tsao (Mesona procumbens Hemsl.) Gow-Chin Yen, Chien-Ya Hung, and Yen-Ju Chen

Downloaded by UNIV OF BATH on July 4, 2016 | http://pubs.acs.org Publication Date: August 5, 2003 | doi: 10.1021/bk-2003-0859.ch014

Department of Food Science, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, Republic of China

The reactive oxygen species scavenging activity of Hsian-tsao (Mesona procumbens Hemsl.) and its protective effect against oxidative damage in Chang liver cells were investigated. Water extracts of Hsian-tsao (WEHT) had antioxidant activity in linoleic acid peroxidation system. WEHT exhibited a positive concentration-dependent scavenging effect on DPPH radical, superoxide anion, hydrogen peroxide, nitric oxide and peroxyl radical. WEHT inhibited lipid peroxidation in Chang liver cells induced by hydrogen peroxide in a concentration -dependent manner. WEHT could inhibit 89% of oxidative DNA damage induced by hydrogen peroxide in Chang liver cells, under a concentration of 100 μg/mL, and the phenolic compounds of Hsian-tsao had a 18-41% inhibitory effect. Seven phenolic compounds (kaempferol, apigenin, caffeic acid, protocatechuic acid, syringic acid, vanillic acid and p -hydrobenzoic acid) were isolated and identified from the extracts of Hsian-tsao. Caffeic acid, kaempferol and protocatechuic acid had marked inhibitory effects on lipid peroxidation in Chang liver cells induced by hydrogen peroxide. The water extracts of Hsian-tsao reduced, by 78%, intracellular reactive oxygen species in Chang liver cells at a concentration of 100 μg/mL, and these species were decreased by 31 and 52% by 10 μΜ caffeic acid and kaempferol, respectivity. Thus, caffeic acid and kaempferol, which had the highest contents of phenolic compounds in the extracts of Hsian-tsao, could be the active components responsible for the antioxidant activity of Hsian-tsao.

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© 2003 American Chemical Society Ho et al.; Oriental Foods and Herbs ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Introduction Reactive oxygen species (ROS), such as superoxide anion radical (0 * ~), singlet oxygen ( 0 ), hydroxyl radical (*OH), peroxyl radical (ROO ), hydrogen peroxide, and nitric oxide (NO*), play important roles in certain clinical diseases and the process of aging (/). They are generated by normal metabolisms or induced by environmental pollution and living styles (2). The generation of ROS and the antioxidant defense systems are normally approximately in balance, but when this balance is tipped, oxidative stress occurs and disease results. The accumulation of excess ROS can cause damage in DNA, lipids, and proteins, and can cause the function of cell metabolism to be lost. An important etiological mechanism of these diseases may be a causal relationship between the presence of oxidants and the generation of lipid hydroperoxides. Recent epidemiological studies indicated that oxidative stress can be partially decreased by increasing the dietary intake of natural antioxidants (3,4). Therefore, it is desirable to find safer, more efficient and more economical dietary antioxidants which can diminish ROS and may prevent aging and related diseases (5). The herb Mesona procumbens Hemsl., called Hsian-tsao in China, is consumed in an herbal drink and jello-type dessert in the Orient. It is also used as an herbal remedy in Chinese folk medicine and is effective against heat-shock, hypertension, diabetes, hepatitis, muscle pain and joint pain. Most studies on this product have focused on its gelation property and proximate composition. In Japan, Hsian-tsao is an ingredient in an SOD (superoxide dismutase) beverage. It has been reported that the herb contains hypoglycemic and hypertension substances, including β-sitosterol, stigmasterol, a - and β-amyrin, oleanolic acid, maslinic and β-sitosterol glycoside (6). Hung and Yen (7) reported that WEHT have an antioxidant activity equal to that of trolox and butylated hydroxyanisole (BHA). Yen et al. (8) indicated that WEHT could reduce UV-C and H 0 induced DNA damage, and that the protective effect might be due to the fact that it contains polyphenol compounds and/or other active components. Recently, Hung and Yen (9) isolated and identified seven phenolic compounds (kaempferol, apigenin, caffeic acid, protocatechuic acid, syringic acid, vanillic acid and p-hydrobenzoic acid) from the extracts of Hsian-tsao. Although Hsiantsao is used as an herbal remedy for hepatitis in Chinese folk medicine, scientific data concerning the protective effect of Hsian-tsao on liver cells against oxidative damage and the contribution of its active compounds are not available. The objectives of this study were to investigate the ROS scavenging activity of 2

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Ho et al.; Oriental Foods and Herbs ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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204 WEHT and to evaluate the protective effect of WEHT and its active phenolic compounds on oxidative damage in Chang liver cells.

Materials and Methods


Reagents and Cell Culture Sun-dried Hsian-tsao (Mesona procumbens Hemsl.) (harvested in 1995) was obtained from a local market in Hua-lian county, Taiwan. The Chang liver cell line (CCRC 60024) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Protocatechuic acid, /?-hydroxybenzoic acid, caffeic acid, vanillic acid, syringic acid, kaempferol and apigenin were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals and cultural medium were of reagent grade.

Preparation of Water Extracts from Hsian-tsao (WEHT) The dried Hsian-tsao was cut into small pieces and ground into a fine power using a mill (RT-08, Rong Tsong, Taichung, Taiwan). The powder was passed through an 80-mesh sieve, collected and sealed in a polyethylene plastic bag, and then stored at 0-4° C for further use. Hsian-tsao (20 g) was extracted with boiling water (400 mL) for 30 min. The extract was filtered through Whatman No. 1filterpaper, and the filtrate was freeze-dried. The extract was dissolved in sterilized water and phenolic compounds standard in 0.1% DMSO/sterilized water for further study.

Determination of Antioxidative Activity The antioxidative activity of Hsian-tsao extracts was determined using the thiocyanate method (10). The percentage of inhibition of peroxidation (%) was calculated as follows: inhibition of peroxidation (%) (capacity to inhibit peroxide formation in linoleic acid at 72 h) = [1 - (A pi 5oonm / A oonm)] *100. sam

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Scavenging Effects on DPPH Radical, Superoxide Anion, Hydrogen Peroxide and Nitric Oxide The effect of WEHT on DPPH radical was estimated according to the method of Shimada et at. (//) by measuring spectrophotometrically at 517 nm. The influence of WEHT on the generation of superoxide in a nonenzymic system was measured by means of spectrophotometric measurement of the product on reduction of nitro blue tetrazolium (12). The method used to detect hydrogen peroxide was based on the peroxidase assay system. Horseradish peroxidase (HRPase) was used, which reacts with hydrogen peroxide to oxidize phenol red into a purplish-red product, as described by RJiinkus and Taylor (13). The method of Marcocciet et al. (14) was used to determine the scavenging effect on nitrite oxide. The reaction mixture was mixed with sulfanilamide and iV-(lnaphthyl)-ethylenediamine dihydrochloride, and the absorbance was measured at 570 nm.

Cell Viability Cell viability was determined on the basis of mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan.

Determination of Lipid Peroxidation in Ceils The lipid peroxidation was determined by measuring thiobarbituric acidreactive substances (TBARS) using a modified method of Ramanathan et al. (15).

Alkaline Single-Cell Gel Electrophoresis (Comet Assay) Comet assay was performed under alkaline conditions following to the methods of Yen et al. (8). The slides were observed using a fluorescent microscope attached to a CCD camera connected to a personal computer based image analysis system (Komet 3.0; Kinetic Imaging Ltd.) For each analysis, 50 individual cells were calculated, and in most cases three separate experiments were conducted for each series. Single cells were analyzed under the fluorescent microscope. The DNA damage was expressed as % Tail DNA = [Tail DNA/(Head DNA + Tail DNA) χ 100. A higher % Tail DNA meant a higher level of DNA damage.


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Reactive Oxygen Species (ROS) Levels Intracellular ROS was measured using afluorescentprobe, DCFH-DA (16). A reaction mixture containing 0.889 mL Chang liver cells (1 xlO cells/mL) and 100 water extracts of Hsian-tsao (final concentrations 10, 50 and 100 μg) or phenolic compounds (final concentration 10 μΜ) was incubated in 24 wells for 20 h. Then, 10 μΐ, hydrogen peroxide (10 mM) was added to the cells simultaneously with 1 μΐ. DCFH-DA (final concentration 5 mM), and the cells were then incubated at 37° C. The DCFfluorescenceintensity was measured using a FLUO star-galaxy fluorescence plate reader (BMG Labtechnologies GmbH Inc., Offenburg, Germany) with the temperature maintained at 37° C. The excitation filter was set at 485 ± 10 nm, and the emission filter was set at 530 ± 12.5 nm. Data points were taken every 5 min for 30 min, and the data were exported to Excel (Microsoft, Seattle, WA) spreadsheet software for analysis.


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Results and Discussion

Antioxidant Activity of WEHT The antioxidant activity of WEHT increased significantly as the concentration increased from 1 to 50 μg/mL; then the antioxidant activity increased more slowly when the concentration exceeded 50 μg/mL. At 200 μg/mL, the capacity to inhibit the peroxidation of linoleic acid was 97%, which was equal to that of BHA (99%). In our previous experiment (17), polyphenols (185 mg/g WEHT), and a lower content of ascorbic acid, α-tocopherol, and βcarotene were determined in WEHT. Recently, Hung and Yen (9) isolated and identified seven phenolic compounds (kaempferol, apigenin, caffeic acid, protocatechuic acid, syringic acid, vanillic acid and p-hydrobenzoic acid) from the extracts of Hsian-tsao. High correlation (r = 0.92) was found between the antioxidant activity and the total polyphenolic content of WEHT. Therefore, polyphenols could be the major antioxidant components responsible for the total antioxidant activity of WEHT. 2


207 Scavenging Effects of WEHT on DPPH Radical, Superoxide Anion, Hydrogen Peroxide and Nitric Oxide Figure 1 shows the scavenging effects of WEHT on DPPH radical, superoxide anion, hydrogen peroxide and nitric oxide. The scavenging activity of WEHT on DPPH radical increased as the concentration of WEHT increased and when IC was 18.7 μg/mL. The correlation coefficients between DPPH radical scavenging activity and antioxidant activity and the content of total polyphenols in WEHT were r = 0.89 and r = 0.83, respectively. The scavenging effect of WEHT on superoxide anion radical increased linearly from 54 to 90% as the concentration increased from 25 to 125 μg/mL when the IC was 16.4 μg/mL. The correlation coefficients between superoxide anion scavenging activity and antioxidant activity and the content of total polyphenols in WEHT were r = 0.90 and r = 0.96, respectively. WEHT showed 71% scavenging ability on hydrogen peroxide at a concentration of 400 50

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Concentration fag/mL) Figure 1. Scavenging effect of water extract from Hsian-tsao on DPPH radical (A), superoxide anion radical (B), hydroxygen peroxide (C) and nitric oxide (D) with different concentration. Each value is meanistandard derivation of three replicate analyses.


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μg/mL with the IC at 227 μ^Γηί. The correlation coefficient between the scavenging activity of hydrogen peroxide and antioxidant activity was r = 0.60. The scavenging ability of WEHT on nitric oxide was 61 and 85% when the concentration was 50 and 200 μg/mL, respectively. IC was 35 μg/mL, indicating that WEHT was a good scavenger on nitric oxide. The correlation coefficients in our study between nitric oxide scavenging activity, and antioxidant activity and the content of total polyphenols were r = 0.82 and r = 0.87, respectively. 50

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Cytotoxicity of WEHT and Isolated Phenolic Compounds in Chang Liver Cells The cytotoxicity of WEHT in Chang liver cells was evaluated based on its effect on cell growth (MTT test). Cell viability was greater than 95% when WEHT (0-1000 μg/mL) was incubated with cells at 37° C for 24 h. In addition, no cytotoxicity was found when Chang liver cells were treated with isolated phenolic compounds of Hsian-tsao under a concentration range of 0-100 μΜ (data not shown).

Effects of WEHT and Isolated Phenolic Compounds on Hydrogen PeroxideInduced Lipid Peroxidation As shown in Table I, the inhibitory effect of WEHT on lipid peroxidation in Chang liver cells increased from 41 to 61% when the concentration increased from 100 mg/mL to 400 mg/mL. This result suggests that WEHT is a good antioxidant, possessing a protective effect that can reduce the oxidative damage on Chang liver cells induced by H 0 . Hung and Yen (9) isolated and identified seven phenolic compounds (kaempferol, apigenin, caffeic acid, protocatechuic acid, syringic acid, vanillic acid and p-hydrobenzoic acid) from the extracts of Hsian-tsao. Therefore, these phenolic compounds in WEHT might play important roles in modulating H 0 -induced lipid peroxidation in Chang liver cells. At a concentration of 10 μΜ, the inhibitory effects of caffeic acid, kaempferol and protocatechuic acid on lipid peroxidation in cells were 44, 31 and 22%, respectively, and the effects of the other compounds were less then 20% (Figure 2). The results indicated that WEHT and its phenolic compounds had protective effects inhibiting lipid peroxidation in cells induced by hydrogen peroxide. 2

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209 Table I. Effect of Water Extract of Hsian-tsao on H 0 -induced Production of Lipid Oxidation Formation in Chang Liver Cells 2

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Inhibition ofperoxidation TBARS (IP %f (nmol/mg protein)" 0 0 1.7710.46 10 16.4613.17" 1.65 ± 0.07 50 29.45 1 5.03 1.39 ± 0.10 100 41.09 + 2.97" 1.15 ±0.06 ^ 200 46.7514.34 0.95 1 0.08 300 50.361 1.28 0.88 ± 0.02 400 60.83 14.85" 0.70 1 0.09 Values are means±SD of triplicate determinations of malondialdehyde. The concentration of the lipid peroxides was calculated by using a molar absorption coefficient of 1.56 χ 10 M" cm" . Inhibition of peroxidation (IP %) = [l-(A i /A |)]xl00. The control containing no added sample represents 100 % lipid peroxidation. The hydrogen peroxide added concentration was 50 μΜ. Values in a column with the different superscripts are significantly different (p

Oriental Foods and Herbs - American Chemical Society

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