Botanical Studies (2012) 53: 55-66.
BIOCHEMISTRY
In vitro antioxidant properties and total phenolic contents of wetland medicinal plants in Taiwan
Yu-Ling HO1, Shyh-Shyun HUANG1, Jeng-Shyan DENG2, Yaw-Huei LIN3, Yuan-Shiun CHANG4, and Guan-Jhong HUANG4*
1Department of Nursing, Hung Kuang University, Sha Lu, Taichung 433, Taiwan
2Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan
3Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan
4School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan
(Received February 10, 2011; Accepted July 20, 2011)
ABSTRACT. The aim of this study was to examine the possible antioxidant activities of the methanol and water extracts of 31 medicinal wetland plants in Taiwan. We assayed for such properties such as: TEAC, DPPH radical scavenging, total polyphenol content, total flavonoid and total flavonol contents using the reduc­ing power method. Our results showed that Rotala rotundifolia, Juncus effusus var. decipiens, Cypems iria, Salix warburgii, Lindernia antipoda, Kyllinga brevifolia, and Typha orientalis possessed both high antioxidant activities and high total polyphenol contents. There was a low correlation between TEAC and total polyphenol content (water extracts, R2=0.14; methanol extracts, R2=0.23) thus eliminating high phenolic content as an im­portant factor in determining the wetland plants' antioxidant capacities. Our results demonstrated that although phytochemicals in the wetland medicinal plants may contribute significantly to their antioxidant activities, these antioxidant activities were not directly related to the polyphenol quantity. Phytochemicals may play key roles in the potent antioxidant activity of wetland medicinal plants. The potential of these easily accessible sources of natural antioxidants should be explored by the pharmaceutical, medical, and health food industries.
Keywords: Antioxidant; Flavonoid; Flavonol; Polyphenol; Wetland medicinal plant.
INTRODUCTION
ach and sweet potato tuberous roots (Huang et al., 2004;
Huang et al., 2005).
It is commonly accepted that reactive oxygen species, such as superoxide (〇2 -), hydroxyl (OH'"), and peroxyl ( OOH, ROO ) radicals, are produced under oxidative stress. Reactive oxygen species play important roles in de­generative or pathological processes, such as aging (Burns et al., 2001), cancer, coronary heart disease, Alzheimer's disease (Diaz et al., 1997), neurodegenerative disorders, atherosclerosis, diabetes, and inflammation (Chen et al., 2006). Several anti-inflammatory, digestive, anti-necrotic, neuroprotective, and hepatoprotective drugs have recently been shown to have antioxidant and/or radical scavenging mechanisms as well (Lin and Huang, 2002). Some natural antioxidants and compounds with radical scavenging ac­tivity have been identified over the last few years, includ­ing echinacoside in Echinaceae root (Hu and Kitts, 2000), anthocyanin (Espin et al., 2000), phenolic compounds (Rice-Evans et al., 1997), and the extracts of water spin-
Medicinal plant parts are commonly rich in phenolic compounds, such as flavonoids, phenolic acids, stilbenes, tannins, coumarins, lignans and lignins. These compounds have multiple biological effects including antioxidant activity (Packer et al., 1999). In vitro experiments on an-tioxidant compounds in higher plants show how they pro­tect against oxidation damage by inhibiting or quenching free radicals and reactive oxygen species (Ali et al., 2008). The role of these compounds as potential antioxidants can be inferred by their similarity to synthetic antioxidants of related structures.
The multifarious natural environment of Taiwan harbors abundant plant resources. Many of these plants, includ­ing those with therapeutic potential, face endangerment. Therefore, we investigated wetland medicinal plants and analyzed their antioxidant activities. In the present study, we collected 31 medicinal wetland plant species that are widely consumed in Taiwan, prepared their water and methanolic extracts, and analyzed their antioxidant activi­ties and polyphenol contents.
*Corresponding author: E-mail: gjhuang@mail.cmu.edu.tw; Tel.: +886-4-22053366 ext. 5508; Fax: +886-4-22083362.
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Botanical Studies, Vol. 53, 2012
MATERIALS AND METHODS
tioxidant equivalent to a 1000 ppm solution of the sample under investigation.
Materials
Butylated hydroxytoluene (BHT), Glutathione (GSH), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 6-Hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox), potassium peroxodisulfate (K2S2O8), Tris (hydroxylmethyl) aminomethane, potassium ferricyanide (K3Fe(CN)6), ferric chloride (FeCl3), catechin, 2,2'-azinobis-(3- ethylbenzothiazoline)-6-sulphonic acid (ABTS), and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Folin-Ciocalteu solution and 95% ethanol were purchased from Merck Co. (Santa Ana, CA, USA). Thirty-one wetland medicinal plants were collected from Taichung, Nantou, and Hsinchu counties in Taiwan. They were identified and authenticated by Dr. Chao-Lin Kuo, Associate Professor and Chairman, Department of Chinese Medicine Resources, China Medical University, Taichung, Taiwan. The medicinal wetland plants studied were all described in the Catalogue of Medicinal Plant Resources in Taiwan, published by the Committee on Chinese Medicine and Pharmacy, Taiwan Department of Health (Lin, 2003).
DPPH radical scavenging antioxidant activity determination
The effects of crude extracts and positive controls (GSH and BHT) on DPPH radicals were estimated based on the method of Yamaguchi et al. (1998). Aliquots (20 |iL) of crude extracts at various concentrations were each mixed with 100 mM Tris-HCl buffer (80 jiL, pH 7.4) and then with 100 jiL of DPPH in ethanol to a final concentration of 250 jiM. The mixture was shaken vigorously and left to stand at room temperature for 20 min in the dark. The absorbance of the reaction solution was measured spectro-photometrically at 517 nm. The percentage of DPPH de-colorization of the samples was calculated according to the equation: % decolorization = [1- (ABS sample /ABS control)] x100. IC50 value was the effective concentration at which DPPH radicals were scavenged by 50% and was obtained by interpolation from linear regression analysis. A lower IC50 value indicated a greater antioxidant activity.
Reducing power measurement
Plant materials methanol extracts preparation
The reducing power of the crude extracts and posi­tive controls (GSH and BHT) were determined according to the method of Yen and Chen (1995). The samples (0, 31.25, 62.5, 125, 250, 500, and 1000 jig/mL) were each mixed with an equal volume of 0.2 M phosphate buffer, pH 6.6, and 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min before an equal volume of 1% TCA was added, and then centrifuged at 5,000 g for 10 min. The upper layer of the solution was mixed with distilled water and 0.1% FeCl3 with a ratio of 1: 1: 2, and the absorbance was measured at 700 nm. Increased ab-sorbance of the reaction mixture indicated an increase in reducing power.
Dried whole herbs (100 g for each sample) were macer­ated with 1L 95% ethanol for 24 hours at room tempera­ture, then filtered and extracted three times. The ethanol extract (3 L) was then evaporated to 10 mL and dried in a vacuum at 40°C. The dried extract was weighed, dissolved in 95% ethanol, and stored at -20°C for further use.
Plant materials water extracts preparation
Dried whole herbs (100 g for each sample) were boiled with 1L distilled water for 1 hour. Filtration and extract collection were performed three times. The resulting de­coction was evaporated to 10 mL and dried in a vacuum at 50°C. The dried extract was weighed, dissolved in distilled water, and stored at -20°C for further use. For each extract, the yield was calculated as a percentage of the dry weight of the whole herbs used (100 g) and the quantity of dry mass obtained after extraction (w/w).
Total polyphenol content determination
Total polyphenol contents of the crude extracts were determined according to the method of Ragazzi and Veronese (1973). 20 μL of each extract (125 μg/mL) was added to 200 μL distilled water and 40 μL of Folin-Ciocalteu reagent. The mixture was allowed to stand at room temperature for 5 min and then 40 μL of 20% sodium carbonate was added to the mixture. The resulting blue complex was then measured at 680 nm. Catechin was used as a standard for the calibration curve. The polyphenol content was calibrated using the linear equation based on the calibration curve. The total polyphenol content was expressed as mg catechin equivalent/g dry weight. The dry weight indicated was the sample dry weight.
TEAC antioxidant activity determination
A TEAC assay was conducted based on the method of Ramos et al. (1999). The ABTS aqueous solution (7 mM) was oxidized with potassium peroxodisulfate (2.45 mM) for 16 hours in the dark at room temperature. The ABTS·+ solution was diluted with 95% ethanol to an absorbance of 0.75 ± 0.05 at 734 nm (Beckman UV-Vis spectrophotometer, Model DU640B). An aliquot (20 μL) of each sample (125 μg/mL) was mixed with 180 μL ABTS·+ solution and the absorbance was read at 734 nm after 1 min. Trolox was used as a reference standard. A standard curve was constructed for Trolox at 0, 15.625, 31.25, 62.5, 125, 250, 500 μM concentrations. TEAC value was expressed as millimolar concentration of Trolox solution, with the antioxidant
Total flavonoid content determination
Total flavonoid contents of the crude extracts were determined according to the method of Lamaison and Carnet (1990). Aliquots of 1.5 mL extracts were each added to an equal volume of 2% AlCl3•6H2O (2 g in 100 mL
HO et al. ― Antioxidant properties and total phenolic contents of wetland medicinal plants in Taiwan
57
methanol) solution. The mixture was vigorously shaken, and the absorbance was read after 10 min of incubation at 430 nm. Rutin was used as the standard for the cali­bration curve. The total flavonoid content was calibrated using the linear equation based on the calibration curve. The total flavonoid content was expressed as mg rutin equivalent/g dry weight. The dry weight indicated was the sample dry weight.
Table 1. The yield of water and methanol extracts of the wet­land medicinal plants.
  Yield (%, w/w)a  
Scientific name
Water extract
Ethanol extract
Acorus gramineus Soland.
12.96
8.37
Avicennia marina (Forsk.) Vierh. -leaf
5.47
5.83
Avicennia marina (Forsk.) Vierh. -root
17.84
24.37
Alisma orientalis (Sam.) Juzep.
43.33
14.84
Alternanthera sessilis (L.) R. Br.
15.91
12.14
Cyperus alternifolius L. subsp. flabelliformis (Rottb.) Kukenthal
11.24
26.76
Commelina communis L.
11.76
14.21
Cyperus difformis L.
23.22
17.84
Cyperus imbricatus Retz.
36.33
9.79
Cyperus iria L.
11.07
7.62
Eichhornia crassipes (Mart.) Solms
14.35
13.05
Echinochloa crus-galli (L.) Beauv.
6.77
6.39
Egeria densa Planch.
20.77
3.42
Euryale ferox Salisb.
14.44
1.43
Eriocaulon sexangulare L.
12.45
2.53
Fimbristylis littoralis Gaud
10.52
3.92
Hedyotis corymbosa (L.) Lam.
21.19
10.66
Hygrophila pogonocalyx Hayata
12.84
6.41
Juncus effusus L. var. decipiens Buchen.
13.04
11.34
Kyllinga brevifolia Rottb.
12.18
4.45
Lindernia antipoda (L.) Alston
63.33
34.03
Marsilea minuta L.
37.03
11.27
Pilea microphylla (L.) Liebm.
4.96
10.76
Phyla nodiflora (L.) Greene
7.86
9.18
Polygonum plebeium R. Br.
45.99
17.31
Pistia stratiotes L.
70.18
21.58
Rotala rotundifolia (Wallich ex Roxb.)
Koehne
4.24
12.31
Spirodela punctata G. F. W. Meyer
13.96
11.42
Salix warburgii O. Seem.
14.63
15.44
Typha orientalis Presl
19.7
0.89
Torulinium odoratum (L.) S. Hooper
39.83
12.76
Total flavonol content determination
The total flavonol contents of the crude extracts were determined according to the method of Arnous et al. (2001). Aliquots of 200 μL extracts were each added to 1 mL of 0.1% p-dimethylaminocinnamaldehyde (DMACA) in methanol/HCl (3:1, v/v). The mixture was vigorously shaken, and the absorbance was read after 10 min of incubation at 640 nm. Catechin was used as the standard for the calibration curve. The total flavonol content was calibrated using the linear equation based on the calibration curve. The total flavonol content was expressed as mg catechin equivalent/g dry weight. The dry weight indicated was the sample dry weight.
Statistical analysis
Experimental results were presented as the mean 士 standard deviation (SD) of three parallel measurements. Statistical analyses were performed by one-way ANOVA, followed by Dunnett's t test. The difference was consid­ered to be statistically significant when the p value was less than 0.05.
RESULTS
Extraction yields
The water and methanol extract yields of the wetland medicinal plants are presented in Table 1. The water ex­tract yields ranged from 4.24% to 70.18%, and the of methanol extract yields ranged from 0.89% to 34.03%. Among the water extracts, Pistia stratiotes produced the highest yield (70.18%), followed by Lindernia antipoda. (63.33%), Polygonum plebeium (45.99%), Alisma ori-entalis (43.33%), and Torulinium odoratum (39.83 %). The highest yield among methanol extracts was obtained from Lindernia antipoda (34.03%), followed by Cyperus alternifolius subsp. flabelliformis (26.76%), Avicennia marina (24.37%), Pistia stratiotes (21.58%) and Cyperus difformis (17.84%).
a On dried weight basis.
Water extractions of the wetland medicinal plants gen­erally yielded more components than methanol extrac­tions. It is worth mentioning that water extraction may allow more hydrogen bonding with phenolic compounds than does methanol.
(Chang et al., 2007a, b). In this assay, ABTS radical mono-cation was generated directly in stable form from potas­sium peroxodisulfate. The radicals were generated before the addition of antioxidants to prevent the interference of compounds, which affected radical formation. This modi­fication made the assay less susceptible to interruptions and prevented the overestimation of antioxidant power
TEAC assay antioxidant activity estimation
The Trolox-equivalent antioxidant capacity (TEAC) as­say is often used to evaluate the total antioxidant power of single compounds and complex mixtures of various plants
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Botanical Studies, Vol. 53, 2012
(Sanchez-Moreno, 2002). The tested samples were only added to the reaction medium once stable absorbance was obtained. Their antioxidant activities were then measured in terms of decolorization. This method is recommended for plant extracts because the maximum wavelength ab­sorption of ABTS at 734 nm eliminates color interference (Awika et al., 2003). The results were expressed as jiM Trolox/mg dry weight of plant material.
followed by Juncus effusus var. decipiens (971.14 ± 49.68 μM Trolox/mg), Cyperus iria (762.04 ± 33.80 μM Trolox/ mg), Salix warburgii (657.57 ± 18.37 μM Trolox/mg) and Kyllinga brevifolia (462.67 ± 9.49 μM Trolox/mg). The plant with the highest antioxidant capacity among the methanol extracts was Juncus effusus var. decipiens (2074.35 ± 116.19 μM Trolox/mg), followed by Salix warburgii (931.45 ± 84.14 μM Trolox/mg), Cyperus iria (769.41 ± 53.57 μM Trolox/mg), Typha orientalis (651.22 ± 14.95 μM Trolox/mg) and Kyllinga brevifolia (342.52 ± 10.91 μM Trolox/mg).
In the TEAC assay, the antioxidant capacities of wetland medicinal plants ranged from 7.52 μM to 1753.41 μM Trolox/mg for the water extracts, and 5.69 μM to 2074.35 μM Trolox/mg for the methanol extracts (Table 2). The differences in antioxidant capacities were very large, up to 233 and 364 fold, respectively. Among the water extracts, Rotala rotundifolia possessed the highest antioxidant capacity (1753.41 ± 76.99 μM Trolox/mg),
Scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radicals
The relatively stable organic radical DPPH is widely used in modeling systems to investigate the scavenging
Table 2. The TEAC of the water and methanol extracts of the wetland medicinal plants.
TEACa
Scientific name and positive controls
                           (μM Trolox/mg ± SD)                           
Water extracted Methanol extracted
GSH
1827.68 ± 76.84
Not detected
BHT
Not detected
11869.41 ± 34.63
Acorus gramineus Soland.
159.52 ± 2.57
225.65 ± 9.45
Avicennia marina (Forsk.) Vierh. -leaf
376.17 ± 10.42
54.15 ± 2.11
Avicennia marina (Forsk.) Vierh. -root
381.85 ± 13.07
177.00 ± 2.13
Alisma orientalis (Sam.) Juzep.
12.33 ± 3.44
19.08 ± 7.96
Alternanthera sessilis (L.) R. Br.
156.38 ± 9.48
148.46 ± 6.64
Cyperus alternifolius L. subsp. flabelliformis (Rottb.) Kukenthal
47.23 ± 3.66
81.77 ± 4.10
Commelina communis L.
113.02 ± 1.96
96.13 ± 7.69
Cyperus difformis L.
132.00 ± 4.83
15.33 ± 16.20
Cyperus imbricatus Retz.
112.35 ± 4.74
27.35 ± 4.81
Cyperus iria L.
762.04 ± 33.80
769.41 ± 53.57
Eichhornia crassipes (Mart.) Solms
102.13 ± 2.66
143.33 ± 6.39
Echinochloa crus-galli (L.) Beauv.
448.98 ± 6.41
39.56 ± 20.28
Egeria densa Planch.
37.10 ± 2.17
5.69 ± 7.58
Euryale ferox Salisb.
14.58 ± 1.11
350.73 ± 4.13
Eriocaulon sexangulare L.
77.58 ± 3.57
222.65 ± 0.86
Fimbristylis littoralis Gaud
311.73 ± 3.71
87.17 ± 5.02
Hedyotis corymbosa (L.) Lam.
283.58 ± 4.45
56.73 ± 7.18
Hygrophila pogonocalyx Hayata
69.40 ± 0.94
43.38 ± 5.03
Juncus effusus L. var. decipiens Buchen.
971.14 ± 49.68
2074.35 ± 116.19
Kyllinga brevifolia Rottb.
462.67 ± 9.49
342.52 ± 10.91
Lindernia antipoda (L.) Alston
320.35 ± 2.80
311.23 ± 16.05
Marsilea minuta L.
94.27 ± 4.82
196.25 ± 13.50
Pilea microphylla (L.) Liebm.
165.44 ± 0.38
248.17 ± 34.50
Phyla nodiflora (L.) Greene
97.04 ± 1.53
86.21 ± 12.58
Polygonum plebeium R. Br.
364.04 ± 1.07
175.44 ± 9.47
Pistia stratiotes L.
7.52 ± 3.64
34.79 ± 2.63
Rotala rotundifolia (Wallich ex Roxb.) Koehne
1753.41 ± 76.99
159.90 ± 15.52
Spirodela punctata G. F. W. Meyer
360.25 ± 7.70
104.17 ± 5.36
Salix warburgii O. Seem.
657.57 ± 18.37
931.45 ± 84.14
Typha orientalis Presl
344.13 ± 5.48
651.22 ± 14.95
Torulinium odoratum (L.) S. Hooper
32.71 ± 0.71
203.67 ± 52.57
aValues represented mean ± S.D. of three parallel measurements (P<0.05).
HO et al. ― Antioxidant properties and total phenolic contents of wetland medicinal plants in Taiwan
59
activities of several natural compounds, such as pheno-lics and anthocyanins, as well as crude mixtures, such as methanol or water extracts from plants. The DPPH radi­cal is scavenged by antioxidants through the donation of electrons forming the reduced DPPH. The color changes from purple to yellow after reduction, and the accompa­nying decrease in absorbance can be quantified at wave­length 517 nm. Table 3 shows the IC50 values for radical-scavenging activities of GSH, BHT and different extract fractions of the wetland medicinal plants using the DPPH colorimetric method.
μg/mL), Avicennia marina -leaf (271.71 ± 1.28 μg/mL), and Polygonum plebeium (301.52 ± 4.62 μg/mL). The positive control glutathione (GSH) had an IC50 value of 71.77 ± 2.09 μg/mL.
For the methanol extracts, Salix warburgii had the lowest IC50 value (59.58 ± 0.33 μg/mL), followed by Juncus effusus var. decipiens (108.95 ± 4.47 μg/mL), Lindernia antipoda (144.61 ± 2.53 μg/mL), Cyperus iria (167.18 ± 0.64 μg/mL), Typha orientalis (208.01 ± 1.46 μg/mL), and Cyperus imbricatus (242.55 ± 3.11 μg/mL). The positive control BHT also had a low IC50 value (139.56 ± 2.96 μg/mL). The above IC50 values showed that Salix warburgii and Juncus effusus var. decipiens demonstrated even higher radical scavenging activities than the positive control in the DPPH assay.
In the DPPH assay conducted on the water extracts, Rotala rotundifolia had the lowest IC50 value among the medicinal plants (94.89 ± 0.31 μg/mL), followed by Salix warburgii (112.69 ± 0.28 μg/mL), Lindernia antipoda (189.14 ± 4.55 μg/mL), Cyperus iria (194.45 ± 0.32
Table 3. The DPPH radical scavenging activity of the water and methanol extracts of the wetland medicinal plants.
DPPH radical scavenging activitya
Scientific name and positive controls
                        
(IC50, μg/mL)                                     
Water extract
Methanol extract
GSH
71.77 ± 2.09
Not detected
BHT
Not detected
139.56 ± 2.96
Acorus gramineus Soland.
896.90 ± 7.60
1045.51 ± 0.69
Avicennia marina (Forsk.) Vierh. -leaf
271.71 ± 1.28
>2,000
Avicennia marina (Forsk.) Vierh. -root
404.19 ± 1.18
713.99 ± 0.24
Alisma orientalis (Sam.) Juzep.
>2,000
>2,000
Alternanthera sessilis (L.) R. Br.
844.69 ± 6.42
946.79 ± 8.39
Cyperus alternifolius L. subsp. flabelliformis (Rottb.) Kukenthal
>2,000
>2,000
Commelina communis L.
>2,000
>2,000
Cyperus difformis L.
1125.67 ± 1.22
489.04 ± 3.82
Cyperus imbricatus Retz.
1882.64 ± 3.92
242.55 ± 3.11
Cyperus iria L.
194.45 ± 0.32
167.18 ± 0.64
Eichhornia crassipes (Mart.) Solms
>2,000
>2,000
Echinochloa crus-galli (L.) Beauv.
548.23 ± 4.62
>2,000
Egeria densa Planch.
>2,000
>2,000
Euryale ferox Salisb.
>2,000
307.35 ± 1.61
Eriocaulon sexangulare L.
>2,000
>2,000
Fimbristylis littoralis Gaud
810.61 ± 6.58
>2,000
Hedyotis corymbosa (L.) Lam.
668.89 ± 8.62
>2,000
Hygrophila pogonocalyx Hayata
1520.06 ± 5.25
>2,000
Juncus effusus L. var. decipiens Buchen.
456.88 ± 3.88
108.95 ± 4.47
Kyllinga brevifolia Rottb.
379.52 ± 2.52
523.55 ± 0.091
Lindernia antipoda (L.) Alston
189.14 ± 4.55
144.61 ± 2.53
Marsilea minuta L.
1400.48 ± 3.2
613.76 ± 1.67
Pilea microphylla (L.) Liebm.
>2,000
423.14 ± 5.61
Phyla nodiflora (L.) Greene
>2,000
789.26 ± 5.84
Polygonum plebeium R. Br.
301.52 ± 4.62
>2,000
Pistia stratiotes L.
>2,000
>2,000
Rotala rotundifolia (Wallich ex Roxb.) Koehne
94.89 ± 0.31
721.89 ± 3.91
Spirodela punctata G. F. W. Meyer
432.20 ± 4.63
1094.73 ± 12.61
Salix warburgii O. Seem.
112.69 ± 0.28
59.58 ± 0.33
Typha orientalis Presl
533.59 ± 4.92
208.01 ± 1.46
Torulinium odoratum (L.) S. Hooper
>2,000
>2,000
aValues represented mean ± S.D. of three parallel measurements (P<0.05).
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Botanical Studies, Vol. 53, 2012
Reducing power measurement
1997). The reducing power of different extract fractions from the wetland medicinal plants are shown in Table 4. Both reduced GSH and BHT were used as the positive controls.
We investigated the reducing capacity of wetland me­dicinal plants by measuring Fe3+-Fe2+ conversion. The re­ducing capacity of a compound may serve as an important indicator of its potential antioxidant activity (Meir et al., 1995). The antioxidant activities of putative antioxidants have been attributed to various mechanisms, such as the prevention of chain initiation, transition metal ion catalyst binding, peroxides decomposition , prevention of contin­ued proton abstraction, and radical scavenging (Diplock,
For the reducing capacity of the water extracts, Salix warburgii had the highest value among the medicinal plants examined (1.64 ± 0.01, Δ700), followed by Rotala rotundifolia (1.61 ± 0.05, Δ700), Lindernia antipoda (1.60 ± 0.07, Δ700), Cyperus iria (1.56 ± 0.01, Δ700), Polygonum plebeium (1.17 ± 0.03, Δ700), and Avicennia marina.-leaf
Table 4. The reducing power of the water and methanol extracts of the wetland medicinal plants.
Reducing power A700a
Scientific name and positive controls
                             (Mean ± SD)                             
Water extract
Methanol extract
GSH
1.80 ± 0.01
Not detected
BHT
Not detested
0.27 ± 0.02
Acorus gramineus Soland.
0.36 ± 0.01
0.04 ± 0.01
Avicennia marina (Forsk.) Vierh. -leaf
1.11 ± 0.01
0.06 ± 0.01
Avicennia marina (Forsk.) Vierh. -root
1.010 ± 0.02
0.36 ± 0.02
Alisma orientalis (Sam.) Juzep.
0.05 ± 0.01
0.15 ± 0.02
Alternanthera sessilis (L.) R. Br.
0.46 ± 0.01
0.19 ± 0.02
Cyperus alternifolius L. subsp. flabelliformis (Rottb.) Kukenthal
0.13 ± 0.01
0.17 ± 0.01
Commelina communis L.
0.23 ± 0.01
0.40 ± 0.01
Cyperus difformis L.
0.39 ± 0.01
0.51 ± 0.02
Cyperus imbricatus Retz.
0.27 ± 0.02
0.41 ± 0.03
Cyperus iria L.
1.56 ± 0.01
0.58 ± 0.03
Eichhornia crassipes (Mart.) Solms
0.17 ± 0.01
0.26 ± 0.01
Echinochloa crus-galli (L.) Beauv.
0.63 ± 0.02
0.03 ± 0.01
Egeria densa Planch.
0.02 ± 0.01
0.04 ± 0.01
Euryale ferox Salisb.
0.07 ± 0.01
0.76 ± 0.12
Eriocaulon sexangulare L.
0.09 ± 0.01
0.32 ± 0.01
Fimbristylis littoralis Gaud
0.40 ± 0.01
0.01 ± 0.01
Hedyotis corymbosa (L.) Lam.
0.552 ± 0.02
0.06 ± 0.00
Hygrophila pogonocalyx Hayata
0.310 ± 0.01
0.09 ± 0.02
Juncus effusus L. var. decipiens Buchen.
0.56 ± 0.01
0.26 ± 0.05
Kyllinga brevifolia Rottb.
0.74 ± 0.01
0.29 ± 0.08
Lindernia antipoda (L.) Alston
1.60 ± 0.07
1.66 ± 0.01
Marsilea minuta L.
0.27 ± 0.01
0.69 ± 0.02
Pilea microphylla (L.) Liebm.
0.08 ± 0.03
0.29 ± 0.02
Phyla nodiflora (L.) Greene
0.33 ± 0.02
0.36 ± 0.03
Polygonum plebeium R. Br.
1.17 ± 0.03
0.36 ± 0.02
Pistia stratiotes L.
0.02 ± 0.01
0.04 ± 0.01
Rotala rotundifolia (Wallich ex Roxb.) Koehne
1.61 ± 0.05
0.25 ± 0.01
Spirodela punctata G. F. W. Meyer
0.71 ± 0.05
0.10 ± 0.04
Salix warburgii O. Seem.
1.64 ± 0.01
1.68 ± 0.01
Typha orientalis Presl
0.58 ± 0.01
0.46 ± 0.02
Torulinium odoratum (L.) S. Hooper
0.09 ± 0.01
0.27 ± 0.05
aValues represented mean ± S.D. of three parallel measurements (P<0.05).
HO et al. ― Antioxidant properties and total phenolic contents of wetland medicinal plants in Taiwan
61
(1.11 ± 0.01, Δ700). The positive control glutathione (GSH) had a high reducing capacity activity of 1.80 ± 0.01, Δ700.
extracts ranged from 0.05 to 14.05 μg CE/mg, and that of the methanol extracts ranged from 0.46 to 14.36 μg CE/mg. The difference in antioxidant capacities was also very large, from 31 up to up to 281 fold. Cyperus iria had the highest flavonol content (14.05 ± 0.88 μg CE/mg), followed by Polygonum plebeium (13.47 ± 1.22 μg CE/mg), and Salix warburgii (5.54 ± 0.18 μg CE/mg) for the water extracts. Cyperus imbricatus had the highest flavonol content (14.36 ± 1.28 μg CE/mg), followed by Cyperus iria (13.41 ± 0.87 μg CE/mg), and Typha orientalis (7.04 ± 0.10 μg CE/mg) for the methanol extracts.
For the methanol extracts, Salix warburgii had the highest reducing capacity value (1.68 ± 0.01, Δ700), followed by Lindernia antipoda (1.66 ± 0.01, Δ700), Euryale ferox (0.76 ± 0.12, Δ700), Marsilea minuta (0.69 ± 0.02, Δ700), Cyperus iria (0.58 ± 0.03, Δ700), and Cyperus difformis (0.51 ± 0.02, Δ700). The positive control BHT also had quite a high reducing capacity (0.27 ± 0.02, Δ700). The results showed that the reducing capacities for radicalscavenging of all the tested wetland medicinal plants were even higher than those of the positive controls.
Relationship between total antioxidant activity and total polyphenol content
Total polyphenol, flavonoid, and flavonol con­tents of the wetland medicinal plants
The correlation coefficients (R2) of total antioxidant activity (TEAC) and total polyphenols of the water and methanol extracts are shown in Figure 1A and 1B. The R2 values of TEAC and total polyphenol content of the water (Figure 1A) and methanol (Figure 1B) extracts were 0.14 and 0.25, respectively. From these statistics, we deter­mined a low correlation between the TEAC and total poly-phenol contents. Linear regression analysis showed a low correlation between antioxidant activity and total phenolic contents. Different wetland medicinal plant species may influence the antioxidant activity as well. High phenolic content is only one of the antioxidant capacity indicators.
The total polyphenol, flavonoid, and flavonol contents of the water and methanol extracts of wetland medicinal plants are shown in Tables 5 and 6, respectively. The total polyphenol content is expressed as μg of catechin equivalent per milligram of dry weight. For the water extracts, the total polyphenol content of the wetland medicinal plants ranged from 17.91 μg to 565.92 μg CE/mg; as for the methanol extract, the total polyphenol content ranged from 7.69 μg CE/mg to 551.50 μg CE/mg, and the difference of antioxidant capacities was also very large, up to 31 and 71 fold. Rotala rotundifolia had a total polyphenol content of 565.92 ± 4.45 μg CE/mg, followed by Lindernia antipoda (389.25 ± 19.12μg CE/mg), Cyperus iria (385.67 ± 5.62 μg CE/mg), Cyperus iria (302.89 ± 21.19 μg CE/mg), Typha orientalis (230.50 ± 1.41 μg CE/mg), and Cyperus difformis (162.04 ± 10.16 μg CE/mg) in their water extracts (Table 5). Salix warburgii had a total polyphenol content of 551.50 ± 17.57 μg CE/mg, followed by Typha orientalis (494.17 ± 10.13 μg CE/mg), Juncus effusus. var. decipiens (489.75 ± 53.28 μg CE/mg), Lindernia antipoda (356.70 ± 17.32 μg CE/mg), Cyperus iria (345.25 ± 9.81 μg CE/mg), and Kyllinga brevifolia (251.25 ± 1.90 μg CE/mg) in their methanol extracts (Table 6).
DISCUSSION
The best antioxidant activities among the 31 wetland medicinal plants screened, based on TEAC assay, DPPH radical scavenging, reducing power, total polyphenol con­tent, total flavonoid content and flavonol content results were: Rotala rotundifolia, Juncus effusus var. decipi-ens, Cyperus iria, Salix warburgii, Lindernia antipoda, Kyllinga brevifolia, and Typha orientalis. The therapeutic properties of Rotala rotundifolia have not been reported in any scientific papers before. This plant possesses antiradia-tion, anti-inflammatory, and antibacterial properties. The present study provided valuable preliminary data through a demonstration of its efficient antioxidant capacity. Isola­tion and characterization of its individual active compo­nents and in vivo relevance await further comprehensive studies.
The total flavonoid content was expressed as μg of rutin equivalent per milligram of dry weight. The total flavonoid contents in the wetland medicinal plant water extracts ranged from 3.11 to 53.92 μg RE/mg, and the total flavonoid contents in their methanol extracts ranged from 1.19 to 72.17 μg RE/mg, furthermore the difference of antioxidant capacities was also very large, up to 17 and 60 fold respectively. Typha orientalis had the highest total flavonoid content (53.92 ± 5.44 μg RE/mg), followed by Rotala rotundifolia (46.24 ± 0.39 μg RE/mg), and Cyperus iria (29.73 ± 0.51 μg RE/mg) in their water extracts. Eriocaulon sexangulare had the highest total flavonoid content (74.55 ± 1.50 μg RE/mg), followed by Polygonum plebeium (72.17 ± 3.33 μg CE/mg), Typha orientalis (71.89 ± 0.42 μg RE/mg), and Salix warburgii (70.34 ± 2.43 μg RE/mg) in their methanol extracts.
Juncus effusus var. decipiens possesses anti-depressant, anti-inflammatory, and antibacterial effects. To our knowl­edge, there were no prior reports on the antioxidant activ­ity of this plant. This study rendered valuable preliminary data through demonstration of its high antioxidant capac­ity. To study the phenolic constituents from the dry stem of Juncus effusus, six phenolic constituents were purified and identified as 7-carboxy-2-hydroxy-1-methyl-5-vinyl-9, 10-dihydrophenanthrene, 2,3-isopylidene-1-O-ferulic acid glyceride, (2S)-2, 3-isopylidene-1-0-p-coumaroyl glycer-ide, dehydroeffusal, p-hydroxybenzaldehyde and luteolin-5,3'-dimethyl ether (Li et al., 2007). Some of these might be antioxidants.
The total flavonol content was expressed as μg of cat-echin equivalent per milligram of dry weight. The total flavonol content of the wetland medicinal plants water
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Botanical Studies, Vol. 53, 2012
Table 5. Total polyphenol, flavonoid, and flavonol content of the water extracts of the wetland medicinal plantsa.
                                             
Water extracted
                            
Scientific name
Polyphenol b
Flavonoida'c
Flavonola- b
Acorus gramineus Soland.
(μg CE/mg) 91.50 ± 1.25
(μg RE/mg) 13.81 ± 4.93
(μg CE/mg) 2.06 ± 0.14
Avicennia marina (Forsk.) Vierh. -leaf
109.25 ± 1.67
27.55 ± 0.24
0.34 ± 0.02
Avicennia marina (Forsk.) Vierh. -root
102.1 ± 7.55
7.78 ± 0.07
0.42 ± 0.01
Alisma orientalis (Sam.) Juzep.
37.04 ± 1.81
3.71 ± 0.05
1.06 ± 0.01
Alternanthera sessilis (L.) R. Br.
137.67 ± 3.81
26.44 ± 0.53
1.56 ± 9.11
Cyperus alternifolius L. subsp. flabelliformis (Rottb.) Kukenthal
78.45 ± 0.93
4.63 ± 0.05
1.23 ± 0.02
Commelina communis L.
113.79 ± 5.76
15.72 ± 0.20
1.04 ± 0.01
Cyperus difformis L.
162.04 ± 10.16
18.96 ± 0.53
1.15 ± 0.01
Cyperus imbricatus Retz.
140.87 ± 7.01
13.70 ± 0.37
1.31 ± 0.01
Cyperus iria L.
385.67 ± 5.62
29.73 ± 0.51
14.05 ± 0.88
Eichhornia crassipes (Mart.) Solms
90.12 ± 7.26
9.97 ± 0.23
1.14 ± 0.01
Echinochloa crus-galli (L.) Beauv.
108.33 ± 9.26
14.88 ± 1.33
2.93 ± 1.98
Egeria densa Planch.
24.66 ± 0.10
5.85 ± 0.09
1.05 ± 0.01
Euryale ferox Salisb.
28.16 ± 0.49
3.28 ± 0.21
1.93 ± 0.02
Eriocaulon sexangulare L.
88.62 ± 0.91
9.57 ± 0.25
1.25 ± 0.02
Fimbristylis littoralis Gaud
87.50 ± 20.02
14.16 ± 0.36
3.13 ± 0.08
Hedyotis corymbosa (L.) Lam.
157.50 ± 7.53
17.04 ± 0.67
0.71 ± 0.01
Hygrophila pogonocalyx Hayata
81.25 ± 9.11
7.86 ± 0.08
1.22 ± 0.01
Juncus effusus L. var. decipiens Buchen.
37.33 ± 10.37
8.79 ± 2.88
0.38 ± 0.01
Kyllinga brevifolia Rottb.
215.92 ± 1.23
9.54 ± 0.44
1.69 ± 0.01
Lindernia antipoda (L.) Alston
389.25 ± 19.12
22.68 ± 0.57
2.23 ± 0.02
Marsilea minuta L.
102.41 ± 6.93
12.42 ± 0.61
3.97 ± 0.15
Pilea microphylla (L.) Liebm.
81.67 ± 3.59
10.00 ± 0.40
0.05 ± 0.01
Phyla nodiflora (L.) Greene
137.04 ± 4.13
16.76 ± 0.10
1.61 ± 0.01
Polygonum plebeium R. Br.
64.10 ± 10.48
17.22 ± 0.25
13.47 ± 1.22
Pistia stratiotes L.
17.91 ± 0.10
3.33 ± 0.04
0.85 ± 0.01
Rotala rotundifolia (Wallich ex Roxb.) Koehne
565.92 ± 4.45
46.24 ± 0.39
4.50 ± 0.27
Spirodela punctata G. F. W. Meyer
67.67 ± 11.58
21.37 ± 1.27
0.81 ± 0.02
Salix warburgii O. Seem.
302.89 ± 21.19
34.20 ± 1.36
5.54 ± 0.18
Typha orientalis Presl
230.50 ± 1.41
53.92 ± 5.44
2.89 ± 0.01
Torulinium odoratum (L.) S. Hooper
43.91 ± 0.55
3.11 ± 0.04
2.39 ± 0.04
aValues represented mean ± S.D. of three parallel measurements.
bData expressed in μg catechin equivalent / mg dry weight (μg CE/mg).
cData expressed in μg rutin equivalent / mg dry weight (μg RE/mg).
Cyperus iria has not been reported in any scientific pa­pers before. This plant possesses rheumatic, antidiuretic, and anti-inflammatory effects. The present study showed first-hand data on the antixodiant capacity of Cyperus iria. Isolation and characterization of its individual active com­ponents and in vivo relevance of such activity await further comprehensive studies.
there have been no prior reports on the antioxidant activity of this plant. This paper studied the antioxidant effects of Salix warburgii for the first time; a bioassay-guided in vitro screen has revealed that a 70% methanol extract of the leaves of Salix matsudana showed considerable inhibitory activity against cyclooxygenases (COX-1 and COX-2) (Li et al., 2008). A subsequent phytochemical study led to the isolation of some compounds: matsudone A, luteolin, isoquercitrin, 7-methoxyflavone, luteolin 7-OTable
Salix warburgii possesses anticoagulant, anti-inflam­matory, and antibacterial properties. To our knowledge,
HO et al. ― Antioxidant properties and total phenolic contents of wetland medicinal plants in Taiwan
63
Table 6. Total polyphenol, flavonoid, and flavonol content of the methanol extracts of the wetland medicinal plantsa.
____________________
Methanol extracted
_______________
Scientific name
Polyphenola,b
Flavonoida,c
Flavonola,b
Acorus gramineus Soland.
(μg CE/mg) 160.58 ± 61.15
(μg RE/mg) 19.98 ± 31.55
(μg CE/mg) 1.38 ± 0.01
Avicennia marina (Forsk.) Vierh. -leaf
7.69 ± 24.50
18.34 ± 24.74
1.49 ± 0.16
Avicennia marina (Forsk.) Vierh. -root
59.76 ± 3.27
42.08 ± 1.22
0.46 ± 0.01
Alisma orientalis (Sam.) Juzep.
37.45 ± 0.28
6.25 ± 1.93
0.94 ± 0.01
Alternanthera sessilis (L.) R. Br.
152.83 ± 11.30
24.07 ± 18.78
3.79 ± 0.38
Cyperus alternifolius L. subsp. flabelliformis (Rottb.) Kukenthal
105.58 ± 13.19
20.51 ± 3.02
3.84 ± 0.05
Commelina communis L.
129.16 ± 4.99
21.21 ± 0.24
1.36 ± 0.01
Cyperus difformis L.
39.01 ± 6.05
15.31 ± 0.68
2.29 ± 0.02
Cyperus imbricatus Retz.
112.07 ± 10.79
26.11 ± 9.91
14.36 ± 1.28
Cyperus iria L.
345.25 ± 9.81
46.88 ± 0.47
13.41 ± 0.87
Eichhornia crassipes (Mart.) Solms
184 ± 19.12
25.60 ± 6.30
3.08 ± 0.06
Echinochloa crus-galli (L.) Beauv.
80.08 ± 8.80
24.21 ± 10.61
2.00 ± 0.38
Egeria densa Planch.
81.79 ± 18.52
12.5.11 ± 5.41
2.95 ± 0.01
Euryale ferox Salisb.
213.58 ± 7.29
16.70 ± 2.31
3.43 ± 0.01
Eriocaulon sexangulare L.
133.02 ± 5.12
74.55 ± 1.50
6.15 ± 0.86
Fimbristylis littoralis Gaud
107.08 ± 4.12
19.16 ± 5.42
2.00 ± 0.37
Hedyotis corymbosa (L.) Lam.
103.67 ± 1.46
26.02 ± 6.49
2.57 ± 0.16
Hygrophila pogonocalyx Hayata
18.05 ± 8.56
1.19 ± 0.53
2.54 ± 1.34
Juncus effusus L. var. decipiens Buchen.
489.75 ± 53.28
30.16 ± 5.07
1.60 ± 0.05
Kyllinga brevifolia Rottb.
251.25 ± 1.90
41.86 ± 2.19
3.85 ± 0.26
Lindernia antipoda (L.) Alston
356.70 ± 17.32
35.21 ± 2.42
2.12 ± 0.01
Marsilea minuta L.
47.81 ± 9.58
12.05 ± 3.84
1.45 ± 0.08
Pilea microphylla (L.) Liebm.
244.08 ± 14.43
14.78 ± 3.38
5.98 ± 0.78
Phyla nodiflora (L.) Greene
29.53 ± 4.71
11.10 ± 1.86
2.68 ± 0.10
Polygonum plebeium R. Br.
44.32 ± 11.38
72.17 ± 3.33
2.82 ± 0.39
Pistia stratiotes L.
51.79 ± 1.12
20.22 ± 2.58
4.97 ± 0.12
Rotala rotundifolia (Wallich ex Roxb.) Koehne
122.92 ± 3.67
28.14 ± 5.48
2.43 ± 0.33
Spirodela punctata G. F. W. Meyer
204.00 ± 4.39
20.84 ± 1.53
3.77 ± 0.29
Salix warburgii O. Seem.
551.50 ± 17.57
70.34 ± 2.43
6.62 ± 0.31
Typha orientalis Presl
494.17 ± 10.13
71.89 ± 0.42
7.04 ± 0.10
Torulinium odoratum (L.) S. Hooper
54.06 ± 11.80
12.27 ± 2.27
3.33 ± 1.56
aValues represented mean ± S.D. of three parallel measurements.
bData expressed in j g catechin equivalent / mg dry weight (μg CE/mg).
cData expressed in j g rutin equivalent / mg dry weight (μg RE/mg).
glucoside, and 4',7-dihydroxyflavone. These isolated compounds were found to possess activities in inhibiting against COX-1 or COX-2.
vivo relevance await further comprehensive studies.
Kyllinga brevifolia possesses analgesic and anti-inflam­matory effects. Oral administration of doses up to 3.0 g/kg did not provoke any toxic symptoms. The toxicity of this plant was observed to be dose dependent and its intraperi-toneal LD50 was found to be 575 mg/kg. It is used in tra­ditional medicine to alleviate stress or as a sedative agent (Hellion-Ibarrola et al., 1999).
Lindernia antipoda possesses analgesic and anti-inflam­matory effects. There have been no prior reports on the antioxidant activity of this plant. This study provided valu­able data by demonstrating the high antioxidant capacity of Lindernia antipoda for the first time. However, isolation and characterization of its active components and their in
64
Botanical Studies, Vol. 53, 2012
assays used in this study measured the oxidative products at the early and final stages of oxidation. Antioxidants have different functional properties, for example quer-cetin, rutin, and catechin can scavenge reactive oxygen species (Liu et al., 2008); p-coumaric acids, on the other hand, inhibit the generation of free radicals and chain-breaking activity (Laranjinha et al., 1995) and metal chela-tion (Van-Acker et al., 1998). These compounds, as well as flavonoids and other organic acids, are highly effective electron donors. However, the components responsible for the antioxidative activities of the wetland medicinal plants are still unclear. Further work must be performed to isolate and identify these components.
In conclusion, the results from these in vitro experi­ments, including ABTS radical monocation scavenging (Table 2), DPPH radical scavenging (Table 3), reducing power method (Table 4), total polyphenol content, total flavonoid content and total flavonol content (Table 5 and 6), demonstrated that phytochemicals in wetland medicinal plants might have significant effects on antioxidant activi­ties. However, the quantity of polyphenols and flavonoids found in the wetland medicinal plant extracts were not directly related to their antioxidant activities. The additive roles of phytochemicals might contribute significantly to the potent antioxidant activity. Hence, some wetland me­dicinal plants could be used as an easy accessible source of natural antioxidants in pharmaceutical and medical in­dustries. For this reason, further work should be performed to isolate and identify the antioxidative components of wetland medicinal plants.
Acknowledgements. This study was supported by a grant CCMP-97-RD-001 from the Committee on Chinese Medicine and Pharmacy, Department of Health, Executive Yuan, Taiwan. And China Medical University (CMU) (CMU99-S-29, CCM-P99-RD-042, and CMU99-COL- 10), Taiwan Department of Heath Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004) and the Cancer Research Center of Excellence (DOH100- TD-C-111-005).
Figure 1. Correlation coefficients (R2) of TEAC and total poly­phenol contents in the water (A) and methanol (B) extracts of the wetland medicinal plants.
Typha orientalis is a commonly used Chinese herbal drug which has been shown to possess blood circulation stimulating, hypertension relieving and nerve soothing ef­fects. There are no prior reports on the antioxidant activity of this plant.
Phenolic compounds, such as flavonoids, phenolic acid and tannins, possess anti-inflammatory, anti-carcinogenic, anti-atherosclerotic, and other properties that may be re­lated to their antioxidant activities (Chung et al., 1998; Wong et al., 2006). Flavonoids and flavonols are two poly-phenolic compounds that play an important role in stabi­lizing lipid oxidation and are associated with antioxidant activity (Yen et al., 1993). Phenolic compounds may con­tribute directly to antioxidative action (Duh et al., 1999). Polyphenolic compounds may have an inhibitory effect on mutagenesis and carcinogenesis in humans when as much as 1.0 g is ingested daily from a diet rich in fruits and veg­etables (Tanaka et al., 1998). The antioxidative activities observed can be attributed to both the different mecha­nisms exerted by different phenolic compounds and to the synergistic effects of different compounds. The antioxidant
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臺灣濕地藥用植物之抗氧化活性和總多酚含量
何玉鈴1 黃世勳1 鄧正賢2 林耀輝3 張永勳4 黃冠中4
1弘光科技大學護理系
2亞洲大學保健營養生技學系
3中央研究院植物暨微生物學研究所
4中國醫藥大學中國藥學暨中藥資源系
本文章研究目的是評估台灣濕地藥用植物之甲醇和水萃取物之抗氧化活性。評估的項目，包括
ABTS清除，清除DPPH自由基，還原力，總多酚含量，總類黃酮類含量、總黃酮醇類含量。結果顯
示，31種濕地藥用植物中以水豬母乳、燈心草、碎米莎草、水柳、泥花草、短葉水蜈蚣和香蒲共七
種，其抗氧化物和多酚類均具不錯之效果和含量。且由抗氧化活性和總多酚含量之線性相關係數結果得
知，水萃取物相關係數為0.14和甲醇萃取物相關係數為0.23 。結果顯示，植物中化學物質含量可能有
助於顯著的抗氧化活性，但這種關係並不一定成正比。濕地藥用植物未來在醫藥和保健食品行業中將可
作為一個容易取得的天然抗氧化劑的來源。
關鍵詞：濕地藥用植物；抗氧化劑；多酚類；黃酮類；黃酮醇類。