• 沒有找到結果。

The Phenolic constituents and free radical scavenging activities of Gynura formosana Kiamnra

N/A
N/A
Protected

Academic year: 2021

Share "The Phenolic constituents and free radical scavenging activities of Gynura formosana Kiamnra"

Copied!
7
0
0

加載中.... (立即查看全文)

全文

(1)

DOI: 10.1002/jsfa.2017

The phenolic constituents and free radical

scavenging activities of Gynura formosana

Kiamnra

Wen-Chi Hou,

1

Rong-Dih Lin,

2

Tzong-Huei Lee,

1

Ying-Hua Huang,

1

Feng-Lin Hsu

1

and Mei-Hsien Lee

1∗

1Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan 2Department of Internal Medicine, Municipal Taipei Ho-Ping Hospital, Taipei 100, Taiwan

Abstract: Gynura formosana Kiamnra (Compositae) is a herbal folk medicine that is a popular vegetable in Taiwan. The free-radical scavenging activities of a 70% aqueous acetone extract from the herb G formosana were evaluated. Bioassay-guided fractionation, column separation on Diaion, Toyopearl HW 40(C), Sephadex LH-20 and MCI CHP20P, and high-performance liquid chromatography (HPLC) were used to isolate for the first time in G formosana four potent phenolics [caffeic acid (I), quercetin 3-O-rutinoside (II), kaempferol 3-O-3-O-rutinoside (III) and kaempferol 3-O-robinobioside (IV)]. The IC50values of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity for compounds I – IV were 6.7, 7.7, 300.3 and 286.7µM, respectively, and, for superoxide radical scavenging activity, they were 187.3, 25.8, 55.3 and 87.4µM, respectively. Using a spin trapping electron spin resonance (ESR) method, caffeic acid (I) and quercetin 3-O-rutinoside (II) exhibited good hydroxyl radical activity. The free radical scavenging activity of G formosana phenolics may improve the economic value of this herb and assist in its development as a health food.

 2004 Society of Chemical Industry

Keywords: free-radical scavenging; Gynura formosana; caffeic acid; quercetin rutinoside; kaempferol

3-O-rutinoside; kaempferol 3-O-robinobioside

INTRODUCTION

Reactive oxygen species (ROS) are highly reactive molecules; they include the hydroxyl radical (.OH), the superoxide anion radical (O2.−), and hydrogen

per-oxide (H2O2). ROS generate metabolic products that

may attack lipids in cell membranes or DNA. Free rad-ical chain reaction processes are associated with several types of biological damage, including DNA damage, carcinogenesis and cellular degeneration related to aging. An imbalance between ROS-generating and -scavenging systems can damage cells. Thus, ROS play an important role in the pathogenesis of human diseases including cancers, arteriosclerosis and car-diovascular diseases.1 Most ROS are scavenged by

endogenous defense systems such as catalase, superox-ide dismutase (SOD), and the peroxidase/glutathione system, but these systems are not completely efficient, making it desirable to isolate exogenous antioxidants from natural sources. Various plant sources have been evaluated for antioxidant activity; the ability of com-ponents of these plants to remove oxidative stresses is of interest in the development of health foods, nutritional supplements and herbal medicines. Recent

studies have also indicated that many plant prod-ucts, including flavonoids, anthraquinones,2 tannins,

proanthocyanidins and other phenolics, as well as substances derived from fruit, vegetables, and vari-ous plant or herbal extracts, have radical-scavenging activity.3,4 Therefore, these substances could be pro-posed as health-beneficial products.5 – 8

Gynura formosana Kiamnra (Compositae) is a herbal

folk medicine that grows at lower altitudes on the north, south and east coasts of Taiwan. The plant is popular as a vegetable and as a treatment for such dis-eases as hypertension, diabetes mellitus and cerebral infarction. The literature has reported the presence of several interesting components in plants of the Gynura genus, such as pyrrolizidine alkaloids, a terpene coumarin, and several new spirostene derivatives.9 – 11

It has also been shown that an ethyl acetate extract of G

formosana contains an optically active chromanone.12

However, there are few reported phytochemical exam-inations of this species. In this study, we iso-lated the phenolic constituents of G formosana and assessed their 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide and hydroxyl radical scavenging activities.

Correspondence to: Mei-Hsien Lee, Graduate Institute of Pharmacognosy, Taipei Medical University, 250 Wu Hsing Street, Taipei 110,

Taiwan

E-mail: Lmh@tmu.edu.tw

(Received 14 November 2003; revised version received 29 March 2004; accepted 24 August 2004) Published online 26 November 2004

(2)

EXPERIMENTAL Plant material

The herbs of G formosana were obtained from a Taipei market in September 2001 and were identified by Mr Mu-Cun Gao, Department of Botany, National Taiwan University. Voucher specimens have been deposited at the Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan.

Chemicals

1,1-Diphenyl-2-picrylhydrazyl (DPPH), phenazine methosulfate (PMS), dihydronicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and fer-rous sulfate (FeSO4·7H2O) were purchased from

Sigma Chemical Co (St Louis, MO, USA). Hydrogen peroxide (H2O2) was obtained from Wako (Osaka,

Japan). The other chemicals and reagents used in the study were high-grade commercial products.

Extraction, isolation and identification

Fresh herbs (5.0 kg) were extracted with 70% aqueous acetone and the extracts were concentrated under reduced pressure. The 70% aqueous acetone extract (300 g) was chromatographed over Diaion HP-20 gel, eluting with water containing increasing concentrations of methanol and finally with a mixture of acetone – water (7:3) to obtain five fractions (Fr A– Fr E). The DPPH free radical scavenging activity was evaluated to identify the active fractions. Repeated chromatography of the fractions with monitoring of bioactivity and HPLC analysis led to the isolation of active constituents. Fractions B and C were applied to Toyopearl HW-40 (C) columns, and elution was with a gradient solvent system of methanol in H2O (H2O→ 20% MeOH → 40% MeOH →

60% MeOH→ 100% MeOH).

Five fractions were obtained from each of the two columns (Fr B-1 – Fr B-5 and Fr 1 – Fr C-5, respectively). Fraction B-1 was then applied to a column packed with Sephadex LH-20 (100µm, Pharmacia Fine Chemicals Co Ltd, Germany) and subjected to semi-preparative HPLC to yield compound I. Fraction C-1 was rechromatographed on a MCI CHP 20P column and eluted with a gradient solvent system of methanol in H2O to give six fractions

(Fr 1 – Fr 6). Fraction 1 and Fr C-2-6 were purified by semi-preparative HPLC to yield compounds II, III and IV.

Caffeic acid (I)

FABMS m/z 181 [M+ H]+; 1H-NMR (methanol-d4, 500 MHz), δ 7.46 (d, J= 15.8 Hz, H-β), 7.01 (d, J= 1.9 Hz, 2), 6.91 (dd, J = 1.9, 8.2 Hz, H-6), 6.76 (d, J= 8.15 Hz, H-5), 6.22 (d, J = 15.8 Hz, H-α); 13C-NMR (methanol-d 4, 125 MHz), δ 115.0 (C-5), 115.7 (C-2), 116.9 (C-α), 122.6 (C-6), 128.1 (C-1), 146.0 (C-3), 149.2 (C-4), 146.8 (C-β), 171.9 (COOH).13

Quercetin 3-O-rutinoside (rutin) (II)

FABMS m/z 611 [M+ H]+;1H-NMR (methanol-d 4, 500MHz), δ 7.66 (1H, d, J = 2.1 Hz, H-2), 7.62 (1H, dd, J= 2.1, 8.5 Hz, H-6), 6.87 (1H, d, J= 8.5 Hz, H-5), 6.40 (1H, d, J= 2.1 Hz, H-8), 6.21 (1H, d, J= 2.1 Hz, H-6), 5.10 (1H, d, J = 7.7 Hz, glc H-1), 4.51 (1H, d, J= 1.6 Hz, rha H-1), 3.79 (1H, dd, J= 3.7, 10.8 Hz, glc H-6), 3.40 (1H, dd, J = 3.0, 10.5 Hz, glc H-6), 1.16 (3H, d, J= 6.0 Hz, rha H-6), 3.27 – 3.80 (sugar proton);13C-NMR (methanol- d4,

125MHz), δ 179.4 (C-4), 166.1 (C-7), 163.0 (C-5), 159.4 (C-9), 158.5 (C-2), 150.0 (C-4), 145.8 (C-3), 135.6 (C-3), 123.6 (C-1), 123.1 (C-6), 117.7 (C-5), 116.1 (C-2), 105.6 (C-10), 104.7 (glc C-1), 102.9 (rha C-1), 100.0 (C-6), 94.9 (C-8), 78.2 (glc C-3), 77.3 (glc C-5), 75.7 (glc C-2), 73.9 (glc C-4), 72.3 (rha C-3), 72.1 (rha C-2), 71.4 (rha C-4), 69.7 (rha C-5), 68.6 (glc C-6), 17.9 (rha C-6).14

Kaempferol 3-O-rutinoside (nicotiflorin) (III)

FABMS m/z 595 [M+ H]+;1H-NMR (methanol-d 4, 500 MHz), δ 8.06 (2H, d, J= 8.9 Hz, H-3, H-5), 6.90 (2H, d, J= 8.9 Hz, H-2, 6), 6.40 (1H, d, J= 2.2 Hz, H-8), 6.21 (1H, d, J = 2.0 Hz, H-6), 5.12 (1H, d, J= 7.4 Hz, glc H-1), 4.53 (1H, d, J = 1.6 Hz, rha H-1), 3.80 (1H, dd, J= 1.0, 10.4 Hz, glc H-6), 3.38 (1H, dd, J= 4.7, 10.8 Hz, glc H-6), 1.11 (3H,

d, J= 6.2 Hz, rha H-6); 3.25–3.62 (sugar proton);

13C-NMR (methanol- d 4, 125 MHz), δ 179.4 (C-4), 166.0 (C-7), 163.0 (C-5), 161.5 (C-4), 159.4 (C-9), 158.6 (C-2), 135.5 (C-3), 132.4 (C-2, C-6), 122.8 (C-1), 116.2 (C-3, C-5), 105.7 (C-10), 104.6 (glc C-1), 102.6 (rha C-1), 100.0 (C-6), 94.9 (C-8), 78.2 (glc C-3), 77.2 (glc C-5), 75.8 (glc C-2), 73.9 (rha C-4), 72.3 (rha C-3), 72.0 (rha C-2), 71.5 (glc C-4), 69.7 (rha C-5), 68.6 (glc C-6), 17.9 (rha C-6).15

Kaempferol 3-O-robinobioside (IV)

FABMS m/z 595 [M+ H]+;1H-NMR (methanol-d 4, 500 MHz), δ 8.09 (2H, d, J= 9.0 Hz, H-2, H-6), 6.88 (2H, d, J= 9.0 Hz, H-3, 5), 6.41 (1H, d, J= 2.0 Hz, H-8), 6.21 (1H, d, J = 2.0 Hz, H-6), 5.03 (1H, d, J= 7.8 Hz, gal H-1), 5.33 (1H, d, J = 1.8 Hz, rha H-1), 3.71 (1H, dd, J= 5.6, 10.2 Hz, gal H-6), 3.38 (1H, dd, J= 6.7, 10.2 Hz, gal H-6), 1.17 (3H,

d, J= 6.2 Hz, rha H-6), 3.49–3.79 (sugar proton);

13C-NMR (methanol- d 4, 125MHz), δ 179.6 (C-4), 166.1 (C-7), 163.0 (C-5), 161.6(C-4), 159.4 (C-9), 158.6 (C-2), 135.7 (C-3), 132.5 (C-2, 6), 122.7 (C-1), 116.1 (C-3, 5), 105.6 (C-10), 105.5 (gal C-1), 101.9 (rha C-1), 100.0 (C-6), 94.9 (C-8), 75.4 (gal C-5), 75.1 (gal C-3), 73.9 (rha C-4), 73.0 (rha C-2), 72.3 (rha C-3), 72.1 (gal C-2), 70.2 (gal C-4), 69.7 (rha C-5), 67.4 (gal C-6), 18.0 (rha C-6).16

HPLC analysis

An LC-10A pump (Shimadzu Corporation Chro-matographic Instruments Division, Kyoto, Japan) was connected to an SPD-6A ultraviolet spectrophoto-metric detector set at UV 280 nm. The column

(3)

consisted of a Licrospher RP-18e, 10.0× 250 mm (Merck, Darmstadt, Germany). The solvent systems were 0.05M H3PO4– 0.05M KH2PO4– acetonitrile

(40:40:20 or 45:45:10). The flow rate was 3.0 ml min−1. The solvent was degassed prior to use.

Gas chromatographic analysis

GC/MS analysis was carried out using a HP-5890/5989B system equipped with a HP-1 capillary column (15 m× 0.25 mm × 0.25µm). Injector and detector temperatures were 250 and 300◦C, respec-tively. The temperature program was 60 – 140◦C (25◦C min−1) and then to 250◦C (10◦C min−1). Helium was used as carrier gas (0.7 bar, 1 ml min−1). The MS was taken at 70 eV. Scanning speed was 0.84 scans s−1and the scanning period was from 40 to 550 s.17Sample volume was 3µl. Split: 1:40.

1, 1-Diphenyl-2-picrylhydrazyl (DPPH) Free radical scavenging activity

The DPPH radical scavenging activity was eval-uated as reported previously.18 Briefly, each test

was conducted by placing a sample and 100µM

DPPH radical in Tris – HCl buffer solution (pH 7.4). After a 20 min incubation period at room temperature in the dark, absorbance was read at 517 nm.

Measurement of superoxide radical scavenging activity

Superoxide radical-scavenging activity was determined by the PMS – NADH superoxide generating system.19

The tested samples were added to a solution contain-ing 80µM PMS, 200µM NBT and 624µM NADH

in phosphate buffer (0.1M, pH 7.4). After

incubat-ing for 2 min at room temperature, absorbance was measured at 560 nm. The superoxide radical scav-enging activity was calculated using the following equation:

Scavenging activity (%)= [1 − (absorbance of sample at 560 nm)/(absorbance of control

at 560 nm)]× 100

Hydroxyl radical scavenging activity determined by electron paramagnetic resonance (EPR) spectrometry

Hydroxyl radicals were generated by the Fenton reaction according to the method of Kohno et al.20

In brief, the solution consisted of 0.1 mM H2O2,

0.1 mM FeSO4, 5 mM

5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and the sample. The spectrum of the DMPO-OH spin adduct was measured after the addition of FeSO4.21 Deionized water was used

instead of the sample solution for control exper-iments. All EPR spectra were recorded at ambi-ent temperature (298 K) on a Bruker EMX-6/1

EPR spectrometer equipped with WIN-EPR Sim-Fonia software (version 1.2). The EPR instru-ment settings were as follows: center field, 348.2± 5.0 mT; microwave power, 2 mW (2.354 GHz); modulation frequency, 100 kHz; modulation ampli-tude, 5 G; time constant, 0.6 s; conversion time, 83 ms.

Data analysis

The data are presented as the mean ± standard deviation (SD) of each triplet test.

RESULTS AND DISCUSSION

The herbs of G formosana were homogenized in 70% acetone. After the evaporation of acetone, the 70% acetone extract was then fractionated with a Diaion HP-20 column. The active fractions were evaluated for DPPH radical-scavenging activity. DPPH is a stable radical that has often been used to evaluate the antioxidant activity of plant extracts.22 When the concentration of the extracts was 25µg ml−1, the scavenging effects were 18.8, 39.3, 73.9, 34.2 and 10.6% for Fr A– E, respectively (Fig 1).

Both activity- and HPLC-directed purification of Fr B and Fr C by column and semi-preparative HPLC methods led to isolation of the principal active constituents. The chemical structures were identified by spectral data [FAB-MS, 1H- and 13C-NMR, heteronuclear multiple-bond correlation

(HMBC), and heteronuclear multiple-quantum cor-relation (HMQC)] and the sugar portions were ana-lyzed by GC-MS spectroscopy. The isolated flavonoid glycosides were methanolysed after methylation and subjected to GC-MS analyses. After comparison with the standards and reports in the literature, com-pounds I– IV were characterized as caffeic acid (I),13 quercetin 3-O-rutinoside (rutin) (II),14 kaempferol 3-O-rutinoside (nicotiflorin) (III),15 and kaempferol 3-O-robinobioside (IV),16respectively. The structures of these compounds are shown in Fig 2; this is the first

Fraction Inhibitory Activity (%) 0 20 40 60 80 100 Fr. A Fr. B Fr. C Fr. D Fr. E

Figure 1. DPPH inhibitory activity of each fraction (25µg ml−1) of

(4)

Figure 2. The structures of the isolated phenolic constituents. 0 50 100 150 200 250 300 Scavenging Activity (%) 0 20 40 60

80 Caffic acid (I)

Quercetin 3-O-rutinoside (II) Kaempferol 3-O-rutinoside (III) Kaempferol 3-O-robinobioside (IV)

Concentration (µg/mL)

Figure 3. The scavenging activity of the four constituents against the

DPPH radical.

time that these substances have been identified from this plant.

The four compounds I– IV were evaluated for their free radical scavenging activities. The four compounds showed dose-dependent curves when evaluated using the DPPH method (Fig 3). The IC50 values of the

DPPH radical were 6.7, 7.7, 300.3, and 286.7µMfor compounds I– IV, respectively (Table 1). The IC50s

of caffeic acid (I) and quercetin 3-O-rutinoside (II) were significantly lower than that of Trolox (IC50,

100.8µM), which was used as a positive control. The DPPH is a stable free radical; the ability to scavenge the DPPH radical is related to proton-donating ability and is one of several methods of antioxidation. The results imply that the antioxidative activity of caffeic

Table 1. DPPH, superoxide, and hydroxyl radical-scavenging

activities of isolated phenolic constituents of Gynura formosana IC50µMa

Compound DPPH Superoxide Hydroxyl

Caffeic acid (I) 6.6 187.3 4.4

Quercetin 3-O-rutinoside (II) 7.7 25.8 7.5 Kaempferol 3-O-rutinoside (III) 300.3 55.3 — Kaempferol 3-O-robinobioside (IV) 286.7 87.4 —

aThe molecular weights of caffeic acid, quercetin 3-O-rutinoside,

kaempferol 3-O-rutinoside, and kaempferol 3-O-robinobioside are 181 (M+ H)+, 611 (M− H)+, 595 (M+ H)+ and 595 (M+ H)+, respectively.

acid (I) and quercetin 3-O-rutinoside (II) may be attributed to their proton-donating ability.

The superoxide radical scavenging activities of the four compounds were determined by the PMS – NADH generating system. Commercial recom-binant human superoxide dismutase was used as a positive control. A560 nm min−1was negatively cor-related (r2= 0.998) with superoxide dismutase added

(20, 40 and 60 units, respectively, for 19.8, 43.0 and 61.3% scavenging activities). The IC50 values were

187.3, 25.8, 55.3, and 87.4µMfor compounds I– IV, respectively (Table 1). The IC50values of compounds

I– IV were equivalent to 1.4, 3.1, 1.5 and 0.9 unitµg−1 recombinant human superoxide dismutase, respec-tively. Superoxide (.O

2−) is the one-electron reduced

(5)

active free radicals that have the potential of reacting with biological cells and inducing tissue damage. The results reveal that the four isolated compounds are scavengers of superoxide radicals and have SOD-like ability.

The scavenging ability of the four compounds against the hydroxyl radical was investigated by EPR spectrometry. Hydroxyl radicals were generated by the Fenton reaction23 and trapped by DMPO

to form DMPO-OH adducts. The intensities of the DMPO-OH spin signal in EPR spectrometry were used to evaluate the scavenging activity of the isolated compounds. When the concentration was 5µM, the four isolated compounds exhibited hydroxyl radical-scavenging activity as shown in Fig 4. Kaempferol rutinoside (III) and kaempferol 3-O-robinobioside (IV) did not have significant inhibitory activities against the hydroxyl radical. Figure 5 shows the scavenging activity against the hydroxyl radical with different concentrations of caffeic acid (I) and quercetin 3-O-rutinoside (II). On the basis of DMPO-OH signal intensities, when the concentrations were

Figure 4. ESR spectra of the scavenging activity of the isolated

constituents at 5µMagainst hydroxyl radicals. (A) Control, (B) caffeic acid (I), (C) quercetin 3-O-rutinoside (II), (D) kaempferol

3-O-rutinoside (III), and (E) kaempferol 3-O-robinobioside (IV).

1, 2, 5 and 7µM, caffeic acid produced 31, 49, 72 and

78% decreases in hydroxyl radical levels (I). When the concentrations were 5, 7, 10 and 15µM, quercetin

3-O-rutinoside (II) caused decreases in hydroxyl radicals

of 37, 48, 60 and 87%, respectively. The IC50values

of caffeic acid (I) and quercetin 3-O-rutinoside (II) were 4.4 and 7.5µM, respectively (Table 1).

Hydroxyl radicals are among the strongest free radicals; they have damaging effects on living cells. They produce other kinds of cell-damaging free radicals and oxidizing agents,24 which can attack

DNA to cause strand scission. In biochemical sys-tems, superoxide radical is converted by superoxide dismutase to hydrogen peroxide, which can subse-quently generate extremely reactive hydroxyl radi-cals in the presence of certain transition metal ions such as iron or copper, especially iron,25 or by UV photolysis.26 In this experiment, caffeic acid (I) showed the most active effect; the EPR signal was significantly decreased in comparison to the con-trol. Fig 5 provides the first piece of evidence that the four isolated compounds exhibited scavenging activity against the hydroxyl radical as shown by EPR spectrometry.

The four isolated compounds are phenolics. Com-pounds II, III and IV are related to 3-substituted flavone glycosides. There are two sugars in the struc-tures of these three flavonoids. The sugar constituents of compounds II and III are rhamnose and glucose; compound IV includes rhamnose and galactose. The aglycon portion of compound II is quercetin, and that of compounds III and IV is kaempferol. A compar-ative analysis of the antioxidant activity of flavonoid glycosides and their flavonoid aglycones has demon-strated that the aglycone form can confer a higher antioxidant activity. Caffeic acid (I) and quercetin

3-O-rutinoside (II) showed good activity against DPPH

and hydroxyl radicals. This may be attributed to the structure of catechol; the phenolic portions of the structures may be important determinants of antioxi-dant activity.

CONCLUSION

In Taiwan, G formosana is a cultured and popular vegetable; it is also used as a folk medicine. This study demonstrated that a 70% aqueous acetone extract of G formosana has free-radical scavenging activity. It is well known that free radicals contribute to the causation of several diseases, such as coronary heart disease, atherosclerosis and various cancers. Phenolic compounds are commonly found in the plant kingdom, and they have been reported to have multiple biological effects, including antioxidant activity.27 – 29

In the current study, we examined the phenolic content of G formosana. Four phenolic constituents from G formosana were identified. None of these phenolic compounds had been previously reported in

G formosana. It would be interesting and worthwhile to

(6)

Figure 5. ESR spectra of the scavenging activity of (A) caffeic acid (I) and (B) quercetin 3-O-rutinoside (II) against hydroxyl radicals. (A) (i) control,

(ii) 1µM, (iii) 2µM, (iv) 5µMand (v) 7µM; (B) (i) control, (ii) 5µM, (iii) 7µM, (iv) 10µMand (v) 15µM.

of G formosana in preventing diseases caused by the overproduction of radicals; this plant may prove to be a useful source of dietary antioxidants in the future.

ACKNOWLEDGEMENTS

We thank Mu-Cun Gao, Department of Botany, National Taiwan University, for plant identification. This study was supported by a grant from the Taipei Medical University (grant no. TMU91-Y05-A126). The authors also express thanks for financial support for this work from the Juridical Person of Yen’s Foundation, Taiwan.

REFERENCES

1 Kawanishi S, Hiraku Y and Oikawa S, Mechanism of guanine-specific DNA damage by oxidative stress and its role in carcinogenesis and aging. Mutat Res 488:65–76 (2001). 2 Traganos F, Dihydroxyanthraquinone and related

bis(substitu-ted) aminoanthraquinones: a novel class of antitumor agents.

Pharmac Ther 22:199–214 (1983).

3 Xie B, Shi H, Chen Q and Ho CT, Antioxidant properties of fractions and polyphenol constituents from green, oolong and black teas. Life Sci 17:77–84 (1993).

4 Formica JV and Regelson W, Review of the biology of quercetin and related bioflavonoids. Food Chem Toxicol 33:1061–1080 (1995).

5 Gantet P and Memelink J, Transcription factors: tools to engineer the production of pharmacologically active plant metabolites. Trends Pharmac Sci 23:563–569 (2002).

6 Vom Endt D, Kijne JW and Memelink J, Transcription factors controlling plant secondary metabolism: what regulates the regulators? Phytochemistry 61:107–114 (2002).

7 Weisburger JH, Approaches for chronic disease prevention based on current understanding of underlying mechanisms.

Am J Clin Nutr 71:1710S –1714S (2000).

8 Nijveldt RJ, van Nood E, van Hoorn DE, Boelens PG, van Norren K and van Leeuwen PA, Flavonoids: a review of probable mechanisms of action and potential applications.

Am J Clin Nutr 74:418–425 (2001).

9 Lin WY, Teng CM, Tsai IL and Chen IS, Anti-platelet aggre-gation constituents from Gynura elliptica. Phytochemistry

53:833–836 (2000).

10 Wiedenfeld H, Kirfel A, Roder E and Will G, Two pyrrolizidine alkaloids from Gynura scandens. Phytochemistry 21:2767–2768 (1982).

11 Bohlmann F and Zdero C, Gynuron, a new terpene-coumarin — derivative as Gynira crepioides. Phytochemistry

16:494–495 (1977).

12 Jong TT and Ju-Yueh CH, An optically active chromanone from

Gynura formosana. Phytochemistry 44:553–554 (1997).

13 Cui CB, Tezuka Y, Kikuchi T, Nakano H, Tamaoki T and Park JH, Constituents of a fern, Davallia mariesii Moore. I. Isolation and structures of davallialactone and a new flavanone glucuronide. Chem Pharm Bull 38:3218–3225 (1990). 14 Lommen A, Godejohann M, Venema DP, Hollman PC and

Spraul M, Application of directly coupled HPLC-NMR-MS to the identification and confirmation of quercetin glycosides and phloretin glycosides in apple peel. Anal Chem

72:1793–1797 (2000).

15 Calzada F, Cedillo-Rivera R and Mata R, Antiprotozoal activity of the constituents of Conyza filaginoides. J Nat Prod

64:671–673 (2001).

16 Hasan A, Ahmed I, Jay M and Voirin B, Flavonoid glycosides and an anthraquinone from Rumex chalepensis. Phytochemistry

(7)

17 Mejanelle P, Bleton J, Goursaud S and Tchapla GA, Identifica-tion of phenolic acids and inositols in balms and tissues from an Egyptian mummy. J Chromatogr A 767:177–186 (1997). 18 Lee MH, Lin RD, Shen LY, Yang LL, Yen KY and Hou WC,

Monoamine oxidase B and free radical scavenging activities of natural flavonoids in Melastoma candidum D. Don. J Agric

Food Chem 49:5551–5555 (2001).

19 Kuo CC, Shih MC, Kuo YH and Chiang W, Antagonism of free-radical-induced damage of adlay seed and its antiproliferative effect in human histolytic lymphoma U937 monocytic cells. J Agric Food Chem 49:1564–1570 (2001).

20 Kohno M, Yamada M, Mitsuta K, Mizuta Y and Yoshikawa T, Spin-trapping studies on the reaction of iron complexes with peroxides and the effects of water-soluble antioxidants. Bull

Chem Soc Japan 64:1447–1453 (1991).

21 Ohsugi M, Fan W, Hase K, Xiong Q, Tezuka Y, Komatsu K, Namba T, Saitoh T, Tazawa K and Kadota S, Active-oxygen scavenging activity of traditional nourishing-tonic herbal medicines and active constituents of Rhodiola sacra.

J Ethnopharmacol 67:111–119 (1999).

22 Hu C and Kitts DD, Studies on the antioxidant activity of Echinacea root extract. J Agric Food Chem 48:1466–1472 (2000).

23 Iinuma S, Naito Y, Yoshikawa T, Takahashi S, Takemura T, Yoshida N and Kondo M, In vitro studies indicating antiox-idative properties of rebamipide. Dig Dis Sci 43:35S –39S (1998).

24 Liu F and Ng TB, Antioxidative and free radical scavenging activities of selected medicinal herbs. Life Sci 66:725–735 (2000).

25 Halliwell B and Gutteridge JM, Formation of thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts: the role of superoxide and hydroxyl radicals. FEBS

Lett 128:347–352 (1981).

26 Keum YS, Park KK, Lee JM, Chun KS, Park JH, Lee SK, Kwon H and Surh YJ, Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng.

Cancer Lett 150:41–48 (2000).

27 Sakihama Y, Cohen MF, Grace SC and Yamasaki H, Plant phenolic antioxidant and prooxidant activities: phenolics-induced oxidative damage mediated by metals in plants.

Toxicol 177:67–80 (2002).

28 Tapiero H, Tew KD, Ba GN and Mathe G, Polyphenols: do they play a role in the prevention of human pathologies?

Biomed Pharm 56:200–207 (2002).

29 Bors W and Michel C, Chemistry of the antioxidant effect of polyphenols. Ann NY Acad Sci 957:57–69 (2002).

數據

Figure 1. DPPH inhibitory activity of each fraction (25 µg ml −1 ) of
Figure 2. The structures of the isolated phenolic constituents. 0 50 100 150 200 250 300Scavenging Activity (%)0204060
Figure 4. ESR spectra of the scavenging activity of the isolated
Figure 5. ESR spectra of the scavenging activity of (A) caffeic acid (I) and (B) quercetin 3-O-rutinoside (II) against hydroxyl radicals

參考文獻

相關文件

The performance guarantees of real-time garbage collectors and the free-page replenishment mechanism are based on a constant α, i.e., a lower-bound on the number of free pages that

(2) Sze-Bi Hsu, Feng-Bin Wang* and Xiao-Qiang Zhao, Global Dynamics of Zooplankton and Harmful Algae in Flowing Habitats, Journal of Differential Equations, Vol. Grover* and

Theorem 3.1, together with some algebraic manipulations, implies that the quantum corrections attached to the extremal ray exactly remedy the defect caused by the classical product

reading scheme, cross-curricular projects and RaC, etc.) in consideration of the pedagogy and connection with the curriculum of English Language from the case study of exemplars

reading scheme, cross-curricular projects and RaC, etc.) in consideration of the pedagogy and connection with the curriculum of English Language from the case study of exemplars

Wang, Solving pseudomonotone variational inequalities and pseudocon- vex optimization problems using the projection neural network, IEEE Transactions on Neural Networks 17

Define instead the imaginary.. potential, magnetic field, lattice…) Dirac-BdG Hamiltonian:. with small, and matrix

35 you use our 100% Liquid Egg Whites, you’ll know that they are fat free, cholesterol free, cage free and stress free. Just twist the lid, pop the top