The American Journal of Chinese Medicine, Vol. 37, No.

4, 815828 2009 World Scientic Publishing Company Institute for Advanced Research in Asian Science and Medicine

Antioxidant and Antiradical Activities of Wu Ling Shen in a Cell Free System

Huey-Jiun Ko, Airong Song, Min-Nan Lai and Lean-Teik Ng
Department of Food Science

National Chiayi University, Chiayi, Taiwan

Qingdao Agricultural University, Qingdao, Shandong, China Kang Jian Biotech Corp., Ltd., Nantou Hsien, Taiwan Department of Agricultural Chemistry National Taiwan University, Taipei, Taiwan

Abstract: The present study aimed to investigate the antioxidant and antiradical activities of Wu Ling Shen, a popular medicinal fungus (Xylaria nigripes) used in traditional Chinese medicine preparations. Two different X. nigripes materials, the cultivated X. nigripes mycelia (XN) and a commercial X. nigripes product (XNP), were used to prepare the aqueous (XNH vs. XNP-H) and ethanol (XN-E vs. XNP-E) extracts for this study. Polyphenol and total polysaccharide contents of these extracts were also examined. Results showed that extracts of XN possessed stronger antioxidant and antiradical activities than XNP in all tested model systems. However, all extracts exhibited a weak activity in metal chelation and reducing power. Total antioxidant activity of XN extracts (IC50 6.20 g/ml for XN-H and 5.41 g/ml for XN-E), but not XNP extracts (IC50 128.13 g/ml for XNP-H and 96.16 g/ml for XNP-E), was more potent than Trolox (IC50 19.64 g/ml) and vitamin C (IC50 26.39 g/ml). XN-E (IC50 5.12 g/ml) and XNP-E (IC50 8.89 g/ml) possessed a relatively similar potency as that of positive controls (IC50 6.94 g/ml for Trolox and 4.25 g/ml for vitamin C) in the superoxide radical scavenging activity. Although the DPPH radical scavenging of XN extracts was weaker than that of Trolox and vitamin C, it was about eight times more potent than that of XNP extracts. In ABTS assay, both XN and XNP extracts exhibited a moderate ABTS radical scavenging activity. Among the different extracts, XN-E showed the highest total avonoid (32.69 mg/g) and phenol (59.75 mg/g) contents, while XNP-H (7.50% w/w) had the highest level in total polysaccharide content. These results conclude that XN-E possesses the most potent antioxidant and antiradical activities, and that these activities could be derived from its high polyphenol content, but not the level of polysaccharides. Keywords: Wu Ling Shen; Xylaria nigripes; Antioxidant; Antiradical; Polyphenols; Polysaccharides.
Correspondence to: Dr. Lean-Teik Ng, Department of Agricultural Chemistry, National Taiwan University, No.1, Sec. 4, Roosevelt Road, Taipei, Taiwan. Tel: (+886) 2-3366-4804, Fax: (+886) 2-3366-9907, E-mail: nglt97@ntu.edu.tw

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Introduction Free radicals are known to be the major cause of various chronic and degenerative diseases, including aging, coronary heart disease, inammation, cancer and others (Slater, 1984; Halliwell, 1997). Consumption of vegetables and fruits, which are rich in antioxidants such as vitamins and phenolic compounds, are believed to be effective in preventing these diseases through reducing oxidative stress and inhibiting lipid peroxidation in biological systems. Recent studies also indicated that therapeutic benets of certain crude drugs of plant origins were derived from their antioxidant activities (Hu et al., 2003; Zou et al., 2004; Wu and Ng, 2008). These antioxidant activities were reported to be correlated with total phenolic content. This nding was also observed in certain medicinal mushrooms (Cheung et al., 2003; Lo and Cheung, 2005). Xylaria nigripes (Koltz.) Sacc. of family Xylariaceae, also known as Wu Ling Shen, is a less studied medicinal fungus. It is found growing in the wild around abandoned termite nests. Traditionally, it is known to possess therapeutic benets, such as treating insomnia and trauma and is also used as a diuretic and nerve tonic (Ma et al., 1999). Despite its popularity, systematic scientic investigations on its chemical and biological properties remain limited; many of its therapeutic properties are still based on traditional beliefs. It remains unknown whether certain therapeutic claims of either the aqueous or ethanolic extract of XN are derived from its antioxidant activities. With the recent increasing interest in the therapeutic potential of XN products, our aim of this study was to conduct a systematic evaluation of the antioxidant and antiradical activities of different XN extracts, which were prepared according to the traditional medical practice; polyphenol and total polysaccharide contents of these extracts were also determined. Materials and Methods Chemicals Nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), ferrozine, ethylenediaminetetraacetic acid (EDTA), linoleic acid, 2,2 -azino-bis[3-ethylbenthiazoline-6-sulfonic acid] (ABTS) and xanthine oxidase were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from MP Biomedicals Inc. (Eschwege, Germany). Vitamin C, dimethylsulphoxide (DMSO) and ferrous chloride were obtained from Wako Pure Chemical Industries (Osaka, Japan). Trolox was obtained from Aldrich Chemical (Milwaukee, WI, USA). All other chemicals used were of analytical grade. Preparation of X. nigripes Extracts Two different X. nigripes materials, namely X. nigripes mycelia (XN) obtained from Kang Jian Biotech Co. (Nantou Hsien, Taiwan) and X. nigripes mycelia product (XNP) purchased from a drug store in Hangzhou (China), were used in this study. To prepare the aqueous

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extract, 100 g of XN and XNP powders were respectively extracted with 1 liter of boiling water for 1 hour. The extract was ltered with lter paper (Advantec No. 1, Japan) while the residue was re-extracted under the same conditions twice. The ltrates were combined, concentrated and lyophilized. The dried powdered extracts named XN-H and XNP-H were collected and stored at 4 C until use. For the ethanol extract, 100 g of XN and XNP powders were respectively soaked with 1 liter of ethanol (95%) at room temperature for 6 days. After ltering the extract with lter paper (Advantec No. 1, Japan), the ltrate collected was concentrated and lyophilized. The dried powdered-extracts named XN-E and XNP-E were collected and stored at 4 C until use. Total Antioxidant Activity Total antioxidant activity of XN and XNP extracts was determined by the thiocyanate method (Mitsuda et al., 1996). In brief, 2 ml of different concentrations of XN and XNP extracts were mixed with 3 ml of linoleic acid emulsion consisting of 2.5 g Tween-20, 2.5 g linoleic acid, and 0.04 M potassium phosphate buffer (pH 7.0). The mixed solution in a glass ask was incubated at 37 C. After reacting with FeCl2 and thiocyanate at several time intervals, the peroxide value was measured at wavelength 500 nm. Trolox and vitamin C were used as positive controls. ABTS Radical Scavenging Activity The scavenging activity of ABTS+ was measured according to the method described by Re et al. (1999) with some modications. Briey, ABTS was dissolved in deionized water to 7 mM in concentration, which was then mixed with 2.45 mM potassium persulfate. The scavenging activity was determined by mixing 180 l ABTS reagent with 40 l of sample, negative (PBS; phosphate buffered saline) or positive controls (i.e. Trolox and vitamin C), followed by measuring the absorbance at 734 nm. Superoxide Radical Scavenging Activity The superoxide anion scavenging activity of XN and XNP extracts was conducted according to the method described by Gulcin (2006). In brief, superoxide radicals were generated in 3 ml of phosphate buffer (0.1 M, pH 7.4) containing 1 ml of NBT (300 M), 1 ml of PMS (120 M), 1 ml of NADH (968 M) and 1 ml of sample at various concentrations. The mixture was spectrophometically measured at 560 nm. Trolox and vitamin C was used as positive controls. DPPH Radical Scavenging Activity DPPH radical scavenging activity was determined according to the method described previously (Wu and Ng, 2008). Briey, 1 ml of 0.1 mM DPPH radical solution was mixed with

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3 ml of various concentrations of XN and XNP extracts in methanol. The mixture was then vortexed vigorously and left for 30 min at 40C in the dark. Methanol was used as the baseline control. The absorbance was measured at 517 nm. Metal Chelating Activity The chelating effect of ferrous ions by the XN and XNP extracts was determined according to the method described previously (Wu and Ng, 2008). In brief, 1 ml of sample at different concentrations was mixed with 3.7 ml of methanol and 0.1 ml of 2 mM FeCl2 . The reaction was initiated by the addition of 0.2 ml of 5 mM ferrozine, followed by vigorous shaking and was left at room temperature for 10 min. The absorbance was measured spectrophotometrically at 562 nm. EDTA, a strong metal chelator, was used as a standard metal chelator agent. Reducing Power The reducing power of XN and XNP extracts was determined according to the method of Oyaizu (1986). In brief, 2.5 ml of each extract at different concentrations was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. After incubating the mixture at 50 C for 20 min, 2.5 ml of 10% trichloroacetic acid was added, followed by centrifuging at 3,000 rpm for 10 min. Five milliliters of the upper layer solution were mixed with 5 ml distilled water and 1 ml of 0.1% FeCl3 . The absorbance of the mixture was then measured at 700 nm. Total Polysaccharide Analysis The assay was carried out according to procedures described by Fang and Zhong (2002) with some modications. In brief, 0.5 g of dried XN and XNP powders were reuxed with 50 ml of boiling water in hot water bath for 30 min. After cooling, the sample was ltered with lter paper (Advantec No. 1, Toyo, Japan) to obtain the ltrate, which was then transferred to a dialysis tube (Molecular weight cutoff 12,00014,000 Daltons; Medicell International Ltd., London, UK). One mg of -amylase and 1 mg of -glucosidase were added to the ltrate before subjecting to dialysis for 3 days. After adjusting the dialysate to a volume of 50 ml with distilled water, 1 ml of diluted dialysate was taken for total polysaccharide measurement by using phenol-sulfuric acid assay (Dubois et al., 1956). Polyphenols Analysis The total avonoid and phenol contents of XN and XNP extracts were determined by the colorimetric method (Zou et al., 2004) and the Folin-Ciocalteu method (Cliffe et al., 1994), respectively.

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Statistical Analysis Data were presented as means standard deviations (SD). Results were evaluated by one way ANOVA, followed by Duncans multiple range tests using the Statistical Analysis System (SAS Institute, Cary, NC, USA). Differences were considered signicant when p < 0.05. Results Total Antioxidant Activity Results showed that both XN extracts (IC50 6.20 g/ml and 5.41 g/ml for XN-H and XN-E, respectively) exhibited stronger total antioxidant activities than XNP extracts (IC50 128.13 g/ml and 96.16 g/ml for XNP-H and XNP-E, respectively) (Table 1). Furthermore, their activities were also more potent than vitamin C (IC50 26.39 g/ml) and Trolox (IC50 19.64 g/ml). At concentration of 10 g/ml, XN-H and XN-E showed a total inhibitory rate of 75.9% and 78.55% respectively, however, for XNP-H and XNP-E, their inhibitory rates remained lower than 50% even at 50 g/ml (Fig. 1). ABTS Radical Scavenging Activity Table 1 shows that all XN and XNP extracts displayed antioxidant activities as they were able to scavenge the ABTS+ radical cation. However, their potency was moderate as demonstrated by the relatively high IC50 values (ranging from 248.21 g/ml to 464.82 g/ml) as
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% Lipid peroxidation inhibition

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0 XN-H (10 g/ml) XN-E (10 g/ml) XNP-H (50 g/ml) XNP-E (50 g/ml)
Figure 1. Total antioxidant activity of different XN (aqueous: XN-H and ethanol: XN-E) and XNP (aqueous: XNPH and ethanol: XNP-E) extracts. Data are presented as percentage of lipid peroxidation inhibition, means SD (n = 3).

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Table 1. IC50 Values of Antioxidant and Antiradical Activities of Different XN and XNP Extracts IC50 Values ( g/ml) Samples XN-H XN-E XNP-H XNP-E Trolox Vitamin C EDTA TAA 6.20 5.41 128.13 96.16 19.64 26.39 ABTS 248.21 464.82 282.67 362.87 29.03 SOD 24.86 5.12 38.45 8.89 6.94 4.25 DPPH 62.07 73.49 487.68 > 500 5.88 48.38 Metal Chelation > 500 > 500 > 500 > 500 54.50 Reducing Power > 500 > 500 > 500 > 500 66.91 23.17

TAA: Total antioxidant activity.

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% ABTS radical scavenging

60

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20

0
XN-H (300 g/ml) XN-E (300 g/ml) XNP-H(300 g/ml) XNP-E (300 g/ml)

Figure 2. ABTS radical scavenging activity of different XN (aqueous: XN-H and ethanol: XN-E) and XNP (aqueous: XNP-H and ethanol: XNP-E) extracts. Data are presented as the percentage of ABTS radical scavenging, means SD (n = 3).

compared to the positive control, Trolox (IC50 29.03 g/ml). Among the different XN and XNP extracts, XN-H (IC50 248.21 g/ml) showed the best ABTS radical scavenging activity, with 55.27% of ABTS scavenging activity at 300 g/ml (Fig. 2). Superoxide Radical Scavenging Activity Figure 3 shows the inhibitory percentage of XN and XNP extracts on superoxide anion radical generation. Results showed that at concentration of 25 g/ml, the percentage of inhibition on superoxide radical generation by XN-H and XN-E was 75% and 83%, respectively, which were comparable to the positive controls Trolox (65%) and vitamin C (80%) at the same concentration. The superoxide scavenging effect was found to increase with increasing

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% Superoxide radical scavenging

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0 XN-H (10 g/ml) XN-E (10 g/ml) XNP-H (25 g/ml) XNP-E (25 g/ml)

Figure 3. Superoxide radical scavenging activity of different XN (aqueous: XN-H and ethanol: XN-E) and XNP (aqueous: XNP-H and ethanol: XNP-E) extracts. Data are presented as the percentage of superoxide radical scavenging, means SD (n = 3).

concentration of XN extracts regardless of the nature of preparation. Based on IC50 values, the activity of superoxide radical scavenging of XN-E (IC50 5.12 g/ml) appeared slightly weaker than that of vitamin C (IC50 4.25 g/ml), but it was stronger than that of Trolox (IC50 6.94 g/ml) (Table 1). XNP-E also exhibited potent superoxide radical scavenging activity (IC50 8.89 g/ml).

DPPH Radical Scavenging Activity Results showed that both XN-H and XN-E, but not XNP extracts, were effective in reducing the stable radical DPPH to the yellow-colored diphenylpicrylhydrazine, indicating that these extracts were active in DPPH radical scavenging and were concentration dependent (Fig. 1). The inhibition rate of XN-H (IC50 62.07 g/ml) was stronger than that of XN-E (IC50 73.49 g/ml), as shown in Table 1. The performance of both XN extracts was better than that of XNP-H (IC50 487.68 g/ml) and XNP-E (IC50 > 500 g/ml), but was weaker than that of Trolox (IC50 5.88 g/ml) and vitamin C (IC50 48.38 g/ml) at the same concentration.

Reducing Power In this study, the yellow color of the test solution changing to various shades of green and blue depends upon the reducing power of each sample. As shown by the IC50 values, both XN and XNP extracts have a weak reducing power (Table 1).

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% DPPH radical scavenging

60

40

20

0
XN-H (100 g/ml) XN-E (100 g/ml) XNP-H (300 g/ml) XNP-E (300 g/ml)

Figure 4. DPPH radical scavenging activity of different XN (aqueous: XN-H and ethanol: XN-E) and XNP (aqueous: XNP-H and ethanol: XNP-E) extracts. Data are presented as the percentage of DPPH radical scavenging, means SD (n = 3).

Metal Chelating Activity Table 1 shows that both XN and XNP extract possessed a weak metal chelating activity. This was demonstrated by the requirement of a higher concentration of extracts to achieve signicant effectiveness in inhibiting the formation of ferrous and ferrozine complex. Besides EDTA, all test samples showed an IC50 value of greater than 500 g/ml.

Total Polysaccharide Content Figure 5 shows the total polysaccharide content in XN and XNP extracts. Results showed that XNP-H (7.50% w/w) possessed the highest total polysaccharide content, followed by XN-H (2.91% w/w), XN-E (2.70% w/w) and XNP-E (1.95% w/w).

Total Flavonoid and Phenol Contents Figures 6(a) and 6(b) show the total avonoid and phenol contents in XN and XNP extracts. The content of total avonoids (mg/g) in X. nigripes extracts, as expressed in rutin equivalents, varied from 5.31 to 32.69 mg/g, while the total phenolic content, as expressed in gallic equivalents, varied between 14.30 and 59.75 mg/g. Interestingly XN-E extract possessed the highest total avonoid (32.69 mg/g) and total phenol (59.75 mg/g) contents. However, a relatively low amount of total avonoids was noted in XN-H (7.32 mg/g), XNP-H (5.31 mg/g) and XNP-E (12.84 mg/g). XNP-E also possessed a relatively low amount of total phenolics (14.30 mg/g).

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Total polysaccharides (% w/w)

0 XN-H XN-E XNP-H XNP-E

Figure 5. Total polysaccharide content of different XN (aqueous: XN-H and ethanol: XN-E) and XNP (aqueous: XNP-H and ethanol: XNP-E) extracts. Data are presented as means SD (n = 3); bars with the different superscript letters were signicantly different at p < 0.05 as analyzed by Duncans multiple range tests.

Discussion The present study has demonstrated that the antioxidant and antiradical activities of XN extracts are generally more potent than XNP extracts in in vitro assays. We also showed that the potency of these extracts differ depending on the solvent used for extraction, suggesting a possible difference in concentrations and type of antioxidative compounds present in these extracts. Both XN-H and XN-E extracts also exhibited a relatively similar or better potency than Trolox and vitamin C in total antioxidant and superoxide radical scavenging activities. These activities could be derived from the presence of higher polyphenol contents, but not the level of polysaccharides. The differences in antioxidant activities, polyphenol and polysaccharide contents between XN and XNP could be due to the variation in culture conditions (Fan et al., 2003; Yang et al., 2003). Many studies have shown that natural antioxidants are able to reduce DNA damage, mutagenesis, carcinogenesis and inhibit pathogenic bacteria growth. These events are often associated with the termination of free radical propagation in biological systems (Covacci et al., 2001; Zhu et al., 2002; Lin et al., 2007). Thus, the antioxidant capacity of crude drugs is widely used as a parameter for evaluating medicinal bioactive components. In this study, we examined the antioxidant activities of XN and XNP extracts, and compared them to that of Trolox (a water soluble form of -tocopherol) and vitamin C. Although the results showed that both XN and XNP extracts possessed a weak activity in metal chelation and reducing power, the potent capacity of XN in free radical scavenging and inhibiting lipid peroxidation could be of considerable interest. The amount of peroxide produced during the initial stages of lipid oxidation was measured as the total antioxidant capacity. Results showed that XN extracts possessed potent total

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(a) Total avonoids

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Concentration (mg/g)

30

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b
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0 XN-H XN-E
(b) Total phenols

XNP-H

XNP-E

80

Concentration (mg/g)

a
60

40

b c
20

0 XN-H XN-E XNP-H XNP-E

Figure 6. Total avonoid and phenol contents of different XN (aqueous: XN-H and ethanol: XN-E) and XNP (aqueous: XNP-H and ethanol: XNP-E) extracts. Data are presented as means SD (n = 3); bars with the different superscript letters were signicantly different at p < 0.05 as analyzed by Duncans multiple range tests.

antioxidant activity, which is 1520 fold stronger than XNP extracts, and about 3 fold and 5 fold more active than Trolox and vitamin C, respectively. This suggests that XN extracts are effective in inhibiting the lipid peroxidation. The phenolic compounds and other chemical components present in these extracts may have suppressed lipid peroxidation

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through chemical mechanisms such as free radical quenching, electron transfer, radical addition or radical recombination. Superoxide anions are precursors to active free radicals that have potential for reacting with biological macromolecules and thereby inducing tissue damages (Halliwell and Gutteridge, 1984). They have also been noted to directly initiate lipid peroxidation (Wickens, 2001), and play important roles in the formation of other ROS such as hydrogen peroxide, hydroxyl radical, and singlet oxygen, which further induce oxidative damage in lipids, proteins, and DNA (Pietta, 2000). Studies have shown that these damages could be prevented through the scavenging of superoxide anion radicals by antioxidants (Yen and Duh, 1994). We showed that XN-E and XNP-E possessed excellent superoxide radical scavenging activity, suggesting that these extracts could be effective in reducing oxidative stress related diseases. DPPH has been widely used to evaluate the free radical scavenging effectiveness of various antioxidant substances (Cotelle et al., 1996; Ozcelik et al., 2003). According to Liu et al. (2007), antioxidant activities of Xylaria sp. could be derived from phenolic compounds such as 2-hydrazino-8-hydroxy-4-phenylquinoline, 3,4-dimethoxyphenol, 2,4bis(1,1-dimethylethyl)phenol, 3,4-di-hydro-8-hydroxy-3-methylisocoumarin and ferruginol. In another study, it was shown that 5,8-dihydroxy-3-methyl-3,4-digydroisocoumarin, a compound isolated from X. nigripes, possessed a stronger DPPH radical scavenging activity, which was noted to be 1.67 and 2.10 times more potent than ascorbic acid and -tocopherol, respectively (Wu, 2001). Although the specic antioxidant compounds were not elucidated in this study, it is possible that these compounds contributed to the potent DPPH scavenging activity of XN extracts. Plant polysaccharides have been demonstrated to possess strong antioxidant properties (Ramarahnam et al., 1995; Hu et al., 2003). According to Liu et al. (1997), mushroom polysaachrides and protein-bound polysaccharides possessed signicant activities in superoxide and hydroxyl radical scavenging activities. Polysaccharides extracted from Grifola frondosa (Lee et al., 2003), Auricularia auricular (Fan et al., 2007) and Ganoderma tsugae (Tseng et al., 2008) were reported to exhibit potent antioxidant properties. Methanolic, hot and cold water extracts of G. tsugae also showed good antioxidant activities (Mau et al., 2005a; 2005b; Tseng and Mau, 2007; Tseng et al., 2008). The DPPH and superoxide anion radical scavenging activities of Ganoderma atrum were reported to increase by their increasing concentration of polysaccharide content (Chen et al., 2008). In this study, although XNP-H possessed a higher level of total polysaccharide content than XN extracts, their antioxidant and antiradical activities were weaker, suggesting that these activities may not be derived from the level of polysaccharide content. Phenolic compounds of plants (Shahidi et al., 1992) and mushrooms (Cheung et al., 2003; Lo and Cheung, 2005) have been shown to possess powerful antioxidants and free radical scavenging activities. Therefore, it is possible that the antioxidant activity of XN is correlated with its total phenolic contents. Phenolic and avonoid compounds have been reported to play an important role in stabilizing lipid oxidation in biological systems (Halliwell and Gutteridge, 1984; Shahidi et al., 1992; Leong and Shui, 2002; Wu and Ng, 2008). In this study, we revealed that XN extracts, especially XN-E, contained a signicant higher level

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of phenolics and avonoids than XNP extracts and other reported medicinal fungus (Liu et al., 2007). It suggests that the high antioxidant and free radical scavenging activities of XN-E may result from the coexistence of phenolic and avonoid-type compounds. This is in agreement with previous reports that the phenolic and avonoid compounds contribute signicantly to the antioxidant activities of mushrooms (Cheung et al., 2003; Liu et al., 2007). In conclusion, this was the rst detailed study of antioxidant and antiradical activities of X. nigripes extracts. We showed that XN extracts possessed antioxidant and antiradical activities at different magnitudes of potency and were more potent than XNP under the tested model systems. The potent antioxidant and free radical scavenging activities of XN-E could be derived from compounds such as avonoids and phenols, but not polysaccharides. Taken together, these antioxidant activities could have contributed, at least partly, to the therapeutic benets of certain traditional claims of XN. To further support the antioxidant benets of XN, isolation of its bioactive compounds coupled with an in vivo study warranted for further investigations. Acknowledgments The authors would like to thank the National Science Council of Taiwan for funding this study under grant number NSC 95-2313-B-002-121-MY3. References
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