International Journal of Medicinal Mushrooms, 17(2): 139–148 (2015)

1391045-4403/15/$35.00 © 2015 Begell House, Inc. www.begellhouse.com

Biological Activities of the Polysaccharides Produced from Different Sources of Xylaria nigripes (Ascomycetes), a Chinese Medicinal FungusMu-Chun Hung,1 Chia-Chun Tsai,1 Tai-Hao Hsu,1,2,* Zeng-Chin Liang,3 Fang-Yi Lin,2 Shih-Liang Chang,1 Wai-Jane Ho,1 & Chang-Wei Hsieh1,†

1Department of Medicinal Botanicals and Health Applications, Da-Yeh University, Datsuen, Changhua, 51591, Taiwan (R. O. C.); 2Department of Bioindustry Technology, Da-Yeh University, Datsuen, Changhua, 51591, Taiwan (R. O. C.); 3Department of Bioresources, Da-Yeh University, Datsuen, Changhua, 51591, Taiwan (R. O. C.)

†Address all correspondence to: Tai-Hao Hsu, Department of Medicinal Botanicals and Healthcare, Da-Yeh University, Datsuen, Changhua 51591, Taiwan (R. O. C.); Tel.: +886-4-8511888, ext 2288; FAX: +886-4- 8511304; E-mail: th4420@gmail.com

*Address all correspondence to: Chang-Wei Hsieh, Department of Medicinal Botanicals and Health Applications, Da-Yeh University, Datsuen, Changhua 51591, Taiwan (R. O. C.); Tel: +886-4-8511888, ext 1790; FAX: +886-4-8511349; E-mail: welson@mail.dyu.edu.tw

ABSTRACT: Xylaria nigripes, a local rare medicinal fungus, has multi-antioxidant activities owing to its water extraction as shown by previous research. However, the main indicator causing the antioxidant effect was not clear, so this research focused on the antioxidant activities from different sources of X. nigripes such as fruiting body polysaccharides, mycelium intracellular polysaccharides, mycelium extracellular polysaccharides, and their deproteinization products. The mycelium intracellular polysaccharide (XnIPS-1) from X. nigripes showed the highest reducing power of antioxidant activity, since it revealed the lowest IC50 values in all the assayed methodologies. The IC50 values of chelating ferrous ion ability, ABTS radical scavenging activity, and DPPH free radical scavenging were 1412, 174.25, and 351.56 μg/mL, respectively. In addition to these results, this research also explored the mechanism between polysaccharides and antioxidants compared by FT-IR analysis. The spectrum shows that the X. nigripes polysaccharide structure changed after the proteins were removed.

KEY WORDS: medicinal mushrooms and fungi, Xylaria nigripes, extracellular polysaccharide, intracellular poly-saccharide, antioxidant

ABBREVIATIONS: XnFB-1: crude polysaccharide of X. nigripes fruiting body; XnFB-2: polysaccharide of X. nigripes fruiting body after deproteinization; XnIPS-1: crude intracellular polysaccharide of X. nigripes mycelium; XnIPS-2: intracellular polysaccharide of X. nigripes mycelium after deproteinization; XnEPS-1: crude extracellular polysaccharide of X. nigripes mycelium; XnEPS-2: extracellular polysaccharide of X. nigripes mycelium after de-proteinization

I. INTRODUCTION

Mushrooms have been used as medical foods and have been part of the human diet for centuries be-cause they contain rich nutrients, such as essential amino acids, vitamins, and minerals.1 Research con-ducted during the last several decades has indicated that mushrooms provide several pharmacological properties, including antioxidant, antitumor, im-munomodulating, and liver protection benefits.2 Be-sides macronutrients, mushrooms contain antioxi-dant properties which are mainly attributed to their

phenolic content and polysaccharides. In recent years, mushroom-based, non-starch polysaccharides have emerged as an important class of bioactive natural products; most mushroom polysaccharides are relatively nontoxic and do not cause major side effect. Thus, mushroom polysaccharides are ideal components in the development of raw materials.1,2

Xylaria nigripes (Klotz.) Cooke (=Podosordar-ia nigripes (Klotz.) P.M.D. Martin, Xylariaceae, As-comycetes), known by its folkloric name Wu-Ling-Shen in traditional Chinese medicine for its fruiting body and sclerotium, is a medicinal fungus which

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is commonly used to treat insomnia, trauma, and as a diuretic and nerve tonic. It grows naturally in termite nests found underground, but although this fungus is associated with termite nests, it has no mutualistic symbiotic relationship with termites. In Taiwan, nine species of the genus Xylaria have been found from nests of Odontotermes formo-sanus, the only known macrotermitine termite.3 Previous studies have shown that X. nigripes pos-sesses a stronger anti-inflammatory activity and its mechanism of action can be mediated by inhibiting iNOS and COX-2 expression via the NF-κB sig-naling pathway.4 Furthermore, it has been reported that Xylaria nigripes possesses good antioxidant and hepatoprotective activities.5–7

Free radicals are produced in the natural me-tabolism of human cells; in normal situations, the human body generates a certain amount of free radicals during biochemical reactions, mostly in the form of reactive oxygen species (ROS). Exces-sive free radicals would damage macromolecules and lead to various related diseases.8

Antioxidant compounds have the ability to trap free radicals and thus inhibit harmful oxida-tive mechanisms. It has been reported that some mushrooms display antioxidant activity.9 Previ-ous study has demonstrated that polysaccharides from Antrodia cinnamomea have antioxidant properties which may involve up-regulation of glutathione S-transferase (GST) activity, mainte-nance of the normal GSH/GSSG ratio, and scav-enging of ROS.10

Previous study has shown that X. nigripes wa-ter extractions have excellent antioxidant effect.4 However, the source of polysaccharides in antioxi-dant activity assay has not been further explored. No comparison of the antioxidant activity of poly-saccharides in X. nigripes mycelium and fruiting body has been made in the relevant literature. The objective of this paper is to evaluate antioxidant activity and chemical characteristics of polysac-charides from different resources included the fruiting body and mycelium (extracellular poly-saccharide and intracellular polysaccharide), and to further explore whether the removal of protein during the extraction and purification process can

result in changes in the antioxidant capacity. The findings can be applied in the future development of health products.

II. MATERIALS AND METHODS

A. Materials

The fruiting body specimens of X. nigripes were supplied by Kang Jian Biotech. Co., Ltd. Nantou, Taiwan. The strain of X. nigripes BCRC34219 used in this study was purchased from the Biore-sources Collection and Research Centre in Hsin-chu, Taiwan.

The fungal species were inoculated in basal medium consisting (g/L) of glucose (30), 6 yeast extract (6), 2 peptone (2), MnSO4 (0.2), Mg-SO4·7H2O (0.5), and KH2SO4 (0.5) at pH 5.8. The inoculated culture flasks were incubated on a shak-er incubator (150 rpm) at 28°C for 7 days.

Ferrozine, peroxidase, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), Ethylenediaminetetraacetic acid (EDTA), azino-bis (3-ethylbenzothiazoline-6-acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were pur-chased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA); potassium bromide (KBr) was purchased from Scharlau Chemie S.A. with IR spectroscopy grade. All other chemicals and re-agents used were of analytical grade.

B. Extraction of Crude Polysaccharide from X. nigripes Fruiting Body

The extraction of crude polysaccharide from the fruiting body was determined by the method of Sun et al. with minor modifications.11 The fruiting body of X. nigripes was ground in a blender to powder and then extracted three times with 80% alcohol at 75°C for 6 hours to defat and remove some colored materials and oligosaccharides. The precipitate was then separated from the superna-tant; each dried pretreated sample was added to pure water in a ratio of 1:20, then extracted at 95°C for 2.5 hours, after which the aqueous extract was filtered through Whatman filter paper. The residue

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was extracted two times. The water extraction so-lutions were combine and precipitated by the addi-tion of 95% alcohol to a final concentration of 80% (v/v), left at 4°C over night. The precipitates were collected by centrifugation (5000 rpm for 8 min at 4°C) and lyophilized. The extract was named XnFB-1.

C. Extraction of Crude Intracellular Polysaccharide from X. nigripes Mycelium

The inoculated culture flasks were separated by filtration into two parts of mycelium and broth. The extracted crude intracellular polysaccharide from the mycelium was determined using the same methods as that for the extraction from the X. ni-gripes fruiting body; the intracellular polysaccha-ride was named XnIPS-1.

D. Precipitation of Crude Extracellular Polysaccharide from X. nigripes Mycelium Broth

Precipitation of the crude extracellular polysac-charide from X. nigripes was determined by the method of Fan et al. with minor modifications.12 The broth of X. nigripes mycelium was added to 95% alcohol to a final concentration of 80% (v/v), left at 4°C overnight. The precipitates were collected by centrifugation (5000 rpm for 8 min at 4°C) and lyophilized. The product was named XnEPS-1.

E. Sevag Method

Purification of the polysaccharide by the Sevag method was determined by the method of Hou et al. with minor modifications.13 XnFB-1, XnIPS-1, and XnEPS-1 were dissolved in pure water, respec-tively. The Sevag agent (chloroform added to n-butanol at a ratio of 4:1) was added to the polysac-charide solutions to a final concentration of 20% (v/v), followed by intense shaking for 5 min and centrifugation (5000 rpm for 5 min at 4°C). The supernatants were lyophilized and named XnFB-2,

XnIPS-2, and XnEPS-2, respectively.

F. Determination of Total Polysaccharide by Phenol–Sulfuric Acid Method

The total polysaccharide was determined as in Al-balasmeh et al.14 Two milliliters of the extracted polysaccharide solutions were mixed with 1 mL of 5% phenol; 5 mL of sulfuric acid was then added to the tubes and vortexed for 30 sec. The tubes were allowed to stand for 20 min and then placed for 10 min in a water bath at room temperature for color development. Light absorption at 490 nm was recorded on a UV-spectrophotometer (Hita-chi U-3010, Japan). Reference solutions were pre-pared in an identical manner as above, except that the 2 mL polysaccharide solution was replaced by pure water, and the standard curve was determined by glucose.

G. FT-IR Spectrum Analysis

The samples were incorporated into KBr (spec-troscopic grade) and pressed into a 1 mm pellet. Spectra were recorded in the absorbance mode from 400 to 4000 cm–1 using a Shimadzu FT-IR 8400S model.

H. Assay of Chelating Ferrous Ion Ability

The metal chelating activities were determined by the method of Nadaroglu et al. with minor modifi-cations.15 Each sample, at a different concentration (2 mL), was mixed with 1.7 mL of pure water and 0.1 mL of 2mM FeCl2 and mixed vigorously. The reaction was initiated by the addition of 0.2 mL of 5mM ferrozine; the mixture was shaken vigorously and left standing at room temperature for 10 min. Pure water was used as blank and EDTA used as positive control. The chelating ferrous ion ability was determined by measuring the absorbance at 562 nm using a spectrophotometer. The chelating ferrous ion ability was calculated as [(absorbance of control–absorbance of sample)/absorbance of control]×100%.

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I. Assay of ABTS+ Scavenging Activity

The total antioxidant activities of all the samples of polysaccharide were determined by the method of Re et al. with minor modifications.16 The ABTS radical cation (ABTS+) solution was produced by reacting ABTS solution (7 mM) with potassium persulfate (2.45 mM); after incubation at room temperature it was kept in the dark for 12 h. The ABTS+ solution was then diluted with 95% alcohol to obtain an absorbance of 0.70±0.02 at 735 nm. Each sample was added to ABTS+ solution and mixed vigorously. The reaction mixture was allowed to stand at room temperature for 6 min. The total antioxidant activity was determined by measuring the absorbance at 734 nm using a spec-trophotometer. Distilled water was used as a blank and the L-ascorbic acid was used as positive con-trol. ABTS+ radical scavenging activity was calcu-lated as (1–absorbance of sample/absorbance of control)×100%.

J. Reducing Power

The reducing power of all the samples of polysac-charide was determined by the method of Oyaizu and Gülçin et al. with minor modifications.17 An amount of pure water, 0.75 mL, was added in the sample (1 mL) at various concentrations, then mixed with sodium phosphate buffer (0.2M, pH 6.6, 1 mL) and potassium ferricyanide (1%, 1 mL). After the mixture was incubated at 50°C for 20 min, trichloroacetic acid (10%, 1 mL) was added. Pure water was used as a blank. The reducing power was determined by measuring the absorbance at 700 nm using a spectrophotometer. The reducing power was calculated as (Absorbance of sample–Absorbance of control)×multiple of dilution.

K. DPPH Radical Scavenging Assay

The DPPH radical scavenging abilities were deter-mined by the method of Yamaguchi et al.18 with mi-nor modifications. A 1 mL DPPH solution (0.1mM, in 50% alcohol solution) was incubated with vary-ing concentrations of the sample. The reaction

mixture was shaken well and incubated for 20 min at room temperature. The DPPH radical scaveng-ing ability was determined by measuring the absor-bance at 517 nm using a spectrophotometer. Dis-tilled water was used as a blank and the L-ascorbic acid used as positive control. DPPH radical scav-enging activity was calculated as (1–Absorbance of sample/Absorbance of control)×100%.

L. Statistical Analysis

All results are expressed as mean ± SD. Differenc-es among the groups were subjected to a one-way ANOVA (analysis of variance) followed by Dun-can’s multiple range. Statistical significance was accepted when the p-value was less than 0.05.

III. RESULTS AND DISCUSSION

A. X. nigripes Polysaccharide Analysis

All polysaccharides were isolated according to published methods from the X. nigripes. The total polysaccharide contents were 75%, 78.5%, 35.7%, 43.8%, 44.4%, and 53.5% for XnFB-1, XnFB-2, XnIPS-1, XnIPS-2, XnEPS-1, and XnEPS-2, re-spectively. XnFB-2 had the highest total polysac-charide content and XnIPS-1 was the poorest. All results showed that the total polysaccharide con-tent would rise after deproteinization.

The FT-IR spectrum showed the structural characterization of X. nigripes polysaccharide, ranging from 4000 to 400 cm–1 as shown in Fig. 1. The attributions of the main absorptions for poly-saccharides were characteristic of glycosidic structures and were related to hydroxyl groups’ stretching vibration (3400–3500 cm–1). The bond in the region of 2919 cm–1 was the characteristic absorption of the C-H bond stretching vibration. The specific band at 1057 cm–1 was due to the py-ranose ring.19 Absorption at 1150 cm–1 indicated C-O-C stretching. The bond at 1020 cm–1 was for C-O stretching, and the bond between 1000 and 1080 cm–1 was characteristic for the presence of β-glucans due to O-substituted glucose residues.

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1992).20 The FT-IR of polysaccharide isolated from X. nigripes contained an absorption band at 890 cm–1 assigned to the β-glycosidic linkage, and a band at 920 cm–1 attributable to the α-linkage. Finally, the FT-IR spectrums of X. nigripes poly-saccharides were very alike both in position and shape of the characteristic absorption peaks.21 Because of the complexity of the polysaccharide structure, only some of the structure information was identified here. The detailed accurate structure of the polysaccharides needs to be further studied by NMR and methylation analysis.

After deproteinization by the Sevag method, the total polysaccharide content rose above six polysaccharides, with contact between the original polysaccharide and the polysaccharide after depro-teinization; the peaks at 1635, 1520, and 1412 cm–1 vanished and the noise became less in this range in the spectrum. These three peaks may represent the peptide linkage cross-linking the structure between the polysaccharide chain and the protein.B. Assay of Chelating Ferrous Ion Ability

The chelating metal ions effect was proven to be related to antioxidant activities. Transition metals like Fe2+, Cu2+, Pb2+, and Co2+ in the human body may have some reaction that produces the O2– free radical, which may attack the human body or cells. Because of their high reactivity, transition metals were known as the most important lipid oxidation pro-oxidant. Fe2+ is known as the classic pro-oxi-dant due to its high reactivity.22

The high chelating effects on metal ions indi-cate high antioxidant activity. Ferrozine can form a complex with ferrous ions. In the presence of chelating agents such as EDTA and polysaccha-rides, the complex formation is disrupted, and thus a decrease in the red color of the complex can be measured.22

The effects of the chelating ferrous ion abil-ity of the six samples are given in Fig. 2. The polysaccharide extracts of six samples from X. ni-gripes exhibited the ability to chelate ferrous ions in a dose-dependent manner. The effect of the six samples on the reducing power was in the follow-ing order: XnIPS-1>XnIPS-2>XnFB-2>XnFB- 1>XnEPS-1>XnEPS-2. The percentages were 67.56%, 62.13%, 49.03%, 48.29%, 36.15%, and 32.03%, respectively, at a concentration of 2.5 mg/mL. The chelating ferrous ion ability of EDTA was 20.73 μg/mL. In the previous research the use of fungus extract polysaccharide by hot water and ly-ophilization, named ABMP-F, showed an IC50 of ABMP-F of about 420 μg/mL, better than the six samples of X. nigripes.24 Different uronic acid con-tent may cause this situation. Intracellular polysac-charide from X. nigripes proved to have the highest

FIGURE 1. FT-IR spectra of the Xylaria nigripes poly-saccharides from the bottle were XnFB-1, XnFB-2, XnIPS-1, XnIPS-2, XnEPS-1, and XnEPS-2, respec-tively, which were obtained from the fruiting body and mycelium. FT-IR assignments, wave number (cm–1): 3400–3500 shows hydroxyl groups’ stretching vibration, 2919 shows characteristic absorption of the C-H bond stretching vibration, 1057 was due to the pyranose ring, 890 was assigned to the β-glycosidic linkage, and a band at 920 was attributable to the α-linkage.

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chelating ferrous ion ability and antioxidant poten-tial, since it revealed the lowest IC50 values in a chelating ferrous ion ability assay (Table 1).

Some reports indicated that hydroxyl groups on the polysaccharide extracted from mushrooms were available to chelate the metal ions such as Fe2+.25 Ferrous ions were the most effective pro-ox-idant in the food system.26 Given these results, the chelating effect of X. nigripes polysaccharides on ferrous ions could affect the activities of scaveng-ing free radicals to protect the human body against oxidative damage.46

C. Assay of ABTS Radical Scavenging Activity

The ABTS radical scavenging activity, measured by the change of absorbance, has become a wide-ly used method for detecting antioxidant activ-ity of chemical components.27 It was equivalent to estimating the total antioxidant activity of a potential antioxidant of complex samples.28 The scavenging activity on the ABTS assay can be used in aqueous solution just like water-soluble polysaccharides.29

The effects of the ABTS radical scavenging

activity of the six samples are given in Fig. 3. The polysaccharide extracts of the six samples from X. nigripes exhibited dose-dependent ABTS radical scavenging activity. The IC50 values of the ABTS radical scavenging activity of the six samples (XnFB-1, XnFB-2, XnIPS-1, XnIPS-2, XnEPS-1, and XnEPS-2) were 840.78, 1833.47, 174.25, 231.88, 1571.33, and 1372.69 μg/mL, respectively. The IC50 of L-ascorbic acid was 37.38 μg/mL. In the previous research, the use of ultrasonic-assisted extract of the polysaccharide named HPs showed an IC50 of about 165 μg/mL, which was similar to that of XnIPS-1.30 The re-sults were almost identical for the two effects of the polysaccharide extraction process. With a higher concentration of X. nigripes polysaccha-ride, the antioxidant activity and its concentration showed a positive linear relationship. This may be attributed to the hydrogen protons of the chemical structure of the polysaccharide combined with the ABTS cation radical.

The total antioxidant activity suggests the electron-donating capacity of X. nigripes polysac-charide. Thus it may act as a radical chain termina-tor by transforming reactive free radicals into more stable nonreactive products.

FIGURE 2. Chelating ferrous ion ability of XnFB-1, XnFB-2, XnIPS-1, XnIPS-2, XnEPS-1, and XnEPS-2.

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D. Reducing Power

Reducing power could be used as an indicator to survey the potential of antioxidant capacity be-cause of the close relationship between antioxidant activity and reducing power.31

The reducing power of the six samples is giv-en in Fig. 4. The polysaccharide extracts of the six samples from X. nigripes exhibited dose-depen-dent reducing power. The effect of the six samples on the reducing power took the following order: XnIPS-1>XnIPS-2>XnEPS-1>XnFB-1>XnEPS- 2>XnFB-2; the absorbances were 0.550, 0.336, 0.295, 0.200, 0.177, and 0.150 at a concentration of 2500 μg/mL, respectively. All the X. nigripes polysaccharides had a positive linear relation with

the absorbance, depending on the concentration. The best was XnIPS-1, which was 3.6 times higher than XnFB-2.

The reducing power assay was based on the ability of X. nigripes polysaccharide to reduce the yellow ferric form into the blue ferrous form by donating electrons.32 Due to the measured absor-bance by spectrophotometer at 700 nm, the change in color was seen as linearly related to the reducing power of the electron-donating compounds.

E. DPPH Radical Scavenging Assay

The DPPH free radical assay was widely used to determine the free radical scavenging abilities of various natural compounds for convenience and

TABLE 1. The IC50 Value of the Six Polysaccharides from Xylaria nigripes in Antioxidant AssaysSample XnFB-1 XnFB-2 XnIPS-1 XnIPS-2 XnEPS-1 XnEPS-2Chelating ferrous activity (μg/mL)

— — 1412.38 1756.61 — —

ABTS+ scavenging activity (μg/mL)

840.78 1833.47 174.25 231.88 1571.33 1372.69

DPPH scavenging activity (μg/mL)

349.04 1330.52 351.56 1854.30 454.68 1314.77

FIGURE 3. ABTS radical scavenging activity of XnFB-1, XnFB-2, XnIPS-1, XnIPS-2, XnEPS-1, and XnEPS-2.

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reproducibility. By measuring the decrease in DPPH radical absorption after the reaction with X. nigripes polysaccharides, it can be expressed as the activity of the compounds. The effects of the DPPH radical scavenging activity of the six sam-ples are given in Fig. 5. The polysaccharide ex-tracts of the six samples from X. nigripes exhibited dose-dependent DPPH radical scavenging activ-ity. The IC50 values of the DPPH radical scaveng-ing activity of the six samples (XnFB-1, XnFB-2, XnIPS-1, XnIPS-2, XnEPS-1, and XnEPS-2) were 349.04, 1330.52, 351.56, 1854.30, 454.68, and 1314.77 μg/mL, respectively. The IC50 of L-ascor-bic acid was 27.18 μg/mL. Within the test dosage range, XnFB-1 and XnIPS-1 had similar IC50 val-ues. The antioxidant experiments showed XnIPS-1 to have the highest activity.

The possible mechanism could be the X. ni-gripes polysaccharide providing protons to scav-enge the DPPH free radical. The DPPH free radi-cal is a long-lived organic nitrogen radical with a purple color. This purple radical was reduced by antioxidant compounds to the yellow form.33 The scavenging activity can be determined by the dis-coloration of the DPPH solution via monitored

measuring of the absorbance decrease at 517 nm until the absorbance remains stable in organic so-lutions.34

IV. CONCLUSIONS

This study is the first research to indentify the poly-saccharide form of X. nigripes compared with the fruiting body and mycelium in antioxidants. The mycelium intracellular polysaccharide (XnIPS-1) showed the highest antioxidant activity. Next is the polysaccharide of the fruiting body (XnFB-1). Last is the extracellular polysaccharide (XnEPS-1). The intracellular polysaccharide may have the functional groups such as the hydroxyl, carbon-hydrogen bond, and the C-O bond in FT-IR spec-trum analysis. The total polysaccharide content rose after deproteinization in the overall samples with lowered antioxidant activities. The possibility will be the peptide bonds, which link the structure between the polysaccharide and protein, affecting the result of decreasing the activities. Most of the production methods were based on the cultivation of mushrooms on a solid substrate in a controlled-temperature greenhouse. For the production of

FIGURE 4. Reducing power of XnFB-1, XnFB-2, XnIPS-1, XnIPS-2, XnEPS-1, and XnEPS-2.

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bioactive compounds for intracellular polysaccha-ride by submerged mycelia culture of a mushroom X. nigripes, production should be carried out un-der sterile and well-controlled conditions. In this study, we found the mycelium intracellular poly-saccharides from X. nigripes have the highest an-tioxidant activities. This could be beneficial to the antioxidant protection system in health appilica-tions, which would protect the human body against oxidative damage.

ACKNOWLEDGMENT

This work was supported by funds from the Na-tional Science Council, Executive Yuan, Taiwan, R. O. C. under the grant number NSC101-2632-E-212-001-MY3.

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