Isolation, characterization of melanin derived from Ophiocordyceps sinensis, an entomogenous fungus endemic to the Tibetan Plateau

Journal of Bioscience and Bioengineering VOL. 113 No. 4, 474 – 479, 2012 www.elsevier.com/locate/jbiosc

Isolation, characterization of melanin derived from Ophiocordyceps sinensis, an entomogenous fungus endemic to the Tibetan Plateau Caihong Dong and Yijian Yao⁎ Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, P.O. Box 2714, Beijing 100101, China Received 1 September 2011; accepted 4 December 2011 Available online 18 January 2012

Melanins are pigments of high molecular weight formed by oxidative polymerization of phenolic or indolic compounds. In this present study, a black pigment was isolated from the fermentation broth of Ophiocordyceps sinensis, an entomogenous fungus which is endemic to the Tibetan Plateau by alkaline extraction, acid hydrolysis, and repeated precipitation. It was designed as melanin since the physical and chemical properties including its ultraviolet (UV) and infrared (IR) spectra of the black pigment conformed to the characteristic of melanin and similar to the commercial synthetic melanin. The antioxidant activity of melanin derived from O. sinensis was evaluated. They showed much stronger scavenging abilities on 1,1-diphenyl2-picrylhydrazyl (DPPH•) and the chelating ability on ferrous ions than that of the water extract from the mycelia of O. sinensis, with IC50 value 18.51 ± 0.85 μg/ml and 2.58 ± 0.26 μg/ml, separately. This is the first report of melanin from O. sinensis and will be helpful for the study on the physiology and the artificial cultivation of this fungus, an endangered species. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Ophiocordyceps sinensis; Melanin; Isolation; Antioxidant; DPPH• radicals]

Melanins are dark-brown to black pigments of macromolecules formed by oxidative polymerization of phenol and/or indolic compounds, which widely exist in animals, plants and microorganisms. They showed a broad spectrum of biological roles, including antioxidant (1,2), antitumor activity (3), antivenin activity (4), anti-virus (5), liverprotecting activity (6,7) and radio protective (8) etc. They are widely used in medicine, pharmacology, cosmetics and other fields. The study on fungal melanin has been lasted for many years (9) and many fungi constitutively synthesize melanin (10). Melanin is not essential for fungal growth and development, but have been reported to act as “fungal armour” and function in the protection of fungi against environmental stress such as UV radiation, temperature extremes (11). Some melanized fungi inhabit remarkably extreme environments including high altitude, Arctic and Antarctic regions (8,12). Melanin also was showed as a virulence factor for a number of plant and human pathogenic fungi (13,14). Black pigments have been extracted and identified as melanin from fungal sources, such as Aspergillus nidulans (Eidam) G. Winter (1), Tuber melanosporum Vittad. (15), Agaricus bisporus (J.E. Lange) Pilát (16), Auricularia auricula (L.) Underw. (17) and Hypoxylon archeri Berk. (18) etc. They were found to conform to the physical and chemical properties of melanins: high molecular weight; insolubility in water, aqueous acid and common organic solvents; decolourization by oxidizing agents (NaOCl and H2O2) and a positive reaction for polyphenols. The absorption spectra of their alkaline solutions show ⁎ Corresponding author. Tel.: + 86 10 64807496; fax: + 86 10 64807518. E-mail address: [email protected] (Y. Yao).

no maxima or minima in the ultraviolet or visible ranges and the plots of log absorbance vs. wavelength give essentially straight lines with negative gradients (19). Ophiocordyceps sinensis (Berk.) G. H. Sung, J. M. Sung, Hywel-Jones & Spatafora (≡ Cordyceps sinensis (Berk.) Sacc.), an entomogenous fungus, is one of the best known traditional Chinese medicines and health foods. The fungus parasitizes larvae of moths (Lepidoptera), especially Hepialus armoricanus Oberthür, and converts each larva into a sclerotium, from which the stroma and fruit-body grows (20). The complex including the fungal stroma and the sclerotium, which appears as larva body owing to the intact exoskeleton of the insect, has been used as a health food and traditional medicine to invigorate the lung and nourish the kidney in China for hundreds of years, at least from the 17th century (21). It has been officially classified as a drug in the Chinese Pharmacopoeia since 1964 (22) and listed as an endangered species under the second class of state protection (Order No. 4 of the State Forestry Administration and Ministry of Agriculture: The List of the Wild Plants under the State Emphasized Protection. Available from: http://www.gov.cn/gongbao/content/2000/ content_60072.htm). Ophiocordyceps sinensis is endemic to the Tibetan Plateau, the roof of the world, and may be found only from above 3000 m in altitude (23,24). Strong ultraviolet radiation, hypoxia and cold environment are the most important ecological factors, which have profoundly effected on plateau organism survival. As a native organism of the plateau which is constantly challenged by the harsh environmental stress during their long evolutional history, O. sinensis must have formed its own strategies and mechanisms in adaptation to severe

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altitude. Since melanin can function in the protection of fungi against environmental stress such as UV radiation, temperature extremes, whether O. sinensis can produce the pigment is intriguing. To the best of our knowledge, the melanin from O. sinensis has not been reported so far. The antioxidant activity of melanin from different sources, e.g., from Aspergillus nidulans (1), Pleurotus cystidiosus (25), tea (2) and Cryptococcus neoformans Vuill. (10) has been studied. It seemed that melanin has strong antioxidant activity. O. sinensis has been reported to have antioxidant properties, however, the responsible component is not very clear (26,27). The study on whether the melanin from O. sinensis is partially responsible for the antioxidant activity will be helpful for the pharmacological investigation on O. sinensis. In the present study, melanic pigments derived from O. sinensis were isolated and identified and the antioxidant activity was studied. In the first step, the black pigment was extracted from fermentation broth of O. sinensis and designed as melanin through its physical and chemical properties including ultraviolet and infrared spectra. Subsequently, the antioxidant activities of the melanin extracted from O. sinensis were evaluated by scavenging abilities on DPPH• and chelating ability. As far as we know, this is the first report of melanin extracted from O. sinensis. It will be helpful for the study on the physiology and the artificial cultivation of this fungus, an endangered species.

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Characteristics of melanin from O. sinensis Physical and chemical characteristics of melanin from O. sinensis were obtained according to different procedures (29). Primary characteristics of melanin determined were: solubility in water, aqueous acid and common organic solvents; oxidative bleaching by means of KMnO4, K2Cr2O7, NaOCl and H2O2; and positive reaction for polyphenols. Ultraviolet–visible (UV) absorption spectra were obtained with a Nanodrop ND 1000 spectrophotometer and NanoDrop 2.4.7c software (NanoDrop Technologies Inc., Wilmington, DE, USA). Infrared (IR) spectra were recorded on a Perkin Elmer spectrometer (Model 1600 FT). Total phenol groups were assayed quantitatively by absorbance at 760 nm using Folin–Ciocalteu reagent following the method of Singleton et al. (30) with modification. In short, to 6.0 ml of distilled water, 0.1 ml of sample and 0.5 ml of Folin–Ciocalteu's phenol reagent was mixed, followed by the addition of 1.5 ml of 20% sodium carbonate(w/v) and the volume was made up to 10.0 ml with distilled water. After incubation for 30 min at 25°C, the absorbance was measured at a wavelength of 760 nm on a Unico-2100 spectrophotometer (Shanghai, China). The concentration of phenol groups was calculated from a standard curve obtained by subjecting various amounts of gallic acid to the same treatment as the test samples. Scavenging effect on DPPH• radicals of melanin from O. sinensis The effect of melanin from O. sinensis on DPPH• radicals was studied following the method of Blois (31) with some modifications. A 0.5 mM solution of DPPH• in ethanol and in 0.05 M acetate buffer (pH = 5.5) were prepared. Extracting solution of 0.1 ml at different concentrations was mixed with 2 ml of acetate buffer, 1.9 ml of absolute ethanol and 1 ml of DPPH• solution. The mixture was shaken immediately after adding DPPH• and allowed to stand at room temperature in dark for 30 min. The decrease in absorbance at 517 nm was then measured. BHA and α-tocopherol were used as positive controls and the sample solution without DPPH• was used as sample blank. The radical scavenging activity was measured as a decrease in the absorbance of DPPH• and was calculated using the following equation:

MATERIALS AND METHODS Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH•), ferrozine, synthetic melanin, gallic acid and Folin–Ciocalteu's phenol reagent were purchased from SigmaAldrich; α-tocopherol from Lancaster (Morecambe, UK); ethylenediaminetetraacetic acid (EDTA) from Amresco (OH, USA); hydrogen peroxide (H2O2), sodium hypochlorite (NaOCl) and sodium carbonate from Beijing Chemical Reagents Company (Beijing, China); butylated hydroxytoluene (BHT) from China National Pharmaceutical Group Shanghai Chemical Reagents Company (Shanghai, China); All reagents were of analytical grade. Strains and conditions for submerged culture A strain (CGMCC No. 2793) originally isolated from fresh specimen collected from Sichuan, China, in this laboratory. The identity of the strain was confirmed by means of both morphological and molecular methods. The Internal Transcribed Spacer (ITS1-5.8S-ITS2) of nuclear ribosomal DNA (nrDNA) was amplified and sequenced from the culture. The sequence of the DNA fragment (EU570923) was compared with a data set generated in this laboratory containing ITS sequences from dried specimens and living strains of O. sinensis obtained from various regions of the Tibetan Plateau. The strain was maintained on potato dextrose agar (PDA) supplemented with 5% wheat bran and 0.5% peptone at 4°C. The strain was first incubated on the same medium as for the stock at 18°C for 60 days in Petri dish. Seed cultures were grown in 500-ml Erlenmeyer flasks containing 100 ml of liquid medium, inoculated with a 5-mm agar disc from the 60-day culture. The flasks were rotated at 100 rpm, at 18°C for 15 days and then 100 ml medium in 500-ml Erlenmeyer flask was inoculated with 10 ml of seed culture mycelium and incubated on a rotary shaker at 100 rpm and 18°C for 35 days (28). The culture liquid was centrifuged for 20 min at 8000 × g and the resulting supernatant filtered through Whatman no. 1 filter paper was used for melanin extraction. Isolation and purification of melanin from O. sinensis The melanin in the culture medium was extracted following the method of (29) with some modification. The culture solutions were acidified to pH 1.5 with 6 M HCl and allowed to stand overnight to precipitate the polymer. The melanin precipitates were recovered by centrifugation at 6000 × g for 15 min, washed with 0.0 l M HC1 and distilled water, and lyophilized. The extracted melanin was purified by acid hydrolysis, organic solvent (chloroform, ethyl acetate and ethanol) treatment and repeated precipitation. The samples of the melanin were hydrolysed with 7 M HC1 at 100°C for 2 h (15), then filtered, and the filter residue was washed with distilled water. The non-hydrolysable melanin was washed sequentially with chloroform, ethyl acetate and ethanol and then dried. The solid matter was re-dissolved in 1 M KOH and mixture was filtered. The supernatant was acidified with 1 M HC1and the filter residue was washed with water. The precipitation was repeated four times. Each precipitation included acidification of the melanin solution by the addition of HCl to pH 1.5 and filtered. Time profiles of cell growth and melanin production of O. sinensis in liquid culture Thirty Erlenmeyer flasks were prepared for the determination of the growth curve of O. sinensis in liquid culture. The conditions were performed as our previous report (28). Mycelium was harvested from three flasks every 5 days and measured by mycelial dry weight. The final pH value in the culture was read at the time of harvesting. After sampling, the fermentation supernatant was stored at − 20°C and then thawed for analyses of residual sugar and isolation of melanin. Residual sugar level was assayed by phenol-sulphuric acid method.

Scavenging activityð%Þ ¼

Ab−ðAs−AsbÞ  100 Ab

ð1Þ

Where Ab, As and Asb are the absorbances at 517 nm of DPPH• of the blank, melanin or positive control, and sample blank respectively. Ferrous ion chelating activity assay of melanin from O. sinensis The chelating activity of melanin on ferrous ion was measured as reported by Decker and Welch (32). One millilitre of extracts (0.3125–10 μg/ml) was mixed with 3.7 ml of deionizer water and then the mixture was reacted with ferrous chloride (2 mM, 0.1 ml) and ferrozine (5 mM, 0.2 ml) for 20 min. The absorbance at 562 nm was determined spectrophotometrically. EDTA was used as positive control and chelating activity on ferrous ion was calculated using the following equation, with Ab as the absorbance of the blank without melanin or EDTA and As as the absorbance in the presence of the melanin or EDTA. Inhibition ð%Þ ¼

Ab−As  100 Ab

ð2Þ

Statistical analysis Experimental results recorded were means ± standard deviation of triple determinations. The data were analysed by one-way analysis of variance (ANOVA). Tests of significant differences were determined by Duncan's multiple range tests at p = 0.05 or independent sample T-test (p = 0.05). The IC50 values were calculated by using median-effect analysis and CalcuSyn software (Biosoft). Results were processed by SPSS 11.0 (SPSS Inc.) and Origin 7.0 (OriginLab Corporation).

RESULTS Time profile of cell growth and melanin production of O. sinensis in liquid culture In submerged cultures, the lag phase lasted for 10 days followed by the exponential phase for about 15 days (Fig. 1), with a maximum mycelial dry weight of 23.45 g l− 1 at day 25. The stationary phase only lasted 2 days and soon turned to death phase. The melanin in the medium could be detected at day 15 and then the yield of melanin increased until day 35. After that, the production of melanin kept stable. Initial pH value of the medium increased during the lag and exponential phases to 7.8 and then decreased slightly by the end of incubation. The kinetics of residual sugar decreased sharply at the exponential growth phase and then declined slowly until the batch was terminated (Fig. 1). Characterization of the pigment The average yield of crude melanin was 1.775 g per 1000 ml fermentation broth of O. sinensis. After further purification with organic solvents (chloroform, ethyl acetate and ethyl alcohol), acid hydrolysis and repeated precipitation,

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J. BIOSCI. BIOENG.,

8

2.0

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26 24 22

4

16

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14 12 2

10 8 6

-1

-1

-1

18

Melanin production (g l )

6

1.5

Residual sugar(g l )

pH

7

Mycelial dry weight(g l )

20

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0.5

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4 2 5

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Culture days FIG. 1. Growth and melanin production curve of Ophiocordyceps sinensis during the liquid culture. The symbols represent the following samples: closed triangles, mycelial dry weight; closed squares, final pH; closed squares, residual sugar; open stars, melanin production.

7.95% yields (based on the crude melanin) was obtained, which itself contained 45% total phenol measured by Folin–Ciocalteau phenol reagent. The physical and chemical properties of the amorphous black pigment extracted from O. sinensis were compared with synthetic melanin from Sigma. There was great similarity between these two pigments. Both of them were insoluble in both water and organic solvents (ethanol, hexane, acetone, benzene and chloroform), dissolved only in alkali, precipitated below pH 3, bleached by H2O2, KMnO4, K2Cr2O7, and gave a positive reaction for polyphenols by producing flocculent brown precipitate with FeCl3. The tiny difference was found in the reaction with FeCl3: melanin from O. sinensis showed flocculent relatively quickly than the synthetic melanin. The Ultraviolet–visible spectrum for melanin extracted from O. sinensis showed the similar characteristics to the spectrum of synthetic melanin from Sigma (Fig. 2). Solutions of melanin (0.2 mg/ml) in 0.1 M potassium hydroxide exhibited strong optical absorbance in Ultraviolet region and decreased progressively as the wavelength increased. The plots of log absorbance versus wavelength gave lines with negative slopes (Fig. 3). The slopes for the melanin extracted from O. sinensis and synthetic melanin from Sigma were −0.0019 and −0.0017, respectively.

The IR spectra for melanin from O. sinensis and synthetic melanin from Sigma were very similar to each other, though small differences did occur (Fig. 4). Both the spectra of the two samples display: (i) a strong, broad band at 3400 cm− 1, attributed to stretching vibrations of OH and NH2 groups; (ii) strong band at 1710 cm− 1 caused by the vibration of C=O of COOH and 1620 cm− 1 due to the vibration of aromatic C=C and COO− groups. The major difference was seen in the wavelength of 2929 cm− 1, where melanin from O. sinensis has a peak caused by CH2 and CH3, and also by NH group oscillation, whereas the synthetic melanin showed no peak. Scavenging effect on DPPH• radicals DPPH• radical scavenging activity of the melanin from O. sinensis from 5 to 80 μg/ml was compared with that of BHT, a synthetic antioxidant and α-tocopherol, a natural antioxidant (Fig. 5). The scavenging effect was evident at all of the tested concentrations and increased with the increasing concentration. The IC50 value of the melanin from O. sinensis was calculated as 18.51 ± 0.85 μg/ml. The free radical scavenging effect of melanin was even slightly higher than BHT and α-tocopherol over the concentration of 40 μg/ml, though there was no significant difference.

0

Log absorbance

-0.5 -1 -1.5 -2 -2.5 -3 200

300

400

500

600

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800

Wavelength (nm) FIG. 2. UV spectra of melanic pigments derived from Ophiocordyceps sinensis. The concentration of alkaline solution of melanin used was 0.2 mg/ml. Line a, synthetic melanin from Sigma. Line b, purified melanin extracted from Ophiocordyceps sinensis.

FIG. 3. Linear plots of optical density against wavelength. The concentration of alkaline solution of melanin used was 0.2 mg/ml. Solid lines represent synthetic melanin with negative slope − 0.0017 and dash lines represent purified melanin extracted from O. sinensis with negative slope − 0.0019.

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110 100

100 Chelating effects (%)

Transmittance (%)

90 80 70

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60 3421.98

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1706.49 1618.56

40 30

10 4000

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1624.73

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0 1000

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Fe2+ chelating activity Melanin from O. sinensis showed a better ferrous ion chelating ability than EDTA when the concentration was over 2 μg/ml. The IC50 value of the melanin from O. sinensis was calculated as 2.58 ± 0.26 μg/ml and the chelating ability was reached 94.74% at the concentration of 10 μg/ml (Fig. 6). DISCUSSION The importance of melanin for survival and longevity of fungi has been recognized for many years (33) and it was emphasized that fungal melanins merit a higher profile than they often receive in both pathology and ecology in the Mycological Research News (34). Melanins are considered as an effective protective screen of organisms, which gradually evolve and form for resistance to UV and ionizing radiation, temperature extremes in the environment. In this study, we have isolated melanin from O. sinensis, a fungus endemic to the Tibetan Plateau, for the first time. Characterization of the melanin indicated that it possessed physical and chemical properties very similar to synthetic melanin from Sigma Company. The results also suggested that the melanin extracted from O. sinensis have strong antioxidant activities.

. DPPH scavenging effects (%)

90 80 70 60 50 40 30 20 10 10

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0

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(cm-1)

FIG. 4. Infrared spectra of melanin extracted from Ophiocordyceps sinensis (line a) in comparison with synthetic melanin (line b).

0

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1716.81

3403.54

wavenumber

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b

20

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Concentration (µg ml-1) FIG. 5. Scavenging effect on DPPH• radicals of melanin from Ophiocordyceps sinensis. The symbols represent the following samples: closed squares, melanin from Ophiocordyceps sinensis; open squares, BHT; closed triangles, α-tocopherol.

FIG. 6. Ferrous ion chelating activity of melanin of Ophiocordyceps sinensis. The symbols represent the following samples: closed squares, melanin from O. sinensis; open squares, EDTA.

In most cases fruit bodies or cultured mycelium have been used for isolation of fungal melanins, such as T. melanosporum (15), A. auricula (17) and H. archeri (18). However, there is almost no melanin formation in the natural fruit bodies or submerged cultured mycelium of O. sinensis. This suggested that this melanin was extracellular melanin, which was formed by secreting phenol oxidases into the medium to oxidize phenolic compounds of various origins or secreting phenols into the medium, where they are autoxidized or are oxidized by enzymes released from the fungus (33). It is reported that A. nidulans can produce melanin in the culture medium with the yield 175 μg per ml medium (1), 10 times less than the yield in this study. Due to the high chemical diversity of biological sources for melanin production, there is no common standard method for melanin isolation and purification. In this study, a method has been developed for melanin isolation and purification from the fermentation broth of O. sinensis. The acid hydrolysis was employed for purification of the melanin against carbohydrates and proteins. Organic solvents were used to wash away lipids. Multiple precipitations were employed to sequester the melanin from low molecular weight polyphenols and to improve the homogeneity. Melanin extracted from O. sinensis showed all the physical and chemical properties common to synthetic melanin and other natural melanin (29,35). The nature of the pigment was further confirmed by its spectral properties. Its ultraviolet and visible spectrum was typical of the absorption profile of melanin (Fig. 2). It absorbed strongly in the UV region and progressively less as the wavelength increased. This is due to the presence of many complex conjugated structures in the melanin molecule (36). The decrease in the absorption with increasing wavelength is almost linear plots (Fig. 3). Though it was reported that the slope was not useful for distinguishing among different types of melanin (33), the gradients −0.0019 and −0.0017 for melanin of O. sinensis and synthetic melanin, respectively, are similar to those found for the melanin extracted (35) from the aleuriospores of Epicoccum nigrum Link (−0.0015), from sclerotia of Colletotrichum coccodes S. Hughes (− 0.0026). IR spectra also presented all the basic characteristics of synthetic melanin, taken as the reference material. Some minor difference found in 2920 cm− 1, which also existed in the crude melanin and non-hydrolysable melanin from T. melanosporum (15) and melanin from tea (37), probably due to aliphatic impurities. It was reported that there are dihydroxyphenylalanine (DOPA), glutaminyl-4-hydroxybenzene (GDHB), catechol and dihydroxynaphthalene (DHN) melanins in fungi according to their different

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precursors (11). The best characterized fungal melanin is dihydroxynaphtalene (DHN) melanin and ascomycetous fungi usually produce DHN melanin. However, in this study, melanin pigment from O. sinensis possesses similar structure with DOPA melanin. Then we performed the specific DHN melanogenesis inhibitor (tricyclazole at 1–10 ppm) test and we didn't find the inhibition on melanin synthesis in O. sinensis (data not shown). Though the ascomycetous fungi Tuber and Neurospora crassa are reported to synthesise DOPA melanin (33), to ascertain whether the melanin from O. sinensis is DOPA melanin, it is necessary to further analyse the enzyme and intermediate product during the melanin synthetic pathway (38), which is our ongoing project in this laboratory. The scavenging activity on DPPH•, a stable free radical of the extracted melanin from O. sinensis was studied. The activity reached more than 80% at the concentrations over 40 μg/ml as shown in Fig. 4, which was slightly higher than that of BHT and α-tocopherol, the commercial antioxidants. Yao et al. (39) also reported the strong DPPH• scavenging activity of melanin from Testae of Armeniaca vulgaris var. ansu at the concentration over 200 μg/ml. The IC50 value was calculated as 18.51 ± 0.85 μg/ml, significantly lower than that of melanin from Lachnum singerianum (Dennis) W.Y. Zhuang & Z. Wang (40). The activity of melanin from O. sinensis in the present study was much more effective than that of the water extract from the mycelia of O. sinensis reported by this group (26). The effective components of scavenging free radicals of the water extract of O. sinensis were also discussed, it cannot be concluded that any certain compound such as polysaccharides and mannitol and cordycepin has a main effect on the antioxidant activity of O. sinensis. Here it was concluded that melanin may be another effective component responsible for the scavenging free radicals. To understand the antioxidant activity better, the chelating power of melanin from O. sinensis in relation to Fe2+ was studied. Melanin from O. sinensis also showed a much better ferrous ion chelating ability than that of the water extract (26). It was reported that fungal melanins have a high biosorptive capacity for a variety of metal ions due to melanin contain various functional groups which provide an array of multiple nonequivalent binding sites for metal ions (41). This may explain the strong chelating power. It was believed that the metal binding of fungal melanins could confer a survival advantage. If the metals are toxic to the organism, the melanin can prevent their entry to the cell. If the metals are essential to cell physiology and are present in low amounts in the environment, then melanin may concentrate them in a manner that makes them more available to the cell (11). It was accepted that the antioxidants can reduce the damage induced by ionizing radiation (42,43). O. sinensis is inhabited in the Tibetan Plateau and may be found only from above 3000 m in altitude (23,24), where the background radiation levels are much higher than that at sea level. The mechanism why O. sinensis can endure the strong radiation has not been reported yet as far as we know. The effect of melanin enhancing the survival of fungi can be mainly due to its function as an extracellular redox buffer which can neutralize oxidants generated by environmental stress. The relation between the melanin and the mechanism was underway in this laboratory. Melanin also represents virulence factors for several pathogenic fungi (44). As an entomogenous fungus, whether melanin is the virulence factor for this fungus to the larvae is another intriguing problem since the artificial or semi-artificial cultivation of this species is so attractive and troublesome. ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (NSFC 31100014), the National Science and Technology Supporting Projects 2007BAI32B03 and 2008BADA1B06 operated by the Ministry of Science and Technology of China, the National Science Funds for Distinguished Young Scholars from the National Natural Science

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