Food Chemistry 141 (2013) 34243427

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Food Chemistry
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Short communication

Antioxidant polyketide phenolic metabolites from the edible mushroom Cortinarius purpurascens
Ming-Sheng Bai a,b,1, Chen Wang a,1, Shi-Chun Zong c, Ming Lei a, Jin-Ming Gao a,
a

Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, College of Science, Northwest A&F University, Yangling, Shaanxi 712100, China College of Life Sciences, Ningxia University, Yinchuan, Ningxia 750021, China c Division of Medical Clinic, Xian University of Posts and Telecommunications, Xian, Shaanxi 710061, China
b

a r t i c l e

i n f o

a b s t r a c t
Phytochemical investigation of the ethyl acetate extract of the edible macrofungus, Cortinarius purpurascens, led to the isolation of nine anthraquinone-related pigments, citreorosein 6,8-dimethyl ether (1), physcione (2), rufoolivacin (3), rufoolivacin C (4), rufoolivacin D (5), leucorufoolivacin (6), verbindung cr11 (7), verbindung cr60 (8) and 1-Hydroxy-3-methyl-2-isopropanyl-6,8-dimethoxyanthraquinone (9). The structures of these isolated compounds were characterised by spectroscopic methods and comparison with published data. Among the tested compounds, 36 exhibited potent DPPH radical-scavenging activity with IC50 values in the range of 38 lg/ml. The results indicated that the fungus is a possible source of natural products with potential antioxidant activity. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 16 January 2013 Received in revised form 15 April 2013 Accepted 21 May 2013 Available online 31 May 2013 Keywords: Cortinarius purpurascens Antioxidant activity Anthraquinones Polyketides Fungal pigments

1. Introduction It is well-known that food-derived antioxidants exhibit strong protective effects against a variety of chronic health problems, such as cancer, ageing and cardiovascular diseases (Eklund et al., 2005; Jang et al., 1997; Katalinic, Milos, Kulisic, & Jukic, 2006), and that they are believed to be harmless sources of natural antioxidants. In particular, great interest has arisen in the possibility that naturally occurring antioxidants from edible materials may slow ageing and reduce the risk of ageing-related degenerative diseases (Pham-Huy, He, & Pham-Huy, 2008). Numerous antioxidants, such as phenolic compounds (e.g., polyphenols, avonoids, and tannins), carotenoids, organic acid, and polysaccharides, discovered in various natural sources have already been utilised in foods and medicines (Heleno et al., 2012; Kayashima & Katayama, 2002; Lv et al., 2012; Mau, Lin, & Chen, 2002). Edible and medicinal mushrooms are nutritionally functional foods and important sources of physiologically benecial and non-toxic medicines. They have been used in folk medicine throughout the world since ancient times. (Gao, 2006). Recently, these fungi have been reported to possess a variety of bioactivities, such as antitumor, antimicrobial, hypoglycemic, and antioxidant (Li et al., 2006; Xiao et al., 2012; Xu, Beelman, & Lambert, 2012).

Phenolic compounds (Chen, Feng, Huang, & Su, 2012; Cheung & Cheung, 2005; Kang et al., 2007) and polysaccharides (Li et al., 2006; Xiao et al., 2012; Xu et al., 2012) are very often thought to be responsible for the antioxidant activity of fungi. The basidiomycete Cortinarius purpurascens Fr. (Family: Cortinariaceae), which is an edible wild mushroom distributed in Helan Mountain, Ningxia Province, is one of the most popular edible fungi in China. It also enjoys the reputation of the meat plant due to its excellent eating quality and nutritional composition. However, very little is known about the chemical and biological functions of this mushroom. In the course of our ongoing search for naturally occurring bioactive metabolites from the higher fungi (Gao et al., 2007; Shi, Li, Gao, & Zhang, 2011a,b; Yang et al., 2012), chemical studies of the fruiting bodies of C. purpurascens led to the isolation and characterisation of nine known, polyketide-derived, anthraquinone pigments 19. Herein, we describe the isolation, structural elucidation and antioxidant properties of these pigments.

2. Materials and methods 2.1. General Melting point was determined on an XRC-1 micro-melting point apparatus and uncorrected. 1H and 13C NMR spectra were recorded on a Bruker DRX-500 spectrometer using CDCl3 or MeOH-d4 as solvents, with TMS as an internal standard, d in ppm, J in Hz. Electrospray ionisation (ESI) mass spectra were acquired in the positive

Corresponding authors. Tel.: +86 29 87092515.

1

E-mail address: jinminggao@nwsuaf.edu.cn (J.-M. Gao). These authors contributed equally to this work.

0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.05.099

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ion mode on a API QSTAR Pulsar 1 spectrometer. Column chromatography (CC) was carried out on silica gel (200300 mesh, Qingdao Marine Chemical Ltd., Qingdao, PR China), Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), and RP-18 gel (40 63 lm, Merck, Darmstadt, Germany). Fractions were monitored by TLC, and spots were visualised by heating silica gel plates sprayed with 10% H2SO4 in ethanol and with UV light at 254 nm. tert-Butylhydroquinone (TBHQ) was purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from SigmaAldrich Chemie GMBH (Steinheim, Germany). All the other chemicals and solvents used in this study were of analytical grade. 2.2. Fungal material The fruiting bodies of C. purpurascens were collected from Helan Mountain in Ningxia Autonomous Region, China, in August 2008, and authenticated by Prof. Yan-yun Chen, College of Life Sciences, Ningxia University. The voucher specimens were deposited at the College of Science, Northwest A&F University, China. 2.3. Extraction and isolation The air-dried and ground fruit-bodies of C. purpurascens (2.5 kg) were ultrasonically extracted three times, with 15 l of methanol at room temperature, then concentrated in vacuo, at 45 C in a rotary vacuum evaporator. The resulting crude residue (11 g), was partitioned successively between H2O and petroleum ether, ethyl acetate (EtOAc), and then n-butanol to yield three soluble extracts, namely petroleum ether (1.4 g), EtOAc (7.5 g), n-butanol (1.2 g) parts, and the remaining residue (0.9 g). The resulting extracts were subjected to the DPPH radical-scavenging assay and the petroleum ether, EtOAC, n-butanol and aqueous parts presented particular radical scavenging capacity (Table 1). The most active EtOAc portion selected was subjected to silica gel CC and eluted with increasing polarities of a mixture of CHCl3 and methanol (MeOH), and 14 fractions were collected according to their TLC analyses. Fraction 1 was separated by silica gel column and eluted with petroleum ether/acetone (8/2, v/v) and then further puried by Sephadex LH-20 column with MeOH to yield compounds 1 (120 mg), 2 (30 mg), 3 (15 mg) and 9 (4 mg). Fraction 2 was separated by silica gel CC and eluted with CHCl3/acetone (40/1, v/v) and then further puried by Sephadex LH-20 gel column with MeOH to give compounds 4 (12 mg) and 5 (8 mg). Fraction 14 was separated by silica gel CC and eluted with CHCl3/acetone (40/1, v/v) and then further puried by a Sephadex LH-20 column using MeOH to furnish compounds 6 (10 mg) and 7 (10 mg). Compound 8 (17 mg) was obtained from fraction 12 by Sephadex LH-20 column with MeOH, followed by silica gel CC and eluted with CHCl3/acetone (40/1, v/v). Citreorosein 6,8-dimethyl ether (1): orange needles; 1H and 13C NMR data was in agreement with those reported (Gill & Steglich, 1987); ESI-MS m/z: 315.4 [M+H]+. Physcione (2): yellow needles; mp 194195 C; 1H and 13C NMR data were consistent with reported values (Zan, Bau, Bao, & Li, 2008); ESI-MS m/z: 285.2 [M+H]+. Rufoolivacin A (3): red amorphous powder; 1H and 13C NMR data agreed well with published data (Gao et al., 2010); ESI-MS m/z: 556 [M]+. Rufoolivacin C (4): red amorphous powder; mp 305308 C; 1H and 13C NMR data matched literature value (Gao et al., 2010); ESI-MS m/z: 557 [M+H]+. Rufoolivacin D (5): red amorphous powder; mp 354357 C; 1H and 13C NMR data were identical to those reported (Gao et al., 2010); ESI-MS m/z: 573 [M+H]+.

Table 1 DPPH scavenging activities of the extracts and compounds 18 from C. purpurascens. Test material Petroleum ether extract Ethyl acetate extract n-Butanol extract Aqueous extract Citreorosein 6,8-dimethyl ether 1 Physcione 2 Rufoolivacin 3 Rufoolivacin C 4 Rufoolivacin D 5 Leucorufoolivacin 6 Verbindung cr11 7 Verbindung cr60 8 Compound 9 TBHQ IC50 (lg/ml) 307 8.06 55.8 0.13 194 2.44 368 1.78 154 2.65 230 3.58 4.65 0.02 8.63 0.10 7.50 0.12 3.88 0.05 68.2 0.65 36.5 0.60 Not tested 3.72 0.05

Leucorufoolivacin (6): yellow powder; 1H and 13C NMR data were in accord with reported data (Frde, 1994); ESI-MS m/z: 543 [M+H]+. Verbindung cr11 (7): yellow powder; 1H and 13C NMR data matched literature value (Frde, 1994); ESI-MS m/z: 543 [M+H]+. Verbindung cr60 (8): dark brown powder; 1H and 13C NMR data agreed well with literature value (Frde, 1994); ESI-MS m/z: 543 [M+H]+. 1-Hydroxy-3-methyl-2-isopropanyl-6,8-dimethoxyanthraquinone (9): orange red needle; 1H and 13C NMR data agreed well with literature data (Zan et al., 2008); ESI-MS m/z: 341 [M+H]+. 2.4. DPPH radical-scavenging assay DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay was carried out according to the method described by Sharma and Bhat (2009) with some modications (Mishra, Ojha, & Chaudhury, 2012; Yu & Zhou, 2005). Briey, the reaction was started by adding 1 ml of 0.2 mM DPPH methanol solution to the extracts in DMSO (or pure compounds in methanol) at different concentrations, with a nal volume of 4 ml. After gentle mixing and 30 min of standing at room temperature, the absorbance of the resulting solutions was measured at 517 nm with a spectrophotometer (Thermal, England). TBHQ was used as a positive reference. The DPPH scavenging capacity of each crude extract was calculated using the following equation:

Scavenging capacity % A0 A1 Ae=A0 100

where A0, A1 and Ae stand for the absorbance of the control, antioxidant extract and blank, respectively. The DPPH scavenging capacity of each pure compound was calculated using the following equation:

Scavenging capacity % A0 A1=A0 100

where A0 and A1 stand for the absorbance of the control and antioxidant compound, respectively. All the antioxidant measurements were performed in triplicate under six different concentrations, and the data was expressed as the mean standard deviation (SD). IC50 values were calculated using probit regression and expressed in lg, indicating the concentration of the antioxidant samples necessary to quench 50% of the free radicals in the reaction mixture. 2.5. Statistical analyses All the statistical analyses were conducted using SPSS (Statistical Program for Social Sciences, SPSS Corporation, Chicago, IL) version 15.0 for Windows and a probability value (P) less than 0.05 was accepted as statistically signicant.

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H3C
7 6

O
8 8a

O
9

OH
1 9a 2 3 4a 4

OH

OH

H 3C

OH

CH3 CH3

O CH3

10a

10

OH

O CH3 O 2

O 1

CH3

O CH3 O 9

CH3

H 3C
7'

O
8' 8'a

OH
1' 2' 3'

O CH3 CH3 O
4 9a 1 3

H3C

OH

O CH3

H3C

OH

O CH3

H 3C

O O

6' 5' 5 6 7 8a 8

4'a 4' 10 10a 9

H3C
CH3

O O

CH3 CH3 O

H3C H3C

O O

CH3 CH2OH O

H 3C

4a

H 3C

H 3C

OH 3

H3C O

OH 4

H3C

OH 5

H3C

OH

O CH3

H3C

O CH3
H 3C

H 3C

OH

O CH 3

H3C H3C

O O

CH3 CH3

H3C H3C

O O

CH3 CH3

O O

CH 3 CH 3

H 3C

H3C

OH 6

OH

H3C

O 7

OH

H 3C

O 8

OH

Fig. 1. Chemical structures of metabolites 18 from C. purpurascens.

3. Results and discussion 3.1. Isolation and characterisation of pure pigments The powdered fruiting bodies of C. purpurascens were extracted with methanol and concentrated under vacuum. The resulting crude residue was partitioned successively with petroleum ether, ethyl acetate (EtOAc), and n-butanol to afford three soluble extracts, and the remaining aqueous layer. These extracts were subjected to a DPPH free radical-scavenging assay, of which the EtOAc extract was found to show the best activity (IC50 = 55.76 lg/ml). Accordingly, this active extract was further fractionated by way of normal-phase silica gel and Sephadex LH-20 gel chromatographic methods, providing compounds 19. On the basis of spectroscopic analyses including 1H and 13C NMR spectroscopy and ESIMS, the structures of the isolated compounds were identied as citreorosein 6,8-dimethyl ether (1) (Gill & Steglich, 1987), physcione (2) (Zan et al., 2008), rufoolivacin (3) (Gao et al., 2010), rufoolivacin C (4) (Gao et al., 2010), rufoolivacin D (5) (Gao et al., 2010), leucorufoolivacin (6) (Frde, 1994), verbindung cr11 (7) (Frde, 1994), verbindung cr60 (8) (Frde, 1994), and 1-hydroxy3-methyl-2-isopropanyl-6,8-dimethoxyanthraquinone (9) (Zan et al., 2008) (Fig. 1). It should be noted that six of these metabolites, 38, belong to the rare class of rufoolivacin compounds, which consist of 1-naphthol and 1,4- or 1,2-anthraquinone through the 40 ,10-coupled aryl linkage. This is the rst report of the isolation of these anthraquinone pigments from the higher fungus C. purpurascens.

3.2. DPPH scavenging capacity The four extracts obtained and the isolated compounds 18 were evaluated for their DPPH radical scavenging capacity through the determination of the IC50 values, and the results are shown in Table 1. Among the four extracts, the EtOAc extract was found to be the most potent DPPH radical scavenger, showing an IC50 value of 55.76 lg/ml, while the aqueos extract was the least active. As shown in Table 1, the anthraquinones 1 and 2 had little or no activity, with IC50 values of >150 lg/ml when compared with TBHQ, a well-known antioxidant used as a positive control (IC50 = 3.72 lg/ ml). In the case of the fully aromatic rufoolivacin-type compounds 36, 6 (non-quinoid rufoolivacin) and 3 (the para-quinoid rufoolivacin) (IC50 = 3.88 and 4.65 lg/ml, respectively) showed approximately twofold better than the ortho-quinoid isomers 4 and 5 (IC50 = 7.50 and 8.63 lg/ml, respectively) in radical scavenging ability, which suggests that the presence of para-quinoid unit may be required for activity. Compunds 6 and 3 exhibited very similar activity to the control TBHQ. Furthermore, 6 and 3 displayed much higher properties than 7 and 8, while 6 was slightly stronger than 3 since 6 contained an additional hydroxyl group at C-1 compared to 3. The signicant discrepancies in their radical scavenging activities were likely due to the position and number of hydroxyls at positions C-1, C-10 and C-9 in the aromatic ring. On the other hand, this may be because of the role of a conjugated double bond at the para-position for the stabilisation of the phenoxyl radical in the radical scavenging process. These ndings revealed that the relative order in DPPH scavenging potency for

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these compounds was found to be: 6 > 3 > 5 > 4 > 8 > 7, with compound 6 exerting the highest activity.

4. Conclusions In summary, nine known anthraquinone derivatives 19, including six rare rufoolivacin compounds (49) and three simple analogs (13) were rst isolated from the fruiting bodies of C. purpurascens. Within the limits of the compounds examined, the structureactivity relationship might be related to the position of hydroxyl substitution, and to the 1,2- or 1,4-anthraquinone moiety. Taking into consideration that this mushroom is used as an edible material, it is clearly important to reveal its natural antioxidatants. These results may serve as a scientic foundation for development and utilisation efforts of this macrofungus. Acknowledgements This work was nancially supported by the Forestry Research Foundation for the Public Service Industry of China (201204603) and the National Natural Science Foundation of China (30370019). References
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