Chemical constituents from the fungus Monascus purpureus and their antifungal activity

Phytochemistry Letters 4 (2011) 372–376

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Chemical constituents from the fungus Monascus purpureus and their antifungal activity Ming-Jen Cheng a,1,*, Ming-Der Wu a,1, Ih-Sheng Chen b, Min Tseng a, Gwo-Fang Yuan a a b

Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsinchu 300, Taiwan School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 June 2011 Received in revised form 25 July 2011 Accepted 4 August 2011 Available online 18 August 2011

One new cyclohex-2-enone derivative, purpureusone (1), together with seven known compounds (2–8) were isolated from the n-BuOH-soluble fraction of the 95% EtOH extract of the red yeast rice fermented with the yellow mutant of the fungus Monascus purpureus BCRC 38038. Their structures were characterized by direct interpretation of their spectroscopic methods, including 1D NMR (1H, 13C NMR, DEPT), 2D NMR (COSY, NOESY, HSQC and HMBC), and HRESIMS. Purpureusone (1) contains a cyclohex-2enone skeleton connected with one g-lactone ring, one octanoyl, and 2-oxopentyl side chains. Some isolates were evaluated for their antifungal effect against Candida albicans and Saccharomyces cerevisiae using a TLC bioautographic method. Compound 1 showed antifungal inhibitory activity in vitro. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Monascus purpureus Red yeast rice Metabolites Cyclohex-2-enone derivative Purpureusone Antifungal activity

1. Introduction Red yeast rice, a fermented product from Monascus species, which is also known as red mold rice, koji, red koji, anka, angkak, and ben-koji, has been used in Asia for over 1000 years as a common food additive for improving the color of food and preserving meat and fish (Ma et al., 2000). It is also applied for medicinal purposes to aid food digestion (Mudgett, 2000) and blood circulation (Ma et al., 2000). Several secondary metabolites such as pigments (Blanc et al., 1994), polyketide analogs (monacolins) (Wild et al., 2002), g-aminobutyric acid (GABA) (Juzlova´ et al., 1996), dimerumic acid (Juzlova´ et al., 1996), monascodilon (Hossain et al., 1996) and the mycotoxin citrinin (Juzlova´ et al., 1996), have been identified. Traditionally many of these metabolites have been verified to possess several functions including antihypertensive (Tsuji et al., 1992), antihypolipidemic (Endo, 1979, 1985; Martinokova et al., 1995), and antibacterial activity (Wong and Bau, 1977). Earlier chemical investigations of this species mainly focused on its polar pigment constituents (Blanc et al., 1994; Juzlova´ et al., 1996; Akihisa et al., 2005; Nozaki et al., 1991; Blanc et al., 1995; Sato et al., 1997; Jongrungruangchok

* Corresponding author. Tel.: +886 3 5223191x568; fax: +886 3 5224171. E-mail addresses: [email protected], [email protected] (M.-J. Cheng). 1 Both authors contributed equally to this work.

et al., 2004; Huang et al., 2008), and several nonpigment compounds (Wild et al., 2002, 2003; Juzlova´ et al., 1996; Akihisa et al., 2004) have also been obtained. In our previous work, we have reported over fifteen constituents, together with their biological activity from the mycelium of M. pilosus BCRC 38072 (Cheng et al., 2008). In the course of our search for potential diverse secondary metabolites from natural fungal sources, the 95% EtOH extract of Monascus purpureus BCRC 38038 showed antifungal activities against Candida albicans and Saccharomyces cerevisiae using direct bioautography in a TLC bioassay (Rahalison et al., 1991; Patino˜ and Cuca, 2010), and the aim of this study was the isolation of its metabolites and to evaluate their bioactivity. Investigation of its nBuOH-soluble fraction of the 95% EtOH extract of a yellow mutant of the red yeast rice fermented with the title fungus led to the isolation of one new cyclohex-2-enone derivative, purpureusone (1), together with seven known compounds (2–8). We report herein the details of isolation, structure elucidation and biological activity of the isolates.

2. Results and discussion The 95% EtOH extract prepared from the red yeast rice fermented with the fungus M. purpureus BCRC 38038 was partitioned between n-BuOH and H2O. The n-BuOH layer was subjected repeatedly to column chromatography on RP-18 and silica gel CC to afford eight compounds, the structures of which

1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.08.003

M.-J. Cheng et al. / Phytochemistry Letters 4 (2011) 372–376

O 21

19

15

17

H

H

14

7

6

3a

O

9

7a

1 2

8

373

O

4 13

5

10

12 11

O

O

Fig. 1. Chemical structure of purpureusone.

were elucidated by 1D and 2D NMR spectra and comparison with literature data (Fig. 1). Compound 1 was isolated as a yellow syrup and gave the molecular formula C23H34O5, with seven unsaturated degrees by positive HRESIMS [M+Na]+ at m/z 413.2301 (calcd 413.2304), which was supported by its NMR data (Table 1). The UV spectrum absorption lmax (MeOH) at 250 nm, and the IR absorption bands at 1770 (g-lactone), 1708 (conjugated CO) and 1640 (vinyl) cm1 functionalities, as well as the observation of the featuring carbon resonances [dC 194.4 (C-4), 131.8 (C-6), and 150.7 (C-5)] in the 13C NMR spectrum (Table 1), revealed the presence of an a,bunsaturated carbonyl functionality (cyclohex-2-enone moiety) in 1. In the 1H NMR spectrum of 1, one allylic Me [dH 1.73 (3H, s, H-5)], three sets of a-methylene protons of two ketones [dH 2.66 (1H, dt, J = 18.0, 7.2 Hz, H-15), 2.90 (1H, dt, J = 18.0, 7.2 Hz, H-15), 2.57 (2H, t, J = 7.6 Hz, H-10), 3.54 (1H, d, J = 17.4 Hz, H-8), and 3.72 (1H, d, J = 17.4 Hz, H-8)], two sets of b-methylene protons of two ketones [dH 1.58 (2H, m, CH2-11 and H-16)], two aliphatic CH groups [dH 4.27 (1H, d, J = 13.2 Hz, H-1), 3.08 (1H, ddd, J = 13.2, 11.4, 4.2 Hz, H7a)], one set of CH2 protons [dH 2.51 (1H, dd, J = 18.0, 4.2 Hz, Heq-7), and 2.90 (1H, dd, J = 18.0, 11.4 Hz, Hax-7)], four aliphatic CH2 protons [dH 1.31 (8H, m, H-17–20)], and two terminal Me moieties

Table 1 1 H NMR and 13C NMR data (CD3COCD3, 600 and 150 MHz) of compound 1, d in ppm, J in Hz. No.

Purpureusone

d Ca 1 2 3a 4 5 Me–5 6 7 7a 8

55.4 171.2 84.1 194.4 150.7 11.7 131.8 33.7

dH d s s s s q s t

43.9 d 48.8 t

9 10 11 Me–12 Me–13 14 15

206.0 45.3 17.7 13.8 17.3 203.6 43.5

s t t q q s t

16 17 18 19 20 Me–21

23.3 29.8 29.9 31.9 23.1 14.1

t t t t t q

4.27 (1H, d, J = 13.2)

1.73 (3H, s) 2.51 2.62 3.08 3.54 3.72

(1H, (1H, (1H, (1H, (1H,

dd, J = 18.0, 4.2, Heq) dd, J = 18.0, 11.4, Hax) ddd, J = 13.2, 11.4, 4.2) d, J = 17.4), d, J = 17.4)

2.57 1.58 0.87 1.47

(1H, (2H, (3H, (3H,

t, J = 7.6) m) t, J = 7.6) s)

2.66 2.90 1.58 1.31 1.31 1.31 1.31 0.90

(1H, (1H, (2H, (2H, (2H, (2H, (2H, (3H,

dt, J = 18.0, 7.2) dt, J = 18.0, 7.2) m) m) m) m) m) t, J = 7.5)

a Multiplicity was determined by DEPT experiments (s = quaternary, d = methine, t = methylene, q = methyl).

[dH 0.90 (3H, t, J = 7.5 Hz, CH3-21) and 0.87 (3H, t, J = 7.6 Hz, CH312)] were observed. Twenty-three 13C NMR signals (Table 1) corresponding to seven quaternary carbons, two CH, ten CH2, and four CH3 groups were observed from the 13C NMR and DEPT spectra. The 13C NMR and DEPT spectra exhibited the presence of four C5 5O carbonyl functions including one a,b-unsaturated C5 5O group [dC 194.4 (C-4)], one lactone C5 5O group [dC 171.2 (C-2)], and two saturated ketone groups [dC 203.6 (C-14) and 206.0 (C-9), one C5 5C bond [dC 131.8 (C-6) and 150.7 (C-5)], and one methyl attached to an oxygen-bearing carbon group [dC 17.3 (C-13)]. Since five out of seven unsaturation equivalents were accounted for by the above-mentioned 13C NMR data, 1 was inferred to have two rings (ring A as a six-membered and ring B as a five-membered ring). In addition, rings A and B were further determined as a cyclohex-2-enone skeleton combined with one g-lactone ring by the following HMBC and COSY analyses. Analysis of the 1H–1H COSY spectrum [dH 4.27 (H-1)/3.08 (H-7a)/2.51, 2.62 (H-7)] and by the aid of HMBC correlations of H-13 to C-3a, C-7a and C-4, of H-7a to C-3a and C-2, of H-7 to C-3a and C-7a, and of CH3-5 to C-4, C-5 and C-6, which yielded a cyclohex-2-enone moiety that was fused to the g-lactone unit at C-3a/7a. This allowed the skeleton of 1 to be determined as 5,13-dimethyl-3a,7a-dihydro-3H,4H-benzofuran2,4-dione (rings A and B). HMBC correlations from dH 3.54/3.72 (H8), 2.57 (H-10), 1.58 (H-11) to dC 206.0 (C-9), and from dH 4.27 (H1), 3.08 (H-7a), 2.66/2.90 (H-15), 1.58 (H-16) to dC 203.6 (C-14) together with the 1H–1H COSY spectrum [dH 2.57 (H-10)/1.58 (H11), dH 1.58 (H-11)/0.87 (H-12), dH 2.66, 2.90 (H-15)/1.58 (H-16), dH 1.58 (H-16)/1.31 (H-17), dH 1.31 (H-20)/0.90 (H-21)] established the presence of 2-oxopentyl (C3H7COCH2–, from H-8 to H-12) and octanoyl moieties (C7H15CO–, from C-14 to CH3-21), respectively. Finally, the HMBC contacts between CH2-8 (dH 3.54/3.72) with C-6 (dc 131.8), and C-5 (dc 150.7) led to the direct connection of the 2oxopentyl group at C-6. The HMBC also showed correlations between CH2-15 (dH 2.66/2.90) with C-1 (dC 55.4), as well as between H-7a (dH 3.08) with C-14 (dC 203.6) which enabled us to verify the connection of the octanoyl group to the g-lactone ring at C-1. The other key correlations of HMBC are illustrated in Fig. 2. The relative configuration of 1 was derived by a NOESY spectrum (Fig. 2) in combination with biogenetic considerations and comparison with similar compounds (Wild et al., 2003), the relative configuration of which was based on a NOESY analyses. NOE contacts for Me-13/H-1, Me-13/Hax-7, H-1/H-7a, H-7a/Hax-7, and H-1/Hax-7 disclosed that Me-13, H-1, H-7a, and Hax-7 were on the same side of the molecular plane, tentatively assumed as the borientation. The H-7a/CH2-15 and CH2-7/CH2-8 NOE cross-peaks report on a preferred conformation of the octanoyl and 2oxopentyl groups around the C-1/C-14 and C-6/C-8 bonds, respectively. On the other hand, the NOE cross peaks between H-8 and Me-5, between H-10 and H-11, and between H-10 and H12 were also observed in Fig. 2. Based on the information from the 1 H NMR, COSY, and NOESY spectra, a computer-generated 3D structure was obtained by using the abovementioned molecular modeling program with MM2 force-field calculations for energy

M.-J. Cheng et al. / Phytochemistry Letters 4 (2011) 372–376

374

O 7 21

19

17

10

8 6

14 15

1

O

11

7a

2

12

9

3a

O

5

O

4 13

O H

H

O

H

H

O O

O O

O

O O

O O

Fig. 2. Key COSY (—), NOESY ($) and HMBC (!) correlations of 1.

minimization (Supplementary data Fig. 3). The calculated distances between H-1/H-13 (2.781 A˚), H-13/Hax-7 (2.673 A˚), H-1/H7a (2.221 A˚), H-7a/Hax-7 (2.384 A˚), and H-7a/H-13 (2.359 A˚) are less than 4.00 A˚, this is consistent with the well-defined NOESY (Fig. 2) observed for each of the proton pairs. Consequently, the relative configuration of C-1, C-7a, and C-3a, was assigned as rel(1S,7aS,3aR). Thus, the structure of 1 was unambiguously determined as (1S*,7aS*,3aR*)-5,13-dimethyl-1-octanoyl-6-(2oxopentyl)-3a,7a-dihydro-3H,4H-benzofuran-2,4-dione, and named purpureusone. Additionally our study afforded seven known compounds, identified as monascin (2) (Li et al., 2006), ankaflavin (3) (Li et al., 2006), ergosterol peroxide (4) (Sgarbi et al., 1997), syringaldehyde (5) (Chen et al., 1999), syringic acid (6) (Chen et al., 1999), p-anisic acid (7) (Mounetou et al., 1998), and linolenic acid (8) (Chang et al., 2000). The structures of these known compounds were established by comparison of their spectroscopic data with literature data. The antifungal activities against C. albicans and S. cerevisiae by direct bioautography in a TLC bioassay were conducted for four compounds (1–4) isolated from this study, while the amount of other compounds available was too low to allow testing (Table 2) (Rahalison et al., 1991; Patino˜ and Cuca, 2010). The minimum amount of compound 1 required for the inhibition of fungal growth was appreciable at 1 mg for S. cerevisiae and at 5 mg for C. albicans.

Compound 1 showed a potent effect against the yeasts, C. albicans and S. cerevisiae, respectively. Compounds 2 and 3 exhibited moderate fungitoxicity towards C. albicans and S. cerevisiae, showing a detection limit of 25 and 25 mg, respectively. Compound 4 showed weak fungitoxicity towards C. albicans and S. cerevisiae, showing a detection limit of 50 and 100 mg, respectively. Compound 1 is promising as it has antifungal activity against S. cerevisiae similar to that of the positive control (mycostatin < 1 mg). In conclusion, we focused on the minor secondary metabolites appearing in the n-BuOH-soluble fraction of a 95% EtOH extract of

Table 2 Antifungal activity of isolated compounds 1–4. Compounds

1 2 3 4 Mycostatinb a b

Antifungal activitya Candida albicans

Saccharomyces cerevisiae

5 25 25 50 1

1 25 25 100 1

Minimal amount (mg) of compound to inhibit growth on silica gel TLC plates. Positive control.

M.-J. Cheng et al. / Phytochemistry Letters 4 (2011) 372–376

the red yeast rice fermented by M. purpureus BCRC 38038. Through a comprehensive investigation, the n-BuOH-soluble fraction was repeatedly subjected to column chromatography on silica gel to furnish one new metabolite (1) and seven known ones (2–8). Four isolates (1–4) were evaluated for their antifungal activities against C. albicans and S. cerevisiae using direct bioautography assay. Compound 1 showed significantly antifungal activity, whereas 2 displayed moderate inhibitory effect. To the best of our knowledge, this is the first report on the metabolites isolated from Monascus sp. evaluated their antifungal activity by direct bioautography in a TLC bioassay, compared to the known biological activities of some of Monascus metabolites in red yeast rice previously (Endo, 1979, 1985; Endo et al., 1986; Tsuji et al., 1992; Martinokova et al., 1995). It is likely to have some relevance for the medicinal properties of red yeast preparations. Compound 1 has a related g-lactone attached to a cyclohexenone ring and alkyl side chain, compared with the similar azaphilone structure monascin (Li et al., 2006), revealing that 1 is a new type of azaphilone derivative in nature products. From the results described herein in comparison to those described in literature concern various metabolites from Monascus species (Wild et al., 2002, 2003; Juzlova´ et al., 1996; Nozaki et al., 1991; Blanc et al., 1995; Sato et al., 1997; Akihisa et al., 2004, 2005; Jongrungruangchok et al., 2004; Huang et al., 2008), we infer that the genus Monascus species would be a rich source of structurally diverse natural products.

375

3.3. Extraction and isolation

3. Experimental

The dried red yeast rice of M. purpureus BCRC 38038 (1 kg) were extracted five times with 95% EtOH at room temperature to yield a EtOH syrup extract, which was partitioned between n-BuOH: H2O (1:1) to provide an n-BuOH-soluble fraction (2 g) and an H2Osoluble fraction (4.2 g). The n-BuOH-soluble fraction (2 g) was subjected to silica gel CC and eluted with CH2Cl2–MeOH gradient system (20:1, 10:1; 5:1, 1:1; 0:1) to obtain fractions A–D. Fraction A (0.25 g) was applied to a silica gel column eluting with CH2Cl2: EtOAc (30:1) to give three fractions (A.1–A.3). Fraction A.1 (15.0 mg) was purified by preparative TLC (RP-18) developed with MeOH to give 8 (2.8 mg). Fraction A.3 (25 mg) was subjected to silica gel CC, eluting gradually with CH2Cl2 and acetone to gain 1 (4.3 mg) and 2 (4.1 mg). Fraction B (0.42 g) was submitted to a silica gel column with a gradient system of n-hexane-acetone to yield 6 fractions (B.1–B.6). Fraction B.2 (50.4 mg) was purified by preparative TLC developed with n-hexane: EtOAc (2:1) to afford 3 (3.6 mg). Fractions B.3 (25 mg) was purified by RP-18 MPLC eluting with MeOH: H2O (1:1) to gain 4 (3.8 mg). Fraction B.5 was subjected to silica gel CC eluting with n-hexane-acetone gradient system to afford three fractions (B.5.1–B.5.3). Fraction B.5.1 was purified by RP-18 MPLC eluting with MeOH: H2O (5:1) to afford 5 (1.3 mg). Fraction D (0.56 g) was applied to silica gel CC eluting with CH2Cl2 followed by increasing concentrations of acetone in CH2Cl2 to furnish five fractions (D.1– D.5). Fraction D.1 was further purified by preparative TLC (RP-18) developed with acetone: H2O (4:1) to give 6 (2.3 mg) and 7 (2.5 mg).

3.1. General experimental procedures

3.4. Purpureusone (1)

Optical rotations were recorded on a Jasco P-1020 polarimeter. UV spectra were run on a Jasco V-530 UV/VIS spectrophotometer. IR spectra (KBr or neat) were obtained on a Genesis II FTIR spectrophotometer. 1D (1H, 13C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra, using chloroform-d1 or acetone-d6 as solvents, were acquired on Varian Mercury-400 (400 MHz for 1H NMR, 100 MHz for 13C NMR) and Varian VNMRS-600 (600 MHz for 1 H NMR, 150 MHz for 13C NMR) spectrometers. Chemical shifts were internally referenced to the solvent signals in chloroform-d1 (1H, d 7.26; 13C, d 77.0) or acetone-d6 (1H, d 2.05; 13C, d 205.1). Lowresolution and high-resolution ESIMS spectra were determined on a Bruker APEX-II mass spectrometer. Column chromatography (CC) was performed with 70–230 or 230–400 mesh silica gel (Merck) and Spherical C18 100A Reversed Phase Silica Gel (RP-18) (particle size: 20–40 mm) (SILICYCLE). Preparative thin-layer chromatography was conducted on silica gel 60 F-254 (Merck) and RP-18 F254S (Merck).

 Yellow syrup; ½a25 D þ 17:6 (c 0.06, acetone); IR (Neat) nmax 1770 (g-lactone), 1708 (conjugated C5 5O), 1640 (vinyl) cm1; 1H 13 (CD3COCD3, 600 MHz) and C NMR (CD3COCD3, 150 MHz), see Table 1; ESIMS: m/z 413 [M+Na]+; HRESIMS: m/z 413.2301 [M+Na]+ (calcd for C23H34O5Na, 413.2304).

3.2. Cultivation and preparation of red yeast rice M. purpureus BCRC 38038 was used throughout this study, and specimens deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute. M. purpureus BCRC 38038 was maintained on potato dextrose agar (PDA) and the strain was cultured on PDA slants at 25 8C for 1 week and then the spores were harvested by sterile water. The spores (5  105) were seeded into 300 ml shake flasks containing 50 ml RGY medium (3% rice starch, 7% glycerol, 2% polypeptone, 3% soybean powder, 0.2% MgSO4, 0.2% NaNO3), and cultivated with shaking (150 rpm) at 25 8C for 3 days. After the mycelium enrichment step, an inoculum mixing 100 ml mycelium broth and 100 ml RGY medium was inoculated into plastic boxes (25 cm  30 cm) containing 1 kg sterile rice and cultivated at 25 8C for producing red yeast rice and above mentioned RGY medium was added for maintaining the growth. After 21 days of cultivation, the red yeast rice was harvested, and used as a sample for further extraction.

3.5. Antifungal activity The antifungal activity of the tested compounds against C. albicans (BCRC 21538) and S. cerevisiae (BCRC 20822) was determined using the bioautographic technique (Rahalison et al., 1991; Patino˜ and Cuca, 2010). The microorganisms used in the antifungal assay have been maintained at the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. Ten microliters (10 ml) of the solutions were prepared, in different concentrations, corresponding to 100, 50, 25, 10, 5, 2 and 1 mg of pure compounds. After eluting with CH2Cl2:EtOAc (4:1), the plates were sprayed with a spore suspension of fungi and incubated for 72 h in the darkness in a moistened chamber at 25 8C. The antifungal agent, mycostatin, was chosen in this study for positive controls at 1 mg.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2011.08.003. References Akihisa, T., Mafune, S., Ukiya, M., Kimura, Y., Yasukawa, K., Suzuki, T., Tokuda, H., Tanabe, N., Fukuoka, T., 2004. (+)- and (–)-syn-2-Isobutyl-4-methylazetidine2,4-dicarboxylic acids from the extract of Monascus pilosus-fermented rice (redmold rice). J. Nat. Prod. 67, 479–480. Akihisa, T., Tokuda, H., Yasukawa, K., Ukiya, M., Kiyota, A., Sakamoto, N., Suzuki, T., Tanabe, N., Nishino, H., 2005. Azaphilones, furanoisophthalides, and amino acids from the extracts of Monascus pilosus-fermented rice (red-mold rice) and their chemopreventive effects. J. Agric. Food Chem. 53, 562–565.

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