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Three new constituents from the fungus of Monascus purpureus and their anti-inflammatory activity
Phytochemistry Letters 31 (2019) 242–248
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Three new constituents from the fungus of Monascus purpureus and their anti-inflammatory activity
T
Ho-Cheng Wua, Ming-Jen Chengb, , Ming-Der Wub, Jih-Jung Chenc,d, Yen-Lin Chenb, ⁎⁎ Hsun-Shuo Changa,e, ⁎
a
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsinchu, 300, Taiwan c Faculty of Pharmacy, School of Pharmaceutical Sciences, National Yang-Ming University, Taipei, 112, Taiwan d Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, 404, Taiwan e School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 807, Kaohsiung, Taiwan b
ARTICLE INFO
ABSTRACT
Keywords: Monascus purpureus Eurotiaceae Spiro[isochromane-1,2′-pyran]-4′(3′H)-one Benzenoid Azaphilone Anti-inflammatory
A chemical study on the EtOAc-soluble fraction of the 95% EtOH extract of red yeast rice fermented with the fungus Monascus purpureus BCRC 38110 (Eurotiaceae) has resulted in the isolation of three new compounds, i.e., two new 5′,6′-dihydrospiro[isochromane-1,2′-pyran]-4′(3′H)-one derivatives, designated as monascuspirolide A (1) and monascuspirolide B (2) and one benzenoid derivative, monapurpureusin B (3), together with three known compounds, monaspurpurone (4), monascin (5), and ankaflavin (6). The structures and relative configurations of these compounds were elucidated by spectroscopic analyses, including 1D- and 2D-NMR spectroscopy and mass spectrometry, and by comparison of their spectral data with the literature data of authentic samples. To the best of our knowledge, the former two new compounds (1 & 2) displayed an unusual 5′,6′dihydrospiro[isochromane-1,2′-pyran]-4′(3′H)-one pattern compared to previous Monascus metabolites. Some phytochemicals were evaluated for anti-inflammatory activity through the measurement of nitric oxide (NO) production levels in lipopolysaccharide (LPS)-stimulated murine-derived macrophages RAW264.7 cell lines.
1. Introduction Fungi of the genus Monascus (family Monascaceae, class Eurotiomycetes) have been used to ferment rice in Asia for centuries. Originally, Monascus rice products were used to make rice wine, but red yeast rice, which is also called ang-kak or Hongqu, has also been used as a meat colorant, health food, and a Chinese folk medicine (Ma et al., 2000). However, in Western countries, red yeast rice extracts are mainly used for their cholesterol-lowering effects. The investigation of the chemical components of Monascus spp. dates back to 1973, when Endo et al. isolated monacolin K analogues from M. ruber (Manchand et al., 1973). A review of the literature regarding Monascus species (Akihisa et al., 2005; Ma et al., 2000) reveals that azaphilone, furanoisophthalides, amino acids, and polyketides are the major metabolites. Several types of secondary metabolites from red yeast rice have also been reported, including azaphilone pigments, monacolins, citrinin, dimerumic acid, and γ-aminobutyric acid (Akihisa et al., 2005; Hsu et al., 2010; Jongrungruangchok et al., 2004; Jůzlová
⁎
et al., 1996; Loret and Morel, 2010; Zhu et al., 2012). Recently, a number of reports have demonstrated that these red yeast rice metabolites exhibit a wide variety of biological activities. Monacolins were found to inhibit the activity of the enzyme HMG-CoA, which is involved in cholesterol biosynthesis (Lee et al., 2006; Li et al., 2013; Shi and Pan, 2010). In addition to its cholesterol-lowering effect, red yeast rice has also been reported to have anti-colon-cancer (Hong et al., 2008; Zhu et al., 2012), anti-liver-cancer (Chang et al., 2016), anti-prostate-cancer (Chiu et al., 2013, 2012), and anti-breast-cancer (Lee et al., 2013) activity. Recent studies have demonstrated that the azaphilone pigments of Monascus sp. contribute to its anti-inflammatory activity (Akihisa et al., 2005; Hsu et al., 2013; Yasukawa et al., 1994). In one study, azaphilone pigments were found to be more effective in inhibiting 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear oedema in mice than non-azaphilonoid pigments. Previous investigations of Monascus species have isolated constituents with various skeletons, mainly azaphilones (yellow, orange, and red pigments) and monacolin analogues. However, the pharmacological and toxicological
Corresponding author at: Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsinchu 300, Taiwan. Corresponding author at: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan. E-mail addresses: [email protected] (M.-J. Cheng), [email protected] (H.-S. Chang).
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https://doi.org/10.1016/j.phytol.2018.12.017 Received 14 September 2018; Received in revised form 17 December 2018; Accepted 18 December 2018 Available online 14 May 2019 1874-3900/ © 2018 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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effects of most of the secondary metabolites isolated from Monascus sp. remain unknown or unclear, and many metabolites still have not been identified. Despite a growing chemical library of compounds from Monascus, the phytochemical aspects of this genus have not been comprehensively examined. Accordingly, it is still worthwhile to explore and investigate the components of red yeast rice and their bioactivity. In previous studies, several Monascus species have been sources of bioactive secondary metabolites such as monaspurpurone (tetralone derivative with antifungal activity) from Monascus purpureus BCRC 38113 (Cheng et al., 2010), purpureusone (cyclohex-2-enone derivative with antifungal activity) from Monascus purpureus BCRC 38038 (Cheng et al., 2011), (E)-methyl-4-(2-acetyl-4-oxotridec-1-enyl)-6-propylnicotinate (an anti-inflammatory pyridine alkaloid) from Monascus pilosus BCRC PK-1 (Cheng et al., 2012a), monascusazaphilones A–C (azaphilone derivatives that inhibit NO production) from Monascus purpureus BCRC 38108 (Wu et al., 2013), and monascuskaolin (an azaphilone derivative that inhibits NO production) from Monascus kaoliang BCRC 31506 (Cheng et al., 2012a,b). In the course of our search for diverse secondary metabolites from natural fungal sources, we have screened over 150 species of fungal materials using TLC and HPLC profile analyses. One particular species, M. purpureus BCRC 38110, was identified as having more plentiful metabolites by our preliminary analyses. Monascus purpureus BCRC 38110 is a mutant yellow strain that shows a different morphology from other traditional red mutant Monascus species on potato dextrose agar (PDA) plates. Moreover, it was also found to be an active species in our continuous effort to search for interesting anti-inflammatory agents from natural sources. As part of a series of studies on the nitric oxide (NO) production and inhibitory activity of compounds derived from natural sources, we were especially interested in studying the chemical composition of red yeast rice. The 95% EtOH extract of M. purpureus BCRC 38110 showed inhibitory activity on lipopolysaccharide (LPS)induced nitric oxide (NO) release in RAW 264.7 murine macrophages in our primary screening (ca. 85% inhibition at a concentration of 10 mg/ mL). The aim of this study was to isolate the metabolites in the extract and to evaluate their bioactivity. Six compounds, including two unprecedented 5′,6′-dihydrospiro[isochromane-1,2′-pyran]-4′(3′H)-one: monascuspirolides A & B (1 & 2), one benzenoid derivative, monapurpureusin B (3), and together with three known compounds (4-6) isolated from the EtOAc-soluble fraction of the 95% EtOH extract of red yeast rice fermented with the title fungus. Details of the isolation, structure elucidation, and the anti-inflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production activities of some isolates are described herein.
Table 1 1 H- and 13C-NMR Data (CDCl3, 600 and 150 MHz, resp.) of compounds 1 and 2. δ in ppm, J in Hz. position
1 δC
1 2 3
100.1
4
36.9
4a 5 6 7 8 8a 9 10
143.3 124.4 148.4 133.0 122.4 138.3 21.9 51.8
11 12
204.6 48.6
13
66.5
14 15 16
20.9 83.0
17
85.8
18 19 20 21 22
202.8 22.3 171.9
23 OH-16 OH-17 OH-18
13.9
64.2
63.5
δH (m, J in Hz)
2 δC
δH (m, J in Hz)
100.0 4.09 (dqd, 12.6, 6.0, 4.5, Hax-3) 2.78 (dd,17.4, 4.5, Heq4) 2.80 (dd, 17.4, 12.6, 0.9, Hax-4) 7.36 (s) 7.88 (s) 1.34 (d, 6.0) 2.54 (dd, 14.4, 2.1) 3.00 (d, 14.4) 2.36 (dd like, 15.0, 10.8, Hax-12) 2.52 (dd, 15.0, 3.0, 2.1, Heq-12) 4.28 (dqd, 10.8, 6.0, 3.0, Hax-13) 1.36 (d, 6.0)
64.1 36.2
142.0 129.6 139.8 128.5 126.5 135.4 20.9 51.5 204.8 48.5
66.5 21.9 33.8
72.7 1.33 (s) 4.03 4.25 1.11 4.30 2.88
(dq, 10.5, 7.2) (dq, 10.5, 7.2) (t, 7.2) (br s) (br s)
78.1 200.3 17.4
4.09 (dqd, 11.6, 6.0, 4.2, Hax-3) 2.71 (dd,16.8, 4.2, Heq-4) 2.75 (dd,16.8, 11.6, Hax4) 7.14 (s) 8.07 (s) 1.33 (d, 6.6) 2.48 (dd, 14.4, 2.1) 3.05 (d, 14.4) 2.39 (dd, 14.7, 11.4, Hax12) 2.51 (ddd, 14.7, 3.0, 2.1, Heq-12) 4.26 (dqd, 11.4, 6.0, 3.0, Hax-13) 1.35 (d, 6.0) 2.98 (ddd, 17.1, 11.4, 0.6, , Hax-16) 3.24 (dd, 17.1, 5.7, , Heq16) 4.13 (dd, 11.4, 5.7, Hax17) 1.30 (s)
2.53 (br s) or 3.82 (br s) 3.82 (br s) or 2.53 (br s)
singlet aromatic protons [δH 7.36 (1H, s, H-5) and 7.88 (1H, s, H-8)], one ethoxy group [δH 1.11 (3H, t, J = 7.2 Hz, H-23), 4.03 (dq, J = 10.5, 7.2 Hz, H-22b), 4.25 (dq, J = 10.5, 7.2 Hz, H-22a)], two hydroxyl groups [δH 2.88 (1H, brs, OH-17), 4.30 (1H, brs, OH-16)], three Me groups including one Me group bearing with quaternary carbon [δH 1.33 (3H, s, H-19)] and two tertiary Me [δH 1.34 (3H, d, J = 6.0 Hz, H9), 1.36 (3H, d, J = 6.0 Hz, H-15), two of the AB portion of a typical ABX system signals at [δH 4.09 (1H, dqd, J = 12.6, 6.6, 4.5 Hz, Hax-3), 2.78 (1H, dd, J = 17.4, 4.5 Hz, Heq-4), 2.80 (1H, ddd, J = 17.4, 12.6, 0.9 Hz, Hax-4)] and [δH 4.28 (1H, dqd, J = 10.8, 6.0, 3.0 Hz, Hax-13), 2.36 (1H, dd, J = 15.0, 10.8 Hz, Hax-12), 2.52 (1H, ddd, J = 15.0, 3.0, 2.1 Hz, Heq-12)], and signals of α-methylene protons of one ketone [δH 2.54 (1H, dd, J = 14.4, 2.1 Hz, Heq-10), 3.00 (1H, d, J = 14.4 Hz, Hax10)]. The 13C-NMR and DEPT spectra exhibited the presence of three C]O carbonyl functions including one α,β-unsaturated C]O group (δC 202.8 (C-18)) in inden-1-one core, one ester C]O group (δC 171.9 (C20)), and one saturated ketone group (δC 204.6 (C-11)). Eight out of ten unsaturation equivalents were accounted for by the above-mentioned 13 C-NMR data, 1 was inferred to have two rings (one as a tetrahydropyran and another as a 4H-tetrahydropyrone). In addition, two rings were further determined as a tetrahydropyran skeleton combined with 4H-tetrahydropyrone ring via ketal function (C-1) by the following HMBC and COSY analyses. The observation followed by the COSY
2. Results and discussion Compound 1 was obtained as an optically-active whitish solid with [α]23 D : –41 (c 0.1, CHCl3). The molecular formula was determined as C22H26O8 with an IHD of 10 by HR-ESI-MS (m/z 441.15203 ([M +Na]+, C22H26NaO8+; calcd. 441.15199)), which was in agreement with the 1H- and 13C-NMR data (Table 1). The presence of an inden-1one skeleton was presumed from the UV absorption maxima at 212 (4.34) and 252 (3.99) nm, and a strong IR absorption at 1715 cm−1, as well as the observation of the featuring carbon resonances [δC 122.4, 124.4, 133.0, 138.3, 143.3, 148.4, and 202.8 (C-18)] in the 13C NMR spectrum (Table 1), revealed the presence of an inden-1-one functionality in 1. Its residual IR spectrum showed absorption bands for OH (3482 cm−1), and one ester (1725 cm−1) functionalities, respectively. The 13C-NMR (DEPT) spectrum of 1 (Table 1) exhibited 22 signals: four Me, four CH2 (one being next to an oxygen atom), and four CH (two being next to an oxygen atom), as well as ten quaternary C-atoms, including one peak at δ = 100.1 ppm (C-1) appeared as a strongly deshielded quaternary carbon atom, typical for a ketal group. The 1H-NMR spectrum of 1 exhibited signals attributed to two 243
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C]O group (1720 cm−1), respectively. Structurally, compound 2 was similar to monascuspirolide A, expect that the cyclopenten-1-one was changed to a cyclohexen-1-one ring in compound 2. Three aliphatic protons resonating at δH 2.98 (1H, ddd, J = 17.1, 11.4, 0.6 Hz, Hax-16), 3.24 (1H, dd, J = 17.1, 5.7 Hz, Heq-16), and 4.13 (1H, dd, J = 11.4, 5.7 Hz, Hax-17), were assigned Hax-16, Heq-16, and Hax-17, respectively, on the basis of their multiplicities, coupling constants, and HMBCs as follows: H-17 (δH 4.13)/C-18 (δC 78.1); H-16 (δH 3.24)/C-5 (δC 129.6) and C-6 (δC 139.8) (Fig. 2). The C-20 methyl group was located at C-18, based on the correlations between H-20 (δH 1.30) and C-17 (δC 72.7), C18 (δC 78.1), and C-19 (δC 200.3). The other correlations were similar to those of monascuspirolide A. The relative configuration of stereogenic centers C-17 and C-18 of cyclopenten-1-one ring were solved by the significant correlation of CH3-20/H-17 in the NOESY experiment. Thus, the OH-17 and CH3-20 were assigned as anti-relation form. The absolute configuration of compound 2 was further determined by application of the CD excitation chirality method. The CD spectrum of 2 showed a positive Cotton effect at 215 (Δε + 41.8) nm and a negative Cotton effect at 257 (Δε –7.1) nm, which was closed to that of (R)-2-acetyl-3,6-dihdroxycyclohex-2enone (Zaitsev and Mikhal’chuk, 2001) suggesting the R absolute configuration at C-17. Therefore, the (17R, 18R) absolute configuration was assigned for 2. However, the absolute configuration in C-1 remains undetermined. Compound 2 was identified as a previously unreported natural product and named monascuspirolide B. Monapurpureusin B (3) was obtained as a colorless oil with an [α]23 D : +34 (c 0.13, MeOH). In molecular formula was established as C10H12O4 by HRESIMS, indicating of five degrees of unsaturation. The IR spectrum showed an absorption at 3167 (OH), 1623 (C]O), 1446, 1373 (aromatic ring) cm−1. Its UV spectrum revealed absorption bands at λmax 212, 230, 276 and 314 nm and exhibited bathochromic shift after adding KOH aq. The 1H NMR spectroscopic data of 3 showed one ABX system aromatic ring at [δH 6.32 (d, J = 2.4 Hz, H-3′), 6.44 (dd, J = 8.4, 2.4 Hz, H-5′), and 7.83 (d, J = 8.4 Hz, H-6′)], one oxymethine [δH 4.33, (m, H-3)], one methylene group [δH 2.97 (dd, J = 15.4, 4.8 Hz, H-2a)/3.13 (dd, J = 15.4, 7.8 Hz, H-2b)], and one methyl group at [δH 1.24 (d, J = 6.6 Hz, CH3-4]. From 13C NMR, DEPT, and HSQC experiments, an aromatic ring [δC 104.3, 109.5, 115.1, 134.9, 166.6, and 167.2], one methyl group [δC 24.5 (C-4)], one oxymethine [δC 65.8 (C-3)], one methylene [δC 48.4 (C-2)], and another carbonyl group [δC 205.6 (C-1)] were confirmed. The COSY and the HMBC experiment for 3 revealed a correlation between the carbonyl carbon C-1 (δ 205.6) and the H-6′ proton, confirming that the 3-hydroxybutane-1-one group was located at the C-1′ position. The attachment of the 3-hydroxybutane-1one substituent at C-1′ was confirmed by the correlation between H-2 and H-6′ in NOESY spectrum (Fig. 3). The planar structure of 3 was elucidated as 1-(2,4-dihydroxyphenyl)-3-hydroxybutan-1-one The absolute configuration of 3 was assigned as S from the dextrorotatory optical activity {[α]23 D : +34} by analogy with previous observations on (S)-3-hydroxy-1-phenylbutan-1-one [[α]20 +76.1 (c 1.88, CHCl3)] D (Diehl and Brückner, 2017), and 3-hydroxy-1-phenyl-butan-1-one [[α]25 D +54.2 (c 0.1 CHCl3)] (Contente et al., 2016). Moreover, the observation that H-6' is deshielded by one carbonyl group [δC 205.6 (C1)] further supported the position of each aromatic substitution. The 1H- and 13C-NMR chemical shifts, and key HMBC, and COSY correlations are shown in Figs. 2 and 3. Based on these spectral evidences, the structure of 3 was confirmed and named monapurpureusin B. Additionally, three known compounds were assigned as following: monaspurpurone (4) (Cheng et al., 2010), monascin (5) (Li et al., 2006), and ankaflavin (6) (Li et al., 2006) were readily identified by comparison of their spectral data (UV, IR, 1H NMR, MS) with the data from the corresponding values in the literature, or with authentic samples. The four isolates present in sufficient amounts (1, 2, 4 and 5) were screened by examining their inhibitory effects on LPS-induced inducible
(Fig. 2), HSQC, and HMBC (Fig. 2) spectra of 1 established the presence of the partial three substitutes: A (inden-1-one core), B (tetrahydropyran, C-1 to C-8a), and C (4H-tetrahydropyrone, C-10 to C-15), for skeleton of 1. The entire skeleton of 1 was constructed by the aid of HMBC spectrum (Fig. 2). The HMBC correlation (Fig. 2) from H-8 (δH 7.88) to C-18 (δC 202.8) and C-6 (δC 148.4), H-5 (δH 7.36) to C-16 (δC 83.0), Me-19 group (δH 1.33) to C-17 (δC 85.8) and C-16 (δC 83.0), one hydroxyl group (OH-16) (δH 4.39) to C-16 (δC 83.0), C-6 (δC 148.4) and C-20 (δC 171.9), the terminal methyl group (δH 1.11) to C-22 (δC 63.5), H-22 (δH 4.03/4.25) to C-20 (δC 171.9) revealed the presence of an inden-1-one core connected with an ethyl carboxylate, one methyl group and two hydroxyl groups. The 1H-1H COSY plot [H–4 (δH 2.78/ 2.80)/H–3 (δH 4.09)/Me-9 (δH 1.34)] and by the HMBC correlations of H-4 (δH 2.79) to C-3 (δC 64.2), C-4a (δC 143.3) and C-8a (δC 138.3), H-3 (δH 4.09) to C-1 (δC 100.1), verified the junction of the two substitutes A to B unit at C-4a and C-8a. This allowed the skeleton of 1 to be determined as ethyl 6,7-dihydroxy-3,7-dimethyl-8-oxo-1,3,4,6,7,8-hexahydrocyclopenta[g]isochromene-6-carboxylate (rings A and B). Further confirmation by the COSY spectrum (Fig. 2) [H–12 (δH 2.36/2.52)/ H–13 (δH 4.09)/Me-15 (δH 1.36)] together with HMBC correlations between the H-13 (δH 4.28) and C-11 (δC 204.6), between the H-12 (δH 2.36/2.52) and C-11 (δC 204.6), C-13 (δC 66.5), C-15 (δC 20.9), and between H-10 (δH 2.54/3.00) and C-1 (δC 100.1), and C-11 (δC 204.6), established the presence of a 2-methyltetrahydro-4H-pyran-4-one (substitute C). The carbon C-1 (δC 100.1) of the ketal function is common to both substitutes B and C, suggesting a connection between the partial structures through C-1 as shown in Fig. 1. Furthermore, the attachment of the ring C to C-1 was disclosed according to the HMBC cross-peaks of H-8 with C-1 and of H-10 with C-1 and 8a. The relative configuration of 1 was determined by a combination of the analysis of the coupling constants and by extensive NOESY experiments. The relative configurations of stereogenic centers C-3 and C13 of rings B/C and the acetal spiro center at C-1, connecting the two rings, were determined by a combination of the analysis of the 1H NMR J-values and by extensive NOESY experiments (Fig. 3). NOEs for Me–9 (δH 1.34) /Hax–3 (δH 4.09), and CH2–4 (δH 2.78 (eq)/2.80 (ax)); Me–15 (δH 1.36) /Hax–13 (δH 4.28), and CH2–12 (δH 2.36 (ax)/2.52 (eq)), and Hax-12 (δH 2.36)/Hax-10 (δH 3.00) indicated that Hax–3, Me–15, Hax–12, Heq–4 were on the same side of the molecular plane, tentatively assumed as β-orientation. The Hax–13 was α–oriented which was further confirmed by the NOE Hax–13/Heq–12. The H–8/Hax–10 and Hax–10/ Hax–12 NOESY cross-peaks report on a preferred conformation of the tetrahydropyranone ring around the acetal spiro center, respectively. On the other hand, the NOE cross peaks H–5/CH2–4, and CH2–22/ Me–23 were also observed in Fig. 3. 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 minimization (Fig. 3). The calculated distances between Me–9/Hax–3 (2.521 Å), and CH2–4 (2.598 Å) is less than 4.00 Å, and between Me–15/Hax–13 (2.502 Å), and CH2–12 (2.472 Å) are also less than 4.00 Å; this is consistent with the well-defined NOESY (Fig. 3) observed for each of the proton pairs. Consequently, the relative configuration of C-3, and C-13, was assigned as rel-(3S,13S) (Fig. 3). The other NOESY correlation between OH-16 and Me-19 was also found, indicated that OH-16 and Me19 were on the same side of the ring system. The entire structure of 1 was confirmed by the 13C-NMR, DEPT, COSY, and NOESY experiments and named monascuspirolide A Compound 2 was obtained as an optically whitish solid with an [α]23 D : +10 (c 0.28, CHCl3). The HRESIMS data indicated a molecular formula of C20H24O6, based on the [M+Na]+ ion signal at m/z 383.14634 ([M+Na]+, calc. 383.14651). Analysis of NMR (Table 1), UV, and IR data suggested that the structure of 2 is similar to that of compound 1. The IR spectrum revealed the presence of OH groups (3485 cm−1), one conjugated carbonyl group (1686 cm−1), and one 244
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Fig. 1. Metabolites isolated from M. purpureus BCRC 38110.
nitric oxide synthase (iNOS)-dependent NO production in the murine macrophage cell line RAW 264.7 (see Table 3). Based on the results of our bioactivity assays, the following conclusions can be summarized: a) As shown in Table 3, compared to quercetin (IC50 value 36.9 ± 1.7 μM), which was used as positive control in this study, monascuspirolide A (1) and monascuspirolide B (2) displayed NO inhibitory activity with IC50 values of 17.5 ± 1.0 and 23.5 ± 2.8 μM, respectively. b) Compounds 4 and 5 were less effective NO inhibitory activity, with IC50 values 54.3 ± 4.7 and 61.3 ± 8.1 μM. c) Cytotoxicities were observed in the cells treated with compounds 3 and 4 (cell viability < 70% at 50 μM), whereas the other compounds had no influence on cell viability indicated that the inhibitory activities of LPSinduced NO production by active compounds were not resulted from its cytotoxicity.
Monascus purpureus BCRC 38113 (Cheng et al., 2010), purpureusone (antifungal inhibitory, cyclohex-2-enone derivative) from Monascus purpureus BCRC 38038 (Cheng et al., 2011), (E)-methyl 4-(2-acetyl-4oxotridec-1-enyl)-6-propylnicotinate (anti-inflammatory, pyridine alkaloid) from Monascus pilosus BCRC PK-1 (Cheng et al., 2011), monascusazaphilones A–C (inhibition on NO production, azaphilone derivatives) from Monascus purpureus BCRC 38108 (Wu et al., 2013), and monascuskaolin (anti-NO production, azaphilone) from Monascus kaoliang BCRC 31506 (Cheng et al., 2012a,b). In the course of our search for potential anti-inflammatory metabolites from natural sources, and M. purpureus BCRC 38110 has been found to be one of the active species. In this successive study, we focused on the minor secondary metabolites appearing in the EtOAc-soluble fraction of a 95% EtOH extract of the red mold rice produced by M. purpureus BCRC 38110. The new metabolites 1 & 2, found in this study is novel, naturally occurring compounds. It is worthy to mention that this is the first report of the spiro derivatives containing an indene ring isolated from this Monascus spp. and was found to have moderate anti-inflammatory properties, as determined the measurement of nitric oxide (NO) production levels in lipopolysaccharide (LPS)-stimulated murine-derived macrophages RAW264.7 cell lines. However, the chemical characteristics, as well as the biological activities, of many species remain chemically unexplored or poorly studied. Thus, the
3. Conclusions Reviewing the past literature regarding Monascus species (Akihisa et al., 2004; Akihisa et al., 2005; Ma et al., 2000) reveals that azaphilone, furanoisophthalides, amino acid, and polyketides are the major metabolites. In our previous studies, several species in the Monascus have been sources of bioactive secondary metabolites such as monaspurpurone (antifungal inhibitory, tetralone derivative) from 245
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apparatus (Yanaco, Kyoto, Japan) and were uncorrected. Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Kyoto, Japan), UV spectra were obtained with a Jasco-V-530 UV/vis spectrophotometer (Jasco, Kyoto, Japan), and IR spectra (KBr) were acquired with a Mattson Genesis IITM FTIR spectrophotometer (Mattson Genesis, Mattson, Germany). 1D (1H, 13C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra, were recorded on a Varian Germini-2000 spectrometer (Varian, Inc. Vacuum Technologies, MA, USA) operated at 200 MHz (1H) and 50 MHz (13C), Varian Unityplus-400 spectrometer (Varian, Inc. Vacuum Technologies, MA, USA) operated at 400 MHz (1H) and 100 MHz (13C), Varian Mercuryplus-400 spectrometer (Varian, Inc. Vacuum Technologies, MA, USA) operated at 400 MHz (1H) and 100 MHz (13C), and Varian VNMRS-600 spectrometer (Varian, Inc. Vacuum Technologies, MA, USA) operated at 600 MHz (1H) and 150 MHz (13C). Low-resolution mass spectra were obtained with POLARIS Q Thermo Finnigan (Thermo Fisher Scientific, Chicago, USA), Water ZQ 4000 (Waters, Massachuselts, USA), and VG Quattro GC/MS/ MS/DS (Waters, Massachuselts, USA) mass spectrometers. HRESIMS were recorded on a Bruker APEX II mass spectrometer (Bruker, Karlsruhe, Germany). Silica gel (70–230 and 230–400 mesh; Silicycle, Quebec, Canada) was used for column chromatography (CC), and silica gel 60 F254 (Merck, Darmstadt, Germany) and RP-18 F254S (Merck, Darmstadt, Germany) were used for TLC and preparative TLC. Respectively, visualised with Ce2(SO4)3 aq solution. Futher purification was performed by medium-performance liquid chromatography (MPLC) (ceramic pump: VSP-3050; EYELA, Kyoto, Japan). 4.2. Microorganism and preparation of red mold rice Monascus purpureus BCRC 38110 was obtained from BCRC, FIRDI (Hsinchu, Taiwan) and maintained on potato dextrose agar (PDA; Difco). The strain was cultured on PDA slants at 25 °C for 7 days and then the spores were harvested by sterile water. The spores (5 × 105/ ml) were seeded into 250 ml shake flasks containing 50 ml RGY medium (3% rice starch, 7% glycerol, 1.2% polypeptone, 3% soybean powder, 0.1% MgSO4, 0.2% NaNO3), and cultivated with shaking (150 rpm) at 25 °C 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 1.5 kg sterile rice and cultivated at 25°C for producing red mold rice (RMR; also called beni-koji in Japan). At day 7, 150 ml RGY medium was added for maintaining the growth of cells. After 21 days of cultivation, the RMR was harvested and lyophilized for the extraction of metabolites. 4.3. Extraction and isolation The dried red yeast rice of Monascus purpureus (1.5 kg) were extracted three times with 95% EtOH at room temperature. The ethanol syrup extract was partitioned between EtOAc and H2O (1:1) to afford EtOAc-soluble layer (15.6 g) and H2O-soluble layer. The EtOAc-soluble layer (15.6 g) was subjected to CC (180 g, 70–230 mesh, n-hexane/ EtOAc 100 :1–100% acetone, then wash with 100% methanol) to produce 15 subfractions. Fr. 12 was re-subjected to CC (30 g SiO2, 70–230 mesh; n-hexane/EtOAc 10:1→5:1) to furnish 15 subfractions: Fr. 12.1∼12.15. Fr. 12-10∼Fr. 12-12 were combined and subjected to MPLC (SiO2; CH2Cl2/EtOAc 4:1) to give monaspurpurone (4) (0.9 mg). Monascuspirolide A (1) (2.5 mg) and monapurpureusin B (3) (1.2 mg) were purified by semi-prepared HPLC on RP-18 silica (H2O/ACN 3:2, flow = 2) from Fr. 12-13. Fr. 14 was subjected to MPLC (SiO2; dichloromethane/EtOAc 3:2) to give 11 subfractions: Fr. 14.1∼14.11. Fr. 14-4∼Fr. 14-7 were combined and chromatographed on a reversed phase C-18 column eluted with W/acetone mixture (1:1) to give monascuspirolide B (2) (43.0 mg). Fraction 14.11 (66 mg) was purified by preparative TLC (n-hexane–EtOAc, 3:1) to give monascin (5) (2.1 mg) and ankaflavin (6) (4.7 mg).
Fig. 2. COSY (—) and HMBC (→) correlations of 1–3.
different biological activities of Monascus spp and related diverse secondary metabolites are seems to be worth to be further explored. Further study to realize the different biological activities of Monascus spp and related secondary metabolites are under in progress. 4. Experimental 4.1. General All melting points were determined on a Yanaco micromelting 246
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Fig. 3. Key NOESY (↔) correlations of 1 & 2.
4.3.1. Monascuspirolide A (1): whitish solid [α]23 D : –41 (c 0.1, CHCl3); UV (MeOH) λmax (log ε): 212 (4.34), 252 (3.99) nm; IR (ATR): 3482 (OH), 1725 (ester), 1715 (C]O) cm−1; CD (MeOH): λext 211 (Δε + 7.4), 240 (Δε + 1.1), 310 (Δε –0.9), 393 (Δε + 0.5) nm; HRESI-MS m/z: 441.15203 [M+Na]+ (calcd for C22H26NaO8+, 441.15199); 1H- (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDCl3): see Table 1.
Table 2 1 H- and 13C-NMR Data (acetone-d6, 600 and 150 MHz, resp.) of compound 3. δ in ppm, J in Hz. position
3 δC
4.3.2. Monascuspirolide B (2): whitish solid [α]23 D : +10 (c 0.28, CHCl3). UV (MeOH): λmax (log ε) 212 (4.28), 256 (3.97) nm. IR (ATR): 3485 (OH), 1720 (C = O), 1686 (C]O), 1447, 1384, 1354, 1327 (aromatic ring) cm−1; CD (MeOH): λext 215 (Δε + 41.8), 257 (Δε –7.1), 289 (Δε –7.2) nm; ESI-MS m/z 361 [M +H]+; HR-ESI-MS m/z 383.14634 [M+Na]+ (calcd for C20H24NaO6+, 383.14651); 1H- (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDCl3): see Table 1. 4.3.3. Monapurpureusin B (3): colourless oil [α]23 D : +34 (c 0.13, MeOH). UV (MeOH): λmax (log ε) 212 (4.06), 230 (3.79), 276 (4.00), 314 (3.76) nm. UV (MeOH + KOH): λmax (log ε) 202 (4.07), 250 (3.62), 332 (4.30) nm. IR (ATR): 3167 (OH), 1623 (C] O), 1446, 1373 (aromatic ring) cm−1; ESI-MS m/z 197 [M+H]+; HRESI-MS m/z 219.06270 [M+Na]+ (calcd for C10H12NaO4+, 219.062780); 1H- (600 MHz, acetone-d6) and 13C-NMR (150 MHz, acetone-d6): see Table 2.
1 2
205.6 48.4
3 4 1' 2' 3' 4' 5' 6'
65.8 24.5 115.1 167.2 104.3 166.6 109.5 134.9
δH (m, J in Hz) 2.97 (dd, 15.4, 4.8 Hz, H-2b) 3.13 (1H, dd, J = 15.4, 7.8 Hz, H-2a) 4.33 (m) 1.24 (d, 6.6 Hz) 6.32 (d, 2.4 Hz) 6.44 (dd, 8.4, 2.4 Hz) 7.83 (d, 8.4 Hz)
Gibco BRL Life Technologies, Inc.) supplemented with 10% heat inactivated fetal bovine serum (FBS) and incubated at 37 °C in a humidified 5% CO2 atmosphere with a 96-well flat-bottomed culture plate. After 24 h, the condition medium was replaced with fresh DMEM and FBS. Then the compounds 1 and 2 (0, 1, 5, 10, and 20 μg/mL) were added respectively in the presence of lipopolysaccharide (LPS, 1 μg/mL; Sigma, Cat no: L-2654) and incubated at the same condition for 24 h. The cultured cells were then centrifuged and the supernatants used for NO production measurement. The supernatant was mixed with an equal volume of the Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) ethyl-enediamine dihydrochloride in 2.5% phosphoric acid solution) and incubated for 10 min at room temperature. Nitrite concentration was determined by measuring the absorbance at 540 nm using an ELISA
4.4. Determination of NO production and cell viability assay The murine macrophage cell line RAW264.7 (BCRC 60001 = ATCC TIB-71) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, 247
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Table 3 Inhibitory Effects of the four isolates (1, 2, 5 and 6) from Monascus purpureus BCRC 38110 on LPS-activated NO productions in RAW 264.7 macrophages. Compounds
IC50 (μM)a NO
Monascuspirolide A (1) Monascuspirolide B (2) Monascin (5) Ankaflavin (6) Quercetinb
17.5 ± 1.0 23.5 ± 2.8 54.3 ± 4.7 61.3 ± 8.1 36.9 ± 1.7
Cheng, M.J., Wu, M.D., Chen, I.S., Chen, C.Y., Lo, W.L., Yuan, G.F., 2010. Secondary metabolites from the red mould rice of Monascus purpureus BCRC 38113. Nat. Prod. Res. 24, 1719–1725. Cheng, M.J., Wu, M.D., Chen, I.S., Tseng, M., Yuan, G.F., 2011. Chemical constituents from the fungus Monascus purpureus and their antifungal activity. Phytochem. Lett. 4, 372–376. Cheng, M.J., Wu, M.D., Su, Y.S., Chen, I.S., Yuan, G.F., 2012a. Anti-inflammatory compounds from Monascus pilosus-fermented rice. Phytochem. Lett. 5, 63–67. Cheng, M.J., Wu, M.D., Su, Y.S., Yuan, G.F., Chen, Y.L., Chen, I.S., 2012b. Chemical constituents from the fungus Monascus purpureus and their antifungal activity. Phytochem. Lett. 5, 262–266. Chiu, H.W., Fang, W.H., Chen, Y.L., Wu, M.D., Yuan, G.F., Ho, S.Y., 2012. Monascuspiloin enhances the radiation sensitivity of human prostate cancer cells by stimulating endoplasmic reticulum stress and inducing autophagy. PLoS One 7https://doi.org/10. 1371/journal.pone.0040462. Epub 0042012 Jul 0040463. Chiu, H.W., Chen, M.H., Fang, W.H., Hung, C.M., Chen, Y.L., Wu, M.D., Yuan, G.F., Wu, M.J., Wang, Y.J., 2013. Preventive effects of Monascus on androgen-related diseases: androgenetic alopecia, benign prostatic hyperplasia, and prostate cancer. J. Agric. Food Chem. 61, 4379–4386. Contente, M.L., Serra, I., Brambilla, M., Eberini, I., Gianazza, E., De Vitis, V., Molinari, F., Zambelli, P., Romano, D., 2016. Stereoselective reduction of aromatic ketones by a new ketoreductase from Pichia glucozyma. Appl. Microbiol. Biotechnol. 100, 193–201. Diehl, J., Brückner, R., 2017. Synthesis of enantiomerically pure β-hydroxy ketones via βKeto Weinreb amides by a condensation/asymmetric-hydrogenation/acylation sequence. Eur. J. Org. Chem. 2017, 278–286. Hong, M.Y., Seeram, N.P., Zhang, Y., Heber, D., 2008. Anticancer effects of Chinese red yeast rice versus monacolin K alone on colon cancer cells. J. Nutr. Biochem. 19, 448–458. Hsu, Y.W., Hsu, L.C., Liang, Y.H., Kuo, Y.H., Pan, T.M., 2010. Monaphilones A−C, three new antiproliferative azaphilone derivatives from Monascus purpureus NTU 568. J. Agric. Food Chem. 58, 8211–8216. Hsu, L.C., Liang, Y.H., Hsu, Y.W., Kuo, Y.H., Pan, T.M., 2013. Anti-inflammatory properties of yellow and orange pigments from Monascus purpureus NTU 568. J. Agric. Food Chem. 61, 2796–2802. Johansson, M., Kopcke, B., Anke, H., Sterner, O., 2002. Biologically active secondary metabolites from the ascomycete A111-95. J. Antibiot. 55, 104–106. Jongrungruangchok, S., Kittakoop, P., Yongsmith, B., Bavovada, R., Tanasupawat, S., Lartpornmatulee, N., Thebtaranonth, Y., 2004. Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang. Phytochemistry 65, 2569–2575. Jůzlová, P., Martínková, L., Křen, V., 1996. Secondary metabolites of the fungus Monascus: a review. J. Ind. Microbiol. Biotechnol. 65, 2569–2575. Lee, C.L., Tsai, T.Y., Wang, J.J., Pan, T.M., 2006. In vivo hypolipidemic effects and safety of low dosage Monascus powder in a hamster model of hyperlipidemia. Appl. Microbiol. Biotechnol. 70, 533–540. Lee, C.I., Lee, C.L., Hwang, J.F., Lee, Y.H., Wang, J.J., 2013. Monascus-fermented red mold rice exhibits cytotoxic effect and induces apoptosis on human breast cancer cells. Appl. Microbiol. Biotechnol. 97, 269–1278. Li, L.L., Chen, J.P., Kong, L.Y., 2006. Chemical constituents from Monascus anka. Chin. J. Nat. Med. 4, 32–35. Li, X., Liu, C., Duan, Z., Guo, S., 2013. HMG-CoA reductase inhibitors from Monascusfermented rice. J. Chem. 2013, 6. Loret, M.O., Morel, S., 2010. Isolation and structural characterization of two new metabolites from Monascus. J. Agric. Food Chem. 58, 1800–1803. Ma, J., Li, Y., Ye, Q., Li, J., Hua, Y., Ju, D., Zhang, D., Cooper, R., Chang, M., 2000. Constituents of red yeast rice, a traditional Chinese food and medicine. J. Agric. Food Chem. 48, 5220–5225. Manchand, P.S., Whalley, W.B., Chen, F.C., 1973. Isolation and structure of ankaflavin: a new pigment from Monascus anka. Phytochemistry 12, 2531–2532. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Shi, Y.C., Pan, T.M., 2010. Anti-diabetic effects of Monascus purpureus NTU 568 fermented products on streptozotocin-induced diabetic rats. J. Agric. Food Chem. 58, 7634–7640. Wu, M.D., Cheng, M.J., Yech, Y.J., Chen, Y.L., Chen, K.P., Yang, P.H., Chen, I.S., Yuan, G.F., 2013. Monascusazaphilones A–C, three new azaphilone analogues isolated from the fungus Monascus purpureus BCRC 38108. Nat. Prod. Res. 27, 1145–1152. Yasukawa, K., Takahashi, M., Natori, S., Kawai, K.I., Yamazaki, M., Takeuchi, M., Takido, M., 1994. Azaphilones inhibit tumor promotion by 12-O-tetradecanoylphorbol-13acetate in two-stage carcinogenesis in mice. Oncology 51, 108–112. Zaitsev, V.G., Mikhal’chuk, A.L., 2001. Enantioconvergent synthesis of (−)-(S)- and (+)-(R)-2-acetyl-3,6-dihydroxycyclohex-2-enone starting from rac-6-hydroxy-3methoxycyclohex-2-enone. Chirality 13, 488–492. Zhu, L., Yau, L.F., Lu, J.G., Zhu, G.Y., Wang, J.R., Han, Q.B., Hsiao, W.L., Jiang, Z.H., 2012. Cytotoxic dehydromonacolins from red yeast rice. J. Agric. Food Chem. 60, 934–939.
a The IC50 values are represented as means ± SD based on three independent experiments. b Quercetin was used as a positive control.
plate reader (μ Quant) (Johansson et al., 2002). The percentage of NO inhibition of the test compound was calculated as following: Inhibitory rate (%) = (1 – (LPS/sample – untreated)/(LPS – untreated)) x 100. All tests were run in triplicate and averaged. The data were expressed as a mean of three experiments. The software SigmaPlot was used for determining the IC50 values. A MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay was used to determine cell viability. The assay was modified from that of Mosmann (1983). The test is based upon the selective ability of living cells to reduce the yellow soluble salt, MTT, to a purple-blue insoluble formazan. MTT (Merck; dissolved in phosphatebuffered saline at 5 mg/mL) solution was added onto the attached cells mentioned above (10 μl per 100 μl culture) and incubated at 37 °C for 4 h. Then, DMSO was added and amount of colored formazan metabolite formed was determined by absorbance at 550 nm. The optical density of formazan formed in control (untreated) cells was taken as 100% viability. Conflict of interest Authors declare no conflict of interest. Acknowledgments The authors thank Senior Technician Mrs. Chyi-Jia Wang of Center for Resources, Research and Development (CRRD) of Kaohsiung Medical University (KMU) for measuring the 2D-NMR data. This work was kindly supported by the Food Industry Research and Development Institute (FIRDI) and partially supported by Ministry of Science and Techology, R.O.C. (MOST-106-2320-B-037-014). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2018.12.017. References 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. Chang, W.T., Chuang, C.H., Lee, W.J., Huang, C.S., 2016. Extract of Monascus purpureus CWT715 fermented from Sorghum liquor biowaste inhibits migration and invasion of SK-Hep-1 human hepatocarcinoma cells. Molecules 21, 1691.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Three new constituents from the fungus of Monascus purpureus and their anti-inflammatory activity
T
Ho-Cheng Wua, Ming-Jen Chengb, , Ming-Der Wub, Jih-Jung Chenc,d, Yen-Lin Chenb, ⁎⁎ Hsun-Shuo Changa,e, ⁎
a
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsinchu, 300, Taiwan c Faculty of Pharmacy, School of Pharmaceutical Sciences, National Yang-Ming University, Taipei, 112, Taiwan d Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, 404, Taiwan e School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 807, Kaohsiung, Taiwan b
ARTICLE INFO
ABSTRACT
Keywords: Monascus purpureus Eurotiaceae Spiro[isochromane-1,2′-pyran]-4′(3′H)-one Benzenoid Azaphilone Anti-inflammatory
A chemical study on the EtOAc-soluble fraction of the 95% EtOH extract of red yeast rice fermented with the fungus Monascus purpureus BCRC 38110 (Eurotiaceae) has resulted in the isolation of three new compounds, i.e., two new 5′,6′-dihydrospiro[isochromane-1,2′-pyran]-4′(3′H)-one derivatives, designated as monascuspirolide A (1) and monascuspirolide B (2) and one benzenoid derivative, monapurpureusin B (3), together with three known compounds, monaspurpurone (4), monascin (5), and ankaflavin (6). The structures and relative configurations of these compounds were elucidated by spectroscopic analyses, including 1D- and 2D-NMR spectroscopy and mass spectrometry, and by comparison of their spectral data with the literature data of authentic samples. To the best of our knowledge, the former two new compounds (1 & 2) displayed an unusual 5′,6′dihydrospiro[isochromane-1,2′-pyran]-4′(3′H)-one pattern compared to previous Monascus metabolites. Some phytochemicals were evaluated for anti-inflammatory activity through the measurement of nitric oxide (NO) production levels in lipopolysaccharide (LPS)-stimulated murine-derived macrophages RAW264.7 cell lines.
1. Introduction Fungi of the genus Monascus (family Monascaceae, class Eurotiomycetes) have been used to ferment rice in Asia for centuries. Originally, Monascus rice products were used to make rice wine, but red yeast rice, which is also called ang-kak or Hongqu, has also been used as a meat colorant, health food, and a Chinese folk medicine (Ma et al., 2000). However, in Western countries, red yeast rice extracts are mainly used for their cholesterol-lowering effects. The investigation of the chemical components of Monascus spp. dates back to 1973, when Endo et al. isolated monacolin K analogues from M. ruber (Manchand et al., 1973). A review of the literature regarding Monascus species (Akihisa et al., 2005; Ma et al., 2000) reveals that azaphilone, furanoisophthalides, amino acids, and polyketides are the major metabolites. Several types of secondary metabolites from red yeast rice have also been reported, including azaphilone pigments, monacolins, citrinin, dimerumic acid, and γ-aminobutyric acid (Akihisa et al., 2005; Hsu et al., 2010; Jongrungruangchok et al., 2004; Jůzlová
⁎
et al., 1996; Loret and Morel, 2010; Zhu et al., 2012). Recently, a number of reports have demonstrated that these red yeast rice metabolites exhibit a wide variety of biological activities. Monacolins were found to inhibit the activity of the enzyme HMG-CoA, which is involved in cholesterol biosynthesis (Lee et al., 2006; Li et al., 2013; Shi and Pan, 2010). In addition to its cholesterol-lowering effect, red yeast rice has also been reported to have anti-colon-cancer (Hong et al., 2008; Zhu et al., 2012), anti-liver-cancer (Chang et al., 2016), anti-prostate-cancer (Chiu et al., 2013, 2012), and anti-breast-cancer (Lee et al., 2013) activity. Recent studies have demonstrated that the azaphilone pigments of Monascus sp. contribute to its anti-inflammatory activity (Akihisa et al., 2005; Hsu et al., 2013; Yasukawa et al., 1994). In one study, azaphilone pigments were found to be more effective in inhibiting 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear oedema in mice than non-azaphilonoid pigments. Previous investigations of Monascus species have isolated constituents with various skeletons, mainly azaphilones (yellow, orange, and red pigments) and monacolin analogues. However, the pharmacological and toxicological
Corresponding author at: Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsinchu 300, Taiwan. Corresponding author at: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan. E-mail addresses: [email protected] (M.-J. Cheng), [email protected] (H.-S. Chang).
⁎⁎
https://doi.org/10.1016/j.phytol.2018.12.017 Received 14 September 2018; Received in revised form 17 December 2018; Accepted 18 December 2018 Available online 14 May 2019 1874-3900/ © 2018 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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effects of most of the secondary metabolites isolated from Monascus sp. remain unknown or unclear, and many metabolites still have not been identified. Despite a growing chemical library of compounds from Monascus, the phytochemical aspects of this genus have not been comprehensively examined. Accordingly, it is still worthwhile to explore and investigate the components of red yeast rice and their bioactivity. In previous studies, several Monascus species have been sources of bioactive secondary metabolites such as monaspurpurone (tetralone derivative with antifungal activity) from Monascus purpureus BCRC 38113 (Cheng et al., 2010), purpureusone (cyclohex-2-enone derivative with antifungal activity) from Monascus purpureus BCRC 38038 (Cheng et al., 2011), (E)-methyl-4-(2-acetyl-4-oxotridec-1-enyl)-6-propylnicotinate (an anti-inflammatory pyridine alkaloid) from Monascus pilosus BCRC PK-1 (Cheng et al., 2012a), monascusazaphilones A–C (azaphilone derivatives that inhibit NO production) from Monascus purpureus BCRC 38108 (Wu et al., 2013), and monascuskaolin (an azaphilone derivative that inhibits NO production) from Monascus kaoliang BCRC 31506 (Cheng et al., 2012a,b). In the course of our search for diverse secondary metabolites from natural fungal sources, we have screened over 150 species of fungal materials using TLC and HPLC profile analyses. One particular species, M. purpureus BCRC 38110, was identified as having more plentiful metabolites by our preliminary analyses. Monascus purpureus BCRC 38110 is a mutant yellow strain that shows a different morphology from other traditional red mutant Monascus species on potato dextrose agar (PDA) plates. Moreover, it was also found to be an active species in our continuous effort to search for interesting anti-inflammatory agents from natural sources. As part of a series of studies on the nitric oxide (NO) production and inhibitory activity of compounds derived from natural sources, we were especially interested in studying the chemical composition of red yeast rice. The 95% EtOH extract of M. purpureus BCRC 38110 showed inhibitory activity on lipopolysaccharide (LPS)induced nitric oxide (NO) release in RAW 264.7 murine macrophages in our primary screening (ca. 85% inhibition at a concentration of 10 mg/ mL). The aim of this study was to isolate the metabolites in the extract and to evaluate their bioactivity. Six compounds, including two unprecedented 5′,6′-dihydrospiro[isochromane-1,2′-pyran]-4′(3′H)-one: monascuspirolides A & B (1 & 2), one benzenoid derivative, monapurpureusin B (3), and together with three known compounds (4-6) isolated from the EtOAc-soluble fraction of the 95% EtOH extract of red yeast rice fermented with the title fungus. Details of the isolation, structure elucidation, and the anti-inflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production activities of some isolates are described herein.
Table 1 1 H- and 13C-NMR Data (CDCl3, 600 and 150 MHz, resp.) of compounds 1 and 2. δ in ppm, J in Hz. position
1 δC
1 2 3
100.1
4
36.9
4a 5 6 7 8 8a 9 10
143.3 124.4 148.4 133.0 122.4 138.3 21.9 51.8
11 12
204.6 48.6
13
66.5
14 15 16
20.9 83.0
17
85.8
18 19 20 21 22
202.8 22.3 171.9
23 OH-16 OH-17 OH-18
13.9
64.2
63.5
δH (m, J in Hz)
2 δC
δH (m, J in Hz)
100.0 4.09 (dqd, 12.6, 6.0, 4.5, Hax-3) 2.78 (dd,17.4, 4.5, Heq4) 2.80 (dd, 17.4, 12.6, 0.9, Hax-4) 7.36 (s) 7.88 (s) 1.34 (d, 6.0) 2.54 (dd, 14.4, 2.1) 3.00 (d, 14.4) 2.36 (dd like, 15.0, 10.8, Hax-12) 2.52 (dd, 15.0, 3.0, 2.1, Heq-12) 4.28 (dqd, 10.8, 6.0, 3.0, Hax-13) 1.36 (d, 6.0)
64.1 36.2
142.0 129.6 139.8 128.5 126.5 135.4 20.9 51.5 204.8 48.5
66.5 21.9 33.8
72.7 1.33 (s) 4.03 4.25 1.11 4.30 2.88
(dq, 10.5, 7.2) (dq, 10.5, 7.2) (t, 7.2) (br s) (br s)
78.1 200.3 17.4
4.09 (dqd, 11.6, 6.0, 4.2, Hax-3) 2.71 (dd,16.8, 4.2, Heq-4) 2.75 (dd,16.8, 11.6, Hax4) 7.14 (s) 8.07 (s) 1.33 (d, 6.6) 2.48 (dd, 14.4, 2.1) 3.05 (d, 14.4) 2.39 (dd, 14.7, 11.4, Hax12) 2.51 (ddd, 14.7, 3.0, 2.1, Heq-12) 4.26 (dqd, 11.4, 6.0, 3.0, Hax-13) 1.35 (d, 6.0) 2.98 (ddd, 17.1, 11.4, 0.6, , Hax-16) 3.24 (dd, 17.1, 5.7, , Heq16) 4.13 (dd, 11.4, 5.7, Hax17) 1.30 (s)
2.53 (br s) or 3.82 (br s) 3.82 (br s) or 2.53 (br s)
singlet aromatic protons [δH 7.36 (1H, s, H-5) and 7.88 (1H, s, H-8)], one ethoxy group [δH 1.11 (3H, t, J = 7.2 Hz, H-23), 4.03 (dq, J = 10.5, 7.2 Hz, H-22b), 4.25 (dq, J = 10.5, 7.2 Hz, H-22a)], two hydroxyl groups [δH 2.88 (1H, brs, OH-17), 4.30 (1H, brs, OH-16)], three Me groups including one Me group bearing with quaternary carbon [δH 1.33 (3H, s, H-19)] and two tertiary Me [δH 1.34 (3H, d, J = 6.0 Hz, H9), 1.36 (3H, d, J = 6.0 Hz, H-15), two of the AB portion of a typical ABX system signals at [δH 4.09 (1H, dqd, J = 12.6, 6.6, 4.5 Hz, Hax-3), 2.78 (1H, dd, J = 17.4, 4.5 Hz, Heq-4), 2.80 (1H, ddd, J = 17.4, 12.6, 0.9 Hz, Hax-4)] and [δH 4.28 (1H, dqd, J = 10.8, 6.0, 3.0 Hz, Hax-13), 2.36 (1H, dd, J = 15.0, 10.8 Hz, Hax-12), 2.52 (1H, ddd, J = 15.0, 3.0, 2.1 Hz, Heq-12)], and signals of α-methylene protons of one ketone [δH 2.54 (1H, dd, J = 14.4, 2.1 Hz, Heq-10), 3.00 (1H, d, J = 14.4 Hz, Hax10)]. The 13C-NMR and DEPT spectra exhibited the presence of three C]O carbonyl functions including one α,β-unsaturated C]O group (δC 202.8 (C-18)) in inden-1-one core, one ester C]O group (δC 171.9 (C20)), and one saturated ketone group (δC 204.6 (C-11)). Eight out of ten unsaturation equivalents were accounted for by the above-mentioned 13 C-NMR data, 1 was inferred to have two rings (one as a tetrahydropyran and another as a 4H-tetrahydropyrone). In addition, two rings were further determined as a tetrahydropyran skeleton combined with 4H-tetrahydropyrone ring via ketal function (C-1) by the following HMBC and COSY analyses. The observation followed by the COSY
2. Results and discussion Compound 1 was obtained as an optically-active whitish solid with [α]23 D : –41 (c 0.1, CHCl3). The molecular formula was determined as C22H26O8 with an IHD of 10 by HR-ESI-MS (m/z 441.15203 ([M +Na]+, C22H26NaO8+; calcd. 441.15199)), which was in agreement with the 1H- and 13C-NMR data (Table 1). The presence of an inden-1one skeleton was presumed from the UV absorption maxima at 212 (4.34) and 252 (3.99) nm, and a strong IR absorption at 1715 cm−1, as well as the observation of the featuring carbon resonances [δC 122.4, 124.4, 133.0, 138.3, 143.3, 148.4, and 202.8 (C-18)] in the 13C NMR spectrum (Table 1), revealed the presence of an inden-1-one functionality in 1. Its residual IR spectrum showed absorption bands for OH (3482 cm−1), and one ester (1725 cm−1) functionalities, respectively. The 13C-NMR (DEPT) spectrum of 1 (Table 1) exhibited 22 signals: four Me, four CH2 (one being next to an oxygen atom), and four CH (two being next to an oxygen atom), as well as ten quaternary C-atoms, including one peak at δ = 100.1 ppm (C-1) appeared as a strongly deshielded quaternary carbon atom, typical for a ketal group. The 1H-NMR spectrum of 1 exhibited signals attributed to two 243
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C]O group (1720 cm−1), respectively. Structurally, compound 2 was similar to monascuspirolide A, expect that the cyclopenten-1-one was changed to a cyclohexen-1-one ring in compound 2. Three aliphatic protons resonating at δH 2.98 (1H, ddd, J = 17.1, 11.4, 0.6 Hz, Hax-16), 3.24 (1H, dd, J = 17.1, 5.7 Hz, Heq-16), and 4.13 (1H, dd, J = 11.4, 5.7 Hz, Hax-17), were assigned Hax-16, Heq-16, and Hax-17, respectively, on the basis of their multiplicities, coupling constants, and HMBCs as follows: H-17 (δH 4.13)/C-18 (δC 78.1); H-16 (δH 3.24)/C-5 (δC 129.6) and C-6 (δC 139.8) (Fig. 2). The C-20 methyl group was located at C-18, based on the correlations between H-20 (δH 1.30) and C-17 (δC 72.7), C18 (δC 78.1), and C-19 (δC 200.3). The other correlations were similar to those of monascuspirolide A. The relative configuration of stereogenic centers C-17 and C-18 of cyclopenten-1-one ring were solved by the significant correlation of CH3-20/H-17 in the NOESY experiment. Thus, the OH-17 and CH3-20 were assigned as anti-relation form. The absolute configuration of compound 2 was further determined by application of the CD excitation chirality method. The CD spectrum of 2 showed a positive Cotton effect at 215 (Δε + 41.8) nm and a negative Cotton effect at 257 (Δε –7.1) nm, which was closed to that of (R)-2-acetyl-3,6-dihdroxycyclohex-2enone (Zaitsev and Mikhal’chuk, 2001) suggesting the R absolute configuration at C-17. Therefore, the (17R, 18R) absolute configuration was assigned for 2. However, the absolute configuration in C-1 remains undetermined. Compound 2 was identified as a previously unreported natural product and named monascuspirolide B. Monapurpureusin B (3) was obtained as a colorless oil with an [α]23 D : +34 (c 0.13, MeOH). In molecular formula was established as C10H12O4 by HRESIMS, indicating of five degrees of unsaturation. The IR spectrum showed an absorption at 3167 (OH), 1623 (C]O), 1446, 1373 (aromatic ring) cm−1. Its UV spectrum revealed absorption bands at λmax 212, 230, 276 and 314 nm and exhibited bathochromic shift after adding KOH aq. The 1H NMR spectroscopic data of 3 showed one ABX system aromatic ring at [δH 6.32 (d, J = 2.4 Hz, H-3′), 6.44 (dd, J = 8.4, 2.4 Hz, H-5′), and 7.83 (d, J = 8.4 Hz, H-6′)], one oxymethine [δH 4.33, (m, H-3)], one methylene group [δH 2.97 (dd, J = 15.4, 4.8 Hz, H-2a)/3.13 (dd, J = 15.4, 7.8 Hz, H-2b)], and one methyl group at [δH 1.24 (d, J = 6.6 Hz, CH3-4]. From 13C NMR, DEPT, and HSQC experiments, an aromatic ring [δC 104.3, 109.5, 115.1, 134.9, 166.6, and 167.2], one methyl group [δC 24.5 (C-4)], one oxymethine [δC 65.8 (C-3)], one methylene [δC 48.4 (C-2)], and another carbonyl group [δC 205.6 (C-1)] were confirmed. The COSY and the HMBC experiment for 3 revealed a correlation between the carbonyl carbon C-1 (δ 205.6) and the H-6′ proton, confirming that the 3-hydroxybutane-1-one group was located at the C-1′ position. The attachment of the 3-hydroxybutane-1one substituent at C-1′ was confirmed by the correlation between H-2 and H-6′ in NOESY spectrum (Fig. 3). The planar structure of 3 was elucidated as 1-(2,4-dihydroxyphenyl)-3-hydroxybutan-1-one The absolute configuration of 3 was assigned as S from the dextrorotatory optical activity {[α]23 D : +34} by analogy with previous observations on (S)-3-hydroxy-1-phenylbutan-1-one [[α]20 +76.1 (c 1.88, CHCl3)] D (Diehl and Brückner, 2017), and 3-hydroxy-1-phenyl-butan-1-one [[α]25 D +54.2 (c 0.1 CHCl3)] (Contente et al., 2016). Moreover, the observation that H-6' is deshielded by one carbonyl group [δC 205.6 (C1)] further supported the position of each aromatic substitution. The 1H- and 13C-NMR chemical shifts, and key HMBC, and COSY correlations are shown in Figs. 2 and 3. Based on these spectral evidences, the structure of 3 was confirmed and named monapurpureusin B. Additionally, three known compounds were assigned as following: monaspurpurone (4) (Cheng et al., 2010), monascin (5) (Li et al., 2006), and ankaflavin (6) (Li et al., 2006) were readily identified by comparison of their spectral data (UV, IR, 1H NMR, MS) with the data from the corresponding values in the literature, or with authentic samples. The four isolates present in sufficient amounts (1, 2, 4 and 5) were screened by examining their inhibitory effects on LPS-induced inducible
(Fig. 2), HSQC, and HMBC (Fig. 2) spectra of 1 established the presence of the partial three substitutes: A (inden-1-one core), B (tetrahydropyran, C-1 to C-8a), and C (4H-tetrahydropyrone, C-10 to C-15), for skeleton of 1. The entire skeleton of 1 was constructed by the aid of HMBC spectrum (Fig. 2). The HMBC correlation (Fig. 2) from H-8 (δH 7.88) to C-18 (δC 202.8) and C-6 (δC 148.4), H-5 (δH 7.36) to C-16 (δC 83.0), Me-19 group (δH 1.33) to C-17 (δC 85.8) and C-16 (δC 83.0), one hydroxyl group (OH-16) (δH 4.39) to C-16 (δC 83.0), C-6 (δC 148.4) and C-20 (δC 171.9), the terminal methyl group (δH 1.11) to C-22 (δC 63.5), H-22 (δH 4.03/4.25) to C-20 (δC 171.9) revealed the presence of an inden-1-one core connected with an ethyl carboxylate, one methyl group and two hydroxyl groups. The 1H-1H COSY plot [H–4 (δH 2.78/ 2.80)/H–3 (δH 4.09)/Me-9 (δH 1.34)] and by the HMBC correlations of H-4 (δH 2.79) to C-3 (δC 64.2), C-4a (δC 143.3) and C-8a (δC 138.3), H-3 (δH 4.09) to C-1 (δC 100.1), verified the junction of the two substitutes A to B unit at C-4a and C-8a. This allowed the skeleton of 1 to be determined as ethyl 6,7-dihydroxy-3,7-dimethyl-8-oxo-1,3,4,6,7,8-hexahydrocyclopenta[g]isochromene-6-carboxylate (rings A and B). Further confirmation by the COSY spectrum (Fig. 2) [H–12 (δH 2.36/2.52)/ H–13 (δH 4.09)/Me-15 (δH 1.36)] together with HMBC correlations between the H-13 (δH 4.28) and C-11 (δC 204.6), between the H-12 (δH 2.36/2.52) and C-11 (δC 204.6), C-13 (δC 66.5), C-15 (δC 20.9), and between H-10 (δH 2.54/3.00) and C-1 (δC 100.1), and C-11 (δC 204.6), established the presence of a 2-methyltetrahydro-4H-pyran-4-one (substitute C). The carbon C-1 (δC 100.1) of the ketal function is common to both substitutes B and C, suggesting a connection between the partial structures through C-1 as shown in Fig. 1. Furthermore, the attachment of the ring C to C-1 was disclosed according to the HMBC cross-peaks of H-8 with C-1 and of H-10 with C-1 and 8a. The relative configuration of 1 was determined by a combination of the analysis of the coupling constants and by extensive NOESY experiments. The relative configurations of stereogenic centers C-3 and C13 of rings B/C and the acetal spiro center at C-1, connecting the two rings, were determined by a combination of the analysis of the 1H NMR J-values and by extensive NOESY experiments (Fig. 3). NOEs for Me–9 (δH 1.34) /Hax–3 (δH 4.09), and CH2–4 (δH 2.78 (eq)/2.80 (ax)); Me–15 (δH 1.36) /Hax–13 (δH 4.28), and CH2–12 (δH 2.36 (ax)/2.52 (eq)), and Hax-12 (δH 2.36)/Hax-10 (δH 3.00) indicated that Hax–3, Me–15, Hax–12, Heq–4 were on the same side of the molecular plane, tentatively assumed as β-orientation. The Hax–13 was α–oriented which was further confirmed by the NOE Hax–13/Heq–12. The H–8/Hax–10 and Hax–10/ Hax–12 NOESY cross-peaks report on a preferred conformation of the tetrahydropyranone ring around the acetal spiro center, respectively. On the other hand, the NOE cross peaks H–5/CH2–4, and CH2–22/ Me–23 were also observed in Fig. 3. 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 minimization (Fig. 3). The calculated distances between Me–9/Hax–3 (2.521 Å), and CH2–4 (2.598 Å) is less than 4.00 Å, and between Me–15/Hax–13 (2.502 Å), and CH2–12 (2.472 Å) are also less than 4.00 Å; this is consistent with the well-defined NOESY (Fig. 3) observed for each of the proton pairs. Consequently, the relative configuration of C-3, and C-13, was assigned as rel-(3S,13S) (Fig. 3). The other NOESY correlation between OH-16 and Me-19 was also found, indicated that OH-16 and Me19 were on the same side of the ring system. The entire structure of 1 was confirmed by the 13C-NMR, DEPT, COSY, and NOESY experiments and named monascuspirolide A Compound 2 was obtained as an optically whitish solid with an [α]23 D : +10 (c 0.28, CHCl3). The HRESIMS data indicated a molecular formula of C20H24O6, based on the [M+Na]+ ion signal at m/z 383.14634 ([M+Na]+, calc. 383.14651). Analysis of NMR (Table 1), UV, and IR data suggested that the structure of 2 is similar to that of compound 1. The IR spectrum revealed the presence of OH groups (3485 cm−1), one conjugated carbonyl group (1686 cm−1), and one 244
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Fig. 1. Metabolites isolated from M. purpureus BCRC 38110.
nitric oxide synthase (iNOS)-dependent NO production in the murine macrophage cell line RAW 264.7 (see Table 3). Based on the results of our bioactivity assays, the following conclusions can be summarized: a) As shown in Table 3, compared to quercetin (IC50 value 36.9 ± 1.7 μM), which was used as positive control in this study, monascuspirolide A (1) and monascuspirolide B (2) displayed NO inhibitory activity with IC50 values of 17.5 ± 1.0 and 23.5 ± 2.8 μM, respectively. b) Compounds 4 and 5 were less effective NO inhibitory activity, with IC50 values 54.3 ± 4.7 and 61.3 ± 8.1 μM. c) Cytotoxicities were observed in the cells treated with compounds 3 and 4 (cell viability < 70% at 50 μM), whereas the other compounds had no influence on cell viability indicated that the inhibitory activities of LPSinduced NO production by active compounds were not resulted from its cytotoxicity.
Monascus purpureus BCRC 38113 (Cheng et al., 2010), purpureusone (antifungal inhibitory, cyclohex-2-enone derivative) from Monascus purpureus BCRC 38038 (Cheng et al., 2011), (E)-methyl 4-(2-acetyl-4oxotridec-1-enyl)-6-propylnicotinate (anti-inflammatory, pyridine alkaloid) from Monascus pilosus BCRC PK-1 (Cheng et al., 2011), monascusazaphilones A–C (inhibition on NO production, azaphilone derivatives) from Monascus purpureus BCRC 38108 (Wu et al., 2013), and monascuskaolin (anti-NO production, azaphilone) from Monascus kaoliang BCRC 31506 (Cheng et al., 2012a,b). In the course of our search for potential anti-inflammatory metabolites from natural sources, and M. purpureus BCRC 38110 has been found to be one of the active species. In this successive study, we focused on the minor secondary metabolites appearing in the EtOAc-soluble fraction of a 95% EtOH extract of the red mold rice produced by M. purpureus BCRC 38110. The new metabolites 1 & 2, found in this study is novel, naturally occurring compounds. It is worthy to mention that this is the first report of the spiro derivatives containing an indene ring isolated from this Monascus spp. and was found to have moderate anti-inflammatory properties, as determined the measurement of nitric oxide (NO) production levels in lipopolysaccharide (LPS)-stimulated murine-derived macrophages RAW264.7 cell lines. However, the chemical characteristics, as well as the biological activities, of many species remain chemically unexplored or poorly studied. Thus, the
3. Conclusions Reviewing the past literature regarding Monascus species (Akihisa et al., 2004; Akihisa et al., 2005; Ma et al., 2000) reveals that azaphilone, furanoisophthalides, amino acid, and polyketides are the major metabolites. In our previous studies, several species in the Monascus have been sources of bioactive secondary metabolites such as monaspurpurone (antifungal inhibitory, tetralone derivative) from 245
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apparatus (Yanaco, Kyoto, Japan) and were uncorrected. Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Kyoto, Japan), UV spectra were obtained with a Jasco-V-530 UV/vis spectrophotometer (Jasco, Kyoto, Japan), and IR spectra (KBr) were acquired with a Mattson Genesis IITM FTIR spectrophotometer (Mattson Genesis, Mattson, Germany). 1D (1H, 13C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra, were recorded on a Varian Germini-2000 spectrometer (Varian, Inc. Vacuum Technologies, MA, USA) operated at 200 MHz (1H) and 50 MHz (13C), Varian Unityplus-400 spectrometer (Varian, Inc. Vacuum Technologies, MA, USA) operated at 400 MHz (1H) and 100 MHz (13C), Varian Mercuryplus-400 spectrometer (Varian, Inc. Vacuum Technologies, MA, USA) operated at 400 MHz (1H) and 100 MHz (13C), and Varian VNMRS-600 spectrometer (Varian, Inc. Vacuum Technologies, MA, USA) operated at 600 MHz (1H) and 150 MHz (13C). Low-resolution mass spectra were obtained with POLARIS Q Thermo Finnigan (Thermo Fisher Scientific, Chicago, USA), Water ZQ 4000 (Waters, Massachuselts, USA), and VG Quattro GC/MS/ MS/DS (Waters, Massachuselts, USA) mass spectrometers. HRESIMS were recorded on a Bruker APEX II mass spectrometer (Bruker, Karlsruhe, Germany). Silica gel (70–230 and 230–400 mesh; Silicycle, Quebec, Canada) was used for column chromatography (CC), and silica gel 60 F254 (Merck, Darmstadt, Germany) and RP-18 F254S (Merck, Darmstadt, Germany) were used for TLC and preparative TLC. Respectively, visualised with Ce2(SO4)3 aq solution. Futher purification was performed by medium-performance liquid chromatography (MPLC) (ceramic pump: VSP-3050; EYELA, Kyoto, Japan). 4.2. Microorganism and preparation of red mold rice Monascus purpureus BCRC 38110 was obtained from BCRC, FIRDI (Hsinchu, Taiwan) and maintained on potato dextrose agar (PDA; Difco). The strain was cultured on PDA slants at 25 °C for 7 days and then the spores were harvested by sterile water. The spores (5 × 105/ ml) were seeded into 250 ml shake flasks containing 50 ml RGY medium (3% rice starch, 7% glycerol, 1.2% polypeptone, 3% soybean powder, 0.1% MgSO4, 0.2% NaNO3), and cultivated with shaking (150 rpm) at 25 °C 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 1.5 kg sterile rice and cultivated at 25°C for producing red mold rice (RMR; also called beni-koji in Japan). At day 7, 150 ml RGY medium was added for maintaining the growth of cells. After 21 days of cultivation, the RMR was harvested and lyophilized for the extraction of metabolites. 4.3. Extraction and isolation The dried red yeast rice of Monascus purpureus (1.5 kg) were extracted three times with 95% EtOH at room temperature. The ethanol syrup extract was partitioned between EtOAc and H2O (1:1) to afford EtOAc-soluble layer (15.6 g) and H2O-soluble layer. The EtOAc-soluble layer (15.6 g) was subjected to CC (180 g, 70–230 mesh, n-hexane/ EtOAc 100 :1–100% acetone, then wash with 100% methanol) to produce 15 subfractions. Fr. 12 was re-subjected to CC (30 g SiO2, 70–230 mesh; n-hexane/EtOAc 10:1→5:1) to furnish 15 subfractions: Fr. 12.1∼12.15. Fr. 12-10∼Fr. 12-12 were combined and subjected to MPLC (SiO2; CH2Cl2/EtOAc 4:1) to give monaspurpurone (4) (0.9 mg). Monascuspirolide A (1) (2.5 mg) and monapurpureusin B (3) (1.2 mg) were purified by semi-prepared HPLC on RP-18 silica (H2O/ACN 3:2, flow = 2) from Fr. 12-13. Fr. 14 was subjected to MPLC (SiO2; dichloromethane/EtOAc 3:2) to give 11 subfractions: Fr. 14.1∼14.11. Fr. 14-4∼Fr. 14-7 were combined and chromatographed on a reversed phase C-18 column eluted with W/acetone mixture (1:1) to give monascuspirolide B (2) (43.0 mg). Fraction 14.11 (66 mg) was purified by preparative TLC (n-hexane–EtOAc, 3:1) to give monascin (5) (2.1 mg) and ankaflavin (6) (4.7 mg).
Fig. 2. COSY (—) and HMBC (→) correlations of 1–3.
different biological activities of Monascus spp and related diverse secondary metabolites are seems to be worth to be further explored. Further study to realize the different biological activities of Monascus spp and related secondary metabolites are under in progress. 4. Experimental 4.1. General All melting points were determined on a Yanaco micromelting 246
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Fig. 3. Key NOESY (↔) correlations of 1 & 2.
4.3.1. Monascuspirolide A (1): whitish solid [α]23 D : –41 (c 0.1, CHCl3); UV (MeOH) λmax (log ε): 212 (4.34), 252 (3.99) nm; IR (ATR): 3482 (OH), 1725 (ester), 1715 (C]O) cm−1; CD (MeOH): λext 211 (Δε + 7.4), 240 (Δε + 1.1), 310 (Δε –0.9), 393 (Δε + 0.5) nm; HRESI-MS m/z: 441.15203 [M+Na]+ (calcd for C22H26NaO8+, 441.15199); 1H- (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDCl3): see Table 1.
Table 2 1 H- and 13C-NMR Data (acetone-d6, 600 and 150 MHz, resp.) of compound 3. δ in ppm, J in Hz. position
3 δC
4.3.2. Monascuspirolide B (2): whitish solid [α]23 D : +10 (c 0.28, CHCl3). UV (MeOH): λmax (log ε) 212 (4.28), 256 (3.97) nm. IR (ATR): 3485 (OH), 1720 (C = O), 1686 (C]O), 1447, 1384, 1354, 1327 (aromatic ring) cm−1; CD (MeOH): λext 215 (Δε + 41.8), 257 (Δε –7.1), 289 (Δε –7.2) nm; ESI-MS m/z 361 [M +H]+; HR-ESI-MS m/z 383.14634 [M+Na]+ (calcd for C20H24NaO6+, 383.14651); 1H- (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDCl3): see Table 1. 4.3.3. Monapurpureusin B (3): colourless oil [α]23 D : +34 (c 0.13, MeOH). UV (MeOH): λmax (log ε) 212 (4.06), 230 (3.79), 276 (4.00), 314 (3.76) nm. UV (MeOH + KOH): λmax (log ε) 202 (4.07), 250 (3.62), 332 (4.30) nm. IR (ATR): 3167 (OH), 1623 (C] O), 1446, 1373 (aromatic ring) cm−1; ESI-MS m/z 197 [M+H]+; HRESI-MS m/z 219.06270 [M+Na]+ (calcd for C10H12NaO4+, 219.062780); 1H- (600 MHz, acetone-d6) and 13C-NMR (150 MHz, acetone-d6): see Table 2.
1 2
205.6 48.4
3 4 1' 2' 3' 4' 5' 6'
65.8 24.5 115.1 167.2 104.3 166.6 109.5 134.9
δH (m, J in Hz) 2.97 (dd, 15.4, 4.8 Hz, H-2b) 3.13 (1H, dd, J = 15.4, 7.8 Hz, H-2a) 4.33 (m) 1.24 (d, 6.6 Hz) 6.32 (d, 2.4 Hz) 6.44 (dd, 8.4, 2.4 Hz) 7.83 (d, 8.4 Hz)
Gibco BRL Life Technologies, Inc.) supplemented with 10% heat inactivated fetal bovine serum (FBS) and incubated at 37 °C in a humidified 5% CO2 atmosphere with a 96-well flat-bottomed culture plate. After 24 h, the condition medium was replaced with fresh DMEM and FBS. Then the compounds 1 and 2 (0, 1, 5, 10, and 20 μg/mL) were added respectively in the presence of lipopolysaccharide (LPS, 1 μg/mL; Sigma, Cat no: L-2654) and incubated at the same condition for 24 h. The cultured cells were then centrifuged and the supernatants used for NO production measurement. The supernatant was mixed with an equal volume of the Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) ethyl-enediamine dihydrochloride in 2.5% phosphoric acid solution) and incubated for 10 min at room temperature. Nitrite concentration was determined by measuring the absorbance at 540 nm using an ELISA
4.4. Determination of NO production and cell viability assay The murine macrophage cell line RAW264.7 (BCRC 60001 = ATCC TIB-71) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, 247
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Table 3 Inhibitory Effects of the four isolates (1, 2, 5 and 6) from Monascus purpureus BCRC 38110 on LPS-activated NO productions in RAW 264.7 macrophages. Compounds
IC50 (μM)a NO
Monascuspirolide A (1) Monascuspirolide B (2) Monascin (5) Ankaflavin (6) Quercetinb
17.5 ± 1.0 23.5 ± 2.8 54.3 ± 4.7 61.3 ± 8.1 36.9 ± 1.7
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a The IC50 values are represented as means ± SD based on three independent experiments. b Quercetin was used as a positive control.
plate reader (μ Quant) (Johansson et al., 2002). The percentage of NO inhibition of the test compound was calculated as following: Inhibitory rate (%) = (1 – (LPS/sample – untreated)/(LPS – untreated)) x 100. All tests were run in triplicate and averaged. The data were expressed as a mean of three experiments. The software SigmaPlot was used for determining the IC50 values. A MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay was used to determine cell viability. The assay was modified from that of Mosmann (1983). The test is based upon the selective ability of living cells to reduce the yellow soluble salt, MTT, to a purple-blue insoluble formazan. MTT (Merck; dissolved in phosphatebuffered saline at 5 mg/mL) solution was added onto the attached cells mentioned above (10 μl per 100 μl culture) and incubated at 37 °C for 4 h. Then, DMSO was added and amount of colored formazan metabolite formed was determined by absorbance at 550 nm. The optical density of formazan formed in control (untreated) cells was taken as 100% viability. Conflict of interest Authors declare no conflict of interest. Acknowledgments The authors thank Senior Technician Mrs. Chyi-Jia Wang of Center for Resources, Research and Development (CRRD) of Kaohsiung Medical University (KMU) for measuring the 2D-NMR data. This work was kindly supported by the Food Industry Research and Development Institute (FIRDI) and partially supported by Ministry of Science and Techology, R.O.C. (MOST-106-2320-B-037-014). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2018.12.017. References 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. Chang, W.T., Chuang, C.H., Lee, W.J., Huang, C.S., 2016. Extract of Monascus purpureus CWT715 fermented from Sorghum liquor biowaste inhibits migration and invasion of SK-Hep-1 human hepatocarcinoma cells. Molecules 21, 1691.
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