Secondary Metabolites with Antifungal Activities from Mangrove Derived Fungus Monascus purpureus WMD2424

The mold Monascus, also called red yeast rice, anka, or koji, has been used as the natural food coloring agent and food additives for more than 1000 years in Asian countries. It has also been used in Chinese herbology and traditional Chinese medicine due to its easing digestion and antiseptic effects. However, under different culture conditions, the ingredients in Monascus-fermented products may be changed. Therefore, an in-depth understanding of the ingredients, as well as the bioactivities of Monascus-derived natural products, is important. Here, through the thorough investigation into the chemical constituents of M. purpureus wmd2424, five previously undescribed compounds, monascuspurins A–E (1–5), were isolated from the EtOAc extract of mangrove-derived fungus Monascus purpureus wmd2424 cultured in RGY medium. All the constituents were confirmed via HRESIMS and 1D- and 2D-NMR spectroscopy. Their antifungal activity was also evaluated. Our results showed that four constituents (compounds 3–5) possessed mild antifungal activity against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae. It is worth mentioning that the chemical composition of the type strain Monascus purpureus wmd2424 has never been studied.


Introduction
Throughout human history, food has been used to satisfy hunger and provide nutrition. Nowadays, food can be widely used not only to eliminate diseases, but also to improve the quality of life. Finding beneficial food resources from the wisdom of your ancestors is a fairly effective strategy.
Despite plants, fungi (e.g., Actinomucor spp., Amylomyces spp., Rhizopus spp., Monascus spp., Neurospora spp., Aspergillus spp., Penicillium spp., Torulopsis spp., Trichosporon spp., and Zygosaccharomyces spp.) also take an important place in producing various food products in fermented forms [1]. Fungi of the genus Monascus (Monascaceae) have been used to ferment rice in Asia for centuries. It has been widely utilized as food additives, natural food coloring agent, food antiseptic, and healthy food for nearly two thousand years [2,3]. The production of red yeast rice was used as a Chinese folk medicine, recorded in old Chinese literature as a means of easing digestion and soothing pain. Monascus first became known in the West back in 1884, when van Tieghem introduced the usage of red powder (Monascus ruber) in Java local populations. Until 1979, Endo et al. isolated monacolin K analogues from M. ruber and opened up the investigation of ingredients and bioactivities from Monascus [4]. Monacolin K is the same compound as cholesterol-lowering medicine  As part of our continuing efforts to explore the chemical diversity of marine fungal metabolites, Monascus purpureus WMD2424, fermented using RGY medium (3% rice starch, 7% glycerol, 1.5% polypeptone, 3% soybean powder, 0.2% MgSO 4 , and 0.2% NaNO 3 ), was investigated. The scaled-up fermentation and extensive chromatographic separation of the EtOAc extract resulted in the isolation of 5 new metabolites, monascuspurins A-E (1)(2)(3)(4)(5), and their antifungal activity was also evaluated. Herein, we report the structural determination of the new compounds ( Figure 2) and the bioactivities of these compounds.

Taxonomic Identification (Phenotypic and Genotypic Data) of Monascus purpureus wmd2424
The sample WMD2424 is a filamentous fungal strain collected from the Chiayi mangrove wetland, inoculated in CYA medium, and cultured at 25 °C for 7 days. The diameter of the colony on the CYA plate is 15 mm, and the front color of the colony is reddish orange; the colony is velutinous, without radial grooves (sulcate), exudate (exudate), and soluble pigment (soluble pigment); the back of the colony is reddish orange. Observed under an optical microscope, the mycelium has a septate, and the thin wall is colorless; the conidia are colorless, and the wall is smooth; conidia (conidia) grow on the top or lateral hyphae, with several clusters, pear-shaped, and a truncated bottom, 8.1-16.7 × 6.3-15.5 μm in size, with smooth walls that are colorless; the outer walls of the ascomata are

Taxonomic Identification (Phenotypic and Genotypic Data) of Monascus purpureus wmd2424
The sample WMD2424 is a filamentous fungal strain collected from the Chiayi mangrove wetland, inoculated in CYA medium, and cultured at 25 • C for 7 days. The diameter of the colony on the CYA plate is 15 mm, and the front color of the colony is reddish orange; the colony is velutinous, without radial grooves (sulcate), exudate (exudate), and soluble pigment (soluble pigment); the back of the colony is reddish orange. Observed under an optical microscope, the mycelium has a septate, and the thin wall is colorless; the conidia are colorless, and the wall is smooth; conidia (conidia) grow on the top or lateral hyphae, with several clusters, pear-shaped, and a truncated bottom, 8.1-16.7 × 6.3-15.5 µm in size, with smooth walls that are colorless; the outer walls of the ascomata are light red but all immature; the same condition was found after 14 days of culture, and only one ascoma was found. The fruit contains ascospores, and the ascospores have a smooth, colorless outer wall and a broad oval shape, with a size of 4.3-5.6 × 3.8-4.8 µm. Partial sequence analysis of the β-tubulin gene fragment was carried out. The total length of the sequence was 1019 bp, compared with the GenBank database, and analyzed and judged with reference to the taxonomic literature. The results showed that the sequence similarity with Monascus purpureus wmd2424 was 99.88% (838/839). The strain was identified as Monascus purpureus according to the colony culture morphology, microstructural characteristics and partial sequence analysis of β-tubulin gene fragment.  (Table 1), 2D-NMR (Figures 3 and 4), IR, and UV spectra showed that compound 1 was a xanthene derivative similar to xanthonoides as monasxanthone A [35]. The molecular weight of 1 is 30 units more than 1 and showed another proton peak at δ H 4.25 (1H, m, H-16) and 3.21 (3H, s, OCH 3 -16), suggesting the existence of a methoxy group in 1. The NMR spectra of 1 represent a 4-methoxy-2-oxopentyl moiety at C-5 position in 1 instead of a pentan-2-one in monasxanthone A. Thus, the structure of 1 was elucidated as 7-acetyl-4,8-dihydroxy-1-(4-methoxy-2-oxopentyl)-4-methyl-4,4a-dihydro-3H-xanthen-3-one and named monascuspurin A. The relative configuration of 1 was deduced from the NOESY spectrum ( Figure 4). The absolute configuration of 1 was further established as (8S,8bR,16S), for the experimental electronic circular dichroism (ECD) curve was in line with its theoretical curve, which was calculated by using the time-dependent density functional theory (TD-DFT) approach [36] (Figure 5).    , which was in agreement with the 1 H-and 13 C-NMR data ( Table 1). The UV spectrum absorption λ max (MeOH) at 242 nm, and a strong IR absorption at 1675 cm −1 , as well as the observation of the featuring carbon resonances [δ C 132.1 (C-8a), 146.2 (C-4a), and 195.1 (C-8)] in the 13 C-NMR spectrum (Table 1) Table 2) corresponding to six quaternary C-atoms (including three carbonyl groups, one oxygenated quaternary carbon), one OCH, two olefinic carbons, four CH 2 , and four CH 3 groups were observed in the 13 C-NMR and DEPT spectra.    The 1 H-and 13 C-NMR spectra of 3 (Table 1) were similar to those of monaphilone A [31]; the major difference was the presence of signals for an γ-lactone attached to C-3a and 7a in 3, instead of signals for a 4H-pyran group in monaphilone A [31]. HMBC correlations between the H-atom signals at δH 4.89/5.05 (CH2(1)) and the C-atom signals at δC 198.3 (C-7) once indicated that the γ-lactone was located at C-3a and 7a of the cyclohex-2-enone ring. The relative configuration of 3 was derived using a NOESY spectrum ( Figure 4) and a comparison with similar compounds [31], the relative configuration of which was based on a NOESY analyses. No NOEs for H-5/Meax-8 and Hax-4 indicated that Me-8 and Hax-4 were on the same side of the molecular plane, tentatively assumed as α-orientation.

Structure Elucidation of Compounds
The H-5 was occupied at axial β-oriented, which was further confirmed by the NOE H-5/Heq-4. The relative configuration at C-5 and 6 were determined to be (5S*,6S*) based on the correlation between the [α]D value and the known configuration at C-5/C-6 for monaphilone A type derivatives [31]. In order to determine the absolute configuration of 3, the theoretical electronic circular dichroism (ECD) spectra of 4 possible stereoisomers were calculated using a time-dependent density-functional theory (TDDFT) calculation, and the calculated ECD curve of (5S,6S) revealed good agreement with the experimental spectrum of 2 ( Figure 5). Therefore, the absolute configuration of 3 was assigned as (5S,6S) and named as monascuspurin C.
Compound 4 was obtained as colorless oil. The molecular formula was determined as C18H16N2O3 on the basis of the [M+Na] + peak at m/z 331.10588 (calcd. 331.10586 for C18H16NaN2O3) in its HR-ESI-MS. The UV absorptions (λmax 220, 252, and 312 nm) confirmed the presence of a pyridine moiety [38]. IR absorption bands were assigned to amide (3400 cm −1 ), multiple carbonyls C=O (1712 and 1656 cm −1 ) ,and the pyridine ring (1589, 1535, and 1458 cm −1 ) functional groups. Twelve indices of hydrogen deficiency (IHD) were The 1 H-and 13 C-NMR spectra of 2 (Tables 1 and 2) were similar to those of monaphilone C [31], except that an acetoxy moiety at C-6 of 2 replaced a 2-oxoheptyl moiety at C-6 of monaphilone C. The planner structure of 2 was confirmed using the COSY and HMBC experiments ( Figure 3). The stereochemistry of 2 was proposed on the basis of the NOESY experiments ( Figure 4). The H-6/CH 3 -12 has no correlation in the NOESY spectrum ( Figure 4) represented acetoxy group and CH 3 -12 are syn-form. The physicochemical data and NMR spectra of compound 2 and the known compound monapurpureusone [31] are similar; the only difference is that the specific rotation of monapurpureusone is negative, while the specific rotation of 2 is positive, and it can be inferred that the two are stereoisomers. On comparing the reference to the (6S,7R)-configuration of FK17-P2b (α] 26 D : + 26.0 (c 0.1, MOH)) [37], the relative configuration of 2 can be assigned as rel-(6S,7R)configuration, and named monascuspurin B. The absolute configuration of 2 was defined via a comparison of the experimental and calculated ECD data ( Figure 5). Therefore, the absolute configuration of 2 was undoubtedly determined as (6S,7R).   1H, m, H-5)]. Eighteen C-atom signals ( Table 2) corresponding to six quaternary C-atoms, one CH, nine CH 2 , and two CH 3 groups, were observed in the 13 C-NMR and DEPT spectra. Since four out of six unsaturation equivalents were accounted for via the above-mentioned 13 C-NMR data, 1 was inferred to have two rings (one as a six-membered and another as a five-membered ring). In addition, two rings were further determined as a cyclohex-2-enone skeleton combined with one γ-lactone ring via the detail HMBC and COSY analyses.
The 1 H-and 13 C-NMR spectra of 3 (Table 1) were similar to those of monaphilone A [31]; the major difference was the presence of signals for an γ-lactone attached to C-3a and 7a in 3, instead of signals for a 4H-pyran group in monaphilone A [31]. HMBC correlations between the H-atom signals at δ H 4.89/5.05 (CH 2 (1)) and the C-atom signals at δ C 198.3 (C-7) once indicated that the γ-lactone was located at C-3a and 7a of the cyclohex-2enone ring. The relative configuration of 3 was derived using a NOESY spectrum ( Figure 4) and a comparison with similar compounds [31], the relative configuration of which was based on a NOESY analyses. No NOEs for H-5/Me ax -8 and H ax -4 indicated that Me-8 and H ax -4 were on the same side of the molecular plane, tentatively assumed as α-orientation.
The H-5 was occupied at axial β-oriented, which was further confirmed by the NOE H-5/H eq -4. The relative configuration at C-5 and 6 were determined to be (5S*,6S*) based on the correlation between the [α] D value and the known configuration at C-5/C-6 for monaphilone A type derivatives [31]. In order to determine the absolute configuration of 3, the theoretical electronic circular dichroism (ECD) spectra of 4 possible stereoisomers were calculated using a time-dependent density-functional theory (TDDFT) calculation, and the calculated ECD curve of (5S,6S) revealed good agreement with the experimental spectrum of 2 ( Figure 5). Therefore, the absolute configuration of 3 was assigned as (5S,6S) and named as monascuspurin C.
The 1 H-and 13 C-NMR spectra of 4 (Table 1) were similar to those of monascopyridine C and D [38]; the major difference was the presence of signals for ABC system aromatic ring attached between C-5 and C-6 in 4, instead of signals for an alkyl groups in monascopyridine C and D. HMBC correlations between the H-atom signals at δ H 8.04 (H-13) and the C-atom signals at δ C 151.0 (C-6), and 143.5 (C-4a) and δ H 7.70 (H-14) and the C-atom signals at δ C 126.7 (C-5), indicated that the ABC system aromatic ring was bounded at C-5 and 6. The other key correlations of HMBC were illustrated in Figure 3.
On the basis of the evidence, the entire structure of 4 was confirmed and named monascuspurin D. The relative configuration at C-7 was determined to be 7R based on the correlation between the [α] 26 D :+ 15.9 (c 0.01, CHCl 3 ) and the known configuration at C-7 for (R)-2-hydroxy-2-methylcyclohexanone derivatives [39]. The absolute configuration of 4 was defined via a comparison of the experimental and calculated ECD data ( Figure 5). Therefore, the absolute configuration of 4 was determined as 7R.
Compound 5 was obtained as an optically active oil.  235 and 285 nm) confirmed the presence of a benzenoid nucleus. The bands at 3400, 1780, 1695, and 1615/1577 cm −1 in the IR spectrum revealed the presence of a hydroxyl group, γ-lactone, and aromatic ring, respectively. Nine indices of hydrogen deficiency (IHD) were determined from the molecular formula, 13 C-NMR (Table 1), and DEPT spectra. The 1 H-NMR and 13 C-NMR spectra ( Table 2) of 5 were similar to those of ankaflavin [9], except that a 2-ethylphenol group of 5 replaced a (E)-6-(prop-1-en-1-yl)-2H-pyran group at C-4a-C-8a of ankaflavin. Further confirmation using the HMBC correlations (Figure 3 The dextrorotatory optical activity of 5, gathered from the NOESY spectrum (Figure 4), indicates that Hax-5 is correlated to H-12 and H-13, and H-6 has no NOE contacts with Hax-5, H-12, and H-13. It can be concluded that Hax-5, H-12, and H-13 are on the same side, and H-6, H-12, and H-13 are on the opposite side, and once again it indicated that the relative configuration of 5 is (6R,7R,13S), as in the case of ankaflavin [9]. In order to determine the absolute configuration of 5, the theoretical ECD spectra of all possible stereoisomers were calculated using the TDDFT calculation, and the calculated ECD curve of the isomer (6R,7R,13S) revealed a good agreement with the experimental one ( Figure 5). Therefore, the absolute configuration of 5 was assigned as (6R,7R,13S)-form and named as monascuspurin E.

Discussion
Red yeast rice has been used in food and traditional Chinese medicine since ancient times. In recent years, research has also found that red yeast rice bacteria can produce many active secondary metabolites. In order to further explore the efficacy of different strains of red yeast rice and expand the application range of red yeast rice, in this study, a strain wmd2424 was isolated from the mangrove forest in Chiayi Wetland, and the strain was identified as Monascus purpureus via the results of colony culture morphology, microstructural characteristics, and partial sequence analysis of the β-tubulin gene fragment. After liquid fermentation using RGY medium, extraction with ethyl acetate, and analysis of its metabolites, a total of six new compounds were obtained.
To the best of our knowledge, this is the first report of isoquinoline-type metabolites from the edible fungi genus Monascus. These results demonstrate that Monascus produces unique and diverse metabolites in different fermentation conditions and soil-derived collections. Therefore, in a special ecological environment, more natural products with biological activity may be found by searching for Monascus species.

Biological Studies
Culture broth from M. purpureus wmd2424 was tested for antifungal activity against the following fungi: Aspergillus niger (BCRC-31512), Penicillium italicum (BCRC-30567), Candida albicans (BCRC-21538), and Saccharomyces cerevisiae (BCRC-20822). The antifungal data are shown in Table 3 and the clinically used antifungal drug ketoconazole was employed as a positive control. Table 3. Antifungal activity of five sufficient compounds isolated from the culture broth of A. punica 04107M (diameter of the zone of growth-inhibitory fungicidal zone is given in mm, including the diameter of the disk, which is 8 mm). Inhibitory zone diameter (mm); ± inhibitory zone; positive control (STD): ketoconazole. Each value represents the mean ± SD.

Test
Our results indicate that compounds 3-5 have moderate antifungal activity compared to ketoconazole, with 1 being weaker. From the results of the antifungal tests, the following conclusions can be drawn about these isolates: (a) within the novel strain, the 2,3-dimethylcyclohex-2-en-1-one (compound 2) and γ-lactone  (Table 3).
The inhibitory activity of compounds 3-5 against A. niger, P. italicum, C. albicans, and S. cerevisiae was further tested using the method described in the experimental section (Table 4). Compound 2 has inhibitory activity against S. cerevisiae, with MIC values of 43.45 µg/mL. Compound 3 has inhibitory activity against C. albicans, with an MIC value of 32.87 µg/mL. Compound 4 was found to have moderate inhibitory activity against the A. niger, and C. albican strains with MIC values ranging from 29.65 and to 58.43 µg/mL. They were less biologically active than the reference compound, ketoconazole, which had MIC values of 4.10, 5.34, 10.88, and 3.57 µg/mL against A. niger, P. italia, C. albicans, and S. cerevisiae, respectively. In this bioassay, no antifungal activity (MIC > 100) was observed for compound 5 at concentrations below 100 µg/mL.

Microorganism, Cultivation, and Preparation of the Strain
This WMD2424 strain was isolated from the mangrove wetland collected in Chiayi County, Taiwan, using HV agar and cultured at 28 • C for 3 weeks. A voucher specimen was immersed in 15% glycerol-water solution at −80 • C and deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI). Analysis of the ITS rDNA using the BLAST database screening provided a 99.9% match with Monascus purpureus, whose sequence has been submitted to GenBank.
To each 500-mL flask containing 150 mL of liquid RGY medium (3% rice starch, 7% glycerol, 1.5% polypeptone, 3% soybean powder, 0.2% MgSO4, and 0.2% NaNO 3 ) were added 10 mL of fungal inocula and incubated at 25 • for 2 weeks on a rotary shaker at the speed of 100 circles/min without illumination. A total of 14.0 L of fungal fermented broth was harvested and then filtered to remove fungal mycelium.

Computational Methods
The theoretical ECD curves of compounds 1-5 were calculated by using Gaussian 09, Revsion E.01. software. Conformational searches were performed using Spartan'14 software with the Molecular Merck force field (MMFF). ECD spectra of conformers with a Boltzmann distribution over 2% were calculated via the TD-DFT method at the B3LYP/6.311+G (d,p) level in MeOH. According to a Gaussian band shape with a 0.2 eV exponential halfwidth from the dipole-length dipolar and rotational strengths, the theoretical ECD spectra were generated using the SpecDis 3.0.

Antifungal Activity Assays
The assays tested for the presence of microorganisms. The in vitro antifungal activity of compounds 1-5 was tested against a panel of laboratory control strains belonging to the Bioresource Collection and Research Center (BCRC) in Hsinchu, Taiwan, namely, the fungal organisms Aspergillus niger (BCRC-31512), Penicillium italicum (BCRC-30567), Candida albicans (BCRC-21538), and Saccharomyces cerevisiae (BCRC-20822).

Via Disk Diffusion Assay
Antifungal susceptibility testing of the isolated compounds was performed with the following strains: Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae using the disk diffusion method and the following CLSI guidelines were applied: M44-A and M44-S2 for yeasts [40,41] and M-51P for filamentous fungi. A standard disk of ketoconazole was used as a positive control, while a disk imbued with 50 µL of pure DMSO was used as a negative control. The diameters of the inhibition zones were measured in millimeters by means of a slide caliper. Each test was performed in triplicate, and the results were analyzed for statistical significance [40][41][42].

Via Broth Dilution Assay
The MIC determination for the antifungal assay was performed according to the Clinical and Laboratory Standard Institute (CLSI) using the broth dilution assay method [43][44][45]. Extract stock solutions and partitions were prepared in 5% DMSO, and twofold serial dilutions were prepared in RPMI in 96-well microtiter plates (Corning Incorporated, Corning, NY, USA). The final concentrations ranged from 0.98 to 2.000 g mL −1 . Test organisms (100 µL) were added to each well in microtiter plates. The growth control contained medium and inoculum. Blank controls contained medium only. The microtiter plates were then incubated at 35 • C and the endpoints were read after 48 h. The lowest concentration for each test compound at which color change occurred was recorded as its primary MIC value. The average of primary values from three individual tests were calculated, and the average was taken as the final MIC value for each of the test compounds.

Conclusions
Red yeast rice is a well-known material which has been widely used for decades, but the chemistry and bioactivity of the constituents are still not so clear. Previous investigation of Monascus species had isolated different skeleton constituents, mainly azaphilones and monacolin analogs. However, some minor compounds such as benzenoid derivatives or other types of compounds from Monascus species have received less attention. Accordingly, it is still worth investigating the ingredients and bioactivity of red yeast rice.
In this report, we committed to explore unusual skeleton compounds in M. purpureus wmd2424, and successfully found new xanthonoid, cyclohexenone, γ-lactone, isoquinoline, and azaphilone skeleton compounds. Xanthonoids are yellow pigments in a C 6 -C 1 -C 6 system and restricted in a few families of higher plants, some fungi and lichens, and has seldom been found in Monascus spp. [46] This is the second report of isolating xanthonoids from Monascus spp, which represent different yellow azaphilone pigments (monascin, ankaflavin) from this genus. The structures of these isolates were determined using spectroscopic experiments. The BuOH soluble fraction from the M. purpureus wmd2424 fermentation broth was tested for antifungal activities. Our results indicated that compounds 3-5 displayed moderate antifungal activities against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae. It is worth mentioning that the chemical composition of M. purpureus wmd2424 has never been studied. The result indicated M. purpureus wmd2424 could produce more metabolites with extensive antifungal activity, and that its metabolites in other mediums were worth being studied further.