Monasnicotinates A–D, Four New Pyridine Alkaloids from the Fungal Strain Monascus pilosus BCRC 38093

Four new pyridine derivatives, monasnicotinates A–D (1–4) were isolated from the red yeast rice of Monascus pilosus BCRC 38093. Their structures were elucidated on the basis of physicochemical evidence, in-depth NMR spectroscopic analysis, and high-resolution mass spectrometry. Their inhibitory effects on NO production was also evaluated.


Introduction
Monascus-fermented rice (ang-kak, red koji) has been used for centuries as a natural food colorant and traditional medicine in oriental countries. The species of Monascus produced secondary metabolites such as pigments [1], monacolin K [2], γ-aminobutyric acid [3], dimerumic acid [4], and citrinin [5]. Several secondary metabolites from Monascus sp. have been found to have some beneficial pharmacological effects in decreasing blood pressure [6], lowering plasma cholesterol OPEN ACCESS levels [2,7,8] and antibacterial activity [9]. In Taiwan, M. purpureus, M. pilosus, and M. ruber, are the common species used to make Monascus-fermented red rice, which contains some red pigments and physiological biological active metabolites. M. pilosus is one of the fungi traditionally used in food items in south China, Taiwan, Japan, Korea, Indonesia and other eastern countries. Many metabolites were identified from the Monascus species in previous studies [1,5,[10][11][12][13][14][15][16][17][18][19], but knowledge of their biological or toxicological effects is limited. Therefore, characterization of the secondary metabolites of Monascus and their functionality still remain unclear and are worthy of examination.

Results and Discussion
Compound 1, obtained as a yellowish oil, had the molecular formula C 21 H 27 NO 4 , requiring nine degrees of unsaturation, as determined by HR-ESI-MS data [m/z 380.1837 ([M+Na] + ; calc. 380.1835)] in combination with its 1 H-NMR, 13 C-NMR and DEPT data. The IR spectrum revealed the presence of multiple carbonyls (1712, 1668 cm -1 ), one of which was by UV spectrum analysis (λ max 253, 280 and 330 nm) in conjugation with a pyridine.

Fungal Material
Monascus pilosus BCRC 38093 was used throughout this study, and specimens deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI).

Fungal Material
BCRC 38093 was 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 × 10 5 ) were seeded into 300 mL shake flasks containing 50 mL RGY medium (3% rice starch, 7% glycerol, 1.1% polypeptone, 3% soybean powder, 0.1% MgSO 4 , 0.2% NaNO 3 ), and cultivated with shaking (150 rpm) at 25 °C for 3 days. After the mycelia enrichment step, an inoculum mixing 100 mL mycelia broth and 100 mL RGY medium was inoculated into plastic boxes (25 cm × 30 cm) containing 1 kg sterile rice and cultivated at 25 °C for producing red yeast rice. At day 7, 150 mL RGY medium was added for maintaining the growth of cells. After 28 days of cultivation, the red yeast rice was harvested and lyophilized for metabolites extraction.

Biological Assay
Determination of Nitric Oxide Production. The murine macrophage cells RAW264.7 (BCRC 60001 = ATCC TIB-71) were transferred to 96-well plates at a density of 1x10 5 cell/well. After 24 hr incubation, the cells were stimulated with 1 μg/mL of LPS (Sigma, Cat no: L-2654) for 24 hr in the presence or absence of the compounds (0, 1, 5, 10 and 20 μg/mL) tested. As a parameter of NO synthesis, nitrite concentration was measured in the supernatant of RAW264.7 cells by the Griess reagent [1:1 mixture of 1% sulfanilamide and 0.1% N-(1-naphthyl)ethyl-enediamine dihydrochloride, each in 2.5% phosphoric acid solution] in a 96-well plate, and incubated for 10 min at room temperature. Nitrite concentration was determined by measuring the absorbance at 540 nm using an ELISA plate reader (μ Quant) [20]. All tests were run in triplicate and averaged. The data were expressed as a mean of three experiments. Statistical comparisons were carried out the Student's t-test for paired values.
Determination of cell viability. The cell viability was assessed using a MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, Merck KGaA, Damstadt, Germany]-based colorimetric assay, as previously described [21]. After sampling the supernatant for the NO assay, 100 μL of fresh medium containing 0.5 mg/mL of MTT was added to each well and incubated for 3 hr at 37 °C. The medium was then removed and the violet for mazan crystals in the viable cells were dissolved in dimethyl sulfoxide. The absorbance of each well was then read at a wavelength of 540 nm using microplate reader (μ Quant, Bio-TEK instruments INC).

Conclusions
In this study, we focused on the minor secondary metabolites appearing in the EtOAc-soluble fraction of a 95% EtOH extract of the red yeast rice produced by Monascus purpureus BCRC 38093. Four new natural metabolites 1-4 were found in this study. In the in vitro NO production inhibitory assay, isolates 1, 3, and 4 showed stronger inhibition on NO production with IC 50 value of 6.95, 8.34, and 9.42 μg/mL respectively, while showing no cytotoxicity to normal cells at the same concentration.