Hindawi Publishing Corporation Journal of Chemistry Volume 2013, Article ID 872056, 6 pages http://dx.doi.org/10.1155/2013/872056 Research Article HMG-CoA Reductase Inhibitors from Monascus-Fermented Rice Xuemei Li, Chunli Liu, Zhenwen Duan, and Shuren Guo Beijing Peking University WBL Biotechnology Co., Ltd., Beijing 100094, China Correspondence should be addressed to Xuemei Li; [email protected] Received 3 August 2013; Revised 11 October 2013; Accepted 22 October 2013 Academic Editor: Marjana Novic Copyright © 2013 Xuemei Li et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Seven compounds were isolated from Monascus-fermented rice by column chromatography with silica gel and semiprep HPLC. eir structures were elucidated by extensive spectroscopic methods. All compounds displayed HMG-CoA reductase inhibitory potential, among them compound 7 exhibited strong inhibition with IC 50 value comparable with lovastatin. In this study, two compounds (1 and 2) were obtained from natural source for the first time. 1. Introduction Monascus-fermented rice, also called red yeast rice, red Koji or “Hongqu”, is commonly used in Asian food and medicine for centuries. Its pharmaceutical function was stated that “Hongqu” promotes “digestion and blood circulation, can strengthen the spleen and dry the stomach.” by Li Shizhen, the great pharmacologist of the Ming Dynasty [1]. Since monacolin K was first isolated from Monascus ruber by Endo in 1979, a series of monacolins, such as monacolin J, monacolin L, monacolin X and monacolin M, have been found and disclosed to be potent 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMG-CoA reduc- tase) inhibitors [2–5]. Aſter that dihydromonacolin-MV and dehydromonacolin-MV2 isolated from Monascus sp. have been characterized for their antioxidant action [6, 7]. Non- monacolin secondary metabolites have also been identified so far [8], but it is still unclear whether the cholesterol- lowering effect of Monascus-fermented rice is due solely to the monacolin K content or if other monacolins, sterols, and isoflavones contribute to its cholesterol-lowering effect. In our search for HMG-CoA reductase inhibitory com- pounds from Monascus-fermented rice, we carried out the chemical investigation on Monascus purpureus (M. pur- pureus, CGMCC No. 0272)-fermented rice, resulting in the isolation of seven HMG-CoA reductase inhibitory compounds including (4R,6R)-6-(2-((1S,2S)-2,6-dimethyl- 1,2-dihydro naphthalene-1-yl)-ethyl)-4-hydroxy-tetrahydro- pyran-2-one (1), (4R,6R)-6-(2-(2,6-dimethylnaphthalene- 1-yl)ehtyl-4-hydroxy-tetrahydropyran-2-one (2), 1-monoli- nolein (3), (9Z,12Z)-octadeca-9,12-dienoic acid (4), Monas- cinol (5), Pulchellalactam (6), and Lunatinin (7). eir structures were determined based on spectroscopic data. In our study compounds 1 and 2 were obtained from natural source for the first time, and compounds 3 and 4 were isolated from the genus for the first time. Details of the isolation, structure elucidation, and HMG-CoA reductase inhibitory activities of these compounds are reported here. 2. Materials and Methods 2.1. General. NMR spectra were run on a Bruker Avance III 600 MHz spectrometer. EI-MS and HR-ESI-MS were measured on Agilent 5973N and Bruker micrOTOF-QII mass spectrometers. IR spectra were recorded on Nicolet Nexus- 670 and Nicolet 380 FT-IR spectrometers. UV spectra were measured on a Shimadzu UV2401PC UV/vis spectropho- tometer. Column chromatography (CC) was performed with silica gel (200–300 mesh) and thin-layer chromatography (TLC) was performed on silica gel GF 254 from Qingdao Marine Chemical Inc., China. RP-C18 silica gel (YMC) was purchased from Greenherbs Science & Technology Devel- opment Co., Ltd., China. Sephadex LH-20 (Pharmacia) was purchased from H&E Co., Ltd., China. MPLC separation was performed with Combiflash (ISCO) and HPLC was performed on Agilent 1260 apparatus using 5C18-MS-II column (ODS, 250 mm × 10 mm) and monitored with a
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Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 872056 6 pageshttpdxdoiorg1011552013872056
Research ArticleHMG-CoA Reductase Inhibitors from Monascus-Fermented Rice
Xuemei Li Chunli Liu Zhenwen Duan and Shuren Guo
Beijing Peking University WBL Biotechnology Co Ltd Beijing 100094 China
Correspondence should be addressed to Xuemei Li xuemei0309126com
Received 3 August 2013 Revised 11 October 2013 Accepted 22 October 2013
Academic Editor Marjana Novic
Copyright copy 2013 Xuemei Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Seven compounds were isolated from Monascus-fermented rice by column chromatography with silica gel and semiprep HPLCTheir structures were elucidated by extensive spectroscopic methods All compounds displayed HMG-CoA reductase inhibitorypotential among them compound 7 exhibited strong inhibition with IC
50value comparable with lovastatin In this study two
compounds (1 and 2) were obtained from natural source for the first time
1 Introduction
Monascus-fermented rice also called red yeast rice red Kojior ldquoHongqurdquo is commonly used in Asian food and medicinefor centuries Its pharmaceutical function was stated thatldquoHongqurdquo promotes ldquodigestion and blood circulation canstrengthen the spleen and dry the stomachrdquo by Li Shizhenthe great pharmacologist of the Ming Dynasty [1]
Since monacolin K was first isolated from Monascusruber by Endo in 1979 a series of monacolins such asmonacolin J monacolin L monacolin X and monacolin Mhave been found and disclosed to be potent 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reduc-tase) inhibitors [2ndash5] After that dihydromonacolin-MV anddehydromonacolin-MV2 isolated from Monascus sp havebeen characterized for their antioxidant action [6 7] Non-monacolin secondary metabolites have also been identifiedso far [8] but it is still unclear whether the cholesterol-lowering effect of Monascus-fermented rice is due solely tothe monacolin K content or if other monacolins sterols andisoflavones contribute to its cholesterol-lowering effect
In our search for HMG-CoA reductase inhibitory com-pounds from Monascus-fermented rice we carried out thechemical investigation on Monascus purpureus (M pur-pureus CGMCC No 0272)-fermented rice resulting inthe isolation of seven HMG-CoA reductase inhibitorycompounds including (4R 6R)-6-(2-((1S 2S)-26-dimethyl-12-dihydro naphthalene-1-yl)-ethyl)-4-hydroxy-tetrahydro-pyran-2-one (1) (4R 6R)-6-(2-(26-dimethylnaphthalene-
1-yl)ehtyl-4-hydroxy-tetrahydropyran-2-one (2) 1-monoli-nolein (3) (9Z12Z)-octadeca-912-dienoic acid (4) Monas-cinol (5) Pulchellalactam (6) and Lunatinin (7) Theirstructures were determined based on spectroscopic data Inour study compounds 1 and 2 were obtained from naturalsource for the first time and compounds 3 and 4 wereisolated from the genus for the first time Details of theisolation structure elucidation and HMG-CoA reductaseinhibitory activities of these compounds are reported here
2 Materials and Methods
21 General NMR spectra were run on a Bruker AvanceIII 600MHz spectrometer EI-MS and HR-ESI-MS weremeasured onAgilent 5973N andBrukermicrOTOF-QIImassspectrometers IR spectra were recorded on Nicolet Nexus-670 and Nicolet 380 FT-IR spectrometers UV spectra weremeasured on a Shimadzu UV2401PC UVvis spectropho-tometer Column chromatography (CC) was performed withsilica gel (200ndash300mesh) and thin-layer chromatography(TLC) was performed on silica gel GF
254from Qingdao
Marine Chemical Inc China RP-C18 silica gel (YMC) waspurchased from Greenherbs Science amp Technology Devel-opment Co Ltd China Sephadex LH-20 (Pharmacia) waspurchased from HampE Co Ltd China MPLC separationwas performed with Combiflash (ISCO) and HPLC wasperformed on Agilent 1260 apparatus using 5C18-MS-IIcolumn (ODS 250mm times 10mm) and monitored with a
2 Journal of Chemistry
DAD detector NADPH and HMG-CoA were purchasedfrom Merck and Sigma respectively Cholestyramine wasobtained from Nanjing Lifecare Pharmaceutical Lovstatinwas purchased from China National Institutes for Foodand Drug Control All other chemicals were obtained fromBeijing Chemical Reagent Co Ltd All organic solvents wereof analytical purity and purchased from Beijing ChemicalReagent Co Ltd China
22 Biological Material Monascus-fermented rice was pre-pared from cooked rice inoculated with M purpureus(CGMCC No 0272) produced by Beijing Peking Univer-sity WBL Biotechnology Co Ltd The Monascus-fermentedrice was dried with a drying oven at 80∘C and the driedMonascus-fermented rice was pulverized with a pulverizer
TheMonascus purpureuswas identified by China GeneralMicrobiological Culture Collection Center (CGMCC) anddeposited in CGMCC
23 Extraction and Isolation Dried and powdered Monas-cus-fermented rice (4 kg) was successively ultrasonicextracted 3 times with petroleum ether and ethyl acetate toyield petroleum ether crude extract (82 g) and ethyl acetatecrude extract (170 g) The petroleum ether crude extractwas subjected to silica gel column chromatography elutedwith a gradient mixture of petroleum ether-ethyl acetate(50 50 to 0 100) to yield 2 fractions (P1-P2) Fr P1 (54 g)was further subjected to a silica gel column eluted withpetroleum ether-ethyl acetate (75 25) to yield 3 fractions(P1A-P1C) Fraction P1C (205 g) was chromatographed overa C18 column eluted with 75 MeOH to give 3 fractions(P1C1-P1C3) P1C1 subfraction (103 g) was purified by gelchromatography over Sephadex LH-20 eluted with CH
2Cl2-
MeOH (1 1) and further purified by MPLC using 75ndash85MeOH to afford compound 5 (54mg) and compound 6(50mg) Fr P2 (20 g) was further subjected to a silica gelcolumn eluted with petroleum ether-ethyl acetate (25 75)to yield 3 fractions (P2A-P2C) P2B subfraction (85 g)was purified by gel chromatography over Sephadex LH-20eluted with CH
2Cl2-MeOH (1 1) and further purified by
HPLC using 90 MeOH to afford compound 3 (14mg)Ethyl acetate crude extract (170 g) was subjected to silicagel column chromatography eluted with a gradient mixtureof petroleum ether-ethyl acetate (50 50 to 0 100) to yield2 fractions (E1-E2) Fr E2 (48 g) was further subjected to asilica gel column eluted with petroleum ether-ethyl acetate(33 67) to yield 4 fractions (E2A-E2D) Fr E2B (108 g)was chromatographed on MPLC eluted with CH
2Cl2-ethyl
acetate-MeOH (30 30 1) to yield 4 fractions (E2B1-E2B4)E2B1 subfraction (38 g) was purified by gel chromatographyover Sephadex LH-20 eluted with CH
2Cl2-MeOH (2 1) and
further purified by HPLC using 66 acetonitrile-MeOH(1 1) in H
2O to afford compound 1 (48mg) and compound 2
(18mg) E2B2 subfraction (40 g) was purified by C18 columnchromatography eluted with 82 acetonitrile-MeOH (1 1)in H2O and further purified by gel chromatography over
Sephadex LH-20 eluted with CH2Cl2-MeOH (2 1) to afford
compound 7 (56mg) Fr E2C (47 g) was chromatographed
over a Sephadex LH-20 column (CH2Cl2-MeOH 6 4) and
afforded five subfractions (E2C1-E2C5) E2C4 subfraction(049 g) was purified by C18 column chromatography elutedwith 85 MeOH and further purified by HPLC using ACNto afford compound 4 (67mg)
24 HMG-CoA Reductase Inhibitory Assay
241 Preparation of Microsomes Male Sprague-Dawley rats(100ndash200 g) were killed by cervical dislocation at or nearthe peak of the daily circadian rhythm in reductase activ-ity In most experiments rats were fed a diet containing5 cholestyramine for 4 days prior to the preparation ofmicrosomes Livers were excised rinsed in ice-cold bufferA (KESD buffer) containing 10mM potassium phosphate2mM EDTA 250mM sucrose and 1mMDTT (pH68) Thelivers were then minced through a tissue press and homog-enized in three volumes of buffer A per g of liver by fourstrokes with the loose pestle of a Dounce homogenizer andone stroke with the tight pestle Mitochondria and cell debriswere sedimented by centrifugation two times at 12000 g for15min at 4∘C Crude microsomes were prepared from thetwo 12000 g supernatants by sedimentation at 105000 g for90min at 4∘CThe pellet was resuspended in buffer A (1mLgliver) and resedimented at 105000 g for 90min at 4∘C [9]The washed microsomal pellets were quickly-frozen in anacetone-dry ice bath and stored at minus80∘C prior to use
242 Solubilization of HMG-CoA Reductase For optimalsolubilization of the reductase the frozen microsomes wereallowed to thaw at room temperature before addition ofan equal volume of 50 glycerol in buffer B containing02M sucrose 01MKCl 008Mpotassiumphosphate 2mMpotassium EDTA and 10mM DTT (pH 72) preheated to37∘CThe suspension was rehomogenized with 10 downwardpasses of a hand-driven all-glass Potter-Elvehjem homoge-nizer and then incubated at 37∘C for 60min The suspensionwas diluted threefold with buffer B preheated to 37∘C toa final glycerol concentration of 83 rehomogenized with10 downward passes of the glass homogenizer pestle andcentrifuged at 105000 g for 60min at 25∘C The supernatantcontaining solubilized HMG-CoA reductase was removedand used immediately for enzyme purification [10]
243 Purification of HMG-CoA Reductase The solubilizedenzyme was fractioned with saturated ammonium sulfatesolution and the protein precipating between 35 and 50ammonium sulfate was dissolved in buffer B [11]
244 Assay of HMG-CoA Reductase The activity of HMG-CoA reductase was determined at 37∘C in total volume of200120583L HMG-CoA reductase 10120583L was preincubated withbuffer C (KCl 200mM potassium phosphate 160mM EDTA4mM DTT 10mM pH68) for 5min at 37∘C and the reac-tion was initiated by adding 15 120583L of cofactor-substrate solu-tion (200120583M NADPH and 50 120583M HMG-CoA in buffer C)and 5 120583L of sample solution (dissolved in 50DMSO) or 5120583Lof 50DMSO (negative control) [12]The dynamic change of
Journal of Chemistry 3
O
OHO
6 5 4a
17
8 8a
43
2
H
1
O
OHO
H
2
OOO
O1211
10
13
8
91415 6 5 4
3
2
17
OH1617
1819
2021H
5
HN O
234
5
6
O
OH
OH
OH
4 6
788a
3 54a
O
12
7
OH
O4
O
OHO
H
H
Monacolin L
O
O
18 16 14 11 8 4 2 1
3
OHOH6
2998400
1998400
1998400 2
998400
2998400
3998400
1998400
1998400
3998400
3998400
4998400
5998400
6998400
7998400
Figure 1 Structures of compounds 1ndash7 and monacolin L
OD340
was detected with VerSamax ELISAmicroplate readerand the rate of the change within 5min was used to evaluatethe activity of HMG-CoA reductase and then to evaluate theinhibition activity of each sample
3 Results and Discussion
Repeated column chromatography of the petroleumether extract and ethyl acetate extract of Monascus-fermented rice afforded seven known compounds (1-7see Figure 1) The compounds were identified as (4R6R)-6-(2-((1S 2S)-26-dimethyl-12-dihydro naphthalene-1-yl)-ethyl)-4-hydroxy- tetrahydropyran-2-one (1) [13 14] (4R6R)-6-(2-(26-dimethylnaphthalene-1-yl)ehtyl-4-hydroxyte-trahydropyran-2-one (2) [15] 1-monolinolein (3) [16](9Z12Z)-octadeca-912-dienoic acid (4) [17] Monascinol (5)[18] Pulchellalactam (6) [19] and Lunatinin (7) [20 21] onthe basis of comparison of their NMR data and HR-ESI-MSor EI-MS data with those reported in the literature It isworthwhile to point out that compounds 1 and 2were isolatedfrom natural source for the first time and compounds 3and 4 were isolated from the genus for the first time Allisolated compounds were evaluated for their ability to inhibitHMG-CoA reductase
31 Chemistry Compound 1was obtained as a colorless gumThe HR-ESI-MS gave a pseudo molecular ion peak at mz3231492 [M + Na]+ consistent with a molecular formula ofC19H24O3 The UV spectrum showed maximum absorption
at 2290 and 2658 nm Its IR spectrum revealed absorptionbands at 3380 (hydroxyl) and 1712 cmminus1 (carbonyl) The 1Hand 13C NMR data of 1 were similar to those of monacolinL previous reported [22] Comparing the molecular formulaand NMR data of 1 with those of monacolin L it was foundthat 1 absent four hydrogens and had two more aromaticdouble bond signals at 120575 1273693 (1H d J = 78Hz H-7) with 1362 (C-6) and 1276696 (1H d J = 78Hz H-8) with 1357 (C-8a) In addition the chemical shift of 6-Me at 120575 229 (3H s) was moved obviously downfield Thetwo aromatic double bonds were located between C-5 andC-4a based on the HMBC correlations of H-7C5 C-8aand 6-Me H-8C-4a C-8a C-1 and C-7 (see Figure 2)Additionally the optical rotation at [120572]25D + 315 (c 0111CH2Cl2) is similar to that of monacolin L whose optical
rotation at [120572]25D + 16416 (c 06 CH2Cl2MeOH = 2 1)
suggesting that the chiral carbons C-1 C-2 C-31015840 and C-51015840 of compound 1 also had S S R R-configurations It isfurther confirmed by the NOESY correlations (see Figure 2)observed between H-1 (120575H 258) with H-2 (120575H 272) H-31015840
4 Journal of Chemistry
O
OHO
H
Figure 2 Key HMBC (rarr ) and NOESY (harr) correlations incompound 1
with H-1 (120575H 258) and H-31015840 with H-2 (120575H 272) SimilarlyNOESY cross peaks between 2-Me (120575H 106) with H-71015840 (120575H158 and 176) H-71015840 (120575H 158 and 176) with H-51015840 (120575H 456)Thus the structure of 1 was identified as (4R 6R)-6-(2-((1S 2S)-26-dimethyl-12-dihydro-naphthalene-1-yl)-ethyl)-4-hydroxy-tetrahydropyran-2-one This compound was re-ported by Greenspan et al and Stokker [13 14] The 1H and13C NMR data was assigned unambiguously by HSQC andHMBC experiments (see Tables 1 and 2) Compound 1 wasobtained from natural source for the first time
Compound 2 was obtained as a colorless gum The HR-ESI-MS gave a pseudo molecular ion peak at mz 3211484[M+Na]+ consistent with amolecular formula of C
19H22O3
The UV spectrum showed maximum absorption at 2318 and2816 nm Its IR spectrum revealed absorption bands at 3434(hydroxyl) and 1712 cmminus1 (carbonyl)TheUV andNMR dataof 2 were similar to those of 1 but showed one more doublebond signals at 120575 1342 (C-1) and 1322 (C-2) and absenttwo hydrogens (H-1 and H-2) and the downfield chemicalshift of 2-Me at 120575 249 (3H s) indicating that 2 was 12-twodehydrogenated 1 Additionally the optical rotation at [120572]25D+ 112 (c 006 CH
2Cl2) is similar to that of monacolin L
suggesting that the chiral carbonsC-31015840 andC-51015840 of compound2 also had R R-configurations Thus the structure of 2 wasdetermined as (4R 6R)-6-(2-(26-dimethylnaphthalene-1-yl)ehtyl-4-hydroxy-tetrahydropyran-2-one This compoundwas reported byOka et al [15]The 1Hand 13CNMRdata wasassigned unambiguously by HSQC and HMBC experiments(see Tables 1 and 2) Compound 2 was also obtained fromnatural source for the first time
Compound 3 colorless oil HR-ESI-MS mz 3772698[M+Na]+ 1H NMR (600MHz CDCl
3) 120575 534 (4H m H-
9 10 12 and 13) 417 (1H dd J = 114 36Hz H-11015840b) 413 (1Hdd J = 114 60Hz H-11015840a) 391 (1H m H-21015840) 369 (1H ddJ = 114 36Hz H-31015840b) 359 (1H dd J = 114 60Hz H-31015840a)276 (2H m H-11) 233 (2H t J = 78Hz H-2) 204 (4H mH-8 and H-14) 162 (2H m H-3) 131 (14H m H-4 to H-7and H-15 to H-17) 088 (3H t J = 72Hz H-18) 13C NMR(150MHz CDCl
3) 120575 1745 (C-1) 1304 (C-10) 1302 (C-9)
1282 (C-13) 1280 (C-12) 704 (C-21015840) 653 (C-11015840) 635 (C-31015840)343 (C-2) 317 (C-16) 297 (C-7) 295 (C-6) 293 (C-5) 292
Table 1 1H NMR (600MHz) data for 1 2 and monacolin L (120575 inppm 119869 in Hz)
No 1a 2a Monacolin Lb
1 258 (m) mdash 140 (m)2 272 (m) mdash 231 (m)3 574 (dd 96 30) 727 (d 84) 572 (m)4 635 (dd 96 24) 756 (d 84) 587 (m)4a mdash mdash mdash5 686 (brs) 758 (brs) 539 (m)6 mdash mdash 232 (m)7 693 (d 78) 734 (dd 90 12) 146 (m) 153 (m)8 696 (d 78) 794 (d 90) 120 (m) 181 (m)8a mdash mdash 205 (m)11015840 mdash mdash mdash
21015840 255 (m) 268 (ddd 174 36 18) 244 (dd 150 84)266 (dd 180 54) 277 (dd 174 48) 255 (dd 150 48)
31015840 428 (m) 440 (m) 420 (m)41015840 163 (m) 186 (m) 182 (m) 200 (m) 162 (m) 178 (m)51015840 456 (m) 486 (m) 376 (m)61015840 142 (m) 166 (m) 192 (m) 198 (m) 160 (m) 165 (m)71015840 158 (m) 176 (m) 316 (m) 337 (m) 130 (m) 173 (m)2-Me 106 (d 78) 249 (brs) 099 (dd 72 18)6-Me 229 (s) 250 (brs) 090 (dd 72 30)aCDCl3bCD3OD
(C-4 and C-15) 274 (C-8) 273 (C-14) 258 (C-11) 250 (C-3)227 (C-17) and 142 (C-18) Compound 3 was characterizedas 1-monolinolein by comparison with the literature [16] andwas isolated from the genus for the first time
Compound 4 colorless oil EI-MSmz 280 [M]+ 1HNMR(600MHz CD
3OD) 120575 537 (2H m H-9 and H-10) 531 (2H
m H-12 and H-13) 278 (2H brt J = 72Hz H-11) 227 (2Ht J = 72Hz H-2) 207 (4H m H-8 and H-14) 160 (2H mH-3) 135 (14H m H-4 to H-7 and H-15 to H-17) 091 (3Ht J = 72Hz H-18) 13CNMR (150MHz CD
3OD) 120575 1779 (C-
1) 1311 (C-9 and C-10) 1293 (C-13) 1292 (C-12) 352 (C-2)329 (C-16) 309 (C-7) 307 (C-6) 305 (C-5) 304 (C-4 andC-15) 284 (C-8 and C-14) 268 (C-11) 263 (C-3) 238 (C-17) and 146 (C-18) Compound 4 was identified as (9Z12Z)-octadeca-912-dienoic acid This compound was reported byLuo et al [17]The 1H and 13CNMR data was assigned by 2DNMR data Compound 4was also isolated from the genus forthe first time
Compound 5 yellow oil HR-ESI-MS mz 3831875 [M +Na]+ UV (MeOH) 120582max 2305 and 3885 nm IR ]max cm
minus1
(KBr) 3461 3018 2955 2931 2859 1780 1716 1673 1522 1065756 1H NMR (600MHz CDCl
3) 120575 647 (1H dt J = 150
66Hz H-2) 588 (1H dd J = 150 12Hz H-3) 528 (1H d J= 36Hz H-5) 501 (1H dd J = 126 12Hz H-12b) 469 (1Hd J = 120Hz H-12a) 416 (1H dt J = 90 36Hz H-16) 300(1H td J = 120 42Hz H-8) 273 (1H dd J = 132 30Hz H-15) 272 (1H dd J = 180 36Hz H-7b) 256 (1H dd J = 180
Journal of Chemistry 5
Table 2 13C NMR (150 MHz) data for 1 2 and monacolin L
No 1a 2a Monacolin Lb
1 422 1342 4372 328 1322 3023 1345 1294 13424 1269 1259 12974a 1334 1330 13845 1270 1278 13106 1362 1342 3287 1273 1285 3598 1276 1234 2388a 1357 1304 36511015840 1711 1709 174021015840 387 388 43231015840 627 628 68241015840 361 362 30751015840 767 759 71261015840 338 358 44971015840 226 242 2602-Me 153 201 1446-Me 211 215 218aCDCl3bCD3OD
120Hz H-7a) 185 (3H dd J = 66 12Hz H-1) 154 (2Hm H-17) 154 (1H m H-18b) 140 (3H s H-13) 131 (2H mH-19) 131 (2H m H-20) 131 (1H m H-18a) 089 (3H t J =66HzH-21) 13CNMR (150MHz CDCl
3) 120575 186 (C-1) 1353
(C-2) 1246 (C-3) 1604 (C-4) 1035 (C-5) 1142 (C-6) 310(C-7) 415 (C-8) 832 (C-9) 1909 (C-10) 1512 (C-11) 639(C-12) 177 (C-13) 1753 (C-14) 491 (C-15) 696 (C-16) 352(C-17) 260 (C-18) 317 (C-19) 227 (C-20) and 141 (C-21)Those datawere identical to the literature data formonascinol[18] Thus compound 5 was determined as monascinol
Compound 6 Colorless solid HR-ESI-MS mz 1521056[M + 1]+ UV (MeOH) 120582max 27550 nm 1HNMR (600MHzCDCl
3) 120575 953 (1H brs NH) 586 (1H s H-3) 512 (1H d
119869 = 96Hz H-11015840) 274 (1H m H-21015840) 206 (3H s 4-Me) 109(6H d J = 66Hz 21015840-Metimes2) 13C NMR (150MHz CDCl
3)
120575 1730 (C-2) 1208 (C-3) 1488 (C-4) 1378 (C-5) 1209 (C-11015840) 276 (C-21015840) 230 (21015840-Me times 2) and 120 (4-Me) Compound6 was elucidated as pulchellalactam by comparing with thereference data [19]
Compound 7Off-white amorphous powder HR-ESI-MSmz 2730753 [M + Na]+ UV (MeOH) 120582max 2405sh 24702805 and 3300 nm IR Vmax cm
minus1 (KBr) 3342 3124 29691676 1624 1584 1515 1455 1152 1117 833 1HNMR (600MHzCDCl
3) 120575 1130 (1H s 8-OH) 1077 (1H s 6-OH) 646 (1H s
H-5) 642 (1H s H-4) 397 (1HmH-21015840) 250 (2HmH-11015840)200 (3H s 7-Me) 112 (3H d J =60HzH-31015840) 13CNMR(150MHz CDCl
3) 120575 1662 (C-1) 1544 (C-3) 1015 (C-4) 1363 (C-
4a) 1052 (C-5) 1599 (C-6) 1096 (C-7) 1635 (C-8) 978 (C-8a) 425 (C-11015840) 639 (C-21015840) 233 (C-31015840) and 79 (7-Me) Thestructure of compound 7 was elucidated by extensive 1D and
2D NMR spectroscopy and confirmed by comparison of its1H and 13C NMR data with those of Lunatinin [20 21]
32 HMG-CoA Reductase Inhibitory Activity The com-pounds 1ndash7were tested in vitro for theirHMG-CoA reductaseinhibitory activities by using the method as described inthe experimental part Lovastatin was used as the positivecontrol Compounds 1-2 (IC
50272 and 312 120583gmL) and 6-7
(IC50
280 and 128120583gmL) demonstrated strong HMG-CoAreductase inhibitory activity comparable with the standarddrug lovastatin (IC
50160 120583gmL) Compounds 3ndash5 caused
moderate activity at the highest concentration (400 120583gmL)It indicates that no statin compounds inMonascus-fermentedrice also have strongHMG-CoA reductase inhibitory activity
4 Conclusions
In conclusion we isolated seven compounds fromMonascus-fermented rice and evaluated their HMG-CoA reductaseinhibitory activities in vitro All compounds showed goodHMG-CoA reductase inhibitory activity among them com-pound 7 exhibited strong inhibition with IC
50value compa-
rable with that of the standard drug lovastatinThus no statincompounds in Monascus-fermented rice also have strongHMG-CoA reductase inhibitory activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Compre-hensive platform of Natural drugs and New formulations(2013ZX09402201) and program for Zhongguancun HaidianScience Park enterprise postdoctoral fellowship
References
[1] J Ma Y Li Q Ye et al ldquoConstituents of red yeast rice atraditional Chinese food and medicinerdquo Journal of Agriculturaland Food Chemistry vol 48 no 11 pp 5220ndash5225 2000
[2] A Endo ldquoMonacolin K a new hypocholesterolemic agentproduced by aMonascus speciesrdquoThe Journal of Antibiotics vol32 no 8 pp 852ndash854 1979
[3] A Endo K Hasumi and S Negishi ldquoMonacolins J and L newinhibitors of cholesterol biosynthesis produced by Monascusruberrdquo The Journal of Antibiotics vol 38 no 3 pp 420ndash4221985
[4] A Endo K Hasumi and T Nakamura ldquoDihydromonacolinL and monacolin X new metabolites those inhibit cholesterolbiosynthesisrdquo The Journal of Antibiotics vol 38 no 3 pp 321ndash327 1985
[5] A Endo D Komagata and H Shimada ldquoMonacolin M a newinhibitor of cholesterol biosynthesisrdquoThe Journal of Antibioticsvol 39 no 12 pp 1670ndash1673 1986
[6] M A Dhale S Divakar S U Kumar and G VijayalakshmildquoIsolation and characterization of dihydromonacolin-MV from
6 Journal of Chemistry
Monascus purpureus for antioxidant propertiesrdquoAppliedMicro-biology and Biotechnology vol 73 no 5 pp 1197ndash1202 2007
[7] M A Dhale S Divakar S Umesh-Kumar and G Vijay-alakshmi ldquoCharacterization of dehydromonacolin-MV2 fromMonascus purpureus mutantrdquo Journal of Applied Microbiologyvol 103 no 6 pp 2168ndash2173 2007
[8] X-M Li X-H Shen Z-W Duan and S-R Guo ldquoAdvancesin studies on secondary metabolites from Monascusrdquo ChineseTraditional and Herbal Drugs vol 42 no 5 pp 1018ndash1025 2011
[9] B W Philipp and D J Shapiro ldquoImproved methods for theassay and activation of 3-hydroxy-3-methylglutaryl coenzymeA reductaserdquo Journal of Lipid Research vol 20 no 5 pp 588ndash593 1979
[10] P A Edwards D Lemongello and A M Fogelman ldquoImprovedmethods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductaserdquo Journal of LipidResearch vol 20 no 1 pp 40ndash46 1979
[11] P A Edwards D Lemongello J Kane I Shechter and AM Fogelman ldquoProperties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzymeA reductase and regulation of enzymeactivityrdquoThe Journal of Biological Chemistry vol 255 no 8 pp3715ndash3725 1980
[12] P Li and L Chen ldquoXuezhikang inhibits the activity of HMG-CoA reductase in pig liverrdquo Basic Medical Sciences and Clinicsvol 23 no 5 pp 531ndash534 2003
[13] M D Greenspan J B Yudkovitz A W Alberts L S Argen-bright B H Arison and J L Smith ldquoMetabolism of lovastatinby rat and human liver microsomes in vitrordquo Drug Metabolismand Disposition vol 16 no 5 pp 678ndash682 1988
[14] G E Stokker ldquoSynthesis of the 31015840(S)-hydroxy derivative ofsimvastatinrdquo Bioorganic amp Medicinal Chemistry Letters vol 4no 14 pp 1767ndash1770 1994
[15] HOka A Terahara andA Endo ldquo4-hydroxy-2-pyrone deriva-tives and their pharmaceutical preparationsrdquo Patent No EP0010951 A1 1980
[16] C Hong X Piao and C Lou ldquoIsolation and identificationof chemical constituents from Alisma orientalerdquo Journal ofChongqing Institute of Technology vol 22 no 4 pp 78ndash81 2008
[17] C Luo W Zhang C Sheng C Zheng J Yao and Z MiaoldquoChemical composition and antidiabetic activity of OpuntiaMilpa Alta extractsrdquo Chemistry amp Biodiversity vol 7 no 12 pp2869ndash2879 2010
[18] T Akihisa Y Tadashi S Takashi et al ldquoMonascinol and tumorpromoter activity inhibitors and food containing itrdquo Patent NoJP 2008056618 A 2008
[19] J S Bryans N E A Chessum N Huther A F Parsons andF Ghelfi ldquoMetal-catalysed radical cyclisations leading to N-heterocycles new approaches to gabapentin and pulchellalac-tamrdquo Tetrahedron vol 59 no 33 pp 6221ndash6231 2003
[20] L-L Li J-P Chen and L-Y Kong ldquoChemical constituents ofMonascus ankardquo Chinese Pharmaceutical Journal vol 41 no 15pp 1131ndash1133 2006
[21] Y Yamaguchi R Masuma Y-P Kim R Uchida H Tomodaand S Omura ldquoTaxonomy and secondary metabolites ofPseudobotrytis sp FKA-25rdquoMycoscience vol 45 no 1 pp 9ndash162004
[22] X-M Li X-H Shen Z-WDuan and S-R Guo ldquoA newmona-colin analogue from Xuezhikang capsulerdquo Acta PharmaceuticaSinica vol 46 no 5 pp 564ndash567 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
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Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
2 Journal of Chemistry
DAD detector NADPH and HMG-CoA were purchasedfrom Merck and Sigma respectively Cholestyramine wasobtained from Nanjing Lifecare Pharmaceutical Lovstatinwas purchased from China National Institutes for Foodand Drug Control All other chemicals were obtained fromBeijing Chemical Reagent Co Ltd All organic solvents wereof analytical purity and purchased from Beijing ChemicalReagent Co Ltd China
22 Biological Material Monascus-fermented rice was pre-pared from cooked rice inoculated with M purpureus(CGMCC No 0272) produced by Beijing Peking Univer-sity WBL Biotechnology Co Ltd The Monascus-fermentedrice was dried with a drying oven at 80∘C and the driedMonascus-fermented rice was pulverized with a pulverizer
TheMonascus purpureuswas identified by China GeneralMicrobiological Culture Collection Center (CGMCC) anddeposited in CGMCC
23 Extraction and Isolation Dried and powdered Monas-cus-fermented rice (4 kg) was successively ultrasonicextracted 3 times with petroleum ether and ethyl acetate toyield petroleum ether crude extract (82 g) and ethyl acetatecrude extract (170 g) The petroleum ether crude extractwas subjected to silica gel column chromatography elutedwith a gradient mixture of petroleum ether-ethyl acetate(50 50 to 0 100) to yield 2 fractions (P1-P2) Fr P1 (54 g)was further subjected to a silica gel column eluted withpetroleum ether-ethyl acetate (75 25) to yield 3 fractions(P1A-P1C) Fraction P1C (205 g) was chromatographed overa C18 column eluted with 75 MeOH to give 3 fractions(P1C1-P1C3) P1C1 subfraction (103 g) was purified by gelchromatography over Sephadex LH-20 eluted with CH
2Cl2-
MeOH (1 1) and further purified by MPLC using 75ndash85MeOH to afford compound 5 (54mg) and compound 6(50mg) Fr P2 (20 g) was further subjected to a silica gelcolumn eluted with petroleum ether-ethyl acetate (25 75)to yield 3 fractions (P2A-P2C) P2B subfraction (85 g)was purified by gel chromatography over Sephadex LH-20eluted with CH
2Cl2-MeOH (1 1) and further purified by
HPLC using 90 MeOH to afford compound 3 (14mg)Ethyl acetate crude extract (170 g) was subjected to silicagel column chromatography eluted with a gradient mixtureof petroleum ether-ethyl acetate (50 50 to 0 100) to yield2 fractions (E1-E2) Fr E2 (48 g) was further subjected to asilica gel column eluted with petroleum ether-ethyl acetate(33 67) to yield 4 fractions (E2A-E2D) Fr E2B (108 g)was chromatographed on MPLC eluted with CH
2Cl2-ethyl
acetate-MeOH (30 30 1) to yield 4 fractions (E2B1-E2B4)E2B1 subfraction (38 g) was purified by gel chromatographyover Sephadex LH-20 eluted with CH
2Cl2-MeOH (2 1) and
further purified by HPLC using 66 acetonitrile-MeOH(1 1) in H
2O to afford compound 1 (48mg) and compound 2
(18mg) E2B2 subfraction (40 g) was purified by C18 columnchromatography eluted with 82 acetonitrile-MeOH (1 1)in H2O and further purified by gel chromatography over
Sephadex LH-20 eluted with CH2Cl2-MeOH (2 1) to afford
compound 7 (56mg) Fr E2C (47 g) was chromatographed
over a Sephadex LH-20 column (CH2Cl2-MeOH 6 4) and
afforded five subfractions (E2C1-E2C5) E2C4 subfraction(049 g) was purified by C18 column chromatography elutedwith 85 MeOH and further purified by HPLC using ACNto afford compound 4 (67mg)
24 HMG-CoA Reductase Inhibitory Assay
241 Preparation of Microsomes Male Sprague-Dawley rats(100ndash200 g) were killed by cervical dislocation at or nearthe peak of the daily circadian rhythm in reductase activ-ity In most experiments rats were fed a diet containing5 cholestyramine for 4 days prior to the preparation ofmicrosomes Livers were excised rinsed in ice-cold bufferA (KESD buffer) containing 10mM potassium phosphate2mM EDTA 250mM sucrose and 1mMDTT (pH68) Thelivers were then minced through a tissue press and homog-enized in three volumes of buffer A per g of liver by fourstrokes with the loose pestle of a Dounce homogenizer andone stroke with the tight pestle Mitochondria and cell debriswere sedimented by centrifugation two times at 12000 g for15min at 4∘C Crude microsomes were prepared from thetwo 12000 g supernatants by sedimentation at 105000 g for90min at 4∘CThe pellet was resuspended in buffer A (1mLgliver) and resedimented at 105000 g for 90min at 4∘C [9]The washed microsomal pellets were quickly-frozen in anacetone-dry ice bath and stored at minus80∘C prior to use
242 Solubilization of HMG-CoA Reductase For optimalsolubilization of the reductase the frozen microsomes wereallowed to thaw at room temperature before addition ofan equal volume of 50 glycerol in buffer B containing02M sucrose 01MKCl 008Mpotassiumphosphate 2mMpotassium EDTA and 10mM DTT (pH 72) preheated to37∘CThe suspension was rehomogenized with 10 downwardpasses of a hand-driven all-glass Potter-Elvehjem homoge-nizer and then incubated at 37∘C for 60min The suspensionwas diluted threefold with buffer B preheated to 37∘C toa final glycerol concentration of 83 rehomogenized with10 downward passes of the glass homogenizer pestle andcentrifuged at 105000 g for 60min at 25∘C The supernatantcontaining solubilized HMG-CoA reductase was removedand used immediately for enzyme purification [10]
243 Purification of HMG-CoA Reductase The solubilizedenzyme was fractioned with saturated ammonium sulfatesolution and the protein precipating between 35 and 50ammonium sulfate was dissolved in buffer B [11]
244 Assay of HMG-CoA Reductase The activity of HMG-CoA reductase was determined at 37∘C in total volume of200120583L HMG-CoA reductase 10120583L was preincubated withbuffer C (KCl 200mM potassium phosphate 160mM EDTA4mM DTT 10mM pH68) for 5min at 37∘C and the reac-tion was initiated by adding 15 120583L of cofactor-substrate solu-tion (200120583M NADPH and 50 120583M HMG-CoA in buffer C)and 5 120583L of sample solution (dissolved in 50DMSO) or 5120583Lof 50DMSO (negative control) [12]The dynamic change of
Journal of Chemistry 3
O
OHO
6 5 4a
17
8 8a
43
2
H
1
O
OHO
H
2
OOO
O1211
10
13
8
91415 6 5 4
3
2
17
OH1617
1819
2021H
5
HN O
234
5
6
O
OH
OH
OH
4 6
788a
3 54a
O
12
7
OH
O4
O
OHO
H
H
Monacolin L
O
O
18 16 14 11 8 4 2 1
3
OHOH6
2998400
1998400
1998400 2
998400
2998400
3998400
1998400
1998400
3998400
3998400
4998400
5998400
6998400
7998400
Figure 1 Structures of compounds 1ndash7 and monacolin L
OD340
was detected with VerSamax ELISAmicroplate readerand the rate of the change within 5min was used to evaluatethe activity of HMG-CoA reductase and then to evaluate theinhibition activity of each sample
3 Results and Discussion
Repeated column chromatography of the petroleumether extract and ethyl acetate extract of Monascus-fermented rice afforded seven known compounds (1-7see Figure 1) The compounds were identified as (4R6R)-6-(2-((1S 2S)-26-dimethyl-12-dihydro naphthalene-1-yl)-ethyl)-4-hydroxy- tetrahydropyran-2-one (1) [13 14] (4R6R)-6-(2-(26-dimethylnaphthalene-1-yl)ehtyl-4-hydroxyte-trahydropyran-2-one (2) [15] 1-monolinolein (3) [16](9Z12Z)-octadeca-912-dienoic acid (4) [17] Monascinol (5)[18] Pulchellalactam (6) [19] and Lunatinin (7) [20 21] onthe basis of comparison of their NMR data and HR-ESI-MSor EI-MS data with those reported in the literature It isworthwhile to point out that compounds 1 and 2were isolatedfrom natural source for the first time and compounds 3and 4 were isolated from the genus for the first time Allisolated compounds were evaluated for their ability to inhibitHMG-CoA reductase
31 Chemistry Compound 1was obtained as a colorless gumThe HR-ESI-MS gave a pseudo molecular ion peak at mz3231492 [M + Na]+ consistent with a molecular formula ofC19H24O3 The UV spectrum showed maximum absorption
at 2290 and 2658 nm Its IR spectrum revealed absorptionbands at 3380 (hydroxyl) and 1712 cmminus1 (carbonyl) The 1Hand 13C NMR data of 1 were similar to those of monacolinL previous reported [22] Comparing the molecular formulaand NMR data of 1 with those of monacolin L it was foundthat 1 absent four hydrogens and had two more aromaticdouble bond signals at 120575 1273693 (1H d J = 78Hz H-7) with 1362 (C-6) and 1276696 (1H d J = 78Hz H-8) with 1357 (C-8a) In addition the chemical shift of 6-Me at 120575 229 (3H s) was moved obviously downfield Thetwo aromatic double bonds were located between C-5 andC-4a based on the HMBC correlations of H-7C5 C-8aand 6-Me H-8C-4a C-8a C-1 and C-7 (see Figure 2)Additionally the optical rotation at [120572]25D + 315 (c 0111CH2Cl2) is similar to that of monacolin L whose optical
rotation at [120572]25D + 16416 (c 06 CH2Cl2MeOH = 2 1)
suggesting that the chiral carbons C-1 C-2 C-31015840 and C-51015840 of compound 1 also had S S R R-configurations It isfurther confirmed by the NOESY correlations (see Figure 2)observed between H-1 (120575H 258) with H-2 (120575H 272) H-31015840
4 Journal of Chemistry
O
OHO
H
Figure 2 Key HMBC (rarr ) and NOESY (harr) correlations incompound 1
with H-1 (120575H 258) and H-31015840 with H-2 (120575H 272) SimilarlyNOESY cross peaks between 2-Me (120575H 106) with H-71015840 (120575H158 and 176) H-71015840 (120575H 158 and 176) with H-51015840 (120575H 456)Thus the structure of 1 was identified as (4R 6R)-6-(2-((1S 2S)-26-dimethyl-12-dihydro-naphthalene-1-yl)-ethyl)-4-hydroxy-tetrahydropyran-2-one This compound was re-ported by Greenspan et al and Stokker [13 14] The 1H and13C NMR data was assigned unambiguously by HSQC andHMBC experiments (see Tables 1 and 2) Compound 1 wasobtained from natural source for the first time
Compound 2 was obtained as a colorless gum The HR-ESI-MS gave a pseudo molecular ion peak at mz 3211484[M+Na]+ consistent with amolecular formula of C
19H22O3
The UV spectrum showed maximum absorption at 2318 and2816 nm Its IR spectrum revealed absorption bands at 3434(hydroxyl) and 1712 cmminus1 (carbonyl)TheUV andNMR dataof 2 were similar to those of 1 but showed one more doublebond signals at 120575 1342 (C-1) and 1322 (C-2) and absenttwo hydrogens (H-1 and H-2) and the downfield chemicalshift of 2-Me at 120575 249 (3H s) indicating that 2 was 12-twodehydrogenated 1 Additionally the optical rotation at [120572]25D+ 112 (c 006 CH
2Cl2) is similar to that of monacolin L
suggesting that the chiral carbonsC-31015840 andC-51015840 of compound2 also had R R-configurations Thus the structure of 2 wasdetermined as (4R 6R)-6-(2-(26-dimethylnaphthalene-1-yl)ehtyl-4-hydroxy-tetrahydropyran-2-one This compoundwas reported byOka et al [15]The 1Hand 13CNMRdata wasassigned unambiguously by HSQC and HMBC experiments(see Tables 1 and 2) Compound 2 was also obtained fromnatural source for the first time
Compound 3 colorless oil HR-ESI-MS mz 3772698[M+Na]+ 1H NMR (600MHz CDCl
3) 120575 534 (4H m H-
9 10 12 and 13) 417 (1H dd J = 114 36Hz H-11015840b) 413 (1Hdd J = 114 60Hz H-11015840a) 391 (1H m H-21015840) 369 (1H ddJ = 114 36Hz H-31015840b) 359 (1H dd J = 114 60Hz H-31015840a)276 (2H m H-11) 233 (2H t J = 78Hz H-2) 204 (4H mH-8 and H-14) 162 (2H m H-3) 131 (14H m H-4 to H-7and H-15 to H-17) 088 (3H t J = 72Hz H-18) 13C NMR(150MHz CDCl
3) 120575 1745 (C-1) 1304 (C-10) 1302 (C-9)
1282 (C-13) 1280 (C-12) 704 (C-21015840) 653 (C-11015840) 635 (C-31015840)343 (C-2) 317 (C-16) 297 (C-7) 295 (C-6) 293 (C-5) 292
Table 1 1H NMR (600MHz) data for 1 2 and monacolin L (120575 inppm 119869 in Hz)
No 1a 2a Monacolin Lb
1 258 (m) mdash 140 (m)2 272 (m) mdash 231 (m)3 574 (dd 96 30) 727 (d 84) 572 (m)4 635 (dd 96 24) 756 (d 84) 587 (m)4a mdash mdash mdash5 686 (brs) 758 (brs) 539 (m)6 mdash mdash 232 (m)7 693 (d 78) 734 (dd 90 12) 146 (m) 153 (m)8 696 (d 78) 794 (d 90) 120 (m) 181 (m)8a mdash mdash 205 (m)11015840 mdash mdash mdash
21015840 255 (m) 268 (ddd 174 36 18) 244 (dd 150 84)266 (dd 180 54) 277 (dd 174 48) 255 (dd 150 48)
31015840 428 (m) 440 (m) 420 (m)41015840 163 (m) 186 (m) 182 (m) 200 (m) 162 (m) 178 (m)51015840 456 (m) 486 (m) 376 (m)61015840 142 (m) 166 (m) 192 (m) 198 (m) 160 (m) 165 (m)71015840 158 (m) 176 (m) 316 (m) 337 (m) 130 (m) 173 (m)2-Me 106 (d 78) 249 (brs) 099 (dd 72 18)6-Me 229 (s) 250 (brs) 090 (dd 72 30)aCDCl3bCD3OD
(C-4 and C-15) 274 (C-8) 273 (C-14) 258 (C-11) 250 (C-3)227 (C-17) and 142 (C-18) Compound 3 was characterizedas 1-monolinolein by comparison with the literature [16] andwas isolated from the genus for the first time
Compound 4 colorless oil EI-MSmz 280 [M]+ 1HNMR(600MHz CD
3OD) 120575 537 (2H m H-9 and H-10) 531 (2H
m H-12 and H-13) 278 (2H brt J = 72Hz H-11) 227 (2Ht J = 72Hz H-2) 207 (4H m H-8 and H-14) 160 (2H mH-3) 135 (14H m H-4 to H-7 and H-15 to H-17) 091 (3Ht J = 72Hz H-18) 13CNMR (150MHz CD
3OD) 120575 1779 (C-
1) 1311 (C-9 and C-10) 1293 (C-13) 1292 (C-12) 352 (C-2)329 (C-16) 309 (C-7) 307 (C-6) 305 (C-5) 304 (C-4 andC-15) 284 (C-8 and C-14) 268 (C-11) 263 (C-3) 238 (C-17) and 146 (C-18) Compound 4 was identified as (9Z12Z)-octadeca-912-dienoic acid This compound was reported byLuo et al [17]The 1H and 13CNMR data was assigned by 2DNMR data Compound 4was also isolated from the genus forthe first time
Compound 5 yellow oil HR-ESI-MS mz 3831875 [M +Na]+ UV (MeOH) 120582max 2305 and 3885 nm IR ]max cm
minus1
(KBr) 3461 3018 2955 2931 2859 1780 1716 1673 1522 1065756 1H NMR (600MHz CDCl
3) 120575 647 (1H dt J = 150
66Hz H-2) 588 (1H dd J = 150 12Hz H-3) 528 (1H d J= 36Hz H-5) 501 (1H dd J = 126 12Hz H-12b) 469 (1Hd J = 120Hz H-12a) 416 (1H dt J = 90 36Hz H-16) 300(1H td J = 120 42Hz H-8) 273 (1H dd J = 132 30Hz H-15) 272 (1H dd J = 180 36Hz H-7b) 256 (1H dd J = 180
Journal of Chemistry 5
Table 2 13C NMR (150 MHz) data for 1 2 and monacolin L
No 1a 2a Monacolin Lb
1 422 1342 4372 328 1322 3023 1345 1294 13424 1269 1259 12974a 1334 1330 13845 1270 1278 13106 1362 1342 3287 1273 1285 3598 1276 1234 2388a 1357 1304 36511015840 1711 1709 174021015840 387 388 43231015840 627 628 68241015840 361 362 30751015840 767 759 71261015840 338 358 44971015840 226 242 2602-Me 153 201 1446-Me 211 215 218aCDCl3bCD3OD
120Hz H-7a) 185 (3H dd J = 66 12Hz H-1) 154 (2Hm H-17) 154 (1H m H-18b) 140 (3H s H-13) 131 (2H mH-19) 131 (2H m H-20) 131 (1H m H-18a) 089 (3H t J =66HzH-21) 13CNMR (150MHz CDCl
3) 120575 186 (C-1) 1353
(C-2) 1246 (C-3) 1604 (C-4) 1035 (C-5) 1142 (C-6) 310(C-7) 415 (C-8) 832 (C-9) 1909 (C-10) 1512 (C-11) 639(C-12) 177 (C-13) 1753 (C-14) 491 (C-15) 696 (C-16) 352(C-17) 260 (C-18) 317 (C-19) 227 (C-20) and 141 (C-21)Those datawere identical to the literature data formonascinol[18] Thus compound 5 was determined as monascinol
Compound 6 Colorless solid HR-ESI-MS mz 1521056[M + 1]+ UV (MeOH) 120582max 27550 nm 1HNMR (600MHzCDCl
3) 120575 953 (1H brs NH) 586 (1H s H-3) 512 (1H d
119869 = 96Hz H-11015840) 274 (1H m H-21015840) 206 (3H s 4-Me) 109(6H d J = 66Hz 21015840-Metimes2) 13C NMR (150MHz CDCl
3)
120575 1730 (C-2) 1208 (C-3) 1488 (C-4) 1378 (C-5) 1209 (C-11015840) 276 (C-21015840) 230 (21015840-Me times 2) and 120 (4-Me) Compound6 was elucidated as pulchellalactam by comparing with thereference data [19]
Compound 7Off-white amorphous powder HR-ESI-MSmz 2730753 [M + Na]+ UV (MeOH) 120582max 2405sh 24702805 and 3300 nm IR Vmax cm
minus1 (KBr) 3342 3124 29691676 1624 1584 1515 1455 1152 1117 833 1HNMR (600MHzCDCl
3) 120575 1130 (1H s 8-OH) 1077 (1H s 6-OH) 646 (1H s
H-5) 642 (1H s H-4) 397 (1HmH-21015840) 250 (2HmH-11015840)200 (3H s 7-Me) 112 (3H d J =60HzH-31015840) 13CNMR(150MHz CDCl
3) 120575 1662 (C-1) 1544 (C-3) 1015 (C-4) 1363 (C-
4a) 1052 (C-5) 1599 (C-6) 1096 (C-7) 1635 (C-8) 978 (C-8a) 425 (C-11015840) 639 (C-21015840) 233 (C-31015840) and 79 (7-Me) Thestructure of compound 7 was elucidated by extensive 1D and
2D NMR spectroscopy and confirmed by comparison of its1H and 13C NMR data with those of Lunatinin [20 21]
32 HMG-CoA Reductase Inhibitory Activity The com-pounds 1ndash7were tested in vitro for theirHMG-CoA reductaseinhibitory activities by using the method as described inthe experimental part Lovastatin was used as the positivecontrol Compounds 1-2 (IC
50272 and 312 120583gmL) and 6-7
(IC50
280 and 128120583gmL) demonstrated strong HMG-CoAreductase inhibitory activity comparable with the standarddrug lovastatin (IC
50160 120583gmL) Compounds 3ndash5 caused
moderate activity at the highest concentration (400 120583gmL)It indicates that no statin compounds inMonascus-fermentedrice also have strongHMG-CoA reductase inhibitory activity
4 Conclusions
In conclusion we isolated seven compounds fromMonascus-fermented rice and evaluated their HMG-CoA reductaseinhibitory activities in vitro All compounds showed goodHMG-CoA reductase inhibitory activity among them com-pound 7 exhibited strong inhibition with IC
50value compa-
rable with that of the standard drug lovastatinThus no statincompounds in Monascus-fermented rice also have strongHMG-CoA reductase inhibitory activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Compre-hensive platform of Natural drugs and New formulations(2013ZX09402201) and program for Zhongguancun HaidianScience Park enterprise postdoctoral fellowship
References
[1] J Ma Y Li Q Ye et al ldquoConstituents of red yeast rice atraditional Chinese food and medicinerdquo Journal of Agriculturaland Food Chemistry vol 48 no 11 pp 5220ndash5225 2000
[2] A Endo ldquoMonacolin K a new hypocholesterolemic agentproduced by aMonascus speciesrdquoThe Journal of Antibiotics vol32 no 8 pp 852ndash854 1979
[3] A Endo K Hasumi and S Negishi ldquoMonacolins J and L newinhibitors of cholesterol biosynthesis produced by Monascusruberrdquo The Journal of Antibiotics vol 38 no 3 pp 420ndash4221985
[4] A Endo K Hasumi and T Nakamura ldquoDihydromonacolinL and monacolin X new metabolites those inhibit cholesterolbiosynthesisrdquo The Journal of Antibiotics vol 38 no 3 pp 321ndash327 1985
[5] A Endo D Komagata and H Shimada ldquoMonacolin M a newinhibitor of cholesterol biosynthesisrdquoThe Journal of Antibioticsvol 39 no 12 pp 1670ndash1673 1986
[6] M A Dhale S Divakar S U Kumar and G VijayalakshmildquoIsolation and characterization of dihydromonacolin-MV from
6 Journal of Chemistry
Monascus purpureus for antioxidant propertiesrdquoAppliedMicro-biology and Biotechnology vol 73 no 5 pp 1197ndash1202 2007
[7] M A Dhale S Divakar S Umesh-Kumar and G Vijay-alakshmi ldquoCharacterization of dehydromonacolin-MV2 fromMonascus purpureus mutantrdquo Journal of Applied Microbiologyvol 103 no 6 pp 2168ndash2173 2007
[8] X-M Li X-H Shen Z-W Duan and S-R Guo ldquoAdvancesin studies on secondary metabolites from Monascusrdquo ChineseTraditional and Herbal Drugs vol 42 no 5 pp 1018ndash1025 2011
[9] B W Philipp and D J Shapiro ldquoImproved methods for theassay and activation of 3-hydroxy-3-methylglutaryl coenzymeA reductaserdquo Journal of Lipid Research vol 20 no 5 pp 588ndash593 1979
[10] P A Edwards D Lemongello and A M Fogelman ldquoImprovedmethods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductaserdquo Journal of LipidResearch vol 20 no 1 pp 40ndash46 1979
[11] P A Edwards D Lemongello J Kane I Shechter and AM Fogelman ldquoProperties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzymeA reductase and regulation of enzymeactivityrdquoThe Journal of Biological Chemistry vol 255 no 8 pp3715ndash3725 1980
[12] P Li and L Chen ldquoXuezhikang inhibits the activity of HMG-CoA reductase in pig liverrdquo Basic Medical Sciences and Clinicsvol 23 no 5 pp 531ndash534 2003
[13] M D Greenspan J B Yudkovitz A W Alberts L S Argen-bright B H Arison and J L Smith ldquoMetabolism of lovastatinby rat and human liver microsomes in vitrordquo Drug Metabolismand Disposition vol 16 no 5 pp 678ndash682 1988
[14] G E Stokker ldquoSynthesis of the 31015840(S)-hydroxy derivative ofsimvastatinrdquo Bioorganic amp Medicinal Chemistry Letters vol 4no 14 pp 1767ndash1770 1994
[15] HOka A Terahara andA Endo ldquo4-hydroxy-2-pyrone deriva-tives and their pharmaceutical preparationsrdquo Patent No EP0010951 A1 1980
[16] C Hong X Piao and C Lou ldquoIsolation and identificationof chemical constituents from Alisma orientalerdquo Journal ofChongqing Institute of Technology vol 22 no 4 pp 78ndash81 2008
[17] C Luo W Zhang C Sheng C Zheng J Yao and Z MiaoldquoChemical composition and antidiabetic activity of OpuntiaMilpa Alta extractsrdquo Chemistry amp Biodiversity vol 7 no 12 pp2869ndash2879 2010
[18] T Akihisa Y Tadashi S Takashi et al ldquoMonascinol and tumorpromoter activity inhibitors and food containing itrdquo Patent NoJP 2008056618 A 2008
[19] J S Bryans N E A Chessum N Huther A F Parsons andF Ghelfi ldquoMetal-catalysed radical cyclisations leading to N-heterocycles new approaches to gabapentin and pulchellalac-tamrdquo Tetrahedron vol 59 no 33 pp 6221ndash6231 2003
[20] L-L Li J-P Chen and L-Y Kong ldquoChemical constituents ofMonascus ankardquo Chinese Pharmaceutical Journal vol 41 no 15pp 1131ndash1133 2006
[21] Y Yamaguchi R Masuma Y-P Kim R Uchida H Tomodaand S Omura ldquoTaxonomy and secondary metabolites ofPseudobotrytis sp FKA-25rdquoMycoscience vol 45 no 1 pp 9ndash162004
[22] X-M Li X-H Shen Z-WDuan and S-R Guo ldquoA newmona-colin analogue from Xuezhikang capsulerdquo Acta PharmaceuticaSinica vol 46 no 5 pp 564ndash567 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 3
O
OHO
6 5 4a
17
8 8a
43
2
H
1
O
OHO
H
2
OOO
O1211
10
13
8
91415 6 5 4
3
2
17
OH1617
1819
2021H
5
HN O
234
5
6
O
OH
OH
OH
4 6
788a
3 54a
O
12
7
OH
O4
O
OHO
H
H
Monacolin L
O
O
18 16 14 11 8 4 2 1
3
OHOH6
2998400
1998400
1998400 2
998400
2998400
3998400
1998400
1998400
3998400
3998400
4998400
5998400
6998400
7998400
Figure 1 Structures of compounds 1ndash7 and monacolin L
OD340
was detected with VerSamax ELISAmicroplate readerand the rate of the change within 5min was used to evaluatethe activity of HMG-CoA reductase and then to evaluate theinhibition activity of each sample
3 Results and Discussion
Repeated column chromatography of the petroleumether extract and ethyl acetate extract of Monascus-fermented rice afforded seven known compounds (1-7see Figure 1) The compounds were identified as (4R6R)-6-(2-((1S 2S)-26-dimethyl-12-dihydro naphthalene-1-yl)-ethyl)-4-hydroxy- tetrahydropyran-2-one (1) [13 14] (4R6R)-6-(2-(26-dimethylnaphthalene-1-yl)ehtyl-4-hydroxyte-trahydropyran-2-one (2) [15] 1-monolinolein (3) [16](9Z12Z)-octadeca-912-dienoic acid (4) [17] Monascinol (5)[18] Pulchellalactam (6) [19] and Lunatinin (7) [20 21] onthe basis of comparison of their NMR data and HR-ESI-MSor EI-MS data with those reported in the literature It isworthwhile to point out that compounds 1 and 2were isolatedfrom natural source for the first time and compounds 3and 4 were isolated from the genus for the first time Allisolated compounds were evaluated for their ability to inhibitHMG-CoA reductase
31 Chemistry Compound 1was obtained as a colorless gumThe HR-ESI-MS gave a pseudo molecular ion peak at mz3231492 [M + Na]+ consistent with a molecular formula ofC19H24O3 The UV spectrum showed maximum absorption
at 2290 and 2658 nm Its IR spectrum revealed absorptionbands at 3380 (hydroxyl) and 1712 cmminus1 (carbonyl) The 1Hand 13C NMR data of 1 were similar to those of monacolinL previous reported [22] Comparing the molecular formulaand NMR data of 1 with those of monacolin L it was foundthat 1 absent four hydrogens and had two more aromaticdouble bond signals at 120575 1273693 (1H d J = 78Hz H-7) with 1362 (C-6) and 1276696 (1H d J = 78Hz H-8) with 1357 (C-8a) In addition the chemical shift of 6-Me at 120575 229 (3H s) was moved obviously downfield Thetwo aromatic double bonds were located between C-5 andC-4a based on the HMBC correlations of H-7C5 C-8aand 6-Me H-8C-4a C-8a C-1 and C-7 (see Figure 2)Additionally the optical rotation at [120572]25D + 315 (c 0111CH2Cl2) is similar to that of monacolin L whose optical
rotation at [120572]25D + 16416 (c 06 CH2Cl2MeOH = 2 1)
suggesting that the chiral carbons C-1 C-2 C-31015840 and C-51015840 of compound 1 also had S S R R-configurations It isfurther confirmed by the NOESY correlations (see Figure 2)observed between H-1 (120575H 258) with H-2 (120575H 272) H-31015840
4 Journal of Chemistry
O
OHO
H
Figure 2 Key HMBC (rarr ) and NOESY (harr) correlations incompound 1
with H-1 (120575H 258) and H-31015840 with H-2 (120575H 272) SimilarlyNOESY cross peaks between 2-Me (120575H 106) with H-71015840 (120575H158 and 176) H-71015840 (120575H 158 and 176) with H-51015840 (120575H 456)Thus the structure of 1 was identified as (4R 6R)-6-(2-((1S 2S)-26-dimethyl-12-dihydro-naphthalene-1-yl)-ethyl)-4-hydroxy-tetrahydropyran-2-one This compound was re-ported by Greenspan et al and Stokker [13 14] The 1H and13C NMR data was assigned unambiguously by HSQC andHMBC experiments (see Tables 1 and 2) Compound 1 wasobtained from natural source for the first time
Compound 2 was obtained as a colorless gum The HR-ESI-MS gave a pseudo molecular ion peak at mz 3211484[M+Na]+ consistent with amolecular formula of C
19H22O3
The UV spectrum showed maximum absorption at 2318 and2816 nm Its IR spectrum revealed absorption bands at 3434(hydroxyl) and 1712 cmminus1 (carbonyl)TheUV andNMR dataof 2 were similar to those of 1 but showed one more doublebond signals at 120575 1342 (C-1) and 1322 (C-2) and absenttwo hydrogens (H-1 and H-2) and the downfield chemicalshift of 2-Me at 120575 249 (3H s) indicating that 2 was 12-twodehydrogenated 1 Additionally the optical rotation at [120572]25D+ 112 (c 006 CH
2Cl2) is similar to that of monacolin L
suggesting that the chiral carbonsC-31015840 andC-51015840 of compound2 also had R R-configurations Thus the structure of 2 wasdetermined as (4R 6R)-6-(2-(26-dimethylnaphthalene-1-yl)ehtyl-4-hydroxy-tetrahydropyran-2-one This compoundwas reported byOka et al [15]The 1Hand 13CNMRdata wasassigned unambiguously by HSQC and HMBC experiments(see Tables 1 and 2) Compound 2 was also obtained fromnatural source for the first time
Compound 3 colorless oil HR-ESI-MS mz 3772698[M+Na]+ 1H NMR (600MHz CDCl
3) 120575 534 (4H m H-
9 10 12 and 13) 417 (1H dd J = 114 36Hz H-11015840b) 413 (1Hdd J = 114 60Hz H-11015840a) 391 (1H m H-21015840) 369 (1H ddJ = 114 36Hz H-31015840b) 359 (1H dd J = 114 60Hz H-31015840a)276 (2H m H-11) 233 (2H t J = 78Hz H-2) 204 (4H mH-8 and H-14) 162 (2H m H-3) 131 (14H m H-4 to H-7and H-15 to H-17) 088 (3H t J = 72Hz H-18) 13C NMR(150MHz CDCl
3) 120575 1745 (C-1) 1304 (C-10) 1302 (C-9)
1282 (C-13) 1280 (C-12) 704 (C-21015840) 653 (C-11015840) 635 (C-31015840)343 (C-2) 317 (C-16) 297 (C-7) 295 (C-6) 293 (C-5) 292
Table 1 1H NMR (600MHz) data for 1 2 and monacolin L (120575 inppm 119869 in Hz)
No 1a 2a Monacolin Lb
1 258 (m) mdash 140 (m)2 272 (m) mdash 231 (m)3 574 (dd 96 30) 727 (d 84) 572 (m)4 635 (dd 96 24) 756 (d 84) 587 (m)4a mdash mdash mdash5 686 (brs) 758 (brs) 539 (m)6 mdash mdash 232 (m)7 693 (d 78) 734 (dd 90 12) 146 (m) 153 (m)8 696 (d 78) 794 (d 90) 120 (m) 181 (m)8a mdash mdash 205 (m)11015840 mdash mdash mdash
21015840 255 (m) 268 (ddd 174 36 18) 244 (dd 150 84)266 (dd 180 54) 277 (dd 174 48) 255 (dd 150 48)
31015840 428 (m) 440 (m) 420 (m)41015840 163 (m) 186 (m) 182 (m) 200 (m) 162 (m) 178 (m)51015840 456 (m) 486 (m) 376 (m)61015840 142 (m) 166 (m) 192 (m) 198 (m) 160 (m) 165 (m)71015840 158 (m) 176 (m) 316 (m) 337 (m) 130 (m) 173 (m)2-Me 106 (d 78) 249 (brs) 099 (dd 72 18)6-Me 229 (s) 250 (brs) 090 (dd 72 30)aCDCl3bCD3OD
(C-4 and C-15) 274 (C-8) 273 (C-14) 258 (C-11) 250 (C-3)227 (C-17) and 142 (C-18) Compound 3 was characterizedas 1-monolinolein by comparison with the literature [16] andwas isolated from the genus for the first time
Compound 4 colorless oil EI-MSmz 280 [M]+ 1HNMR(600MHz CD
3OD) 120575 537 (2H m H-9 and H-10) 531 (2H
m H-12 and H-13) 278 (2H brt J = 72Hz H-11) 227 (2Ht J = 72Hz H-2) 207 (4H m H-8 and H-14) 160 (2H mH-3) 135 (14H m H-4 to H-7 and H-15 to H-17) 091 (3Ht J = 72Hz H-18) 13CNMR (150MHz CD
3OD) 120575 1779 (C-
1) 1311 (C-9 and C-10) 1293 (C-13) 1292 (C-12) 352 (C-2)329 (C-16) 309 (C-7) 307 (C-6) 305 (C-5) 304 (C-4 andC-15) 284 (C-8 and C-14) 268 (C-11) 263 (C-3) 238 (C-17) and 146 (C-18) Compound 4 was identified as (9Z12Z)-octadeca-912-dienoic acid This compound was reported byLuo et al [17]The 1H and 13CNMR data was assigned by 2DNMR data Compound 4was also isolated from the genus forthe first time
Compound 5 yellow oil HR-ESI-MS mz 3831875 [M +Na]+ UV (MeOH) 120582max 2305 and 3885 nm IR ]max cm
minus1
(KBr) 3461 3018 2955 2931 2859 1780 1716 1673 1522 1065756 1H NMR (600MHz CDCl
3) 120575 647 (1H dt J = 150
66Hz H-2) 588 (1H dd J = 150 12Hz H-3) 528 (1H d J= 36Hz H-5) 501 (1H dd J = 126 12Hz H-12b) 469 (1Hd J = 120Hz H-12a) 416 (1H dt J = 90 36Hz H-16) 300(1H td J = 120 42Hz H-8) 273 (1H dd J = 132 30Hz H-15) 272 (1H dd J = 180 36Hz H-7b) 256 (1H dd J = 180
Journal of Chemistry 5
Table 2 13C NMR (150 MHz) data for 1 2 and monacolin L
No 1a 2a Monacolin Lb
1 422 1342 4372 328 1322 3023 1345 1294 13424 1269 1259 12974a 1334 1330 13845 1270 1278 13106 1362 1342 3287 1273 1285 3598 1276 1234 2388a 1357 1304 36511015840 1711 1709 174021015840 387 388 43231015840 627 628 68241015840 361 362 30751015840 767 759 71261015840 338 358 44971015840 226 242 2602-Me 153 201 1446-Me 211 215 218aCDCl3bCD3OD
120Hz H-7a) 185 (3H dd J = 66 12Hz H-1) 154 (2Hm H-17) 154 (1H m H-18b) 140 (3H s H-13) 131 (2H mH-19) 131 (2H m H-20) 131 (1H m H-18a) 089 (3H t J =66HzH-21) 13CNMR (150MHz CDCl
3) 120575 186 (C-1) 1353
(C-2) 1246 (C-3) 1604 (C-4) 1035 (C-5) 1142 (C-6) 310(C-7) 415 (C-8) 832 (C-9) 1909 (C-10) 1512 (C-11) 639(C-12) 177 (C-13) 1753 (C-14) 491 (C-15) 696 (C-16) 352(C-17) 260 (C-18) 317 (C-19) 227 (C-20) and 141 (C-21)Those datawere identical to the literature data formonascinol[18] Thus compound 5 was determined as monascinol
Compound 6 Colorless solid HR-ESI-MS mz 1521056[M + 1]+ UV (MeOH) 120582max 27550 nm 1HNMR (600MHzCDCl
3) 120575 953 (1H brs NH) 586 (1H s H-3) 512 (1H d
119869 = 96Hz H-11015840) 274 (1H m H-21015840) 206 (3H s 4-Me) 109(6H d J = 66Hz 21015840-Metimes2) 13C NMR (150MHz CDCl
3)
120575 1730 (C-2) 1208 (C-3) 1488 (C-4) 1378 (C-5) 1209 (C-11015840) 276 (C-21015840) 230 (21015840-Me times 2) and 120 (4-Me) Compound6 was elucidated as pulchellalactam by comparing with thereference data [19]
Compound 7Off-white amorphous powder HR-ESI-MSmz 2730753 [M + Na]+ UV (MeOH) 120582max 2405sh 24702805 and 3300 nm IR Vmax cm
minus1 (KBr) 3342 3124 29691676 1624 1584 1515 1455 1152 1117 833 1HNMR (600MHzCDCl
3) 120575 1130 (1H s 8-OH) 1077 (1H s 6-OH) 646 (1H s
H-5) 642 (1H s H-4) 397 (1HmH-21015840) 250 (2HmH-11015840)200 (3H s 7-Me) 112 (3H d J =60HzH-31015840) 13CNMR(150MHz CDCl
3) 120575 1662 (C-1) 1544 (C-3) 1015 (C-4) 1363 (C-
4a) 1052 (C-5) 1599 (C-6) 1096 (C-7) 1635 (C-8) 978 (C-8a) 425 (C-11015840) 639 (C-21015840) 233 (C-31015840) and 79 (7-Me) Thestructure of compound 7 was elucidated by extensive 1D and
2D NMR spectroscopy and confirmed by comparison of its1H and 13C NMR data with those of Lunatinin [20 21]
32 HMG-CoA Reductase Inhibitory Activity The com-pounds 1ndash7were tested in vitro for theirHMG-CoA reductaseinhibitory activities by using the method as described inthe experimental part Lovastatin was used as the positivecontrol Compounds 1-2 (IC
50272 and 312 120583gmL) and 6-7
(IC50
280 and 128120583gmL) demonstrated strong HMG-CoAreductase inhibitory activity comparable with the standarddrug lovastatin (IC
50160 120583gmL) Compounds 3ndash5 caused
moderate activity at the highest concentration (400 120583gmL)It indicates that no statin compounds inMonascus-fermentedrice also have strongHMG-CoA reductase inhibitory activity
4 Conclusions
In conclusion we isolated seven compounds fromMonascus-fermented rice and evaluated their HMG-CoA reductaseinhibitory activities in vitro All compounds showed goodHMG-CoA reductase inhibitory activity among them com-pound 7 exhibited strong inhibition with IC
50value compa-
rable with that of the standard drug lovastatinThus no statincompounds in Monascus-fermented rice also have strongHMG-CoA reductase inhibitory activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Compre-hensive platform of Natural drugs and New formulations(2013ZX09402201) and program for Zhongguancun HaidianScience Park enterprise postdoctoral fellowship
References
[1] J Ma Y Li Q Ye et al ldquoConstituents of red yeast rice atraditional Chinese food and medicinerdquo Journal of Agriculturaland Food Chemistry vol 48 no 11 pp 5220ndash5225 2000
[2] A Endo ldquoMonacolin K a new hypocholesterolemic agentproduced by aMonascus speciesrdquoThe Journal of Antibiotics vol32 no 8 pp 852ndash854 1979
[3] A Endo K Hasumi and S Negishi ldquoMonacolins J and L newinhibitors of cholesterol biosynthesis produced by Monascusruberrdquo The Journal of Antibiotics vol 38 no 3 pp 420ndash4221985
[4] A Endo K Hasumi and T Nakamura ldquoDihydromonacolinL and monacolin X new metabolites those inhibit cholesterolbiosynthesisrdquo The Journal of Antibiotics vol 38 no 3 pp 321ndash327 1985
[5] A Endo D Komagata and H Shimada ldquoMonacolin M a newinhibitor of cholesterol biosynthesisrdquoThe Journal of Antibioticsvol 39 no 12 pp 1670ndash1673 1986
[6] M A Dhale S Divakar S U Kumar and G VijayalakshmildquoIsolation and characterization of dihydromonacolin-MV from
6 Journal of Chemistry
Monascus purpureus for antioxidant propertiesrdquoAppliedMicro-biology and Biotechnology vol 73 no 5 pp 1197ndash1202 2007
[7] M A Dhale S Divakar S Umesh-Kumar and G Vijay-alakshmi ldquoCharacterization of dehydromonacolin-MV2 fromMonascus purpureus mutantrdquo Journal of Applied Microbiologyvol 103 no 6 pp 2168ndash2173 2007
[8] X-M Li X-H Shen Z-W Duan and S-R Guo ldquoAdvancesin studies on secondary metabolites from Monascusrdquo ChineseTraditional and Herbal Drugs vol 42 no 5 pp 1018ndash1025 2011
[9] B W Philipp and D J Shapiro ldquoImproved methods for theassay and activation of 3-hydroxy-3-methylglutaryl coenzymeA reductaserdquo Journal of Lipid Research vol 20 no 5 pp 588ndash593 1979
[10] P A Edwards D Lemongello and A M Fogelman ldquoImprovedmethods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductaserdquo Journal of LipidResearch vol 20 no 1 pp 40ndash46 1979
[11] P A Edwards D Lemongello J Kane I Shechter and AM Fogelman ldquoProperties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzymeA reductase and regulation of enzymeactivityrdquoThe Journal of Biological Chemistry vol 255 no 8 pp3715ndash3725 1980
[12] P Li and L Chen ldquoXuezhikang inhibits the activity of HMG-CoA reductase in pig liverrdquo Basic Medical Sciences and Clinicsvol 23 no 5 pp 531ndash534 2003
[13] M D Greenspan J B Yudkovitz A W Alberts L S Argen-bright B H Arison and J L Smith ldquoMetabolism of lovastatinby rat and human liver microsomes in vitrordquo Drug Metabolismand Disposition vol 16 no 5 pp 678ndash682 1988
[14] G E Stokker ldquoSynthesis of the 31015840(S)-hydroxy derivative ofsimvastatinrdquo Bioorganic amp Medicinal Chemistry Letters vol 4no 14 pp 1767ndash1770 1994
[15] HOka A Terahara andA Endo ldquo4-hydroxy-2-pyrone deriva-tives and their pharmaceutical preparationsrdquo Patent No EP0010951 A1 1980
[16] C Hong X Piao and C Lou ldquoIsolation and identificationof chemical constituents from Alisma orientalerdquo Journal ofChongqing Institute of Technology vol 22 no 4 pp 78ndash81 2008
[17] C Luo W Zhang C Sheng C Zheng J Yao and Z MiaoldquoChemical composition and antidiabetic activity of OpuntiaMilpa Alta extractsrdquo Chemistry amp Biodiversity vol 7 no 12 pp2869ndash2879 2010
[18] T Akihisa Y Tadashi S Takashi et al ldquoMonascinol and tumorpromoter activity inhibitors and food containing itrdquo Patent NoJP 2008056618 A 2008
[19] J S Bryans N E A Chessum N Huther A F Parsons andF Ghelfi ldquoMetal-catalysed radical cyclisations leading to N-heterocycles new approaches to gabapentin and pulchellalac-tamrdquo Tetrahedron vol 59 no 33 pp 6221ndash6231 2003
[20] L-L Li J-P Chen and L-Y Kong ldquoChemical constituents ofMonascus ankardquo Chinese Pharmaceutical Journal vol 41 no 15pp 1131ndash1133 2006
[21] Y Yamaguchi R Masuma Y-P Kim R Uchida H Tomodaand S Omura ldquoTaxonomy and secondary metabolites ofPseudobotrytis sp FKA-25rdquoMycoscience vol 45 no 1 pp 9ndash162004
[22] X-M Li X-H Shen Z-WDuan and S-R Guo ldquoA newmona-colin analogue from Xuezhikang capsulerdquo Acta PharmaceuticaSinica vol 46 no 5 pp 564ndash567 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
4 Journal of Chemistry
O
OHO
H
Figure 2 Key HMBC (rarr ) and NOESY (harr) correlations incompound 1
with H-1 (120575H 258) and H-31015840 with H-2 (120575H 272) SimilarlyNOESY cross peaks between 2-Me (120575H 106) with H-71015840 (120575H158 and 176) H-71015840 (120575H 158 and 176) with H-51015840 (120575H 456)Thus the structure of 1 was identified as (4R 6R)-6-(2-((1S 2S)-26-dimethyl-12-dihydro-naphthalene-1-yl)-ethyl)-4-hydroxy-tetrahydropyran-2-one This compound was re-ported by Greenspan et al and Stokker [13 14] The 1H and13C NMR data was assigned unambiguously by HSQC andHMBC experiments (see Tables 1 and 2) Compound 1 wasobtained from natural source for the first time
Compound 2 was obtained as a colorless gum The HR-ESI-MS gave a pseudo molecular ion peak at mz 3211484[M+Na]+ consistent with amolecular formula of C
19H22O3
The UV spectrum showed maximum absorption at 2318 and2816 nm Its IR spectrum revealed absorption bands at 3434(hydroxyl) and 1712 cmminus1 (carbonyl)TheUV andNMR dataof 2 were similar to those of 1 but showed one more doublebond signals at 120575 1342 (C-1) and 1322 (C-2) and absenttwo hydrogens (H-1 and H-2) and the downfield chemicalshift of 2-Me at 120575 249 (3H s) indicating that 2 was 12-twodehydrogenated 1 Additionally the optical rotation at [120572]25D+ 112 (c 006 CH
2Cl2) is similar to that of monacolin L
suggesting that the chiral carbonsC-31015840 andC-51015840 of compound2 also had R R-configurations Thus the structure of 2 wasdetermined as (4R 6R)-6-(2-(26-dimethylnaphthalene-1-yl)ehtyl-4-hydroxy-tetrahydropyran-2-one This compoundwas reported byOka et al [15]The 1Hand 13CNMRdata wasassigned unambiguously by HSQC and HMBC experiments(see Tables 1 and 2) Compound 2 was also obtained fromnatural source for the first time
Compound 3 colorless oil HR-ESI-MS mz 3772698[M+Na]+ 1H NMR (600MHz CDCl
3) 120575 534 (4H m H-
9 10 12 and 13) 417 (1H dd J = 114 36Hz H-11015840b) 413 (1Hdd J = 114 60Hz H-11015840a) 391 (1H m H-21015840) 369 (1H ddJ = 114 36Hz H-31015840b) 359 (1H dd J = 114 60Hz H-31015840a)276 (2H m H-11) 233 (2H t J = 78Hz H-2) 204 (4H mH-8 and H-14) 162 (2H m H-3) 131 (14H m H-4 to H-7and H-15 to H-17) 088 (3H t J = 72Hz H-18) 13C NMR(150MHz CDCl
3) 120575 1745 (C-1) 1304 (C-10) 1302 (C-9)
1282 (C-13) 1280 (C-12) 704 (C-21015840) 653 (C-11015840) 635 (C-31015840)343 (C-2) 317 (C-16) 297 (C-7) 295 (C-6) 293 (C-5) 292
Table 1 1H NMR (600MHz) data for 1 2 and monacolin L (120575 inppm 119869 in Hz)
No 1a 2a Monacolin Lb
1 258 (m) mdash 140 (m)2 272 (m) mdash 231 (m)3 574 (dd 96 30) 727 (d 84) 572 (m)4 635 (dd 96 24) 756 (d 84) 587 (m)4a mdash mdash mdash5 686 (brs) 758 (brs) 539 (m)6 mdash mdash 232 (m)7 693 (d 78) 734 (dd 90 12) 146 (m) 153 (m)8 696 (d 78) 794 (d 90) 120 (m) 181 (m)8a mdash mdash 205 (m)11015840 mdash mdash mdash
21015840 255 (m) 268 (ddd 174 36 18) 244 (dd 150 84)266 (dd 180 54) 277 (dd 174 48) 255 (dd 150 48)
31015840 428 (m) 440 (m) 420 (m)41015840 163 (m) 186 (m) 182 (m) 200 (m) 162 (m) 178 (m)51015840 456 (m) 486 (m) 376 (m)61015840 142 (m) 166 (m) 192 (m) 198 (m) 160 (m) 165 (m)71015840 158 (m) 176 (m) 316 (m) 337 (m) 130 (m) 173 (m)2-Me 106 (d 78) 249 (brs) 099 (dd 72 18)6-Me 229 (s) 250 (brs) 090 (dd 72 30)aCDCl3bCD3OD
(C-4 and C-15) 274 (C-8) 273 (C-14) 258 (C-11) 250 (C-3)227 (C-17) and 142 (C-18) Compound 3 was characterizedas 1-monolinolein by comparison with the literature [16] andwas isolated from the genus for the first time
Compound 4 colorless oil EI-MSmz 280 [M]+ 1HNMR(600MHz CD
3OD) 120575 537 (2H m H-9 and H-10) 531 (2H
m H-12 and H-13) 278 (2H brt J = 72Hz H-11) 227 (2Ht J = 72Hz H-2) 207 (4H m H-8 and H-14) 160 (2H mH-3) 135 (14H m H-4 to H-7 and H-15 to H-17) 091 (3Ht J = 72Hz H-18) 13CNMR (150MHz CD
3OD) 120575 1779 (C-
1) 1311 (C-9 and C-10) 1293 (C-13) 1292 (C-12) 352 (C-2)329 (C-16) 309 (C-7) 307 (C-6) 305 (C-5) 304 (C-4 andC-15) 284 (C-8 and C-14) 268 (C-11) 263 (C-3) 238 (C-17) and 146 (C-18) Compound 4 was identified as (9Z12Z)-octadeca-912-dienoic acid This compound was reported byLuo et al [17]The 1H and 13CNMR data was assigned by 2DNMR data Compound 4was also isolated from the genus forthe first time
Compound 5 yellow oil HR-ESI-MS mz 3831875 [M +Na]+ UV (MeOH) 120582max 2305 and 3885 nm IR ]max cm
minus1
(KBr) 3461 3018 2955 2931 2859 1780 1716 1673 1522 1065756 1H NMR (600MHz CDCl
3) 120575 647 (1H dt J = 150
66Hz H-2) 588 (1H dd J = 150 12Hz H-3) 528 (1H d J= 36Hz H-5) 501 (1H dd J = 126 12Hz H-12b) 469 (1Hd J = 120Hz H-12a) 416 (1H dt J = 90 36Hz H-16) 300(1H td J = 120 42Hz H-8) 273 (1H dd J = 132 30Hz H-15) 272 (1H dd J = 180 36Hz H-7b) 256 (1H dd J = 180
Journal of Chemistry 5
Table 2 13C NMR (150 MHz) data for 1 2 and monacolin L
No 1a 2a Monacolin Lb
1 422 1342 4372 328 1322 3023 1345 1294 13424 1269 1259 12974a 1334 1330 13845 1270 1278 13106 1362 1342 3287 1273 1285 3598 1276 1234 2388a 1357 1304 36511015840 1711 1709 174021015840 387 388 43231015840 627 628 68241015840 361 362 30751015840 767 759 71261015840 338 358 44971015840 226 242 2602-Me 153 201 1446-Me 211 215 218aCDCl3bCD3OD
120Hz H-7a) 185 (3H dd J = 66 12Hz H-1) 154 (2Hm H-17) 154 (1H m H-18b) 140 (3H s H-13) 131 (2H mH-19) 131 (2H m H-20) 131 (1H m H-18a) 089 (3H t J =66HzH-21) 13CNMR (150MHz CDCl
3) 120575 186 (C-1) 1353
(C-2) 1246 (C-3) 1604 (C-4) 1035 (C-5) 1142 (C-6) 310(C-7) 415 (C-8) 832 (C-9) 1909 (C-10) 1512 (C-11) 639(C-12) 177 (C-13) 1753 (C-14) 491 (C-15) 696 (C-16) 352(C-17) 260 (C-18) 317 (C-19) 227 (C-20) and 141 (C-21)Those datawere identical to the literature data formonascinol[18] Thus compound 5 was determined as monascinol
Compound 6 Colorless solid HR-ESI-MS mz 1521056[M + 1]+ UV (MeOH) 120582max 27550 nm 1HNMR (600MHzCDCl
3) 120575 953 (1H brs NH) 586 (1H s H-3) 512 (1H d
119869 = 96Hz H-11015840) 274 (1H m H-21015840) 206 (3H s 4-Me) 109(6H d J = 66Hz 21015840-Metimes2) 13C NMR (150MHz CDCl
3)
120575 1730 (C-2) 1208 (C-3) 1488 (C-4) 1378 (C-5) 1209 (C-11015840) 276 (C-21015840) 230 (21015840-Me times 2) and 120 (4-Me) Compound6 was elucidated as pulchellalactam by comparing with thereference data [19]
Compound 7Off-white amorphous powder HR-ESI-MSmz 2730753 [M + Na]+ UV (MeOH) 120582max 2405sh 24702805 and 3300 nm IR Vmax cm
minus1 (KBr) 3342 3124 29691676 1624 1584 1515 1455 1152 1117 833 1HNMR (600MHzCDCl
3) 120575 1130 (1H s 8-OH) 1077 (1H s 6-OH) 646 (1H s
H-5) 642 (1H s H-4) 397 (1HmH-21015840) 250 (2HmH-11015840)200 (3H s 7-Me) 112 (3H d J =60HzH-31015840) 13CNMR(150MHz CDCl
3) 120575 1662 (C-1) 1544 (C-3) 1015 (C-4) 1363 (C-
4a) 1052 (C-5) 1599 (C-6) 1096 (C-7) 1635 (C-8) 978 (C-8a) 425 (C-11015840) 639 (C-21015840) 233 (C-31015840) and 79 (7-Me) Thestructure of compound 7 was elucidated by extensive 1D and
2D NMR spectroscopy and confirmed by comparison of its1H and 13C NMR data with those of Lunatinin [20 21]
32 HMG-CoA Reductase Inhibitory Activity The com-pounds 1ndash7were tested in vitro for theirHMG-CoA reductaseinhibitory activities by using the method as described inthe experimental part Lovastatin was used as the positivecontrol Compounds 1-2 (IC
50272 and 312 120583gmL) and 6-7
(IC50
280 and 128120583gmL) demonstrated strong HMG-CoAreductase inhibitory activity comparable with the standarddrug lovastatin (IC
50160 120583gmL) Compounds 3ndash5 caused
moderate activity at the highest concentration (400 120583gmL)It indicates that no statin compounds inMonascus-fermentedrice also have strongHMG-CoA reductase inhibitory activity
4 Conclusions
In conclusion we isolated seven compounds fromMonascus-fermented rice and evaluated their HMG-CoA reductaseinhibitory activities in vitro All compounds showed goodHMG-CoA reductase inhibitory activity among them com-pound 7 exhibited strong inhibition with IC
50value compa-
rable with that of the standard drug lovastatinThus no statincompounds in Monascus-fermented rice also have strongHMG-CoA reductase inhibitory activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Compre-hensive platform of Natural drugs and New formulations(2013ZX09402201) and program for Zhongguancun HaidianScience Park enterprise postdoctoral fellowship
References
[1] J Ma Y Li Q Ye et al ldquoConstituents of red yeast rice atraditional Chinese food and medicinerdquo Journal of Agriculturaland Food Chemistry vol 48 no 11 pp 5220ndash5225 2000
[2] A Endo ldquoMonacolin K a new hypocholesterolemic agentproduced by aMonascus speciesrdquoThe Journal of Antibiotics vol32 no 8 pp 852ndash854 1979
[3] A Endo K Hasumi and S Negishi ldquoMonacolins J and L newinhibitors of cholesterol biosynthesis produced by Monascusruberrdquo The Journal of Antibiotics vol 38 no 3 pp 420ndash4221985
[4] A Endo K Hasumi and T Nakamura ldquoDihydromonacolinL and monacolin X new metabolites those inhibit cholesterolbiosynthesisrdquo The Journal of Antibiotics vol 38 no 3 pp 321ndash327 1985
[5] A Endo D Komagata and H Shimada ldquoMonacolin M a newinhibitor of cholesterol biosynthesisrdquoThe Journal of Antibioticsvol 39 no 12 pp 1670ndash1673 1986
[6] M A Dhale S Divakar S U Kumar and G VijayalakshmildquoIsolation and characterization of dihydromonacolin-MV from
6 Journal of Chemistry
Monascus purpureus for antioxidant propertiesrdquoAppliedMicro-biology and Biotechnology vol 73 no 5 pp 1197ndash1202 2007
[7] M A Dhale S Divakar S Umesh-Kumar and G Vijay-alakshmi ldquoCharacterization of dehydromonacolin-MV2 fromMonascus purpureus mutantrdquo Journal of Applied Microbiologyvol 103 no 6 pp 2168ndash2173 2007
[8] X-M Li X-H Shen Z-W Duan and S-R Guo ldquoAdvancesin studies on secondary metabolites from Monascusrdquo ChineseTraditional and Herbal Drugs vol 42 no 5 pp 1018ndash1025 2011
[9] B W Philipp and D J Shapiro ldquoImproved methods for theassay and activation of 3-hydroxy-3-methylglutaryl coenzymeA reductaserdquo Journal of Lipid Research vol 20 no 5 pp 588ndash593 1979
[10] P A Edwards D Lemongello and A M Fogelman ldquoImprovedmethods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductaserdquo Journal of LipidResearch vol 20 no 1 pp 40ndash46 1979
[11] P A Edwards D Lemongello J Kane I Shechter and AM Fogelman ldquoProperties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzymeA reductase and regulation of enzymeactivityrdquoThe Journal of Biological Chemistry vol 255 no 8 pp3715ndash3725 1980
[12] P Li and L Chen ldquoXuezhikang inhibits the activity of HMG-CoA reductase in pig liverrdquo Basic Medical Sciences and Clinicsvol 23 no 5 pp 531ndash534 2003
[13] M D Greenspan J B Yudkovitz A W Alberts L S Argen-bright B H Arison and J L Smith ldquoMetabolism of lovastatinby rat and human liver microsomes in vitrordquo Drug Metabolismand Disposition vol 16 no 5 pp 678ndash682 1988
[14] G E Stokker ldquoSynthesis of the 31015840(S)-hydroxy derivative ofsimvastatinrdquo Bioorganic amp Medicinal Chemistry Letters vol 4no 14 pp 1767ndash1770 1994
[15] HOka A Terahara andA Endo ldquo4-hydroxy-2-pyrone deriva-tives and their pharmaceutical preparationsrdquo Patent No EP0010951 A1 1980
[16] C Hong X Piao and C Lou ldquoIsolation and identificationof chemical constituents from Alisma orientalerdquo Journal ofChongqing Institute of Technology vol 22 no 4 pp 78ndash81 2008
[17] C Luo W Zhang C Sheng C Zheng J Yao and Z MiaoldquoChemical composition and antidiabetic activity of OpuntiaMilpa Alta extractsrdquo Chemistry amp Biodiversity vol 7 no 12 pp2869ndash2879 2010
[18] T Akihisa Y Tadashi S Takashi et al ldquoMonascinol and tumorpromoter activity inhibitors and food containing itrdquo Patent NoJP 2008056618 A 2008
[19] J S Bryans N E A Chessum N Huther A F Parsons andF Ghelfi ldquoMetal-catalysed radical cyclisations leading to N-heterocycles new approaches to gabapentin and pulchellalac-tamrdquo Tetrahedron vol 59 no 33 pp 6221ndash6231 2003
[20] L-L Li J-P Chen and L-Y Kong ldquoChemical constituents ofMonascus ankardquo Chinese Pharmaceutical Journal vol 41 no 15pp 1131ndash1133 2006
[21] Y Yamaguchi R Masuma Y-P Kim R Uchida H Tomodaand S Omura ldquoTaxonomy and secondary metabolites ofPseudobotrytis sp FKA-25rdquoMycoscience vol 45 no 1 pp 9ndash162004
[22] X-M Li X-H Shen Z-WDuan and S-R Guo ldquoA newmona-colin analogue from Xuezhikang capsulerdquo Acta PharmaceuticaSinica vol 46 no 5 pp 564ndash567 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 5
Table 2 13C NMR (150 MHz) data for 1 2 and monacolin L
No 1a 2a Monacolin Lb
1 422 1342 4372 328 1322 3023 1345 1294 13424 1269 1259 12974a 1334 1330 13845 1270 1278 13106 1362 1342 3287 1273 1285 3598 1276 1234 2388a 1357 1304 36511015840 1711 1709 174021015840 387 388 43231015840 627 628 68241015840 361 362 30751015840 767 759 71261015840 338 358 44971015840 226 242 2602-Me 153 201 1446-Me 211 215 218aCDCl3bCD3OD
120Hz H-7a) 185 (3H dd J = 66 12Hz H-1) 154 (2Hm H-17) 154 (1H m H-18b) 140 (3H s H-13) 131 (2H mH-19) 131 (2H m H-20) 131 (1H m H-18a) 089 (3H t J =66HzH-21) 13CNMR (150MHz CDCl
3) 120575 186 (C-1) 1353
(C-2) 1246 (C-3) 1604 (C-4) 1035 (C-5) 1142 (C-6) 310(C-7) 415 (C-8) 832 (C-9) 1909 (C-10) 1512 (C-11) 639(C-12) 177 (C-13) 1753 (C-14) 491 (C-15) 696 (C-16) 352(C-17) 260 (C-18) 317 (C-19) 227 (C-20) and 141 (C-21)Those datawere identical to the literature data formonascinol[18] Thus compound 5 was determined as monascinol
Compound 6 Colorless solid HR-ESI-MS mz 1521056[M + 1]+ UV (MeOH) 120582max 27550 nm 1HNMR (600MHzCDCl
3) 120575 953 (1H brs NH) 586 (1H s H-3) 512 (1H d
119869 = 96Hz H-11015840) 274 (1H m H-21015840) 206 (3H s 4-Me) 109(6H d J = 66Hz 21015840-Metimes2) 13C NMR (150MHz CDCl
3)
120575 1730 (C-2) 1208 (C-3) 1488 (C-4) 1378 (C-5) 1209 (C-11015840) 276 (C-21015840) 230 (21015840-Me times 2) and 120 (4-Me) Compound6 was elucidated as pulchellalactam by comparing with thereference data [19]
Compound 7Off-white amorphous powder HR-ESI-MSmz 2730753 [M + Na]+ UV (MeOH) 120582max 2405sh 24702805 and 3300 nm IR Vmax cm
minus1 (KBr) 3342 3124 29691676 1624 1584 1515 1455 1152 1117 833 1HNMR (600MHzCDCl
3) 120575 1130 (1H s 8-OH) 1077 (1H s 6-OH) 646 (1H s
H-5) 642 (1H s H-4) 397 (1HmH-21015840) 250 (2HmH-11015840)200 (3H s 7-Me) 112 (3H d J =60HzH-31015840) 13CNMR(150MHz CDCl
3) 120575 1662 (C-1) 1544 (C-3) 1015 (C-4) 1363 (C-
4a) 1052 (C-5) 1599 (C-6) 1096 (C-7) 1635 (C-8) 978 (C-8a) 425 (C-11015840) 639 (C-21015840) 233 (C-31015840) and 79 (7-Me) Thestructure of compound 7 was elucidated by extensive 1D and
2D NMR spectroscopy and confirmed by comparison of its1H and 13C NMR data with those of Lunatinin [20 21]
32 HMG-CoA Reductase Inhibitory Activity The com-pounds 1ndash7were tested in vitro for theirHMG-CoA reductaseinhibitory activities by using the method as described inthe experimental part Lovastatin was used as the positivecontrol Compounds 1-2 (IC
50272 and 312 120583gmL) and 6-7
(IC50
280 and 128120583gmL) demonstrated strong HMG-CoAreductase inhibitory activity comparable with the standarddrug lovastatin (IC
50160 120583gmL) Compounds 3ndash5 caused
moderate activity at the highest concentration (400 120583gmL)It indicates that no statin compounds inMonascus-fermentedrice also have strongHMG-CoA reductase inhibitory activity
4 Conclusions
In conclusion we isolated seven compounds fromMonascus-fermented rice and evaluated their HMG-CoA reductaseinhibitory activities in vitro All compounds showed goodHMG-CoA reductase inhibitory activity among them com-pound 7 exhibited strong inhibition with IC
50value compa-
rable with that of the standard drug lovastatinThus no statincompounds in Monascus-fermented rice also have strongHMG-CoA reductase inhibitory activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Compre-hensive platform of Natural drugs and New formulations(2013ZX09402201) and program for Zhongguancun HaidianScience Park enterprise postdoctoral fellowship
References
[1] J Ma Y Li Q Ye et al ldquoConstituents of red yeast rice atraditional Chinese food and medicinerdquo Journal of Agriculturaland Food Chemistry vol 48 no 11 pp 5220ndash5225 2000
[2] A Endo ldquoMonacolin K a new hypocholesterolemic agentproduced by aMonascus speciesrdquoThe Journal of Antibiotics vol32 no 8 pp 852ndash854 1979
[3] A Endo K Hasumi and S Negishi ldquoMonacolins J and L newinhibitors of cholesterol biosynthesis produced by Monascusruberrdquo The Journal of Antibiotics vol 38 no 3 pp 420ndash4221985
[4] A Endo K Hasumi and T Nakamura ldquoDihydromonacolinL and monacolin X new metabolites those inhibit cholesterolbiosynthesisrdquo The Journal of Antibiotics vol 38 no 3 pp 321ndash327 1985
[5] A Endo D Komagata and H Shimada ldquoMonacolin M a newinhibitor of cholesterol biosynthesisrdquoThe Journal of Antibioticsvol 39 no 12 pp 1670ndash1673 1986
[6] M A Dhale S Divakar S U Kumar and G VijayalakshmildquoIsolation and characterization of dihydromonacolin-MV from
6 Journal of Chemistry
Monascus purpureus for antioxidant propertiesrdquoAppliedMicro-biology and Biotechnology vol 73 no 5 pp 1197ndash1202 2007
[7] M A Dhale S Divakar S Umesh-Kumar and G Vijay-alakshmi ldquoCharacterization of dehydromonacolin-MV2 fromMonascus purpureus mutantrdquo Journal of Applied Microbiologyvol 103 no 6 pp 2168ndash2173 2007
[8] X-M Li X-H Shen Z-W Duan and S-R Guo ldquoAdvancesin studies on secondary metabolites from Monascusrdquo ChineseTraditional and Herbal Drugs vol 42 no 5 pp 1018ndash1025 2011
[9] B W Philipp and D J Shapiro ldquoImproved methods for theassay and activation of 3-hydroxy-3-methylglutaryl coenzymeA reductaserdquo Journal of Lipid Research vol 20 no 5 pp 588ndash593 1979
[10] P A Edwards D Lemongello and A M Fogelman ldquoImprovedmethods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductaserdquo Journal of LipidResearch vol 20 no 1 pp 40ndash46 1979
[11] P A Edwards D Lemongello J Kane I Shechter and AM Fogelman ldquoProperties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzymeA reductase and regulation of enzymeactivityrdquoThe Journal of Biological Chemistry vol 255 no 8 pp3715ndash3725 1980
[12] P Li and L Chen ldquoXuezhikang inhibits the activity of HMG-CoA reductase in pig liverrdquo Basic Medical Sciences and Clinicsvol 23 no 5 pp 531ndash534 2003
[13] M D Greenspan J B Yudkovitz A W Alberts L S Argen-bright B H Arison and J L Smith ldquoMetabolism of lovastatinby rat and human liver microsomes in vitrordquo Drug Metabolismand Disposition vol 16 no 5 pp 678ndash682 1988
[14] G E Stokker ldquoSynthesis of the 31015840(S)-hydroxy derivative ofsimvastatinrdquo Bioorganic amp Medicinal Chemistry Letters vol 4no 14 pp 1767ndash1770 1994
[15] HOka A Terahara andA Endo ldquo4-hydroxy-2-pyrone deriva-tives and their pharmaceutical preparationsrdquo Patent No EP0010951 A1 1980
[16] C Hong X Piao and C Lou ldquoIsolation and identificationof chemical constituents from Alisma orientalerdquo Journal ofChongqing Institute of Technology vol 22 no 4 pp 78ndash81 2008
[17] C Luo W Zhang C Sheng C Zheng J Yao and Z MiaoldquoChemical composition and antidiabetic activity of OpuntiaMilpa Alta extractsrdquo Chemistry amp Biodiversity vol 7 no 12 pp2869ndash2879 2010
[18] T Akihisa Y Tadashi S Takashi et al ldquoMonascinol and tumorpromoter activity inhibitors and food containing itrdquo Patent NoJP 2008056618 A 2008
[19] J S Bryans N E A Chessum N Huther A F Parsons andF Ghelfi ldquoMetal-catalysed radical cyclisations leading to N-heterocycles new approaches to gabapentin and pulchellalac-tamrdquo Tetrahedron vol 59 no 33 pp 6221ndash6231 2003
[20] L-L Li J-P Chen and L-Y Kong ldquoChemical constituents ofMonascus ankardquo Chinese Pharmaceutical Journal vol 41 no 15pp 1131ndash1133 2006
[21] Y Yamaguchi R Masuma Y-P Kim R Uchida H Tomodaand S Omura ldquoTaxonomy and secondary metabolites ofPseudobotrytis sp FKA-25rdquoMycoscience vol 45 no 1 pp 9ndash162004
[22] X-M Li X-H Shen Z-WDuan and S-R Guo ldquoA newmona-colin analogue from Xuezhikang capsulerdquo Acta PharmaceuticaSinica vol 46 no 5 pp 564ndash567 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
6 Journal of Chemistry
Monascus purpureus for antioxidant propertiesrdquoAppliedMicro-biology and Biotechnology vol 73 no 5 pp 1197ndash1202 2007
[7] M A Dhale S Divakar S Umesh-Kumar and G Vijay-alakshmi ldquoCharacterization of dehydromonacolin-MV2 fromMonascus purpureus mutantrdquo Journal of Applied Microbiologyvol 103 no 6 pp 2168ndash2173 2007
[8] X-M Li X-H Shen Z-W Duan and S-R Guo ldquoAdvancesin studies on secondary metabolites from Monascusrdquo ChineseTraditional and Herbal Drugs vol 42 no 5 pp 1018ndash1025 2011
[9] B W Philipp and D J Shapiro ldquoImproved methods for theassay and activation of 3-hydroxy-3-methylglutaryl coenzymeA reductaserdquo Journal of Lipid Research vol 20 no 5 pp 588ndash593 1979
[10] P A Edwards D Lemongello and A M Fogelman ldquoImprovedmethods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductaserdquo Journal of LipidResearch vol 20 no 1 pp 40ndash46 1979
[11] P A Edwards D Lemongello J Kane I Shechter and AM Fogelman ldquoProperties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzymeA reductase and regulation of enzymeactivityrdquoThe Journal of Biological Chemistry vol 255 no 8 pp3715ndash3725 1980
[12] P Li and L Chen ldquoXuezhikang inhibits the activity of HMG-CoA reductase in pig liverrdquo Basic Medical Sciences and Clinicsvol 23 no 5 pp 531ndash534 2003
[13] M D Greenspan J B Yudkovitz A W Alberts L S Argen-bright B H Arison and J L Smith ldquoMetabolism of lovastatinby rat and human liver microsomes in vitrordquo Drug Metabolismand Disposition vol 16 no 5 pp 678ndash682 1988
[14] G E Stokker ldquoSynthesis of the 31015840(S)-hydroxy derivative ofsimvastatinrdquo Bioorganic amp Medicinal Chemistry Letters vol 4no 14 pp 1767ndash1770 1994
[15] HOka A Terahara andA Endo ldquo4-hydroxy-2-pyrone deriva-tives and their pharmaceutical preparationsrdquo Patent No EP0010951 A1 1980
[16] C Hong X Piao and C Lou ldquoIsolation and identificationof chemical constituents from Alisma orientalerdquo Journal ofChongqing Institute of Technology vol 22 no 4 pp 78ndash81 2008
[17] C Luo W Zhang C Sheng C Zheng J Yao and Z MiaoldquoChemical composition and antidiabetic activity of OpuntiaMilpa Alta extractsrdquo Chemistry amp Biodiversity vol 7 no 12 pp2869ndash2879 2010
[18] T Akihisa Y Tadashi S Takashi et al ldquoMonascinol and tumorpromoter activity inhibitors and food containing itrdquo Patent NoJP 2008056618 A 2008
[19] J S Bryans N E A Chessum N Huther A F Parsons andF Ghelfi ldquoMetal-catalysed radical cyclisations leading to N-heterocycles new approaches to gabapentin and pulchellalac-tamrdquo Tetrahedron vol 59 no 33 pp 6221ndash6231 2003
[20] L-L Li J-P Chen and L-Y Kong ldquoChemical constituents ofMonascus ankardquo Chinese Pharmaceutical Journal vol 41 no 15pp 1131ndash1133 2006
[21] Y Yamaguchi R Masuma Y-P Kim R Uchida H Tomodaand S Omura ldquoTaxonomy and secondary metabolites ofPseudobotrytis sp FKA-25rdquoMycoscience vol 45 no 1 pp 9ndash162004
[22] X-M Li X-H Shen Z-WDuan and S-R Guo ldquoA newmona-colin analogue from Xuezhikang capsulerdquo Acta PharmaceuticaSinica vol 46 no 5 pp 564ndash567 2011
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
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