HMG-CoA Reductase Inhibitors from Monascus-Fermented Rice

Seven compounds were isolated from Monascus-fermented rice by column chromatography with silica gel and semiprep HPLC. Their 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.


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-3methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors [2][3][4][5].After that dihydromonacolin-MV and dehydromonacolin-MV2 isolated from Monascus sp. have been characterized for their antioxidant action [6,7].Nonmonacolin secondary metabolites have also been identified so far [8], but it is still unclear whether the cholesterollowering 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.

HMG-CoA Reductase Inhibitory Assay
2.4.1.Preparation of Microsomes.Male Sprague-Dawley rats (100-200 g) were killed by cervical dislocation at or near the peak of the daily circadian rhythm in reductase activity.In most experiments rats were fed a diet containing 5% cholestyramine for 4 days prior to the preparation of microsomes.Livers were excised, rinsed in ice-cold buffer A (KESD buffer) containing 10 mM potassium phosphate, 2 mM EDTA, 250 mM sucrose, and 1 mM DTT (pH 6.8).The livers were then minced through a tissue press and homogenized in three volumes of buffer A per g of liver by four strokes with the loose pestle of a Dounce homogenizer and one stroke with the tight pestle.Mitochondria and cell debris were sedimented by centrifugation two times at 12,000 g for 15 min at 4 ∘ C. Crude microsomes were prepared from the two 12,000 g supernatants by sedimentation at 105,000 g for 90 min at 4 ∘ C. The pellet was resuspended in buffer A (1 mL/g liver) and resedimented at 105,000 g for 90 min at 4 ∘ C [9].The washed microsomal pellets were quickly-frozen in an acetone-dry ice bath and stored at −80 ∘ C prior to use.

Solubilization of HMG-CoA Reductase.
For optimal solubilization of the reductase, the frozen microsomes were allowed to thaw at room temperature before addition of an equal volume of 50% glycerol in buffer B, containing 0.2 M sucrose, 0.1 M KCl, 0.08 M potassium phosphate, 2 mM potassium EDTA, and 10 mM DTT (pH 7.2), preheated to 37 ∘ C. The suspension was rehomogenized with 10 downward passes of a hand-driven, all-glass Potter-Elvehjem homogenizer and then incubated at 37 ∘ C for 60 min.The suspension was diluted threefold with buffer B preheated to 37 ∘ C to a final glycerol concentration of 8.3%, rehomogenized with 10 downward passes of the glass homogenizer pestle, and centrifuged at 105,000 g for 60 min at 25 ∘ C. The supernatant containing solubilized HMG-CoA reductase was removed and used immediately for enzyme purification [10].

Purification of HMG-CoA
Reductase.The solubilized enzyme was fractioned with saturated ammonium sulfate solution and the protein precipating between 35% and 50% ammonium sulfate was dissolved in buffer B [11].OD 340 was detected with VerSamax ELISA microplate reader, and the rate of the change within 5 min was used to evaluate the activity of HMG-CoA reductase, and then to evaluate the inhibition activity of each sample.

HMG-CoA Reductase Inhibitory
Activity.The compounds 1-7 were tested in vitro for their HMG-CoA reductase inhibitory activities by using the method as described in the experimental part.Lovastatin was used as the positive control.Compounds 1-2 (IC 50 272 and 312 g/mL) and 6-7 (IC 50 280 and 128 g/mL) demonstrated strong HMG-CoA reductase inhibitory activity comparable with the standard drug lovastatin (IC 50 160 g/mL).Compounds 3-5 caused moderate activity at the highest concentration (400 g/mL).It indicates that no statin compounds in Monascus-fermented rice also have strong HMG-CoA reductase inhibitory activity.

Conclusions
In conclusion, we isolated seven compounds from Monascusfermented rice and evaluated their HMG-CoA reductase inhibitory activities in vitro.All compounds showed good HMG-CoA reductase inhibitory activity, among them compound 7 exhibited strong inhibition with IC 50 value comparable with that of the standard drug lovastatin.Thus, no statin compounds in Monascus-fermented rice also have strong HMG-CoA reductase inhibitory activity.