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Alcohol metabolism causes an excessive accumulation of liver lipids and inflammation, resulting in liver damage. The yellow pigments monascin (MS) and ankaflavin (AK) of Monascus purpureus -fermented rice were proven to regulate ethanol-induced damage in HepG2 cells, but the complete anti-inflammatory and anti-fatty liver mechanisms in the animal model are still unclear. This study explored the roles of MS and AK in improving alcoholic liver injury. MS and AK were simultaneously fed to evaluate their effects and mechanisms in C57BL/6J mice fed the Lieber-DeCarli liquid alcohol diet for 6 weeks. The results indicated that MS and AK significantly reduced the serum aspartate aminotransferase and alanine aminotransferase activity, as well as the total liver cholesterol and triglyceride levels. The histopathological results indicated that MS and AK prevented lipid accumulation in the liver. MS and AK effectively enhanced the activity of antioxidant enzymes and reduced the degree of lipid peroxidation; AK was particularly effective and exhibited a superior preventive effect against alcoholic liver injury and fatty liver. In addition to inhibiting the phosphorylation of the MAPK family, MS and AK directly reduced TNF-α, IL-6, and IL-1β levels, thereby reducing NF-κB and its downstream iNOS and COX-2 expressions, as well as increasing PPAR-γ, Nrf-2, and HO-1 expressions to prevent liver damage. MS and AK also directly reduced TNF-α, IL-6, and IL-1β expression, thereby reducing the production of NF-κB and its downstream iNOS and COX-2, and increasing PPAR-γ, Nrf-2, and HO-1 expressions, preventing alcohol damage to the liver.

Background/purposes: Monascin (MS) and ankaflavin (AK) produced by Monascus purpureus NTU 568 were proven to show excellent hypolipidemic effects in our previous studies; however, the mechanism is still unclear., Methods: This study used MS, AK, and monacolin K as test substances and performed tests on rats fed high-fat and high-cholesterol diet for 8 weeks. The lipid levels and the related protein levels of the rats were assessed to understand the effects of MS, AK, and monacolin K on lipid metabolism., Results: MS and AK lowered low-density lipoprotein cholesterol (LDL-C) and preserved high-density lipoprotein cholesterol contents. MS and AK inhibited acetyl-coenzyme A acetyltransferase, microsomal triglyceride transfer protein, and apolipoprotein (apo) B-100 expression, thereby preventing LDL assembly. In addition, enhanced LDL-receptor expression increased the transport of LDL-C to the liver for metabolism. MS and AK also significantly increase apo A1 expression, which facilitates high-density lipoprotein cholesterol formation., Conclusion: Monascus-fermented MS and AK can perform blood lipid regulation via the suppression of LDL-C assembly and stimulation of apo A1 expression in liver., (Copyright © 2016. Published by Elsevier B.V.)

Yellow pigments monascin (MS) and ankaflavin (AK) are secondary metabolites derived from Monascus-fermented products. The hypolipidemic and anti-inflammatory effects of MS and AK indicate that they have potential on preventing or curing nonalcoholic fatty liver disease (NAFLD). Oleic acid (OA) and high-fat diet were used to induce steatosis in FL83B hepatocytes and NAFLD in mice, respectively. We found that both MS and AK prevented fatty acid accumulation in hepatocytes by inhibiting fatty acid uptake, lipogenesis, and promoting fatty acid beta-oxidation mediated by activating peroxisome proliferator-activated receptor (PPAR)-α and AMP-activated kinase (AMPK). Furthermore, MS and AK significantly attenuated high-fat diet-induced elevation of total cholesterol (TC), triaceylglycerol (TG), free fatty acid (FFA), and low density lipoprotein-cholesterol (LDL-C) in plasma. MS and AK promoted AMPK phosphorylation, suppressed the steatosis-related mRNA expression and inflammatory cytokines secretion, as well as upregulated farnesoid X receptor (FXR), peroxisome proliferator-activated receptor gamma co-activator (PGC)-1α, and PPARα expression to induce fatty acid oxidation in the liver of mice. We provided evidence that MS and AK act as PPARα agonists to upregulate AMPK activity and attenuate NAFLD. MS and AK may be supplied in food supplements or developed as functional foods to reduce the risk of diabetes and obesity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Monascus-fermented monascin and ankaflavin are found to strongly inhibit differentiation and lipogenesis and stimulate lipolysis effects in a 3T3-L1 preadipocyte model, but the in vivo regulation mechanism is unclear. This study uses obese rats caused by a high-fat diet to examine the effects of daily monascin and ankaflavin feeding (8 weeks) on antiobesity effects and modulation of differentiation, lipogenesis, and lipid absorption. The results show that monascin and ankaflavin had a significant antiobesity effect, which should result from the modulation of monascin and ankaflavin on the inhibition of differentiation by inhibiting CCAT/enhancer-binding protein β (C/EBPβ) expression (36.4% and 48.3%) and its downstream peroxisome proliferator-activated receptor γ (PPARγ) (55.6% and 64.5%) and CCAT/enhancer-binding protein α (C/EBPα) expressions (25.2% and 33.2%) and the inhibition of lipogenesis by increasing lipase activity (14.0% and 10.7%) and decreasing heparin releasable lipoprotein lipase (HR-LPL) activity (34.8% and 30.5%). Furthermore, monascin and ankaflavin are the first agents found to suppress Niemann-Pick C1 Like 1 (NPC1L1) protein expression (73.6% and 26.1%) associated with small intestine tissue lipid absorption. Importantly, monascin and ankaflavin are not like monacolin K, which increases creatine phosphokinase (CPK) activity, known as a rhabdomyolysis indicator.

Monacolin K has long been considered a major effective component in the hypolipidemic functions of Monascus . Monacolin K also serves as a well-known hypolipidemic medication, but its side effect myopathy is a concern. Monascin and ankaflavin, the yellow pigments produced by Monascus species, have been proven to possess hypolipidemic functions; however, no studies have compared the hypolipidemic effects of monascin, ankaflavin, and monacolin K under the same dosages. In this study, the equal dosages of monascin, ankaflavin, and monacolin K were oral administrated to hamsters fed a high cholesterol diet for 6 weeks. Comparison of the displayed hypolipidemic and anti-atherosclerosis effects was performed, in addition to an investigation into the inducement of side effect. The results indicated that monascin and ankaflavin were similar to monacolin K in significantly reducing total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels in serum and lipid plaque (p < 0.05) in the heart aorta. In addition, ankaflavin achieved the effects of serum TC and TG reduction, with no significant difference as compared to those effects of monacolin K (p > 0.05). However, as compared to monacolin K, ankaflavin possessed more significant effects on the prevention of fatty liver and lipid plaque accumulation in heart aorta. More importantly, monascin significantly enhanced high-density lipoprotein cholesterol (HDL-C) concentrations, while monacolin K displayed the opposite effect. Regarding the side effect, monacolin K also raised elevated creatinine phosphokinase (CPK) activity, which was highly correlated with rhabdomyolysis development, while monascin and ankaflavin did not induce such a side effect. In conclusion, MS and AK had the potential to be developed as hypolipidemic agents without rhabdomyolysis development.

Ankaflavin (AK) is an active compound having anti-inflammatory, anti-cancer, antiatherosclerotic, and hypolipidemic effects. We have previously reported that AK acts as an antioxidant and antidiabetic drug; however, the mechanism by which AK prevents diabetes remains unknown. Hyperglycemia is associated with protein glycation, which produces advanced glycation end-products (AGEs). Methylglyoxal (MG)-a metabolite of carbohydrates-is believed to cause insulin resistance by inducing inflammation and pancreas damage. In this work, diabetes was induced in Wistar rats (4 weeks of age) by treating them with MG (600 mg/kg bw) for 4 weeks. We observed that AK (10mg/kg bw) exerted peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity, thereby enhancing insulin sensitivity (as indicated by hepatic GLUT2 translocation, PTP1B suppression, and glucose uptake) by downregulating blood glucose and upregulating pancreatic and duodenal homeobox-1 and Maf-A expression and increasing insulin production in MG-induced rats. However, these effects were abolished by the administration of GW9662 (PPARγ antagonist), but the expression of hepatic heme oxygenase-1 (HO-1) and glutamate-cysteine ligase (GCL) was not suppressed in MG-induced rats. Therefore, the nuclear factor erythroid-related factor-2 (Nrf2) activation was investigated. AK did not affect hepatic Nrf2 mRNA or protein expression but significantly increased Nrf2 phosphorylation (serine 40), which was accompanied by increased transcriptional activation of hepatic HO-1 and GCL. These data indicated that AK protected rats from oxidative stress resulting from MG-induced insulin resistance. In contrast, these effects were not detected when the rats were treated with the antidiabetic drug rosiglitazone (10mg/kg bw). Moreover, we found that AK did not inhibit the generation of AGEs in vitro; however, the glutathione (GSH) levels in liver and pancreas of MG-induced rats were elevated in rats administered AK. Therefore, we believe that GSH may lower the MG level, which attenuates the formation of AGEs in the serum, kidney, liver, and pancreas of MG-induced rats. We also found that AK treatment reduced the production of inflammatory factors, such as tumor necrosis factor-α and interleukin-1β. Taken together, the results of our mechanistic study of MG-induced rats suggest that the protective effects of AK against diabetes are mediated by the upregulation of the signaling pathway of Nrf2, which enhances antioxidant activity and serves as a PPARγ agonist to enhance insulin sensitivity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The role of inflammation-induced oxidative stress in the pathogenesis and progression of chronic inflammatory airways diseases has received increasing attention in recent years. Nuclear factor-erythroid 2 related factor 2 (Nrf-2) is the primary transcription factor that regulates the expression of antioxidant and detoxifying enzymes. In this study, yellow pigment ankaflavin (AK), derived from Monascus-fermented products, elevated nuclear Nrf-2 protein translocation in both the A549 lung cell line and the lungs of ovalbumin (OVA)-challenged mice. Furthermore, AK increased the mRNA expression of antioxidant enzymes regulated by Nrf-2, leading to a reduction in allergen-driven airway inflammation, mucus cell hyperplasia, and eosinophilia in OVA-challenged mice. Additionally, AK prevented T-cell infiltration and Th2 cytokines including interleukin (IL)-4, IL-5, and IL-13 generation in bronchial alveolar lavage fluid. The adhesion molecules ICAM-1, VCAM-1, and eotaxin were substantially reduced by AK treatment. Importantly, the inhibitory effect of AK on adhesion molecule production and immune cell infiltration was abolished by Nrf-2 small interfering RNA. This is the first study to illustrate that AK acts as a novel Nrf-2 activator for modulating the oxidative stress pathway to improve the lung injury and ameliorate the development of airway inflammation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

An azaphilonidal derivative monaphilone A (MA) was recently isolated from the fermented products of Monascus purpureus NTU 568 by our laboratory. We report here the exploration of apoptosis-related and anti-inflammatory properties of MA and ankaflavin (AK) by some experiments about inducing death of human laryngeal carcinoma cell line HEp-2 and reducing inflammatory responses on murine macrophage RAW 264.7 cells. We employed a ssDNA enzyme-linked immunosorbent assay (ELISA) kit to investigate the nuclear changes of early apoptosis induced by AK and MA on HEp-2 cells and used a western blot and an enzyme activity assay to demonstrate the activation of caspase-3, caspase-8, and caspase-9 by MA and AK. Our studies revealed that AK and MA may decrease lipopolysaccharide (LPS)-induced inflammatory responses, including nitrite productions and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW 264.7 cells. All evidence support that azaphilonidal derivatives from M. purpureus NTU 568, such as AK and MA, are suitable for the development of chemotherapy or chemopreventive agents.

The Monascus fermentation product red mold rice (RMR) has been found to contain the cholesterol-lowering agent monacolin K (MK) in both its lactone (MKL) and acid (MKA) forms and the mycotoxin citrinin (CT). The yellow pigments in RMR, namely, monascin (MS) and ankaflavin (AK), have been reported to exhibit antimetastatic and antiangiogenic activities. Currently, MK and these yellow pigments are usually detected in RMR by different analytical methods that are inconvenient, expensive, and time-consuming. The goal of this study was to establish a rapid, synchronous analytical method for determination of the MKA, MKL, MS, AK, and CT levels in RMR. MKA, MKL, MS, AK, and CT were extracted by the same extraction method, then separated by RP-HPLC with a C18 column. The effluent from the column was passed through a photodiode array detector and then introduced directly into a fluorescence detector. The results showed that high recovery rates of MKA, MKL, MS, AK, and CT are possible if RMR powder is extracted with 75% ethanol (10 mL) at 80 degrees C for 30 min. With regard to the optimal conditions of the HPLC, the peaks of MKA, MKL, MS, AK, and CT can be clearly separated from any noise peaks by isocratic elution with a mobile phase comprising 0.05% trifluoroacetic acid in acetonitrile-water (62.5 + 37.5, v/v).

The aim of the present work is to investigate the effects of Monascus secondary metabolites, monascin (MS) and ankaflavin (AK), on cell proliferation, adipogenesis, lipolysis and heparin-releasable lipoprotein lipase (HR-LPL) in 3T3-L1 preadipocyte. MS and AK inhibit the proliferation of 3T3-L1 cells in a dose-dependent fashion. At 8 μg/mL concentration MS inhibits proliferation for 80.5% after 48 h, whereas the value for AK is 69.2%. Adipogenesis is inhibited by MS and AK without dose-dependency. Triglyceride is decreased 37.1% and 41.1% respectively by treating 0.125 μg/mL MS and AK. Adipocyte-specific transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein β (C/EBPβ), C/EBPδ and C/EBPα mRNA levels are measured by real-time polymerase chain reaction. The expression of the four transcriptional factors analyzed (PPARγ, C/EBPβ, C/EBPδ and C/EBPα) is reduced at the initial and the middle period. At the later period, there is no effect on the expression of PPARγ and C/EBPα by treating MS and AK. Furthermore, both MS and AK increase basal lipolysis of mature adipocytes by 113.2% and 278.3% upregulation, respectively. And both MS and AK reduce the activity of HR-LPL, by 45.3% and 58.1% reduction, respectively. This study reveals for the first time that Monascus secondary metabolites, MS and AK, can prevent the differentiation of preadipocyte and stimulate basal lipolysis of mature adipocytes, avoiding the accumulation of lipid.

Monascus-fermented red mold dioscorea (RMD) has been proven to possess greater hypolipidemic effect than red mold rice (RMR) even though they include equal levels of cholesterol-lowering agent monacolin K. However, higher concentrations of yellow pigments (monascin and ankaflavin) were found in RMD than in RMR. In this study, purified monascin and ankaflavin were administered to hyperlipidemic hamsters for 8 weeks, respectively, to test whether these two compounds were novel hypolipidemic ingredients. In the statistical results, monascin and ankaflavin showed significant effect on lowering cholesterol, triglyceride, and low-density lipoprotein cholesterol levels in serum, as well as aorta lipid plaque (p < 0.05). Importantly, monascin and ankaflavin, unlike monacolin K, were able to perform up-regulation rather than down-regulation on high-density lipoprotein cholesterol (HDL-C) levels in serum. This finding not only explained why RMD showed greater hypolipidemic and HDL-C-raising effect than RMR but also proved that monascin and ankaflavin would act as novel and potent hypolipidemic ingredients.