Introduction

Red mold rice (RMR) is a fermented food product that is produced by inoculating Monascus into steamed rice, and it is a common food item in China. Red mold-fermented products have been used in Asia for centuries to enhance the flavor of food, as well as serving as a traditional medicine for the treatment of digestive disorders, vascular function, and blood circulation (Lee et al. 2006a; Ma et al. 2000). Red mold-fermented products are now considered to be functional foods, and they have been developed as commercial capsules for cardiovascular disease prevention. Monascus species produce several bioactive metabolites, and the secondary metabolites that are produced include pigments (red pigments: monascorubramine and rubropunctanin; orange pigments: monascorubrin and rubropunctanin; yellow pigments: ankaflavin (AK) and monascin (MS)) (Wong and Bau 1977; Wild et al. 2002), antioxidant compounds (e.g., isoflavones, dimerumic acid, phenols, and tannins) (Akihisa et al. 2005; Aniya et al. 2000), polyketide monacolins (antiobesity, anti-inflammatory, antidiabetic, and antioxidative stress-related metabolites, such as azaphilones, furanoisophthalides, and amino acids) (Su et al. 2003; Akihisa et al. 2005; Lee et al. 2006b), and γ-aminobutyric acid (GABA, a neurotransmitter and hypotensive agent) (Su et al. 2003; Juslova et al. 1996; Ma et al. 2000). Oxidative stress is increased during diabetes and caused damage to organisms. Reactive oxygen species (ROS) and the inflammatory response are generated as a result of hyperglycemia, which causes many of the secondary complications of diabetes (West 2000). Previous studies revealed that the level of blood glucose was decreased in experimental rats fed with Monascus-fermented products (Chen and Liu 2006); triglyceride (TG) and total cholesterol (TC) levels were decreased (Shi and Pan 2010a). The diabetic rats showed higher ROS and lower antioxidant enzyme (glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT)) activities in pancreas when treated with red mold-fermented products (Shi and Pan 2010b). These results indicated that red mold-fermented products not only regulate hyperglycemia but also provide the prevention effects of hyperglycemia-induced oxidative stress. The aforementioned Monascus-fermented products and their metabolites may be of great benefit for the amelioration of diabetes symptoms and their development.

Diabetes, oxidative stress, and diabetic complications

Diabetes mellitus is a metabolic disorder that is characterized by an elevated blood glucose concentration and the inadequate secretion or activity of endogenous insulin (Singh et al. 2008). Diabetes leads to hyperglycemia, and it also causes hyperlipidemia, hyperinsulinemia, hypertension, and atherosclerosis (Sowers et al. 2001; Beckman et al. 2002; Sepici et al. 2004). The etiology of this disease is not well defined, but environmental factors, autoimmune disease, and viral infections have been implicated (Like et al. 1979; Kataoka et al. 1983; Shewade et al. 2001; Maritim et al. 2003). Several studies have suggested that a hyperglycemia-induced overproduction of superoxide seems to be activated in all pathways involved in the pathogenesis of diabetes complications (Fig. 1) (Robertson et al. 2003; Ceriello 2006). In vivo evidence supports the major contribution of hyperglycemia to the production of oxidative stress and the acute endothelial dysfunction of patients with diabetes (Ceriello 2006). Free radical-caused oxidative stress and oxidative damage to tissues are common endpoints of age-related or chronic diseases, such as atherosclerosis, diabetes, Alzheimer’s disease, and rheumatoid arthritis (Baynes and Thorpe 1999; Tuppo and Forman 2001; Hadjigogos 2003). Abnormally high levels of free radicals and the loss of antioxidant defense mechanisms lead to damage to the cellular organelles and enzymes, increased lipid peroxidation, DNA damage, and protein derivatives, and the development of insulin resistance (West 2000; Maritim et al. 2003). Vascular function, like impaired endothelium-dependent vasodilatation, has been in diabetic animal models (Mayhan 1989) and has found endothelial dysfunction (Johnstone et al. 1993). Normal or diabetic animals exposed to exogenous hyperglycemia subsequently have exhibited attenuated endothelium-dependent relaxation, an endothelial dysfunction (Kawano et al. 1999). Free radicals generation-induced oxidative stress has produced the hyperglycemia-dependent endothelial dysfunction; it makes diabetes and its complication severe (Diederich et al. 1994).

Fig. 1
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Hyperglycemia presents excessively high concentrations of glucose for reactive oxidative species formation and oxidative stress (Robertson et al. 2003)

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Microvascular and cardiovascular oxidative stress increases during the development of diabetes complications (Coleman 2001; Maritim et al. 2003). The mechanisms whereby increased oxidative stress leads to the activation of the five major pathways involved in the pathogenesis of complications are as follows: increased formation of advanced glycation end products (AGEs) (Scivittaro et al. 2000), polyol pathway flux (Chung et al. 2003), increased expression of the AGEs receptor and its activating ligands, overactivation of the hexosamine pathway (Horal et al. 2004), and protein kinase C activation (Tuttle et al. 2009; Giacco and Brownlee 2010). Increased intracellular ROS activation occurs in a number of proinflammatory pathways (Bulua et al. 2011), which has been implicated in inflammatory diseases including rheumatoid arthritis, type 1 diabetes (Chen et al. 2008), and multiple sclerosis (Gilgun-Sherki et al. 2004). Overexpression of antioxidant enzymes in transgenic diabetic mice, such as SOD, prevents diabetic nephropathy (Ceriello et al. 2000), retinopathy (Lopes de Jesus et al. 2008), and cardiomyopathy (Singal et al. 2001). Understanding the relationships among oxidative stress, diabetes, and its complications will aid the discovery of novel therapeutic treatments for the prevention of diabetic complications.

Beneficial effects of red mold-fermented products on blood glucose management

Red mold products fermented by Monascus purpureus NTU 568 produce secondary metabolites such as monacolin K, MS, AK, and GABA, with potent hypolipidemic and antihypertensive effects that have been characterized in our previous studies (Lee et al. 2006a, b; Wu et al. 2009). We studied the preventive and beneficial effects of M. purpureus NTU 568 fermented red mold products on diabetic animals (Shi and Pan 2010a, b; Shi et al. 2011). As discussed below, results from two of these studies might help to explain the main mechanism whereby red mold-fermented products ameliorate the development of diabetes by lowering the levels of blood glucose and lipid profiles in diabetic animals.

Diabetes symptom modification via blood glucose reduction

Red mold-fermented products are known to have a role in the regulation of blood glucose and insulin resistance (Chang et al. 2006; Chen and Liu 2006; Shi and Pan 2010a). Red mold-fermented products can also lower blood cholesterol levels, control cardiovascular complications, and glucose homeostasis (Journoud and Jones 2004; Chang et al. 2006). They are considered useful for the treatment of diabetes. The studies revealed that feeding experimental rats with 150 mg/kg of Monascus-fermented products after 12 h of fasting led to the blood glucose level decreasing by 19.4% after 90 min compared with a control group, while the insulin level increased by 60.2% (Chen and Liu 2006). The mediation of acetylcholine (ACh, an inhibitor of choline uptake) release from the nerve terminals to enhance insulin secretion by red mold-fermented products may also be considered (Chen and Liu 2006). Gluconeogenesis augmentation is a major factor affecting plasma glucose increases in diabetic animals (Consoli et al. 1989). Previous studies showed that the phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels in the liver of diabetic rats were inhibited by oral treatment with red mold-fermented products for 14 days (Chang et al. 2006). Red mold-fermented products may act directly or indirectly via endogenous substances to modify hepatic PEPCK gene expression. After 8 weeks of feeding diabetic rats with different types of red mold-fermented products (RMR, red mold adlay and red mold dioscorea (RMD)), it was found that all types of red mold products reduced the blood glucose levels (Shi and Pan 2010a). Moreover, the degraded lipid profiles of TG and TC were improved by red mold-fermented product treatments (Table 1) (Shi and Pan 2010b). Red mold-fermented products can increase the release of ACh from nerve terminals, decrease hepatic gluconeogenesis, and increase insulin secretion, which lowers the blood glucose activity.

Table 1 Serum triglyceride (TG) and cholesterol (TC) levels in diabetic rats were ameliorated by red mold-fermented products treatment
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Inhibiting hyperglycemia-increased oxidative stress and inflammation via antioxidation and anti-inflammatory effects

Red mold-fermented products that contain a variety of antioxidants are mentioned in an ancient Chinese pharmacopoeia of medicinal food and herbs. The antioxidant action of traditional foods has been investigated, and it was shown that Monascus anka and Monascus ruber have a strong antioxidant action (Aniya et al. 1999). M. anka and M. ruber scavenged over 60% of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and inhibited lipid peroxidation at a concentration of 1.6% (Aniya et al. 2000). The radical-scavenging, iron-chelating, and DNA-protection activities of liquid fermentation products derived from Monascus pilosus were significantly higher when grown in a garlic-containing medium (Kuo et al. 2006). The addition of garlic to the culture medium significantly increased the antioxidant activities of M. pilosus fermentation products in terms of DPPH (50% inhibition at a concentration of 4.62%), superoxide (50% inhibition at a concentration lower than 5.0%), and hydrogen peroxide (50% inhibition at a concentration lower than 0.1%) scavenging activity, iron-chelating activity, as well as protecting against lipid peroxidation and DNA damage (Kuo et al. 2006).

Throughout the experimental period (8 weeks), diabetic rats had higher ROS levels (12.1–65.8%) and lower activities of SOD (18.2–35.7%), CAT (26.4–34.9%), and GR (9.0–30.0%) in the pancreas compared with rats treated with red mold-fermented products (Shi and Pan 2010b). Moreover, nitric oxide (leading to oxidative stress) production and endothelin-1 (upregulated in diabetes) levels were improved by red mold-fermented product treatment (Shi and Pan 2010b).

The RMD contained 3,572.7 mg/kg MS and 2,444.3 mg/kg AK higher than RMR (3,099.7 mg/kg MS and 1,048.8 mg/kg AK) (Shi and Pan 2010c). RMD has greater hypolipidemic, antidiabetic, and antioxidant effects than traditional RMR in experimental animals where it reduces oxidative stress and the anti-inflammatory response (Lee et al. 2007b; Shi and Pan 2010a, b). Diabetic rats treated with RMD for 6 weeks had higher activity levels of SOD, GR, glutathione peroxidase (GPx), and CAT in the pancreas compared with diabetic control rats (Table 2) (Shi et al. 2011). The islet inflammatory process in diabetic rats exhibited an increased islet cytokine (interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α) expression (Ehses et al. 2009). The increased production of IL-6 and interferon (IFN)-γ by STZ-induced diabetic rats was identified as autoimmune diabetes (Ishihara and Hirano 2002). RMD inhibited the diabetes-induced elevation in the levels of IL-1β and TNF-α in the pancreas, and it ameliorated pancreatic β-cell damage (Fig. 2) caused by STZ (Shi et al. 2011). It was demonstrated that red mold-fermented products possess several treatment-oriented properties, including the control of hyperglycemia, antioxidative stress function, as well as anti-inflammatory and cytoprotective effects.

Table 2 Antioxidative activity of red mold dioscorea in the pancreas of experimental rats
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Fig. 2
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Immunohistochemical evaluation on pancreas (×400). Immunohistochemical staining was carried out using avidin-biotinylated horseradish peroxidase kit. NC normal control group, DC diabetic control group, RMD diabetic animals with red mold dioscorea treatment (Shi et al. 2011)

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Red mold secondary metabolites with antidiabetic and related beneficial effects γ-aminobutyric acid (GABA)

GABA is an amino acid transmitter that is present in the inhibitory neurons of the central nervous system, which is synthesized from glutamic acid by glutamic acid decarboxylase (Fig. 3) (Gerber and Hare 1980; Pipeleers et al. 1985). GABA has several well-known physiological functions, including antihypertensive and diabetic hyperglycemia prevention activities (Wu et al. 2009; Soltani et al. 2011). Several manufactured functional foods have a high GABA content: GABA-enriched rice germ by soaking in water (Komatsuzaki et al. 2007), GABA-enriched brown rice by high pressure treatment and germination (Kinefuchi et al. 1999), and red mold rice containing the Monascus fungus (Rhyu et al. 2000). Some studies have isolated and identified the GABA-rich Monascus strains and irradiated them with UV or modified substrates to raise their GABA production (Chuang et al. 2011; Jiang et al. 2011). The importance of GABA for the function of hormonal secretion has been reported (Cavagnini et al. 1977; Sorenson et al. 1991). GABA agonists have been used to modify the blood glucose levels of diabetic rats and increase the plasma insulin concentrations to levels similar to those of non-diabetic animals (Gomez et al. 1999). These experiments demonstrated that GABA and GABA receptor agonist drugs act on the endocrine pancreas in vivo, ultimately increasing the insulin levels and decreasing the blood glucose levels of diabetic rats (Gomez et al. 1999).

Fig. 3
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The chemical structure of γ-aminobutyric acid (GABA)

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Type 1 diabetes is an autoimmune disease that is characterized by the infiltration of the pancreatic islets with T lymphocytes and macrophages, where consequent loss of β-cells requires β-cell restoration and immune suppression therapy (Eizirik et al. 2009; Lehuen et al. 2010; Soltani et al, 2011). Research has indicated that GABA exerts antidiabetic effects by acting on the islet β-cells and the immune system. GABA leads to membrane depolarization and the activation of PI3-K/Akt-dependent pathways, which restores β-cell mass and reverses diabetes. GABA has β-cell regenerative and immune inhibitory effects on islet cell function, and it regulates glucose homeostasis (Soltani et al. 2011).

Monascin (MS)

It was reported that MS is the major constituent of the azaphilonoid pigments found in extracts of RMR (Fig. 4) (Hsu et al. 2010). MS is a potential cancer-preventive agent for combating chemical and environmental carcinogenesis. It is also an anti-inflammatory agent that inhibits the 12-O-tetradecanoylphorbol 13-acetate-induced inflammatory response in mice (Akihisa et al. 2005). In previous studies, we showed that the RMD may have hypolipidemic and hypoglycemic effects via its secondary metabolite MS (Lee et al. 2010b; Shi and Pan 2010a). Chronic inflammation in muscle tissue is linked with type 2 diabetes, insulin resistance, and diabetic complications. Peroxisome proliferator-activated receptor (PPAR, a member of the nuclear receptor family of transcription factors) ligands have been reported to activate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Bulhak et al. 2009). Studies have indicated that MS inhibited the p-JNK activity and prevented PPAR-γ phosphorylation via its PPAR-γ activity and the PI3K/Akt pathway (Lee et al. 2011). MS treatment of C2C12 cells may elevate PPAR-γ mRNA expression and prevent PPAR-γ phosphorylation. Moreover, the use of a PPAR-γ antagonist (GW9662) to block PPAR-γ activation in C2C12 cell indicated that MS may be an agonist of PPAR-γ, which improves insulin resistance (Lee et al. 2011).

Fig. 4
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The chemical structure of monascin (MS)

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The mechanisms whereby MS exerts its in vivo action were tested using animal and Caenorhabditis elegans models (Shi et al. 2012). The nematode C. elegans has been an important animal model for studying the molecular mechanisms of drug effects and disease pathogenesis. In animal experiments, we found that the levels of blood glucose, serum insulin, TG, TC, high-density lipoprotein, and the activities of antioxidant enzymes were ameliorated by MS treatment in STZ-induced diabetic rats (Shi et al. 2012). DAF-16/FOX (Forkhead box) proteins are a family of transcription factors that are involved in metabolism, stress resistance, and antioxidative defense in C. elegans and mammals (Henderson and Johnson 2001; Murphy et al. 2002). Studies have indicated that MS induced the hepatic mRNA levels of FOXO1, FOXO3a, catalase, and MnSOD in diabetic rats and enhanced the expression of small heat shock protein, glutathione S-transferase, and SOD-3 in C. elegans (Fig. 5) (Shi et al. 2012). Mechanistic studies in cells, rats, and C. elegans suggest that the protective effects of MS are mediated via the regulation of the FOXO/DAF-16-dependent insulin signaling pathway and the AKT pathway, by inducing the expression of stress response/antioxidant genes, regulating PPAR-γ, and inhibiting JNK activation (Lee et al. 2011; Shi et al. 2012).

Fig. 5
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Effects of monascin (MS) on the expressions of heat shock protein (HSP)-16.2, superoxide dismutase (SOD)-3, and glutathione S-transferase (GST)-4 in Caenorhabditis elegans. Transgenic worms were incubated with 1 μM MS or 0.1% DMSO as the solvent control at 22 °C. Total GFP fluorescence for each whole worm was quantified by Image-Pro Plus software. Data shown are the average number of pixels in the transgenic C. elegans (n = 20) at each indicated treatment. Data are presented as the mean ± SD (Shi et al. 2012)

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Safety

Red mold-fermented products may be contaminated by citrinin, which is regarded as a toxic secondary metabolite of Aspergillus and Penicillium species to damage the kidneys and liver. (Hetherington and Raistrick 1931). The lethal dose (LD50) of citrinin has been reported to be about 35–58 mg/kg for oral administration to a mouse, 50 mg/kg to a rat, and 134 mg/kg to a rabbit (Hanika an Carlton 1994). The toxicity evaluations of Monascus-fermented products for an experimental period of as long as 4 months have shown no toxicity effects (Li et al. 1998).

Studies on increasing the level of monacolin K and decreasing the level of citrinin have been investigated by several laboratories (Chen and Hu 2005; Wang et al. 2004). The previous study has developed a post-process to remove citrinin yet retain monacolin K in the RMR preparation (Lee et al. 2007a). On the basis of the findings from the 90-day animal test with citrinin (1, 2, 10, 20, and 200 ppm) treatment, the no-observable-adverse-effect level (NOAEL) is 200 ppm citrinin for male Wistar rats (Lee et al. 2010a). Investigations are focused on the conditions of red mold-fermented production to a lower citrinin concentration.

Conclusion

Monascus-fermented rice is mentioned in an ancient Chinese pharmacopoeia of medicinal food. A product fermented with M. purpureus NTU 568 has been used to ameliorate hyperlipidemia, hypertension, diabetes, obesity, and Alzheimer’s disease. The novel product RMD was found to contain higher amounts of antioxidative and anti-inflammatory substances, GABA, and MS, compared with traditional RMR, and it also had greater potential for ameliorating insulin resistance and diabetes. Table 3 shows that red mold-fermented products and its secondary metabolites utilize a different preventive mechanism for diabetes, and a hypothesis regarding the preventative activity of red mold-fermented products and its secondary metabolites is presented in Fig. 6. Several mechanisms, PI3-K/Akt-dependent pathway and FOXO/DAF-16 transcription factors activation, explaining how Monascus species-fermented products ameliorate diabetes and related oxidative stress are available at present, but its underlying functional ingredients and its deep mechanisms remain elusive. Therefore, future studies should be focused on the isolation of functional ingredients and investigations of their mechanisms in different animal models, which will support the development of useful therapies for diabetes and its complications.

Table 3 Comparisons between the reduced levels of risk factors for diabetes and its complications caused by red mold-fermented products and its secondary metabolites
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Fig. 6
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Hypothesis proposing of the preventative approach of red mold-fermented products and its secondary metabolites with diabetic oxidative stress, inflammatory response, and insulin resistance

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