Red Yeast Rice: A Systematic Review of the Traditional Uses, Chemistry, Pharmacology, and Quality Control of an Important Chinese Folk Medicine

Red yeast rice (RYR), a Chinese traditional folk medicine produced by the fermentation of cooked rice kernels with a Monascaceae mold, Monascus purpureus, has long been used to treat blood circulation stasis, indigestion, diarrhea, and limb weakness in East Asian countries. This article provides a systematic review of the traditional uses, chemistry, biological activities, and toxicology of RYR to highlight its future prospects in the field of medicine. The literature reviewed for this article was obtained from the Web of Science, Elsevier, SciFinder, PubMed, CNKI, ScienceDirect, and Google Scholar, as well as Ph.D. and M.Sc. dissertations, published prior to July 2019. More than 101 chemical constituents have been isolated from RYR, mainly consisting of monacolins, pigments, organic acids, sterols, decalin derivatives, flavonoids, polysaccharides, and other compounds. Crude extracts of RYR, as well as its isolated compounds, possess broad pharmacological properties with hypolipidemic, anti-atherosclerotic, anti-cancer, neurocytoprotective, anti-osteoporotic, anti-fatigue, anti-diabetic, and anti-hypertensive activities. However, further studies are needed to characterize its diverse chemical constituents and the toxicological actions of the main bioactive compounds. New pharmacological trials addressing the overlooked traditional uses of RYR, such as in the treatment of indigestion and diarrhea, are required.


PHARMACOLOGICAL ACTIvITIeS
RYR has been the subject of several pharmacological investigations due to its various ethnomedicinal uses. Studies have demonstrated that RYR exhibits a wide range of biological properties with hypolipidemic, anti-atherosclerotic, anti-cancer, neurocytoprotective, hepatoprotective, anti-osteoporotic, anti-fatigue, anti-diabetic, anti-obesity, immunomodulatory, anti-inflammatory, and anti-hypertensive activities. Among these, hypolipidemic and anti-atherosclerotic activities are most pronounced given that RYR is thought to play curative roles in resolving turbidity, invigorating blood circulation, and resolving blood stasis. These effects are summarized in Table 2 and discussed in greater detail in the following sections. The

Hypolipidemic effect
Hyperlipidemia is the bane of current dietary patterns and rather torpid lifestyles. As a result, the search for supplements that can lower the triglyceride (TG) and cholesterol levels has gained significant momentum. Statins have been used to eliminate vascular occlusions, but with increasing reports of side effects, alternatives are being pursued (Patel, 2016). In this regard, the hypolipidemic potential of RYR has been consistently proved with reliable experimental results (Gerards et al., 2015). In dyslipidemia patients, RYR, which was administered as a dietary supplement (4-48 mg/kg), significantly decreased TG, total Frontiers in Pharmacology | www.frontiersin.org December 2019 | Volume 10 | Article 1449 cholesterol (TC), and low density lipoprotein cholesterol (LDL-C) levels, and increased the high density lipoprotein cholesterol (HDL-C) level (Heber et al., 1999;Becker et al., 2009;Bruno et al., 2018). However, further studies that follow patients for longer periods of time are needed to investigate the effects RYR on the risk factors and treatment of various chronic diseases.
Frontiers in Pharmacology | www.frontiersin.org December 2019 | Volume 10 | Article 1449 TG, and LDL-C levels by up-regulating lipoprotein lipase and low density lipoprotein receptor mRNA expression in the liver (Chen, 2004). The oral administration of RYR yellow, red, and orange pigments also markedly alleviated disturbances in lipid metabolism; ameliorated serum lipid levels; suppressed hepatic lipid accumulation and steatosis; and promoted fecal cholesterol, triacylglycerol, and bile acid excretion. The mechanisms of action involve an up-regulation of the mRNA levels of farnesoid X receptor and peroxisomeproliferator-activated receptor-gamma, the main receptors for the metabolism of cholesterol and homeostasis of bile acids (Zhou et al., 2019). However, some of these results did not show a dose-effective relationship, and some of these studies lacked positive controls. RYR is often combined with other products that can effectively reduce lipid levels, such as berberine (Millan et al., 2016), policosanols (Guardamagna et al., 2009), coenzyme Q 10 , plant stanols and sterols (Feuerstein and Bjerke, 2012), olive extracts (Verhoeven et al., 2015), and curcumin (Derosa et al., 2018). Among these, berberine and policosanols have been extensively investigated due to their lipid-lowering properties, in which an increase in LDL receptor half-life and expression and an increase in insulin sensitivity via the activation of AMP-activated protein kinase (AMPK) (Spigoni et al., 2017) were observed. Bifidobacterium longum, a probiotic strain showing potent hypolipidemic effects, has also been combined with RYR in nutraceutical formulations given that alterations in gut microbiota were found to be involved in the pathogenesis of systemic diseases related to hypercholesterolemia (Weis, 2018;Ruscica et al., 2019). Additionally, several Chinese medicines have been used with RYR to treat hyperlipidemia, such as Tao He Cheng Qi Tang consisting of Prunus persica semen, Cinnamomum cassia ramulus, Rheum palmatum rhizome, natrii sulfas, and Glycyrrhiza uralensis rhizome (Yang, 2017). However, the possible interactions, synergistic effects, and underlying mechanisms of RYR in combination with other ingredients from poly-herbal preparations remain unknown and should be further investigated.

Anti-Atherosclerotic Activity
The inhibition of cholesterol synthesis is effective for the primary and secondary prevention of atherosclerotic diseases (Li et al., 2005b). Experimental studies in animal and cell models have demonstrated that RYR, as well as Xuezhikang (XZK, its pure extract), has anti-atherosclerotic activity. An eight-week administration of XZK (600-1,200 mg/kg) to C57BL/6 mice was found to significantly and dose-dependently inhibit vulnerable plaque progression, decrease the plaque area, and suppress lesional endoplasmic reticulum stress (Shen et al., 2017). In another study involving atherosclerotic rats fed a high-cholesterol diet, the oral administration of RYR at 120 mg/kg significantly reduced the plaque size, stabilized the plaque, protected the endothelium, and decreased the number of lipid droplets and cholesterol calculi, and lowered the levels of high sensitivity C-reaction protein (Hs-CRP), IL-6, and TNF-α, suggesting that the antiatherosclerotic activity of RYR may be related to inflammatory signaling pathways . The administration of aqueous and ethanol extracts of RYR to homocysteine-treated human aortic smooth muscle cells (HASMCs) (1-160 μg/ml) for 24 h reduced TNF-α-induced metalloproteinase (MMP)-2 and MMP-9 expression. It also reduced nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS) formation, supporting the notion that RYR may have a potential application in the treatment of atherosclerosis (Lin et al., 2011). Unfortunately, the chemical analyses of XZK and the aqueous and ethanol extracts of RYR were not performed in these studies. Based on the significant decrease in the viability of HASMCs treated with relatively high concentrations of RYR (160 μg/ml), attention should be paid to the possible side-effects in clinical applications.

Anti-Cancer Activity
Cancer, a major disease, is the leading cause of death throughout the world, and developing effective anti-cancer therapies remains a challenge for those in medical research (Zhu et al., 2018). Previous studies have reported that RYR supplements have anti-cancer activity. The oral administration of RYR (120 mg/kg, qd) to breast cancer patients for 12 weeks significantly decreased serum tumor biomarker (CA125, CA153) levels and improved their life quality and immune function after chemotherapy and resection surgery (Zhong, 2017). Although this study showed that RYR possesses anti-cancer activity, only a single dose was used, thereby limiting information on the dose-dependent effect.
In SCID tumor mice induced by human prostate cancer cells, the administration of powdered RYR significantly reduced the oral tumor volume and decreased the serum prostate-specific antigen level, as well as the gene expression of enzymes involved in androgen synthesis (HSD3B2, AKR1C3, and SRD5A1) (Hong et al., 2011). Thus, clinical studies of RYR use for the prevention of prostate cancer in men undergoing active surveillance of the disease should be considered. Studies have reported that polysaccharides, monacolins, and pigments from RYR are responsible for the anti-cancer activity of RYR. The administration of RYR polysaccharides (at a very high dose of 800 mg/kg) to replanted S 180 tumor mice for 2 weeks remarkably inhibited tumor growth and improved body weight (Ding, 2007). However, this trial lacked a positive control as well as an evaluation of serum biochemical indicators of the tumor. In K-562, SK-OV-3, and SNU-1 cells, the administration of rubropunctatin, a red pigment, and RYR-derived monacolin L (1.25-40 μg/ml) for 48 h exhibited very strong inhibitory effects against cancer cell proliferation, and the effect of rubropunctatin was comparable with that of anti-cancer drugs cis-platinum, taxol, and 10-hydroxy-camptothecin in their IC 50 values. In this study, the authors demonstrated that RYR at least partially exerted its anti-cancer effects through telomerase-mediated inhibition of rubropunctatin and monacolin L-induced apoptosis (Xu et al., 2017). However, both studies failed to discuss the effectiveness of RYR-derived monacolin and pigments using animal models, and more in-depth studies should be carried out to further develop these compounds into anti-cancer drugs.
Neurocytoprotective Activity RYR extracts have also been shown to possess neurocytoprotective activity. In rats treated with amyloid β (Aβ), an amino acid associated with Alzheimer's disease, the oral administration of RYR (151-755 mg/kg) for 4 weeks could markedly reverse the memory deficits in the water maze and passive avoidance tasks, as well as prevent Aβ40 infusion and damage in the hippocampus and cortex, which are known involve in the increase of thiobarbituric acid reactive substances and ROS. Using human neuroblastoma IMR32 cells exposed to a high concentration of cholesterol, the authors also reported that RYR down-regulated Aβ40 formation and deposition by suppressing cholesterolinduced β-secretase activity and apolipoprotein E expression. RYR also mediated the proteolysis of amyloid precursor protein (APP) into a soluble APP α-fragment with neuroprotective activity in the hippocampus . However, further studies are needed to confirm the neurocytoprotective effect using low doses of RYR suitable for human administration. Lovastatin derivatives are also responsible for the neurocytoprotective effect of RYR. The administration of one lovastatin-derived compound, designated 3f (12.5-100 μmol/L), for 24 h significantly reduced 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, reduced caspase-3, -8, and -9 activities, and lowered intracellular calcium concentrations elevated by 6-OHDA in a concentration-dependent manner, without inhibiting the production of ROS (Lin et al., 2015). Studies on the mechanism of action of compound 3f are being conducted.

Hepatoprotective effect
RYR extracts exhibit a strong hepatoprotective effect both in alcoholic fatty liver and non-alcoholic fatty liver disease (NAFLD) mice models. In chronic alcohol-induced mice, the oral administration of powdered RYR (307.5-1,537.5 mg/ kg) for 5 weeks significantly attenuated the increased levels of serum transaminases (aspartate aminotransferase and alanine aminotransferase), hepatic TGs, and TC. Furthermore, RYR elevated the hepatic antioxidant activity that reduced hepatic cell damage (steatosis) and decreased tissue inflammatory cytokine levels (Cheng and Pan, 2011). These findings suggested that RYR may represent a novel, protective strategy against alcoholic liver disease by attenuating oxidative stress, inflammatory responses, and steatosis. However, the authors of this study did not perform comparisons using a positive control. The administration of RYR extracts (XZK at 300 mg/kg) to NAFLD mice for 6 weeks significantly ameliorated dyslipidemia and fat accumulation in the liver; improved insulin resistance; ameliorated oxidative stress; lessened hepatic steatosis, necro-inflammation, and collagen deposition; and reversed abnormalities in the aminotransferase level. The mechanism of action of RYR likely involves inhibition of the hepatic expression of TNF-α (Hong et al., 2007). These results suggested that RYR may have a potential clinical application in the treatment of NAFLD. In this study, however, the optimal dose, constituents, and side effects of RYR were not assessed.

Anti-Osteoporotic Activity
Osteoporosis is a common health problem characterized by low bone mass and structural deterioration of the bone, resulting in an increased susceptibility to fractures (Zhang et al., 2018b). Several studies have reported the protective role of RYR in osteoporosis. In rats with ovariectomy-induced bone loss and osteoblast cells, the administration of ethanol extracts from RYR (at a very high dose of 1.56-6.24 g/kg) for 20 weeks significantly increased the bone mineral density and decreased the levels of bone turnover markers in vivo, including osteocalcin and tartrate-resistant acid phosphatase. Moreover, its administration improved the viability of osteoblasts and enhanced the mRNA and protein expression of bone morphogenetic protein (Bmp) 2 and Bmp4, suggesting that RYR may be useful in preventing and treating osteoporosis . However, future studies should confirm this anti-osteoporotic effect under clinical conditions with low doses of RYR that are suitable for human administration. In rabbits with bone defects and UMR 106 cells, the administration of RYR extracts significantly promoted new bone formation in vivo, enhanced bone optical density, and increased alkaline phosphatase activity in vitro, suggesting that RYR is a natural product that can potentially treat bone defects and osteoporosis (Wong and Rabie, 2008). However, additional evidence from randomized controlled trials is required to identify other regulatory mechanisms that may be responsible for the anti-osteoporotic effects. The bioactive constituents of these extracts also remain unknown.

Anti-Fatigue Activity
RYR extracts exhibit strong anti-fatigue capability. The oral administration of RYR (24 mg/kg) to dyslipidemia patients for 4 weeks was found to significantly reduce muscle fatigue and preserve physical activity (Xue et al., 2017). Nevertheless, only a single dose of RYR was used in this study. In a Wistar rat model, the administration of powdered RYR (at a very high dose of 1-5 g/kg) for 4 weeks significantly extended the swimming time for the rats, effectively delayed the lowering of the glucose level in the blood, prevented the increase in lactate and blood urea nitrogen concentrations, and decreased the contribution of exerciseinduced oxidative stress, suggesting that RYR has anti-fatigue activity and may potentially be a useful pharmacological agent (Wang et al., 2006). However, the main shortcomings of this study were that the dose of RYR was too high and no mechanism of action was provided. RYR extracts are abundant monacolins and pigments. The administration of MK (10-90 mg/kg) or ethanol-soluble red pigments (50-200 mg/kg) to mice for 30 days significantly improved the hepatic glycogen level, decreased the serum urea nitrogen level, and increased the swimming time in an endurance test, indicating that MK and red pigments are responsible for the anti-fatigue activity of RYR (Chen, 2004). However, future studies are needed to identify the mechanism of action of these constituents.

Anti-Diabetic Activity
Diabetes is assuming epidemic proportions across the world (Lam and LeRoith, 2012). The best approach to control hyperglycemia and mitigate diabetes is dietary intervention, rather than a reliance on drugs (Patel, 2016). In this regard, the possible role of RYR was investigated. The administration of RYR (50-350 mg/ kg) to streptozotocin-induced diabetic rats for 2 weeks markedly decreased the plasma glucose level, reversed hyperphagia, and decreased the mRNA level of phosphoenolpyruvate carboxykinase in the liver in a dose-dependent manner, indicating that RYR decreased hepatic gluconeogenesis and lowered the plasma glucose level in diabetic rats (Chang et al., 2006). A similar study has reported that RYR could promote the release of acetylcholine from nerve terminals, which in turn stimulated muscarinic M3 receptors in pancreatic cells and augmented insulin release in order to lower the plasma glucose level (Chen and Liu, 2006). In another study, the administration of RYR (300 mg/kg) to diabetic mice for 8 weeks significantly decreased the blood glucose level by improving glucose tolerance and insulin secretion, protected islets from hyperglycemic injury, and inhibited the expression of key factors in oxidative stress, including 8-OHdG, 4-HNE, and gp91phox, indicating that the effects of RYR on oxidative stress may at least partly account for the improved insulin secretion of pancreatic islets in diabetes . However, this evidence is still tenuous; no detailed clinical trials involving RYR supplementation have been performed, and no chemical constituents of RYR responsible for the anti-diabetic activity have been presented. Furthermore, several studies also failed to include positive controls and dose-dependent effect analyses.

Anti-Obesity Activity
Obesity is defined as an excess of white adipose tissue, which is associated with a higher risk of developing diabetes and cardiovascular disease (Marques et al., 1998). RYR has been shown to possess anti-obesity activity. In mice fed a high-fat diet, the administration of RYR (at a very high dose of 4-20 g/ kg) significantly lowered weight gain and fat pad mass, which was accompanied by smaller fat cells. The anti-obesity effects of RYR were mainly derived from the lipolytic activity and mild anti-appetite potency of RYR. In 3T3-L1 cells, the investigators demonstrated that RYR extracts suppressed the proliferation and differentiation of preadipocytes, which may have inhibited new adipocyte formation or hyperplasia in adipose tissue . The main shortcoming in this study was the very high dose of RYR, which will restrict the clinical application of RYR in the treatment of obesity.

Anti-Inflammatory Activity
Inflammation signifies an agitated metabolic and immunological system (Odegaard and Chawla, 2013), and anti-inflammatory drugs that can restore the homeostasis of a perturbed system are in great demand. Various anti-inflammatory drugs exist, although many associate with adverse effects (Patel, 2016). RYR has been reported to possess an inflammation-quenching effect. In RAW264.7 cells induced by lipopolysaccharide, the administration of azaphilonoid derivatives isolated from RYR (5-20 μg/ml), including monapurfluores A and B, monascopyridines C and D, and monasfluores A and B, significantly inhibited the release of the inflammatory mediator nitric oxide (NO) from macrophages (Hsu et al., 2010). NO has been reported to be a signaling agent for various inflammatory diseases; therefore, its suppression is likely to ameliorate irritation (Patel, 2016). However, this study failed to include a positive control, and other inflammatory indicators, such as IL-1β, TNF-α, and IL-6, should be studied in the future. In mice fed a high-cholesterol diet, the administration of XZK (300 mg/kg) for 6 weeks significantly reduced the levels of the inflammatory transcription factors TNF-α and IL-6, and attenuated renal injury by managing the abnormal lipid levels and the consequent stress (Ding et al., 2014). This study also lacked a positive control, and the effects of the various doses were not assessed.

Anti-Hypertensive Activity
Hypertension, commonly recognized as a silent killer, is the most common cardiovascular disease and a major risk factor for atherosclerosis, metabolic syndrome, renal dysfunction, myocardial infarction, heart attack, and stroke, which are the most prevalent causes of death in industrialized countries (Williams, 2009;Wang et al., 2010). As many drugs have side effects, healthy diets are expected to prevent or alleviate the complications. The potential of RYR, in combination with other bioactive components, to manage the problem is discussed below. In grade 1 essential hypertension patients, the administration of a nutraceutical formulation containing RYR, folic acid, coenzyme Q 10 , Orthosiphon stamineus (a tropical herb from the Lamiaceae family), policosanol, and berberine significantly reduced the mean 24-h systolic and diastolic blood pressure levels (Trimarco et al., 2012). However, the ratios between RYR and the other constituents are not optimized based on the antihypertensive activity. In spontaneously hypertensive rats, the intravenous bolus administration of RYR ethanol extracts (10-50 mg/kg) elicited biphasic and potent hypotensive and cardiac inhibitory effects, as well as a significant decrease in sympathetic vasomotor activity . Further studies are needed to identify the chemical constituents in RYR ethanol extracts responsible for the anti-hypertensive effects. Metaanalysis of the existing literature showed that RYR, together with conventional therapy and statins, can lower the systolic blood pressure, without any significant adverse side effects (Patel, 2016;Xiong et al., 2017). Taken collectively, the results are encouraging but limited. Although meaningful reductions in blood pressure have been observed, evidence for the use RYR in the treatment of hypertension is weak based on the available data. Thus, more rigorous, high-quality trials are required to provide stronger evidence.

Other Activities
Polysaccharides from RYR have been shown to possess immunomodulatory activity. In a mice model, the administration of RYR-derived polysaccharides (50-300 mg/ kg) significantly improved the phagocytic activity of abdominal cavity macrophages, prompted the formation of the peripheral blood E-rose loop, increased the transformation rate of lymphocytes, and enhanced nonspecific immunity (Zhang et al., 2008). However, this study failed to include a positive control, and the relationship between polysaccharide structure and immunomodulatory activity should be further investigated. In laying hens, the administration of RYR significantly increased the laying rate, Haugh units, and albumen height, as well as decreased the yolk cholesterol content, suggesting that RYR plays important roles in improving the production and quality of eggs (Sun et al., 2015). Further studies are required to determine the level of citrinin, a nephrotoxin in RYR, in the serum and eggs of hens in order to meet a lower citrinin level with regard to RYR production.
Methane (CH 4 ), a greenhouse gas that is 25 times more effective than carbon dioxide in global warming, is emitted during enteric fermentation in ruminants, accounting for approximately 10% of the total anthropogenic CH 4 (Holter and Young, 1992;Hristov et al., 2013). In a castrated Boer crossbred goat model, the administration of RYR significantly reduced enteric CH 4 emissions, serum lipid levels, and the growth of several archaeal species (e.g. Methanobrevibacter) in the rumen. Therefore, as a natural fermentation product, RYR can reduce enteric methane emissions in goats (Wang et al., 2016). However, caution should be taken when considering the digestibility of protein and organic matter, and further studies are needed to evaluate its effects in different animals with various diets as well as its effects on animal health and food safety.

PHARMACOKINeTIC STUDIeS
MK (also known as lovastatin), categorized as a class II compound according to the Biopharmaceutics Classification System (BCS), is mainly responsible for the blood cholesterol lowering effect of RYR (Wu and Benet, 2005). MK exhibits poor oral bioavailability (<5%) because of its low solubility (1.3 μg/ mL in water), extensive metabolism in the gut and liver, and transmembrane efflux via the drug transporter P-glycoprotein (Serajuddin et al., 1991;Chen et al., 2005). Its bioavailability can be improved by increasing the dissolution rate and/or decreasing pre-systemic clearance (Neuvonen et al., 2008;Wu et al., 2011). In Caco-2 cells, extracts of RYR (LipoCol Forte, XZK, and Cholestin) were more effective in inhibiting the activities of CYP450 enzymes and P-glycoprotein, and showed higher lovastatin absorption and dissolution rates than that of lovastatin alone (Chen et al., 2012a;. Moreover, in human studies, the authors demonstrated that volunteers receiving LipoCol Forte capsules or powder exhibited higher area under the plasma concentration-time curve and C max (maximum plasma concentration) values for both lovastatin and its active metabolite, lovastatin acid, and lower T max (time to reach the peak concentration) values than those receiving lovastatin tablets, suggesting that the oral bioavailability of lovastatin is significantly improved when combined with RYR as a result of the higher dissolution rate . In this regard, the increased dissolution rate seen with RYR products might enable lovastatin (a BCS class II drug) to function as a BCS class I drug. Additionally, Leone and colleagues (Leone et al., 2016) prepared a new formulation that combined 60% gelatin with 40% alginate. They observed a delayed release of lovastatin from RYR, a prolonged inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and decreased cholesterol synthesis. However, further studies are needed to explore the underlying mechanism of actions, as well as the pharmacokinetics of the other bioactive compounds present in RYR, including monacolins and pigments such as rubropunctamine, monascorubramine, and ankaflavin.

QUALITY CONTROL
To assess and control the quality of RYR, the Chinese Pharmacopoeia suggests using morphology, ultraviolet-visible spectrophotometry, thin-layer chromatography, and highperformance liquid chromatography (HPLC). MK can be used as a marker of quality in the official monograph of the RYR as well as in the label claim of several commercial RYRrelated products. The MK content of RYR should be more than 0.22% by HPLC (Chinese Pharmacopoeia, 2015). However, the European Commission has not legislated on the limits for RYR supplements and there is no standardization in European countries, while the Food and Drug Administration (FDA) has stated in 2007 that RYR products containing MK are identical to a drug, and therefore, subject to regulation (FDA, U.S, 2007). With regard to therapeutic efficacy, a 2011 European Food Safety Authority (EFSA) report confirmed that the daily intake of 10 mg of MK was beneficial in maintaining normal cholesterol levels (EFSA, 2011). However, manufactures of RYR products rarely disclose the levels of MK and the active ingredients are not standardized.
In RYR extracts, the content of active ingredients, which includes MK, pigments, phenolic compounds, and flavonoids, varies in quality with the fermentation process, extraction procedures used, strain of M. purpureus, and storage conditions. The MK concentration in RYR can be increased by the mixedculture fermentation of rice with two different Monascus species (M. purpureus and M. ruber), with the optimized fermentation process parameters including a pH of 6.03 at a temperature of 29.46°C for 13.89 days. This process predicts 2.83 mg/g and yields 2.80 mg/g of MK/gram of fermented rice with 98.93% accuracy (Panda et al., 2010). Moreover, the supplementation of the fermentation substrate with nitrogen sources influences the pigment production efficiency of RYR. For instance, the addition of orange peels and sunflower meal to solid-state fermentation (SSF) cultures of M. purpureus can enhance pigment production in RYR, yielding 9 absorbance units (AU)/per g of dry fermented substrate (gdfs) (Kantifedaki et al., 2018). Babitha and colleagues (Babitha et al., 2007) reported that the addition of monosodium glutamate to jackfruit seed SSF of M. purpureus improved the production of red (30.8 AU/gdfs) and yellow (25.5 AU/gdfs) pigments, followed by the addition of soybean meal, peptone, and chitin powder. In addition, a combination of light and bacteria can be used to enhance secondary metabolite production during RYR fermentation. The fermentation of RYR with Bacillus subtilis, a rod-shaped, Gram-positive endospore-forming bacterium, under blue light-emitting diodes enhanced the production of phenolic compounds (68.4 ± 1 mg GAE/g DW) and flavonoids (51.7 ± 1 mg QE/g DW) compared to white light and darkness (Elumalai et al., 2019). Further studies should focus on the molecular mechanisms and optimization of conditions for the commercialscale production of these secondary metabolites. Under optimal extraction conditions with 45% ethanol, 1.5% phosphate, and an extraction time of 70 min, 91.6% of citrinin was removed and 79.5% of MK was retained in the final RYR extract (Lee et al., 2007a). M. purpureus strains are commonly used to enhance the production of MK and pigments in RYR. Using rigorous physicochemical mutagenesis technologies involving ultravioletlithium chloride, microwave heating, and genome shuffling mutations, two mutant M. purpureus strains, strain 183-3 with a highly stable capacity to produce pigment and strain R"-30 with a highly stable capacity to produce MK, were obtained (Xu, 2014;Li, 2016). In addition, Li et al. reported that the MK content in RYR significantly decreased under conditions of high humidity, high temperature (75% RH, 60°C), and sunlight, indicating that MK in RYR is light-and heat-sensitive, and RYR should be stored in the cool and dark place (Li et al., 2005a).
The area in which RYR is produced also influences its quality. RYR is now produced in more than 120 cities in 15 countries in Asia, Europe, and North America, such as China, Japan, the United States, Germany, Italy, Thailand, India, Korea, and Belgium. The general geographical distribution of RYR is shown in Figure S2. In studies that used UHPLC-DAD-QToF-MS to determine the MK content in 26 brands of RYR from four large retail chains in the United States (i.e. GNC, Walgreens, Walmart, and Whole Foods), it was reported that the MK content was highly variable, ranging more than 60-fold from 0.09 to 5.48 mg per 1,200 mg (Cohen et al., 2017). In another study that involved LC-PDA-MS, Huang and colleagues found that RYR from cities in Fujian Province, especially Gutian, Nanping, and Pinnan, possessed a higher MK content compared to six other habitats in China (Huang et al., 2006). These results indicate that the MK content in RYR extracts significantly differs between samples from different habitats. Thus, the MK content should be compared across RYR products, uniform legislation should be proposed, and strict quality control of RYR extracts should be implemented.
Citrinin is a polyketide secondary metabolite found in food and feed that is produced by several fungi, including M. purpureus (Bezeria da Rocha et al., 2014;Kiebooms et al., 2016). It is a mycotoxin known to cause kidney damage as well as disrupt metabolic processes in the liver . Citrinin, as a component, has been involved in several controversies related to the quality and safety of RYR products, because up to 80% of RYR products may contain this mycotoxin (Heber et al., 2001). The EFSA has set a level of no concern for citrinin-caused nephrotoxicity at 0.2 μg/kg body weight per day, but it has acknowledged the need for more reliable data on the citrinin level in food and feed before it can undertake exposure studies and risk assessment (Marley et al., 2016). Different analytical methods have been developed for the quantification of citrinin in a variety of samples in the last few years, including LC-FDA (Marley et al., 2016), UHPLC-MS/ MS (Kiebooms et al., 2016), UHPLC-DAD-QToF-MS (Avula et al., 2014), LC-DAD/FLD-MS n , microspherebased flow cytometric immunoassays (MFCI) , and a multi-commutated fluorometric optosensor technology (Jimenez Lopez et al., 2014). The maximum allowed level of citrinin as a contaminant in RYR is 200 ppb in Japan, but the European Union has recommended a limit of 100 ppb Nigovic et al., 2013). However, many commercial RYR products do not meet the criteria, and the highest citrinin concentration detected by HPLC was 140 mg/kg (Ji et al., 2015). More in-depth studies are necessary to arrive at more accurate conclusions and to extrapolate data for risk assessment with respect to the prevalence of citrinin in our food chain and its potential impact on human health.

SAFeTY AND ADveRSe eFFeCTS
According to the currently available results of in vitro and in vivo studies, RYR does not seem to be no mutagenic or toxic. In an albino rat model, the administration of acute doses of RYR at 0.5-5.0 g/kg body weight did not cause toxicity or mortality. Similarly, the administration of RYR at a level of 2.0-12.0% (w/w) for 14 weeks did not produce any significant changes in the food intake or body weight of the treated rats compared to control rats (Kumari et al., 2009). In an Ames test, the equivalent of up to 1 mg of ethanol extract from RYR per plate exhibited no genotoxicity toward Salmonella typhimurium strains TA 98, TA 100, and TA 102. Moreover, it has been reported that high levels of RYR exhibited no toxicity, following subchronic administration at levels up to 1,000 mg/kg of body weight for 28 and 90 consecutive days in the rats (Yu et al., 2008).
However, RYR does have adverse effects because MK and citrinin associate with an increased risk of myopathy (Lapi et al., 2008;Polsani et al., 2008;Dobremez et al., 2018), symptomatic hepatitis (Roselle et al., 2008), peripheral neuropathy (Kumari et al., 2013), erectile dysfunction (Liu and Chen, 2018), and anaphylactic reactions (Hipler et al., 2002). Among these, the prevalence rates of myopathy and hepatitis are the highest. Using Italy's WHO-UMC system, CIOMS/RUCAM score, and WHO-Vigibase, 52 out of 1,261 studies reported 55 adverse reactions to RYR dietary supplements from April 2002 to September 2015, and myopathy and hepatitis accounted for 52.73% of the total adverse reactions (Mazzanti et al., 2017). Although RYR supplementation appears promising for patients with hypercholesterolemia or hyperlipidemia or those at high risk for cardiovascular events, there is insufficient regulation of RYR-containing supplements in China, the United States, and European countries. Until there is a standardized system in place for product formulation and manufacturing, and the amount of MK in a given RYR supplement is clearly stated, the effectiveness and safety of these products will remain in question (Dujovne, 2017;Peng et al., 2017). Thus, realworld vigilance should be used, and it should include consumers, clinicians, and policymakers to promote the proper use of RYR. Furthermore, randomized controlled trials should be carried out to test the safety and efficacy of each RYR preparation.
The safety profile of RYR supplements is highly similar to that of statins (Mazzanti et al., 2017). Some statins, including lovastatin (MK in RYR), are metabolized by the cytochrome P450 enzyme CYP3A4 and it is known that coadministration of drugs that inhibit CYP3A4 can increase the plasma half-lives of these statins and the risk of myotoxicity (Mastaglia and Needham, 2012). CYP3A4 inhibitors reported to interact with statins and to cause rhabdomyolysis include clarithromycin (Grunden and Fisher, 1997), verapamil (Choi et al., 2010), cyclosporine (Olbricht et al., 1997), diltiazem (Azie et al., 1998), itraconazole (Neuvonen and Jalava, 1996), and azithromycin (Grunden and Fisher, 1997). Statininduced rhabdomyolysis may result from increased exposure to the drug, plausible mechanisms for the interaction include increased absorption of lovastatin by competition at gut P-glycoprotein or decreased excretion of statin due to competition for renal tubular secretion or interaction at hepatic CYP3A4 sites (DiGregorio and Pasikhova, 2009). Co-administration of a statin and a fibrate drug also increases the rhabdomyolysis risk, and gemfibrozil in particular should be avoided in patients receiving lovastatin (Franssen et al., 2009). Therefore, the healthcare providers need to heighten their awareness and screening for potential drug-drug interaction in patients consuming RYR products, and consider early monitoring of liver function and signs of muscle injury. In parallel, consumers should be informed that MK contained in RYR is identical to lovastatin, and need to be discouraged from using RYR preparations as self-medication, particularly if they have experienced previous adverse reactions to statins.

CONCLUSIONS AND FUTURe PeRSPeCTIveS
RYR is widely used in prescriptions, as well as an alternative medicine and a food supplement, in Asia, the United States, and European countries. This review summarized the current information on the traditional uses, chemistry, pharmacology, pharmacokinetics, quality control, and toxicity of RYR. In classical Chinese herbal textbooks and the Chinese Pharmacopoeia, RYR is commonly used for resolving turbidity, invigorating blood circulation, and resolving blood stasis. Pharmacological studies have revealed that RYR possesses many biological properties with hypolipidemic, antiatherosclerotic, anti-cancer, neurocytoprotective, hepatoprotective, anti-osteoporotic, anti-fatigue, anti-diabetic, anti-obesity, immunomodulatory, anti-inflammatory, anti-hypertensive, and anti-microbial activities. The results of these pharmacological investigations support the traditional use of RYR. Furthermore, more than 101 compounds have been isolated from RYR. Among these, monacolins and pigments are the most abundant and the major bioactive constituents in RYR.
Outstanding progress has been made in the chemistry and pharmacology of RYR. However, several gaps in knowledge will require additional studies. Firstly, the systematic data on the toxicology and drug interactions of RYR extracts are limited, and there are only a few studies documenting the toxic effects of RYR. Presently, the evidence is insufficient for RYR to be interpreted as toxic. Thus, a comprehensive toxicological study of both the bioactive extracts and isolated constituents from RYR is urgently needed. Furthermore, due to its potential in vitro and in vivo toxic effects, the clinical application of RYR should be restricted until more definitive studies demonstrate its safety, quality, and efficacy. Secondly, although monacolins in RYR have been found to possess pharmacological activities that are similar to those in pigments, such as hypolipidemic activity, the mechanisms underlying the absorption, distribution, metabolism, and excretion, as well as the synergistic or antagonistic effects between the two constituents, are unknown; thus, they should be further studied. Moreover, other bioactive constituents of RYR, such as organic acids, sterols, decalin derivatives, and flavonoids, should also be examined for their potential use in the development of drugs and as a food supplement. Thirdly, well-designed pharmacodynamic and pharmacological studies should be designed, and comprehensive investigations should be conducted to identify the relevant compounds responsible for the pharmacological effects and the potential mechanisms. Fourthly, although the pharmacological properties support the traditional uses of RYR in the treatment of blood circulation stasis, bone defects, and limb weakness, additional pharmacological studies are needed to address its use in the remediation of indigestion and diarrhea. Finally, the possible interactions, safety profile, synergistic or antagonistic effects, and underlying mechanisms between the bioactive compounds present in RYR and other combined used products remain unknown and should be further studied.
In conclusion, future studies should focus on the absorption, distribution, metabolism, and excretion of monacolins and their interactions with pigments, which would promote our understanding of the underlying mechanisms of the various biological activities of RYR. Further research should also consider the pharmacological activities that were overlooked with regard to the traditional uses of RYR, especially in the treatment of gastrointestinal diseases, such as indigestion and diarrhea. Finally, more efforts are needed in order to gain new insights into the toxicological effects of the main active compounds and the quality control of RYR based on its diverse chemical constituents.

DATA AvAILABILITY STATeMeNT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
BZ, JH, and LQ conceived the review. BZ, GY, and FQ wrote the manuscript. JW collected the literatures. QZ edited the manuscript. All the authors have seen and agreed on the finally submitted version of the manuscript.