A Review of Red Yeast Rice, a Traditional Fermented Food

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Review A Review of Red Yeast Rice, a Traditional Fermented Food in Japan and East Asia: Its Characteristic Ingredients and Application in the Maintenance and Improvement of Health in Lipid Metabolism and the Circulatory System

Hiroyuki Fukami 1,*, Yuki Higa 1 , Tomohiro Hisano 1, Koichi Asano 1, Tetsuya Hirata 1 and Sansei Nishibe 2

1 Central R&D Laboratory, KOBYASHI Pharmaceutical Co., Ltd., Ibaraki 567-0057, Japan; [email protected] (Y.H.); [email protected] (T.H.); [email protected] (K.A.); [email protected] (T.H.) 2 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari 061-0293, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-80-3452-9483; Fax: +81-72-640-0137

Abstract: Red yeast rice has been used to produce alcoholic beverages and various fermented foods in China and Korea since ancient times; it has also been used to produce tofuyo (Okinawan- style fermented tofu) in Japan since the 18th century. Recently, monacolin K (lovastatin) which has cholesterol-lowering effects, was found in some strains of Monascus fungi. Since statins have Citation: Fukami, H.; Higa, Y.; been used world-wide as a cholesterol-lowering agent, processed foods containing natural statins Hisano, T.; Asano, K.; Hirata, T.; are drawing attention as materials for primary prevention of life-style related diseases. In recent Nishibe, S. A Review of Red Yeast years, large-scale commercial production of red yeast rice using traditional solid-state fermentation Rice, a Traditional Fermented Food in has become possible, and various useful materials, including a variety of monascus pigments Japan and East Asia: Its Characteristic (polyketides) that spread as natural pigments, in addition to statins, are produced in the fermentation Ingredients and Application in the Maintenance and Improvement of process. Red yeast rice has a lot of potential as a medicinal food. In this paper, we describe the history Health in Lipid Metabolism and the of red yeast rice as food, especially in Japan and East Asia, its production methods, use, and the Circulatory System. Molecules 2021, ingredients with pharmacological activity. We then review evidence of the beneﬁcial effects of red 26, 1619. https://doi.org/10.3390/ yeast rice in improving lipid metabolism and the circulatory system and its safety as a functional food. molecules26061619 Keywords: red yeast rice; Monascus fungi; solid-state fermentation; monacolin; statin; LDL-cholesterol; Academic Editors: Alessia Fazio and polyketide; pigment; lipid metabolism; cardiovascular system Pierluigi Plastina

Received: 28 December 2020 Accepted: 5 February 2021 1. Introduction: Overview of Red Yeast Rice, Its History, Production Method, and Use Published: 15 March 2021 1.1. The History behind Koji (Yeast Grain) and Red Yeast Rice (Red Koji)

Publisher’s Note: MDPI stays neutral Every country in the world has its own traditional food culture, and quite a few with regard to jurisdictional claims in countries have their own brewed foods, made using different fermentation mechanisms. published maps and institutional afﬁl- In contrast to western countries, where malt and fruit juice are used for fermentation, in iations. Eastern and Southeastern Asia, fermented foods are produced using rice and beans malted with ﬁlamentous fungus such as Aspergilli. Fungi are widely used since the areas have a climate of high temperatures and humidities. Koji is made by breeding molds that can be used for food fermentation on grains such as rice, wheat, and soybeans. Miso, soy-sauce, vinegar, mirin (sweet sake), pickles, sake Copyright: © 2021 by the authors. Koji Licensee MDPI, Basel, Switzerland. (Japanese rice wine), and distilled spirit are all made from various grains which play a This article is an open access article central role in “Wasyoku”, a food culture in Japan. distributed under the terms and There are various types of koji and koji molds (also called koji fungi): yellow koji (made conditions of the Creative Commons with A. oryzae), used to produce miso, soy-sauce, mirin and sake; black koji (made with A. Attribution (CC BY) license (https:// awamori), used to produce awamori (Okinawan distilled spirit); and black koji or white koji creativecommons.org/licenses/by/ (made with A. Kawachii), used to produce syochuu (Japanese distilled spirits). Red yeast rice 4.0/). (red yeast grain, red koji, Beni-Koji), made with Monascus fungi, has been used to produce

Molecules 2021, 26, 1619. https://doi.org/10.3390/molecules26061619 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 20

Molecules 2021, 26, 1619 2 of 20

(made with A. Kawachii), used to produce syochuu (Japanese distilled spirits). Red yeast rice (red yeast grain, red koji, Beni-Koji), made with Monascus fungi, has been used to pro- Tofunyuduce Tofunyu(fermented (fermented bean curd) bean incurd) Taiwan in Taiwan and China and Ch sinceina ancientsince ancient times andtimes has and also has been also usedbeen toused produce to producetofuyo tofuyo(Okinawan (Okinawan style fermentedstyle fermented bean curd)bean incurd) Japan. in Japan. MonascusMonascus fungifungi produceproduce redred pigmentspigments andand thethe kojikoji producedproduced withwith MonascusMonascus fungifungi exhibitexhibit aa deepdeep redred colorcolor (Figure(Figure1 1).). These These red red pigments pigments have have been been used used industrially industrially since since thethe 1950s1950s inin Japan.Japan. ForFor example,example, theythey havehave been usedused inin processedprocessed fishfish pastepaste products,products, suchsuch asas crab-flavored-fishcrab-flavored-fish cakescakes andand processedprocessed meatmeat products,products, suchsuch asas hamham andand sausages. Recently,Recently, theythey havehave beenbeen widelywidely usedused inin variousvarious foodsfoods includingincluding bread,bread, confectionariesconfectionaries andand beverages,beverages, suchsuch asas amazakeamazake (fermented(fermented ricerice drink).drink).

Figure 1. Appearance of red yeast rice. Figure 1. Appearance of red yeast rice. Red yeast rice is recorded in Bencao Gangmu (Compendium of Materia Medica), a representative book on Chinese medicine, written in 1596 which has been highly valued Red yeast rice is recorded in Bencao Gangmu (Compendium of Materia Medica), a since ancient times in China. Lovastatin (monacolin K), which has cholesterol lowering representative book on Chinese medicine, written in 1596 which has been highly valued effects, was found as an active ingredient from Monascus fungi [1–3]. Statins such as since ancient times in China. Lovastatin (monacolin K), which has cholesterol lowering lovastatin and its analogs are used world-wide as serum cholesterol lowering agents which effects, was found as an active ingredient from Monascus fungi [1–3]. Statins such as lovas- are used as first-line drugs for atherosclerotic diseases. Evidence for the beneficial and tatin and its analogs are used world-wide as serum cholesterol lowering agents which are pleiotropic effects of statins has been reported, and further accumulation of evidence is used as first-line drugs for atherosclerotic diseases. Evidence for the beneficial and plei- expected in the future. Red yeast rice also contains lovastatin, and although it is currently otropic effects of statins has been reported, and further accumulation of evidence is ex- used as a functional food, it could be used in the prevention of life-style related diseases pected in the future. Red yeast rice also contains lovastatin, and although it is currently to reduce metabolic syndrome and the metabolic domino, through the improvement of used as a functional food, it could be used in the prevention of life-style related diseases dietary habits. to reduceIn this metabolic study, we syndrome review Monascus and the fungi:metabolic their domino, history, through production the improvement methods of red of yeastdietary rice, habits. foods made using red yeast rice, characteristic ingredients, evidence of potential beneficialIn this effects study, to improvewe review lipid Monascus metabolism fungi: and their the history, circulatory production system, andmethods safety. of red yeast rice, foods made using red yeast rice, characteristic ingredients, evidence of potential 1.2.beneficial Monascus effects Fungi to improve and Red Yeast lipid Rice metabolism and the circulatory system, and safety. Monascus fungi are taxonomically classified into Hemiascomycetes; they are filamentous fungi1.2. Monascus belonging Fungi to theand AscomycetesRed Yeast Riceclass, similar to Aspergillus, which are used in the productionMonascus of miso fungi, soy-sauce are taxonomically and sake. TheseclassifiedMonascus into Hemiascomycetesfungi are called; they red koji aremold filamen- (or redtous koji fungi fungi) belonging because to theythe Ascomycetes produce red class pigments;, similar approximately to Aspergillus, which 20 strains are used have in been the identifiedproduction so of far miso (Table, soy-sauce1). Most and of thesake.Monascus These Monascusfungi were fungi found are called in red red yeast koji ricemold and (or brewedred koji foods fungi) or because beverages they produced produce with red red pigments; yeast rice approximately in China, Taiwan, 20 strains or Korea have from been the secondidentified half so of far the (Table 1920s to1). the Most first of half the of Monascus the 1930s fungi [4]. About were 50found strains in red of Monascus yeast ricefungi and arebrewed preserved foods inor thebeverages Biological produced Resource with Center, red ye Departmentast rice in China, of Biotechnology, Taiwan, or Korea National from Institutethe second of Technologyhalf of the 1920s andEvaluation to the first (NITE)half of the in Japan 1930s (Table [4]. About2)[5]. 50 strains of Monascus fungi are preserved in the Biological Resource Center, Department of Biotechnology, Na- tional Institute of Technology and Evaluation (NITE) in Japan (Table 2) [5].

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Table 1. Typical Monascus fungi and their isolation source.

Strain Source M. purpureus Red yeast rice, malted rice (miquzi in Chinese) (China, Korea, Taiwan) M. anka Red yeast rice (Taiwan), malted rice of red nyufu M. anka var. rubellus Lees from the making process of red Laojiu (Chinese alcoholic beverage) M. barkeri Malted rice for samutu-syu (Chinese red alcoholic beverage) M. albidus Chantofu (Shanghai) M. araneosus Malted rice for gaoliangilu (Chinese distilled spirit, Northeastern China) M. furiginosus Malted rice (Guizhou Province, China) M. major Malted rice (Fuzhou, China) M. albidus var. glaber Malted rice (Fuzhou, China) M. pilosus Malted rice for gaoliangilu (Fengtian, China) M. rubropanctatus Powdered malted rice for medicinal wine (Incheon, Korea) M. pubigerus Malted rice for gaoliangilu (Liaoyang, China) M. rubinosus Malted rice (Guangdong Province) M. serorubescens Red funyu (Hong Kong) M. vitreus Red funyu (Hong Kong) M. kaoliang Malted rice for gaoliangilu (Taiwan) M. ruber Silage, soil, rotten fruit etc. M. paxi Dead branch and leaves of plants

Table 2. Representative Monascus fungi preserved at the Biological Resource Center (NBRC), NITE in Japan.

Strain M. anka Nakazawa et Sato NBRC 4478 M. purpreus Went NBRC 4513 M. anka Nakazawa et Sato (T. Hasegawa) NBRC 6540 M. major Sato NBRC 4485 M. ruber van Tieghem NBRC 4492 M. pubigerus Sato NBRC 4521 M. araneosus Sato NBRC 4482 M. rubiginosus Sato NBRC 4484 M. anka var. rubellus Sato NBRC 5965 M. ruber van Tieghem (S. Udagawa) NBRC 9203 M. pilosus Sato (FAT) NBRC 4520 M. fuliginosus Sato NBRC 4483 M. pilosus Sato NBRC 4480 M. paxii Lingelsheim NBRC 8201 M. vitreus Sato NBRC 4532 M. vitreus Sato (J. Nicot) NBRC 7537 M. albidus Sato NBRC 4489 M. anka var. rubellus Sato (H. Iizuka) NBRC 6085 M. serorubescens Sato NBRC 4487 M. albidus var. glaber Sato NBRC 4486 M. serorubescens Sato (FAT) NBRC 4525

1.3. Production of Red Yeast Rice Red yeast rice is documented as Chinese herbal cuisine that improves blood circulation in Nichiyo-Honzo, a Chinese herbology book written by Duan Wu in 1329. Red yeast rice was also called tan-giku, and its production method is described in Tiangong Kaiwu, a compendium on industry, agriculture and artisanry, written by Song Ying in the 17th century in China (Figure2)[ 4,6]. In brief, after soaking in water for 7 days, polished rice is steamed, and the lees of Shaoxing wine are added as seed fungi and cultured in a tile room for about 1 week. Interestingly, the method is very similar to that used in the production of yellow koji in Japan. Monascus fungi (red koji fungi) have low proliferative ability, and about 1 week is required for the production of red yeast rice, though only 2 days are required for Molecules 2021, 26, x FOR PEER REVIEW 4 of 20

compendium on industry, agriculture and artisanry, written by Song Ying in the 17th century in China (Figure 2) [4,6]. In brief, after soaking in water for 7 days, polished rice is

Molecules 2021, 26, 1619 steamed, and the lees of Shaoxing wine are added as seed fungi and cultured in a tile room4 of 20 for about 1 week. Interestingly, the method is very similar to that used in the production of yellow koji in Japan. Monascus fungi (red koji fungi) have low proliferative ability, and about 1 week is required for the production of red yeast rice, though only 2 days are re- quiredthat of for yellow that kojiof yellow. The long-term koji. The long-term culture increases culture theincreases chance the of contaminationchance of contamination by various bygerms; various therefore, germs; therefore, the area of the each area production of each production process isprocess kept thoroughly is kept thoroughly clean, and clean, the andaseptically the aseptically cultured cultur red yeasted red rice yeast is used rice asis used the seed as the fungus. seed fungus.

FigureFigure 2. 2. ProductionProduction method method of of red red yeast yeast rice rice described described in in“Tiangong “Tiangong Kaiwu Kaiwu”. Rice”. Rice inoculated inoculated with with MonascusMonascus fungifungi is di- is vided between several bamboo trays, which are placed on shelves to maintain good air circulation. Ambient air during divided between several bamboo trays, which are placed on shelves to maintain good air circulation. Ambient air during the culture is a key factor. The room in which the shelves are placed must be wide and there must be a high ceiling. The the culture is a key factor. The room in which the shelves are placed must be wide and there must be a high ceiling. The room must also be facing south to avoid afternoon sunlight and its temperature should be controlled. Rice inoculated with theroom fungi must should also bebefacing mixed south up and to avoiddown afternoon3 times every sunlight 2 hours. and its temperature should be controlled. Rice inoculated with the fungi should be mixed up and down 3 times every 2 h. Red yeast rice itself has been used in the coloring of foods, meat in particular, in Red yeast rice itself has been used in the coloring of foods, meat in particular, in China and Taiwan since ancient times. Red pigments extracted and isolated from red yeast China and Taiwan since ancient times. Red pigments extracted and isolated from red yeast rice have been produced as natural pigments on an industrial scale since 1945. Since the rice have been produced as natural pigments on an industrial scale since 1945. Since the carcinogenicity of synthetic red pigments was discovered, the consumption of natural pig- carcinogenicity of synthetic red pigments was discovered, the consumption of natural ments (red yeast rice pigment) made by Monascus fungi has increased. pigments (red yeast rice pigment) made by Monascus fungi has increased. The traditional method for the production of red yeast rice-related foods was the The traditional method for the production of red yeast rice-related foods was the solid-statesolid-state culture culture method method described described above; above; however, however, the the pigments pigments are arenow now industrially industri- Molecules 2021, 26, x FOR PEER REVIEW 5 of 20 producedally produced by simple by simple processes processes of extraction of extraction and concentration and concentration from red from yeast red rice, yeast prolif- rice, eratedproliferated by an byaerated an aerated and agitated and agitated culture culture method method (liquid-state (liquid-state culture) culture) (Figure (Figure 3) [7].3 )[ 7].

Traditional solid-state fermentation method since ancient

Inoculation of Monascus fungi Polished rice Sterilization Culture Drying Grinding Product Water

Liquid-state fermentation method Inoculation of Monascus fungi Culture components Jar fermenter Sterilization Culture Sparation Red yeast rice solution Water

Filtration Concentration Flush heat pasteurization Freeze drying Product

Figure 3. Solid-state/liquid-state culture culture method method of of red red yeast yeast rice. rice.

1.4. Brewed Foods Produced Using Red Yeast Rice Red yeast rice has been widely used as a material for the production of anchu (Chi- nese red wine), tofunyu (fermented bean curd), and a coloring agent for foods or a preservative for meat in China and Taiwan. The production technique for red Laojiu (Chinese alcohol beverage) originated in a Fijian Province in China and was introduced into Taiwan about 200 years ago. It is said to be the origin of the present-day Taiwanese red alcoholic beverage called Hon-ru-chu that is popular at festivals and wedding ceremonies [4]. Tofunyu, also called funyu or nyuhu, is a flavored food produced using mold proliferated on bean cakes which are salted and then matured by soaking in unrefined sake [8,9]. Fermented bean curd produced using red yeast rice is often called red funyu, and it is said to have originated the period about 1500 years ago in China. Red funyu is now produced in Jiangsu, Zhejiang, Sichuan, Hong Kong and Taiwan. Brewed foods in East Asia were introduced to Japan sometime long ago. Tofuyo, a type of fermented bean curd (Figure 4), is a fermented food from Okinawa which is similar to red funyu in China and Taiwan. Tofuyo was introduced to Japan as “a fragrant and sweet/tasty food that improved digestive function and was effective in the treatment of various diseases” according to Gyozen Honzo (Edible plants of Okinawa), a book on dietary plants compiled in 1832 [4]. It was highly valued as a nutritional food and side dish in the 1800s. At present, tofuyo is produced by a novel method using aged sake, awamori (Okina- wan distilled spirit).

Figure 4. Tofuyo, a food that has been passed down the generations in Okinawa, Japan.

Funyu has been eaten for more than 1000 years in China. In Japan, it is prepared using miso produced with red yeast rice. In 1985, a Monuscus fungi was used to brew miso prepared by a traditional method, and it has been in use ever since [10].

Molecules 2021, 26, x FOR PEER REVIEW 5 of 20

Traditional solid-state fermentation method since ancient

Inoculation of Monascus fungi Polished rice Sterilization Culture Drying Grinding Product Water

Liquid-state fermentation method Inoculation of Monascus fungi Culture components Jar fermenter Sterilization Culture Sparation Red yeast rice solution Water

Filtration Concentration Flush heat pasteurization Freeze drying Product Molecules 2021, 26, 1619 5 of 20 Figure 3. Solid-state/liquid-state culture method of red yeast rice.

1.4.1.4. Brewed Foods Produced UsingUsing RedRed YeastYeast RiceRice RedRed yeastyeast ricerice has has been been widely widely used used as as a materiala material for for the the production production of anchu of anchu(Chinese (Chi- rednese wine), red wine),tofunyu tofunyu (fermented (fermented bean curd) bean, curd) and a, and coloring a coloring agent foragent foods for orfoods a preservative or a preserva- for meattive for in meat China in and China Taiwan. and Taiwan. TheThe productionproduction techniquetechnique forfor redred LaojiuLaojiu (Chinese(Chinese alcoholalcohol beverage)beverage) originatedoriginated inin aa FijianFijian ProvinceProvince inin ChinaChina andand waswas introducedintroduced intointo TaiwanTaiwan aboutabout 200200 yearsyears ago.ago. ItIt isis saidsaid toto bebe thethe originorigin ofof thethe present-daypresent-day Taiwanese Taiwanese red red alcoholic alcoholic beverage beverage called calledHon-ru-chu Hon-ru-chuthat that is popularis popular at festivalsat festivals and an weddingd wedding ceremonies ceremonies [4]. [4]. TofunyuTofunyu,, alsoalso calledcalled funyufunyu oror nyuhunyuhu,, isis aa ﬂavoredflavored foodfood produced using mold prolifer- atedated onon beanbean cakes which are salted and then matured by soaking in unreﬁnedunrefined sake [[8,9].8,9]. FermentedFermented beanbean curdcurd producedproduced usingusing redred yeastyeast ricerice isis oftenoften calledcalled redred funyufunyu,, andand itit isis saidsaid toto havehave originatedoriginated thethe periodperiod aboutabout 15001500 yearsyears agoago inin China.China. RedRed funyufunyu isis nownow producedproduced inin Jiangsu,Jiangsu, Zhejiang,Zhejiang, Sichuan,Sichuan, HongHong KongKong andand Taiwan.Taiwan. BrewedBrewed foodsfoods inin EastEast AsiaAsia werewere introducedintroduced toto JapanJapan sometimesometime longlong ago.ago. Tofuyo,Tofuyo, aa typetype ofof fermentedfermented beanbean curdcurd (Figure(Figure4 4),), is is a a fermented fermented food food from from Okinawa Okinawa which which is is similar similar toto redred funyufunyu inin ChinaChina andand Taiwan.Taiwan. Tofuyo waswas introducedintroduced toto JapanJapan asas “a“a fragrantfragrant andand sweet/tastysweet/tasty food food that that improved improved digestive func functiontion and was effective inin thethe treatmenttreatment ofof variousvarious diseases”diseases” accordingaccording totoGyozen Gyozen HonzoHonzo (Edible(Edible plantsplants ofof Okinawa),Okinawa), aa bookbook onon dietarydietary plantsplants compiled in 1832 1832 [4]. [4]. It It was was highly highly valued valued as as a nutritional a nutritional food food and and side side dish dish in the in the1800s. 1800s. At present, At present, tofuyotofuyo is producedis produced by a novel by a method novel method using aged using sake aged, awamorisake, awamori (Okina- (Okinawanwan distilled distilled spirit). spirit).

FigureFigure 4.4. Tofuyo,, aa foodfood thatthat hashas beenbeen passedpassed downdown thethe generationsgenerations inin Okinawa,Okinawa, Japan.Japan.

FunyuFunyu has been been eaten eaten for for more more than than 1000 1000 years years in China. in China. In Japan, In Japan, it is prepared it is prepared using usingmiso producedmiso produced with red with yeast red yeastrice. In rice. 1985, In 1985,a Monuscus a Monuscus fungifungi was used wasused to brew to brew miso misopre- preparedpared by bya traditional a traditional method, method, and and it has it has been been in use in use ever ever since since [10]. [10 ]. Anchu has been produced in Okinawa since the 18th century, according to a written record and products list [11]. For many years, it has been produced in China using yeast rice on which Monascus fungi are proliferated. During 1850–1900, it is said, red rice and steamed red rice cakes wrapped in bamboo leaves were a traditional Okinawan food [12]. In China,

a book called “Honzo-Jyushin”, published in 1751, described a method for preparing red rice cake as follows: rice was mixed with red yeast rice and steamed, and the resultant red rice cake could be offered for food. Red rice is prepared by steaming rice grains after they have been mixed with red yeast rice. Red rice cakes are prepared by mixing red yeast rice soaked in sake and mashed in advance. The red yeast rice pigments mentioned above have been used in processed fish paste products (including crab-flavored fish paste cakes), jam, tomato ketchup, sweetened bean jam, stewed octopus, salmon roe, and processed meat products such as ham and sausage. In addition, red yeast rice pigments are used for coloring bread, confectionaries including rice confectionaries, and beverages, including amazake, and they are also widely used as pigments for foods in Asia and Europe [6,13]. Molecules 2021, 26, 1619 6 of 20

2. Polyketides and Other Metabolites Produced in Monascus Fungi 2.1. Polyketides Identified in Red Yeast Rice The compendium “Tiangong Kaiwu” describes fish meat as “generally spoilable, but its quality can be kept by smearing red yeast rice on its surface, even in hot conditions; no fly or maggot will approach, even 10 days later: red yeast rice is a truly miracle agent”. This description indicates that red yeast rice has sterilizing or bacteriostatic action to prevent deterioration of fish meat caused by bacterial contamination (it may prevent fly swarming by other mechanisms). The custom to use red yeast rice in the preservation of foods is still used today; it is often used in the preservation of pork and chicken in Taiwan. Recent studies have demonstrated that M. purpureus produces substances with antibacterial activity against Bacillus, Streptococcus, and Pseudomonas species. Some of these substances are considered to be pigments. “Bencao Gangmu” (Compendium Materia Medica, vol. 25) written by Li Shizhen in 1590 describes a number of medicinal effects of red yeast rice, including that red yeast rice improves blood circulation, helps digestion, activates splenic function, cures diarrhea, heals bruises and injuries, enhances blood health, and protects a woman just after childbirth from blood stasis. Red yeast rice pigments (polyketides) are called azaphilone pigments, and the fol- lowing have been isolated and identified: monascin, ankaflavin, and monascinol (yellow pigments); rubropunctamine and monascorubramine (purple pigments); and rubropunc- tatin and monascorubrin (red pigments) (Figure5)[ 4,7,14]. Furthermore, it has been reported that red yeast rice produces polyketide derivatives such as monascumic acid and γ-amino butyric acid (GABA) etc. [15–17]. Monascus pigments are known to have antibacterial activity and anti-cancer effects and GABA to have blood pressure-regulating effect [18–20]. It has also been reported that fermented rice bran with Monascus increases phenolic acid and enhances antioxidant activity [21,22]. In addition, organic acids, amino Molecules 2021, 26, x FOR PEER REVIEWacids, sterols, decalin compound derivatives, flavonoids, lignans, coumarins, terpenoids,7 of 20 and polysaccharides are reported as components contained in red yeast rice [23].

FigureFigure 5. 5.Major Major pigments pigments (polyketides (polyketides oror azaphiloneazaphilone pigments) pigments) produced produced by by MonascusMonascus fungi.fungi.

AA potent potent inhibitor inhibitor of of cholesterol cholesterol synthesis,synthesis, monacolinmonacolin K (lovastatin) was discovered in 1979in 1979 in a in strain a strain of Monascus of Monascusby by Endo Endo et et al. al. [1 –[1–3,24].3,24]. Other Other active active substances substances withwith a sim- similar structureilar structure to monacolin to monacolin K have K have since since been been found found (Figure (Figure6)[ 6)25 [25,26].,26]. Monacolins Monacolins have have two forms,two forms, acid oracid lactone or lactone form, form, depending depending on theon the conditions conditions of of the the solution, solution, particularly particularly the pHthe (Figure pH (Figure7)[27 7)]. [27].

Figure 6. Monacolins (polyketides) produced by Monascus fungi.

Figure 7. Lactone and acid forms of monacolin K.

MoleculesMolecules 2021 2021, ,26 26, ,x x FOR FOR PEER PEER REVIEW REVIEW 77 ofof 2020

FigureFigure 5. 5. Major Major pigments pigments (polyketides (polyketides or or aza azaphilonephilone pigments) pigments) produced produced by by Monascus Monascus fungi. fungi.

AA potent potent inhibitor inhibitor of of cholesterol cholesterol synthesis, synthesis, monacolin monacolin K K (lovastatin) (lovastatin) was was discovered discovered inin 1979 1979 in in a a strain strain of of Monascus Monascus by by Endo Endo et et al. al. [1–3,24]. [1–3,24]. Other Other active active substances substances with with a a sim- sim- Molecules 2021, 26, 1619 ilarilar structure structure to to monacolin monacolin K K have have since since been been found found (Figure (Figure 6) 6) [25,26]. [25,26]. Monacolins Monacolins have have7 of 20 twotwo forms, forms, acid acid or or lactone lactone form, form, depending depending on on the the conditions conditions of of the the solution, solution, particularly particularly thethe pH pH (Figure (Figure 7) 7) [27]. [27].

FigureFigureFigure 6. 6. Monacolins Monacolins 6. Monacolins (polyketides) (polyketides) (polyketides) produced produced produced by by Monascus byMonascusMonascus fungi. fungi.fungi.

FigureFigureFigure 7. 7. Lactone Lactone 7. Lactone and and andacid acid acid forms forms forms of of monacolin monacolin of monacolin K. K. K. 2.2. Production of Metabolites, Including Representative Polyketides in Traditional Solid-State Fermentation Endo et al. also studied on red yeast rice that contained natural statins and favored by the Japanese in their diet. They examined color, taste and fragrance of various red yeast rice. Useful strains of Monascus fungi were bred, based on productivity of red yeast rice pigments and monacolin K. Recently, many attempts on breedings of Monascus strains and production conditions for efﬁcient production of monacolin K have been reported [28]. The developed red yeast rice was produced using a traditional solid-state fermentation method [7]. M. pilosus is frequently used as a health food in Japan; we studied changes in its metabolite contents during growth. Steam-sterilized rice was inoculated with M. pilosus NITE BP-412, and a solid-state culture was started when the water content reached 42%. The culture was grown for 43 days; a temperature of 30 ◦C was used for the ﬁrst 4 days and then 22 ◦C for the remaining time; the change in the contents of metabolites was then analyzed. The change in appearance of the red yeast rice during the 43 days is shown in Figure8 and demonstrates the gradual change in color of the rice from white to red. The change in monacolin K content in the red yeast rice during fermentation was measured: The contents of both the lactone and acid form time-dependently increased (Figure9). The contents of three azaphilone pigments (monascin, monascinol, and rubropunctamine) in red yeast rice were measured: The monascin content reached its peak 20 days after the start of the culture. Monascinol reached its peak 30 days after the start of the culture, and rubropunctamine began to increase about 10 days after the start of the culture and reached its peak at about 30 days. All pigments plateaued after they peaked (Figure 10). The time course change in the contents of two amino acids (GABA and monascumic acid) in red yeast rice during fermentation was also examined. The GABA content increased, with the peak occurring about day 20, and the level gradually decreased thereafter. The Molecules 2021, 26, x FOR PEER REVIEW 8 of 20

2.2. Production of Metabolites, Including Representative Polyketides in Traditional Solid-State Fermentation Endo et al. also studied on red yeast rice that contained natural statins and favored by the Japanese in their diet. They examined color, taste and fragrance of various red yeast rice. Useful strains of Monascus fungi were bred, based on productivity of red yeast rice pigments and monacolin K. Recently, many attempts on breedings of Monascus strains and production conditions for efficient production of monacolin K have been reported. [28]. The developed red yeast rice was produced using a traditional solid-state fermentation method [7]. M. pilosus is frequently used as a health food in Japan; we studied changes in its metabolite contents during growth. Steam-sterilized rice was inoculated with M. pilosus NITE BP-412, and a solid-state culture was started when the water content reached 42%. The culture was grown for 43 days; a temperature of 30 °C was used for the first 4 days and then 22 °C for the remaining time; the change in the contents of metabolites was then analyzed. The change in appearance of the red yeast rice during the 43 days is shown in Figure 8 and demonstrates the gradual change in color of the rice from white to red. The change in monacolin K content in the red yeast rice during fermentation was measured: The contents of both the lactone and acid form time-dependently increased (Figure 9). The contents of three azaphilone pigments (monascin, monascinol, and rubropunctamine) in red yeast rice were measured: The monascin content reached its peak 20 days after the start of the culture. Monascinol reached its peak 30 days after the start of the culture, and rubropunctamine began to increase about 10 days after the start of the culture and reached its peak at about 30 days. All pigments plateaued after they peaked (Figure 10). The time Molecules 2021, 26, 1619 course change in the contents of two amino acids (GABA and monascumic acid) in8 ofred 20 yeast rice during fermentation was also examined. The GABA content increased, with the peak occurring about day 20, and the level gradually decreased thereafter. The monas- monascumiccumic acid content acid content also increased, also increased, with the with peak the on peak day on 20, day and 20, the and level the plateaued level plateaued there- thereafterafter (Figure (Figure 11). 11).

Molecules 2021, 26, x FOR PEER REVIEW 9 of 20

Figure 8. Time course change in appearanceappearance ofof redred kojikoji,, whichwhich reddensreddens duringduring fermentation.fermentation.

monacolin K lactone

400 300 200

100 0 135791113151719212325272931333537394143 Concentration (mg/100 g) (mg/100 Concentration Days in culture

monacolin K hydroxyl acid 400 300 200 100 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 Concentration (mg/100 g) (mg/100 Concentration Days in culture

FigureFigure 9. 9. TimeTime course course change change in in the the content content of of lacton lactonee form form and and acid acid form form of ofmonacolin monacolin K in K red in red yeastyeast rice rice during during fermentation, fermentation, qu quantiﬁedantified using using the the method method specif speciﬁedied by by the the Korea Korea Food Food and and Drug Drug AdministrationAdministration (KFDA) (KFDA) [29]. [29].

monascin

101.E+07×106

8.E+068×106

4.E+064×106

absorbance at 391 nm 0.E+000 1 3 5 7 9 1113151719212325272931333537394143 Days in culture monascinol

101.E+07×106

8.E+068×106

4.E+064×106

0.E+000 Absorbance at at Absorbance 388 nm 1 3 5 7 9 1113151719212325272931333537394143 Days in culture

rubropunctamine

101.E+07×106

8.E+068×106

4.E+064×106

0.E+00 Absorbance at 538 nm 0 1 3 5 7 9 1113151719212325272931333537394143 Days in culture

Figure 10. Time course change in the contents of 3 azaphilone pigments in red yeast rice during fermentation, quantified according to the reference method [30].

Molecules 2021, 26, x FOR PEER REVIEW 9 of 20

monacolin K lactone

400 300 200 100 0 135791113151719212325272931333537394143 Concentration (mg/100 g) (mg/100 Concentration Days in culture

monacolin K hydroxyl acid 400 300 200 100 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 Concentration (mg/100 g) (mg/100 Concentration Days in culture

Figure 9. Time course change in the content of lactone form and acid form of monacolin K in red Molecules 2021, 26, 1619 yeast rice during fermentation, quantified using the method specified by the Korea Food and 9Drug of 20 Administration (KFDA) [29].

monascin

101.E+07×106

8.E+068×106

4.E+064×106

absorbance at 391 nm 0.E+000 1 3 5 7 9 1113151719212325272931333537394143 Days in culture monascinol

101.E+07×106

8.E+068×106

4.E+064×106

0.E+000 Absorbance at at Absorbance 388 nm 1 3 5 7 9 1113151719212325272931333537394143 Days in culture

rubropunctamine

101.E+07×106

8.E+068×106

4.E+064×106

0.E+00 Absorbance at 538 nm 0 1 3 5 7 9 1113151719212325272931333537394143 Days in culture Molecules 2021, 26, x FOR PEER REVIEW 10 of 20

FigureFigure 10. 10. TimeTime course course change change in in the the contents contents of of3 azaphilone 3 azaphilone pigments pigments in red in red yeast yeast ricerice during during fermentation,fermentation, quantified quantiﬁed according according to to the the reference reference method method [30]. [30].

monascumic acid 3.E+083×108

2.E+082×108 Intensity

0.E+000 (m/z = [M+H]+:216.25) = (m/z 135791113151719212325272931333537394143 Days in culture

GABA 2.E+072×107

1.E+071×107 Intensity

0.E+000 (m/z = [M+H]+:104.12) = (m/z 135791113151719212325272931333537394143 Days in culture

FigureFigure 11. 11. TimeTime course course change change in inthe the contents contents of 2 of amino 2 amino acids acids (GABA (GABA and monascumic and monascumic acid) acid)in redin red yeast yeast rice riceduring during fermentation, fermentation, quanti quantiﬁedfied by liquid by liquid chromatography-mass chromatography-mass spectrometry spectrometry (LC- MS)(LC-MS) [16,17]. [16 ,17].

TheThe results suggestsuggest that thatM. M. pilosus pilosusfought fought against against foreign foreign enemies, enemies, utilizing utilizing monascumic monas- cumicacid as acid its antibacterial as its antibacterial activity activity in the ﬁrstin the half first of theirhalf of growth their growth and monacolin and monacolin K with itsK withcholesterol its cholesterol synthesis synthesis inhibiting inhibiting activity inactivity the second in the half second of their half growth. of their GABAgrowth. could GABA be couldused asbe a used nitrogen as a sourcenitrogen in source the second in the half second of growth, half of when growth, nutrients when nutrients become deﬁcient; become deficient; however, we suggest GABA was mainly used to fight against foreign enemies since it decreased in the middle of the second half of the growth phase. Based on these findings, we expect further improvements to traditional solid-state fermentation methods will lead to the development of useful materials in the production of red yeast rice.

3. Effectiveness of Red Yeast Rice: Its Functional Ingredients and Physiological Ac- tions on Lipid Metabolism and the Circulatory System 3.1. Monacolins and Their Cholesterol Lowering Effect Monacolins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, a key enzyme regulating hepatic cholesterol synthesis, and exert potent serum cholesterol lowering effects (Figure 12).

HMG-CoA reductase Acetyl CoA HMG-CoA × Mevalonic acid Squalene Lanosterol Cholesterol

Red yeast rice

Figure 12. Biosynthetic pathway of cholesterol synthesis and the action of red yeast rice in it.

Lovastatin and its analogs (including their derivatives) have been used as medicines since 1987 by Japanese and foreign pharmaceutical companies. Statins inhibit HMG-CoA reductase, lower hepatic cholesterol biosynthesis and elevate the expression of hepatic LDL-receptors to maintain cholesterol homeostasis, which in turn enhances uptake of LDL-cholesterol into the liver and decreases blood cholesterol levels [31–33]. LDL-cholesterol is known as bad cholesterol because it is involved in atheroma formation and causes atherosclerosis. Since statins have excellent LDL-cholesterol-lowering effects, they are used as first-line drugs for patients with hypercholesterolemia, and several world-wide large-scale clinical trials have been conducted on statins. These trials have demonstrated

Molecules 2021, 26, x FOR PEER REVIEW 10 of 20

monascumic acid 3.E+083×108

2.E+082×108 Intensity

0.E+000 (m/z = [M+H]+:216.25) = (m/z 135791113151719212325272931333537394143 Days in culture

GABA 2.E+072×107

1.E+071×107 Intensity

0.E+000 (m/z = [M+H]+:104.12) = (m/z 135791113151719212325272931333537394143 Days in culture

Figure 11. Time course change in the contents of 2 amino acids (GABA and monascumic acid) in red yeast rice during fermentation, quantified by liquid chromatography-mass spectrometry (LC- MS) [16,17].

The results suggest that M. pilosus fought against foreign enemies, utilizing monas- Molecules 2021, 26, 1619 cumic acid as its antibacterial activity in the first half of their growth and monacolin10 of K 20 with its cholesterol synthesis inhibiting activity in the second half of their growth. GABA could be used as a nitrogen source in the second half of growth, when nutrients become deficient; however, we suggest GABA was mainly used to fight against foreign enemies sincehowever, it decreased we suggest in the GABA middle was of mainlythe second used half to ﬁghtof the against growth foreign phase. enemiesBased on since these it findings,decreased we in expect the middle further of improvements the second half to of traditional the growth solid-state phase. Based fermentation on these methods ﬁndings, willwe expectlead to furtherthe development improvements of useful to traditional materials solid-state in the production fermentation of red methods yeast rice. will lead to the development of useful materials in the production of red yeast rice. 3. Effectiveness of Red Yeast Rice: Its Functional Ingredients and Physiological Ac- 3. Effectiveness of Red Yeast Rice: Its Functional Ingredients and Physiological tionsActions on Lipid on Lipid Metabolism Metabolism and and the theCirculatory Circulatory System System 3.1.3.1. Monacolins Monacolins and and Their Their Cholesterol Cholesterol Lowering Lowering Effect Effect MonacolinsMonacolins are are inhibitors ofof 3-hydroxy-3-methylglutaryl-CoA3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) (HMG-CoA) reductase, reductase,a key a enzymekey enzyme regulating regulating hepatic hepatic cholesterol cholesterol synthesis, synthesis, and exert and potentexert potent serum serum cholesterol cho- lesterollowering lowering effects (Figureeffects (Figure12). 12).

HMG-CoA reductase Acetyl CoA HMG-CoA × Mevalonic acid Squalene Lanosterol Cholesterol

Red yeast rice

FigureFigure 12. 12. BiosyntheticBiosynthetic pathway pathway of of cholesterol cholesterol synt synthesishesis and and the the action action of of red red yeast yeast rice rice in in it. it.

LovastatinLovastatin and and its its analogs analogs (including (including their their derivatives) derivatives) have have been been used used as as medicines medicines sincesince 1987 1987 by by Japanese Japanese and and foreign foreign pharmaceutical pharmaceutical companies. companies. Statins Statins inhibit inhibit HMG-CoA HMG-CoA reductase,reductase, lower lower hepatic hepatic cholesterol cholesterol biosynth biosynthesisesis and and elevate elevate the the expression expression of of hepatic hepatic LDL-receptorsLDL-receptors to maintainmaintain cholesterolcholesterol homeostasis, homeostasis, which which in turnin turn enhances enhances uptake uptake of LDL- of LDL-cholesterolcholesterol into into the liverthe liver and and decreases decreases blood blood cholesterol cholesterol levels levels [31– [31–33].33]. LDL-cholesterol LDL-choles- terolis known is known as bad as bad cholesterol cholesterol because because it isit is involved involved in in atheroma atheroma formationformation and causes causes atherosclerosis.atherosclerosis. Since statinsstatins have have excellent excellent LDL-cholesterol-lowering LDL-cholesterol-lowering effects, effects, they they are usedare usedas first-line as first-line drugs drugs for patients for patients with hypercholesterolemia,with hypercholesterolemia, and several and several world-wide world-wide large- scale clinical trials have been conducted on statins. These trials have demonstrated not only large-scale clinical trials have been conducted on statins. These trials have demonstrated their efficacy on cardiovascular disease such as cardiac infarction and cerebral stroke but also various actions such as potential preventive action on bone fracture. Statins prevented cardiovascular events by 25–30% in clinical trials assessing primary and secondary preventive effects on coronary artery disease, indicating that they are essential medicines for patients with cardiovascular disease [34,35]. Since it has been reported recently that statins show pleiotropic effects, such as a restorative effect on vascular endothelial functions and anti-inflammatory effects, an accumulation of evidence for their therapeutic and preventive effects on various diseases is expected [36,37]. Clinical studies in Japan have also been conducted on processed food containing red yeast rice produced using the traditional solid-state fermentation described in Section 2.2[38–40]. In normal healthy volunteers with plasma LDL-cholesterol levels of 120 mg/dL or higher, long-term feeding of a processed food product containing 100 or 200 mg of red yeast rice produced by solid-state fermentation significantly lowered plasma levels of LDL-cholesterol and total cholesterol compared to those in volunteers fed the placebo at 2–8 weeks after starting the feeding. The lowered levels returned to the initial values after the end of red yeast rice intake. No significant differences in HDL-cholesterol and triglyc- eride levels and safety evaluation items among the 3 groups were found (Figure 13)[38]. In addition, in patients with hyperlipidemia where it was not improved during 1-month- diet therapy according to an NCEP panel, repetitive intake of a processed food product containing red yeast rice at 1 g/day significantly lowered levels of total cholesterol and LDL-cholesterol by about 10% [40]. Reports of clinical trials in Europe also show significant improvements in hypercholesterolemia by ingestion of red yeast rice containing 3–10 mg of monacolin K [41,42]. Molecules 2021, 26, x FOR PEER REVIEW 11 of 20

not only their efficacy on cardiovascular disease such as cardiac infarction and cerebral stroke but also various actions such as potential preventive action on bone fracture. Statins prevented cardiovascular events by 25–30% in clinical trials assessing primary and secondary preventive effects on coronary artery disease, indicating that they are essential medicines for patients with cardiovascular disease [34,35]. Since it has been reported recently that statins show pleiotropic effects, such as a restorative effect on vascular endothelial functions and anti-inflammatory effects, an accumulation of evidence for their therapeutic and preventive effects on various diseases is expected [36,37]. Clinical studies in Japan have also been conducted on processed food containing red yeast rice produced using the traditional solid-state fermentation described in Section 2.2. [38–40]. In normal healthy volunteers with plasma LDL-cholesterol levels of 120 mg/dL or higher, long-term feeding of a processed food product containing 100 or 200 mg of red yeast rice produced by solid-state fermentation significantly lowered plasma levels of LDL-cholesterol and total cholesterol compared to those in volunteers fed the placebo at 2–8 weeks after starting the feeding. The lowered levels returned to the initial values after the end of red yeast rice intake. No significant differences in HDL-cholesterol and triglyc- eride levels and safety evaluation items among the 3 groups were found (Figure 13) [38]. In addition, in patients with hyperlipidemia where it was not improved during 1-month- diet therapy according to an NCEP panel, repetitive intake of a processed food product containing red yeast rice at 1 g/day significantly lowered levels of total cholesterol and LDL-cholesterol by about 10% [40]. Reports of clinical trials in Europe also show signifi- Molecules 2021, 26, 1619 cant improvements in hypercholesterolemia by ingestion of red yeast rice containing11 3-10 of 20 mg of monacolin K [41,42].

FigureFigure 13. 13. EffectEffect of of intake intake of of red red yeast yeast rice rice produced produced by by solid-state solid-state ferm fermentationentation in in normal normal healthy healthy volunteersvolunteers whose whose plasma plasma cholesterol cholesterol levels levels were were 120 120 mg/dL mg/dL or higher. or higher. Each Eachbar indicates bar indicates the the ± meanmean ± standardstandard deviation.deviation. *: *: Significant Significant difference difference from start ofof ingestioningestion ( p(p< < 0.05, 0.05, paired paired ANOVA). ANOVA).#: Significantly #: Significantly different from differ theent placebo from the group placebo (p < group 0.05, multiple (p < 0.05, comparison multiple comparison using Bonferroni). using Bonferroni). Recently, we compared the pharmacokinetics of monacolin K in blood after oral administrationRecently, we of compared purified monacolinthe pharmacokinetics K (lactone of form, monacolin 99% purity) K in blood andred after yeast oral ricead- ministration of purified monacolin K (lactone form, 99% purity) and red yeast rice pro- Molecules 2021, 26, x FOR PEER REVIEWproduced by solid-state fermentation in male SD rats (7 weeks old). Most of the monacolin12 of 20 ducedK in the by plasma solid-state was detectedfermentation in its in acid male form: SD bothrats (7 in weeks the monacolin old). Most K-administered of the monacolin group K inand the the plasma red yeast was ricedetected -administered in its acid group. form: Surprisingly,both in the monacolin the maximum K-administered level (C max) group and andarea the under red curveyeast rice (AUC) -administered of plasma monacolin group. Surprisingly, K concentration the maximum 4 hours after level administration (C max) and areaweretion underwere several several curve times (AUC)times higher higher of inplasma the in redthe monacolin yeastred yeast rice-administered Krice-administered concentration group 4 hoursgroup than after than in theadministra- in the purified puri- monacolinfied monacolin K-administered K-administered group group (Figure (Figure 14, Table 14, Table3). These 3). These results results suggest suggest that red that yeast red riceyeast contains rice contains ingredients ingredients that that enhance enhance the the absorption absorption of monacolinof monacolin K K into into blood. blood. TheThe medicinal benefit benefit of red yeast rice is considered to be different to thatthat ofof monacolinmonacolin K alone. Furthermore, our recent experiments show that monacolin L, monascinol, and alone. Furthermore, our recent experiments show that monacolin L, monascinol, and monascodilone also also have have HMG-C HMG-CoAoA reductase reductase activity. activity. Since Since red redyeast yeast rice ricecontains contains var- variousious ingredients, ingredients, synergistic synergistic effects effects areare expected. expected.

Monacolin K lactone concentration Monacolin K hydroxy acid concentration 6.0 450.0 Monacolin K group (n =3) 400.0 Monacolin K group (n =3) 5.0 Red yeast rice group (n =3) 350.0 Red yeast rice group (n =3) 4.0 300.0 250.0 3.0 200.0 2.0 150.0 100.0

1.0 (ng/ml) Concentration Concentration (ng/ml) 50.0 0.0 0.0 01234 01234 Time (h) Time (h)

FigureFigure 14.14. Plasma concentrations of lactone form and acid formform ofof monacolinmonacolin KK afterafter singlesingle administrationadministration ofof puriﬁedpurified monacolinmonacolin KK oror red red yeast yeast rice rice (40 (40 mg/kg mg/kg monacolin monacolin K orK doseor dose of redof red yeast yeast rice rice equivalent equivalent to 40 to mg/kg 40 mg/kg monacolin monacolin K) in K) male in SDmale rats SD (7 rats weeks (7 weeks old). Eachold). Each bar indicates bar indicates the mean the mean± standard ± standard deviation, deviation, quantiﬁed quantified according according to the to reference the reference method method [43]. [43].

Table 3. Pharmacokinetic parameters of the lactone form and acid form of monacolin K in plasma after single administration of purified monacolin K or red yeast rice (40 mg/kg monacolin K or dose of red yeast rice equivalent to 40 mg/kg monacolin K). Peak analysis was performed according to the reference method [44].

PK parameter of Monacolin K Lactone PK Parameter Of Monacolin K Hydroxy Acid Monacolin K Administration Group Monacolin K Administration Group PK Parameter 101 102 103 Mean S.D. PK Parameter 101 102 103 Mean S.D. t1/2(h) N.C. N.C. N.C. - - t1/2(h) 2.7 1.7 N.C. 2.2 - Tmax(h) 1.0 1.0 N.C. 1.0 - Tmax(h) 1.0 1.0 2.0 1.3 0.6 Cmax(ng/ml) 3.11 1.85 N.C. 2.48 - Cmax(ng/ml) 72.6 113 55.5 80.4 29.5 AUC0-4h(ng·h/ml) 3.11 1.85 N.C. 2.48 - AUC0-4h(ng·h/ml) 143 274 153 190 73 Red yeast rice administration group Red yeast rice administration group PK parameter 201 202 203 Mean S.D. PK parameter 201 202 203 Mean S.D. t1/2(h) N.C. N.C. N.C. - - t1/2(h) 1.4 1.1 0.98 1.1 0.2 Tmax(h) 1.0 1.0 1.0 1.0 0.0 Tmax(h) 1.0 1.0 1.0 1.0 0.0 Cmax(ng/ml) 4.32 4.03 3.54 3.96 0.39 Cmax(ng/ml) 254 349 406 336 77 AUC0-4h(ng·h/ml) 7.20 5.22 6.46 6.29 1.00 AUC0-4h(ng·h/ml) 457 470 632 520 98 S.D.: Standard deviation

Molecules 2021, 26, 1619 12 of 20

Table 3. Pharmacokinetic parameters of the lactone form and acid form of monacolin K in plasma after single administration of purified monacolin K or red yeast rice (40 mg/kg monacolin K or dose of red yeast rice equivalent to 40 mg/kg monacolin K). Peak analysis was performed according to the reference method [44].

PK Parameter of Monacolin K Lactone PK Parameter Of Monacolin K Hydroxy Acid Monacolin K Administration Group Monacolin K Administration Group PK Parameter 101 102 103 Mean S.D. PK Parameter 101 102 103 Mean S.D.

t1/2 (h) N.C. N.C. N.C. - - t1/2 (h) 2.7 1.7 N.C. 2.2 - Tmax (h) 1.0 1.0 N.C. 1.0 - Tmax (h) 1.0 1.0 2.0 1.3 0.6 Cmax (ng/mL) 3.11 1.85 N.C. 2.48 - Cmax (ng/mL) 72.6 113 55.5 80.4 29.5 AUC0–4h (ng·h/mL) 3.11 1.85 N.C. 2.48 - AUC0–4h (ng·h/mL) 143 274 153 190 73 Red yeast rice administration group Red yeast rice administration group PK parameter 201 202 203 Mean S.D. PK parameter 201 202 203 Mean S.D. t1/2 (h) N.C. N.C. N.C. - - t1/2 (h) 1.4 1.1 0.98 1.1 0.2 Tmax (h) 1.0 1.0 1.0 1.0 0.0 Tmax (h) 1.0 1.0 1.0 1.0 0.0 Cmax (ng/mL) 4.32 4.03 3.54 3.96 0.39 Cmax (ng/mL) 254 349 406 336 77 AUC0–4h (ng·h/mL) 7.20 5.22 6.46 6.29 1.00 AUC0–4h (ng·h/mL) 457 470 632 520 98 S.D.: Standard deviation

3.2. Improving Effects of Red Yeast Rice on Chyle in Blood and Increased Viscosity Caused by Hyperlipidemia In general, continuous intake of a high-fat or high-cholesterol diet causes hyperlipidemia and the blood develops chyle, a white cloudy substance which mainly consists of Molecules 2021, 26, x FOR PEER REVIEW 13 of 20 lipoproteins. Chylemia is regarded as a risk factor for circulatory diseases, and its suppression is considered to be important for risk reduction. Recently, we conducted an experiment in which male Japanese white rabbits (10–12 weeks old) were fed a diet containing 0.25% intocholesterol a red yeast for 2rice-treated weeks to produce group, in chylemia. which powdered The rabbits red wereyeast thenrice was allocated orally intoadminis- a red teredyeast for rice-treated 3 weeks or group, non-treated in which group. powdered Blood redsamples yeast were rice wascollected orally from administered both groups for for3 weeks plasma or biochemical non-treated examinations group. Blood samplesand measurement were collected of plasma from bothturbidity. groups Macroscopic for plasma observationsbiochemical examinationsshowed lower and plasma measurement turbidity ofin plasmathe red turbidity.yeast rice-treated Macroscopic group observations compared withshowed the lowernon-treated plasma group, turbidity and biochemical in the red yeast analysis rice-treated revealed group a statistically compared significant with the increasenon-treated in light group, transmittance and biochemical (Figure analysis 15) and revealed decrease a statisticallyin plasma total signiﬁcant cholesterol increase levels in inlight the transmittancered yeast rice-treated (Figure group15) and co decreasempared with in plasma the non-treated total cholesterol group. levels in the red yeast rice-treated group compared with the non-treated group.

Figure 15. Difference in transmittance of plasma between red yeast rice-treated (2 weeks) and Figure 15. Difference in transmittance of plasma between red yeast rice-treated (2 weeks) and non- non-treated groups of male Japanese white rabbits (10–12 weeks old) fed a high-cholesterol diet. treated groups of male Japanese white rabbits (10–12 weeks old) fed a high-cholesterol diet. A decreaseA decrease in turbidity in turbidity in the in red the yeast red yeastrice-treated rice-treated group groupis apparent. is apparent. Each bar Each indicates bar indicates the mean the ± standardmean ± standarddeviation.deviation. Significant Signiﬁcant differences differences were tested were by tested Student's by Student’s t-test. Plasmat-test. Plasmacloudiness cloudiness was evaluatedwas evaluated by measuring by measuring the transmittance the transmittance at a wavelength at a wavelength of 660 of nm 660 using nm using an absorptiometer. an absorptiometer.

ItIt has has also also been been reported reported that that hyperlipidemia hyperlipidemia induces induces metabolic metabolic abnormality abnormality of lipo- of proteinslipoproteins and an and increase an increase in plasma in plasma viscosity, viscosity, which which in turn in turnelevates elevates the risk the of risk cardiac of cardiac and cerebrovascular diseases [45–47]. Lovastatin or ezetimibe, anti-hyperlipidemic drugs, have been reported to decrease plasma viscosity [48,49]. In our study, male Japanese white rabbits (14 weeks old) were fed a diet containing 0.25% cholesterol (HC diet) for 3 months as a hyperlipidemic animal model. During the 3-month feeding period, the control group was fed the HC diet alone, and the red yeast rice-treated group was fed the HC diet with red yeast rice at 500 mg/kg body weight. Blood biochemical analyses, performed during the feeding period, included measurements of plasma turbidity, viscosity and cholesterol content of lipoproteins. Plasma turbidity was improved, and total cholesterol and LDL- cholesterol plasma levels were reduced in the red yeast rice-treated group between months 1–3 of the experimental period, when compared to the control group. Further- more, 3 months after the start of administration, the low-density cholesterol content of lipoproteins and plasma viscosity were still lower in the red yeast rice-treated group compared with the control group (Figure 16). In particular, the cholesterol contents in chylomicrons and VLDL were significantly lowered (Figure 17). These results indicate that ingredients in red yeast rice suppressed the increase in plasma viscosity by decreasing the content of large (low-density) lipoproteins through a reduction in VLDL release and clearance of chylomicrons.

Molecules 2021, 26, 1619 13 of 20

and cerebrovascular diseases [45–47]. Lovastatin or ezetimibe, anti-hyperlipidemic drugs, have been reported to decrease plasma viscosity [48,49]. In our study, male Japanese white rabbits (14 weeks old) were fed a diet containing 0.25% cholesterol (HC diet) for 3 months as a hyperlipidemic animal model. During the 3-month feeding period, the control group was fed the HC diet alone, and the red yeast rice-treated group was fed the HC diet with red yeast rice at 500 mg/kg body weight. Blood biochemical analyses, performed during the feeding period, included measurements of plasma turbidity, viscosity and cholesterol content of lipoproteins. Plasma turbidity was improved, and total cholesterol and LDL-cholesterol plasma levels were reduced in the red yeast rice-treated group between months 1–3 of the experimental period, when compared to the control group. Furthermore, 3 months after the start of administration, the low-density cholesterol content of lipoproteins and plasma viscosity were still lower in the red yeast rice-treated Molecules 2021, 26, x FOR PEER REVIEWgroup compared with the control group (Figure 16). In particular, the cholesterol14 contents of 20 in chylomicrons and VLDL were signiﬁcantly lowered (Figure 17). These results indicate Molecules 2021, 26, x FOR PEER REVIEW 14 of 20 that ingredients in red yeast rice suppressed the increase in plasma viscosity by decreasing the content of large (low-density) lipoproteins through a reduction in VLDL release and clearance of chylomicrons. Control group Red yeast rice group 1.50 Control group Red yeast rice group 1.48 1.50 ## 1.461.48 # ## 1.441.46 # 1.421.44 1.401.42 1.381.40

Plasma viscosity (cp) 1.361.38 1.34 Plasma viscosity (cp) 1.36 1.321.34 1.301.32 04812 1.30 Time (weeks) 04812 Figure 16. Time course changesTime (weeks) in plasma viscosity in a control group and a red yeast rice-treated group in hypercholesterolemic model rabbits. Each bar indicates the mean ± standard deviation. Figure 16. TimeTime course changes changes in in plasma plasma viscosity viscosity in in a a control control group group and and a ared red yeast yeast rice-treated rice-treated Significant differences between control group and red yeast group were tested by Student's t-test group in hypercholesterolemic model rabbits. Each bar indicates the mean ± standard± deviation. (#:group p < 0.05, in ##: hypercholesterolemic p < 0.01). Plasma viscosity model rabbits.was measured Each bar using indicates a plate the and mean cone viscometerstandard accord- deviation. Significant differences between control group and red yeast group were tested by Student's t-test ingSigniﬁcant to the reference differences method between [50]. control group and red yeast group were tested by Student’s t-test (#: (#:p < p 0.05, < 0.05, ##: ##:p < p 0.01).< 0.01). Plasma Plasma viscosity viscosity was was measured measured using using a plate a plate and and cone cone viscometer viscometer according accord- to ing to the reference method [50]. the reference method [50].

Control group Red yeast rice group Control group Red yeast rice group 80 300 70 Control group Red yeast rice group Control group Red yeast rice group 80 250300 6070 ## ## 200250 ## 5060 ## ## ## ## ## 40 150200 ## 50 ## ## 30 ## 40 150 CM-C (mg/dL) CM-C 100

2030 VLDL-C (mg/dL)

CM-C (mg/dL) CM-C 100 10 50

20 VLDL-C (mg/dL) 010 050 04812 04812 0 Time (weeks) 0 Time (weeks) 04812 04812 Time (weeks) Figure 17. Time course change in contents of cholesterol in plasma low-density lipoproteins (chylomicron-cholesterolTime (weeks) and Figure 17. Time course change in contents of cholesterol in plasma low-density lipoproteins (chylomicron-cholesterol and VLDL-cholesterol) obtained from a red yeast rice-treated and control groups of hypercholesteremic model rabbits. Each bar VLDL-cholesterol) obtained from a red yeast rice-treated and control groups of hypercholesteremic model rabbits. Each Figure 17. Time course± change in contents of cholesterol in plasma low-density lipoproteins (chylomicron-cholesterol and barindicates indicates the the mean meanstandard ± standard deviation. deviation. Signiﬁcant Significant differences differences between between control control group group and red and yeast red groupyeast weregroup tested were by VLDL-cholesterol) obtained from a red yeast rice-treated and control groups of hypercholesteremic model rabbits. Each testedStudent’s by Student'st-test (##: t-testp < 0.01).(##: p Plasma< 0.01). lipoproteinPlasma lipoprotein cholesterol cholesterol was quantiﬁed was qu accordingantified according to the reference to the reference method [ 51method]. bar indicates the mean ± standard deviation. Significant differences between control group and red yeast group were [51]. tested by Student's t-test (##: p < 0.01). Plasma lipoprotein cholesterol was quantified according to the reference method [51]. Red yeast rice has the potential to prevent vascular disease associated with hyper- lipidemiaRed oryeast reduce rice thehas riskthe potentialfor these todise preventases, throughvascular suppression disease associated of atheroma with hyper- for- mationlipidemia and improveor reduce blood the risk fluidity, for these by lowering diseases, levels through of chylomicrons suppression and of atheromaVLDL, lipo- for- proteinsmation thatand areimprove the main blood components fluidity, by of loweringplasma chyle. levels of chylomicrons and VLDL, lipoproteins that are the main components of plasma chyle. 3.3. Promising Effects of Ingredients in Red Yeast Rice on Risk Reduction for Circulatory Diseases3.3. Promising Effects of Ingredients in Red Yeast Rice on Risk Reduction for Circulatory DiseasesThe evidence presented above suggests red yeast rice could reduce the risk of vascular diseasesThe evidence associated presented with hyperlipidemia; above suggests however, red yeast redrice yeast could rice reduce also the contain risk of statins vascu- andlar variousdiseases other associated ingredients with hyperlipidemia;which are effect ivehowever, in the circulatory red yeast rice system. also Tablecontain 4 sum-statins marizesand various the potential other ingredients effects of monacolin which are K effect (lovastatin)ive in the on circulatory circulatory system. diseases, Table including 4 sum- cardiacmarizes disease. the potential There effectshas been of monacolin a lot of evid K (lovastatin)ence reported on circulatoryon the favorable diseases, actions including of cardiac disease. There has been a lot of evidence reported on the favorable actions of

Molecules 2021, 26, 1619 14 of 20

Red yeast rice has the potential to prevent vascular disease associated with hyperlipidemia or reduce the risk for these diseases, through suppression of atheroma formation and improve blood ﬂuidity, by lowering levels of chylomicrons and VLDL, lipoproteins that are the main components of plasma chyle.

3.3. Promising Effects of Ingredients in Red Yeast Rice on Risk Reduction for Circulatory Diseases The evidence presented above suggests red yeast rice could reduce the risk of vascular diseases associated with hyperlipidemia; however, red yeast rice also contain statins and various other ingredients which are effective in the circulatory system. Table4 summarizes the potential effects of monacolin K (lovastatin) on circulatory diseases, including cardiac disease. There has been a lot of evidence reported on the favorable actions of statins, including their HMG-CoA reductase inhibiting activity which suppresses atheroma formation in atherosclerosis [52–54], improving effects on blood circulation [55,56], and pleiotropic effects on nitric oxide (NO) production which plays a pivotal role in homeostasis of the cardiovascular system and the NO synthetase–NO pathway [57–59]. Effects of lovastatin on atherogenic signaling cascades [60] have also been reported [61–63]. There have also been similar favorable reports of red yeast rice extracts, such as upregulation of constitutive NO synthetase expression, the increase in plasma levels of nitric oxides and improvement of abnormal hemorheology in an atherosclerotic rat model induced by high-cholesterol diet feeding [64]. These effects are considered to be beneﬁcial in the prevention of circulatory diseases and improvement in blood circulation.

Table 4. Actions of monacolin K (lovastatin) on circulatory system and related ﬁelds.

Evaluation Target Study Subjects or Study Materials Effect of Monacolin K Ref. Decrease in number of plaques in the aorta and Rabbits fed on a high-lipid diet [52] lung artery Inhibition of platelet aggregation, macrophage foam Patients with hypercholesterolemia [53] Atheroma formation cell formation and LDL oxidation Human umbilical vein endothelial Inhibition of NF-KB activation by CRP [54] cells (HUVECs) Hypertensive model mice Increase in renal blood ﬂow [55] Blood circulation Several studies using Increase in cerebral blood ﬂow and cerebral [56] experimental animals vasomotor response Increase of IL-1 induced production of nitrite Cardiac myocytes (Increase in NOS expression and NO production [61] mediated by Rho inhibition) NOS signaling pathway Squamous epithelial cancer cells Induction of expression of Rho family proteins [62] Mesenchymal stem cells derived from Activation (phosphorylation) of AI3K/Akt pathway [63] rat bone marrow and MEK 1/2 pathway

Ingredients that are known to be effective in red yeast rice, other than statins, include GABA, which exerts a hypotensive effect through reduction of adrenergic vasoconstriction by its suppressive effect on the release of sympathetic nerve transmitters mediated by presynaptic GABA receptors [65,66]. In addition, GABA promotes sodium excretion from the kidneys and suppresses increases in blood pressure [67,68]. Oxidative stress is also regarded as a therapeutic target related to atherosclerosis. Reactive oxygen inactivates NO and at the same time damages vascular endothelial cells and reduces NO production. It has also been suggested that plasma LDL suppresses NOS activity in vascular endothelial cells by hydroxyl radicals and the like, thereby reducing NO production and promoting arteriosclerosis. [69,70]. Lovastatin inhibits oxidation of LDL, and this improves the vascular endothelial function (vasoconstrictor reaction) in hyperlipidemia, and in particular, the combined use of an antioxidant can signiﬁcantly Molecules 2021, 26, 1619 15 of 20

improve the function [64,71]. Antioxidant activity has also been reported in red yeast rice pigments [72,73]: monascin and ankaﬂavin suppressed LDL-cholesterol oxidation [74]. In addition, a previous clinical study reported that a red yeast rice extract reduced plasma levels of oxidized LDL, an index of oxidative stress, and lipoprotein-associated phospholi- pase A2 activity [75]. Red yeast rice made from brown rice, black rice, and red rice has also been shown to improve vascular endothelial function with increased NO production [76].

Molecules 2021, 26, x FOR PEER REVIEW Monascin and ankaﬂavin are reported to act as the agonist for peroxisome proliferator-16 of 20 activated receptors (PPARs), e.g., PPARα [77]. PPARs that are involved in metabolism of carbohydrates, lipids, and energy are also closely involved in development of life-style related diseases, such as obesity and atherosclerosis. PPARα agonists (ﬁbrates) have been related diseases, such as obesity and atherosclerosis. PPARα agonists (fibrates) have been clinically used as antihyperlipidemic drugs. clinically used as antihyperlipidemic drugs. Vascular function can be improved by increasing NO, suppressing oxidative stress, Vascular function can be improved by increasing NO, suppressing oxidative stress, hypertension, obesity, etc. [78–80]. It is expected that ingredients contained in red yeast hypertension, obesity, etc. [78–80]. It is expected that ingredients contained in red yeast rice, such as monacolin K (lovastatin), GABA, and red yeast rice pigments (azaphilone rice, such as monacolin K (lovastatin), GABA, and red yeast rice pigments (azaphilone pigments) exert exert synergic synergic or or additive additive effects effects to tolower lower the the risk risk for foratherosclerosis atherosclerosis and andother other circulatory diseases diseases (Figure (Figure 18). 18).

Figure 18. ExpectedExpected effect effect on on the the circulatory circulatory system system of ofred red yeast yeast rice rice and and its components. its components.

4. Safety of of Red Red Yeast Yeast Rice Rice Red yeastyeast ricerice produced produced properly properly by by traditional traditional solid-state solid-state fermentation fermentation using usingMonascus fungiMonascus is considered fungi is considered to be safe to for befood safe for use; food not use; only not because only because of its long of its history long history within the Asianwithin dietthe Asian but also diet because but also redbecause yeast red rice yeast manufacturers rice manufacturers have made have everymade effortevery effort to ensure itsto ensure safety its using safety various using various tests, including tests, including mutagenicity, mutagenicity, acute acute toxicity, toxicity, and chronicand chronic toxicity tests.toxicity The tests. processed The processed food containingfood containing red yeastred yeast rice rice used used in in a previousa previous clinical clinical trialtrial has beenhas been commercially commercially used used for for more more than than 20 20 years years in in Japan, Japan, and and no no problematic problematic cases cases have beenhave been reported reported from from a safety a safety point point of of view. view. Furthermore, since since red red yeast yeast rice rice pigments pigments industrially produced produced with with MonascusMonascus fungifungi are are approved approved as food as food additives, additives, their their safety safety is is institutionally secured secured by by various various toxici toxicityty tests tests based based on onlegal legal standards standards [7]. [7]. Of the the MonascusMonascus fungi,fungi, M.M. pilosus pilosus, M., M. ruber, ruber and, and M.M. purpureus purpureus are aremainly mainly used used for food for food in Japan, Taiwan, Taiwan, and and China, China, respectively. respectively. It Ithas has been been reported reported that that the thegene gene encoding encoding citrinin, a a mycotoxin mycotoxin causing causing renal renal toxicity, toxicity, is isfunctional functional in some in some strains strains of M. of purpureusM. purpureus and M. ruber ruber [81,82].[81,82]. We We recently recently conducted conducted whole-ge whole-genomenome analysis analysis of these of these three three strains strains of MonascusMonascus fungifungi usingusing aa nextnext generationgeneration sequencersequencer andand demonstrateddemonstrated thatthat M.M. pilosuspilosus was unablewas unable to generate to generate citrinin citrinin [83 ].[83]. This This ﬁnding finding indicates indicates that, that, of of the the three three commercially commercially used Monascusused Monascusfungi, fungi, only onlyM. pilosus M. pilosusdoes does not generatenot generate the mycotoxin,the mycotoxin, citrinin. citrinin. Consequently, Conse- quently, based on its long history and the results of recent studies, red yeast rice is a safe material as long as its prepared and used properly.

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based on its long history and the results of recent studies, red yeast rice is a safe material as long as its prepared and used properly.

5. Conclusions In paragraph 1, we introduced that red yeast rice has been a prized medicinal food in East Asia for about 1500 years, and has been widely used as a food and pigment in recent years. In paragraph 2, we introduced the metabolites of red yeast rice, mainly polyketide compounds. These ingredients vary depending on the type of Monascus and the culture/manufacturing method, but even today, red yeast rice produced by tradi-tional solid fermentation is widely used as a health food material and we showed the changes in the amounts of the typical components during the solid-state fermentation process. In the future, it is expected to develop red yeast rice materials that has more beneficial balances of ingredients in the safe ancient manufacturing method. In Section3, we introduced mainly introduced the findings on the usefulness of red yeast rice on lipid metabolism and circulatory system. The data from our animal studies described in Sections 3.1 and 3.2 suggest that in addition to monacolin K, other compo-nents of red yeast rice may improve lipid metabolism. However, it is a future issue to conduct experiments with more appropriate controls to clarify in more detail whether the effects other than LDL-cholesterol lowering effect are due to the red yeast rice component alone or the combination of monacolin K and red yeast rice components. In addition, the findings of the effects of statins, pigments, and GABA on the circulatory system as typical components were introduced in Section 3.3, but the involvement of various components reported to contain in red yeast rice, such as sterols, decalin compounds, the derivatives, flavonoids, lignans, coumarins, terpenoids, polysaccha-rides and phenolic acids is unknown [23]. Further elucidations of the relationship be-tween each of components of red yeast rice are expected. Here, we introduced the typical lipid metabolism and effects of monacolin K (lovastatin) on the lipid metabolism and circulatory system, but recent reports have shown that monacolin K is also useful for the treatment of neurological disorders, cancer, etc. and these studies are also being highlighted [84]. Furthermore, various functionalities such as anti-cancer effect, neuroprotective effect, liver protective effect, osteoporosis improving effect, anti-diabetic effect, anti-obesity effect, anti-fatigue ef-fect, and anti-inflammatory effect have been reported for red yeast rice [23]. It has been suggested that various components other than monacolin K are involved in these actions in a complex manner. In the future, it is expected that evidence will be accumulated and will help improve the quality of life in a wide range beyond the prevention of lifestyle-related diseases. In conclusion, red yeast rice is promising as a functional food material that can maintain and improve health in addition to preventing lifestyle related diseases. As a food material, it has high medicinal properties, various functions and safety. It is expected that new values will be elucidated in the future.

Author Contributions: Conceptualization, H.F.; investigation and resources, Y.H., T.H. (Tomohiro Hisano) and K.A.; data curation, Y.H., T.H. (Tomohiro Hisano) and K.A.; writing—original draft preparation, H.F.; writing—review and editing, S.N., T.H. (Tetsuya Hirata), Y.H. and H.F.; visualiza- tion, H.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank Yongsu Kim, Kensuke Tachiki., Makoto Tamesada, Takuya Makino, and Yuki Ozeki for their assistance in experiments, discussions, and literature information. We are grateful to Tazuru Kikkawa of Kamakura Techno-Science, Inc., for performing the pharmacokinetic analysis; Tsutomu Tajikawa from the Department of Mechanical Engineering, Faculty of Engineering Science, Kansai University, for implementation of plasma viscosity experiments; and Skylight Biotech Molecules 2021, 26, 1619 17 of 20

Inc., for analysis of lipoproteins. The authors report no conflicts of interest in the preparation of this paper. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Conflicts of Interest: The authors declare no conflict of interest. No funding body had a role in the writing of this review. Sample Availability: Samples of the compounds are not available from the authors.

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