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Abstract It is difficult to extract beneficial components from Monascus pigments due to their complex composition. The low purity of Monascus pigment preparations limits their further application in research and production. Therefore， this study was conducted to optimize the conditions for extraction and isolation of Monascus pigments from red yeast rice， to improve the purity of Monascus pigment preparations. Three fractions were isolated using column chromatography from red yeast rice， they were red， orange and yellow respectively. Then， the three fractions were analyzed for composition and purity using thin layer chromatography and liquid chromatographymass spectrometry （LCMS）. The chromatograms revealed that the yellow and orange fractions had complex compositions that were difficult to be separated， while the red fraction consisted of fewer components. Finally， a single component was isolated from the red fraction using a thin layer chromatography plate， identified as monascorubramine according to its molecular mass.
Key words Monascus pigments; Separation; Purification; Monascorubramine
As fungi that are beneficial to humans， Monascus spp. can be found in trees， soils and sediments. The genus Monascus belongs to Family Elaphomycetaceae， Order Eurotiales， Class Eurotiomycetes， Division Ascomycota in Kingdom Fungi[1]. So far， more than 20 Monascus spp. have been found in various fermented foods， soils and decaying fruits.
The species of Monascus are mostly saprophytic fungi that prefer an acidic environment with an optimum pH range of 3.5 to 5 and an optimum temperature range of 32 to 35. They can also survive in 10% ethanol[2]. In laboratory， Monascus spp. are often cultured in Malt Extract Agar （MEA） medium. They are different in the color of hyphae， which are mostly white at first， then turn into light red， purple and so on.
Monascus pigments are the main metabolites of Monascus spp. At present， there are 10 Monascus pigments whose chemical structure have been established， six of which are soluble in alcohol， and the other four are soluble in water[3]. Monascus pigments are a mixture of polyketone compounds. There are three major types of Monascus pigments： red， orange， and yellow depending on the color. Among them， six alcoholsoluble pigments can be used as functional food colorants， they are rubropunctamine （red）， monascorubramine （red）， monascorubrine （orange）， rubropunctatine （orange）， ankaflavine （yeallow）， and monascine （yellow）[4]. Materials and Methods
Materials and reagents
Red yeast rice was purchased from Shandong Zhonghui Biotechnology Co.， Ltd. Absolute ethanol （AR）， quartz sand （100-200 mesh）， ammonium acetate， chloroform （AR）， magnesium sulfate， nhexane （AR）， ethyl acetate （AR） and methanol （chromatographic grade） were all bought from CINC High Purity Solvents （Shanghai） Co.， Ltd.
Instruments and equipment
Highspeed pulverizer was purchased from Zhejiang Yongkang Xian Medical Instruments Factory， ultrasonic cleaner from Jiekang Washing Electric Co.， Ltd， vacuum filter from Zhengzhou Greatwall Scientific Industrial and Trade Co.， Ltd.， GF254 thin layer chromatography silica gel analysis plate （25 mm ≠ 75 mm） from Qingdao Shenghai Fine Silica Gel Chemical Co.， Ltd， high performance liquid chromatograph from Shimadzu Corporation， and liquid chromatographymass spectrometry instrument from Agilent Technologies.
Extraction of Monascus pigment
Red yeast rice sample was ground into a fine powder using a pulverizer. Then， 20 g of the powder was collected， soaked in 200 ml of 70% ethanol in a beak， and sonicated at 40 for 30 min[5].
The mixture was vacuumfiltered to remove impurities， and the filtrate was rotary evaporated to nearly dryness， dissolved in 70% ethanol， transferred to a plate， dried in an oven at 40， and finally scraped off with a knife.
Separation of Monascus pigments by column chromatography
Pretreatment of silica gel
The silica gel to be used was dried in an oven at 120 for 30 min， transferred to a dry beaker， and soaked for 20 min in a solvent that had been prepared to elute yellow component[6].
Sample processing
One gram of dried sample was weighed， dissolved in a proper volume of ethanol in a beaker. After 5 g of silica gel powder was added and well mixed， the sample was rotary evaporated to nearly dryness， transferred to another dry beaker and further dried in an over at 40.
Column preparation
The tube was rinsed with tap water and distilled water before it was dried in an oven. Column was prepared by wet method and equilibrated for 3 to 4 h. Finally， an appropriate amount of magnesium sulfate was added onto the top of silica gel.
Sample loading
The solid sample （at an amount of 1/60 to 1/30 of the silica gel） to be separated was gently added to the top surface of the stationary phase along the tube wall， without disturbing the stationary phase. Elution
The column was first eluted with the mixture of nhexane and ethyl acetate （8≥2， v≥v） at a flow rate of 1- 2 ml/min， till fractions of different colors appeared[7]， they were yellow， orange， light red， red and dark red from the bottom to the top of the column. After the yellow fraction was eluted out and collected， the eluent was replaced by the mixture of ethyl acetate and methanol （9≥1， v≥v） to collect the orange fraction. Finally， the column was eluted with methanol to collect the red fraction.
Detection and separation of Monascus pigments by thin layer chromatography
The red， orange and yellow fractions collected above were subjected to thin layer chromatography on a silica gel plate to initially identify the composition of each fraction.
Each sample was applied as a series of spots （1-3 mm in diameter） 1 cm apart from the short edge of the plate. After multiple times of tests， the optimal solvent for separating the component in each fraction was determined： the mixture of nhexane and ethyl acetate （8≥2， v≥v） for yellow fraction， ethyl acetate for orange fraction， the mixture of ethyl acetate and methanol （6≥4， v≥v） for red fraction.
The single components of the red fraction was scraped off with a knife， dissolved in methanol， and centrifuged to remove impurities.
Determination of Monascus pigments by high performance liquid chromatography
Pretreatment of sample
Each fraction of Monascus pigments collected by column chromatography was centrifuged， blown to dryness with a gentle stream of nitrogen， dissolved in 40% methanol and passed through a 0.45 um organic filter[8].
Chromatographic conditions
All determinations were performed at ambient temperature of 20-25 using a Kromasil C18 （250 mm≠4.6 mm， 5 m） column. The mobile phase A was acetic acidammonium acetate buffer （pH = 5）， and the mobile phase B was methanol. The column effluent was monitored at 264 nm. The injection volume was 20 l with a flow rate of 1 ml/min.
Determination of Monascus pigments by mass spectrometry
Monascus pigment sample was dissolved in methanol and passed through 0.45 m organic filter membrane before being subjected to mass spectrometry.
Mass spectrometry was performed under the conditions as follows： Gas Temp 350， Drying Gas 10 L/min Nebulizer 35 psig， VCap 3500 V， Fragmentor 150 v， 100-1000 m/z[9].
Results and Analysis
The yellow， orange and red fractions of Monascus pigments collected by column chromatography were further analyzed by highperformance liquid chromatography （Fig. 1， Fig. 2 and Fig. 3）. It was found that the chromatograms of the yellow and orange fractions had multiple peaks and baseline noise， suggesting they had complex components which were difficult to be separated. In contrast， there were only two major peaks in the chromatogram of red fraction， so the components of this fraction were further separated.
The red fraction was further separated with the mixture of chloroform， methanol and water （5≥3≥1， v≥v≥v） on a thin layer chromatography plate， and two spots were obtained. The retention factor （Rf） value was 0.34 for spot 1 and 0.38 for spot 2， respectively. Liquid chromatography-mass spectrometry （LCMS） analysis revealed that spot 1 had simpler composition. The single component had a molecular mass 397， which was the same as that of monascorubramine previously reported[10]. The monascorubramine sample we isolated had a purity of 90%. Fig. 4 and 5 show the chromatogram and mass spectrum of monascorubramine.
Conclusions
The conditions for extraction， separation and purification of Monascus pigments from red yeast rice were optimized in this study. In brief， Monascus pigments were separated by column chromatography and thin layer chromatography， and identified by liquid chromatographymass spectrometry. Finally， a single Monascus pigment component with a high purity above 90% was obtained， which was identified as monascorubramine. The results may provide a theoretical basis for the research of application of Monascus pigments.
References
[1]ZHOU WB， JIA RB， LI Y， et al. Research progress of components， biological activity and application of Monascus pigment[J]. China Brewing， 2016， 35（7）： 6-10.
[2]JIA J， ZHANG J. Research and application of Monascus spp.[J]. Academic Periodical of Farm Products Processing， 2005（1）： 25-28.
[3]CHEN J， WU YZ， ZHAO XY， et al Research progress on genes related three main polyketides biosynthetic pathway in functional Monascus[J]. China Brewing， 2014， 33（8）： 10-14.
[4]CUI L. Extraction of Monascus pigment and preparation and detection of yellow components[D]. Taian， Shandong Agricultural University， 2008.
[5]SHI H， CHEN DD， HU X. Study on the extraction of monascus pigment[J]. Journal of Hubei Normal University （Natural Science）， 2004， 24（4）： 69-72.
[6]XU W， FAN ZC， MA SH. Separation of Monascus pigments by column chromatography and inhibition comparison of isolated components[J]. Liquor Making， 2010， 37（6）： 49-52.
[7]LI MQ， CHEN YZ， PAN Y. Separation and purification of effective components in functional Monascus pigment by column chromatography[J]. China Brewing， 2011， 30（5）： 41-45.
[8]SUN YJ， HU ZZ， GAO B. Isolation and identification of Monascus strains and determination of Monascus pigment[J]. China Brewing， 2011， 30（1）： 52-54.
[9]LIAN X， LIU L， DONG S， et al. Two new Monascus red pigments produced by Shandong Zhonghui Food Company in China[J]. European Food Research and Technology， 2015， 240（4）： 719724.
[10]YANG Y， CHEN D， DA WY， et al. Monacolin and Monascus spp. and Monascus pigment[J]. Bulletin of Biology， 2017， 52（7）： 13.
Key words Monascus pigments; Separation; Purification; Monascorubramine
As fungi that are beneficial to humans， Monascus spp. can be found in trees， soils and sediments. The genus Monascus belongs to Family Elaphomycetaceae， Order Eurotiales， Class Eurotiomycetes， Division Ascomycota in Kingdom Fungi[1]. So far， more than 20 Monascus spp. have been found in various fermented foods， soils and decaying fruits.
The species of Monascus are mostly saprophytic fungi that prefer an acidic environment with an optimum pH range of 3.5 to 5 and an optimum temperature range of 32 to 35. They can also survive in 10% ethanol[2]. In laboratory， Monascus spp. are often cultured in Malt Extract Agar （MEA） medium. They are different in the color of hyphae， which are mostly white at first， then turn into light red， purple and so on.
Monascus pigments are the main metabolites of Monascus spp. At present， there are 10 Monascus pigments whose chemical structure have been established， six of which are soluble in alcohol， and the other four are soluble in water[3]. Monascus pigments are a mixture of polyketone compounds. There are three major types of Monascus pigments： red， orange， and yellow depending on the color. Among them， six alcoholsoluble pigments can be used as functional food colorants， they are rubropunctamine （red）， monascorubramine （red）， monascorubrine （orange）， rubropunctatine （orange）， ankaflavine （yeallow）， and monascine （yellow）[4]. Materials and Methods
Materials and reagents
Red yeast rice was purchased from Shandong Zhonghui Biotechnology Co.， Ltd. Absolute ethanol （AR）， quartz sand （100-200 mesh）， ammonium acetate， chloroform （AR）， magnesium sulfate， nhexane （AR）， ethyl acetate （AR） and methanol （chromatographic grade） were all bought from CINC High Purity Solvents （Shanghai） Co.， Ltd.
Instruments and equipment
Highspeed pulverizer was purchased from Zhejiang Yongkang Xian Medical Instruments Factory， ultrasonic cleaner from Jiekang Washing Electric Co.， Ltd， vacuum filter from Zhengzhou Greatwall Scientific Industrial and Trade Co.， Ltd.， GF254 thin layer chromatography silica gel analysis plate （25 mm ≠ 75 mm） from Qingdao Shenghai Fine Silica Gel Chemical Co.， Ltd， high performance liquid chromatograph from Shimadzu Corporation， and liquid chromatographymass spectrometry instrument from Agilent Technologies.
Extraction of Monascus pigment
Red yeast rice sample was ground into a fine powder using a pulverizer. Then， 20 g of the powder was collected， soaked in 200 ml of 70% ethanol in a beak， and sonicated at 40 for 30 min[5].
The mixture was vacuumfiltered to remove impurities， and the filtrate was rotary evaporated to nearly dryness， dissolved in 70% ethanol， transferred to a plate， dried in an oven at 40， and finally scraped off with a knife.
Separation of Monascus pigments by column chromatography
Pretreatment of silica gel
The silica gel to be used was dried in an oven at 120 for 30 min， transferred to a dry beaker， and soaked for 20 min in a solvent that had been prepared to elute yellow component[6].
Sample processing
One gram of dried sample was weighed， dissolved in a proper volume of ethanol in a beaker. After 5 g of silica gel powder was added and well mixed， the sample was rotary evaporated to nearly dryness， transferred to another dry beaker and further dried in an over at 40.
Column preparation
The tube was rinsed with tap water and distilled water before it was dried in an oven. Column was prepared by wet method and equilibrated for 3 to 4 h. Finally， an appropriate amount of magnesium sulfate was added onto the top of silica gel.
Sample loading
The solid sample （at an amount of 1/60 to 1/30 of the silica gel） to be separated was gently added to the top surface of the stationary phase along the tube wall， without disturbing the stationary phase. Elution
The column was first eluted with the mixture of nhexane and ethyl acetate （8≥2， v≥v） at a flow rate of 1- 2 ml/min， till fractions of different colors appeared[7]， they were yellow， orange， light red， red and dark red from the bottom to the top of the column. After the yellow fraction was eluted out and collected， the eluent was replaced by the mixture of ethyl acetate and methanol （9≥1， v≥v） to collect the orange fraction. Finally， the column was eluted with methanol to collect the red fraction.
Detection and separation of Monascus pigments by thin layer chromatography
The red， orange and yellow fractions collected above were subjected to thin layer chromatography on a silica gel plate to initially identify the composition of each fraction.
Each sample was applied as a series of spots （1-3 mm in diameter） 1 cm apart from the short edge of the plate. After multiple times of tests， the optimal solvent for separating the component in each fraction was determined： the mixture of nhexane and ethyl acetate （8≥2， v≥v） for yellow fraction， ethyl acetate for orange fraction， the mixture of ethyl acetate and methanol （6≥4， v≥v） for red fraction.
The single components of the red fraction was scraped off with a knife， dissolved in methanol， and centrifuged to remove impurities.
Determination of Monascus pigments by high performance liquid chromatography
Pretreatment of sample
Each fraction of Monascus pigments collected by column chromatography was centrifuged， blown to dryness with a gentle stream of nitrogen， dissolved in 40% methanol and passed through a 0.45 um organic filter[8].
Chromatographic conditions
All determinations were performed at ambient temperature of 20-25 using a Kromasil C18 （250 mm≠4.6 mm， 5 m） column. The mobile phase A was acetic acidammonium acetate buffer （pH = 5）， and the mobile phase B was methanol. The column effluent was monitored at 264 nm. The injection volume was 20 l with a flow rate of 1 ml/min.
Determination of Monascus pigments by mass spectrometry
Monascus pigment sample was dissolved in methanol and passed through 0.45 m organic filter membrane before being subjected to mass spectrometry.
Mass spectrometry was performed under the conditions as follows： Gas Temp 350， Drying Gas 10 L/min Nebulizer 35 psig， VCap 3500 V， Fragmentor 150 v， 100-1000 m/z[9].
Results and Analysis
The yellow， orange and red fractions of Monascus pigments collected by column chromatography were further analyzed by highperformance liquid chromatography （Fig. 1， Fig. 2 and Fig. 3）. It was found that the chromatograms of the yellow and orange fractions had multiple peaks and baseline noise， suggesting they had complex components which were difficult to be separated. In contrast， there were only two major peaks in the chromatogram of red fraction， so the components of this fraction were further separated.
The red fraction was further separated with the mixture of chloroform， methanol and water （5≥3≥1， v≥v≥v） on a thin layer chromatography plate， and two spots were obtained. The retention factor （Rf） value was 0.34 for spot 1 and 0.38 for spot 2， respectively. Liquid chromatography-mass spectrometry （LCMS） analysis revealed that spot 1 had simpler composition. The single component had a molecular mass 397， which was the same as that of monascorubramine previously reported[10]. The monascorubramine sample we isolated had a purity of 90%. Fig. 4 and 5 show the chromatogram and mass spectrum of monascorubramine.
Conclusions
The conditions for extraction， separation and purification of Monascus pigments from red yeast rice were optimized in this study. In brief， Monascus pigments were separated by column chromatography and thin layer chromatography， and identified by liquid chromatographymass spectrometry. Finally， a single Monascus pigment component with a high purity above 90% was obtained， which was identified as monascorubramine. The results may provide a theoretical basis for the research of application of Monascus pigments.
References
[1]ZHOU WB， JIA RB， LI Y， et al. Research progress of components， biological activity and application of Monascus pigment[J]. China Brewing， 2016， 35（7）： 6-10.
[2]JIA J， ZHANG J. Research and application of Monascus spp.[J]. Academic Periodical of Farm Products Processing， 2005（1）： 25-28.
[3]CHEN J， WU YZ， ZHAO XY， et al Research progress on genes related three main polyketides biosynthetic pathway in functional Monascus[J]. China Brewing， 2014， 33（8）： 10-14.
[4]CUI L. Extraction of Monascus pigment and preparation and detection of yellow components[D]. Taian， Shandong Agricultural University， 2008.
[5]SHI H， CHEN DD， HU X. Study on the extraction of monascus pigment[J]. Journal of Hubei Normal University （Natural Science）， 2004， 24（4）： 69-72.
[6]XU W， FAN ZC， MA SH. Separation of Monascus pigments by column chromatography and inhibition comparison of isolated components[J]. Liquor Making， 2010， 37（6）： 49-52.
[7]LI MQ， CHEN YZ， PAN Y. Separation and purification of effective components in functional Monascus pigment by column chromatography[J]. China Brewing， 2011， 30（5）： 41-45.
[8]SUN YJ， HU ZZ， GAO B. Isolation and identification of Monascus strains and determination of Monascus pigment[J]. China Brewing， 2011， 30（1）： 52-54.
[9]LIAN X， LIU L， DONG S， et al. Two new Monascus red pigments produced by Shandong Zhonghui Food Company in China[J]. European Food Research and Technology， 2015， 240（4）： 719724.
[10]YANG Y， CHEN D， DA WY， et al. Monacolin and Monascus spp. and Monascus pigment[J]. Bulletin of Biology， 2017， 52（7）： 13.