The Separation And Purification Of Red Yeast Rice Pigments And The Analysis Method Of Ten Pigments

The Monascus pigments(MPs),one of the Monascus fermented secondary metabolites,is a good edible nature pigments,has been used as food colorants for more than 1000 years in China,Japan and other Southeastern Asian countries.The MPs have many functions such as anti-inflammatory,antibacterial and cholesterol-lowering effects.The MPs is an azaphilone mixture,which usually include red,yellow and orange pigments.It is important to establish a finger profile analysis method of the natural Monascus pigments for the quality control and differentiate it from chemical modification of Monascus pigments.It is also helpful to light stability and hue research on MPs.In this work,the research aims are as follows:(1)purified and further structural identification of Monascus pigments.(2)optimized a HPLC method to simultaneously analyze the isolated pigments.The analytical HPLC is used to establish a method for the analysis of pigment,which provides a convenient and feasible quick detection method for the screening and identification of pigments in MPs samples.After screening,the fermentation products of the NM7 which comes from our laboratory and the Gutian 4000E buying from market were selected to purify the target pigments.The Monascus pigments crude was acquired from two kinds of MPs raw materials with ultrasonic assisted ethanol extraction.Preparative HPLC experiments were performed to purified the target pigments using Agilent 1260 series Liquid Chromatography system on a cosmosil Packed column(20×250 mm,5 ?m).The separated pigments chromatographic peak were respectively collected,then concentrated with vacuum rotary evaporation,nitrogen blowing and freeze-drying methods.After these steps,ten different pigments were obtained,which is named as:RA,RB,MFA,MFB,Y1,Y2,R1,R2,O1 and O2,respectively.R1,R2,RA and RB were red pigments.Y1,Y2,MFA and MFB were yellow pigments.O1 and O2 were orange pigments.The purities of target pigments were identified more than 90%with analytical HPLC.The pigments accurate molecular weights were measured using the LC-MS-IT-TOF.The results are as follows:RA 397.1886,RB 425.2204,MFA 356.1652,MFB 384.1926,Y1358.1778,Y2 386.2090,R1 353.1623,R2 381.1938,O1 354.1503 and O2 382.1805.The structures characterization was determined with an AV-400 NMR spectrometer.R1,R2,Y1,Y2,O1,O2,MFA and MFB were identified as Monascorubramine,Rubropunctamine,monascin,ankaflavin,Monascorubin,Rubropunctatin,Monasfluore A and Monasfluore B by comparing MS and 1H-NMR spectra NMR data with those reported in literature,respectively.The structures of RA and RB were identified by comparing MS and 1H-NMR spectra,13C-NMR spectra,and 2D spectra(HMQC,HMBC,COSY),respectively.Finally,a HPLC detection method for simultaneously detection of 10 pigments was optimized using a Eclipse Plus C18(4.6×250 mm,5 ?m).The mobile phase was composed by water and acetonitrile with gradient elution(the detection method of HPLC-2),the flow rate and detection wave length was set at 1mL/min and 410 nm,respectively.