用次生代谢物指纹图谱法区分不同用途红曲的可行性探索
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摘要
针对红曲药材传统功效研究基础非常薄弱、流通市场上红曲药材质量一致性差的现状,本文以实地采集的道地产区在内、来源准确的红曲为研究对象,以其活性次生代谢物为重点探针,首次探索建立了能区分不同用途红曲的超高效液相色谱法:采用Capcell Core AQ柱, 100 mm×4.6 mm, 2.7μm; PDA (200～650 nm,提取:237 nm)和ELSD检测器串联;流动相为含0.1%甲酸的水(A)-乙腈(B)系统,流速为0.5 mL?min-1,梯度洗脱(0～15 min, 50%B→85%B; 15～16 min, 85%B→50%B并维持至21 min)。所建方法的各指标均符合中国药典规定。样品考察发现:(1)相同用途的红曲含许多共性次生代谢物,不同来源的色曲和酿造红曲的指纹图谱相似性大于0.90;而不同用途的红曲在次生代谢物种类、含量方面有明显差异,用PCA (principal component analysis,主成分分析)能予以明确区分;(2)色曲和酿造红曲中均未检出洛伐他汀;含洛伐他汀的红曲,不同企业样品的指纹图谱和洛伐他汀含量会有差异,甚至差异显著,需要结合活性研究对其工艺进行科学规范;(3)传统"健脾消食、活血化瘀"用红曲,与道地产区的酿造红曲指纹图谱非常相似。上述结果表明:本文所建的红曲次生代谢物指纹图谱法能有效区分不同用途红曲,为揭示红曲药材的不同疗效与其次生代谢物之间的关系、进而科学规范并提高流通市场上红曲药材的质量奠定了坚实基础。
Though red yeast rice(RYR) has been used as medicine for centuries, few study has been reported about its biological activities related to traditional medicinal application and marketed RYR showed poor consistency in quality. In this study, with comprehensive investigation of their production processes and field acquisition samples including those from genuine producing area, an ultra performance liquid chromatographic(UPLC)method was firstly established to discriminate RYR for different applications based on their secondary metabolites fingerprint. It was performed on a CAPCELL CORE AQ column(100 mm × 4.6 mm, 2.7 μm), with PDA(range:200-650 nm, extracted: 237 nm) and ELSD detection. The mobile phase used was water(A) and acetonitrile(B)both containing 0.1% formic acid at gradient elution(0-15 min, 50% B→85% B(linear); 15-16 min, 85 % B→50% B(linear) and maintained until 21 min), with a flow rate of 0.5 mL?min-1. The method established was fully validated in agreement with guidelines of Chinese Pharmacopeia. Common metabolites were found in RYR for same application and the fingerprints of RYR for food coloring or brewing from various manufacturers had similarities above 0.90. Meanwhile, significant differences were observed among the fingerprints for various applications and discrimination could be achieved by principal component analysis(PCA). Lovastatin was absence in RYRs for food coloring or brewing, and the fingerprint of traditional medicinal RYR was similar to that of RYR for brewing. However, standardization was required for RYR containing lovastatin because of their significant differences from various manufacturers in fingerprints and lovastatin content. The results demonstrated the feasibility to discriminate RYR for different applications by the secondary metabolites fingerprint method established in this study, which provides a scientific basis to investigate the relationship between biological activities of medicinal RYR and their corresponding secondary metabolites, and further aid their quality standardization and improvement.
Though red yeast rice(RYR) has been used as medicine for centuries, few study has been reported about its biological activities related to traditional medicinal application and marketed RYR showed poor consistency in quality. In this study, with comprehensive investigation of their production processes and field acquisition samples including those from genuine producing area, an ultra performance liquid chromatographic(UPLC)method was firstly established to discriminate RYR for different applications based on their secondary metabolites fingerprint. It was performed on a CAPCELL CORE AQ column(100 mm × 4.6 mm, 2.7 μm), with PDA(range:200-650 nm, extracted: 237 nm) and ELSD detection. The mobile phase used was water(A) and acetonitrile(B)both containing 0.1% formic acid at gradient elution(0-15 min, 50% B→85% B(linear); 15-16 min, 85 % B→50% B(linear) and maintained until 21 min), with a flow rate of 0.5 mL?min-1. The method established was fully validated in agreement with guidelines of Chinese Pharmacopeia. Common metabolites were found in RYR for same application and the fingerprints of RYR for food coloring or brewing from various manufacturers had similarities above 0.90. Meanwhile, significant differences were observed among the fingerprints for various applications and discrimination could be achieved by principal component analysis(PCA). Lovastatin was absence in RYRs for food coloring or brewing, and the fingerprint of traditional medicinal RYR was similar to that of RYR for brewing. However, standardization was required for RYR containing lovastatin because of their significant differences from various manufacturers in fingerprints and lovastatin content. The results demonstrated the feasibility to discriminate RYR for different applications by the secondary metabolites fingerprint method established in this study, which provides a scientific basis to investigate the relationship between biological activities of medicinal RYR and their corresponding secondary metabolites, and further aid their quality standardization and improvement.
引文
[1]Endo A.Monacolin K,a new hypocholesterolemic agent pro‐duced by a Monascus species[J].J Antibiot,1979,32:852-854.
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[10]Lu Z,Kou W,Du B,et al.Effect of Xuezhikang,an extract from red yeast Chinese rice,on coronary events in a Chinese popula‐tion with previous myocardial infarction[J].Am J Cardiol,2008,101:1689-1693.
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[13]Fu JQ,Zhang HS,Yao JC.Chinese Red Yeast Rice and Related Practical Techniques(中国红曲及其实用技术)[M].Wuhan:Wuhan University of Technology Press,2017:169-214,38-77.
[14]Tsukahara M,Shinzato N,Tamaki Y,et al.Red yeast rice fermen‐tation by selected Monascus sp.with deep-red color,lovastatin production but no citrinin,and effect of temperature-shift cultiva‐tion on lovastatin production[J].Appl Biochem Biotechnol,2009,158:476-482.
[15]Miyake T,Uchitomi K,Zhang MY,et al.Effects of the principal nutrients on lovastatin production by Manascus pilosus[J].Biosci Biotechnol Biochem,2006,70:1154-1159.
[16]Huang J,Liao NQ,Li HM.Linoleic acid enhance the production of moncolin K and red pigments in Monascus ruber by acti‐vating mokH and mokA,and by accelerating cAMP-PkA path‐way[J].Int J Biol Macromol,2018,109:950-954.
[17]Said MF,Brooks J,Chisti Y.Optimal C:N ratio for the produc‐tion of red pigments by Monascus ruber[J].World J Microbiol Biotechnol,2014,30:2471-2479.
[18]Chen MH,Ji DW,Ma RJ,et al.Effects of different substrates on the important metabolites of Monascus fermented products[J].China Brewing(中国酿造),2013,32:22-24.
[19]Yang Y,Zheng RJ,Hua YY,et al.High-performance liquid chromatographic analysis of lovastatin(lactone)and lovastatin acid in red yeast rice[J].Chin J Health Lab Technol(中国卫生检验杂志),2014,24:1714-1717.
[2]Endo A,Husumi K,Nakamura T,et al.Dihydromonacolin L and monacolin X,new metabolites that inhibit cholesterol biosyn‐thesis[J].J Antibiot,1985,38:321-327.
[3]Endo A,Husumi K,Negishi S.Monacolin J and L,new inhibi‐tors of cholesterol biosynthesis produced by Monoscus ruber[J].J Antibiot,1985,38:420-422.
[4]Endo A,Komagata D,Shimada H.Monacolin M,a new inhibitor of cholesterol biosynthesis[J].J Antibiot,1986,39:1670-1673.
[5]Endo A.Monacolin K,a new hypocholesterolemic agent that specifically inhibits 3-hydroxy-3-methylglutaryl coenzyme Areductase[J].J Antibiot,1980,33:334-336.
[6]Endo A.Chemistry,biochemistry and pharmacology of HMG-CoA reductase inhibitors[J].Klin Wochenschr,1988,66:421-427.
[7]Huang CF,Li TC,Lin CC,et al.Efficacy of Monascus purpu‐reus Went rice on lowering lipid ratios in hypercholesterolemic patients[J].Eur J Cardiovasc Prev Rehabil,2007,14:438-440.
[8]Lin CC,Li TC,Lai MM.Efficacy and safety of Monascus purpureus Went rice in subjects with hyperlipidemia[J].Eur JEndocrinol,2005,153:679-686.
[9]Gheith O,Sheashaa H,Abdelsalam M,et al.Efficacy and safety of Monascus purpureus Went rice in children and young adults with secondary hyperlipidemia:a preliminary report[J].Eur JInternal Med,2009,20:e57-e61.
[10]Lu Z,Kou W,Du B,et al.Effect of Xuezhikang,an extract from red yeast Chinese rice,on coronary events in a Chinese popula‐tion with previous myocardial infarction[J].Am J Cardiol,2008,101:1689-1693.
[11]Dujovne CA.Red yeast rice preparations:are they suitable substitutions for statins?[J].Am J Med,2017.DOI:10.1016/j.amjmed.2017.05.013.
[12]Lindsey C,Andrea G,Atanaz Z,et al.Review of red yeast rice content and current food and drug administration oversight[J].JClin Lipidol,2013,7:117-122.
[13]Fu JQ,Zhang HS,Yao JC.Chinese Red Yeast Rice and Related Practical Techniques(中国红曲及其实用技术)[M].Wuhan:Wuhan University of Technology Press,2017:169-214,38-77.
[14]Tsukahara M,Shinzato N,Tamaki Y,et al.Red yeast rice fermen‐tation by selected Monascus sp.with deep-red color,lovastatin production but no citrinin,and effect of temperature-shift cultiva‐tion on lovastatin production[J].Appl Biochem Biotechnol,2009,158:476-482.
[15]Miyake T,Uchitomi K,Zhang MY,et al.Effects of the principal nutrients on lovastatin production by Manascus pilosus[J].Biosci Biotechnol Biochem,2006,70:1154-1159.
[16]Huang J,Liao NQ,Li HM.Linoleic acid enhance the production of moncolin K and red pigments in Monascus ruber by acti‐vating mokH and mokA,and by accelerating cAMP-PkA path‐way[J].Int J Biol Macromol,2018,109:950-954.
[17]Said MF,Brooks J,Chisti Y.Optimal C:N ratio for the produc‐tion of red pigments by Monascus ruber[J].World J Microbiol Biotechnol,2014,30:2471-2479.
[18]Chen MH,Ji DW,Ma RJ,et al.Effects of different substrates on the important metabolites of Monascus fermented products[J].China Brewing(中国酿造),2013,32:22-24.
[19]Yang Y,Zheng RJ,Hua YY,et al.High-performance liquid chromatographic analysis of lovastatin(lactone)and lovastatin acid in red yeast rice[J].Chin J Health Lab Technol(中国卫生检验杂志),2014,24:1714-1717.