Metabolic Profiling Preliminary Study On Isoflavonoids In Pueraria Lobata Based On Two Dimensional High Performance Thin Layer Chromatography

The application of metabolomics in the research of medicinal plants is helpful,not only revealing the mechanism of formation, transformation and relationship of theeffective components in the plant body, but also understanding regional distribution ofplants by studying metabolites of plants in differente co-environment, to identify ofgenuine medicinal materials and to guide medicinal material cultivation. It will play ahuge role in development of the traditional Chinese medicine.Metabonomics analysis methods are varied. High performance thin layerchromatography (HPTLC) has been widely used for separation and identification ofnatural products, and its main advantages are rapidity, simplicity, and highsample-throughput. Normally, one-dimensional TLC can achieve the separation ofmixture. But to a mixture of complex ingredient, especially plant extract, simpleone-dimensional thin layer chromatography is difficult to achieve the ideal ofseparation effect, so complex separation procedures like two-dimensional thin layerchromatography (2D-HPTLC) were developed.Pueraria radix is a kind of plant resource for traditional Chinese medicine andfood. The depth studies had confirmed that isoflavoniods were biologically activeingredients of P. radix. So far, more than30kinds of isoflavoniods from P. radix arereported. Meanwhile, biosynthesis pathway of flavoniods was very clear andsynthesis-related enzyme and genes of flavoniods were separaton and identification.So isoflavoniods of P. radix is a model system of secondary metabolites for study ofmetabolism.In this paper, P. lobata (Willd) Ohwi and P. thomsonii Benth serve as the researchsystem, the metabolic profiling platform of isoflavoniods in P.lobata based on2D-HPTLC was implored. By analysing the metabolic profiling platform onisoflavoniods in P.lobata and P.thomsonii Benth from different producing areas and indifferent growth years, try to reveal accumulation regularity of isoflavoniods. thisstudy not only researching of accumulation regularity of isoflavoniods in P. lobataand P. thomsonii Benth from different producing areas and in different growth years,also playing an important significance to perfecting metabolomics methodology. Themain results are as following: Compares the effects of2D-HPTLC with different adsorbents and differentmobile phases in this paper, and2D-HPTLC technology platform was determinedPloyamide for adsorbent; Mobile phase system: the first one-dimensional for water:n-butanol: acetone:1,4-Dioxane=70:15:10:5(v/v); the two-dimensional forbenzene: methanol: butanone: water=55:25:23:2(v/v), the effective assay ofisoflavoniods was realized and the metabolic profiling analysis method ofisoflavoniods was established.2D-HPTLC pattern show that: content of common spots of P. lobata and P.thomsonii Benth have clear difference. This suggests that the weight per unitisoflavnoid in P. thomsonii Benth is well below the P. lobata’s. Growing years have amajor effect on the isoflavoniods content of P. lobata. With the age of growth, most ofisoflavoniods have decrease, but some increase.2D-HPTLC patterns of P. lobatasamples from different producing areas are similar. There was a significant differencein isoflavoniods content, for example, the contents of puerarin、3’-methoxypuerarin ordaidzin, from high to low, were Juhuashan, Shucheng three year, Jinzhai three year,Wuhu heng gang; but the content of daidzein rom high to low, were Juhuashan, zhaithree year, Shucheng three year,Wuhu heng gang;2D-HPTLC patterns of P.thomsoniiBenth samples from different producing areas are similar. There was certaindifference in isoflavoniods content, for example, the content of puerarin or daidzin,from high to low, were Gange no.5, Jangxi Jian, Ningguo no.2, Wu hu nanling.There were14compounds extracted, isolated and purified from P.radix cvShuchen, they were detected by HPLC and their structures were determined mainlyby means of NMR, ESI-MS techniques. Compound1was determined as3’-methoxypuerarin; Compound2was determined as daidzin; Compound3was determined asononin; Compound4was determined as genistin; Compound5was determined assissotrin; Compound6was determined as daidzein; Compound7was determined aspuerarin; Compound8was determined as genistein8-C-glucoside; Compound9wasdetermined as formononetin8-C-apiosyl-(1-6)glupyanside; Compound10wasdetermined as8-C-β-glucofuranosyl-4’,7-dihydroxy-isoflavone; Compound11wasdetermined as formononetin; Compound12was determined as4’,7-Di-O-Methyldaidzein; Compound13was determined as (+)-puerol B-2’’-O-glucopyranoside;Compound14was determined as formononetin8-C-xyl-(1-6)glupyanside. AndCompound10、12was isolated from Pueraria.family for the first time. There were6compounds extracted, isolated and purified from callus cultures ofAnhui Shuchen P.radix: daidzin (Compound2); ononin (Compound3); genistin(Compound4); daidzein (Compound6); puerarin (Compound7); Compound15wasdetermined as genistein.There are significant differences in the content and component of isoflavonoidsfrom P. lobata cell suspension cultures and P. lobata, P. lobata is relatively highcontent in puerarin,3’-methoxypuerarin and other glycosides; glycosides of P. lobatacell suspension including daidzin and genistin; aglycons including genistein anddaidzein.