Study On The Method For The Quality Control Of Trifolium Pratense Cv.minshan Based On HPLC Fingerprint And GC-MS Technology

Trifolium pratense cv.minshan is a unique pasture species in Gansu.It has extremely high medicinal and nutritional value,which is rich in isoflavones,amino acids and other substances.It is a natural medicinal plant with great development prospects.This research mainly studies the quality control methods of Trifolium pratense cv.minshan and provides a scientific basis for the quality control of it.The main research contents are as follows:(1)The identification method of Trifolium pratense cv.minshan was established.It was found that the flowers,stems and leaves of Trifolium pratense cv.minshan have non-glandular hairs and square crystals of calcium oxalate,which can be used as the microscopic characteristics of it;With biochanin A and formononetin as reference substances,silica gel as the stationary phase,and petroleum ether: acetone=2:1(V/V)as the mobile phase,a thin-layer chromatographic identification method for Trifolium pratense cv.minshan was established.The results show that the established thin-layer chromatography method has good separation ability for biochanin A and formononetin in the sample,and the method has good stability and reproducibility.Microscopic features combined with thin-layer chromatography can identify Trifolium pratense cv.minshan.(2)The quality control method of HPLC fingerprint of Trifolium pratense cv.minshan was established.A single rotation method was used to optimize the HPLC fingerprinting conditions,and 0.2% formic acid and acetonitrile were used as mobile phases for gradient elution.With biochanin A,ononin,formononetin,genistein,daidzin,daidzein and galycosin-7-glucoside as the index components,the methodology was validated.Taking the common peaks of 12 batches of Trifolium pratense L.samples from 4 producing areas as objects,principal component analysis and cluster analysis were carried out to determine the sources of Trifolium pratense L.samples from different producing areas.It provides a reference method for the quality control of Trifolium pratense cv.minshan,and lays a foundation for the subsequent study of the "spectral effect" relationship.(3)GC-MS was used to determine the composition of the volatile oil of Trifolium pratense cv.minshan,and a GC-MS method for the determination of free amino acids from Trifolium pratense cv.minshan was established,the GC-MS method for the determination of volatile oil and amino acids of Trifolium pratense cv.minshan was applied to the determination of volatile oil and amino acids of Trifolium pratense L.from other regions and compared.We used GC-MS to determine the volatile oil composition of Trifolium pratense L.from different origins,and calculated the proportion of each volatile compound in the total volatile oil using the peak area normalization method.The results show that the volatile oil components of Trifolium pratense cv.minshan are the most abundant,and the volatile oil components of Trifolium pratense cv.e’xi are the simplest.A single rotation method was used to optimize the amino acid derivation conditions;the United Nations Food and Agriculture Organization/World Health Organization amino acid model was used to carry out amino acid evaluation of Trifolium pratense L.from different origins.The results showed that the nutritional value of Trifolium pratense cv.minshan was relatively high.