Identification And Quality Standard Study On The Main Species Of The Complex Primitive Tibetan Medicine Rubus Stem

Tibetan medicine Rubus Stem is derived from a variety of plants of Rubus genus in Rosaceae,and mainly distributed in Sichuan,Tibet,Qinghai,Gansu,Yunnan and other places provinces.It can be used to treat pneumonia,influenza,bronchitis and other diseases,and is a traditional Tibetan medicine medicine.At present,there are some problems in Tibetan medicine Rubus Stem,such as unclear origin,difficult identification of medicine village and low standard level of Tibetan medicine.These problems affect the safe and reasonable application of Tibetan medicine Rubus Stem in clinical practice.Objectives1.Textual research on the botanic origin plants of Tibetan medicine Rubus Stem.2.To establish the identification method for the main species of Tibetan medicine Rubus Stem,and provide scientific basis for improving its quality standard.Methods:1.Study on the botanic origin plants: By systematically consulting literatures and standards,the name,nature and flavor,effect,classification,original plant of Tibetan medicine Rubus Stem were investigated.And analyzing the main application of medicinal materials about Tibetan medicine Rubus Stem.2.Research on identification method based on infrared spectrum and DNA barcode:(1)Fourier transform infrared spectrometer was used to collect the infrared date of of the main species of Tibetan medicine Rubus Stem(Rubus subornatus Focke,Rubus biflorus Buch.-Ham.ex Smith,Rubus niveus Thunb.Pre-processing methods were applied to optimize the spectrum and then the spectral data were further analyzed by chemometrics,such as HCA,PCA and OPLS-DA.(2)rbcl F/R,rbcl 1F/724 R and rpo C 2F/4R were used to identify the 3 main species of Tibetan medicine Rubus Stem.To evaluate the ability of each sequence to identify different primitive plants of Tibetan medicine Rubus Stem,and then the candidate sequences were used to identify the commercial medicinal materials of Tibetan medicine Rubus Stem.3.The 3 main species were studied by characters and microscopic identification,and the water content,total cross content,acid insoluble sexual content and extract were examined according to the method of Chinese Pharmacopoeia(2020 edition).Based on UPLC-Q Exactive Orbitrap-HRMS,the chemical constituents of the main species of Tibetan medicine Rubus Stem were analyzed,and TLC method was established by selecting representative constituents.Results:1.In combination with the frequency of herbal records and the coincidence degree of morphological description,it can be inferred that Rubus biflorus Buch.-Ham.ex Smith,Rubus subornatus Focke,Rubus subornatus var.melanadenus Focke,Rubus phoenicolasius Maxim.and Rubus niveus Thunb.were the main species of Tibetan medicine.2.Infrared spectrum and DNA barcode identification methods of Tibetan medicine Rubus Stem were established:(1)In the second derivative spectrum,the band 400-1800 cm-1 was the identification region of the 3 varieties.PCA and OPLS-DA methods were able to quickly distinguish the 3 varieties.(2)The rbcl F/R sequence had a better identification effect.Through BLAST comparison of NCBI database,phylogenetic tree construction and Gen Bank sequence alignment,we determined the primitive plants of medicinal materials.Infrared spectroscopy was suitable for the identification of fresh samples and DNA barcoding was suitable for the identification of medicinal materials.3.Formulation of quality standard for identifying Tibetan medicine Rubus Stem:(1)Identification characteristics and microscopic identification: Rubus biflorus Buch.-Ham.ex Smith had a greenish-brown or yellowish-brown surface,with lavender or pink residual pith,and Rubus niveus Thunb.had circular stem marks on its stem.In comparison with Rubus subornatus var.melanadenus Focke and Rubus biflorus Buch.-Ham.ex Smith,the xylem vessels of Rubus niveus Thunb.were scattered singly or one or two together.The cluster crystals in Rubus biflorus Buch.-Ham.ex Smith powder were smaller and more pointed,and the solitary crystals in Rubus niveus Thunb.powder were found.(2)Identified 22 compounds from 3 main varieties,and catechins were selected as reference substance to establish TLC identification method.(3)Established the corresponding limit requirements(moisture content 8.5% or less,the total ash content 1.8% or less,acid insoluble ash content 0.6 % or less,30% alcohol soluble extract 12% or higher).Conclusion:This paper preliminarily inferred the main species of Tibetan medicine Rubus Stem by textual research.By using Fourier transform infrared spectroscopy and DNA barcoding technology,a new method for the identification of Tibetan medicine Rubus Stem was established.And through pharmacognosy study,the quality standard of Tibetan medicine Rubus Stem was preliminarily established.The results of this study can provide a basis for the quality standard improvement and follow-up research of Tibetan medicine Rubus Stem.