A Preliminary Study On The Quality Standard Of Southwestern National Medicine Gangxiang

ObjectiveGangxiang,derived from Dalbergia yunnanensis Franch.of the legume containing resinous roots,rhizomes and canes,is mainly produced in Yunnan,Sichuan,Guizhou and other places,and also distributed in Southeast Asia such as Myanmar.Field investigations have found that in the southwest ethnic minority areas,they are traditionally used to make tea and soak in wine,or single oral administration is used to treat cold,cough,asthma,acute and chronic gastroenteritis and cardiovascular diseases,as well as external use to treat skin ulcers,injuries,burns,bites from snakes and insects,etc.The local Taoist temples and the people have always used Gangxiang as a sacrifice and incense to avoid filth.Gangxiang is a common fragrant medicine recorded in medicinal books in the southwest region,such as "Xichang Chinese Herbal Medicine" and "Sichuan Chinese Herbal Medicine".As a common national medicine in the southwest region,Gangxiang is rich in resources and has definite clinical effects.It has potential development and utilization prospects and economic value.In order to further develop new resources of Dalbergia medicinal plants,this article intends to conduct a preliminary study on the quality standards of Gangxiang,including:pharmacognosy research,inspection items and extract determination,volatile oil and total flavonoid content determination,volatile oil composition and multiple differences in origin Statistical Analysis.Finally,a draft of the quality standard for Gangxiang was formulated to provide a theoretical basis for the comprehensive development and utilization of Gangxiang.Methods1.The morphological research and variety identification of Gangxiang original plantCompare the specimens of the Chinese Flora and the China Virtual Herbarium,and consult relevant experts in Dalbergia spp.to conduct research on the original plant morphology of Gangxiang;Use DNA molecular identification technology to identify the original plant of Gangxiang.2.Characteristics and microscopic identificationUsing visual,hand-touching,nose-smelling,mouth-tasting and other character identification methods,we make the research and summary description of the shape,size,surface,texture,cross-section,odor and other characteristics of Gangxiang samples.Using traditional microscopy method such as paraffin sections to study the characteristics of the three-dimensional section,powder and dissociated tissue of Gangxiang.3.Chemical composition and thin-layer chromatography inspectionSystematic pre-examination and inspection of the chemical components of Gangxiang through precipitation reaction and color reaction;Established the thin-layer identification system of Gangxiang by layer chromatography.4.Inspection items and determination of extract contentWith refering to the ash determination method,moisture determination method,and extract determination method under the four general provisions of the 2015 edition of"Chinese Pharmacopoeia",the total ash,moisture and extract content of 36 batches of Gangxiang samples were determined,and the above items were formulated based on the measurement results Content limit.5.Determination of the content of volatile oil and total flavonoidsWith refering to the volatile oil determination method "2204" in the 2015 edition of"Chinese Pharmacopoeia",the content of the volatile oil was determined.With rutin as the reference substance and aluminum chloride as the developer,UV-visible spectrophotometry was used to determine total flavonoid content.6.GC-MS analysis of volatile oil components and multivariate statistical analysis of differences in production areaThe vapor distillation method was used to extract the volatile oil from the samples of Gangxiang.Gas chromatography-mass spectrometry(GC-MS)was used to identify and analyze the chemical components of the volatile oils of Gangxiang.Finally,use SIMCA-P software to perform principal component(PCA)analysis,orthogonal partial least squares discriminant(OPLS-DA)analysis,and use VIP value and Independent-Samples T Test to filter the variables,so as to screen out obvious differential variables and their markers are used to identify the different origins of Gangxiang.Results1.The original plants of Gangxiang are large vines,sometimes in the form of large shrubs or small trees.Pinnate compound leaves,13 to 25 small leaves,nearly leathery,oblon.Cymes are terminal or born in the upper leaf axils.Flowers are slightly dense.bracteoles ovate,calyx bell-shaped,corolla white.9 Stamens,short style.The pods are thin leathery.oblong or elliptical,with obvious reticulation on the seed part.Most of the seeds are one,and 2-3 are rare,with kidney-shaped.Flowering season is from April to May.The online Blast result of the target band(rbcl sequence)amplified by the total DNA of the fresh leaves by PCR is 100%similar to the Dalbergia yunnanensis Franch.’s rbcl sequence in genebank,and the E value is 0.In addition,after matching with the plant gene bank of Dalbergia experts Shijin Li and Tieyao Tu,the results are consistent with Dalbergia yunnanensis Franch..Therefore,the original plant of Gangxiang is Dalbergia yunnanensis Franch..2.Gangxiang is in the shape of long cylinder,flat block or irregular block,with different sizes.The surface has fine wood grain and rich color.Knife cut marks are obvious,and those with oily feet show oil marks,and sometimes yellow-white wood remains incomplete.The texture is hard and sinks into water,with obvious small holes and irregular textures on the section.The smell is aromatic,the taste is slightly astringent and sweet.The buried Gangxiang is brown-black or dark-brown,and there is often remaining mud.3.The cross-section of the root of the wood ray is 1 to 3 rows of cells,and the ducts are mostly single and occasionally in groups.The axial parenchyma is ribbon-shaped and arranged alternately with fiber bundles.The tangential longitudinal section of the wood ray is slightly superimposed,with 1～3 rows of cells in width and 5～24 cells in height;the ducts are short-segmented ducts with marginal pits;the wood parenchyma cells are rectangular or sharp at one end,and the walls are thickened like beads.The radial longitudinal section of the wood ray is a horizontal band,which is a special-shaped cell ray,which intersects the wood fiber perpendicularly.The three sections of the stem are microscopically similar to the roots.The powder and dissociated tissues mainly contain wood fiber cells,marginal pits ducts,wood ray cells,wood parenchyma cells.4.The pre-test results of the chemical composition show that Gangxiang may contain sugars,organic acids,phenols,tannins,flavonoids,etc.in addition to the volatile oil.TLC identification results show that the system of n-hexane-ethyl acetate-formic acid(13:6:1.6)has good reproducibility and resolution,which can provide a basis for the authenticity identification of Gangxiang.5.The test results of inspection items and extract content showed that the total ash content of 36 batches of Gangxiang samples ranged from 0.38%to 3.95%,with an average of 1.57%;the moisture content ranged from 4.34%to 14.02%,with an average of 9.62%;The 80%ethanol extract content ranged from 11.23%to 55.99%,with an average value of 31.98%.According to the experimental results,it is proposed that the total ash content of the bark should not exceed 4.5%,the moisture content should not exceed 14.5%,and the 80%ethanol extract content should not be less than 11.0%.6.The volatile oil content of 36 batches of Gangxiang ranges from 0.30%to 3.59%,with an average value of 1.25%.The proposed extraction rate of the volatile oil of Gangxiang shall not be less than 0.3%;the range of total flavonoids content is between 0.80%and 7.19%.The average value is 4.03%,and the total flavonoids content of different origins and specifications of Gangxiang has certain differences.7.GC-MS component analysis showed that the main component of the volatile oil of Gangxiang was neroli,and the other main components were 2-Methylene-5α-cholestan-3β-ol,Humulene epoxide II,Weideol,Farnesol,Bisabolol,etc.In addition,the use of multivariate statistical analysis based on the volatile oil components can distinguish the different origins of Gangxiang to a certain extent and provide a reference for the identification of its origin.ConclusionThe pharmaceutical research of Gangxiang shows that its source is clear,its properties and microscopic characteristics are outstanding;the established thin-layer unfolding system has simple and safe reagent composition,and the experimental operation is simple and feasible.The proposed inspection items,extract and volatile oil content limits are reasonable and reliable.The main component of the volatile oil of Gangxiang is neroliol.Using the SIMCA-P software to perform a multivariate statistical analysis of the volatile oil components can preliminarily distinguish the differences in production areas.Based on the above projects,this subject drafted a draft of the quality standard for Gangxiang,which provides a theoretical basis for the quality control of Gangxiang,and lays the foundation for the development of new resources of ethnic medicine.