The Establishment Of Quality Standard For Cyanotis Arachnoidea C.b.clark Of Yi Herbs And Study On Its Main Components Promoting Wounding Healing

Objective: Cyanotis arachnoidea C.B.Clark is one of the commonly used medicinal materials of Yi ethnic groups.It has the effects of warming the meridians,clearing collaterals,removing dampness and relieving pain,mainly used for rheumatism,relieving dampness and reducing swelling,preventing deficiency and heat,diabetes,wound healing,etc.With the wide application of Cyanotis arachnoidea C.B.Clark in various fields,its demand is expanding,the cultivation area of medicinal materials is gradually expanding and dispersing to Yunnan,Sichuan,Fujian,Guangdong,Taiwan and other places.However,at present,the basic research on Cyanotis arachnoidea C.B.Clark is very weak at home and abroad,and there is no quality control index for main characteristic ingredients or active ingredients,let alone a systematic quality control standard system,which seriously restricts the rational development and wide utilization of Cyanotis arachnoidea C.B.Clark.Therefore,it is urgent to carry out in-depth research on Cyanotis arachnoidea C.B.Clark,further explore the pharmacodynamic material basis and mechanism of Cyanotis arachnoidea C.B.Clark to provide accurate and scientific basis for the quality control and wide application of medicinal materials.Methods:(1)Establishment of quality standard for Cyanotis arachnoidea C.B.Clark of Yi herbs: The ecological environment of Cyanotis arachnoidea C.B.Clark in Sichuan,Yunnan and Fujian were investigated,and 32 batches of Cyanotis arachnoidea C.B.Clark were sampled from 3 producing areas.Then,We focus on making wax leaf specimens and physical specimens;Observing and describing the characters of medicinal materials;Carrying out the microstructure and powder microscopic identification of medicinal parts.In this study,We also established a TLC method for the identification of 20-hydroxyecdysone,Completed the inspection items and the content determination of the extract.At the same time,HPLC method was used to establish the fingerprint of Cyanotis arachnoidea C.B.Clark to analyze the difference of herb from different producing areas combined with stoichiometry.the contents of three main phytosteroids components(20-hydroxyecdysone,ajugasterone C,2-o-acetyl-20-hydroxyecdysone)were determined.Thus,we preliminarily draw up the draft quality standard of Cyanotis arachnoidea C.B.Clark.(2)Study on wound healing activity of main components in Cyanotis arachnoidea C.B.Clark of Yi herbs: we established mouse fibroblast(L929)model in vitro.MTT was used to detect the effects of three main phytosteroids on the viability of L929 to further determine the effect on the proliferation of fibroblast at the effective concentration range.Cell scratch method was used to evaluate the effect of components on the migration of fibroblasts.Meanwhile,using Western blot for detecting the content change of TGF-β,VEGF and b FGF,so as to comprehensively and systematically evaluate the effect and mechanism of active ingredients on promoting wound healing.Results:(1)The macroscopical plant morphology,medicinal material characters and microscopic characteristics of Cyanotis arachnoidea C.B.Clark were described accurately.The contents of water,total ash,acid insoluble ash and extracts of 32 batches of Cyanotis arachnoidea C.B.Clark ranged from 8.28 to 12.73%,8.02 to 11.56%,3.41 to 5.02%,18.45%to 24.56%.20-hydroxyecdysone was selected as TLC,chloroform-methanol-water-formic acid(7:3:0.5:0.05)was utilized as developing agent for the qualitative identification of Cyanotis arachnoidea C.B.Clark.The spot separation effect was good with moderate specific shift.The fingerprint similarity of 32 batches of Cyanotis arachnoidea C.B.Clark was above 0.9.The results of stoichiometric analysis showed that the 32 batches of Cyanotis arachnoidea C.B.Clark could be divided into three categories,and most of the samples from each producing area were distributed in different quadrants,showing regional differences.20-hydroxyecdysone and 2-o-acetyl-20-hydroxyecdysone were determined as the main components affecting the quality of herb.Further evaluation of quality differences among different producing areas showed that yunnan producing area had higher comprehensive scores.The contents of 20-hydroxyecdysone,ajugasterone C,2-o-acetyl-20-hydroxyecdysone varied from 14.6905 to 37.1789 mg/g,1.6250 to 4.7236mg/g,0.2405 to 8.7820mg/g,respectively.The content of 20-hydroxyecdysone and ajugasterone C was higher in Yunnan,and the content of 2-o-acetyl-20-hydroxyecdysone was higher in Sichuan.(2)20-hydroxyecdysone,ajugasterone C and 2-o-acetyl-20-hydroxyecdysone all could improve the activity of fibroblasts in a certain concentration range of 0.1～100 μmol/L,0.1～150 μmol/L and 0.1～100 μmol/L,respectively.Further experimental study proved that the three phytosterones could promote the proliferation of fibroblasts in different degrees,particularly,20-hydroxyecdysone had the best proliferative activity.Meanwhile,20-hydroxyecdysone,ajugasterone C and 2-o-acetyl-20-hydroxyecdysone also promoted the migration of fibroblasts to some extent.Furthermore,20-hydroxyecdysone and2-o-acetyl-20-hydroxyecdysone can significantly increase the expression of TGF-β,VEGF and b FGF,which can promote wound repair.However,ajugasterone C did not significantly increase the expression of TGF-β,VEGF and b FGF.Conclusion:This study established a draft quality standard for Cyanotis arachnoidea C.B.Clark.Secondly,it was found that 20-hydroxyecdysone,ajugasterone C and2-o-acetyl-20-hydroxyecdysone all could play a certain role in promoting wound healing,however,20-hydroxyecdysone and 2-o-acetyl-20-hydroxyecdysone have more significant effects.above studies have improved the quality control of Cyanotis arachnoidea C.B.Clark and explained its pharmacodynamic material basis,further provided accurate and scientific basis for the rational development and clinical use of medicinal materials.