Based On Microscopic Quantification And Biopotency Of Yam Medicinal Materials Specifications

Commercial specifications and grades of Traditional Chinese medicinal materials(TCMMs)reflect the quality of TCMM.With the changes of medicinal market,it is necessary to improve or establish a new quality standard to meet the needs of the current market.Dioscoreae Rhizoma(abbreviated as DR)was first recorded in "Shen Nong's Herbal Classic",and it is the rhizome of Dioscorea opposita Thunb.(abbreviated as DT).In current market,Dioscoreae Rhizoma is mainly devided into two commercial specifications,which are Dioscorea opposita Thunb.cv.Tiegun(abbreviated as TG)that has a higher price and non-Dioscorea opposita Thunb.cv.Tiegun(abbreviated as NTG)with a relatively lower price.The genuine TG and NTG both come from the same species but different price,which means that the qualities between the two specifications may be different.To explore the difference between TG and NTG,17 batches of DR which came from 9 various provinces were collected.Firstly,we drew the micro-digitial platform into our research to distinguish the difference of microstructure between different varieties of DT.Furthermore,the biological activities of various DT were tested from the perspective of biological potency.It was hoped that this research could provide some theoretical basis for the formulation of commercial specifications.Our research contained two experimental parts,which were the research of microstructure quantification and bioliogical activity.The main contents and consequence were as follows:1.Dioscorea opposita Thunb.is the only source of Dioscoreae Rhizoma stipulated by"Chinese pharmacopoeia".While in medicinal market,Dioscorea persimilis Prain et Burkill,Dioscorea alata L.and Dioscorea fordii Prain et Burkill were also used as DR.We collected 17 batches of DR,and among these 3 batches were identified as Dioscorea alata L.from the appearance.In microscopic identification experiment,we found that the biggest difference between DT and Dioscorea alata L.was the existence of cambium in the middle column sheath.In addition,the microstructures from the same species while different varieties were similar,they all contained starch granules,parenchyma cells,vascular bundle,mucus cells with needle crystal,and so on.2.In the texture description of DR,"Ben Cao Yuan Shi" said that "a solid texture made a good medicine".And,in field investigation,the growers also said that the texture of TG was"fine grain","hard and tough".Furthermore,from the cross section of DR,we also observed that the interparticle density was different from varieties.For example,TG got a more intensive particle compared with NTG from Hebei and Shanxi provinces.In addition,we all know that appearance is the embodiment of internal characteristics,so we decided to verify this difference of interparticle density from the microscopic point.In this study,more than one hundred paraffin sections from various DR were made,and they were all scanned into whole slide images(WSIs)which were easier to be observed by an instrument of digital slice scanner.Then,the quantitative statistic of main microscopic characteristics was made,and also the cluster analysis,significance test were used to distinguish various DR.The results of cluster analysis based on the distribution density of vascular bundles showed that different batches of DR were divided into two categories which were TG and NTG,and the clustering result was consistent with the market.Furthermore,the results of significance test displayed that the distribution density of vascular bundles from these two types of DR showed significant difference.It meant that we had identified the difference in texture from various DR successfully in the aspect of micro level.Thus,this study provided some scientific basis for the texture description in sensory evaluation of DR.3.In this study,TG was used as a model drug.We established a method preliminarily by using High content Screening(HCS)to determine the ability of RAW 264.7 to engulf GFP-E.coli(Escherichia coli labeled by Green Fluorescent Protein)effected by water extracts of DR.The method was established by examining the time of administration,the amount of bacteria added and the time of bacterial stimulation.And the best method was:12h's administration time,50 times' bacteria and 1.5h's stimulation time.Then,we tested the repeatability of this method,and the RSD was 2.31%.Moreover,compared with the group without drugs,at the concentration of 0.156-1.25mg/ml,the water extract of DR could promote the proliferation and improve the average phagocytosis rate of every single RAW264.7 cell.And,the samples have the strongest effect on proliferation and phagocytosis when the concentration is 1.25mg/ml.The above demonstrated that,we had established a method to test the phagocytic activity,and it was suitable for the biological activity study of TG.In addition,TG could not only promote cell proliferation,but also improve the phagocytic ability of every single RAW264.7 in vitro.4.CCK-8 assay and HCS were used to test the cell proliferation activity and phagocytic activity of RAW264.7 macrophages,and then the biological potency was measured according to the qualitative response parallel method to better evaluate the difference of TG and NTG.The results of CCK-8 assay indicated that all these samples increased the cell proliferation activity of RAW264.7 macrophages.And the results of HCS showed that every sample could increase cell phagocytic activity at a certain concentration.RAW264.7 cells could engulf the GFP-E.coli,and the images of HCS reflected the situation of phagocytosis clearly.In addition,the results of biological potency displayed that the biopotency value of varying samples was from 39.56 to 100,and the average biopotency value was significantly higher than NTG(P<0.05),which was consistent with the saying that "TG has a better quality".In summary,we verified the difference between TG and NTG from two experimental parts,which were microstructure quantification and biological potency,and the results were consistent with the market segmentation of DR.It was hoped that this study could provide some theoretical basis for the specification of Dioscoreae Rhizoma.