| Zhongjiefeng?Sarcandra glabra?medicinal herbs have unique resources and are distributed only in southern Asia and East Asia.They are widely distri-buted in the south of the Yangtze River in China,belonging to a featured whole plant Chinese herbal medicine in Lingnan.They are rich in resources and are widely distributed.Also,they are have strong vitality and can be planted artificially,therefore they have great marketing and medicinal potential.Research found that Zhongjiefeng has antiinfluenza virus activity.The extracted eleutheroside B1and rosmarinic acid 4-O-?-D-glucoside?rosmarinic acid glycoside?from Zhongjiefeng have been proved to have anti-influenza virus effects in vitro translation.On the basis of this,the multi-index quality control of the medicinal materials from different habitats and the different medicinal sites of Zhongjiefeng medicinal materials was carried out.According to the distribution of the content of each compound in different parts,eleutheroside B1and rosmarinic acid glycoside were extracted,isolated and purified as a new antiviral active site,laying the foundation for subsequent pharmacodynamic research and development of new antiviral agents.It also provides a reference for the development of new quality control standards of Zhongjiefeng and its comprehensive quality control.ObjectionMolecular biopharmaceutical methods were used to identify the collected fresh herbs from different habitats and to determine the accuracy of the collected samples.HPLC method was used to determine the chlorogenic acid,eleutheroside B1,and glycosides in Zhongjiefeng and in its different medicinal sites.The content of sofraxidin,rosmarinic acid glycoside and rosmarinic acid were determined.The quality of multi-indicator components in different habitats was analyzed to show the content distribution of each compound in different parts.The research on the extraction and purification process of active sites with significant anti-influenza effect in roots and stem of zhongjiefeng etiodendron has provided the foundation for pharmacological research and development of new anti-influenza virus preparations.Methods1.The fresh herbs of zhongjiefeng in different regions of Guangdong,Guangxi,Fujian,Hunan,were collected,respectively.After pretreatment,the leaves were extracted by using the modified CTAB method of our laboratory for DNA extraction.PCR was performed after detection by 0.1%agarose gelelectrop ophoresis and then samples were sent to Beijing Qingke Biotechnology Co.,Ltd.for the detection of the ITS sequence.GeneStudio software was used for sequence splicing,and 10 pairs of swollen wind samples were compared and analyzed with MEGA 5 to construct the Neighbor-joining phylogenetic tree.2.Selection of chlorogenic acid,eleutheroside B1,sofraxidin,rosmarinic acid4-O-?-D-glucoside?rosmarinate acid glycoside?,and rosmarinic acid as indicators establish HPLC assays.Methodological validation of HPLC analysis methods including:linearity and range,precision,repeatability,stability,and sample recovery.3.Differences in contents of major compounds in different habitats and in the roots,stems and leaves of zhongjiefeng:10 batches of herbs from different regions in Guangdong,Guangxi,Hunan,and Sichuan were collected,and 5 indicators of zhongjiefeng and different medicinal sites were detected by HPLC,and the differences in the chemical composition of the whole plant and its roots,stems,and leaves from sources of different origins and the differences in the contents ofIndex components in roots,stems,and leaves of the roots from the same origin were compared.4.The roots and stems of zhongjiefeng were selected as the medicinal materials for extraction and purification of the antiviral active sites of entella eleutheroside B1 and rosmarinic acid glycosides in the roots and stems of zhongjiefeng.After single-factor experiments,the optimum extraction process was determined,and the purification process was studied on the basis of orthogonal experiments using D101 macroporous resin.5.The root of scallopia root with high content of eleutheroside B1 was selected as the extraction medicinal material,extracted according to the preferred antiviral active site extraction technology,and then the extract was separated and purified by macroporous resin column chromatography and then used for preparative liquid chromatography.After purification,the fractions after purification were purified and purified,and the HPLC peak area was normalized to calculate the purity.Results1.Ten different batches of experimental samples from four provinces of Guangdong,Guangxi,Fujian,and Hunan collected in this study were performed by means of plant molecular methods.The original basal primordium was originated from Sarcandra glabra,indicating that the collected samples were zhongjiefeng.2.A method was established for the determination of five index components in zhongjiefeng at the same wavelength by HPLC method.After several optimizations,the peak shape and resolution of the five compounds were all good,and the RSDs of the peak areas detected by the precision and stability tests were all less than 3%,the RSD of each compound in the reproducibility was less than 2%,and the average recovery was 98%102%.3.?1?The five chemical components and their total contents in the same medical site?Whole plant,root,stem and leaf?from different habitats were different.Overall,the content of rosmarinic acid and rosmarinic acid glucoside was different.The difference is the largest.For example,the content of rosmarinic acid in Shixing,Shaoguang,Guangdong,is about 10 times that of the wild zhongjiefeng in Yong Zhou,Hunan;and the content of rosmarinic acid glucoside and rosmarinic acid in the stems of swollen stems from Shixing,Shaoguang,Guangdong,is about 15 times that of wild zhongjiefeng stems in Rong County,Yulin,Guangxi;The content of rosmarinic acid is approximately30 times higher than that of the wild zhongjiefeng leaf in Dao County,Hunan Province.?2?The content of each compound in different medicinal parts of the same origin was different.Compared with other parts,the content ofeleutheroside B1 in root was higher than other parts,and the content of rosmarinic acid glucoside in stem was higher than other parts.The content of rosmarinic acid in leaves is higher than other parts.4.The optimum extraction process for root and stem of zhongjiefeng was as follows:the root and stem of herbaceous stalk root was added by ultrason-ication,and was extracted for twice in 15 times amount of 50%ethanol,1 hour for each time.And the filtrate was mixed and then concentrated to a certain volume under reduced pressure.;the optimal purification method is:the pretreated D101 macroporous resin column was prepared for wet packing,with the column diameter ratio of 1:5.After the flowing water run out,the sample began loading with a volume ratio of 2:1.The extracted rhizome of rhizome was 0.1 g.mL-1,and the sample flow rate was 2.5h×BV-1.When the adsorption equilibrium was reached,it was eluted with 3 times as column volumes of deionized water,and then eluted with 3 times of column volumes of 25%ethanol at an elution flow rate of 2.5h×BV-1.The next 1/3 stream fraction of the 1st column volume?10 mL?was received,and the first 1/3 stream of the third column volume?10 mL?was combined with the second column volume's received fraction.5.After separation and purification by macroporous resin,the content of quercitrin in the higher purity elution fraction can reach 70%-90%.After these fractions are combined and concentrated,the liquid phase is purified and refined,and the HPLC detection is performed to puncture the eleutheroside B1.The purity of eleutheroside B1can reach more than 98%,and can be used as a standard method for mass quantitative control of zhongjiefeng.Conciusion1.Using the laboratory modified CTAB method,the molecular biology method was used to identify zhongjiefeng medicinal materials.The ITS2 sequences of10 batches of zhongjiefeng medicinal materials were identical,indicating that the collected medicinal materials all belonged to Sarcandra glabra.This method is simple,rapid,and accurate,therefore can be used as a method for the identification of zhongjiefeng herbs,and the origin is not the major reson that cause the large difference in the ingredient content of zhongjiefeng.2.From the analysis of the differences in the main chemical components in the whole plant and the different medicinal sites of different habitats of zhongjiefeng,the main reason for the large differences in the compounds was whether the source of the zhongjiefeng was medicinal or wild,and overall,The content and total content of various compounds in the herbs of zhongjiefeng was higher than that of wild herbs.From the analysis of the differences in the main chemical components between the herbs from different habitats and the different medicinal parts of the same origin,it can be seen that the eleutheroside B1 and rosmarinic acid glucoside which have anti-influenza virus effects are mainly concentrated in the roots and stems of zhongjiefeng.In the after extraction,separation and purification experiments of bronchial eleutheroside B1and rosmarinic acid glucoside in the zhongjiefeng herb,the leaves with high content of impurities and pigments should be removed,and roots and stems of zhongjiefeng should be used as the extraction herbs.3.The extraction and purification of active components of eleutheroside B1and rosemary glucosides with anti-influenza virus effect in the roots and stems of scallopia were 0.250 and 0.043 g.mL-1in the active part,respectively.At some sites the purity could reach about 70%using the Peak area normalization method.The process is simple,stable and low cost,therefore can be used for the enrichment of antiviral sites in swollen wind and its further development and utilization.4.The preparation process of stablocyrosine in the roots of tumor root is simple and stable,and the separation and purification effect is obvious,and the higher purity eleutheroside B1 compound can be obtained,which provides a reference for the preparation of a new standard for the determination of zhongjiefeng. |
PDF Full Text Request