| Purpose:Based on the previous research about the pharmacodynamic material basis of Belamcanda chinensis(L)DC.in anti-inflammatory,antitussive and mechanism of action.And we found that it has been widely distributed since ancient times by consulting the herbal classics and modern literature,and the content of pharmacology active ingredient has differences,causing the status quo of the clinically effective heterogeneity.The project refers to zoning standards for seven geographical areas of Chinese,dividing ancient origins and modern origins of Belamcanda chinensis by longitude and latitude,and collecting medicinal materials for experiments in them.The pharmacodynamic material basis of Belamcanda chinensis was taken as the research object,measuring medicinal component content from different areas Chinese medicines which gave play to anti-inflammatory and antitussive by using HPLC.Relating the content determination results to climate,altitude,temperature,precipitation,sunshine duration of local areas and harvesting time,growth years of Chinese medicines,analysing initially the effects of above-mentioned elements on the medicinal composition content.To combine the content determination results with the best ratio,estimating initially the main pharmacological activities of Belamcanda chinensis from different producing areas,in order to provide reference for clinical application of medicinal materials.Method:1 Chemical reference substance studiesThe chemical reference substances used in this experiment include:tectoridin,iristectorigenin A,iristectorigenin B,iridin,tectorigenin,iristectorigenin B,iristectorigenin A,irigenin,irisflorentin,dichotomitin.Among them,three chemical reference substances tectorigenin,irigenin,and dichotomitin were made by the laboratory.1.1 Study on physicochemical properties and structure of chemical reference substancesAccording to the relevant provisions of National Technical Specifications for Pharmaceutical Standard Substances,examining the three chemical reference substances tectorigenin,irigenin,and dichotomitin which extracted by laboratory.And determining whether them meet the requirements of the chemical reference substance for content determination.Observing its characters,determining its melting point and investigating its solubility by methanol,dimethyl sulfoxide,chloroform three solvents of different polarities.Confirming chemical compound types roughly by UV,estimating functional group types roughly by IR,deciding the molecular weight of chemical compounds by HR-ESI-MS,and confirming the arrangement and distribution of the perssad roughly by 1H-NMR and 13C-NMR.Confirming the molecular structure of chemical reference substances by both of them.1.2 Purity study of chemical reference substancesTesting impurity by TLC,HPLC,HPLC-DAD scan.And calculating the content of chemical reference substances by international commonly used Mass Balance Method.2 Selecting the experimental medicineAccording to the ancient textual research,modern literatures,network access and other information preliminary confirming initially wild and cultivation situation in ancient producing areas,modern main producing areas and GAP herbal medicine cultivation bases.Refering to zoning standards for seven geographical areas of Chinese,dividing areas by longitude and latitude.Based on geographical location,climate,altitude and other factors,the producing areas are selected from the above areas.And collecting representative samples of experimental medicines on the basis of different harvest time and growth years.3 Study on preliminary evaluation of Belamcanda chinensis from different areas3.1 Study on medicinal component content of Belamcanda chinensis from different producing areasThe anti-inflammatory medicinal components of Belamcanda chinensis,tectoridin,iristectorigenin A,iristectorigenin B,iridin,tectorigenin,iristectorigenin B,iristectorigenin A,irigenin,and antitussive medicinal components irisflorentin,dichotomitin were taken as the research objects.Using HPLC,the gradient elution was carried out with 0.1%phosphoric acid aqueous solution,acetonitrile and 0.5%methyl-beta-cyclodextrin-0.1%phosphoric acid aqueous solution as mobile phases and 265nm as detection wavelength.The linear range,precision,repeatability,stability and recovery rate of the determination method were further investigated.The determination method was used to determine medicinal component contents in 33batches of Belamcanda chinensis from 12 producing areas.Cluster analysis and comparative analysis were carried out on the determination results to preliminarily explore the difference of the content of medicinal components in Belamcanda chinensis.And investigating the effect on medicinal component contents by climate,altitude,temperature,precipitation,sunshine duration of local and harvesting time,growth years of Chinese medicines.3.2 Study on evaluation of Belamcanda chinensis from different producing areas based on optimum ratioBy comparing the flavone aglycones medicinal component content obtained by measurement with the optimal ratio to preliminarily determine the suitable producing area of Belamcanda chinensis for antitussive.Results:1 Results of chemical reference studiesThe three chemical reference substances which made by laboratory were all isoflavones through UV identification.And their structures were confirmed by spectroscopic analysis as 5,7,4'-trihydroxy-6-methoxy-isoflavones,5,7,3'-trihyd roxy-6,4',5'-triethoxy-isoflavones,5,3'-dyhydroxy-6,7-methylenedioxy-4',5'-dimetho xy-isoflavones.Combined with the information of character,solubility and the melting point,we identified tectorigenin,irigenin,dichotomitin.They were intended to be pure substance by TLC distinguish,HPLC measure and HPLC-DAD scan.The chromatographic peak purity of chemical reference substances obtained by Area Normalization Method and Self Control Method,were 99.69%,99.57%,99.22%,water content were 0.99%,0.96%,0.70%,and the contents quantified by Mass Balance Method were 98.70%,98.61%,98.52%.2 Results of selection of experimental medicinal materialsBased on the textual research of the ancient producing areas and modern producing areas of Belamcanda chinensis,network search and research,overall consideration geographical location,climate,altitude and other factors,we had chosen the producing areas of experiment from seven areas,which were Hainan Changhua,Guizhou Qianxi,Sichuan Chengdu,Sichuan Abazhou,Hunan Changsha,Hubei Enshi,Hubei Huanggang,Henan Nanyang,Anhui Houzhou,Shanxi Shangluo,Hebei Anguo,Liaoning Shenyang.Further,33 batches of samples of experimental medicinal materials were selected according to different harvest time and growth years of medicinal materials.3 Results of the medicinal component contents of Belamcanda chinensis from different producing areasBased on the content determination results of tectoridin,iristectorigenin A,iristectorigenin B,iridin,tectorigenin,iristectorigenin B,iristectorigenin A,irigenin,in anti-inflammatory pharmacological activity and irisflorentin,dichotomitin in antussive pharmacological activity,we found that there were significant differences in the anti-inflammatory and antitussive medicinal component contents of Belamcanda chinensis from different producing areas by One-way analysis of variance.In 33batches Belamcanda chinensis,the anti-inflammatory medicinal component content was the highest in Belamcanda chinensis of Sichuan Abazhou(83.31mg.g-1),next were Belamcanda chinensis of Hainan Changhua(48.73mg.g-1)and Belamcanda chinensis of Hunan Changsha(41.38mg.g-1),and the lowest in Belamcanda chinensis of Shanxi Shangluo(10.47mg.g-1).The difference between the highest and lowest was nearly five times.The antitussive medicinal component content was the highest in Belamcanda chinensis of Henan Nanyang(2.010mg.g-1?3.540mg.g-1),next were Belamcanda chinensis of Guizhou Qianxi(2.190mg.g-1?2.476mg.g-1)and Belamcanda chinensis of Hubei Huanggang(1.644mg.g-1?2.714mg.g-1),the lowest in Belamcanda chinensis of Sichuan Abazhou(0.2235mg.g-1).The difference between the highest and lowest was nearly 17 times.The original data of 10 medicinal component contents in Belamcanda chinensis from different producing areas were used for systematic clustering of 33 batches of samples.There were two kinds of categories,and the Chinese medicines which from the same producing areas were clustered in the same categorie.Among them,the anti-inflammatory medicinal component contents in Hainan Changhua,Sichuan Chengdu,Sichuan Abazhou,Hunan Changsha and Hubei Huanggang were relatively high(28.32mg.g-1?83.31mg.g-1),and their contents in the total component content of the proportion were relatively high(93.82%?99.73%),thus they were bounded for the?categorie.The antitussive medicinal component contents in Guizhou Qianxi,Hunan Enshi,Henan Nanyang,Anhui Haozhou,Shangxi Sahngluo,Hebei Anguo,Liaoning Shenyang were relatively high(0.9053mg.g-1?3.540mg.g-1)and their contents in the total component content of the proportion were relatively high(4.48%?15.13%),thus they were bounded for the?categorie.The results showed that the sunshine duration may have an effect on the antitussive medicinal component contents,the precipitation may have an influence on the antitussive medicinal component contents,and temperature has no exact effect on the medicinal component contents.By comparing the medicinal component contents of Belamcanda chinensis which collected in different months in the same producing area,anti-inflammatory medicinal component contents in autumn harvest period were higher than that in spring harvest period,and showed a dynamic change.From September to March of the next year,the contents gradually decreased to the lowest and then increased,and reached the peak in April and then gradually decreased.From September to May of the next year,the antitussive medicinal component contents showed a trend of decreasing first,then increasing and then decreasing.The content of flavone glycosides did not change significantly in autumn,but the content was higher than that in the spring of the next year.The content rose in March of the next year,and then gradually fell in April.The content of flavone aglycones did not change significantly in autumn,but increased gradually from March of the next year and then tended to be stable.By comparing the medicinal component contents of Belamcanda chinensis from the same producing area with different growth years,the anti-inflammatory medicinal component contents of Belamcanda chinensis varied obviously with the increase of growth years,showing a trend of increasing first and then decreasing,and the content of 3-year old Belamcanda chinensis was the highest.By comparing the medicinal component contents of Belamcanda chinensis from the same producing area with different growth years,the anti-inflammatory medicinal component contents of Belamcanda chinensis varied obviously with the increase of growth years,showing a trend of increasing first and then decreasing,and the content of 3-years old Belamcanda chinensis was the highest.4 Preliminary evaluation of Belamcanda chinensis based on optimum ratioBy comparison,a total of 11 batches Belamcanda chinensis from different producing areas were close to the optimal ratio of antitussive.Conclusion:1 According to the relevant provisions of the Technical Specifications for National Pharmaceutical Standard Substantials,the three kind of chemical reference substances,tectorigenin,irigenin,dichotomitin,made by the laboratory,were identified as single pure substances.And the content was above 98%,which meet the purity requirements of chemical reference substances for content determination.2 According to the textual research of herbals,modern literature,Internet access and research,33 batches of experimental medicinal materials were selected from 12producing areas of medicinal materials in seven regions of China based on factors such as geographical location,climate and altitude,and selected according to different harvest time and growth years of medicinal materials,which were representative to some extent.However,due to the COVID-19 outbreak situation,the original plan on the basis of the preliminary research,the on-site multi-plot collection for 2 to 3 years could not be carried out as scheduled,resulting in too small experimental sample size,which may bring errors to the experimental results.3 One-way ANOVA was used to analyze the medicinal component contents of Belamcanda chinensis from different producing areas.Further clustering showed that the the anti-inflammatory medicinal component contents of Belamcanda chinensis were higher in Aba Prefecture of Sichuan Province,Changhua Prefecture of Hainan Province,Changsha City of Hunan Province,Huanggang City of Hubei Province and Chengdu of Sichuan Province,which grew in 102°19?E?114°7?E,28°54?N?31°54?N from southwest to central China.It was speculated that warm and humid climate conditions might be beneficial to the accumulation of anti-inflammatory medicinal components.The highest anti-inflammatory medicinal component contents was found in the plateau area growing at an altitude of more than 2600 meters,it was speculated that sufficient light might be beneficial to the accumulation of anti-inflammatory medicinal components.The contents of antitussive components in the area of104°15'E?120°21'E,32°18'N?34°05'N in Qinling Mountains was relatively high.It was suggested that the transitional climate between the north and the south may be more conducive to the accumulation of antitussive medicinal components.The content of antitussive medicinal components of medicinal herbs growing in mountainous areas was relatively high,so it was speculated that rainfall might be beneficial to the accumulation of antitussive medicinal components.The anti-inflammatory medicinal component contents and antitussive medicinal component contents of Belamcanda chinensis which growing in 38°?42°mid-latitude plain area were relatively low,so it was speculated that low temperature might be harmful to the growth of medicinal materials.In conclusion,it was preliminarily believed that the medicinal components of Belamcanda chinensis have certain geographical and climatic correlation.4 The study on the medicinal component contents of Belamcanda chinensis from the same producing area showed that sunshine hours may effect the anti-inflammatory medicinal component contents,precipitation may mainly effect the antitussive medicinal component contents,and the correlation between temperature and the content of active components was not significant.However,due to the small sample size of the analysis,the specific influence trend needs to be further studied.5 Through the comparative analysis of Belamcanda chinensis with different harvest time and different growth years,it was found that the change of medicinal component contents were closely related to the growth period of medicinal materials.The best harvest time of Belamcanda chinensis in Nanyang,Henan was roughly in autumn or April in spring,and the best harvest time of Belamcanda chinensis in Qianxi,Guizhou was about 3 years.However,the growing period of medicinal materials from different producing areas were affected by many factors,such as climate and temperature,and there were some differences,so the specific situation should be further studied and analyzed in combination with regional factors,climate factors and geographical factors.6 By comparing the medicinal component contents of 33 batches of Belamcanda chinensis from 12 producing areas with the optimal ratio of antitussive relieving,the Belamcanda chinensis from Hainan Changhua,Guizhou Qianxi,Henan Nanyang,Anhui Haozhou,Shanxi Shangluo and Hebei Anguo were the closed to the best ratio.The origin of medicinal materials was mostly distributed in the Qinling Mountains,thus these areas were considered to be the first choice of producing areas of Belamcanda chinensis for the treatment of the cough disease.7 The medicinal component contents of Belamcanda chinensis in this topic were regarded as examining index to evaluate different producing areas of medicinal materials,preliminary thought the medicinal component content has the diversity of producing areas and the efficacy are different,in order to ensure the clinical efficacy of homogeneity producing areas factors should be listed as consideration scope. |
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