| Purpose:BeLamcanda chinensis?L?DC.is a commonly used TCM with the effects of clearing heat and detoxification,dissolving phlegm and eliminating pharynx.In the previous project group study,it was clear that the main anti-inflammatory components were tectorigenin and irigenin,and the main antitussive components were irisflorentin and dichotom.According to the investigation of Chinese medicinal materials from the main producing areas in China,it was found that the contents of the above components of Belamcanda chinensis varied greatly among different producing areas,Belamcanda chinensis of Hunan did not even contain dichotom,the main antitussive ingredient,resulting in the effect of clinical practice unstable.On the basis of the previous studies,this topic used TLC,HPLC and other analytical techniques to preliminarily study the differences of 22 batches of Belamcanda chinensis collected from 7 producing areas,expected to find a method to distinguish Belamcanda chinensis from different producing areas.At the same time,the data obtained from the experimental study were used to identify the main origin of Belamcanda chinensis suitable for anti-inflammatory or cough relief,and to provide scientific basis for the clinical application of medicinal materials.Method:1 Selection of medicinal materials for researchBased on the classical medical books,modern literature,network information,market research and previous project research,after consulting and analyzing,the medicinal materials for experimental research on this topic were selected.2 Contrastive study of TLC based on chemical componentsFlavonoids are the main components of Belamcanda chinensis in anti-inflammation,anti-virus,cough and bacteriostasis.In the TLC,the former project group used Belamcanda chinensis reference material as the standard to identify the medicinal materials from different origins.The results showed that because of the high content of flavonoids,the spots of flavonoids were darker,which affected the observation of other spots,so it was not suitable for the study of the differences of medicinal materials from different origins.Therefore,according to the chemical constituents of Belamcanda chinensis reported in modern literature,taking Belamcanda chinensis from GAP as a standard,the ethanol leaching concentrated solution was extracted by petroleum ether,ethyl acetate and water-saturated n-butanol in turn to obtain different polarity Belamcanda chinensis test solution.Based on the number and clarity of spots on the thin layer after deployment,the deployment system?petroleum ether-ethyl acetate,trichloromethane-ethyl acetate,trichloromethane-methanol,etc.?,thin layer materials(silica gel G,silica gel GF254,polyamide,etc.),chromogenic reagents?10%ethanol sulfate solution,aluminium trichloride ethanol solution,vanillin sulfuric acid solution,etc.?were investigated and TLC identification methods for different polarity test solutions of Belamcanda chinensis were established respectively.By comparing the number of thin layer spots and Rf values of 22 batches of Belamcanda chinensis from 7 producing areas in different polarity test solutions,the differences of medicinal materials from different producing areas were compared.3 Contrastive study of HPLC fingerprint based on chemical compositionModern studies have shown that Belamcanda chinensis contains terpenoids,steroids,volatile oils and flavonoids.Because of the high content of flavonoids,the response value of chromatographic peaks is high around the corresponding wavelength,and the peak of flavonoids is sealed,so the peak of non-flavonoids is masked or depressed.In this special study,according to the chemical composition of the herbs,the extracts were divided into non-flavonoids and flavonoids for fingerprint analysis by chemical extraction method.In the study of fingerprint of non-flavonoid compounds in test solution,the detection wavelength?210nm,280nm,310nm?,mobile phase?acetonitrile-water,acetonitrile-0.1%phosphoric acid aqueous solution?,column temperature?25?,30?,40??,brand of chromatographic column?DIKMA,phenomenex,Dalian Yilite?and preparation method?separation of alumina column chromatography,ultrasonic extraction of petroleum ether,soxhlet extraction of petroleum ether?of test solution were investigated by means of high performance liquid chromatography?HPLC?and DAD detector.The optimum chromatographic conditions and preparation method of test solution were determined.To determine the common peak areas as the evaluation index,through the precision,repeatability,stability and other methods,the fingerprint analysis method of non-flavonoids by HPLC was established.The similarity evaluation software provided by the Pharmacopoeia Committee and cluster analysis method were used to study the differences of medicinal materials from different origins.In the study of fingerprint of flavonoid compounds in test solution,The number of chromatographic peaks and the degree of separation of flavonoids were taken as the evaluation indexes,the mobile phase?acetonitrile-0.1%phosphoric acid aqueous solution,acetonitrile-0.1%phosphoric acid water-0.55%methyl?-cyclodextrin-0.1%phosphoric acid aqueous solution?,column temperature?25?,40??,column brand?DIKMA,phenomenex,Dalian Elite?were investigated,and determined the optimum chromatographic conditions.To determine the common peak areas as the evaluation index,through the precision,repeatability,stability and other methods,the fingerprint analysis method of flavonoids by HPLC was established.The similarity evaluation software provided by the Pharmacopoeia Committee and cluster analysis method were used to study the differences of medicinal materials from different origins.4 Contrastive study based on the content of active componentsPreliminary research results of modern literature and project group showed that the main anti-inflammatory components of Belamcanda chinensis were as follows:tectorigenin,irigenin,iristectorigenin A,iristectorigenin B,in addition,tectoridin,iristectorin A,iristectorin B,iridin exerts its therapeutic effect by transforming into tectorigenin and other corresponding aglycones in vivo;and the main antitussive active ingredients were irisflorentin and dichotom.Based on the study of fingerprint of flavonoids,the linearity,precision,repeatability,stability and recovery of additions were investigated with reference substances:tectoridin,iristectorin A,iristectorin B,iridin,tectorigenin,iristectorigenin B,iristectorigenin A,irigenin,irisflorentin,dichotom,acetonitrile-0.1%phosphoric acid-0.55%methyl beta-cyclodextrin-0.1%phosphoric acid solution as mobile phase and 260nm as detection wavelength.It was determined that the method could simultaneously determine the content of10 anti-inflammatory and antitussive isoflavones such as tectorigenin.The content determination results and cluster analysis method were used to study the differences of medicinal materials from different origins.5 Comparative study on the contents of anti-inflammatory and antitussive effective components of Belamcanda chinensis from different originsThis topic was based on the establishment of the method of the content determination by HPLC.By comparing the content of anti-inflammatory and anti-cough isoflavones and statistical analysis,the origin of Belamcanda chinensis suitable for anti-inflammatory and anti-cough effects was determined by using the established method of content determination of monomers of anti-inflammatory and anti-cough active ingredients.Result:1 Selection results of medicinal materialsConsulting the relevant literature and the database of the Ministry of Science and Technology,The National Science and Technology Plan of the 9th five-year and 10th five-year entrusts the construction of the Belamcanda chinensis's GAP base to the Professor Liu Hegang of Hubei University of Traditional Chinese Medicine.After communicating with the professor Liu Hegang about the construction of the base,we decided to choose the Belamcanda chinensis planted by Teacher Guo meiyuan from six maple trees village,Tuanfeng County,Huanggang City,Hubei Province as the reference materials for research.According to the classical medical books,the origin of the Belamcanda chinensis was first recorded in Tao Hongjing's"Medical Doctors",the book says"Growing in Nanyang Field",and the later generations of herbs were described in this way as the origin.Therefore,consulting the relevant planting literature,the products of Belamcanda chinensis planting base at the end of Dabie Mountain in Tongbai county of Nanyang city were selected as medicinal materials.According to the sales and planting situation of Belamcanda chinensis in China,the main distribution centers in China are located in Hebei Anguo and Hunan,which are respectively qi-Belamcanda chinensis and xiang-Belamcanda chinensis,accounting for 70%of the domestic market,therefore,Belamcanda chinensis cultivated in Hubei Anguo and Hunan Province was selected as medicinal materials for research.According to the domestic modern literature report and network information,taking Nanyang city as the center,the Belamcanda chinensis cultivated in several nearby provinces--Anhui,Guizhou and Sichuan were selected as medicinal materials for research.In summary,this experiment selected 7 provinces of Henan,Hebei,Hubei,Hunan,Anhui,Guizhou and Sichuan,and 22 batches of Belamcanda chinensisl were used as research objects.2 The result of contrastive study of TLC based on chemical componentsThe experimental results of study on TLC qualitative difference of extracts of small polarity from Belamcanda chinensis by petroleum ether extraction showed that:Different developing agents?petroleum ether-ethyl acetate,petroleum ether-acetone,petroleum ether-acetone and cyclohexane-trichloromethane-ethyl acetate?and different chromogenic reagents?vanillin-concentrated sulfuric acid test solution,10%ethanol sulfate test solution?were used for TLC.The results showed that:Using the method of extending petroleum ether?60?-90??-acetone?2:1?to 4cm,then cyclohexane-trichloromethane-ethyl acetated?14:5:3?to 8cm,using 10%ethanol sulfate solution to developed color,the sample extracted from Belamcanda chinensis petroleum ether could obtain good analytical results.Twenty-two batches of Belamcanda chinensis were developed and coloured at the same dosage.There were 2-6 spots in six batches of samples from Hebei,4-5 spots in two batches from Henan,5-7 spots in five batches from Hubei,4-5 spots in three batches from Hunan,5-6 spots in three batches from Anhui,5-7 spots in two batches from Sichuan and 7 spots in Guizhou.Although there were differences in the number of spots and the depth of colour among Belamcanda chinensis from different origins,there were also great differences among Belamcanda chinensis from the same origin.The analysis suggests that the transformation of components or the difference of collection time may be responsible for this.The experimental results of study on TLC qualitative difference of extracts of medium polarity from Belamcanda chinensis by ethyl acetate showed that:Different developing agents?petroleum ether-ethyl acetate,trichloromethane-ethyl acetate and trichloromethane-methanol?and different chromogenic reagents?aluminum trichloride ethanol test solution,10%ethanol sulfate test solution and 2%ferric chloride test solution?were used for TLC.The results showed that:Using the method of trichloromethane-ethyl acetate-glacial acetic acid?16:4:0.2?,using aluminum trichloride ethanol test solution to develop color,the sample extracted from Belamcanda chinensis petroleum ether could obtain good analytical results.Twenty-two batches of Belamcanda chinensis were developed and coloured at the same dosage.The results showed that Belamcanda chinensis samples from different areas can be divided into three categories:The first type was samples from Hunan Province,there was no yellow-white spot at Rf 0.4;The second type was samples from Henan and Hebei Province with a yellow-white spot at Rf 0.4,and Rf 0.9 had no blue-white spot;The third type was samples from Hubei,Anhui and Guizhou Province with a yellow-white spot at Rf 0.4,and Rf 0.9 had a blue-white spot.The established method can quickly and accurately differentiate Belamcanda chinensis from different origins.The experimental results of study on TLC qualitative difference of extracts of large polarity from Belamcanda chinensis by water saturated n-butanol showed that:Different developing agents?trichloromethane-ethylacetate,trichloromethane-methanol,chloroform-butanone-methanol?and different chromogenic reagents?aluminum trichloride ethanol test solution,10%ethanol sulfate test solution and 2%ferric chloride test solution?were used for TLC.The results showed that:Using the method of trichloromethane-butanone-methanol?10:3:3?,using aluminum trichloride ethanol test solution to develop color,the sample extracted from Belamcanda chinensis water-saturated n-butanol could obtain good analytical results.22 batches of Belamcanda chinensis,5 batches in Hebei,2 batches in Henan,2 batches in Sichuan,1 batch in Guizhou,4 batches in Hubei,and 2 batches in Anhui.There were 4 spots in each batch in Hubei and Anhui,and 3 spots in three batches in Hunan.Although there were some differences in the number of Belamcanda chinensis in different areas,the representation was not strong.3 The result of contrastive study of HPLC fingerprint based on chemical compositionIn the study of fingerprint of non-flavonoid compounds in test solution,the detection wavelength?210nm,280nm,310nm?,mobile phase?acetonitrile-water,acetonitrile-0.1%phosphoric acid aqueous solution?,column temperature?25?,30?,40??,brand of chromatographic column?DIKMA,phenomenex,Dalian Yilite?and preparation method?column chromatography,ultrasonic extraction of petroleum ether,soxhlet extraction of petroleum ether?of test solution were investigated.The results showed that:Instrument was Agilent 1100 high performance liquid chromatography;chromatographic column was Diammonsil C18?2?5?m 250×4.6mm?DIKMA?;mobile phase was A:0.1%phosphoric acid aqueous solution,B:acetonitrile;current speed was 1.0mL·min-1;detection wavelength was210nm;column temperature was 40?,using the gradient elution system.Good analytical results could be obtained.The number of peaks in liquid chromatography was large and the separation degree was good.The precision,reproducibility and stability of non-flavonoids were investigated,and the fingerprint analysis method of non-flavonoids by HPLC was established.The similarity evaluation software provided by the Pharmacopoeia Committee and cluster analysis method were used to study the differences of medicinal materials from different origins.The results showed that 22 batches of Belamcanda chinensis could be divided into two categories:0.9-1.0 and 0.8-0.9.Among them,the similarity of Belamcanda chinensis from Hubei Province except 6#,21#in Hebei and 11#in Hunan was 0.8-0.9,while that of Hebei,Henan,Hunan,Anhui,Guizhou and Sichuan was 0.9-1.0.Cluster analysis could not distinguish Belamcanda chinensis from different habitats very well.11 common peak areas were studied and the results showed that the area of common peaks of No.1,No.2and No.3 of Belamcanda chinensis in Hubei,one batch in Hebei and one batch in Hunan decreased in an equal order,it was suggested that this trend could be used in the next step to enlarge the sample size for further comparative study.In the study of fingerprint of flavonoid compounds in test solution,the mobile phase?acetonitrile-0.1%phosphoric acid aqueous solution,acetonitrile-0.1%phosphoric acid water-0.55%methyl?-cyclodextrin-0.1%phosphoric acid aqueous solution?,column temperature?25?,40??,column brand?DIKMA,phenomenex,Dalian Elite?were investigated.The results showed that:Instrument was Agilent 1100 high performance liquid chromatography;chromatographic column was SinoChrom ODS-BP 5?m 250×4.6mm?Dalian Elite?;mobile phase was A:0.1%phosphoric acid aqueous solution,B:acetonitrile,C:0.55%methyl?-cyclodextrin-0.1%phosphoric acid aqueous solution;current speed was1.0mL·min-1;detection wavelength was 265nm;column temperature was 40?,using the gradient elution system.Sample solution extracted by ultrasonic extraction of 80%ethanol for30 minutes of Belamcanda chinensis could obtain good analytical results.The precision,reproducibility and stability of flavonoids were investigated,and the fingerprint analysis method of flavonoids by HPLC was established.The similarity evaluation software provided by the Pharmacopoeia Committee and cluster analysis method were used to study the differences of medicinal materials from different origins.Similarity evaluation results showed that 22 batches of dried samples could be divided into three categories:from 0.95 to 1.0,from0.85 to 0.95 and from 0.75 to 0.85.The first kind of similarity was between 0.95 and 1.0,it was samples from Hubei Province except 6#.The second kind of similarity was between 0.85and 0.95,include Henan,Hunan,Hebei and Guizhou samples;and the third kind of similarity was between 0.75 and 0.85,which was Anhui and Sichuan samples.Cluster analysis of 12common peak areas could cluster Belamcanda chinensis from Hunan into one group and Belamcanda chinensis from Hubei into one group.12 common peak areas were studied and the results showed the area of No.1 peak?>5000?of Belamcanda chinensis from Hunan Province were more than twice that of other places,while the area of No.12 peak?>10?was much smaller than that of other places.4 The result of contrastive study based on the content of active componentsOn the basis of fingerprint analysis of flavonoids,anti-inflammatory and cough-relieving active ingredients or their transformed:tectoridin,iristectorin A,iristectorin B,iridin,tectorigenin,iristectorigenin B,iristectorigenin A,irigenin,irisflorentin,dichotom were used as control substances.Through experimental investigation such as the linear range,precision,repeatability,stability and recovery of sample addition.The results of 22 batches of samples were compared and analyzed:Content analysis of total flavone glycosides?the sum of tectoridin,iristectorin A,iristectorin B,iridin?and total flavone aglycones?the sum of tectorigenin,iristectorigenin B,iristectorigenin A,irigenin,irisflorentin,dichotom?:Using the content of total glycosides and total aglycones,cluster analysis could separate Belamcanda chinensis from Hunan into one group,of which total glycosides account for 90%and aglycones account for 10%.The proportion of total glycosides in Belamcanda chinensis from other habitats was about50%-80%.The content of total glycosides in Belamcanda chinensis from Hunan was higher than that from other habitats and was stable,so it could be clustered into one group.The content analysis of glycosides?tectoridin,iristectorin A,iristectorin B,iridin?showed that Hunan samples could be clustered into one group by using the content of four glycosides and cluster analysis;Hubei samples and the one of Hebei samples could be clustered into one group.Among them,the proportion of Belamcanda chinensis in Hunan Province?80%?was much higher than that in other areas?25%-40%?.Therefore,it could be clustered into one group.The proportion of Belamcanda chinensis in Hubei Province and a batch of Belamcanda chinensis in Hebei Province?60%?was lower than that in Hunan Province,but higher that in other areas,so it could also be clustered into one group.Cluster analysis of the contents of reciprocal compounds?tectoridin+tectorigenin,iristectorin A+iristectorigenin A,iristectorin B+iristectorigenin B,iridin+irigenin+irisflorentin+dichotom?showed that Hunan samples could be separately classified into one group,while Hubei and one batch of Hebei samples could be classified into one group.Among them,the ratio of the sum value of the content of tectoridin and tectorigenin was 75%in Hunan Province,which was about three times as much as that in other areas?20%-25%?.So it could be clustered into one group.The ratio of the additive value of tectoridin and tectorigenin content in Belamcanda chinensis from Hubei and one batch of Hebei was about40%,which was lower than Belamcanda chinensis from Hunan,but higher than Belamcanda chinensis from other producing areas,so it could also be clustered into one group.5 Comparative Study on the Contents of Anti-inflammatory and Antitussive Effective Components of Belamcanda chinensis from Different OriginsIn this part,the content of 22 batches of anti-inflammatory and anti-cough Isoflavones of Belamcanda chinensis were studied by using the established method for the determination of monomers of anti-inflammatory and anti-cough active ingredients.The results showed that the contents of anti-inflammatory and antitussive active ingredients of Belamcanda chinensis from different habitats were quite different.The contents of anti-inflammatory active ingredients in Belamcanda chinensis from Hunan and Hubei were the highest,about40.8073.34 mg·g-11 and 27.7137.94 mg·g-1.Taking the mean value of anti-inflammatory content of each producing area as variable and taking Hunan and Hubei as test values respectively,the content of anti-inflammatory active ingredients in Belamcanda chinensis from 7 producing areas was tested by T test.The results showed that the content of anti-inflammatory active ingredients in Belamcanda chinensis from Hunan and Hubei was significantly higher than that in other producing areas?P<0.05?.Belamcanda chinensis from Anhui and Hubei had the highest content of effective components in relieving cough,about2.0902.405 mg·g-11 and 1.5722.674 mg·g-1,Belamcanda chinensis from Hunan were the lowest,about 0.8221.262 mg·g-1,and most of them were irisflorentin,and the content of high active dichotom were very low.Taking the mean value of relieving cough content of each producing area as variable and taking Anhui,Hubei and Hunan as test values respectively,the content of relieving cough active ingredients in Belamcanda chinensis from7 producing areas was tested by T test.The results showed that the content of relieving cough active ingredients in Belamcanda chinensis from Anhui and Hubei was significantly higher than that in other producing areas?P<0.05?,Hunan was lower than that in other producing areas?P<0.05?.Conclusion:1 In the study of TLC qualitative identification difference,the TLC identification method of neutral polar extract obtained by ethyl acetate extraction can be used to distinguish Belamcanda chinensis from other medicinal materials from Hunan province.Belamcanda chinensis from Hunan is the origin of the lowest content of cough-stopping active ingredients in all medicinal materials,so it can be used in clinical practice to determine whether Belamcanda chinensis can treat cough-related diseases related to infection.2 In the study of the difference of the fingerprint of non-flavonoids by HPLC,the established method could be used to distinguish Belamcanda chinensis in Hunan,one batch in Hubei from other habitats,by means of the difference of the peak areas of the chromatographic peaks of medicinal materials from different habitats,similarity evaluation combine with the characteristics of the common peak areas.This method,combined with TLC qualitative identification,can quickly and accurately distinguish Belamcanda chinensis from Hunan and Hubei.3 In the study of the difference of the fingerprint of flavonoids by HPLC,the established method could be used to distinguish Belamcanda chinensis from Hunan,Hubei and other habitats,by means of the difference of the peak areas of the chromatographic peaks of medicinal materials from different habitats,cluster analysis and similarity evaluation combine with the characteristics of the common peak areas.4 In the determination of the content of flavonoids by HPLC,the method of fingerprint of flavonoids is used.The content of 10 isoflavones in Belamcanda chinensis from different habitats can be used as the standard.Combining with cluster analysis,the method of distinguishing Hubei Belamcanda chinensisl,Hunan Belamcanda chinensisl from other habitats.5 The results of anti-inflammatory and anti-cough determination of medicinal materials from different producing areas show that 22 batches of Belamcanda chinensisl materials in 7provinces,Hubei Belamcanda chinensisl and Hunan Belamcanda chinensisl have the highest anti-inflammatory content,which is the first choice for treating infectious diseases;Hubei Belamcanda chinensisl and Anhui Belamcanda chinensisl have the highest cough-reducing content,which is the first choice for the treatment of infectious cough disease.6 This topic is an auxiliary project for the national 13th five-year major new drug innovation project.The purpose is to determine the specific identification of the use of medicinal materials in the research of innovative Chinese medicine new drugs.Through the above research,it is basically possible to determine the identification method of high quality Belamcanda chinensisl Chinese medicinal materials.Due to sample collection range,batch and time constraints,the method may have certain defects that need to be further supplemented. |
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