| Objective:Under the guidance of traditional Chinese medicine(TCM)theory and the active components alignment theory,To study the active components alignment(ACA)of ZhiziBopi Decoction(ZBD),and to explore the effects of compatibility,dosage,and dosage form on the ACA,To provide a scientific basis for establishing a quality evaluation system of ACA that corresponds to clinical efficacy,and to provide the preparation of ACA standard substances and the development of ACA traditional Chinese medicine.Methods:This article uses ZhiziBopi decoction as research carrier,the clinical basis of ZBD was retrospectived through literature traceability,according to the principle of prescription,dosage form,solubility and clinical curative effect,we selected 10 components that researched maturity,have clear pharmacological effects,and are related to clinical efficacy:geniposide,genipinlgentiobioside,magnoflorine,liquiritin,glycyrrhizic acid,berberine hydrochloride,palmatinechloride,isliquiritin,crocin Ⅰ,crocin Ⅱ as ZBD active components alignment,using HPLC-DAD multi-wavelength method[column for Syncronis C18,250mm*4.6mm,5μm;mobile phase is acetonitrile(A)and phosphoric acid of 0.2%(B);elution sequence:0～20min,12%～20%A,20～40min,20%～23%A,40～70min,23%～25%A,70～100min,25%～55%A,100～105min,55%～12%A;Flow rate is 0.8mL/min,column temperature is 30℃,sample volume is 5μL;detection wavelength of 238nm is geniposide-genipinlgentiobioside-magnoflorine-liquiritin,265nm is glycyrrhizic acid-berberine hydrochloride-palmatinechloride,360nm is isliquiritin,440nm is crocin I and crocinⅡ]to determine the content of ACA,using data analysis method to analyze the internal relationship between the ACA and compatibility,dosage,and dosage forms.Results:1.The analysis on the active components alignment of ZhiziBopi Decoction①Selection of ZhiziBopi Decoction:According to the formula,dosage form and efficacy of theZBD,the ACA corresponding to its efficacy were selected:geniposide,genipinlgentiobioside,magnoflorine,liquiritin,glycyrrhizic acid,berberine hydrochloride,palmatinechloride,isliquiritin,crocin Ⅰ,crocin Ⅱ.Methodological review:10 kinds of ACA have high specificity and no impurity interference;A good linear relationship(r2≥ 0.9992)was exhibited in the corresponding linear range.The average recoveries ranged from 96.53 to 100.25%with RSD less than 3%.The RSDs in the precision,repeatability,and stability tests were all less than 1.5%.2.Study on the Influencing Factors of ACA of ZhiziBopi Decoction①Formulation factor study:The dosage form has a significant effect on the ACA of ZBD(P<0.05).With the extension of boiling time,The ACA of Fructus Gardeniae,Phellodendron Amurense and Glycyrrhizae Radix increased significantly in 10-20min,After 20 min,there was an increase in content after reduction,and the final content was approximately similar to that at 20 min.The trend of total amount of ACA is the same as Gardenia jasminoides ACA,and its content from high to low(mg/g):60min(39.526±0.821)>20 min(38.728±0.263)>30 min(38.192±0.307)>50 min(37.560± 0.731)>10 min(34.715±0.322)>40 min(33.494±0.525).With different extraction methods,the total amount of ACA when extracting by reflux is greater than the ultrasonic method;With different extraction solvents,the order of the total amount of ACA when extracted by reflux is:30%EtOH(287.265±0.945)>60%EtOH(242.904±0.647)>90%EtOH(241.764±0.856)>H2O(100.364±0.746),The order of the total amount of ACA in the extraction method is:30%EtOH(169.897±0.653)>90%EtOH(144.123±0.787)>60%EtOH(122.717±0.836)>H2O(25.180±0.367),The dissolution amount of ACA was largest in 30%ethanol.②Compatibility factors study:After the compatibility of Fructus Gardeniae,Phellodendron Amurense and Glycyrrhizae Radix,the content of ACA significantly compared with the single herb(P<0.05).The content of ACA of Fructus Gardeniae(mg/g):geniposide(51.551±0.784)-genipinlgentiobioside(11.444±0.232)-crocin I(5.999±0.291)-crocin II(0.597±0.053),The total amount of ACA after compatibility decreased by 12.5%;the ACA of Phellodendron Amurense(mg/g):magnoliaine(5.390±0.359)-berberine hydrochloride(5.147±0.545)-palmatine hydrochloride(1.873±0.057),decreased the total amount of ACA after compatibility by 9.21%;The ACA of Glycyrrhizae Radix(mg/g):glycyrrhizin(8.621±0.251)-isoliquiritin(0.702±0.094)-glycyrrhizic acid(14.612±0.847),The total amount of ACA after compatibility rose by 8.17%,and the content of ACA of ZhiziBopi decoction:Geniposide(44.998±0.625)-genipinlgentiobioside(9.707±0.245)-crocetin Ⅰ(5.599±0.352)-crocetin Ⅱ(0.5861±0.012)-magnoline(4.567±)0.324)-berberine hydrochloride(3.294±0.21 1)-palmatine hydrochloride(1.406±0.196)-glycyrrhizin(8.265±0.614)-isoliquiritin(0.833±0.055),glycyrrhizic acid(16.883±0.602).When the compatibility components were changed,the total amount ACA of Fructus Gardeniae,the total amount ACA of Phellodendron Amurense and the total amount of Glycyrrhizae Radix were all decreased(P<0.05).The total amount of ACA was changed as follows:Raw Fructus Gardeniae-Raw Phellodendron Amurense-Fry Glycyrrhizae Radix(43.651±0.625)>Fry Fructus Gardeniae-Raw Phellodendron Amurense-Fry Glycyrrhizae Radix(39.993 ±0.547)>Raw Fructus Gardeniae-Salt Phellodendron Amurense-Fry Glycyrrhizae Radix(38.603 ± 0.258)>Raw Fructus Gardeniae-Wine Phellodendron Amurense-Fry Glycyrrhizae Radix(38.442 ±0.335)>Raw Fructus Gardeniae-Raw Phellodendron Amurense-Raw Glycyrrhizae Radix(37.713 ± 0.681)>Scorched Fructus Gardeniae-Raw Phellodendron Amurense-Fry Glycyrrhizae Radix(13.961 ± 0.414).③Dose factor study:a,Changes dose of single-factory Fructus Gardeniae,and the ACA of Fructus Gardeniae,Phellodendron Amurense,Glycyrrhizae Radix and ZBD were significantly changed(P<0.05).The dosages of Phellodendron Amurense and Glycyrrhizae Radix were not changed,When the compatibility doses of Fructus Gardeniae were 1g,3g,6g,10g,12g,15g and 18g respectively,the total amount ACA of Fructus Gardeniae and Glycyrrhizae Radix showed an upward trend.The ACA amount of Phellodendron Amurense showed a trend of increasing first and then decreasing;the total amount ACA of ZBD increased,which was in turn:14.023±0.812,23.162±0.536,31.632±0.385,39.535±0.625,42.837±0.724,47.392±0.048,52.826±0.326,The ACArange of Phellodendron Amurense,Glycyrrhizae Radix and Fructus Gardeniae is1:3.6:10-1:1.8:7。b.Changes dose of single-factory Phellodendron Amurense,and the ACA of Fructus Gardeniae,Phellodendron Amurense,Glycyrrhizae Radix and ZBD were significantly changed(P<0.05).When the dose of Fructus Gardeniae and Glycyrrhizae Radix was fixed and the compatibility dose of Phellodendron Amurense changed from 1g to 15g,the total amount ACA of Glycyrrhizae Radix decreased;the total amount ACA of Phellodendron Amurense rose;The total amount ACA of ZBD increased at first and then decreased,and the maximum value was found at the dose of 6g.The ACA range of Phellodendron Amurense,Glycyrrhizae Radix and Fructus Gardeniae is:1:2.7:6～1:1.6:4.3。When the dose of Phellodendron Amurense was 1,3,and 6g,the total amount ACA of ZBD was 38.024±0.749,41.155±0.385,and 41.257±0.564.When the Phellodendron Amurense dose was 9,12,and 15g,the total amount ACA of ZBD is low:34.475±0.460,28.43±0.843,23.234±0.590.c.Changes dose of single-factory Glycyrrhizae Radix,and the ACA of Fructus Gardeniae,Phellodendron Amurense,Glycyrrhizae Radix and ZBD were significantly changed(P<0.05)..The dose of Fructus Gardeniae and Phellodendron Amurense remained the same,and the compatibility doses of Glycyrrhizae Radix were 1g,3g,6g,9g,12g and 15g respectively,The total amount ACA of Fructus Gardeniae and Phellodendron Amurense decreased with the increase of the dosage of Glycyrrhizae Radix;The total amount ACA of Glycyrrhizae Radix gradually increased;the total amount ACA of CBD showed a decreasing trend,followed by 50.479±0.326,46.901±0.585,41.431±0.650,37.76±0.865,34.54±0.840,and 32.29±0.492.The ACA range of Phellodendron Amurense,Glycyrrhizae Radix and Fructus Gardeniae is:1:0.9:5.3 to 1:2:7.Conclusion:1.The analysis on the active components alignment of ZhiziBopi DecoctionThe 10 kinds of ACA of Zhizibopi Decoction related to its clinical efficacy were preliminarily identified:geniposide,genipinlgentiobioside,magnoflorine,liquiritin,glycyrrhizic-acid,berberin-e hydrochloride,palmatine hydrochloride,isliquiritin,crocin I,crocin Ⅱ,established a multi-wavelength HPLC-DAD simultaneous determination of 10 kinds of ACA,this method has a better specificity,precision and reproducibility are applicable to the quality control of the ZBD and its single herb.2.Study on the Influencing Factors of ACA of ZhiziBopi Decoction①Formulation factor study:a.decocting time of the decoction influences the content of ACA,most of the components in the ZBD increase with the decocting time prolonging;some components tend to be stable after reaching a certain content;After a few components reach a certain amount,heating continues to decrease.Therefore,the maximum dissolution rate of all the medicinal components cannot be ensured at one time point or is not destroyed.When boiling time is 20min,the total amount of ACA is close to the maximum value at 60min,which is consistent with the requirement of traditional decoction time.b.Change the extraction method and solvent,and ACA have also changed.When the refluxing method was used to extract the ACA of ZBD,the dissolution of each component was greater than the ultrasonic method,indicating that the dissolution rate of ACA was greatly influenced by temperature;the solubility of ACA at a certain concentration was greater than that in water.30%ethanol is beneficial to the dissolution of ACA of FG and PA.60%ethanol is beneficial to the dissolution of GR.Therefore,changes in the method of preparation of the dosage form will lead to changes in the effective substances,which will affect the clinical efficacy.②Compatibility factors study;There were significant differences in the content of the ACA before and after the compatibility of FG,PA and GR,indicating that the ACA of the single herb in the compound were related to the compatibility,and the clinical efficacy of ZBD is achieved through compatibility.The removal of any one of the prescription drugs will have an effect on the ACA of ZBD.b.Change the species of Chinese Herbal Medicine,the content of ACA changes,indicating that different processed products of the same medicinal material represent different traditional Chinese medicines and have different clinical curative effects.The root cause lies in the differences in the ACA and should be distinguished.When the compatibility consisted of Raw Fructus Gardeniae,Raw Phellodendron Amurense ang Fry Glycyrrhizae Radix,the total amount of ACA was the largest,which verified the rationality of the traditional composition of ZBD.③Dose factor study:When Changing the dose of the single herb in ZBD,the ACA have significant differences with the traditional composition of ZBD.This indicates that the dose affects the degree of interaction between the composition components,thereby affecting the content and proportion of the ACA.Different doses and proportions of ACA represent different traditional Chinese medicines,and their clinical effects are not the same.Therefore,prescriptions cannot be arbitrarily added or subtracted during clinical use.The amount of prescriptions should be reasonably adjusted according to the patient’s condition.In summary,the selection of TCM quality control indicators should not be based on a few individual components or a certain type of component,and should be based on the ACA which reflect the complete of Chinese medicine as the index;when compatibility,dosage,and dosage form of TCM are changed,Changes in the content and proportion of ACA,therefore,the process of Chinese medicine preparation should be operated strictly in accordance with the standard process flow,and clinical prescriptions should not change the composition and doses of TCM,As the TCM has been clinically verified for thousands of years,its safety and effectiveness are irreplaceable.Changing the original compatibility,dosage,and dosage form of the TCM means that the clinical efficacy has changed,not only failing to achieve the desired therapeutic effect,but also it will cause waste of resources. |
