| ObjectiveUltrafine Granular Powder(UGP)of Chinese medicine is a new type of Chinese medicine pieces,which have three application methods:decoction,brewing,and suspension.Among them,the suspension application is a full component application.A randomized controlled clinical trial of the traditional decoction of Danggui Buxue Decoction and the suspension of UGP in the treatment of patients with blood deficiency syndrome had shown that the application of the suspension of UGP had the equivalent by reduction in dose.However,there is still a lack of relevant data about its material basis research to support the clinical research results.This study carried out a study on the chemical components of the Danggui Buxue Decoction.Established an in vitro material basis analysis and detection method for traditional decoction pieces(TDP)and UGP of Danggui Buxue Decoction under different application methods.Compared the similarities and differences of in vitro material basis between its traditional decoction and the suspension of UGP to provide evidence of the material basis for the clinical research results.And compared the similarities and differences of in vitro material basis of three application methods of UGP to guide the rational and efficient application of UGP of Danggui Buxue Decoction in clinical practice.It also provides ideas and reference methods for in vitro material basis analysis of similarities and differences under different application methods for similar products.Methods1.Establishment of database of chemical constituents.of Danggui Buxue Decoction.The UGP of Danggui Buxue Decoction were extracted by system solvent ultrasonic extraction method with different polar solvents in sequence to obtain different polar extraction parts(petroleum ether,ethyl acetate,n-butanol,water part).Established a gas chromatography-mass spectrometry(GC-MS)to analyze and identify the chemical components of petroleum ether extraction part of the UGP of Danggui Buxue Decoction.Establish a liquid chromatography-mass spectrometry(LC-MS)to analyze and identify the chemical components of ethyl acetate extraction part,n-butanol extraction part and water extraction part of the UGP of Danggui Buxue Decoction.2.Screened of material basis analysis methods for the TDP and the UGP of Danggui Buxue Decoction in different applications.(1)Screened of analytical methods for the study of the material basis of the decoction of TDP and the suspension of UGP:The first method was the LC-MS method directly analyzed and identified the components of the suspension of UGP and the decoction of TDP of Danggui Buxue Decoction(Prepared 3 batches of samples of each type of Danggui Buxue Decoction.Each batch of samples should be done 3 copies in parallel).The second method was used the ultrasonic stirring extraction method,the suspension of UGP and the decoction of TDP of Danggui Buxue Decoction were extracted with petroleum ether,ethyl acetate and the water-saturated n-butanol in sequence to obtain the different polar extraction parts of them(Prepared 3 batches of samples of each type of Danggui Buxue Decoction.Each batch of samples should be done 3 copies in parallel).Then identified the components of petroleum ether extraction parts by the GC-MS.And identified the components of ethyl acetate extraction parts,the water-saturated n-butanol extraction parts and the remaining water parts after extraction by the LC-MS.Compared the components information obtained under the two analytical detection methods of direct injection analysis and each different polar extraction parts injection analysis.To select the method with more comprehensive component analysis information as the subsequent comparison of the similarities and differences of the material basis of the traditional decoction of TDP and the suspension of UGP of Danggui Buxue Decoction.(2)Screened of analytical methods for the study of the material basis of the decoction,brewing liquid and suspension of UGP:The first method was the LC-MS method directly analyzed and identified the components of the decoction,brewing liquid and suspension of UGP of Danggui Buxue Decoction(Prepared 3 batches of samples of application method.Each batch of samples should be done 3 copies in parallel).The second method was extracted the decoction,brewing liquid and suspension of UGP of Danggui Buxue Decoction with petroleum ether,ethyl acetate and the water-saturated n-butanol in sequence to obtain the different polar extraction parts of them(Prepared 3 batches of samples of application method.Each batch of samples should be done 3 copies in parallel).Then identified the components of petroleum ether extraction parts by the GC-MS.And identified the components of ethyl acetate extraction parts,the water-saturated n-butanol extraction parts and the remaining water parts after extraction by the LC-MS.Compared the components information obtained under the two analytical detection methods of direct injection analysis and each different polar extraction parts injection analysis.To select the method with more comprehensive component analysis information as the subsequent comparison of the similarities and differences of the material basis of different application methods of UGP of Danggui Buxue Decoction.3.Compared the material basis of the traditional decoction of Danggui Buxue Decoction and the suspension of UGP.Principal Component Analysis(PCA)and Orthogonal Partial Least Squares Discrimination Analysis(OPLS-DA)were used to compare the LC-MS(and GC-MS)analysis data of the traditional decoction of Danggui Buxue Tang and the suspension of UGP,and to evaluate the category and content similarities and differences of material basis.4.Compared the material basis of three application methods of UGP of Danggui Buxue Decoction.Principal Component Analysis(PCA)and Orthogonal Partial Least Squares Discrimination Analysis(OPLS-DA)were used to compare the LC-MS(and GC-MS)analysis data of the decoction,brewing liquid and suspension of UGP of Danggui Buxue Tang,and to evaluate the category and content similarities and differences of material basis.Results1.Establishment of database of chemical constituents of Danggui Buxue Decoction:A total of 35 components in the petroleum ether extraction part of Danggui Buxue Decoction were identified by GC-MS.UPLC-Q/TOF-MSE analysis method combined with UNIFI mass spectrometry data analysis software identified 127 components in the ethyl acetate part,148 components in the n-butanol part and 148 components in the water part of Danggui Buxue Decoction.After removing the repeated components,a total of 221 components in the Danggui Buxue Decoction compound were preliminarily identified,mainly flavonoids,phthalides,and triterpene saponins.2.Screened of material basis analysis methods for the TDP and the UGP of Danggui Buxue Decoction in different applications:(1)Only 47 components identified by direct injection analysis of the traditional decoction of Danggui Buxue Decoction and the suspension of UGP(the frist method).97 components identified by the sample injection analysis of different polar parts(the second method),which the component information was significantly richer than the frist method.The second method was selected to analyze and compare similarities and differences of material basis of the traditional decoction of Danggui Buxue Decoction and the suspension of UGP.(2)47 components identified by direct injection analysis of the decoction,brewing liquid,and suspension of UGP of Danggui Buxue Decoction(the frist method).A total of 97,96 and 97 components were identified respectively by sampling analysis of extracts from different polar parts of the decoction,brewing liquid and suspension of UGP(the second method),which the component information was significantly richer than the frist method.The second method was selected to analyze and compare similarities and differences of material basis of different application methods of UGP of Danggui Buxue Decoction.3.Compared the material basis of the traditional decoction of Danggui Buxue Decoction and the suspension of UGP:The decoction of TDP and the suspension of UGP contain the same type of chemical components,but the content of each component was different.The response value of 64 components(mainly flavonoids and triterpene saponins)in the suspension of UGP were higher than those in the decoction of TDP.The response value of 16 components(mainly flavonoids)in the decoction of TDP were higher than those in the suspension of UGP.PCA and OPLS-DA analysis could divide the decoction of TDP and the suspension of UGP of Danggui Buxue Tang into two groups.Among them,4 phthalide components such as Z-ligustilide and Z-butylidenephthalide were the differential markers of the petroleum ether extraction part.15 components(mainly flavonoids)such as Calycosin-7-O-β-D-glucopyranoside,Ononin were the differential markers of the ethyl acetate extraction parts.10 components(mainly flavonoids)such as Calycosin-7-O-β-D-glucoside-6"-O-malonate,Adenosine were the differential markers of the water-saturated n-butanol extraction parts.4.Compared the material basis of three application methods of UGP of Danggui Buxue Decoction:The three application methods(decoction,brewing liquid and suspension)of UGP contain the same type of chemical components,but the content of each component was different.The response value of 57 components(mainly flavonoids and triterpene saponins)in the suspension were higher than those of the decoction,and the response value of 18 components(mainly flavonoids)in the decoction were higher than those of the suspension.The response value of 65 components(mainly flavonoids and triterpene saponins)in the suspension were higher than those of the brewing liquid,and the response value of 10 components(mainly flavonoids)in the brewing liquid were higher than those of the suspension.PCA analysis could divide the decoction,brewing liquid and suspension of UGP of Danggui Buxue Decoction into two groups,of which the decoction and brewing liquid of UGP were a group.The OPLS-DA analysis could divide the decoction and suspension of UGP of Danggui Buxue Decoction into two groups.Among them,4 phthalide components such as Z-ligustilide and Z-butylidenephthalide were the differential markers of the petroleum ether extraction parts of the decoction and the suspension of UGP.18 components(mainly flavonoids)such as Calycosin-7-O-β-D-glucopyranoside,Calycosin were the differential markers of the ethyl acetate extraction parts of the decoction and the suspension of UGP.10 components(mainly flavonoids and triterpene saponins)such as Calycosin,Adenosine were the differential markers of the water-saturated n-butanol extraction parts of the decoction and the suspension of UGP.The OPLS-DA analysis could divide the brewing and suspension of UGP of Danggui Buxue Decoction into two groups.Among them,5 components(mainly phthalides)such as Z-ligustilide and Z-butylidenephthalide were the differential markers of the petroleum ether extraction parts of the brewing and the suspension of UGP.17 components(mainly flavonoids and triterpene saponins)such as Formononetin,Calycosin were the differential markers of the ethyl acetate extraction parts of the brewing and the suspension of UGP.10 components(mainly triterpene saponins and flavonoids)such as Calycosin,Adenosine were the differential markers of the water-saturated n-butanol extraction parts of the brewing and the suspension of UGP.Conclusion1.The established GC-MS and LC-MS analytical methods can be used for the separation and identification of components in the different polar extraction parts of Danggui Buxue Decoction.2.The components information obtained by the the different polar extraction parts injection analysis of each drug solution is more abundant and comprehensive than the direct injection analysis of the drug solution.The analysis and detection method can more scientifically and comprehensively reflect the similarities and differences of the material basis between the TDP and the UGP of Danggui Buxue Decoction under different application methods.3.The material basis of the suspension of UGP can be entered into the body and utilized in the body is significantly richer than the traditional decoction,and the main content difference markers are flavonoids,phthalides and triterpene saponins,which provides the basic basis in vitro for the application of suspension of UGP of Danggui Buxue Decoction can achieve the equivalent by reduction in dose.4.The material basis of the suspension of UGP can be entered into the body and utilized in the body is significantly richer than the decoction and brewing,and the main content difference markers are flavonoids,triterpene saponins and phthalides.In the clinical application of UGP,from the perspective of full and efficient utilization of the pharmacodynamic components,it is suggested that the suspension stirred into coffee-like should be applied as far as possible. |
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