Study On The Composition Changes Of Sweated And Crude Radix Dipsaci And The Effect On The Pharmacodynamics Based On The Spectral Relationship

Radix Dipsaci is the root of Dipsacus asper Wall.ex Henry^[1].It has the function of reinforcing liver and kidney,strengthening bones and stop uterine bleeding.The traditional processing in origins is called“sweating”.Some scholars have found that after“sweating”,the contents of main chemical components were changed.But whether composition change affect its efficacy is still lack of strong evidence.There are also studies showed that"sweating"can reduce the volatile oil and alkaloid chemical components of Radix Dipsaci,increase toxic and harmful components,which can also cause secondary pollution of medicinal materials.Therefore,the rationality and necessity of"sweating"are not yet clear and there is a need for in-depth research.Objective1.To compare the changes in components of sweated and crude Radix Dipsaci.2.To discuss the effect of“sweating”on the efficacy.3.To study the material basis of pharmacodynamic before and after sweatingMethods1.UPLC-Triple-TOF/MS system was used to analyze the components differences of sweated and crude Radix Dipsaci.Liquid phase conditions were as follows:Agilent Eclipse XDB-C18（250 mm×4.6mm,5.0μm）columns;The mobile phase consisted of 0.1%formic acid in water-acetonitrile;Gradient elution:0-45 min,6%-20%acetonitrile;45-90 min,20-35%acetonitrile;90-110 min,35-70%acetonitrile;The column temperature was room temperature and the volume flow rate was 0.8 m L/min.The UV detection wavelength was 215 nm.Mass spectrometry conditions were as follows:UPLC-Triple-TOF 5600+TOF LC/MS for Negative ion scan mode;Scanning range:m/z 100-1500;Nebulizer gas（GS1）:50 psi;Nebulizer gas（GS2）:50 psi;Curtain gas（CUR）:35 psi;Ion source temperature（TEM）:550℃;Ion source voltage（IS）:-4500 V;First scan:to cluster voltage（DP）:100 V;focusing voltage（CE）:10V;Secondary scan:MS data was acquired using TOF MS～Product Ion～IDA mode;CID energies were 20,40 and60V.2.20 batches of crude and sweated samples were selected respectively,and carry out gradient elution in acetonitrile-0.05%phosphoric acid,with flow velocity1.0m L·min^-1,column temperature 25℃,recording duration 120 min,temperature30℃.The characteristic fingerprinting of crude and sweated samples were established by using Evaluation System of the Similarity of TCM Chromatographic fingerprinting Version 2.0 software published by the National Pharmacopoeia Commission.3.48-month-old male SD rats were selected,and randomly divided them into sweated Radix Dipsaci group,crude Radix Dipsaci group,model control group,and sham operation group.Tibial fracture modeling were established for the first three groups and last group for sham operation.After 24hours,administered 1 time each day for the does of per 100g body weight/1ml.After administration for 4 days,7 days,14 days and 28 days,blood samples were taken for the detection of serum BMP-2,hemorrheology and blood biochemical indexes.Tibia tissues were taken for x-ray and mechanical examination.4.MG-63 cells and osteoblasts were co-cultured with decoction and serum comtained sweated and crude Radix Dipsaci respectively.The cell proliferation was detected by MTT method and ALP activity of osteoblasts was detected by nitrophenyl phosphate method.5.The DAD detector was used to establish the HPLC fingerprints of sweated and crude Radix Dipsaci decoction,and the correlation coefficient was established by gray relational analysis.Results1.In this experiment,liquid chromatography-mass spectrometry（LC-MS/MS）was used to speculate 52 compounds of sweated and crude Radix Dipsaci according to the Scifinder and Reaxy databases and literatures.2.The chromatograms of crude and sweated samples were compared,and 35 common chromatographic peaks in the fingerprints were obtained.The peaks of 7,8,9,10,18,19,20,21,27,and 29 were identified as loganic acid,chlorogenic acid,caffeic acid,loganin,Isochlorogenic acid B,Isochlorogenic acid A,cantleyoside,Isochlorogenic acid C,Asperosaponin X and AsperosaponinⅥrespectively.The content of most of them were reduced after“sweating”.3.Based on the experimental data,it was found that BMP-2,hemorheological indexes,blood biochemical parameters,ALP activity and the maximum fracture resistance of tibial of crude Radix Dipsaci group were not inferior to the sweated Radix Dipsaci group.4.Both decoction and drug-contained serum could significantly promote the proliferation of MG-63 cells and osteoblasts（P<0.01）,and significantly increase the ALP activity of osteoblasts（P<0.01）.Pharmacological methods of the same dose concentration of crude group generally better than or non-inferior to the sweated group.5.The chemical components of peaks 29 and 8 were highly correlated with the proliferation and differentiation of osteoblasts and MG-63 cells,and the correlations were all above 0.7.The top ranked features of relevance of these three pharmacodynamic indicators were the peaks 29,8,10,35,27,21,9.Combined with the previous analysis of liquid chromatography-mass spectrometry,it could be inferred these peaks were Asperosaponin VI,Chlorogenic acid,loganin,Asperosaponin IV isomers,Dictyosaponin X,Isochlorogenic acid C and Caffeic acid.Conclusion1.liquid chromatography-mass spectrometry was used to presume 52 compounds of crude and sweated Radix Dipsaci,which provided a comprehensive material basis for Radix Dipsaci and laid a foundation for fingerprint studies.2.loganic acid,chlorogenic acid,caffeic acid,loganin,Isochlorogenic acid A,cantleyoside,Isochlorogenic acid C and AsperosaponinⅥcontained in Radix Dipsaci were reduced after“sweating”.3.Both sweated and crude Radix Dipsaci could significantly promote the healing of tibial fractures in rats,and indicators of crude Radix Dipsaci group were not inferior to the sweated group.Both decoction and serum contained sweated and crude Radix Dipsaci could promote the proliferation of MG-63 cells and osteoblasts,and improve the ALP activity of osteoblasts.Crude Radix Dipsaci group were generally better than or non-inferior to the sweated Radix Dipsaci group at the same dose.4.Asperosaponin VI,chlorogenic acid,loganin,Asperosaponin IV isomers,Dictyosaponin X,Isochlorogenic acid C,caffeic acid may be the main material basis that affect cell proliferation and differentiation.Combined with previous studies,the contents of these ingredients were almost all decreased after“sweating”.5.The comprehensive results of the overall pharmacological experiments,in vitro experiments,and spectral-effect relationships of pharmacodynamics and fingerprints suggest that it is worth further study if Radix Dipsaci needs"sweating".