Research On Effective Material Basis Of Niuhuang Shangqing Pill For Treating "Internal Fire"

Niuhuang Shangqing Pill(NSP)consists of 19 crude drugs including Bovis Calculus Artifactus,Gardeniae Fructus,and Rhei Radix et Rhizoma which can clear away heat and purg fire,disperse wind and relieve pain,and it is a commonly used and representative prescription to treating “internal fire” in clinical practice of Traditional Chinese Medicine(TCM).As a big TCM preparation,it is a big challenge to analyze the effective substance of NSP,which is also blocking its basic research and quality control.In this study,NSP was used as the sample to screen the effective segments in the treatment of “internal fire” by using a mice model with excessive-heat syndrome based on TCM tongue diagnosis and anti-Streptococcus pneumoniae(S.pneumoniae)model based on microcalorimetry.By using comprehensive two-dimensional chromatography(LC×LC)menthod,multi-dimensional fingerprints of the effective segments of NSP were established.Then,the chemometrics methods such as principal component analysis(PCA),orthogonal partial least squares(OPLS),and orthogonal partial least squares discriminant analysis(OPLS-DA)were used to analyze the multidimensional spectral-activity relationship to determine the antibacterial activity peaks group.Qualitative identification by MS was used to identify the active compunents eventually.This research was to explore and establish the method for analyzing the effective substance of TCM preparation,and to reveal the effective material basis of NSP for treating “internal fire”.The primary results are as follows:1.NSP(made with NSP powder)were divided into segments of 5%,30%,60% and 95% ethanol by polarity.Ultra-high performance liquid chromatography(UPLC)analysis shows that there were only small overlaps between different segments.Mice model with excessive heat syndrome showed that 30% ethanol was the effective segment.2.10 batches of commercial NSP were processed by the above method.Anti-S.pneumoniae test based on microcalorimetry was used to screen effective components.The results showed that the different segments have antibacterial effect variously.60% ethanol segment is relatively stronger.Further analysis on this segment of 10 batches of NSP showed that the antibacterial power is as follows: S7> S9> S10> S6> S8> S4> S2> S1> S3> S5.3.A three-dimensional(3D)fingerprint method based on LC×LC was established,the peak capacity of the established LC×LC method has considerable improvents over traditional one-dimensional method.3D fingerprints of 30% and 60% ethanol segments of 10 batches of NSP were obtained.Seventeen common peaks(X1-X17)were identified by comparing the comprehensive two-dimensional contour plots,retention time and ultraviolet absorption characteristics of each chromatographic peak for 60% ethanol segments of 10 batches of NSP.4.PCA,OPLS,OPLS-DA were used to analyze the spectrum-activity relationship between 3D fingerprints and the antibacterial activity data of 60% ethanol segments.Results showed that compound X17,X14,X15 and X10 were the main antibacterialactivity peaks.LC×LC-Triple-Quadrupole-MS and UPLC-Q-TOF-MS were used to analyze the activity peaks,and three of them were identified by comparison of standard reference: Baicalein(X10),Wogonin(X14),Glycyrrhizic acid(X15).Conclusion: the 30% and 60% ethanol segment of NSP were the effective segments for treatment of “internal fire”(excessive heat syndrome mice model)and anti-S.pneumoniae(based on microcalorimetry).Compound X17(unknown),Wogonin(X14),Glycyrrhizic acid(X15)and Baicalein(X10)probably are the main active ingredients of 60% ethanol segment of NSP to inhibit the growth of S.pneumoniae.The peak capacity of 3D fingerprint based on the established LC×LC method is obviously better than the traditional one-dimensional method.Combined with the chemometrics methods such as PCA,OPLS,OPLS-DA,the proposed method can be used for rapid screening and identifying effective components in TCM preparations.