Study On Fingerprint Of Dachaihu Granule

It is wellknown that the quality control(QC)of traditional Chinese medieine(TCM)is one of the key tasks of TCM modernization.Chemical fingerprinting of TCM is already internationally accepted as an efficient QC technique,which can properly express the intrinsic quality of the herbs and preparations.Dachaihu Granule(DCHG)is derived from a well-known traditional Chinese formula named "Dachaihu decoction" which was founded by ZhongJing Zhang,a famous medical scientist during the Eastern Han Dynasty of China.The formula is a combination of eight medicinal materials,including Bupleuri Radix,Scutellariae Radix,Rhei Radix et Rhizome,Aurantii Fructus lmmaturus.Paeoniae Radix Alba,Pinelliae Rhizoma Praeparatum Cum Zingibere et Alumine,Zingiberis Rhizome Recens and Jujubae Fructus.Recent study showed that DCHG had a significant effect on curing acute or chronic cholecystitis in clinical practice.In this study,the QC methods of DCHG,DCHG intermediate and six raw medicinal materials including Bupleuri Radix,Scutellariae Radix,Rhei Radix et Rhizome,Aurantii Fructus Immaturus,Paeoniae Radix Alba,Zingiberis Rhizome Recens were studied.Fingerprint of DCHG,intermedidate and six raw medicinal materials were carried out as reference to the requirements of "the fingerprints of the technical requirements of traditional Chinese medicine injections(Provisional)",which guaranteed the stability and homogeneity of DCHG.intermediates and raw medicinal materials.A method of HPLC-DAD also has been developed for the simultaneously qualitative and quantitative analysis of ten bioactive compounds in DCHG.The main content could be concluded as follow:1.Fingerprints of raw medicinal materials of DCHG were studied.QC of medicinal materials is considered as the foundation of that of DCHG.Fingerprints of six most important materials of Bupleuri Radix,Scutellariae Radix,Rhei Radix et Rhizome,Aurantii Fructus Immaturus,Paeoniae Radix Alba,Zingiberis Rhizome Recens were studied by HPLC.More than 10 batches of above raw medicinal materials were collected from different areas respectively.Sample preparation method and chromatographic conditions including extraction solvent,extraction methods,extraction time,mobile phase,elution gradient,column detection,and wavelength were investigated and optimized to establish the HPLC fingerprint of the six raw medicinal materials.By the Similarity Evalution System for Chromatographic Fingerprint of TCM(Version 2004A)recommended by State Pharmacopoeia Commossion,similarity evalution was taken for comparision of each raw medicinal materials.The similarity of six raw medicinal materials was over 0.9,and the total peaks in fingerprints were marked,which could provide a basic for QC of the above raw medicinal materials.2.The establishment of fingerprint of DCHG and DCHG intermediate,and the correlation between raw materials,intermediate products and DCHG.Fingerprint of DCHG was studied,and HPLC was introduced to analyze DCHG samples.Extraction solvent,extraction method and extraction time were investigated and optimized,and finally choose methanol as the extraction solvent,sonicated for 20 minutes as the sample preparation method.While mobile phase,elution gradient,column detection,wavelength and column temperature etc.were also investigated and optimized.The HPLC fingerprint of DCHG was established with column Kromasil C18(250 mm × 4.6 mm,5 ?m).The mobile phase was ACN(A)-0.05%H3PO4/H2O(B),and the gradient elution was applied in chromatographic separation.The flow rate was 1 mL-min-1,the detection wavelength was 230 nm,the column temperature was 30?,and the injection volumn was 10 ?L.Verified by methodological,precision,reproducibility and stability are well,and 21 total peaks including baicalin,paeoniflorin,naringin,hesperidin,hesperidin,chrysophanol,etc.were marked in the total model of DCHG fingerprint.The similarity of 10 batches DCHG was over 0.97,which was in line with the requirements of fingerprints.Fingerprint of DCHG intermediate was studied with the reference to the DCHG fingerprint.Sample preparation methods and chromatographic conditions were the same with DCHG.After method validation texts,24 total peaks including baicalin,paeoniflorin,naringin,hesperidin,hesperidin,chrysophanol,etc.were marked in the total model of DCHG intermediate fingerprint and the similarity of 10 batches DCHG intermediate was over 0.96.Through the establishement of the fingerprint of the intermediates,QC of intermediate products in the production process was achieved.The correlation between raw materials,intermediate products and DCHG was studied.Sample preparation methods and chromatographic conditions of eight raw medicinal materials and intermediate products were the same with DCHG,and the main peaks in DCHG fingerprint were assigned.The research results show that DCHG intermediate fingerprint was almost the same with DCHG fingerprint,and the peaks in the DCHG mainly come from Scutellariae Radix,Rhei Radix et Rhizome.Aurantii Fructus Immaturus,Paeoniae Radix.Alba.Bupleuri Radix,but.Aurantii Fructus Immaturus,Paeoniae Radix.Alba and Zingiberis Rhizome Recens played a less important role in the fingerprint of DCHG.3.It was proposed that QC should be conducted base on the effectual segments of TCM,and a high-performance liquid chromatographic(HPLC)method with diode array detector(DAD)for simultaneous determination of ten bioactive compounds in DCHG was developed.The detection wavelength was set at 230 nm for paeoniflorin.254 nm for aloe-emodin.rhein.emodin.chrysophanol and physcion.280 nm for naringin.hesperidin.neohesperidin and baicalin.The above ingredients had a good separation in the same chromatographic conditions and it provided the basis for the QC for DCHG.A comprehensive quality control approach of DCHG based on fingerprinting and content determination was developed according to the above works.This paper not only developed the quality control method of medicinal materials,intermediate and DCHG,which has laid the foundation for the estalishment of quality standard of DCHG,but also studied the relationship between materials,intermediate and DCHG,tracked characteristic fingerprint peaks,which provides the basis for the interpretation of TCM.