Studies On HPLC Fingerprints Of Da Chaihu Decoction And Guifu Dihuang Prescription

Quality control is the critical problem affecting the development of TCMmodernization. Due to the complexity of the components of TCM, the establishmentof a comprehensive and integrated quality standard with layers of TCM is quiteessential. Fingerprint is the very integral characterization mode expressingthecomplex system of TCM.1、Studies on HPLC Fingerprints of Da Chaihu DecoctionDa Chaihu decoction, consisting of Radix Bupleuri, Peony, Pinellia, Ginger,Jujube, Fructus Aurantii Immaturus, Scutellaria Baicalensis Georgi and Rhubarb, isone famous traditional prescription of TCM. According to Treatise on FebrileDiseases, blend Radix Bupleuri (24g), Scutellaria BaicalensisGeorgi (9g), Pinellia(9g), Peony (9g), Fructus Aurantii Immaturus (12g), Ginger (15g), Rhubarb (6g) and4Jujubes and add2400mL of water. Then boil the mixture by intense fire and thenuse slow fire to concentrate the water to1200mL. Filter the medicine residues andcontinue to concentrate the decoction to600mL. The final decoction was processedby precipitation with70%ethanol and the70%ethanol and coarse polysaccharidefractions of Da Chaihu decoction were acquired in the end.After vacuum freeze drying, the medium polarity fraction of Da Chaihudecoction was dissolved with50%methanol to prepare a sample for HPLC analysis.After groping the experimental conditions and methodology validation, the fingerprintof the70%ethanol fraction of Da Chaihu decoction was established. Then comparethat with those of the single herb medicine so that the source of the characteristicchromatographic peaks in the fingerprint of Da Chaihu decoction could be confirmed.As for the coarse polysaccharide fraction of Da Chaihu decoction, its fingerprintwas established by the method of overall hydrolysis with TFA, pre-columnderivatization with PMP and detection with the HPLC techniques. By comparation tothe fingerprint of the standard substance, each chromatographic peakwas identified.For further quality research on Da Chaihu decoction and effective supervisionfrom the origin, we established the respective fingerprints of the70%ethanol fractionfrom Fructus Aurantii Immaturus, Fructus Aurantii and Rhubarb, identified the main chromatographic peaks with the Ion Trap LC/MS method and then did some relevantanalysis and evaluation on the quality of some commercial Chinese herbalmedicine.The following conclusion is made:1. most of the commercially availableFructus Aurantii Immaturus and Fructus Aurantii are authentic herbs produced byShanxi province. However, despite the same origin, the analysis result of thefingerprints of the chemical components suggests that some of the commercialFructus Aurantii Immaturus doesn’t contain naringin and neohesperidin, which aretwo major neoflavonoids working as the important efficacy parts. Meanwhile,synephrine acts as the index of detection of Fructus Aurantii Immaturus according toChinese Pharmacopoeia. But the method of single detection index fails to discriminatethose Fructus Aurantii Immaturus mentioned as above.2. Fructus Aurantii Immaturusand Fructus Aurantii weren’t clearly distinguished until Song Dynasty. In spite of thedifferences in efficacy, their chemical components are generally consistent and theirfingerprints have very high similarity, leading to the failure to distinguish them withsimilarity analysis software. But after experimental verification, we find that FructusAurantii Immaturus and Fructus Aurantii can be distinguished by both systematicclustering and principal component analysis.3. Most of the commercial Rhubarborigins from Gansu province and Qingdao. Their fingerprints of chemical componentshave good stability and high similarity. But of all the identified samples, there existsone batch of Rhubarb with several individually large chromatographic peaks and canbe distinguished from other samples. However, according to Chinese Pharmacopoeia,the total amount of aloe-emodin, rheinic acid, chrysophanol and emodin monomethylether functions as the index of the quality control of Rhubarb, which is difficult toidentify the individual Rhubarb sample above. From the above conclusions, thechoice and supervision of single herb medicine is also very important to the qualitycontrol of Da Chaihu decoction.2、Studies on HPLC Fingerprints of Guifu Dihuang PrescriptionGuifu Dihuang prescription consists of8single herbs including preparedRehmannia root, Yam, Cornus, PoriaCocos, RhizomaAlismatis, Cortex Moutan,processed Radix AconitiLateralis and Cinnamon, with a relative proportion of8:4:4:3:3:3:1:1. Besides the most regular pills like the water honey pills andconcentrated pills, the dosage forms of Guifu Dihuang prescription also includes oralsolution, capsule and so on.Though indifferent dosage forms,the composites of thecompound prescription are basically same. The content of loganin and paeonolis chosen as the indicator of the quality control of the commercial Guifu Dihuangprescription medicines.But such method is hard to reflect the integrity and complexityof TCM. Therefore, the fingerprints of the chemical components in Guifu Dihuangprescription were established, on which the evaluation on the quality of thecommercial Guifu Dihuang prescription drugs was based.After Guifu Dihuang prescription mixtures were processed by grinding, differentextraction methods were investigated and finally absolute ethyl alcohol was chosen tobe used as the extraction solvent with two reasons:1. the extraction ratio of theabsolute ethyl alcoholsolvent was high and more comprehensive chemicalcomponents could be extracted.2. as for the commercial pills, auxiliary materials hadthe least affection on the extraction with absolute ethyl alcohol as the solvent.To mostpossibly avoid the influence on chemical components by the pretreatment, theextraction solution was evaporated to dryness and redissolved as the sample directlyused in HPLC analysis. Then the fingerprint of the chemical components in theethanol fractions of Guifu Dihuang prescription was established. Through thecomparation with the fingerprints of single herbs,6of the main chromatographicpeaks were identified with the method of Ion Trap LC/MS.Guifu Dihuang prescription pills are one of the most common drugs in themarket and are used widely in clinical treatment. On the basis of the establishedfingerprints, qualities of10batches of Guifu Dihuang condensed pills produced by10different manufacturers were assessed. According to the result of similarity analysisand that of the systematic clustering and principal component analysis, the conclusionis quality of commercial Guifu Dihuang pills has good consistence with that of thetraditional Guifu Dihuang prescription, despite some differnces. At the same time,differences to varying extents exist between commercial Guifu Dihuang pills, whichmay be related to different sources of single herb and large-scale production processesof different manufacturers.3、ConclusionThe coarse polysaccharide fractions of Guifu Dihuang prescription were got byextraction with distilled water and precipitation with ethanol after crude herbs weregrinded. Afteroverall hydrolysis with TFA, pre-column derivatization with PMP anddetection with the HPLC techniques, the fingerprint of the coarse polysaccharidefractions was established. Furthermore, the main chromatographic peaks wereidentified through contrast to the fingerprint of the standard preparation. Similarly, qualities of10batches of Guifu Dihuang condensed pills produced by10differentmanufactures were reevaluated.According to a series of data process results, goodquality consistence between commercial Guifu Dihuang pills and the traditional GuifuDihuang prescription and good stability of the polysaccharide quality of commercialGuifu Dihuang pills are determined.The fingerprints established in this article were all chromatographic peaksextracted in a wide range of wavelength and maximum absorption peaks of all thesubstances in that wavelength range can be reflected in one single fingerprint, whichcan remedy the drawbacks of insufficient information and inconvenience broughtrespectively by single and multiple wavelength detecting methods. And furthermore,it can avoid the error from the fingerprint fusion technology of multiple wavelengths,which hasn’t been reported in previous literatures on fingerprint.As a conclusion, fingerprint establishment of chemical components of Da ChaiHu decoction, Fructus Aurantii Immaturus, Fructus Aurantii, Rhubarband GuifuDihuang prescription were completed in this research, based on which integralevaluation of Fructus Aurantii Immaturus, Fructus Aurantii, Rhubarb and GuifuDihuang prescription with multiple data processing methods was done.The scientificand elaborate results suggest fingerprint can woks as a foundation for qualityevaluation. Besides that, fingerprint method can also be applied to the production,manufacture and storage of herbs, as well as the raw materials, intermediate, finishedand current products of compound prescription, in order to find the quality aberranceand deficiency in time and avoid the sub-quality products into the market.