Digital Fingerprints Of Xianggui Huazhuo Capsule And Its Quality Control

Xianggui Huazhuo capsules is a prescription for the clinical applicationof the First Hospital of Hebei Medical University, years of experience side bycinnamon, earth rhubarb, patchouli. Its efficacy are considered to be aromaticXingpi, clear dampness cloud of power. After attending various diseases dueto remain entirely evil, spleen and internal injuries caused by long loose stoolor mucus, bowel distension, anorexia or fever, greasy tongue coating, slipperyor see yellowish or white, pulse slow; Mainly it is used to chronicenteritis,fungal enteritis caused by spleen wet storm treatment. There are somereports about its preparation process, quality control and toxicology. In thepaper, we reported the further studies about its chemical composition, qualitycontrol standards and determination in order to get a desired effective,scientific quality control.This paper reported the research of the Xianggui Huazhuo capsules bycombining of TLC fluorescence scanning, HPLC, and GC-MS methods. Weestablished the fingerprints for Xianggui Huazhuo capsules and its volatile oils.TLC and fluorescence scanning mothods for determination of three maincomponents.In this study, we combined natural medicine chemistry, analyticalchemistry, and pharmacy to study the chemical composition, qualityassessment and control standards of Xianggui Huazhuo capsules, which willlead a meaningful exploration for traditional Chinese medicine capsules.Part one: Studies on the fingerprints of Xianggui Huazhu CapsulesObjective: To establish HPLC fingerprints of Xianggui Huazhu capsules,get reference fingerprint and compare the fingerprints of Xianggui Huazhucapsules collected from different batches. Methods:1Optimization of the extraction method: In order to obtain satisfactoryextraction efficiency, extraction method, extraction solvent and extraction timewere investigated.2Optimization of the chromatographic conditions: Chromatographicconditions such as mobile phase systems, wavelength and column temperaturewere tested for optimization of the chromatographic conditions.3System suitability test: Under the above conditions, resolution andtheoretical plate number the peak of cinnamic aldehyde was calculated.4Methodology: Precision, reproducibility and stability were determined.5Establishment of fingerprint: The fingerprints from8samples of thesolution of Xianggui Huazhuo capsules were ananlysed to get their relevantsimilarity.Results:1Optimization of the extraction method: The dried powders of radixglehniae samples were accurately weighed and extracted by heating refluxwith75%methanol solution for30min.2Optimization of the chromatographic conditions: The analytical columnwas DiamonsilTMC18(250mm×4.6mm,5μm). The flow rate was1.0mL·min-1and the column temperature was30℃.The detection wavelengthwas set at280nm and the sample injection volume was20μl. The mobilephase for HPLC analysis consisted of methanol-acetonitrile-aqueous aceticacid (0.1%) using gradient elution.3System suitability test: Under above conditions, the peak of cinnamicaldehyde was separated well with the resolution of more than1.5and about8000of theoretical plate.4Precision: Designating the major peaks for reference peak, the RSDvalues of relative retention time and peak areas were less than0.48%and1.01%. Reproducibility: Designating the major peaks for reference peak, theRSD values of relative retention time and peak areas were less than0.89%and2.58%. Stability: Designating the major peaks for reference peak, the RSD values of relative retention time and peak areas were less than0.76%and2.33%. Description of the test solution stable within24h.5Establishment of fingerprint and data analysis:We have got18commonpeaks in reference fingerprint from10batches of Xianggui Huazhu Capsules.Used software for similarity analysis, the results showed that the similaritiesof8batches of Xianggui Huazhu Capsules were above0.9.Conclusion: The analytical method of HPLC fingerprint of XiangguiHuazhu Capsules was developed and validated. Relative retention system andthe software for similarity analysis were applied to evaluate the variability ofthose fingerprints from different sources. The method is able to effectivelycontrol the quality of Xianggui Huazhu Capsules.Part two: Studies on the simultaneous determination of3coumarins inXianggui Huazhu Capsules by HPLCObjective: To establish a method for simultaneous determination of3major coumarins in Xianggui Huazhu Capsules, namely cinnamic aldehyde,pogostone and emodin. To raise a new method for effective evaluation ofscience and quality control Xianggui Huazhuo capsules raise new methods.Methods:1Optimization of the extraction method: In order to obtain satisfactoryextraction efficiency, extraction method, extraction solvent and extraction timewere investigated.2Optimization of the chromatographic conditions: Chromatographicconditions such as mobile phase systems, wavelength and column temperaturewere tested for optimization of the chromatographic conditions.3System suitability test: Under the above conditions, resolution andtheoretical plate number the peak of cinnamic aldehyde was calculated.4Methodology: The standard curves of cinnamic aldehyde, pogostone,emodin were established respectively, calibration curves, precision, recoveryand stability were determined.5Sample analysis: Under above-mentioned conditions, the contents of3 coumarins in10batches of Xianggui Huazhuo capsules were determined.Results:1Optimization of the extraction method: The dried powder of radixglehniae samples was accurately weighed and extracted by heating reflux with75%methanol solution for30min.2Optimization of the chromatographic conditions: The analytical columnwas DiamonsilTMC18(250mm×4.0mm,5μm). The flow rate was1.0mL·min-1and the column temperature was30℃. The detection wavelength was set at280nm and the sample injection volume was20μl. The mobile phase forHPLC analysis consisted of methanol-acetonitrile-aqueous acetic acid (0.1%)using gradient elution.3System suitability test: Under above conditions, the peak of cinnamicaldehyde was separated well with the resolution of more than1.5and about8000of theoretical plate.4Methodology: The calibration curves of3coumarins have goodlinearity (r＞0.999). Concentration range:2.0~32.0,1.68~26.88,0.98~15.68μg/mL. The intra-day and inter-day precisions calculated as therelative standard deviation were within the range of0.61%~1.17%and0.63%~1.35%. The coumarins showed the overall recoveries ranging from98.80%～99.57%. The analytes were found to be very stable in75%methanolsolution within24h.5Sample analysis: Content of the three main components from10batches of Xianggui Huazhu capsules were24mg/g,11mg/g and0.4mg/grespectively.Conclusion: In this paper, a rapid, reliable and sensitive method wasdeveloped for the determination of bioactive constituentsin by HPLC. It wasthe first time to report the simultaneous quantification of three maincomponents (cinnamic aldehyde, pogostone and emodin) in Xianggui HuazhuCapsules. The newly established method could be applied as a reliable andsensitive quality control procedure for Xianggui Huazhu Capsules. Part three: GC-MS analysis of volatile oil fingerprints from XiangguiHuazhuo capsulesObjective: To establish GC-MS fingerprints of volatile oil fromXianggui Huazhuo and use MS analysis chemical composition of volatile oilfrom Xianggui Huazhuo capsules. To provide a new approach for the effectivecontrol of the quality of scientific evaluation for Xianggui Huazhuo capsules.Methods:1Gas chromatography conditions: fused silica capillary column (30m×0.25mm,0.25μm); high purity helium as the carrier gas, volume flow:1.0mLmin-1, split ratio50:1; feed injector temperature:230℃; temperatureprogram: initial temperature of100℃, keeping2min, with3℃min-1roseto130℃, keeping5min, then10℃min-1rose to210℃, keeping5min.Injection volume:1.0μL, solvent delay2min, gasification chambertemperature of280℃. Quadrupole mass analyzer. MS conditions: electronimpact (EI) ion source, power voltage70eV; ion source temperature of230℃;pre-column pressure65.072kpa; scanning range20~500amu.2Using steam distillation Xianggui Huazhuo capsules volatile oilcomponents, and gas chromatography-mass spectrometry (GC-MS), thechemical composition of volatile oil were separated and identified by capillarycolumn for analysis, peak area normalization a determination of the relativecontent of law.Results: A total of37separate peaks, from which identified29chemicalcomponents, accounting for90.07%of the total volatile oil. The relativecontent of more than5%are trans-cinnamaldehyde (relative content20.77%),cyclohexene (5.49%relative to the content), naphthalene (5.34%relative tothe content), azulene (relative content of6.26%), o-methoxy-cinnamicaldehyde (relative content of8.26%), patchouli alcohol (relative content of10.43%) and long leaf aldehyde (relative content of10.33%).Conclusion:1The volatile oils main ingredient of Xianggui Huazhuo capsules aretrans-cinnamic aldehyde, patchouli alcohol, aldehyde leaves, chamomile blue and o-methoxy cinnamic aldehyde. By references, cis-cinnamic aldehyde,trans-cinnamic aldehyde, o-methoxy cinnamic aldehyde, thujopsis ene, δ-BiLitseae ene, α-pinene are mainly from cinnamon, patchouli Li alcohol,α-patchouli ene, β-patchouli ene, caryophyllene are mainly from patchouli.2In this study, GC-MS were used to nanlyzed and determine the volatilechemical composition from Xianggui Huazhuo capsules. Instead of aqualitative and quantitative indicators of a single active ingredient oringredients analysis, this method is analysis of the steering group of the activeingredient, which more scientific and accurate reflection the inherent qualityof Xianggui Huazhuo capsules. This study established a GC-MS method fordetermination of the chemical constituents of volatile oil from XiangguiHuazhuo capsules, which can be used for quality control and provide ascientific basis for its further study.Part four: The rapid identification of Xianggui Huazhuo capsule by TLCand its main components quantitative by fluorescence TLC scanningObjective: To establish a quick method to discriminate XiangguiHuazhuo capsules by TLC and determine its contents by fluorescence TLCscaning.Methods: Taking the cinnamic, pogostone, emodin as reference, thesample solution and the reference were pointed in the same silica gel G plate.Petroleum ether(60～90℃)：ethyl acetate（7:3）as the eluent, expand, remove,dry, color, and observe the results.（2）Taking the emodin as a reference, thesample solution and the reference were pointed in the same silica gel G plate.Petroleum ether(60～90℃)：ethyl acetate（7:3）as the eluent, expand, remove,dry, calculated the content of the emodin by TLC scaning.Results:1All corresponding spots to the reference can be found in the samples onthe TLC plate.2By comparing the standard curve of emodin, the content of the emodinfrom Xianggui Huazhuo capsule was0.4mg/g. Conclusion: TLC identification method can be used for rapid detection ofXianggui Huazhuo capsule. The determination results were similar to that ofHPLC method. TLC identification is a simple, rapid and specific method,which is suitable for the rapid detection of hospital preparations.