Study On The Quality Control Of Huang-qin

Traditional Chinese medicine (TCM) is a complex mixture and its qualityhas great impact on the clinical effect. Harvesting time and growth years arethe first impact factor of TCM quality. How to determine optimal harvestingtime and growth years has been drawing much attention of scientists in thefields of medical research. Huang-qin (Scutellaria baicalensis Georgi, SG), anatural complex mixture consisting of hundreds of chemical compounds, isone of the common traditional herbs. Therefore, studying only a few bioactivecompounds cannot actually understand the synergy actions of all compositionsand thus cannot faithfully evaluate the quality of Huang-qin. A tri-step IRmacro-fingerprinting method, integrating Fourier transform infraredspectroscopy (FTIR), second derivative IR (SDIR) with two-dimensionalcorrelation infrared spectroscopy (2DCOS-IR), has been applied to study the“whole” chemical information of all compositions in Huang-qin specimensharvested in different seasons and growth years. Moreover, a number ofHuang-qin samples harvested in different seasons have been analyzed withprincipal component analysis based on their FTIR spectra, and subsequentlythey were exactly identified and classified.Shelf-life is one of the critical impact factors of the quality of Huang-qin.At present, national/international standard is still lacking for administering thestorage (e.g., medicinal shelf life) of Huang-qin, which probably results in theusage of Huang-qin preserved for many years. The quality of overdueHuang-qin is highly reduced by the natural physical or chemicaltransformations, such as bitten by insect, mustiness and coloration, and thusthe safety and effectiveness of Huang-qin are affected. Therefore, goodstorage conditions and long shelf-life are important in ensuring the quality ofHuang-qin. In this study, Accelerated storage tests have been applied to studythe shelf-life of Huang-qin, and thus subsequently provide reference forstorage tests. Moreover, the stability of Huang-qin stored at room temperaturewas also investigated and thus it could provide guidance for determining the shelf-life of Huang-qin.Objective:1. IR macro-fingerprinting method combined with principal componentanalysis is used to analyze the changes of constituents in Huang-qin specimensharvested in different seasons. The harvesting seasons of Huang-qin are fastand accurately identified and thus it could provide a scientific basis forplantations and its relevant industries.2. The dynamic variation rule of components in Huang-qin harvested indifferent growth years is investigated by IR macro-fingerprinting method andthus to precisely distinguish its harvesting years. This can be applied to thequality estimation of the clinical medication.3. Accelerated storage tests have been used to rapidly study the shelf-lifeof Huang-qin. Moreover, the whole chemical information of all compositionsand the contents of the flavonoid compounds are investigated by FTIR andHPLC, respectively. The effect of shelf-life on Huang-qin quality is primarilystudied.4. The stability of Huang-qin stored at room temperature is furtherinvestigated by FTIR and HPLC-DAD-MS, based on the results of acceleratedstorage tests. And the study provides reference for the establishment ofshelf-life of Huang-qin.Methods:1. The KBr tablets of the samples are used to obtain their FTIR spectra.And to collect the2D-IR spectra, the tablet was put into the pool of thetemperature controller to record the IR spectra from50℃to120℃with aninterval of10℃.2. The Agilent XDB-C18column (2.1mm×150mm,3.5μm) is used withthe gradient mobile system and the mobile phase of1%glacial aceticacid-acetonitrile, at the detection wavelength of276nm and a flow rate of0.2ml/min. The column temperature is maintained at25℃. The injection volumeof samples and standards is2μl.3. The mass spectrum data is obtained by HPLC-DAD-MS with an electrospray ionization source in negative ion mode. The Agilent XDB-C18column (2.1mm×150mm,3.5μm) is used with the gradient mobile systemand the mobile phase of0.1%formic acid-acetonitrile, at the detectionwavelength of276nm and a flow rate of0.2ml/min. The column temperatureis maintained at25℃. The injection volume of samples and standards is2μl.Results:1. The position and intensity of peaks have obvious differences in the IRspectra of Huang-qin harvested in spring and autumn. The relative intensitiesof the ester carbonyl and the flavonoids of spring Huang-qin are higher than0.38and0.94respectively, while that of autumn Huang-qin are less than0.30and0.90respectively. Furthermore, the above differences are visuallyvalidated by the second derivative infrared spectra and two-dimensionalcorrelation infrared spectroscopy (2DCOS-IR). Moreover, a large amount ofHuang-qin harvested in spring and autumn are exactly identified and classifiedby principal component analysis.2. Ten batches of biennial Huang-qin have similar IR spectra, while thatof annual and perennial Huang-qin have different IR spectra. The estercarbonyl C=O absorption peak at1740cm-1in the IR spectra of biennialHuang-qin samples have significantly higher intensity than those of annualand perennial Huang-qin. Interestingly, flavonoids absorption peaks at1614cm-1of perennial Huang-qin are at the highest intensity. Moreover,Huang-qin samples at different harvest year are different in the shapes ofcarbohydrates absorption peaks at1058cm-1. The herbs harvested in differentgrowth years are quite different from each other in the position of autopeaks.3. By comparing the tri-step IR macro-fingerprints of Huang-qin sampleswith different accelerating storage period, Huang-qin change significantly onthe peaks shape, position and intensities at1658,1614and1069cm-1mainlyattributed to flavonoids and their glycosides. The correlation coefficients ofthe infrared spectra of Huang-qin samples at month0,1,2and3are0.9670,0.8570and0.7308, respectively. The HPLC quantitation results suggest thatthe content of baicalin and wogonoside decrease whereas baicalein and wogonin initially increase and then decrease after two months of storage.4. The positions, shapes and intensities of peaks in the IR spectra ofHuang-qin with different storage period are rather similar.6bioactivecompounds are identified and than of content are mainly steady.Conclusion:1. The amount of flavonoids and esters of spring Huang-qin samples ishigher than that of autumn Huang-qin samples and their carbohydrates aredifferent. The tri-step IR macro-fingerprints method combined with principalcomponent analysis can be used to accurately and fast identify the Huang-qinfrom different harvest seasons.2. Annual and perennial Huang-qin samples are unstable, while that ofbiennial Huang-qin samples are stable. The highest content of Ester carbonylis in biennial Huang-qin, while than of flavonoids is in perennial Huang-qin.Therefore, the constituents and content of Huang-qin samples at differentharvest year have significant different, and thus medicine doses vary should bebased on medicinal growth years.3. Accelerated storage test results have demonstrated that length ofstorage period has a significant impact on the quality of Huang-qin, andsubsequently to provide a scientific basis for Huang-qin shelf-life test.4. Huang-qin specimens stored six months are stable. The quality ofHuang-qin from different storage period can be reflected well by this study.