| Radix Scutellariae is the dry root of Scutellaria baicalensisGeorgi., which belongs to the family of labiate. It mainlycontains flavone components, which are the bioactivecomponent. It has many effects, such as clearing away heat,drying dampness, purging fire, removing toxic substances,arresting bleeding and preventing miscarriage, and so on. RadixScutellariae mainly are produced in Chengde City of HebeiProvince, Baoding City, Fengyang of Shanxi Province, HenanProvince, Shanxi Province, Neimeng and northeast of China,especially in north of Hebei, where the quality of it is best.Radix Scutellariae of north of Hebei is always called as"Scutellaria baicalensis of Rehe", whose rootstalk is firmer;hollow part is smaller; color is more yellow; quality is best.Because of difference of geography and growth environment,the quality is different including planting and wildness fromdifferent places. Variety and complexity of chemistrycomponents of TCM are the physical foundation to developcurative effect. However, traditional quality evaluation of TCMalways only uses one or several components as index, whichalways can't reflect its internal quality comprehensively. Themodern analytical technique is applied to study fingerprints byusing figures to describe the internal chemical information oftraditional Chinese medicine. In this case, fingerprints just are anew method to reflect the differences of description and quantityof chemical information. It can be used to control the quality ofthe traditional Chinese medicine and drugs preparation throughintegrative information. It's a better method ratified in theinternational to control the quality of traditiona1 Chinesemedicine. So it is necessary to develop the fingerprints ofScutellaria baicalensis from Rehe to control quality of itthoroughly.Objective:Establish HPLC fingerprints of Scutellariabaicalensis Georgi. (SBG) from Rehe of Hebei Province; get thecontrol chromatogram and the fingerprints of SBG collectedfrom different producing areas were compared so as to establisha sensitive and specific method for controlling the quality ofSBG.Methods:1. Establishment of fingerprint: (1) Extraction:An optimal extracting condition was chose by comparing theexperimental results. (2) Chromatographic condition: Chooseappropriate column and adjust different formulation andproportion of mobile phase and column temperature in order toestablish a better fingerprint chromatogram. (3) Systemsuitability test: In this chromatographic condition, calculate theresolution and theoretical plate of baicalin peak. (4) In theexperiment of stability, transfer the sample solution fromScutellaria baicalensis to determine the relevant retention timeand peak area at 0, 2, 4, 6, 8, 10, 12, 24, 36 and 48 hour,respectively. (5) In the experiment of precision,take the sametest solution,inject to the apparatus for six times,determine therelevant retention time and peak area, respectively. (6) In theexperiment of reproducibility, prepare one concentration sampleof Scutellaria baicalensis,repeat each concentration solution forsix times in the same way,determine the relevant retention timeand peak area, respectively. (7) prepare different batchesconcentration sample of Scutellaria baicalensis and its caudexesand leaves,inject to HPLC, and get their relevant fingerprintchromatograms. 2. Assay: (1) Preparation of standard curve:prepare a series of the reference solutions of baicalin andwogonin,determine peak areas,then the regression equationwas respectively obtained with the content of baicalin andwogonin as abscissa and the relevant peak area as ordinate. (2)In the experiment of stability,transfer the sample solution fromScutellaria baicalensis to determine the relevant peak area at 0,2, 4, 6, 8, 10, 12, 24, 36 and 48 hour, respectively. (3) In theexperiment of precision,take the same test solution,inject to theapparatus for six times,determine the relevant peak area ofbaicalin and wogonin respectively, then calculate the precision.(4) In the experiment of reproducibility, prepare oneconcentration sample of Scutellaria baicalensis,repeat eachconcentration solution for six times in the same way, determinethe relevant peak area respectively, then calculate the contentand RSD. (5) In the experiment of recovery, transfer 6 shares ofScutellaria baicalensis and add the reference solutions ofbaicalin and wogonin respectively,then detect the content ofbaicalin and wogonin each. (6) Determination of the lowestdetection limitation:Dilute the reference solution of baicalin andwogonin respectively until the value of S/N was more than orequal to 3. The relevant concentration was the lowest detectionlimitation. (7) Assay:Under above-mentioned conditions,determine the content of baicalin and wogonin in differentbatches of Scutellaria baicalensis respectively.Results:1. Establishment of fingerprint (1) Extraction: Themethod of supersonic wave-extraction with methanol for 45mins was simple, quick and stable. (2) The HPLC system wasperformed on a C18 analytical column gradient eluted with amixture consisting of acetonitrile, 0.25%H3PO4 and THF at aflow rate of 1ml/min. The temperature of column was 30?C. TheUV detection wavelength was set at 274 nm. Injection volumewas 10μl. (3) System suitability test: Under the above condition,the peak corresponding to baicalin of the test solution wasseparated well with the resolution of more than 1.0 and about100000 of theoretical plate. (4) The test solution was stable in48 hours. Same baicalin peak as reference peak, the relevantRSD values of relative retention time and relative area werebetween 0.03% ～0.95% and between 0.16% ～1.67%respectively. (5) The precision of sample was good and the RSDvalues of relative retention time and relative area were between0.02%～1.03% and between 0.13%～0.66%, respectively. (6)the reproducibility of sample was good and the RSD values ofrelative retention time and relative area were between 0.02%～0.85% and between 0.49%～2.65%, respectively. (7) Get therelevant fingerprint chromatograms of different batches ofScutellaria baicalensis and its caudexes and leaves. (8) Dataanalysis: Use similarity and Hierarchical clustering analysis toanalyze data, and the results showed that 26 samples wereclassified as three clusters with Pearson correlation. The firstcluster quality of Scutellaria baicalensis was best, including 10batches SBG of Rehe, whose similarity was between 0.99～1.00;The second cluster quality was common, including some SBGfrom different places, whose similarity was between 0.90～0.99;The last cluster their quality was worst, whose similarity wasbetween 0.80～0.90. 2. Assay: (1) Baicalin and wogonin of theregression equation were:Y =0.0984+36.335X,r=0.9999(n=6) and Y=72.58X-0.2366,r=0.9999(n=6), respectively. Theirlinear range were between 1.20 ～168.00μg and between0.218～8.720μg, respectively. (2) The test solution was stable in48 hours and the RSD values of baicalin and wogonin were0.20% and 0.94%, respectively. (3) The precision of sample wasgood and the RSD values of baicalin and wogonin were 0.07%and 0.90%, respectively. (4) In the experiment of reproducibility,the RSD values of baicalin and wogonin were 0.92% and 1.72%,respectively. (5) The average recoveries of baicalin andwogonin were 99.27% and 99.03%, and their RSD values were0.72% and 0.80%, respectively. (6) The lowest detectionlimitations of baicalin and wogonin were 0.122ng and 0.218ng,respectively. (7) The results showed the contents of baicalin andwogonin in SBG of different sources were between 4.99%～18.40% and between 0.05%～0.76%, respectively. However,the content of baicalin in its caudexes and leaves only was0.19%, and there was little wogonin.Conclusion:It is the first time to establish the RP-HPLCfingerprint chromatogram of Scutellaria baicalensis of Rehe ofChengde City of Hebei Province, get reference fingerprintchromatogram, compare the fingerprints of 26 samples fromvarious sources, and study their difference with similarity andHierarchical clustering analysis. At the same time, the contentsof baicalin and wogonin were determined in different batches ofScutellaria baicalensis respectively, and the quality ofScutellaria baicalensis was evaluated generally. This method issensitive,quick, and useful to actualize standardization planting.It can be used for the quality control for Scutellaria baicalensis.Radix Bupleuri is the dry root of Bupleurum chinense DC.,and B. scorzonerifolium Willd., which belongs to the family ofUmbelliferae. It mainly contains naphtha and saikosaponincomponents, which are the bioactive component. It has manyeffects, such as reducing fever by reconciliation, relieving thedepressed liver, soothing the liver and regulating the circulationof qi, invigorating vital function and elevating spleen yang. Butso far, there has been not report that related to the method todetermine the fingerprints of saikosaponin in Bupleurumchinense. In this paper, saikosaponin in Bupleurum chinensewas mainly analyzed with HPLC-ELSD, the HPLC-ELSDfingerprints of Bupleurum chinense from Hebei Province wasset up at the first time, and the fingerprints of Bupleurumchinense collected from different producing areas werecompared so as to establish a sensitive and specific method forcontrolling the quality of Bupleurum chinense.Objective : Establish the HPLC-ELSD fingerprints ofBupleurum chinense from Hebei Province; get the controlchromatogram and the fingerprints of Bupleurum chinensecollected from different producing areas were compared so as toprovide a new method as a comprehesive quality control itemfor Bupleurum chinense.Methods:(1) Extraction: An optimal extracting conditionwas chose by comparing the experimental results, which canextract more components and more quantities. (2)Chromatographic condition: Choose appropriate column andditector, different formulation and proportion of mobile phaseand adjust optimal flow rate and column temperature in order toestablish a better fingerprint chromatogram. (3) Systemsuitability test: In this chromatographic condition, calculate theresolution and theoretical plate of saikosaponin a peak. (4) Inthe experiment of stability, transfer the sample solution fromBupleurum chinense to determine the relevant retention timeand peak area at 0, 2, 4, 6, 8, 10, 12, 24, 36 and 48 hour,respectively. (5) In the experiment of precision,take the sametest solution,inject to the apparatus for six times,determine therelevant retention time and peak area, respectively. (6) In theexperiment of reproducibility, prepare one concentration sampleof Bupleurum chinense,repeat each concentration solution forsix times in the same way,determine the relevant retention timeand peak area, respectively. (7) prepare different batchsconcentration samples of Bupleurum chinense and its caudexesand leaves,inject to HPLC, and get their relevant fingerprintchromatograms.Results : (1) Extraction: The method of supersonicwave-extraction with 5% NH3·H2O methanol for 60 minuteswas simple, quick and stable. (2) The HPLC separation wasperformed on a C18 analytical column gradient eluted with amixture consisting of acetonitrile and water at the flow rate of1ml/min with ELSD detector. The temperature of column was30?C. Injection volume was 20μl. (3) System suitability test:Under the above condition, the peak of saikosaponin a of thetest solution was separated well with the resolution of more than1.5 and about 40000 of theoretical plate. Its relation time wasabout 26 minutes. (4) The test solution was stable in 48 hours.Same the peak of saikosaponin a as reference peak, the relevantRSD values of relative retention time and relative area werebetween 0.13% ～0.86% and between 2.44% ～2.96%respectively. (5) The precision of sample was good and the RSDvalues of relative retention time and relative area were between0.07%～0.46% and between 2.72%～2.94% respectively. (6)the reproducibility of sample was good and the RSD values ofrelative retention time and relative area were between 0.08%～0.50% and between 2.29%～2.93% respectively. (7) Get therelevant fingerprint chromatograms of different batches ofBupleurum chinense and its caudexes and leaves. (8) Dataanalysis: Hierarchical clustering analysis was performed forclassification of samples with relative peak area as the chemicalvariable. The similarity of whole HPLC chromatogram wasanalyzed with computer Program aided, and results showed that21 samples were classified as three clusters with Pearson...
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