| Scutellaria indica, known as Han Xincao, Da Licao and Tie Dengzhan in Chinese, is the dry whole grass of Scutellaria indica Linn., possessing the effect of clearing away heat and toxic material, promote blood circulation and bleeding, and detumescence. Flavonoids, phenolic compounds, amino acid and organic acid have been test or prepared form Scutellaria indica, and the flavonoids were proven to be the bioactive components with the pharmacological effects of antineoplastic, anti-flammatory, antiviral and Arginase II inhibitory, which had made Scutellaria indica more and more attractive to people. However, there are only a few articles reported corresponding to the quality evaluation of Scutellaria indica. And the papers just reported on the physical and chemical identification and determination of single or multiple composition, which can not control the quality of Scutellaria indica comprehensively. Worse still, as a result of the fact that Scutellaria indica was used as an adulterant of Scutellaria barbata for their similiar botanical morphology. Therefore, it is significant to develop a reliable and simple method for improving the quality control level of Scutellaria indica.The chemical fingerprint, a widely acceptable method for the quality control of TCM, had been successfully utilized to the identification of TCM, efficacy evaluation of TCM and quality control of TCM during all process of the production for its characteristics of integrity and metrization. However, most of the common peak in the established fingerprint can not be identified for the lack of reference substances, which had become a gordian kont impeding the comprehensive application of fingerprint. With the development of measuring technology, the technology of liquid chromatography tandem mass spectrometry combining the high separating effiicacy of liquid chromatography with the high sensitivity and high selective of mass spectrometer can successfully solved the problem of time-consuming and strenuosity of obtaining the reference substances. Therefore, in this study a high performance liquid chromatography method was developed to acquire the fingerprint common model of Scutellaria Indica, which was obtained by hierarchical cluster analysis and principal component analysis after the similarity analysis of 16 samples; And then the quantitative analysis of multi-component with single-marker(QAMS) method of 4 flavonoids in Scutellaria indica were established to improve its quality control level from the quantitative view; Finally, the flavonoids in Scutellaria indica were identified by LC-MS/MS to improve the quality control level of Scutellaria from the qualitative view. Part one Study on the HPLC fingerprint of Scutellaria indicaObjective:To establish the the fingerprint of Scutellaria indica by HPLC, and then develop the HPLC fingerprint common model by hierarchical cluster analysis and principal component analysis after the similarity analysis of 16 samples, which could improve the quality control level of Scutellaria indica from the semi-quantitative and semi-qualitative view.Methods:1. The HPLC analysis was performed on an Agilent 1200 series high performance liquid chromatography system. The separation was conducted on an Agilent Eclipse XDB C18 column(4.6 mm× 250 mm, 5 μm). The separation was proceeded at 30 ℃with a flow rate of 1.0 m L·min-1. 0.6% aqueous solution of acetic acid(A), acetonitrile(B) and tetrahydrofuran(C) were used as the mobile phase in gradient elution mode: 0-15-30-38-44-48-52-55-60-65 min, A(86%-79%-71%-65%-63%-58%-56%-48%-43%-43%), B(10%-20%-27%-32%-35%-38%-40%-48%-53%-53%), C(4%-1%-2%-3%-2%-4%-4%-4%-4%-4%). The detection wavelength for fingerprint were 365 nm from 0 to 40 minute and 275 nm from 40 to 65 minute.2. Evaluate the similarity of 16 samples and mark 28 common peaks with the software of Traditional Chinese medicine similarity evaluation system. After then, hierarchical cluster analysis and principal component analysis were used to establish the HPLC fingerprint common model with the peak area of 28 common peaks as variables.Results:1. The HPLC fingerprint of 16 samples were established, and 28 common peaks were marked. The similarities between the samples and the reference chromatogram were 0.996, 0.881, 0.996, 0.994, 0.997, 0.996, 0.998, 0.896, 0.997, 0.998, 0.849, 0.911, 0.895, 0. 993, 0.997 and 0.894.2. 16 samples were separated into 4 group by hierarchical cluster analysis. And S008, S011, S013 and S016 were gathered as the first group; S001, S003, S004, S005, S006, S007, S009, S010, S014 and S015 were gathered for the second group; S002 was gathered as the third group; S012 was gathered as the fourth group. 16 samples were separated into 3 group by principal component analysis. And S008, S011, S013 and S016 were gathered as the first group; S001, S003, S004, S005, S006, S007, S009, S010, S014 and S015 were gathered as the second group; S002 and S012 were gathered as the third group.3. S001, S003, S004, S005, S006, S007, S009, S010, S014 and S015 were selected to establish the fingerprint common model, in which 28 common peaks were marked. The similarities between samples and the reference chromatogram were 0.999, 0.999, 0.998, 0.996, 0.998, 0.999, 0.999, 0.999, 0.998, 0.997.4. There were significant difference between the components and their contents from Scutellaria indica and Scutellaria barbata under the same chromatographic condition.Conclusion:The established HPLC fingerprint common model was accurate and reliable enough to control the quality of Scutellaria indica from the semi-quantitative and semi-qualitative view. Part two Establishment of the QAMS method of 4 components in Scutellaria indicaObjective:To establish the quantitative analysis of multi-component with a single-marker method for the simultaneous determination of Scutellarin, Wogonoside, Apigenin and Wogonin, so as to improve the quality control level of Scutellaria indica.Methods:1. Establish the external standard method(ESM) by precision test, repeatability test and stability test.2. Determine the relative correction factor from Apigenin to the other analytes by determining the mixed reference substance solution with different injection volumes. And then investigated the repeatability of the RCFs by repeatedly measuring the new prepared mixed reference substance solutions.3. Investigate the effect of different factors such instruments, columns, column temperatures, flow rates and detection wavelengths on the RCFs.4. Evaluate the applicability and reliability of the established method by analyzing the relative error of the content results gained by QAMS and ESM, and the chromatograhic peak positioning results.Results:1. The RCFs of Apigenin to Scutellarin, Wogonoside and Wogonin were 2.142, 3.684 and 0.374 obtained by the mean value method, and the RCFs of Apigenin to Scutellarin, Wogonoside and Wogonin were 2.303, 3.717 and 0.380 obtained by the regression equation method. The RSDs of the RCFs were 3.12%, 3.64% and 2.87% in the repeatability test, which mean that the RCFs were repeatable.2. The RSDs of the RCFs obtained with different instruments, different columns, different column temperatures, different flow rates and different detection wavelength were all no bigger than 5%, which mean that the RCFs were with good applicability.3. There was only one sample, in which the relative error of the content of Scutellarin is bigger than 5%. And there was only one sample, in which the relative error of the content of Wogonoside is bigger than 5%.4. The RSDs of the relative retention time of Scutellarin, Wogonoside and Wogonin were 5.33%, 1.69% and 2.17%, and the RSDs of the difference value of retention time of Scutellarin, Wogonoside and Wogonin were-1.28%,-6.49% and 6.74%.Conclusion:There was no significant difference between the established QAMS method and the traditional ESM. And the established QAMS method can be used to the quality control of Scutellaria indica. Part three Study on the chemical components in Scutellaria indica based on LC-MSObjective:Identify the components in Scutellaria indica by LC-MS, so as to improve its quality control level from the qualitative view.Methods:1. The UPLC analysis was performed on an Waters Acquity UPLC system and a Shimadzu UFLC system. The separation was conducted on an Agilent Poroshell 120 SB-C18 column(2.1 mm×100 mm, 2.7 μm). The separation was proceeded at 35 ℃with a flow rate of 0.4 m L·min-1. 0.1% aqueous solution of formic acid(A) and acetonitrile(B) were used as the mobile phase in gradient elution mode: 0-2.5-6-11-15-15.5-22-26-30 min,A(88%-88%-82%-77%-70%-67%-67%-40%-40%), B(12%-12%-18%-23%-30%-33%-33%-60%-60%). The detection wavelength was 275 nm and the injection volume was 2 μL. The condition of the MS spectrometer were as follows: ion source gas 1 55 psi, ion source gas 2 55 psi, curtain gas 35 psi, temperature 500℃, ISVF 5500 V(in positive modal)/ 4500 V(in negative modal).Results:1. The UPLC fingerprint of 10 samples was established, and 32 common peaks were marked.2. 21 flavonosids were identified by Triple TOF 5600 MS spectrometer with the assist of references and standards.Conclusion:The UPLC fingerprint of Scutellaria indica was established, and the chemical components were identified by a hybrid triple quadruple time-of-flight mass spectrometer, which could improve the quality control level of Scutellaria indica from the qualitative view. |
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