| ObjectiveAccording to the results of the preliminary study by our group, total flavonoids are the main active components of Microcos paniculata L.. On this basis, the study proposed a sophisticated method for the fingerprint development of the total flavonoids in Microcos paniculata L. using HPLC, which covered samples collected from different origins and batches. In order to evaluate the anti-hyperlipidemia effect of this herb, combination should be carried out between fingerprint of the total flavonoids and results of the pharmacodynamics experiment. Then, with the help of grey relational grade analysis, peaks in the fingerprint with significant influence on hyperlipidemia were chosen as biomarker of Microcos paniculata L.. By the above mentioned integration, model of the chromatogram-effect relationship on the anti-hyperlipidemia effects of the total flavonoids in Microcos paniculata L. can be constructed, acting as key to further understanding of connections between therapeutic effects and characteristic of the compounds such as structure and amount in TCM. In general, this thesis mainly discussed methodology development that more accurately and comprehensively reflect effects of TCM, providing a more persuasive quality control method than simply determinating all compounds. Methods1、Microcos paniculata L. samples collection:collect the Microcos paniculata L. samples of different habitat and batches. Followed analysis and quality control instructed by Chinese Pharmacopoeia (2010). The qualified samples were stored for further study.2、Isolation and preparation of the total flavonoids in Microcos paniculata L.:total flavonoids in Microcos paniculata L. was extracted by using the extraction methods which is confirmed in the preliminary study by our group and quatitatively determined by UV.3、Establishing fingerprint of the total flavonoids by HPLC:Use Vitexin, Isovitexin and Narcissin as reference for contruction of total flavonoids fingerprint from different resourse by HPLC. Also, detection wavelength, mobile phase and gradient condition, chromatographic column, column tempreture and flow rate was optimized separately to obtain best chromatogram. For comparison, according to the requirements of the chemical fingerprint, the similarity evaluation system software was used to investigate the HPLC fingerprint of the total flavonoids in Microcos paniculata L.. The characteristic peak area was analised by computer with the method of cluster analysis.4、Dectection of anti-hyperlipidemia activity of the total flavonoids in Microcos paniculata L. from different habitat and batches:the anti-hyperlipidemia model made with NIH mice were established by administration of high fat emulsion for7days. Follow by detection of anti-hyperlipidemia activity of the total flavonoids in25samples by taking TC and TG in the serum as the indexes. Record the ultraviolet absorption value of TC and TG at the maximum absorption wavelength and calculate the concentration of TC and TG. Then calculate the reduction rate of TC and TG.5、The study of the chromatogram-effect relationship in term of anti-hyperlipidemia effects of the total flavonoids in Microcos paniculata L.:(1) The grey relational grade analysis on the anti-hyperlipidemia effect of the total flavonoids in Microcos paniculata L.:data of characteristic relative peak area was first transformed by the method of equalization, then combine with the reduction rate of TC and TG. Pretreated data was consecutively processed by grey relational grade analysis, and was calculated to define its contribution value in term of anti-hyperlipidemia effects.(2) Construction of equation representing chromatogram-effect relationship on the anti-hyperlipidemia effects:The same data processed and evaluate in item (1) was chosen in this item, for constructing the equation of chromatogram-effect relationship on the anti-hyperlipidemia effects. By using backward method of multiple linear regression in SPSS17.0. Then, verification of the equation with the data of the last5samples is carried out.(3) Construct the model of chromatogram-effect relationship:This part was fulfilled by using the method of multiple linear regressions in CAMO The Unscrambler V9.7. After Verified by the method of full cross validation, the model was proven to exactly and precisely reflect the effects. Results1、25samples of Microcos paniculata L. of different habitat and batches was collected in the market, and evaluated using the methods in Chinese Pharmacopoeia (2010), with a result of all qualified and can be used in the next step.2、25samples of the total flavonoids of different batches were obtained by using the extraction methods which is confirmed in the preliminary study by our group. The concentration of the total flavonoids of25samples was all above80%.3、The HPLC fingerprint of the total flavonoids in Microcos paniculata L. is constructed and used for determinations of25samples. The characteristic peak in the HPLC fingerprint of the total flavonoids in Microcos paniculata L. showed good separation, characteristics and uniqueness. As results indicated,25samples can be divided into two types. Analyzing any of them into the chromatographic fingerprint similarity evaluation system software, we can get17common peaks including Vitexin, Isovitexin and Narcissi which is already known. Establish the chromatograpfic control fingerprint and calculate the similarity degree of25samples. We find that the similarity of18samples are above0.90,4samples are above0.80and3samples are lower than0.80.4、The25samples of the total flavonoids extracted in Microcos paniculata L. showed significant reduction in TC and TG level in the serum of anti-hyperlipidemia models. Among25samples from different batches, statistics showed significant difference in anti-hyperlipidemia effects.5、(1) The relational grade of the17characteristic peak and the reduction rate are all above0.85, among them there are8characteristic peaks which are highly correlated with the effect of reducing TC, and10characteristic peaks highly correlated with the effect of reducing TG (relational grade>0.90). The Vitexin, Isovitexin and Narcissi which is known are in the first, second and fifth place. According to the relational grade, the sequence of contribution to reducing blood lipid is9#(Vitexin)>10#(Isovitexin)>11#>13#>16#(Narcissi)>14#>15#>1#>7#>6#>4#>5#>8#>2#>3#>17#>12#(P<0.01, R2=0.987). The anti-hyperlipidemia effects of the total flavonoids in Microcos paniculata L. are the result of combined effect of "effective compoment group".(2) In the17characteristic peaks of HPLC fingerprint, there are7of them have the effect of reducing TC by using the method of multiple linear regression. The chromatogram effect relationships is Y1=0.98+0.023X2-0.016X4-0.05Xs-0.134X9+0.12X10-0.042X14+0.108X17(P<0.01, R2=0.958). Among them, X2、X10、X17were positive correlation with TC, and X4、X8、X9、X14were negative correlation with TC. There are12of them have the effect of reducing TG by using the method of multiple linear regression. The chromatogram effect relationships is Y2=0.42-0.024X1-0.039X3-0.051X4+0.109X6+0.058X7-0.238X9+0.167X10+0.05X11-0.075X12-0.089X14-0.061X16+0.058X17,(P<0.01, R2=0.987). Among them, X6、X7、X10、X11、X17were positive correlation with TG, and X1、X3、 X4、X9、X12、X14were negative correlation with TG. In addition, X9, X10, X16are Vitexin, Isovitexin and Narcissi. Transform the last5data of characteristic peak area with the method of equalization, and enter them into the equations, we can get the predictive value of the reduction rate of TC and TG. All the deviation of predictive value and true value are lower than10%. The result indicates that the equations are accurate and liable which can be used to predict the anti-hyperlipidemia effects of the total flavonoids extracted in Microcos paniculata L..(3) By combination of equalization transformed data of characteristic peak area and the reduction rate of TC and TG, then treated with multiple linear regression, construction of chromatogram-effect model on the anti-hyperlipidemia effects was completed. The predictive value was verified by full cross validation, and caculated for the deciation rate:the average error are lower than10%, the average recovery are in the range of95%～105%. The result indicates that the model is accurate and liable which can be used to predict the anti-hyperlipidemia effects of the total flavonoids extracted in Microcos paniculata L.. ConclusionThe study constructed a HPLC fingerprint for the total flavonoids in Microcos paniculata L., confirming the contribution value to the anti-hyperlipidemia effects of the characteristic peaks. The anti-hyperlipidemia effects of the total flavonoids in Microcos paniculata L. are the result of combined effect of "effective compoment group". Therefore, evaluation of Microcos paniculata L. could not be done by single marker constituent. At the same time, the equation and model of chromatogram-effect relationship on the anti-hyperlipidemia effects of the total flavonoids in Microcospaniculata L. has been constructed. They are accurate and liable which can be used to predict the anti-hyperlipidemia effects of the total flavonoids extracted in Microcos paniculata L.. It provides a new thought and method to establish HPLC fingerprint of TCM’s chromatogram-effect relationship, TCM quality control and develop evaluation criteria of pharmacodynamics, and also can be regraded as demonstrative study of anti-hyperlipidemia effects. |
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