HPLC Fingerprint Analysis of Benchmark Sample of Yanghetang / 中国实验方剂学杂志

ObjectiveTo establish a high performance liquid chromatography（HPLC） fingerprint of Yanghetang benchmark sample， and evaluate its quality with chemometric methods， so as to provide a reference for the quality control of this benchmark sample. MethodHPLC was used to establish the fingerprint of Yanghetang benchmark sample with ZORBAX SB-C18 column（4.6 mm×250 mm， 5 μm）， the mobile phase was consisted of acetonitrile（A） -0.05% phosphoric acid aqueous solution （containing 0.05% triethylamine solution）（B） for gradient elution（0-5 min， 2%-3%A； 5-15 min， 3%-5%A； 15-65 min， 5%-30%A； 65-90 min， 30%-70%A）， the flow rate was 1.0 mL·min-1， the column temperature was 35 ℃， and the detection wavelength was 210， 260 nm. Traditional Chinese Medicine（TCM） Chromatographic Fingerprint Similarity Evaluation System （2012 edition） combined with cluster analysis， principal component analysis（PCA） and partial least squares-discriminant analysis（PLS-DA） were used to evaluate the quality differences between different batches of Yanghetang benchmark samples， and to find the main chemical components responsible for the quality differences. ResultHPLC fingerprint of Yanghetang benchmark sample was established， 13 common peaks were identified and attributed to each common peak， including peaks 2 and 8 from Rehmanniae Radix Praeparata， peaks 10 and 11 from Cinnamomi Cortex， peaks 1， 3-6 from fried Sinapis Semen， peak 13 from Ephedrae Herba， and peaks 7， 9， 12 from Glycyrrhizae Radix et Rhizoma. Eight of them were identified by comparing with control substance， which were 5-hydroxymethylfurfural（peak 2）， sinapine thiocyanate（peak 4）， glycyrrhizin（peak 7）， verbascoside（peak 8）， cinnamic acid（peak 10）， cinnamaldehyde（peak 11）， glycyrrhizic acid（peak 12） and ephedrine hydrochloride（peak 13）. The similarities of the HPLC fingerprints of 15 batches of Yanghetang benchmark samples with the control fingerprint were all greater than 0.80. The three chemometric methods could classify the samples into two categories. Eight differential components were screened out， among which 5-hydroxymethylfurfural， sinapine thiocyanate， verbascoside and ephedrine hydrochloride were identified. ConclusionThe established fingerprint analysis method is accurate， stable and reproducible， which basically reflects the overall chemical composition of Yanghetang benchmark sample， and can provide a basis for establishment of quality standards for compound preparations of this famous classical formula.

Investigation of Intestinal Absorption Characteristics of Multi-index Components of Danggui Buxuetang Based on Drug Absorption Simulating System / 中国实验方剂学杂志

ObjectiveTo investigate the intestinal absorption characteristics of multi-index components in Danggui Buxuetang with drug absorption simulating system （DASS） established by everted intestinal sac model. MethodThe intestinal absorption solution at different time points after administration of Danggui Buxuetang was collected and detected by high performance liquid chromatography （HPLC）， acetonitrile （A）-0.2% glacial acetic acid solution （B） was used as the mobile phase for gradient elution （0-16 min， 15%-23%A； 16-20 min， 23%-28%A； 20-25 min， 28%-30%A； 25-30 min， 30%A； 30-35 min， 30%-65%A； 35-45 min， 65%-95%A）， the detection wavelength was 302 nm. HPLC fingerprint of intestinal absorption solution was established and the common peak was calibrated， and the relative cumulative absorption rate of each index component was calculated. The relative cumulative absorption curves of components were fitted with various mathematical models by DDSolver 1.0 to explore the absorption law of different components. ResultThe absorption process of C2 （calycosin-7-glucoside） and C6 in Danggui Buxuetang was in line with zero-order equation， C9 was best fitted by Weibull equation， and the remaining 7 components were in line with Makoid-Banakar equation. C1 with C2， C3， C5， C7 and C10， C2 with C5 and C7， C3 with C4， C5， C7 and C10， C4 with C6 and C10， C5 with C7， C6 with C10， C7 with C10， C8 with C9 were absorbed simultaneously during the absorption process. With the prolongation of time， the overall cumulative absorption rate of Danggui Buxuetang increased. At 120 min， the overall cumulative absorption rate of Danggui Buxuetang exceeded 38%， and reached 49.14% at 180 min. ConclusionTen ingredients in Danggui Buxuetang are absorbed in the jejunum， but absorption law of various components is different， which shows that the intestinal absorption of compound preparations of traditional Chinese medicine （TCM） has multiple characteristics. Intestinal absorption study of TCM compound preparations with chemical composition as the index can reveal some of its absorption law， but it is not complete.

Quality Evaluation of Aurantii Fructus Based on Fingerprint Qualitative Analysis， Multi-component Quantitative Analysis and Chemometrics / 中国实验方剂学杂志

ObjectiveTo establish an integrated method of fingerprint qualitative， multi-component quantitative analysis and chemometrics， and to evaluate the quality attributes and differences of Aurantii Fructus from different production areas and origins. MethodAnalysis was performed on COSMOSIL 5C18-MS-Ⅱ column （4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile-0.2% phosphoric acid solution for gradient elution （0-4 min， 19%A； 4-5 min， 19%-21%A； 5-18 min， 21%A； 18-19 min， 21%-28%A； 19-27 min， 28%A； 27-28 min， 28%-40%A； 28-36 min， 40%A； 36-37 min， 40%-50%A； 37-42 min， 50%-60%A； 42-46 min， 60%-95%A； 46-55 min， 95%-100%A）， the flow rate was 1 mL·min-1， the column temperature was 30 ℃， the detection wavelength was set at 320 nm， and the injection volume was 10 μL. High performance liquid chromatography （HPLC） fingerprints of Aurantii Fructus from different production areas and origins were established. Then， the quality of 26 batches of samples was evaluated by cluster analysis （CA）， principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA）. A method for the determination of 12 components was developed and verified， and a thermal map-based CA of Aurantii Fructus from different production areas and origins was carried out based on the content difference of samples. ResultThe fingerprint and determination methods were well verified. The similarity of HPLC fingerprint of 12 batches of Aurantii Fructus was 0.85-0.996， 20 common peaks were calibrated and 14 of them were assigned. The resolution and linear relationship of 12 components in quantitative analysis were good. The recovery rates were 99.2%-101.0% with RSD≤2.0%. The results of CA， PCA and OPLS-DA indicated that the differentiation of Aurantii Fructus in different production areas was great， and there were differences among different cultivars. ConclusionThe qualitative analysis of fingerprint and quantitative analysis of multiple indexes based on the same chromatographic analysis conditions are convenient， accurate and reliable， and combined with chemometrics， the identification and quality analysis of Aurantii Fructus from different production areas and origins can be realized， which can provide reference for quality control and evaluation of Aurantii Fructus.

Analysis on Quality Transfer Law of Rhei Radix et Rhizoma Steamed with Rice-wine During Processing Based on Correlation Between External Appearance Color and Internal Component / 中国实验方剂学杂志

Objective:The correlation between the appearance color of cooked rhubarb samples and the components characterized by high performance liquid chromatography （HPLC） fingerprint was studied to reveal the quality transfer law in the steaming process of processed products with rice-wine. Method:The visual analyzer was used to analyze the change of the appearance color of cooked rhubarb sample powder at different processing time， the common components and their relative peak areas of processed products with rice-wine were identified by HPLC fingerprint， as well as multivariate statistics and Pearson correlation analysis were used to cluster， discriminate and analyze the appearance color and the component variables in HPLC fingerprint. Result:During the processing of cooked rhubarb， the <italic>a</italic>^* （red-green value） of sample powder had no obvious change， but the <italic>L</italic>^* （lightness value）， <italic>b</italic>^*<italic> </italic>（yellow-blue value） and <italic>E</italic>^*<italic>ab </italic>（total chromaticity value） showed a decreasing trend， and the appearance color changed from bright to dark， from yellow to brown. A total of 46 chromatographic peaks in the fingerprint were identified at 254 nm and 280 nm， and 18 of them were identified by comparison with reference standards. The change trend of <italic>L</italic>^*，<italic> b</italic>^*<italic> </italic>and <italic>E</italic>^*<italic>ab </italic>were positively correlated with the contents of tannins （catechin， epicatechin and ethyl gallate）， stilbene glycoside （<italic>trans</italic>-3，5，4′-trihydroxystyryl-4′-<italic>O</italic>-<italic>β</italic>-<italic>D</italic>-glucoside）， phenylbutanone glycoside of 4′-hydroxyphenyl-2-butanone-4′-<italic>O</italic>-<italic>β</italic>-<italic>D</italic>-［2ʺ-<italic>O</italic>-gallic-6ʺ-<italic>O</italic>-（4ʺ-hydroxy）-cinnamoyl）-glucoside， conjugated anthraquinones （aloe emodin-8-<italic>O</italic>-glucoside， rhein-8-<italic>O</italic>-glucoside， emodin-8-<italic>O</italic>-glucoside） and <italic>ω</italic>-hydroxyemodin （<italic>P</italic><0.05， <italic>P</italic><0.01）， and negatively correlated with the contents of free anthraquinones （emodin， aloe emodin and physcion）. Compared with 254 nm， the similarities of chromatographic peaks at 280 nm was more obvious， and the number of detected common peaks was more， which could reflect more subtle differences in chemical composition. Conclusion:Tannins， stilbene glycosides and phenylbutanone glycosides are strongly correlated with <italic>L</italic>^*， while anthraquinones are strongly correlated with <italic>b</italic>^*， the decrease of <italic>E</italic>^*<italic>ab</italic> is mainly related to the increase of free anthraquinone content and the decrease of catechins， <italic>ω</italic>-hydroxyemodin， stilbene glycosides， conjugated anthraquinone and phenylbutanone glycosides. The change of appearance color index of process samples can reflect the change trend of the contents of medicinal components in cooked rhubarb to some extent.

Quality Evaluation of Citri Sarcodactylis Fructus from Different Origins Based on HPLC Fingerprint and Chemometrics / 中国实验方剂学杂志

Objective:To establish the high performance liquid chromatography （HPLC） fingerprint of Citri Sarcodactylis Fructus， and to search for makers to characterize the quality difference of Citri Sarcodactylis Fructus from different origins coupled with chemometrics. Method:The analysis was performed on a Thermo Hypersil GOLD C₁₈ column （4.6 mm×250 mm， 5 μm） with mobile phase consisted of acetonitrile-0.05% phosphoric acid solution for gradient elution， and the detection wavelength was set at 254 nm. A total of 31 batches of samples were analyzed to establish the HPLC fingerprint of Citri Sarcodactylis Fructus. Similarity evaluation was performed by Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System （2012 edition） to confirm the common peaks， which were identified by comparison of reference substances. On the basis， chemometrics methods were used to analyze and evaluate the quality of Citri Sarcodactylis Fructus from different origins. At the same time， 3 batches of 5 species of decoction pieces from the genus <italic>Citrus</italic> in the family Rutaceae， including Citri Sarcodactylis Fructus， Aurantii Fructus Immaturus， Aurantii Fructus， Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium， were randomly collected for evaluating the effectiveness and reliability of the established HPLC fingerprint of Citri Sarcodactylis Fructus. Result:HPLC fingerprint of Citri Sarcodactylis Fructus was established and 22 common peaks were identified. And seven common peaks among them were identified as 6，7-dimethoxycoumarin， diosmin， hesperidin， byakangelicin， 5，7-dimethoxycoumarin， bergapten and oxypeucedanin. Except for 2 batches of samples， the similarities of fingerprints between other 29 batches of samples were >0.9. The 31 batches of Citri Sarcodactylis Fructus were basically divided into 3 groups by cluster analysis and principal component analysis， which were consistent with the classification of three different producing areas. Eight differential markers were screened by orthogonal partial least squares discriminant analysis and four of them （5，7-dimethoxycoumarin， bergapten， 6，7-dimethoxycoumarin and diosmin） were identified by reference substances. Similarity evaluation of 5 species of decoction pieces from genus <italic>Citrus</italic> in the family Rutaceae was carried out by taking the reference fingerprint of Citri Sarcodactylis Fructus as treference chromatogram， similarity of Citri Sarcodactylis Fructus decoction pieces was 0.892-0.977， and the similarities of the other 4 kinds of decoction pieces were 0.215-0.517. Conclusion:The established fingerprint method is reasonable， effective and accurate for quality control of Citri Sarcodactylis Fructus， the characterization information is more comprehensive combined with chemometrics.

Chemical pattern recognition of YuanZhi San capsule based on FTIR and HPLC analysis / 国际药学研究杂志

Objective: To provide a scientific basis for the quality control of YuanZhi San capsule(YZSC). Methods: Fourier transform infrared spectroscopy(FTIR)combined with second derivative spectra was used to analyze the chemical components of YZSC;high performance liquid chromatography(HPLC)was used to establish the fingerprints of different batches of YZSC, the quality assessment of YZSC was carried out by similarity evaluation, cluster analysis(CA), principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA). Results: The main components of YZSC were carbohydrate, flavonoids, alkaloids, organic acids and saponins. There were 41 common peaks in the fingerprint, seven peaks were identified using reference substance, they were 3, 6'-disinapoylsucrose, ferulic acid, jatrorrhizine hydrochloride, palmatine chloride, berberine, quercetin and β-asarone. The similarity of 10 batches of samples was higher than 0.99. The CA and PCA analysis indicated that there were differences in different batches of YZSC, and they were mainly divided into two categories. By the OPLS-DA, 10 main chemical constituents were found to be the cause of the quality differences in the batches, and the peaks of berberine, 3, 6'-disinapoylsucrose, palmatine chloride, β-asarone, and jatrorrhizine hydrochloride were recognized by comparison with the reference substances. Conclusion: The chemical pattern recognition technology based on the FTIR and HPLC analysis might be used for the quality evaluation of YZSC.