Quality Assessment Of Yuanhu Zhitong Tablet Based On The Component Analysis And Vasoactivity

Yuanhu Zhitong tablet (YZT), a classical herb medication, is consisted of Radix Angelicae dahuricae (baizhi) and Rhizoma Coiydalis (yanhusuo) processed by vinegar. YZT is widely used due to its excellent effect on therapying gastralgia, costalgia, headache and dysmenorrheal by promoting qi and blood circulation. Meanwhile, YZT has been listed in China pharmacopoeia since1985. Chemically, alkaloids in Rhizoma Corydalis and coumarins in Radix Angelicae Dahuricae are considered to be the critical components to play their strong pharmacological roles.At present, only a few bioactive components are chosen to assess the quality of YZT so that the quality assessment could not effectively complete and comprehensively reflect its quality. Therefore, in our work, the fingerprint and multi-component analysis technique were employed to characterize and quantify the constituents of YZT. Under exactly investigating the chemical information, the vasoactivity was evaluated by utilizing the combining method of the intestinal absorption test accompanying with the vasorelaxation assay. As a result, the quality of YZT was comprehensively evaluated from chemical constituents and vasoactivity aspect. Our study provided a scientific basis for developing a scientific and rational quality assessment system of YZT. The main results of our work were as follows:1. The fingerprint analysis of YZT.In present study, the fast and sensitive UPLC/Q-TOF-MS method was develped to conduct the fingerprint analysis for15YZT samples. The consistency among the chromatograms of representative samples reflected the presence of similar chemical constituents. Seventeen common peaks were found among the15YZT samples, and15constituents of them were identified by the MS information and related literatures, namely protopine, a-allocryptopine, coptisine, tetrahydropalmatine, tetrahydroberberine, corydaline, palmatine, berberine, byakangelicol, dehydrocorydaline, oxypeucedanin, imperatorin, phelloptorin/cnidilin, isoimperatorin and osthole respectively. Thus, the overall chemical information in YZT was clearly characterized, which provided improtant and valuable information for quantitative analysis and quality evaluation. At the same time, hierarchical clustering analysis (HCA) was employed to classify the variability of samples and two species were obtained. It could be concluded that the samples from the same factory could be aggregated in the same.2. The multi-component analysis of YZT.The RRLC-QQQ method was proved to be a reliable and effective technique which could simultaneously identify the conformation and quantity of17active components in YZT within9minutes. The identified17active components included scopoletin, protopine, α-allocryptopine, tetrahydropalmatine, coptisine, tetrahydroberberine, corydaline, berberine, byakangelicin, byakangelicol, xanthotoxin, bergapten, pimpinellin, oxypeucedanin, imperatorin, osthole and isoimperatorin. Meanwhile, it was found that the content of17active components occurred obvious change among different batches. Protopine, tetrahydropalmatine, coptisine, corydaline, byakangelicin, imperatorin, and isoimperatorin were detected to have relatively higher content than other detected components. While the mass of scopoletin and osthole were both less than1.00μg/g. The mass of tetrahydropalmatine varied greatly in different batches, and its mass was largest among S15, S13and S12samples (S represent samples), but was lowest among S1-S6samples. The sum content of17constituents achieved the highest in S12, S13and S14samples, while S7-S11samples contained it relatively lower. HCA was performed based on the characteristics of the contents of17marker compounds to evaluate the YZT variation, whose result was quite similar with the result of fingerprint analysis. It was indicated that quantitative analysis also could excellently reflect the rough chemical characteristics. According to the mentioned above, the developed method was demonstrated to be simple, sensitive and reproducible to complete comprehensive evaluation for the quality control of YZT. 3. The research on a combinative method of intestinal absorption test and vasorelaxation assayThe combinative method of intestinal absorption test and vasorelaxation assay was established for assessing bioactivity. Firstly, the absorbed YZP solution was obtained by using the intestinal absorption method, and then the acculmlated solution were applied in thoracic aorta ring experiment for bioassay. The combinative method was found to have many superiors such as precision, repeatability and sensitivity. Meanwhile, the vasorelaxation of absorbed YZP solutions was investigated, and the result found that the relaxation rate could achieve81.53±3.98%,66.85±10.01%and43.64±17.83%at three concentrations (0.16,0.08and0.04g/mL), respectively, indicating that YZP possessed the powerful vasorelaxation on the endothelium-intact aorta precontracted by60mmol/L KCI solution. Besides, the blank intestinal solution was proved no vasorelaxation in the experiment. Therefore, the combinative method may be more reasonable than conventional method in pharmacological research in vitro.4. The analysis of absorbed chemical constituents.Based on the established RRLC-QQQ method, the chemical components of17constituents in YZP were detected. The results showed that17constituents all could be absorbed by gut sacs, and the content of absorption were largest in high concentration extract for all17constituents, while the lowest absorption occurred in low concentration extract, signifying that the absorption presented a concentration-dependent relationship. The extract solution of YZP was discovered to contain seven components with relatively higher content, which are corydaline, coptisine, imperatorin, protopine, isoimperatorin, a-allocryptopine and tetrahydroberberine. However, after the intestinal absorbing process, the components with relatively higher content changed to protopine, corydaline, a-allocryptopine, tetrahydropalmatine, coptisine, byakangelicin and imperatorin, suggesting that the ammount of these constituents in extract solutions differed from those in intestinal absorbed YZP solutions. Byakangelicol and byakangelicin had higher absorption rates of87%and85%than that of other constitutens. While the absorption rates of imperatorin, osthole and isoimperatorin were all less than0.5%, indicating that the absorption rate of each component vary significantly due to their varying absorption mechanism. From the above conclusion, it was reasonable to choose the intestinal absorbed YZP solutions for pharmacological research in vitro rather than using the extract solutions.5. The vasoactive assay of YZT based on the combination method.The vasorelaxations of9YZT samples were evaluated by the mentioned combination method. It was found that all the9samples could produce vasorelaxation, and the vasorelaxation became stronger with increasing the addition content. When the adding dose achieved1600μl, the vasorelaxation of all9YZT samples obtained the maximunm. Among the9YZT samples, the vasorelaxation rates of S12and S13reached to be80%, while the vasorelaxation rates of S7, S9and S11were less than10%. The bioactivity assessment perfectly solved the problem which the chemical assessment could not intuitively reflect the real quality, and provided and objective and rational result.The quality of YZT was comprehensively studied through the fingerprinting technique, multi-component analysis and vasoactive assessment. The fingerprinting analysis could roughly characterize chemical information and identified the authenticity of YZT, and evaluated the consistency and stability according to the chromatography similarities of samples. The multi-component analysis could provide valuable reference for the quality assessment through obtaining the amount information of main constituents. The bioactivity assessment could intuitively reflect the vasoactivity quality of YZT from comprehensively analyzing the vasorelaxation. The established technique could effectively evaluate the overall quality of YZT through identifying the consituents, detecting the amount and assessing vasorelaxation of YZT, and the multiple modes for quality evaluation provided an important and valuable reference for TCM research.