Study On The Model For Efficient Component Recognition Of Calculus Bovis Based On Target Constituents "knock-out&knock-in"

Traditional Chinese Medicines (TCMs) are the valuable asset accumulated through practice by the Chinese nation for thousands of years. And yet, efficient constituents of most TCMs are still not clear due to the their complexcities, especially, the limitation of study ideas and methods. Quality control has been one of the most important and difficult issues for the modernization, while efficient constituents screening supplies the prerequisite and foundation for quality control.Natural Calculus bovis (C. bovis), cattle gallstones, is a precious chinese herbal medicines With anticonvulsants, antioxidant, cholagogue, anti-inflammatory and other pharmacological effects, and used commonly in clinic with heat, phieglm, resuscitation, detoxifieation effect etc. However, it is expensive caused by the limited source. Scientists have developed the substitutes of natural C. bovis, for example, the artificial C. bovis. However, there are still some significant differences between natural C. bovis and those substitutes in chemical compositions and clinical efficacy.This study was based on existing "knock out&knock in" strategy and experiments. And we prelimilarily screened the key efficient constitutents of C. bovis on in-vitro model that was the hydrogen peroxide-induced damage in the human neuroblastoma (SH-SY5Y) cells. All the results are provided a basis for meeting with the requirements of researching the substitutes and quality control standards which are the effectiveness-related, quantitative and accurate, controllable and assessable. The main contents and results of research are summarized as follows:(1) The contents of bile acid (glycocholic acid (GCA), taurocholate sodium (TCANa), cholic acid (CA), ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA).) and bilirubin in C. bovis were determined by Ultra-performance liquid chromatography (UPLC) and analyzed. We found that there are large fluctuations in main chemical components among the contents of those samples. UPLC-MS was used for analysis of chemical constituents and the chemical fingerprints (FP) were established for C. bovi samples. Though the use of making evaluation by principal component analysis (PCA), we found the samples could not be well classified according to their species and sources. The quality control standard need further improvement and optimization.(2) In this paper, the in-vitro model which is the hydrogen peroxide-induced damage in the human neuroblastoma (SH-SY5Y) cells was established and optimized, using the Real-Time Cell Analyzers (RTCA)xCELLigence system and Cell Counting Kit-8(CCK-8). The optimized condition was:concentration of SH-SY5Y cells was4×105cells/mL, concentration of hydrogen peroxide was0.2mM, content of CCK-8was10%, incubation time was2h, determine wavelength was450nm. This model has the advantages of real-time online, trace amounts, efficient and so on. (3) The activity of C. bovis was evaluated. In this study, we investigated the effects of C. bovis on the hydrogen peroxide-induced damage in the SH-SY5Y cells. Natural C. bovis for cell protection ranges are0.03-0.5mg/mL, superior to Cultured C. bovis whose ranges are0.03-0.25mg/mL. However protection ranges of the Artificial C. bovis have large differences. This result might are related to the chemical constituents and content of C. bovis caused by subjective and artificial factors.(4) The"spectra-effect" relationships were established combined with chemical fingerprints, using Partial Least Squares analysis method, and preliminarily screening composition/portion, which are glycocholic acid, cholic acid, hyodeoxycholic acid and unknown components that may be glycodeoxycholic acid or its isomer.(5) Screening and identification of the activity constituents of C. bovis based on the proposed model and the strategy of target constituents "knock-out&knock-in". The activity of samples that were obtained by "knock-out" from the C. bovis, was determined, key pharmacodynamic constituents (KPCs) were elucidated. The results showed that bilirubin, bilirubin conjugate, glycocholic acid and cholic acid are the actived. While chenodeoxycholic acid and hyodeoxycholic acid have no effect, taurocholic acid sodium and deoxycholic acid caused damage on SH-SY5Y cells damaged by hydrogen peroxide-induced.In summary, the model in vitro which is the hydrogen peroxide-induced damage in the human neuroblastoma (SH-SY5Y) cells, based on the RTCA xCELLigence system, was established and optimized. The target constituents were preliminarily screened and identified based on the in vitro model using "spectra-effect" relationships as entry point. These results provided provide some useful study idea and technical support for efficient constituents recognition and effectiveness-related quality control for C. bovis and other TCMs.