Study On Quality Evaluation Method Of Traditional Chinese Medicine Injection Based On Cell Response Spectrum

Adverse drug reactions (ADRs) of Chinese herbal injections (CHIs) occur frequently, and it is of great significance to enhance the level of quality control for the clinical use of CHIs. According to the relevant reports, anaphylactic reactions take the main part of ADRs of CHIs, and70-80%of anaphylactic reactions belong to anaphylactoid reaction. As mast cell degranulation is an important process of anaphylactoid reaction, that biological effect naturally becomes the key to improve the quality of CHIs. Although, the present methods such as electron microscopy observation to study the degranulation can directly obverse the morphological change of cells at the given time, it needs time-consuming processes, say, chemical fixation, staining. The cell response profile technique (CRPT), with the date output carries the characteristics of fingerprint, can record the information of interaction between drug and cells in a real-time, label-free way.This study began with conventional examination i.e. general rules of Chinese Pharmacopeia on preparations for injections based examination (GRCPE) and chemical fingerprinting examination (CFE), and then a new evaluation method, with Shuanghuanglian lyophilized powder (SLP) as model drug, containing conventional chemical and biological evaluation methods was developed. Then, further attention was paid to the chlorogenic acid, for it has raised much controversy, to study the effect of chlorogenic acids on mast cell degranulation. The study was to warn of potential ADRs early and to provide reference to quality control of CHIs, therefore guarantee the safe clinical medication. Our main content of study and experimental results were as follows:1. Development of mast cell degranulation effect based living cell response profiling methodWith the help of real time cell analysis (RTCA) system, invented upon the principle that all living cells possess different electrical impedance, and Compound 48/80as model drug, RBL-2H3cell as model organism, cell index (CI), median inhibitory concentration (EC50), inhibition ratio (I) as evaluating indexes, the effects of various concentration of Compound48/80on RBL-2H3cell degranulation were monitored. The results showed that when the concentration of Compound48/80ranges from10p,g/mL to50μg/mL, the effects of Compound48/80on RBL-2H3display obvious dose-effect relationship and time-effect relationship and the regression equation of inhibition ratio was I=0.0193C-0.048(r>0.99), EC50=33.45μg/mL. Besides, the results were confirmed by conventional in vitro methods such as observation of cell morphology, mast cell degranulation using flow cytometry, measurement of allergy mediators and in vivo methods as evans blue staining, detection of β-hexosaminidase release rate. All those results demonstrated that the CRPT, represented by cell index-reaction time curve, featuring the advantage of ease of operation, high throughput, real-time and on-line, can provide quality control methods for CHIs with powerful technical support.2. Study on mast cell degranulation induced by chlorogenic acidsThe methods-CRPT, observation of cell morphology, conventional pharmacological experiment, measurement of allergy mediators-were adopted to investigate the degranulation effects induced by the CGAs which representing CGA and its isomers and derivatives including caffeic acid (CA), quinic acid (QA), neochlorogenic acid (neo-CGA), cryptochlorogenic acid (crypto-CGA), isochlorogenic acid A (iso-CGA A), isochlorogenic acid B (iso-CGA B), isochlorogenic acid C (iso-CGA C),1,3-dicaffeoylquinic acid (1,3-di-CQA),1,5-dicaffeoylquinic acid (1,5-di-CQA) using the RTCA system and with Compound48/80as positive control, RBL-2H3cell as model organism. The EC50of C48/80, CA, QA, CGA, neo-CGA, crypto-CGA, iso-CGA A, iso-CGA B, iso-CGA C,1,3-di-CQA,1,5-di-CQA were29.64±3.50,294.19±36.51,2411.60±16.8,612.76±29.40,171.71±23.32,80.25±2.60,917.53±33.50,913.01±32.20,919.73±35.60,920.51±34.65,916.75±31.98μg/mL, respectively. The sequence of degranulation effects on RBL-2H3cells was crypto-CGA> neo-CGA> CA>CGA> iso-CGA B>1,5-di-CQA> iso-CGA A> iso-CGA C>1,3-di-CQA> QA. The in vivo and in vitro study of CGA with the help of LC-MS technology indicates that CGA can be isomerized to crypto-CGA and then to neo-CGA and a little CA which all display stronger degranulation effect and the finding, to some extent, might help to explain the discrepancy of the results in literature.A knock-out and knock-in strategy was adopted to further study the sensitization effect of CGAs in the whole SLP. By comparing the sensitization effect of knocked out positive components, i.e. CGA, neo-CGA, crypto-CGA, CA, with negative components, i.e. the components made from knocking out the positive components in the whole SLP and with the whole injection, we found out that neo-CGA, crypto-CGA, CA contribute the enhancement of the sensitization effect of the whole CHI to some extent. In addition, the co-sensitization phenomenon of CGAs with other components in injection suggests the compounds for sensitization effect of SLP is not just CGAs. By adding of CGA, neo-CGA, crypto-CGA to negative components according the ratio of the three components in injection in an increasing manner, the sensitization effect of the whole injection showed a dose-dependent manner.3. Establishment of quality consistency evaluation method for SLP based on cell response profile technique (CRPT)With SHL as model drug, a new method for quality control of CHIs based on bio-assessment was established by adopting the established CRPT.25batches of SLP containing5batches of normal samples,15batches of artificial abnormal samples (5batches of samples kept at room temperature and away from light,5batches of samples kept in a60℃calorstat for10days,5batches of samples kept under1×104lx illumination for10days), and5batches of expired samples were collected and prepared.6,9, and21batches of abnormal SLP were detected by GRCPE, CFE, living cell response profiling examination (LCRPE) respectively.11,22,22,23batches of abnormal SLP were detected by GRCPE+CFE, GRCPE+CRPT, CFE+CRPT, GRCPE+CFE+CRPT respectively, with detection rate as44%,88%,88%, 92%. The results indicate that living cell response profiling examination can improve the detection efficiency of current method (GRCPE+CFE). The result demonstrated that the LTCP can serve as good supplementary method for current quality methods for CHIs, and can find its position in evaluating quality consistency of SLP and other CHIs and early warning of ADRs.In conclusion, the CRPT developed, with the characteristic of real-time and on-line, serving as good supplementary method for GRCPE and CFE, can directly reflects the bioactivity of CHIs and thus better reflects the quality constancy and stability of CHIs. In addition, the study clarified of the characteristics of the effect of chlorogenic acids on mast cell, and provided a new approach and technique support for the enhancing the quality evaluation level and reducing the ADRs of CHIs.