Résumé
OBJECTIVE To establish a method for the simultaneous determination of 10 rhubarb anthraquinones in Compound gentian sodium bicarbonate tablets and the content of rhaponticin，which are the characteristic components of artifacts，and to use the method to evaluate the quality of compound preparation containing Rheum officinale. METHODS The ultra-performance liquid chromatography （UPLC） method was adopted to determine the contents of 10 rhubarb anthraquinones （aloe-emodin-8-O-glucoside， rheinic acid-8-O-β-D-glucoside，emodin-8-O-glucoside，chrysophanol-8-O-β-D-glucoside，emodin monomethyl ether-8-O-β-D-glucoside， aloe-emodin，rheinic acid，emodin，chrysophanol，emodin monomethyl ether） and rhaponticin in 40 batches of Compound gentian sodium bicarbonate tablets from 8 manufacturers. The determination was performed on the Agilent Eclipse Plus C18 column with a mobile phase consisted of acetonitrile-0.1% phosphoric acid solution （gradient elution） at a flow rate of 0.3 mL/min； the column temperature was set at 30 ℃ ，and the injection volume was 5 μL. Combining principal component analysis and cluster analysis to synthesize the results of content determination，the quality of samples from different manufacturers was evaluated. RESULTS All of above 11 components showed favorable linear relationships with peak areas in their respective mass concentration ranges （r≥0.999 3），with RSDs of precision，repeatability and stability 296261547@qq.com less than 3% （n＝6）； average sample recoveries ranged 96.82%-98.92% （RSD≤1.74%，n＝6）； their contents were 0971-8247794。E-mail：304436784@qq.com 0.011 7-0.252 0，0-0.323 3，0.131 3-1.236 6，0.081 1-1.056 2，0.015 2-0.189 8，0.001 8-0.152 3，0-0.255 2，0.001 9-0.223 4，0.054 3-0.303 0，0.022 7-0.172 2，0-2.835 9 mg/g，respectively. The cumulative variance contribution of the first three principal components was 95.533%； the 40 batches of samples can be clustered into 4 categories：samples from enterprises a and d were in a category of their own，samples from enterprises f，b，g and e were in a category，and samples from enterprises c and h were in a category. There were large differences in the content of rhubarb anthraquinone in the samples from 8 manufacturers，and rhaponticin was only detected in the sample from one enterprise. CONCLUSIONS Established UPLC method is stable and reliable； it can be used for the content determination of 10 rhubarb anthraquinones and rhaponticin in Compound gentian sodium bicarbonate tablets.
Résumé
OBJECTIVE Senna and rhubarb are classified as stimulative laxatives, and known to have similar effec?tive constituents, the anthraquinones. Being protected by theβ-glucoside bond, the anthraquinones can reach the intes?tines where they are degraded into complex metabolites by enzymes secreted from the intestinal microbiome. It is these complex metabolites that produce the laxative effects. Then the similarities and differences of action between the anthra?quinones require further elucidation. METHODS Here, we studied metabolites of senna anthraquinones (SAQ), rhubarb anthraquinones (RAQ) and their chemical marker, sennoside A (SA), in a rat diarrhea model. In the in vitro biotransfor?mation experiments, SAQ, RAQ and SA were incubated with rat fecal flora solution and the metabolites produced were analyzed using HPLC. In the in vivo studies, the same compounds were investigated for purgation induction, with mea?surement of histopathology and multiple aquaporins (Aqps) gene expression in six organs. RESULTS SAQ and RAQ had similar principal constituents but could be degraded into different metabolites. A similar profile of Aqps down-regula?tion for all compounds was seen in the colon, suggesting a similar mechanism of action for purgation. However, in the kidneys and livers of the diarrhea-rats, down-regulation of Aqps was found in the RAQ-rats whereas up-regulation of Aqps was seen in the SAQ-rats. Furthermore, the RAQ-rats showed lower aquaporin 2 (Aqp2) protein expression in the kidneys, whilst the SA-rats and SAQ-rats had higher Aqp2 protein expression in the kidneys. This may have implications for side effects of SAQ or RAQ in patients with chronic kidney or liver diseases. CONCLUSION SAQ and RAQ showed similar laxative actions with a similar mechanism, they could display different actions in rat kidneys and livers. We suggest that the clinical usage of senna or rhubarb products should be clarified for patients having chronic kidney or liver diseases.
Résumé
Objective To detect the hepatotoxicity biomarkers using normal human hepatocyte (HepaRG) and high-content screening,and to combine the micronucleus test and single cell gel electrophoresis to estalish a rapid screening platform for in vitro cytotoxitity and genotoxicity.Methods The effects of rhubarb anthraquinones (AQs) on the reactive oxygen species (ROS),intracellular Ca2+ concentration and mitochondrial membrane potential (MMP) in HepaRG cells were studied using appropriate fluorescent probes Hoechst33342、DCFH-DA、Fluo4-AM、Mito Tracker Red CMX Ros and high-content screening methods,and the potential genotoxiciy triggered by AQs were analyzed using the high-content based cytokinesis block micronucleus test and high throughput comet assay.Results The intracellular ROS level of HepaRG cells was significantly elevated by a 24 h treatment with Emodin (25.0 μg/mL),aloe-emodin (25.0 μg/mL) or chrysophanol (50.0 μg/mL),which are dose-concentration dependent (P ＜ 0.05 and 0.01);the intracellular Ca2+ increased and mitochondrial damage were observed with the treatment of aloe-emodin (25.0 μg/mL) and rhein (50.0 μg/mL,P ＜ 0.05 and 0.01).Comparing to control group,Emodin (25.0 μg/mL) induced an increased micronucleus rate (1.59％ ± 0.68 ％,P ＜ 0.01) and significantly higher percentage tail DNA and Olive tail moment (respectively 10.155％ ± 2.17％ and 0.510 ± 0.06,P ＜ 0.05 and 0.01) after 24 h;while the chrysophanol increased the micronucleus rate to 1.29％ ± 0.54％ (P ＜ 0.01) after 72 h.Conelnsion The results on the cytotoxicities and genotoxicities of AQs are consistent with the literatures.In this study,a rapid screening model for both hepatotoxicity and genotoxicity was successfully established,which will help with the early screening during the drug development stage.