Discovery of the active compounds of Smilacis Glabrae Rhizoma by utilizing the relationship between the individual differences in blood drug concentration and the pharmacological effect in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: This study addresses the rapid discovery of the active compounds (the original constituents and/or metabolites) of a traditional Chinese drug, Smilacis Glabrae Rhizoma (SGR). AIM OF THE STUDY: The aim of this study was to develop a new method to find out the active compounds of traditional drugs in vivo. MATERIALS AND METHODS: A method was established to discover and identify the potential active compounds in drug-containing plasma from rats that were orally administered SGR extract, utilizing the relationship between the individual differences in blood drug concentrations in the rats and the resulting differences in pharmacological effect, and the method was denoted as the RID-PE method. For this method, we used high-performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry (LC-MSn) to identify the compounds (the original constituents and metabolites) and to determine the peak areas of the compounds in drug-containing plasma following SGR treatment. The anti-inflammatory effect of SGR was evaluated using a carrageenan-induced inflammatory rat model. According to the percent inhibition of paw edema in each model rat (14 rats total) orally administered SGR extract, the plasma samples from the rats were sorted and divided into 7 groups. Each group consisted of two plasma samples, and their percent inhibition of paw edema were similar to each other. We performed an LC-MSn analysis on 3 plasma groups, which showed large differences in the inhibition rates, with percent inhibitions of 92.7%, 72.4% and 38.4%. The correlation coefficients (r) between the peak area of each compound and the pharmacological effect (inhibition ratio) of SGR in the three groups were analyzed using SPSS software. When the correlation coefficients of the compounds are greater than 0.8 (0.8 < r ≤1), these compounds are strongly and positively correlated with anti-inflammatory activity, making them potential anti-inflammatory active compounds. RESULTS: Fifty-eight potential anti-inflammatory compounds (0.8 < r ≤ 1) from SGR were discovered in model rat plasma using the RID-PE method, 47 of which were considered to be new potentially anti-inflammatory compounds. Among these compounds, four original constituents and 5 isomers of potential anti-inflammatory metabolites were validated to have significant anti-inflammatory effects, and they included astilbin, syringic acid, catechin, coumalic acid, resveratrol-3'-O-glucuronide (RG, isomer of M2 or M3), 3'-O-methyl-(+)-epicatechin-4'-O-glucuronide (CA-1, isomer of M16), 4'-O-methyl-(+)-epicatechin-3'-O-glucuronide (CA-2, isomer of M16), 4'-O-methyl-(+)-epicatechin-7-O-glucuronide (CA-3, isomer of M16) and 3'-O-methyl-(+)-epicatechin-7-O-glucuronide (CA-4, isomer of M16). In addition, four isomers (CA-1-CA-4) were reported to have anti-inflammatory effects for the first time, and CA-3 was a new compound. CONCLUSIONS: The RID-PE method can be used to discover and identify the active constituents and metabolites of SGR systematically and in vivo. Furthermore, these findings enhance our understanding of the metabolism and effective forms of SGR.

[Anti-inflammation effect of Jinlingzi San in rat metabonomics based on 1H-NMR and LC-MS technology].

To further explore the regulatory effect of Jinlingzi San on in vivo inflammatory mechanism during inflammatory treatment, this study adopted 1H-NMR and LC-MS technology to analyze differences in in vivo metabolites of carrageen-induce rat foot swelling model. Besides, biomarkers related to inflammation models of Jinlingzi San in SD rats were discovered to speculate the regulatory mechanism of Jinlingzi San in resisting carrageen-induce inflammation. Through the analysis of detection spectrum, we found 18 biomarkers of metabolites(citrate, pyruvate, malic acid, succinate, glutamate, lysine, tartrate, 2-oxobutyric acid, glycine, guanosine, 9-cis-retinoic acid, triphosphate, inosine 5'-diphosphate, inosine diphosphate, tripolyphosphate, inorganic triphosphate, glycerophosphocholine, 21-deoxycortisol). Relevant pathway analysis results were TCA cycle, pyruvate metabolism, glycine, serine and threonine metabolism, and dicarboxylic acid metabolism. From the metabolic network, we can see that the anti-inflammatory effect of Jinlingzi San can regulate citric acid, succinic acid and glycine content to resist oxygen free radical and reduce body damage by ROS, so as to down-regulate inflammatory factors generated from body tissues and resist inflammation.

[Subacute toxicity metabonomics of Jinlingzi powder based on LC-MS].

To further understand the metabolic characteristics of Jinlingzi powder toxicity effect in rats and explore the effect of Jinlingzi powder on unknown biological pathways in the treatment process. In this experiment, the effect of three doses of Jinlingzi powder decoction on rat liver and kidney was investigated to explore the characteristics and rules of Jinlingzi powder on in vivo metabonomic changes in rats. First, urine and serum samples of the rats were used for LC-MS analysis. Under the XCMS online analysis, 44 differential substances were found in the identification of metabolites. Finally, Metpa was used for metabolic pathways enrichment and analysis, and five related metabolic pathways were obtained: steroid hormone biosynthesis, tryptophan metabolism, pentose and glucuronate interconversions, ascorbate and aldarate metabolism, as well as glutathione metabolism. Metabolic network diagram showed that the toxicity-related pathways were mainly associated with lysine metabolism in living organisms, glucuronic acid conversion, and hormone metabolism, especially the metabolism imbalance of lysine and glutathione would result in the disorder of energy metabolism or oxidative stress regulation, and thus inducing the damage in rats. Subacute toxicity test results for three doses groups (low, middle and high doses) showed that, Jinlingzi powder with doses of 19.7 g•kg⁻¹ and 39.4 g•kg⁻¹ caused obvious toxic effect, indicating Jinlingzi powder could produce toxic effect in vivo in a dose-dependent manner, and cause irreversible damage to the body.

[Nuclear magnetic resonance-based metabonomics for sedative and hypnotic effect of Banxia Houpo decoction].

Insomnia was a common disease, which might be correlated with γ-aminobutyric acid A (GABAA) receptor mechanism, cytokine regulatory mechanism, excitatory amino acid mechanism and hydroxytryptamine (5-HT) receptor mechanism, but the correlations between these independent mechanisms and pathological characterization were still unclear. To further explore the effect of Banxia Houpo decoction on known or unknown biological pathways during treatment of insomnia, the metabonomics method based on ¹H-NMR was developed for detecting the significant changes in metabolomics after the administration with Banxia Houpo decoction in pentobarbital sodium-induced rat sleeping experiment. Serum and urine samples were analyzed by ¹H-NMR. Principal component analysis (PCA) was carried out for endogenous small molecule metabolites in urine and serum. H-NMR spectroscopies and relevant metabolites were found and identified by Simca-p 17.0 (Umet-rics, Umea, Sweden) and Chenomx NMR Suite 7.1 (Chenomx, Inc., Edmonton, Alberta, Canada) software. The result suggests that Banxia Houpo decoction group and indiplon group had significant differences. The load diagram showed the biggest variation metabolites and intergroup significant differences among 10 metabolic substances. According to the experiment, Banxia Houpo decoction group and indiplon group can prolonge the sleeping time of pentobarbital sodium-induced sprague-dawley rats, with a synergistic effect. The significant changes of these biomarkers indicated that the Banxia Houpo decoction could aid sleep by adjusting the content of glutamine, creatine phosphate, 2-oxoglutarate, and reducing the activity of brain nerves.