Chai-Gui Decoction and its representative components ameliorate spontaneous hypertension rats by modulating lipid metabolism and gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypertension coincides with the category of "vertigo" and/or "headache" on the basis clinical manifestations and traditional Chinese medicine (TCM) theory. Chai-Gui Decoction (CGD), which is in usage for relieving "vertigo" and/or "headache", had been demonstrated to be useful in ameliorating hypertension. AIM OF STUDY: This study was planned to investigate the mechanism of CGD and its components in hypertension by using spontaneous hypertension rat (SHR). MATERIALS AND METHODS: CGD extract and its classification component samples (compounds in plasma, CP; compounds in gut, CG; compounds in plasma and gut, CPG) were prepared for animal experiment. SHR rats were induced with CGD extract (3 g/kg/d BW, 5 g/kg/d BW, 15 g/kg/d BW) and CGD-component classes (CP = 19.501 mg/kg/d, CG = 5.240 mg/kg/d, CPG = 24.741 mg/kg/d) for 4 weeks. Blood pressure (BP) and indexes of renin-angiotensin-aldosterone system (RAAS system) were measured. Histopathology was carried out to assess the efficacy of CGD and its components on aorta tissues. Untargeted metabolomics of lipid from rat serum samples were applied by Ultra-High performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and chemometric analysis to explore the relationship between metabolic pathways and hypertension. 16S rRNA gene sequencing of rat colon content and bioinformatics analysis were used to characterize the effects of CGD and its components on the gut microbiota composition of SHR rats. RESULTS: CGD and its component mixtures showed antihypertensive effect on SHR rats, decreased the blood pressure and reduced the aortic wall thickness in SHR rats. CGD and its component mixtures could improve the RAAS in SHR rats, including increase the percentage of angiotensin 1-7 (Ang 1-7), decrease the percentage of angiotensin II (Ang II), and decrease the Ang Ⅱ/Ang 1-7 ratio. CGD and its component mixtures could regulate the metabolome in SHR rats, mainly as decreasing the higher serum levels of Lysophosphatidylcholine (LPC) 16: 0, LPC 20: 4, and LPC 22: 6. In addition, bacteria from family S24-7 were negatively correlated with levels of LPE 16:0, LPE 18:0, LPE 18:1, and LPE 18:2. CONCLUSION: CGD and its component mixtures exhibited antihypertensive effect on SHR rats. The underlying mechanism could be related to modulation on RAAS, LPC metabolism and the bacterial abundance of family S24-7 in gut.

Characterization of the complete chloroplast genome of Salvia leucantha (Lamiaceae).

Salvia leucantha (Lamiaceae) is an important horticultural plant with great ornamental and economic value. Here, we report the complete chloroplast genome of this species. The chloroplast genome was determined to be 151021 bp and the GC contents was 38.0%. The sequence includes a large single copy (LSC) region of 82,262 bp, a small single copy (SSC) region of 17,537 bp, and two separated inverted regions of 25,611 bp each. It contains 130 unique genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Based on the chloroplast genomes data of 26 species in Salvia, our result indicated that S. leucantha, S. tiliifolia, S. hispanica, and S. splendens formed one clade with Bootstrap = 100%. The four species belong to Salvia subgenus Calosphace, and S. leucantha was closely related to Salvia tiliifolia and Salvia hispanica. This result will facilitate population, genetic diversity and phylogenetic studies of S. leucantha.

Determination and Comparison of Short-Chain Fatty Acids in Serum and Colon Content Samples: Alzheimer's Disease Rat as a Case Study.

Short-chain fatty acids (SCFAs) are the main microbial fermentation products from dietary fibers in the colon, and it has been speculated that they play a key role in keeping healthy in the whole-body. However, differences in SCFAs concentration in the serum and colon samples had attracted little attention. In this study, we have optimized the extract and analysis methods for the determination of ten SCFAs in both serum and colon content samples. Methanol and acetonitrile were chosen for extraction of SCFAs from serum and colon content samples, respectively. Biological samples were collected from Alzheimer's disease rats treated by extract of Schisandra chinensis (Turcz.) Baill (SC-extract) were taken as research objects. The results showed that, the relative peak intensities of SCFAs in the colon content from all groups were quite similar, and the trend was identical in the serum samples. Compared with the values in humans, the ratio of ten SCFAs in rat's colon was similar, while the percent of acetate in rat's serum was significantly higher. For therapy of Alzheimer's disease (AD), SC-extract decreased the concentration of butyrate, 3-Methyvalerate, and caproate in the serum samples towards the trend of normal rats. This study may help our understanding of how SCFAs are transported across colonic epithelium in healthy and diseased organisms.

In Vitro and In Vivo Anti-AChE and Antioxidative Effects of Schisandra chinensis Extract: A Potential Candidate for Alzheimer's Disease.

Acetylcholinesterase (AChE) inhibition and antioxidants are two common strategies for the treatment in the early stage of Alzheimer's Disease (AD). In this study, extracts from nine traditional Chinese medical (TCM) herbs were tested for anti-AChE activity by Ellman's microplate assay and cytotoxicity by CCK-8. Based on its excellent AChE inhibition effect and its lowest cytotoxicity, Schisandra chinensis (SC) extract was selected to do the mechanism research. SC extract protected pheochromocytoma (PC12) cells against H2O2-induced toxicity by improving the cell survival rate in a dose-dependent manner. And it also showed significant free radical (DPPH) scavenging activities, ferric reducing antioxidant power (FRAP), and 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging. To confirm these results, the scopolamine-induced mice models were utilized in this study. Compared with the positive drug (piracetam), SC could also exhibit similar effects to alleviate the mice's cognitive deficits. Moreover, in the mice brain samples, the AChE activity and malondialdehyde (MDA) levels of SC-treatment group both showed a reverse as compared to model group. Taken together, these results all suggested that SC extract may be a potential therapeutic candidate for AD.

Saponins from Clematis mandshurica Rupr. regulates gut microbiota and its metabolites during alleviation of collagen-induced arthritis in rats.

Gut microbiota and their metabolites (short-chain fatty acids, SCFAs) are associated with the pathogenesis of rheumatoid arthritis (RA). Total Clematis triterpenoid saponins (CTSs) prepared from Clematis mandshurica Rupr. possess therapeutic benefits for arthritic diseases. However, the poor pharmacokinetic properties of CTSs have obstructed the translation of these natural agents to drugs. Here, we examined the effects of CTSs on arthritis symptoms, gut microbiota and SCFAs in rats with collagen-induced arthritis (CIA). Our results showed that the arthritis index scores of CIA rats treated with CTSs were significantly lower than those of the model group. Most importantly, CTSs moderated gut microbial dysbiosis and significantly downregulated the total SCFA concentration in CIA rats. Compared to the control group, CTSs treatment have no significant side effects on the gut microbiota and SCFA metabolism in normal rats. Two differential analyses (LEfSe and DESeq2) were combined to study the details of the changes in gut microbiome, and twenty-four marker taxa at the genus level were identified via a comparison among control, model and CIA rats treated with high doses of CTSs. In particular, the mostly significantly increased gram-negative (G-) and decreased gram-positive (G+) genera in CIA rats were well restored by CTSs. The observed SCFA concentrations demonstrated that CTSs tend to maintain the balance of the gut microbiota. The data presented herein suggest that CTSs could ameliorate arthritis-associated gut microbial dysbiosis and may be potential adjuvant drugs that could provide relief from the gastrointestinal damage caused as a side effect of commonly used drugs.

Simultaneous determination of short-chain fatty acids in human feces by HPLC with ultraviolet detection following chemical derivatization and solid-phase extraction segmental elution.

Short-chain fatty acids are currently the most studied metabolites of gut microbiota, but the analysis of them, simultaneously, is still challenging due to their unique property and wide concentration range. Here, we developed a sensitive and versatile high-performance liquid chromatography with ultraviolet detection method, using pre-column derivatization and solid-phase extraction segmental elution, for the quantification of both major and trace amounts of short-chain fatty acids in human feces. Short-chain fatty acids were converted to 3-nitrophenylhydrazine-derived analytes, and then solid-phase extraction segmental elution was used for extraction of major analytes and enrichment of trace analytes. The method validation showed limits of quantitation ˂0.04 mM, and coefficient of determination > 0.998 at a wide range of 0.04-8.0 mM. The intra- and interday precision of analytes were all within accepted criteria, and the recoveries were 96.12 to 100.75% for targeted analytes in fecal samples. This method was successfully applied in quantification of eight analytes in human feces, which therefore could provide a sensitive and versatile high-performance liquid chromatography with ultraviolet detection method for precise and accurate quantitation of short-chain fatty acids in human feces.

A chemical derivatization based UHPLC-LTQ-Orbitrap mass spectrometry method for accurate quantification of short-chain fatty acids in bronchoalveolar lavage fluid of asthma mice.

Recent studies have demonstrated the important role of short-chain fatty acids (SCFAs) in the maintenance of homeostasis of respiratory immunity. However, there is still no report focus on the determination of SCFAs level in bronchoalveolar lavage fluid (BALF), the most common sample used for screening biomarkers of the pulmonary diseases. Herein, an ultra-high-performance liquid chromatography with LTQ-Orbitrap mass spectrometer (UHPLC-LTQ-Orbitrap) oriented 3-nitrophenylhydrazine (3-NPH)-based derivatization method was developed for the quantification of SCFAs in BALF. To achieve accurate quantitation, d4-acetate was used as internal standard to compensate for the matrix effects. Method validation showed a good linearity (R2 > 0.9992) with wide concentration range, and the intra-day and inter-day precision for determination of eight SCFAs in BALF samples was ≤ 14.79%. The quantitation accuracy, assessed by relative recoveries, ranged from 90% to 110% for target SCFAs at three concentration levels. Matrix effects ranged from 85% to 115%, and the lower limits of quantification of these targeted SCFAs were varied from 3 to 24 nmol/L. The SCFAs-targeted method was then applied to determine the changed levels in BALF samples from OVA-induced asthma mice and normal mice. In addition, the universality of our developed method was also demonstrated by determining the SCFAs concentrations in feces, serum and lung tissue samples from asthma and normal mice. These results indicate that 3-NPH derivatization based UHPLC-LTQ-Orbitrap provides accurate view of global SCFAs alternation in different samples, giving a support to deduce the origin of SCFAs in lung. The present study is of great importance for understanding the role of SCFAs in modulation of host metabolism and immunity.

Dose-response characteristics of Clematis triterpenoid saponins and clematichinenoside AR in rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine metabolomics.

Clematidis Radix et Rhizoma is a traditional Chinese medicine widely used for treating arthritic disease. Clematis triterpenoid saponins (TS) and clematichinenoside AR (C-AR) have been considered to be responsible for its antiarthritic effects. However, the underling mechanism is still unclear because of their low bioavailability. To address of this issue, metabolomics tools were performed to determine metabolic variations associated with rheumatoid arthritis (RA) and responses to Clematis TS, C-AR and positive drug (Triptolide, TP) treatments. This metabolomics investigation of RA was conducted in collagen-induced arthritis (CIA) rats. Liquid chromatography/mass spectrometry and multivariate statistical tools were used to identify the alteration of serum and urine metabolites associated with RA and responses to drug treatment. As a result, 45 potential metabolites associated with RA were identified. After treatment, a total of 24 biomarkers were regulated to normal like levels. Among these, PC(18:0/20:4), 9,11-octadecadienoic acid, arachidonic acid, 1-methyladenosine, valine, hippuric acid and pantothenic acid etc, were reversed in Clematis TS and C-AR groups. Tetrahydrocortisol was regulated to normal levels in Clematis TS and TP groups, while 3,7,12-trihydroxycholan-24-oic acid was regulated in C-AR and TP groups. Biomarkers like citric acid, p-cresol glucuronide, creatinine, cortolone were reversed in TP group.

Structural characterization and discrimination of Chinese medicinal materials with multiple botanical origins based on metabolite profiling and chemometrics analysis: Clematidis Radix et Rhizoma as a case study.

Traditional Chinese medicines (TCMs)-based products are becoming more and more popular over the world. To ensure the safety and efficacy, authentication of Chinese medicinal materials has been an important issue, especially for that with multiple botanical origins (one-to-multiple). Taking Clematidis Radix et Rhizoma (CRR) as a case study, we herein developed an integrated platform based on metabolite profiling and chemometrics analysis to characterize, classify, and predict the "one-to-multiple" herbs. Firstly, the predominant constituents, triterpenoid saponins, in three Clematis CRR were rapid characterized by a novel UPLC-QTOF/MS-based strategy, and a total of 49 triterpenoid saponins were identified. Secondly, metabolite profiling was performed by UPLC-QTOF/MS, and 4623 variables were extracted and aligned as dataset. Thirdly, by using pattern recognition analysis, a clear separation of the three Clematis CRR was achieved as well as a total number of 28 variables were screened as the valuable variables for discrimination. By matching with identified saponins, these 28 variables were corresponding to 10 saponins which were identified as marker compounds. Fourthly, based on the relative intensity of the marker compounds-related variables, genetic algorithm optimized support vector machines (GA-SVM) was employed to predict the species of CRR samples. The obtained model showed excellent prediction performance with a prediction accuracy of 100%. Finally, a heatmap visualization was employed for clarifying the distribution of identified saponins, which could be useful for phytochemotaxonomy study of Clematis herbs. These results indicated that our proposed platform was a powerful tool for chemical profiling and discrimination of herbs with multiple botanical origins, providing promising perspectives in tracking the formulation processes of TCMs products.