Determining the protective effects of Ma-Mu-Ran Antidiarrheal Capsules against acute DSS-induced enteritis using 16S rRNA gene sequencing and fecal metabolomics.

Ma-Mu-Ran Antidiarrheal Capsules (MMRAC) is traditional Chinese medicine that has been used to treat diarrhea caused by acute enteritis (AE) and bacillary dysentery in Xinjiang (China) for many years. However, the potential therapeutic mechanism of MMRAC for AE and its regulatory mechanism on host metabolism is unclear. This study used fecal metabolomics profiling with GC/MS and 16S rRNA gene sequencing analysis to explore the potential regulatory mechanisms of MMRAC on a dextran sulfate sodium salt (DSS)-induced mouse model of AE. Fecal metabolomics-based analyses were performed to detect the differentially expressed metabolites and metabolic pathways. The 16S rRNA gene sequencing analysis was used to assess the altered gut microbes at the genus level and for functional prediction. Moreover, Pearson correlation analysis was used to integrate differentially expressed metabolites and altered bacterial genera. The results revealed that six intestinal bacteria and seven metabolites mediated metabolic disorders (i.e., metabolism of amino acid, carbohydrate, cofactors and vitamins, and lipid) in AE mice. Besides, ten altered microbes mediated the differential expression of eight metabolites and regulated these metabolisms after MMRAC administration. Overall, these findings demonstrate that AE is associated with metabolic disorders and microbial dysbiosis. Further, we present that MMRAC exerts protective effects against AE by improving host metabolism through the intestinal flora.

Study on the molecular mechanism of anti-liver cancer effect of Evodiae fructus by network pharmacology and QSAR model.

Introduction: Evodiae Fructus (EF) is the dried, near ripe fruit of Euodia rutaecarpa (Juss.) Benth in Rutaceae. Numerous studies have demonstrated its anti-liver cancer properties. However, the molecular mechanism of Evodiae fructus against liver cancer and its structure-activity connection still require clarification. Methods: We utilized network pharmacology and a QSAR (2- and 3-dimensional) model to study the anti-liver cancer effect of Evodiae fructus. First, by using network pharmacology to screen the active substances and targets of Evodiae fructus, we investigated the signaling pathways involved in the anti-liver cancer actions of Evodiae fructus. The 2D-QSAR pharmacophore model was then used to predict the pIC50 values of compounds. The hiphop method was used to create an ideal 3D-QSAR pharmacophore model for the prediction of Evodiae fructus compounds. Finally, molecular docking was used to validate the rationality of the pharmacophore, and molecular dynamics was used to disclose the stability of the compounds by assessing the trajectories in 10 ns using RMSD, RMSF, Rg, and hydrogen bonding metrics. Results: In total, 27 compounds were acquired from the TCMSP and TCM-ID databases, and 45 intersection targets were compiled using Venn diagrams. Network integration analysis was used in this study to identify SRC as a primary target. Key pathways were discovered by KEGG pathway analysis, including PD-L1 expression and PD-1 checkpoint pathway, EGFR tyrosine kinase inhibitor resistance, and ErbB signaling pathway. Using a 2D-QSAR pharmacophore model and the MLR approach to predict chemical activity, ten highly active compounds were found. Two hydrophobic features and one hydrogen bond acceptor feature in the 3D-QSAR pharmacophore model were validated by training set chemicals. The results of molecular docking revealed that 10 active compounds had better docking scores with SRC and were linked to residues via hydrogen and hydrophobic bonds. Molecular dynamics was used to show the structural stability of obacunone, beta-sitosterol, and sitosterol. Conclusion:Pharmacophore 01 has high selectivity and the ability to distinguish active and inactive compounds, which is the optimal model for this study. Obacunone has the optimal binding ability with SRC. The pharmacophore model proposed in this study provides theoretical support for further screening effective anti-cancer Chinese herbal compounds and optimizing the compound structure.

Transcriptomic analysis in Anemone flaccida rhizomes reveals ancillary pathway for triterpene saponins biosynthesis and differential responsiveness to phytohormones.

Anemone flaccida Fr. Schmidt is a perennial medicinal herb that contains pentacyclic triterpenoid saponins as the major bioactive constituents. In China, the rhizomes are used as treatments for a variety of ailments including arthritis. However, yields of the saponins are low, and little is known about the plant's genetic background or phytohormonal responsiveness. Using one-quarter of the 454 pyrosequencing information from the Roche GS FLX Titanium platform, we performed a transcriptomic analysis to identify 157 genes putatively encoding 26 enzymes involved in the synthesis of the bioactive compounds. It was revealed that there are two biosynthetic pathways of triterpene saponins in A. flaccida. One pathway depends on ß-amyrin synthase and is similar to that found in other plants. The second, subsidiary ("backburner") pathway is catalyzed by camelliol C synthase and yields ß-amyrin as minor byproduct. Both pathways used cytochrome P450-dependent monooxygenases (CYPs) and family 1 uridine diphosphate glycosyltransferases (UGTs) to modify the triterpenoid backbone. The expression of CYPs and UGTs were quite different in roots treated with the phytohormones methyl jasmonate, salicylic acid and indole-3-acetic acid. This study provides the first large-scale transcriptional dataset for the biosynthetic pathways of triterpene saponins and their phytohormonal responsiveness in the genus Anemone.

Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo. Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo. Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro. Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

Triterpenoid saponin flaccidoside II from Anemone flaccida triggers apoptosis of NF1-associated malignant peripheral nerve sheath tumors via the MAPK-HO-1 pathway.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue neoplasms that are extremely rare and are frequently associated with neurofibromatosis type 1 patients. MPNSTs are typically fatal, and there is no effective treatment so far. In our previous study, we showed that flaccidoside II, one of the triterpenoid saponins isolated from Anemone flaccida Fr. Schmidt, has antitumor potential by inducing apoptosis. In the present study, we found that flaccidoside II inhibits proliferation and facilitates apoptosis in MPNST cell lines ST88-14 and S462. Furthermore, this study provides a mechanism by which the downregulation of heme oxygenase-1 via extracellular signal-regulated kinase-1/2 and p38 mitogen-activated protein kinase pathways is involved in the apoptotic role of flaccidoside II. This study suggested the potential of flaccidoside II as a novel pharmacotherapeutic approach for MPNSTs.

The Antitumor Effects of Triterpenoid Saponins from the Anemone flaccida and the Underlying Mechanism.

Anemone flaccida Fr. Schmidt, a family of ancient hopanoids, have been used as traditional Asian herbs for the treatments of inflammation and convulsant diseases. Previous study on HeLa cells suggested that triterpenoid saponins from Anemone flaccida Fr. Schmidt may have potential antitumor effect due to their apoptotic activities. Here, we confirmed the apoptotic activities of the following five triterpenoid saponins: glycoside St-I4a (1), glycoside St-J (2), anhuienoside E (3), hedera saponin B (4), and flaccidoside II (5) on human BEL-7402 and HepG2 hepatoma cell lines, as well as the model of HeLa cells treated with lipopolysaccharide (LPS). We found that COX-2/PGE2 signaling pathway, which plays key roles in the development of cancer, is involved in the antitumor activities of these saponins. These data provide the evidence that triterpenoid saponins can induce apoptosis via COX-2/PGE2 pathway, implying a preventive role of saponins from Anemone flaccida in tumor.

[Study on triterpenoid saponins in the rhizome of Anemone hofengensis].

OBJECTIVE: To study the triterpenoid saponins in the rhizome of Anemone hofengensis. METHODS: The constituents were separated with various chromatographic techniques and their structures were elucidated by physicochemical properties and spectral data. RESULTS: Five compounds were isolated and identified as 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-alpha-L-arabino-pyranosyl-oleanolic acid (1), 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 2)-alpha-L-rhamnopyranosyl-oleanolic acid 28-O-alpha-L-rhamnopyranosyl-(1 --> 4) -beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranoside (2), 3-O-alpha-L-rhamnopyranosyl-(1 --> 2) [beta-D-glucopyranosyl-(1 --> 4)]-alpha-L-rhamnopyranosyl-oleanolic acid-28-O-beta-D-glucopyranosyl-(1 --> 6)-beta-D-gluco-pyranoside (3), 3-O-beta-D-glucopyranosyl-(1 --> 2)-beta-D-xylopyranosyl-oleanolic acid 28-O-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranoside (4), oleanolic acid-28-O-alpha-L-rhamnopyra-nosyl-(1 --> 4)-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranoside (5). CONCLUSION: Compound 1 - 5 are isolated from this plant for the first time.

[Studies on triterpenoid saponins in the rhizome of Anemone flaccida].

OBJECTIVE: To study the triterpenoid saponins in the rhizome of Anemone flaccida. METHODS: The constituents were separated with various chromatographic techniques and their structures were elucidated by means of physicochemical properties and the analysis of their spectral datas. RESULTS: Five compounds were isolated and identified as 3-O-beta-D-glucuronypyranosyl-oleanolic acid-28-O-alpha-L-rhamnopyranosyl (1 --> 4)-beta-D-glucopyranosyl(1 --> 6)-beta-D-glucopyra noside (1), 3-O-beta-D-glucuronypyranosyl-oleanolic acid-28-O-beta-D-glucopyranosyl (1 --> 6)-beta-D-glucopyranoside (2), 3-O-alpha-L-rhamnopyranosy (1 --> 2)-beta-D-glucopyranosyl-oleanolic acid-28-O-alpha-L-rhamnopyranosyl (1 --> 4)-beta-D-glucopyranosyl (1 --> 6)-beta-D-glucopyranoside (3), 3-O-alpha-L-rhamnopyranosyl (1 --> 2)-alpha-L-arabinopyrano-syl-oleanolic acid-28-O-alpha-L-rhamnopyranosyl (1 -->4)-beta-D-glucopyranosyl (1 --> 6)-beta-D-glucopyranoside (4), 3-O-alpha-L-rhamnopyranosyl (1 --> 2)-beta-D-xylopyranosyl-oleanolic acid-28-O-alpha-L-rhamnopyranosyl (1 --> 4)-beta-D-glucopyranosyl (1 --> 6)-beta-D-glucopyranoside (5). CONCLUSION: Compound 1 - 4 are isolated from this plant for the first time. Compound 1,2 are isolated from this genus for the first time.

Triterpenoid saponins from Anemone flaccida induce apoptosis activity in HeLa cells.

Five triterpenoid saponins were isolated from Anemone flaccida Fr. Schmidt. Their structures were identified as glycoside St-I4a (1), glycoside St-J (2), anhuienoside E (3), hederasaponin B (4), and flaccidoside II (5). Compounds 1-2 were isolated from Anemone family for the first time, and compounds 3-4 were isolated from this plant for the first time. The inhibitory effects of saponins on proliferation of HeLa cells were studied by MTT assay, the apoptosis-induction activity was observed by cell-cycle analysis and caspase-3 expression assay. The antitumor activities of the saponins were ranked in the following order: 5 > 3 > 4 > 1 > 2. The data presented here indicated that naturally occurring triterpenoid saponins can be regarded as excellent structures for the potential development of new anticancer agents.