Nudifloside, a Secoiridoid Glucoside Derived from Callicarpa nudiflora, Inhibits Endothelial-to-Mesenchymal Transition and Angiogenesis in Endothelial Cells by Suppressing Ezrin Phosphorylation.

Callicarpa nudiflora is a traditional folk medicine in China used for eliminating stasis to subdue swelling. Several compounds from Callicarpa nudiflora have been proved to show anti-inflammatory, haemostasis, hepatitis, and anti-proliferative effects. Tumor endothelial cells play crucial roles in tumor-induced angiogenesis. Recently, it was demonstrated that ECs may be the important source of cancer associated fibroblasts (CAFs) through endothelial to mesenchymal transition (EndoMT). In this study, we evaluated the effects of nudifloside (NDF), a secoiridoid glucoside from Callicarpa Nudiflora, on TGF-ß1-induced EndoMT and VEGF-induced angiogenesis, and the underlying mechanisms were also involved. It was found that NDF significantly inhibited enhanced migration, invasion and F-actin assembly in endothelial cells (ECs) exposed in TGF-ß1. NDF obviously reversed expression of several biomarkers associated with EndoMT and recovered the morphological characteristics of ECs and tube-like structure induced by TGF-ß1. Furthermore, treatment of NDF resulted in a significant destruction of VEGF-induced angiogenesis in vitro and ex vivo. Data from co-immunoprecipitation assay provided the evidence that Ezrin phosphorylation and the interaction with binding protein can be inhibited by NDF, which can be confirmed by data from Ezrin silencing assay. Collectively, the application of NDF inhibited TGF-ß1-induced EndoMT and VEGF-induced angiogenesis in ECs by reducing Ezrin phosphorylation.

Betulinic acid induces apoptosis of HeLa cells via ROS-dependent ER stress and autophagy in vitro and in vivo.

Betulinic acid (BA), a naturally occurring lupane-type triterpenoid, possesses a wide range of potential activities against different types of cancer. However, the molecular mechanisms involved in anti-cervical cancer about BA were rarely investigated. Herein, the role of BA in cervical cancer suppression by ROS-mediated endoplasmic reticulum stress (ERS) and autophagy was deeply discussed. The findings revealed that BA activated Keap1/Nrf2 pathway and triggered mitochondria-dependent apoptosis due to ROS production. Furthermore, BA increased the intracellular Ca2+ levels, inhibited the expression of Beclin1 and promoted the expression of GRP78, LC3-II, and p62 associated with ERS and autophagy. Besides, BA initiated the formation of autophagosomes and inhibited autophagic flux by the co-administration of BA with 3-methyladenine (3-MA) and chloroquine (CQ), respectively. The in vivo experiment manifested that hydroxychloroquine (HCQ) enhanced the apoptosis induced by BA. For the first time, we demonstrated that BA could initiate early autophagy, inhibit autophagy flux, and induce protective autophagy in HeLa cells. Thus, BA could be a potential chemotherapy drug for cervical cancer, and inhibition of autophagy could enhance the anti-tumor effect of BA. However, the interactions of signaling factors between ERS-mediated and autophagy-mediated apoptosis deserve further attention.

Integrating plasma metabolomics and gut microbiome to reveal the mechanisms of Huangqi Guizhi Wuwu Decoction intervene diabetic peripheral neuropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Huangqi Guizhi Wuwu Decoction (HGWD) is a classic traditional Chinese herbal formula from "Synopsis of Golden Chamber," which is used to treat blood stagnation and has been used for alleviating diabetic peripheral neuropathy (DPN) in the clinic. However, the mechanisms of HGWD intervention DPN are still to be discovered. AIM OF THE STUDY: This study aims to explore the mechanism of HGWD intervention DPN by integrating plasma metabolomics and gut microbiome. MATERIALS AND METHODS: BKS Cg-m+/+Leprdb/J (db/db) mice with DPN were at 16 weeks of age. The indices of DPN phenotypes in db/db mice, pathomorphology of the sciatic nerve, intraepithelial nerve fibers (IENF) of the foot pad, levels of blood lipids and oxidative stress, and inflammatory reaction were used to appraise the HGWD efficacy. Finally, the pharmacological mechanisms of HGWD intervening DPN were explored by metabolomics and 16S rRNA gene sequencing. RESULTS: HGWD reversed DPN phenotypes in db/db mice, improved peripheral nerve structure, ameliorated the level of blood lipids and nerve growth factor in plasma, enhanced antioxidant capacity, and alleviated inflammatory responses. Plasma metabolomics disclosed that HGWD remarkably regulated the unusual levels of thirty-seven metabolites involved in sphingolipid metabolism, biosynthesis of unsaturated fatty acids, arachidonic acid metabolism, and amino acid biosynthesis pathways. The gut microbiome showed that nine bacteria were highly correlated with the efficacy of HGWD in DPN. Integrating analysis of microbiome and metabolomics demonstrated that the interaction of four bacteria with four metabolic pathways might be the significant mechanism of HGWD intervention in DPN. CONCLUSIONS: The mediation of gut microbiota and plasma metabolism may be the potential mechanism of HGWD ameliorating DPN in db/db mice. The interaction of Lactobacillus, Alloprevotella, Bacteroides, and Desulfovibio with four metabolic pathways might be the critical mechanism for HGWD treating DPN.

ß-Carboline alkaloids from Picrasma quassioides and their 3D-QSAR study on anti-inflammation in LPS-induced RAW 264.7 cells.

Two new ß-carboline alkaloids (1-2), 1-pyrrolidone propionyl-ß-carboline (1) and 1-(3-hydroxy-2-oxopiperidine-1-ethyl)-4,8-dimethoxyl-ß-carboline (2), named kumujantine W and J respectively, together with ten known compounds (3-12) were isolated from the stems of Picrasma quassioides (D. Don) Benn. Their structures were elucidated from spectral data including 1D and 2D NMR, UV, IR, HR-ESI-MS spectroscopic analysis and ECD calculations as well as by comparison to the reference databases or literature. The anti-inflammatory effects of these alkaloids (1-12) and six other ß-carboline alkaloids (13-18) in LPS-induced RAW 264.7 cells were evaluated by measuring nitric oxide (NO) concentrations. Among them, compounds 1, 3, 6, 15, and 17 could inhibit the secretion of NO, displaying significant anti-inflammatory activity without affecting cell viability in vitro, and 3D-QSAR analysis further revealed the influence of groups on the activity in ß-carboline alkaloids.

New chemical structures and liver-protective activity of the diterpenoids from Callicarpa rubella.

Callicarpa rubella is a characteristic folk herb in the genus Callicarpa, and has abundant ethnobotanical usage as indigenous medicine in Lingnan area of P. R. China. However, the phytochemical and pharmacological properties of C. rubella was rarely investigated. Now, three new diterpenoids, named rubellapene A-C (1-3), along with five known analogues (4-8), were isolated from C. rubella. Their structures were determined by spectroscopic methods, quantum chemical electronic circular dichroism calculations and single-crystal X-ray diffraction analysis. Notably, the norditerpenoids C18 of clerodane type (rubellapene B) was rarely found in the genus Callicarpa. The liver protective effects of all of the isolates (1-8) were evaluated by the changes of cell viability and transaminase content of AST and ALT in H2O2-induced BRL cells. Compound 1, 3-8 exhibited that potent liver protective effects at different levels.

A comparative study of Liandan Xiaoyan Formula metabolic profiles in control and colitis rats by UPLC-Q-TOF-MS combined with chemometrics.

Liandan Xiaoyan Formula (LDXYF) is a traditional Chinese medicine prescription (TCMP) consisting of Herba Andrographis (dried herb of Andrographis paniculata) and Picrasmae ramulus et folium (dried twiggeries and leaves of Picrasma quassioides). It is used to treat diarrhea, acute gastroenteritis, colitis, and dysentery, among other inflammatory gastrointestinal diseases. However, because of less research on the in vitro chemical composition and holistic metabolism of LDXYF, in vivo mechanisms of action and quality control of LDXYF have not yet been fully assessed due to the lack of studies into its bioactive components. In this study, ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was established for comprehensive analysis of chemical compounds of LDXYF and their metabolites in serum and urine samples of control and colitis rats. As a result, totally 94 compounds in LDXYF were unambiguously identified or tentatively characterized. And a total of 91 LDXYF-related xenobiotics were characterized, including 61 (16 prototypes and 45 metabolites) in serum, and 72 (26 prototypes and 46 metabolites) in urine. Besides, we compared the exposure of metabolites in normal and colitis rats by chemometrics and summarize similarities and differences of metabolic pathways of mainly compounds in normal and colitis conditions, and found that in control and colitis conditions, alkaloids predominantly went through phase I reaction combined phase II reaction (hydroxylation and sulfation, hydroxylation and glucuronidation, demethylation and glucuronidation), while the major metabolic reaction of diterpene lactones were phase Ⅱ reactions (glucuronidation, sulfation). And there were no significant differences in metabolic pathways between control and colitis groups, just the exposure of prototype and their metabolites absorbed into serum or excreted through the urine were different, and 17 alkaloids and 6 diterpene lactone prototypes and their metabolites in serum could be considered as potential pharmacodynamic substances. A comprehensive analysis of the compounds and metabolic characteristics of LDXYF was conducted in our study, and the results laid the chemical foundation for further research into effective substances and the action mechanism of LDXYF.

Nigakinone alleviates DSS-induced experimental colitis via regulating bile acid profile and FXR/NLRP3 signaling pathways.

The correlation of bile acid (BA) metabolism disorder with the pathogenesis of ulcerative colitis (UC) is realized nowadays. Farnesoid X receptor (FXR), a controller for BA homeostasis and inflammation, is a promising target for UC therapy. Nigakinone has potential therapeutic effects on colitis. Herein, we investigated the anti-UC effects and mechanism of nigakinone in colitic animals induced by dextran sulfate sodium (DSS). The related targets involved in the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) signaling pathway were measured. BA-targeted metabolomics was employed to reveal the regulatory effects of nigakinone on BA profile in colitis, while expressions of FXR and its mediated targets referring to BA enterohepatic circulation were determined. The critical role of FXR in the treatment of nigakinone for colitis was studied via molecule-docking, dual-luciferase reporter® (DLR™) assays, FXR silencing cells, and FXR knockout mice. Results showed nigakinone attenuated DSS-induced colitis symptoms, including excessive inflammatory response by NLRP3 activation, and injury of the intestinal mucosal barrier. Nigakinone regulated BA disorders by controlling cholesterol hydroxylase and transporters mediated by FXR, then decreased BA accumulation in colon. Molecular-docking and DLR™ assays indicated FXR might be a target of nigakinone. In vitro, nigakinone restrained BA-induced inflammation and cell damage via FXR activation and inhibition of inflammatory cytokines. However, ameliorating effects of nigakinone on colitis were suppressed by FXR knockout or silencing in vivo or in vitro. Taken together, nigakinone ameliorated experimental colitis via regulating BA profile and FXR/NLRP3 signaling pathway.

Diterpenoid alkaloids isolated from Delphinium trichophorum alleviate pulmonary fibrosis via the TGF-ß/Smad pathway in 3T6 and HFL-1 cells.

Delphinium trichophorum Franch (DTF), a species endemic to China, has been widely used for centuries in Tibet as an indigenous medicine for treating cough, pneumonia, and pulmonary fibrosis. Hetisine-type C20-diterpenoid alkaloids have been reported to be characteristic and active ingredients. Herein, five ones with relatively high contents in D. trichophorum, including 2α,11α,13ß-triacetylhetisine (DTF1), trichodelphinine A (DTF2), trichodelphinine D (DTF3), 2α-acetyl-11α,13ß-dihydroxyhetisine (DTF4), and trichodelphinine C (DTF5), were investigated for anti-fibrosis effects using fibroblasts induced by TGF-ß1 or LPS for the first time. The results showed that all five tested compounds decreased hydroxyproline (HYP) levels and inhibited the abnormal proliferation of 3T6 and HFL-1 cells induced by either TGF-ß1 or LPS. Moreover, DTF1 and DTF2 attenuated the production of collagen (Col-1 and Col-3) at relatively low doses, suggesting their higher efficiency among the five alkaloids. Based on large-scale ligand-based pharmacophore modeling, TGFBR1 was screened as a potential target for these tested alkaloids. The molecular docking results also exhibited high-affinity interactions between TGFBR1 and five alkaloids, especially DTF1 and DTF2. Further experiments revealed that DTF1 and DTF2 could inhibit the expression of TGF-ß1 and α-SMA and the phosphorylation of Smad3 and Smad4 while restoring the expression of Smad7 protein. Overall, DTF1 and DTF2 may reduce collagen generation and delay the development of pulmonary fibrosis by inhibiting the activation of the TGF-ß/Smad signaling pathway. Our results provide experimental and theoretical evidence for DTF1 and DTF2 as superior candidates for further development of anti-fibrotic drugs.

Comparative Pharmacokinetics of Three Bioactive Diterpenoids of Rabdosia serra Extract in Normal and Con A-Induced Liver Injury Rats Using UPLC-MS/MS.

Rabdosia serra (Maxim.) Hara (R. serra), one of the source plants of "Xihuangcao", has been widely used as a Chinese folk herb with the concomitant function of both medicine and foodstuff for the prevention and treatment of liver disease. Diterpenoids were considered as the major bioactive components in R. serra, responsible for their effect on hepatoprotection in previous phytochemical and pharmacological studies, while few comparative pharmacokinetic studies have been conducted under the physiological and pathological conditions. To reveal the difference in the pharmacokinetics process of R. serra extract (RSE) in normal and Con A-induced liver injury rats, a rapid ultra-high-pressure liquid chromatography-tandem mass spectrometry method (total running time: 5 min) was established to simultaneously determine three bioactive diterpenoids (enmein, epinodosin, and isodocarpin) in rat plasma. The results showed significant differences in the pharmacokinetic properties of three analytes between the physiological and pathological states. Compared with normal rats, the AUC of the three analytes was remarkably higher in liver injury rats, while the Tmax, T1/2, and MRT were shortened. It indicated that RSE has higher exposure and quicker elimination in liver injury rats than that in normal rats. Our results suggested that the pharmacokinetics of hepatoprotective medications was affected by liver injury, which prospected to provide essential information for guiding the healthcare and clinical application of R. serra in pathological states.

Pharmacokinetic Study of Four Major Bioactive Components of Liandan Xiaoyan Formula in Ulcerative Colitis and Control Rats Using UPLC-MS/MS.

Liandan Xiaoyan Formula (LXF), a classic Traditional Chinese medicine (TCM) formula, is composed of two Chinese herbal medicines for treating bowel disease under the TCM theory. This study aimed to develop a rapid, stable, sensitive, and reliable method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to simultaneously determine four major bioactive components of LXF (andrographolide, dehydroandrographolide, 1-methoxicabony-ß-carboline, 4-methoxy-5-hydroxy-canthin-6-one) in rat serum and evaluate the pharmacokinetic characteristics of LXF in ulcerative colitis (UC) and control rats. After pretreating by protein precipitation with methanol, separation was performed on a UPLC C18 column using gradient elution with a mobile phase consisting of acetonitrile and 0.1% formic acid at a flowing rate of 0.4 ml/min. Detection was performed on Triple-TOF™ 5600 mass spectrometry set at the positive ionization and multiple reaction monitoring (MRM) mode. The validated method showed good linearity (R 2 ≥ 0.9970), the intra- and inter-day accuracy were within ±11.58%, whereas the intra- and inter-day precision were less than 13.79%. This method was validated and applied to compare the pharmacokinetic profiles of the analytes in serum of UC induced by dextran sulphate sodium (DSS) and control rats after oral administration of LXF. The results showed that four major bioactive components of LXF were quickly absorbed after oral administration in both groups, with higher exposure levels in the UC group. This relationship between the active ingredients' pharmacokinetic properties provided essential scientific information for applying LXF in clinical.

Diterpenoid Alkaloids Isolated from Delphinium brunonianum and Their Inhibitory Effects on Hepatocytes Lipid Accumulation.

This research aimed to excavate compounds with activity reducing hepatocytes lipid accumulation from Delphinium brunonianum. Four novel diterpenoid alkaloids, brunodelphinine B-E, were isolated from D. brunonianum together with eleven known diterpenoid alkaloids through a phytochemical investigation. Their structures were elucidated by comprehensive spectroscopy methods including HR-ESI-MS, NMR, IR, UV, CD, and single-crystal X-ray diffraction analysis. The inhibitory effects of a total of 15 diterpenoid alkaloids on hepatocytes lipid accumulation were evaluated using 0.5 mM FFA (oleate/palmitate 2:1 ratio) to induce buffalo rat liver (BRL) cells by measuring the levels of triglyceride (TG), total cholesterol (TC), alanine transaminase (ALT), aspartate transaminase (AST), and the staining of oil red O. The results show that five diterpenoid alkaloids-brunodelphinine E (4), delbruline (5), lycoctonine (7), delbrunine (8), and sharwuphinine A (12)-exhibited significant inhibitory effects on lipid accumulation in a dose-dependent manner and without cytotoxicity. Among them, sharwuphinine A (12) displayed the strongest inhibition of hepatocytes lipid accumulation in vitro. Our research increased the understanding on the chemical composition of D. brunonianum and provided experimental and theoretical evidence for the active ingredients screened from this herbal medicine in the treatment of the diseases related to lipid accumulation, such as non-alcoholic fatty liver disease and hyperlipidemia.

Integrating metabolomics and network pharmacology to reveal the mechanisms of Delphinium brunonianum extract against nonalcoholic steatohepatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Herba Delphinii Brunoniani, a Tibetan Material Medica, derived from the aerial parts of Delphinium brunonianum Royle, possesses efficacy of cooling blood to remove apthogentic heat, and dispelling wind to arrest itching, and has been used for the treatment for liver disease according to Tibetan Medicine Theories in Shel Gong Shel Phreng. However, the mechanisms of action remain unclear. AIM OF THE STUDY: This work aimed to investigate the efficacy mechanism of Delphinium brunonianum extract (DBE) on nonalcoholic steatohepatitis (NASH), a kind of liver disease by integrating serum metabolomics and network pharmacology analysis. MATERIALS AND METHODS: In this study, NASH model mice were established by a high-fat diet. The indexes of lipid accumulation, insulin resistance, and inflammatory reaction were used to evaluate the efficacy of DBE. A combination of UHPLC-QTOF-MS based metabolomics and network pharmacology was established to illustrate the serum biomarkers of NASH mice and to demonstrate the anti-NASH mechanisms of DBE. Serum metabolomics demonstrated potential metabolites and the corresponding metabolic pathways in the efficacy of DBE. Network pharmacology screened the targets of DBE against NASH. Finally, the mechanisms of DBE against NASH were verified by in-vivo pharmacology. RESULTS: Metabolomics revealed that DBE significantly regulated the abnormal levels of twenty-two metabolites, which involved the biosynthesis of unsaturated fatty acids and steroid hormone, linoleic acid metabolism, arachidonic acid metabolism, and alpha-Linolenic acid metabolism pathways. Network pharmacology showed that DBE exhibited anti-NASH effects through regulating the targets of PTGS2, PLA2, ALOX5, ALOX15, FASN, and CYP450. Finally, united pharmacological verification result, we found that the mechanisms of DBE against NASH may be related to the regulation of the unsaturated fatty acids biosynthesis and the arachidonic acid metabolism pathway. CONCLUSIONS: Integrating serum metabolomic and network analysis, we found that DBE might inhibit the pathological process of NASH by regulating the relative targets and the metabolic pathways, which may be a potential mechanism for the anti-NASH efficacy of DBE. This integrated strategy also provided a rational way for revealing the pharmacodynamic mechanisms of multi-components, multi-targets, and multi-pathways in Traditional Chinese Medicine (TCM).

[Analysis of projects funded by NSFC in field of Chinese medicine toxicology from 2012 to 2021].

This study mainly introduced the research on Chinese medicine toxicology funded by the National Natural Science Foundation of China(NSFC) in 2012-2021 and analyzed the research content. Furthermore, key research topics and characteristic research projects were discussed, such as the toxicity mechanism, relationship between toxicity and efficacy, toxicity-alleviating mechanisms, and new technology and methods. The review suggested that researchers should gain an in-depth understanding of the "toxicity" of Chinese me-dicine, turned to characteristic research topics, and build a toxicological research paradigm suited to the characteristics of Chinese medicine in project application.

Authentication and Quality Assessment of Plumeriae Rubrae Flos by UHPLC-QTOF-MS/MS and UHPLC-DAD Combined With Chemometrics.

BACKGROUND: Plumeriae rubrae flos, the dried flowers of Plumeria rubra cv. acutifolia (PRCA), is one of the most important materials of herbal tea in China. Recently, due to the lack of effective quality evaluation standards, it has been found that Plumeria rubra (PR) and Plumeria rubra var. alba (PRVA) were pretended to be PRCA in herbal material markets. OBJECTIVE: To establish an effective method for comprehensive quality assessment on plumeriae rubrae flos, and distinguishing PRCA from its common adulterants, PR and PRVA. METHOD: In this study, a method combined application of ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS), UHPLC with a diode array detector (UHPLC-DAD), and chemometrics was developed for qualitative and quantitative analysis of PRCA, PR, and PRVA, based on their multiple components. RESULTS: A total of 26 components were identified by UHPLC-QTOF-MS/MS, including nine flavonoids, eight iridoids, seven phenolic acids, and two coumarins from PRCA. Quantified fingerprints were established and validated using UHPLC-DAD based on 18 chemical markers in PRCA, PR, and PRVA samples. The multivariate statistical analysis of quantitative results demonstrated clear discrimination of PRCA, PR, and PRVA, which indicated that isoquercetin, luteolin-3'-O-ß-D-glucoside, 15-demethylplumieride P-E-coumarate, and 4-O-beta-glucopyranosyl-cis-coumaric acid could be considered the most obvious characteristic components for distinguishing PRCA from PR and PRVA. CONCLUSIONS: A combination of quantitative fingerprint and chemometrics analysis provided an effective and reliable strategy for the quality control of PRCA. HIGHLIGHTS: The current study was prospected to apply a comprehensive quality control method for plumeriae rubrae flos.

A UPLC-MS/MS-based metabolomics analysis of the pharmacological mechanisms of rabdosia serra against cholestasis.

BACKGROUND: Rabdosia Serra, the dried aerial parts of Rabdosia serra (Maxim.) Hara (RS) from the Labiatae family, is a traditional Chinese herbal medicine called Xihuangcao. Although RS has been found to exert a therapeutic effect on cholestasis, the underlying molecular mechanism remains unclear. PURPOSE: This study was designed to investigate the pharmacological effect and mechanism of RS on cholestatic rats using metabolomics platform. METHODS: Histopathology and biochemical evaluations were performed to determine the therapeutic effect of RS and developed a rapid metabolite detection technology method based on UPLC-MS/MS to perform metabolomics research. Further, quantitative real-time polymerase chain reaction (RT-qPCR) was used to study the effect of RS on the bile acid metabolism pathway at the transcriptional level. RESULTS: RS significantly reduced the bile flow rates in cholestatic rats and decreased the levels of ALT, AST, TBA, T-BIL, and LDH, which were increased in the model group. Histological analysis showed that RS alleviated the liver injury induced by ANIT. Serum metabolomics results revealed 33 of the 37 biomarkers were found to be significantly altered by ANIT, and 26 were considerably changed following treatment with RS. Metabolic pathway analysis revealed four pathways such as primary bile acid biosynthesis, biosynthesis of unsaturated fatty acids, and arachidonic acid and tryptophan metabolism. The bile acid secretion process and the inflammation and oxidative stress processes are the major biochemical reactions following treatment with ANIT and RS. Bile acid-targeted metabolomics study showed that TCA, GCA, GCDCA, and GDCA might be sensitive biomarkers that induced liver injury. we found that treatment with RS regulated the levels of bile acid in the serum and liver and restored the proportion of bile acids, especially CA and conjugated bile acids, such as TCA and GCA, in the bile duct. RS increased the mRNA expression levels of FXR, SHP, BSEP, and MRP2 in livers, and IBABP, OST-α, and OST-ß in the ileum. CONCLUSION: In this study, RS was found to protect the liver by regulating multiple metabolic pathways and promoting the excretion of bile acids. Simultaneously, RS played an essential role in reversing the imbalance of bile acids and protected against cholestasis by regulating the expression of transporters associated with bile acids. We demonstrated the correlation between molecular mechanisms and metabolites, provide a reference for the fabrication of extracts that can be used to treat cholestasis.

Pharmacokinetic study of seven bioactive components of Xiaoyan Lidan Formula in cholestatic and control rats using UPLC-MS/MS.

A rapid, sensitive, and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed to simultaneously determine the major bioactive components of Xiaoyan Lidan Formula (XYLDF) in rat plasma, using sulfamethoxazole as the internal standard (IS). The seven major bioactive components are andrographolide, dehydroandrographolide, enmein, 1-methoxicabony-ß-carboline, 4,5-dimethoxy-canthin-6-one, 4-methoxy-5-hydroxy-canthin-6-one, and 1-hydroxymethyl-ß-carboline. After pretreating by protein precipitation with methanol, separation was performed on a UPLC C18 column using gradient elution with a mobile phase consisting of acetonitrile and 0.1% formic acid at a flowing rate of 0.7 mL/min. Detection was performed on TSQ Quantum mass spectrometry set at the positive/negative ionization and multiple reaction monitoring (MRM) mode. The intra- and inter-day precision were less than 9.8%, whereas the intra- and inter-day accuracy were within ± 13.4%. The method was validated and applied to compare the pharmacokinetic profiles of the analytes in serum of Alpha-naphthylisothiocyanate (ANIT)-induced cholestasis and control rats after oral administration of XYLDF. The results showed remarkable differences in pharmacokinetic properties of the analytes between cholestatic (model) and control groups, thereby providing essential scientific information for better understanding of mechanism of XYLDF and a reference for its clinical applications.

[Analysis of research status based on projects in pharmacokinetics of traditional Chinese medicine funded by National Natural Science Foundation of China].

The pharmacokinetics of traditional Chinese medicine is a subject that studies the dynamic changes of the absorption, distribution, metabolism and excretion of complex components of traditional Chinese medicine, which is an important method for elucidating the pharmacodynamic material basis, action characteristics, and compatibility mechanisms of traditional Chinese medicine. However, given on the fact that traditional Chinese medicine is a multi-dimensional and complex system with multiple components, multiple pathways, multiple targets, and an unclear pharmacodynamic material basis, the research on the pharmacokinetics of traditio-nal Chinese medicine has become a scientific and technological problem. Although the pharmacokinetics of traditional Chinese medicine has achieved remarkable development with the emergence of new theories, methods and technologies, there are still some problems in the application of the research direction of the pharmacokinetics of traditional Chinese medicine judging from the current application of the National Natural Science Foundation of China. Therefore, this article discussed the current research status on pharmacokinetics of traditional Chinese medicines by analyzing the projects funded by the National Natural Science Foundation of China in the past 5 years from 2016 to 2020, mainly including the application and funding analysis, main research contents of the projects in pharmacokinetics of traditional Chinese medicines. And the research hotspots, difficulties and deficiencies were focused in order to provide certain reference for researchers engaged in pharmacokinetics of traditional Chinese medicine.

Network pharmacology-based mechanism prediction and pharmacological validation of Xiaoyan Lidan formula on attenuating alpha-naphthylisothiocyanate induced cholestatic hepatic injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The well-known Chinese prescription, Xiaoyan Lidan Formula (XYLDF), possesses efficiency of heat-clearing, dampness-eliminating and jaundice-removing. It has long been used clinically for the treatment of hepatobiliary diseases due to intrahepatic cholestasis (IHC). However, the mechanism of XYLDF for its therapeutic effects remains elusive. AIM OF THE STUDY: The study aimed to explore the potential targets for liver protective mechanism of XYLDF based on network pharmacology and experimental assays in ANIT-induced cholestatic hepatic injury (CHI) in rats. MATERIALS AND METHODS: On the basis of the 29 serum migrant compounds of XYLDF elucidated by UPLC-TOF-MS/MS, a network pharmacology approach was applied for the mechanism prediction. Systematic networks were constructed to identify potential molecular targets, biological processes, and signaling pathways. And the interactions between significantly potential targets and active compounds were simulated by molecular docking. For the mechanism validation, an ANIT-induced rat model was used to evaluate the effects of XYLDF on CHI according to serum biochemistry, bile flow rates, histopathological examination, and the gene and protein expression including enzymes related to synthesis, export, and import of bile acid in liver and ileum, and those of inflammatory cytokines, analyzed by RT-qPCR and WB. RESULTS: The results of network pharmacology research indicated TNF (TNF-α), RELA (NF-κB), NR1H4 (FXR), and ICAM1 (ICAM-1) to be the important potential targets of XYLDF for cholestatic liver injury, which are related to bile metabolism and NF-κB-mediated inflammatory signaling. And the molecular docking had pre-validated the prediction of network pharmacology, as the core active compounds of XYLDF had shown strong simulation binding affinity with FXR, followed by NF-κB, TNF-α, and ICAM-1. Meanwhile, the effects of XYLDF after oral administration on ANIT-induced CHI in rats exhibited the decreased levels of transaminases (ALT and AST), TBA, and TBIL in serum, raised bile flow rates, and markedly improved hepatic histopathology. Furthermore, consistent to the above targets prediction and molecular docking, XYLDF significantly up-regulated the expression of FXR, SHP, BSEP, and MRP2, and down-regulated CYP7A1 and NTCP in liver, and promoted expression of IBABP and OSTα/ß in ileum, suggesting the activation of FXR-mediated pathway referring to bile acid synthesis, transportation, and reabsorption. Moreover, the lower levels of TNF-α in plasma and liver, as well as the reduced hepatic gene and protein expression of NF-κB, TNF-α, and ICAM-1 after XYLDF treatment revealed the suppression of NF-κB-mediated inflammatory signaling pathway, as evidenced by the inhibition of nuclear translocation of NF-κB. CONCLUSIONS: XYLDF exhibited an ameliorative liver protective effect on ANIT-induced cholestatic hepatic injury. The present study has confirmed its mechanism as activating the FXR-regulated bile acid pathway and inhibiting inflammation via the NF-κB signaling pathway.

5-Hydroxy-4-methoxycanthin-6-one alleviates dextran sodium sulfate-induced colitis in rats via regulation of metabolic profiling and suppression of NF-κB/p65 signaling pathway.

BACKGROUND: 5-Hydroxy-4-methoxycanthin-6-one (PQ-A) is the main active compound in Ramulus et Folium Picrasmae, a Chinese herbal medicine commonly used in colitis treatment. PURPOSE: To clarify PQ-A's role and mechanism in colitis treatment based on a non-targeted metabolomics study. METHODS: Rats with ulcerative colitis (UC) established with 4% dextran sulfate sodium (DSS) were orally treated with PQ-A. Body weight, disease activity index (DAI), colon length, biochemical parameters (MDA and SOD), and histopathological score in colon tissue were measured. A UPLC-Q-TOF-MS/MS approach-based metabolomics analysis was conducted to explore the underlying mechanisms of PQ-A in colitis treatment. Inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-10) concentrations in serum and their protein levels in the colon were determined. CD3 and NF-κB/p65 immunohistochemistry in the colon was semi-quantified. The related protein or mRNA in IKK-NF-κB/p65 signaling pathway was measured by Western blotting or RT-PCR, respectively. Potential molecular interactions between PQ-A and NF-κB/p65 was predicted using DS 2.5 software. RESULTS: PQ-A significantly prevented body weight loss and colonic shortening in colitic rats, and reduced the DAI and histopathologic score as well. PQ-A decreased MDA levels in the UC rat serum and increased those of SOD. Metabolomics results revealed forty-nine differential metabolites as biomarkers of DSS-induced colitis, demonstrating that the path-mechanism of colitis involved the perturbation of eight metabolic pathways, including alpha-linolenic acid and linoleic acid metabolism, sphingolipid metabolism, retinol metabolism, bile acid metabolism, et al. Thirty-six biomarkers were especially reversed to normal-like levels by PQ-A via regulation of alpha-linolenic acid and linoleic acid metabolism, sphingolipid metabolism, and retinol metabolism, which effectively hinted the potential pharmacological mechanism of PQ-A related to NF-κB/p65 inflammatory signaling. Molecular docking results predicted high affinity interaction between PQ-A and NF-κB/p65, involving hydrogen-bond interactions at five amino acid residues, suggesting NF-κB/p65 as a target. PQ-A decreased TNF-α, IL-1ß, and IL-6 concentrations in serum and their protein levels in colon tissue in colitic rats. CD3, MYD88, p-IκBα, NF-κB/p65, and p-NF-κB/p65 expression levels decreased, whereas those of IKKß and IκBα increased in colitic tissue following PQ-A treatment. PQ-A strongly inhibited nuclear translocation of NF-κB/p65. CONCLUSIONS: We provide an overview of PQ-A's possible mechanism of action in colitis treatment based on serum non-targeted metabolomics. PQ-A treatment can protect rats against DSS-induced colitis by suppressing the NF-κB/p65 signaling pathway.