Human UDP-glucuronosyltransferase 1As catalyze aristolochic acid D O-glucuronidation to form a lesser nephrotoxic glucuronide.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. AIM OF THE STUDY: To identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity. MATERIALS AND METHODS: The O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD. RESULTS: AAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-ß-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 µM, 9.05 µM, 3.87 µM, and 7.00 µM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. CONCLUSION: Collectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.

Rationally Engineered CYP3A4 Fluorogenic Substrates for Functional Imaging Analysis and Drug-Drug Interaction Studies.

Cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme-mediated drug metabolism and drug-drug interaction (DDI). Herein, an effective strategy was used to rationally construct a practical two-photon fluorogenic substrate for hCYP3A4. Following two-round structure-based substrate discovery and optimization, we have successfully constructed a hCYP3A4 fluorogenic substrate (F8) with desirable features, including high binding affinity, rapid response, excellent isoform specificity, and low cytotoxicity. Under physiological conditions, F8 is readily metabolized by hCYP3A4 to form a brightly fluorescent product (4-OH F8) that can be easily detected by various fluorescence devices. The practicality of F8 for real-time sensing and functional imaging of hCYP3A4 has been examined in tissue preparations, living cells, and organ slices. F8 also demonstrates good performance for high-throughput screening of hCYP3A4 inhibitors and assessing DDI potentials in vivo. Collectively, this study develops an advanced molecular tool for sensing CYP3A4 activities in biological systems, which strongly facilitates CYP3A4-associated fundamental and applied research studies.

Tongxie-Yaofang formula regulated macrophage polarization to ameliorate DSS-induced colitis via NF-κB/NLRP3 signaling pathway.

BACKGROUND: Macrophages infiltration and activation play multiple roles in maintaining intestinal homeostasis and participate in the occurrence and development of UC. Thus, the restoration of immune balance can be achieved by targeting macrophage polarization. Previous studies have reported that TXYF could effectively ameliorate DSS-induced colitis. However, the underlying mechanisms of TXYF for DSS-induced colitis are still ill-defined. METHODOLOGY: This study was designed to explore the therapeutic effect of TXYF and its regulation in macrophages polarization during DSS-induced mice. In C75BL/6 mice, dextran sulfate sodium (DSS) was used to induce colitis and concomitantly TXYF was taken orally to evaluate its curative effect. In vitro experiment was implemented on BMDMs by lipopolysaccharide, IFN- and ATP. RESULTS: Here, we found that TXYF ameliorated clinical features in DSS-induced mice, decreased macrophages M1 polarization but remarkably increased M2 polarization. Mechanically, TXYF treatment effectively inhibited the activities of nuclear transcription factor NF-κB, which further contributed to the decrease of the inflammasome genes of NLRP3, limiting the activation of NLRP3 inflammasome in vivo and in vitro. CONCLUSION: Our findings demonstrated administration of TXYF can interfere with macrophage infiltration and polarization to improve the symptoms of acute colitis, by repressing NF-κB/NLRP3 signaling pathway activation. This enriches the mechanism and provides new prospect for TXYF in the treatment of colitis.

Methylophiopogonanone A is a naturally occurring broad-spectrum inhibitor against human UDP-glucuronosyltransferases: Inhibition behaviours and implication in herb-drug interactions.

Methylophiopogonanone A (MOA) is an abundant homoisoflavonoid in the Chinese herb Ophiopogonis Radix. Recent investigations revealed that MOA inhibited several human cytochrome P450 enzymes (CYPs) and stimulated OATP1B1. However, the inhibitory effects of MOA on phase II drug-metabolizing enzymes, such as human UDP-glucuronosyltransferases (hUGTs), have not been well investigated. Herein, the inhibition potentials of MOA on hUGTs were assessed. The results clearly demonstrated that MOA dose-dependently inhibited all tested hUGTs including UGT1A1 (IC50 = 1.23 µM), one of the most important detoxification enzymes in humans. Further investigations showed that MOA strongly inhibited UGT1A1-catalysed NHPH-O-glucuronidation in a range of biological settings including hUGT1A1, human liver microsomes (HLM) and HeLa cells overexpressing UGT1A1. Inhibition kinetic analyses demonstrated that MOA competitively inhibited UGT1A1-catalysed NHPH-O-glucuronidation in both hUGT1A1 and HLM, with Ki values of 0.52 and 1.22 µM, respectively. Collectively, our findings expanded knowledge of the interactions between MOA and human drug-metabolizing enzymes, which would be very helpful for guiding the use of MOA-related herbal products in clinical settings.

A TCM formula VYAC ameliorates DNCB-induced atopic dermatitis via blocking mast cell degranulation and suppressing NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: A Traditional Chinese Medicine (TCM) formula (VYAC) consists of three herbs including Viola yedoensis Makino, herb (Violaceae, Viola), Sophora flavescens Aiton, root (Fabaceae, Sophora) and Dictamnus dasycarpus Turcz, root and rhizome (Rutaceae, Dictamnus), has been traditionally prescribed to treat various skin diseases in clinic. AIM OF THE STUDY: This study aims to investigate the therapeutic effects of VYAC on the 2,4-dinitrobenzene (DNCB) induced atopic dermatitis (AD)-like mice and to explore the underlying mechanisms. MATERIALS AND METHODS: VYAC was extracted with 70 % aqueous ethanol and lyophilized powder was used. AD-like mice were challenged by DNCB, VYAC (150 and 300 mg/kg) were oral administration daily from day 7 to day 28. At the end of experiment, the clinical scores were recorded, serum and skin in the dorsal were isolated to evaluate the therapeutic effects of VYAC. RBL-2H3 cells were stimulated with C48/80 for degranulation and plasmids expressing constitutively active form of Syk (Silence or overexpression) were transfected into RBL-2H3 cells to explore the underlying mechanisms in vitro. RESULTS: VYAC significantly ameliorated the cardinal symptoms in the DNCB-induced AD-like mice by repairing the skin barrier function, inhibiting mast cells infiltration, restraining the serum IgE and histamine release and decreasing TNF-α, IL-4 as well as Syk mRNA level in dorsal skin and alleviating inflammation. Besides, VYAC significantly blocked RBL-2H3 cells degranulation, reduced ß-hexosaminidase and histamine release, and suppressed NF-κB pathway. What's more, the degranulation of RBL-2H3 was reduced after Syk silence and increased after Syk overexpression. CONCLUSION: Our findings clearly suggested that VYAC treat AD through inhibiting the inflammatory mediator productions and blocking mast cell degranulation via suppressing Syk mediated NF-κB pathway.

Development of (G3-C12)-mediated camptothecin polymeric prodrug targeting to Galectin-3 receptor against androgen-independent prostate cancer.

The development of small molecule anticancer drugs, with low water solubility and high toxicity, into polymeric prodrugs has developed into a promising strategy in clinical application. In this study, we synthesized a novel G3-C12-mediated esterase-sensitive tumor-targeting polymeric prodrug of camptothecin (CPT), P(OEGMA-co-CPT-co-G3-C12), and explored its anticancer activity against androgen-independent prostate cancer in vitro and in vivo. Compared to free CPT, the multifunctional polymeric prodrug demonstrated improved water solubility and stability, higher intracellular uptake, and enhanced cytotoxicity in DU145 cells in vitro. Furthermore, it displayed an improved accumulation in the tumor and an enhanced anticancer activity in vivo. Hence, P(OEGMA-co-CPT-co-G3-C12) could be a promising drug in the treatment of androgen-independent prostate cancer.

[Spectrum-effect relationship between HPLC fingerprint and free radicals scavenging in Guizhi Shaoyao Zhimu Decoction].

To establish the spectrum-effect relationship between HPLC fingerprint and free radicals activity scavenging in Guizhi Shaoyao Zhimu Decoction( GSZD),and provide a basis for the quality evaluation and modernization of classical prescriptions. Shimadsu GL-science C18 column( 4. 6 mm×250 mm,5 µm) was used with acetonitrile-0. 1% formic acid solution as the mobile phase for gradient elution. The detective wave length was 254 nm; the column temperature was set at 32 ℃; the injection volume was 20 µL; and the flow rate was 1. 0 m L·min-1.10 batches of primary standard samples of GSZD were detected,and their HPLC fingerprint was established by using the similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine( TCM). The activity of scavenging free radicals was studied by 1,1-diphenyl-2-trinitrophenylhydrazine( DPPH) method,and the spectrum-effect relationship was studied by Pearson bivariate correlation analysis. The common mode of GSZD fingerprints was established,and 26 common peaks were marked,with similarities ranging from 0. 929 to 0. 998. Eight of the chromatographic peaks were identified by using the control comparison method: gallic acid,mangiferin,paeoniflorin,glycyrrhizin,asparagus,5-O-methylvisamicin,cinnamic acid,and ammonium glycyrrhetate. Among them,the content changes of No. 14( paeoniside),20,12( mangiferin),13 and 23( cinnamic acid) common peaks were negatively correlated with free radical scavenging activity. The fingerprint showed high precision,repeatability and stability,and the common peaks were well separated,so it can be used for the quality evaluation of GSZD,and could provide reference for further studies on the material basis of GSZD.

Rhizoma coptidis as a Potential Treatment Agent for Type 2 Diabetes Mellitus and the Underlying Mechanisms: A Review.

Diabetes mellitus, especially type 2 diabetes mellitus (T2DM), has become a significant public health burden. Rhizoma coptidis (RC), known as Huang Lian, is widely used for treating diabetes in China. The bioactive compounds of RC, especially alkaloids, have the potential to suppress T2DM-induced lesions, including diabetic vascular dysfunction, diabetic heart disease, diabetic hyperlipidemia, diabetic nephropathy, diabetic encephalopathy, diabetic osteopathy, diabetic enteropathy, and diabetic retinopathy. This review summarizes the effects of RC and its bioactive compounds on T2DM and T2DM complications. Less research has been conducted on non-alkaloid fractions of RC, which may exert synergistic action with alkaloids. Moreover, we summarized the pharmacokinetic properties and structure-activity relationships of RC on T2DM with reference to extant literature and showed clearly that RC has potential therapeutic effect on T2DM.

Coptidis Rhizoma: a comprehensive review of its traditional uses, botany, phytochemistry, pharmacology and toxicology.

CONTEXT: Coptidis rhizome (CR), also known as Huanglian in Chinese, is the rhizome of Coptis chinensis Franch., C. deltoidea C.Y. Cheng et Hsiao, or C. teeta Wall (Ranunculaceae). It has been widely used to treat bacillary dysentery, diabetes, pertussis, sore throat, aphtha, and eczema in China. OBJECTIVES: The present paper reviews the latest advances of CR, focusing on the botany, phytochemistry, traditional usages, pharmacokinetics, pharmacology and toxicology of CR and its future perspectives. METHODS: Studies from 1985 to 2018 were reviewed from books; PhD. and MSc. dissertations; the state and local drug standards; PubMed; CNKI; Scopus; the Web of Science; and Google Scholar using the keywords Coptis, Coptidis Rhizoma, Huanglian, and goldthread. RESULTS: Currently, 128 chemical constituents have been isolated and identified from CR. Alkaloids are the characteristic components, together with organic acids, coumarins, phenylpropanoids and quinones. The extracts/compounds isolated from CR cover a wide pharmacological spectrum, including antibacterial, antivirus, antifungal, antidiabetic, anticancer and cardioprotective effects. Berberine is the most important active constituent and the primary toxic component of CR. CONCLUSIONS: As an important herbal medicine in Chinese medicine, CR has the potential to treat various diseases. However, further research should be undertaken to investigate the clinical effects, toxic constituents, target organs and pharmacokinetics, and to establish criteria for quality control, for CR and its related medications. In addition, the active constituents, other than alkaloids, in both raw and processed products of CR should be investigated.

Cellular stress response mechanisms of Rhizoma coptidis: a systematic review.

Rhizoma coptidis has been used in China for thousands of years with the functions of heating dampness and purging fire detoxification. But the underlying molecular mechanisms of Rhizoma coptidis are still far from being fully elucidated. Alkaloids, especially berberine, coptisine and palmatine, are responsible for multiple pharmacological effects of Rhizoma coptidis. In this review, we studied on the effects and molecular mechanisms of Rhizoma coptidis on NF-κB/MAPK/PI3K-Akt/AMPK/ERS and oxidative stress pathways. Then we summarized the mechanisms of these alkaloid components of Rhizoma coptidis on cardiovascular and cerebrovascular diseases, diabetes and diabetic complications. Evidence presented in this review implicated that Rhizoma coptidis exerted beneficial effects on various diseases by regulation of NF-κB/MAPK/PI3K-Akt/AMPK/ERS and oxidative stress pathways, which support the clinical application of Rhizoma coptidis and offer references for future researches.

Effects of Viola yedoensis Makino anti-itching compound on degranulation and cytokine generation in RBL-2H3 mast cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese herb compound prescription Viola yedoensis Makino Anti-itching Compound (VYAC), which consists of Viola yedoensis Makino, herb, Sophora flavescens Aiton, root, and Dictamnus dasycarpus Turcz, root and rhizome, has been traditionally used to treat various skin allergic inflammatory diseases in clinic. AIM OF THE STUDY: The aim of this study is to investigate the effects of VYAC on degranulation and to determine its anti-inflammatory mechanism in RBL-2H3 mast cells. MATERIALS AND METHODS: VYAC was extracted with water-coction extraction (Shufen et al., 2012). The aqueous extracts were concentrated in vacuum under reduced pressure and lyophilized using a freeze dryer, and lyophilized powder was obtained. MTT was used to evaluate the cytotoxic of VYAC on RBL-2H3 cells. Degranulation was carried out with RBL-2H3 cell model, which was stimulated with A23187 plus PMA. ß-Hexosaminidase and histamine were measured to evaluate degranulation. The mRNA levels of inflammation cytokines (IL-1ß, TNF-α, IL-6, and iNOS) were investigated by RT-PCR to explain the anti-inflammatory mechanism of VYAC. RESULTS: VYAC did not show cytotoxic effect on RBL-2H3 cells in the range of 25-400µg/mL. A higher dose of VYAC (800µg/mL) showed significant cytotoxicity (P<0.05). VYAC could significantly inhibit ß-hexosaminidase and histamine release when treated with 100, 200, and 400µg/mL (P<0.05), but could not significantly inhibit ß-Hexosaminidase and histamine release when treated with 25 and 50µg/mL (p>0.05). The mRNA levels of inflammatory cytokines (TNF-α, IL-1ß, IL-6, and iNOS) could significantly decrease when treated with 200 and 400µg/mL (P<0.05) of VYAC, which were associated with the development of inflammation. CONCLUSIONS: Results showed that VYAC inhibited ß-hexosaminidase and histamine release, which was inhibit A23187 plus PMA stimulated RBL-2H3 cell degranulation and downregulated inflammatory cytokines (IL-1ß, TNF-α, IL-6, and iNOS) expression to block inflammatory development.