A high-affinity fluorescent probe for human uridine-disphosphate glucuronosyltransferase 1A9 function monitoring under environmental pollutant exposure.

Uridine-disphosphate glucuronosyltransferase 1A9 (UGT1A9), an important detoxification and inactivation enzyme for toxicants, regulates the exposure level of environmental pollutants in the human body and induces various toxicological consequences. However, an effective tool for high-throughput monitoring of UGT1A9 function under exposure to environmental pollutants is still lacking. In this study, 1,3-dichloro-7-hydroxy-9,9-dimethylacridin-2(9H)-one (DDAO) was found to exhibit excellent specificity and high affinity towards human UGT1A9. Remarkable changes in absorption and fluorescence signals after reacting with UGT1A9 were observed, due to the intramolecular charge transfer (ICT) mechanism. Importantly, DDAO was successfully applied to monitor the biological functions of UGT1A9 in response to environmental pollutant exposure not only in microsome samples, but also in living cells by using a high-throughput screening method. Meanwhile, the identified pollutants that disturb UGT1A9 functions were found to significantly influence the exposure level and retention time of bisphenol S/bisphenol A in living cells. Furthermore, the molecular mechanism underlying the inhibition of UGT1A9 by these pollutant-derived disruptors was elucidated by molecular docking and molecular dynamics simulations. Collectively, a fluorescent probe to characterize the responses of UGT1A9 towards environmental pollutants was developed, which was beneficial for elucidating the health hazards of environmental pollutants from a new perspective.

Potent Inhibition of Human Cytochrome P450 3A4 by Biflavone Components from Ginkgo Biloba and Selaginella Tamariscina.

CYP3A4-mediated Phase I biotransformation is the rate-limiting step of elimination for many commonly used clinically agents. The modulatory effects of herbal medicines on CYP3A4 activity are one of the risk factors affecting the safe use of drug and herbal medicine. In the present study, the inhibitory effects of nearly hundred kinds of herbal medicines against CYP3A4 were evaluated based on a visual high-throughput screening method. Furthermore, biflavone components including bilobetin (7-demethylginkgetin, DGK), ginkgetin (GK), isoginkgetin (IGK), and amentoflavone (AMF) were identified as the main inhibitory components of Ginkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST), which displayed very strong inhibitory effects toward CYP3A4. The inhibitory effects of these biflavones on clinical drugs that mainly undergo CYP3A4-dependent metabolism were evaluated. The IC 50 of GK toward tamoxifen, gefitinib and ticagrelor were found to be of 0.478 ± 0.003, 0.869 ± 0.001, and 1.61 ± 0.039 µM, respectively. These results suggest the potential pharmacokinetic interactions between the identified biflavones and clinical drugs undergoing CYP3A4-mediated biotransformation. The obtained information is important for guiding the rational use of herbal medicine in combination with synthetic pharmaceuticals.

Triterpenoids from the fruiting bodies of Ganoderma lucidum and their inhibitory activity against FAAH.

Seventeen triterpenoids including four new lanostane triterpenoids (1-3 and 5) were isolated from the fruiting bodies of Ganoderma lucidum by various chromatographic techniques. Their chemical structures were determined by extensive spectroscopic data, including 1D-NMR, 2D-NMR, and HRESIMS. In addition, the spectral data of compound 4 was reported for the first time. In an in vitro bioassay, most isolated triterpenoids could inhibit the hydrolysis activity of fatty acid amide hydrolase (FAAH). Furthermore, there is no cytotoxicity observed for these isolated triterpenoids. Therefore, G. lucidum showed the potential application for anti-neuroinflammation and more FAAH inhibitors may be explored from G. lucidum.

Regioselective hydroxylation of carbendazim by mammalian cytochrome P450: A combined experimental and computational study.

Carbendazim (CBZ), a broad-spectrum pesticide frequently detected in fruits and vegetables, could trigger potential toxic risks to mammals. To facilitate the assessment of health risks, this study aimed to characterize the cytochrome P450 (CYPs)-mediated metabolism profiles of CBZ by a combined experimental and computational study. Our results demonstrated that CYPs-mediated region-selective hydroxylation was a major metabolism pathway for CBZ in liver microsomes from various species including rat, mouse, minipig, dog, rabbit, guinea pig, monkey, cow and human, and the metabolite was biosynthesized and well-characterized as 6-OH-CBZ. CYP1A displayed a predominant role in the region-selective hydroxylation of CBZ that could attenuate its toxicity through converting it into a less toxic metabolite. Meanwhile, five other common pesticides including chlorpyrifos-methyl, prochloraz, chlorfenapyr, chlorpyrifos, and chlorothalonil could significantly inhibit the region-selective hydroxylation of CBZ, and consequently remarkably increased CBZ exposure in vivo. Furthermore, computational study clarified the important contribution of the key amino acid residues Ser122, and Asp313 in CYP1A1, as well as Asp320 in CYP1A2 to the hydroxylation of CBZ through hydrogen bonds. These results would provide some useful information for the metabolic profiles of CBZ by mammalian CYPs, and shed new insights into CYP1A-mediated metabolic detoxification of CBZ and its health risk assessment.

Identification of Escherichia coli β-glucuronidase inhibitors from Polygonum cuspidatum Siebold & Zucc

Abstract Gut bacterial β-glucuronidase (GUS) can reactivate xenobiotics that exert enterohepatic circulation- triggered gastrointestinal tract toxicity. GUS inhibitors can alleviate drug-induced enteropathy and improve treatment outcomes. We evaluated the inhibitory effect of Polygonum cuspidatum Siebold & Zucc. and its major constituents against Escherichia coli GUS (EcGUS), and characterized the inhibitory mechanism of each of the components. Trans-resveratrol 4'-O-β-D-glucopyranoside (HZ-1) and (-)-epicatechin gallate (HZ-2) isolated from P. cuspidatum were identified as the key components and potent inhibitors. These two components displayed strong to moderate inhibitory effects on EcGUS, with Ki values of 9.95 and 1.95 μM, respectively. Results from molecular docking indicated that HZ-1 and HZ-2 could interact with the key residues Asp163, Ser360, Ile 363, Glu413, Glu504, and Lys 568 of EcGUS via hydrogen bonding. Our findings demonstrate the inhibitory effect of P. cuspidatum and its two components on EcGUS, which supported the further evaluation and development of P. cuspidatum and its two active components as novel candidates for alleviating drug-induced damage in the mammalian gut.

Bisfischoids A and B, dimeric ent-abietane-type diterpenoids with anti-inflammatory potential from Euphorbia fischeriana Steud.

Two undescribed ent-abietane-type diterpenoid dimers with nonacyclic backbone formed by intermolecular [4 + 2] cycloaddition into a spirocyclic skeleton, bisfischoids A (1) and B (2), along with a known one fischdiabietane A (3), were identified from Euphorbia fischeriana Steud. Their structures were elucidated by extensive spectroscopic analysis, ECD and NMR calculation combined with DP4+ probability analysis, as well as X-ray diffraction. The anti-inflammatory potential of dimers 1-3 were examined using their inhibitory effects on soluble epoxide hydrolase (sEH), which revealed that 1 and 2 exhibited promising activities with inhibition constant (Ki) of 3.20 and 1.95 µM, respectively. Further studies of molecular docking and molecular dynamics indicated that amino acid residue Tyr343 in the catalytic cavity of sEH was the key site for their inhibitory function.

Natural soluble epoxide hydrolase inhibitors from Alisma orientale and their potential mechanism with soluble epoxide hydrolase.

Inhibition of soluble epoxide hydrolase (sEH) is considered to be an effective treatment for inflammation-related diseases, and small molecules origin from natural products show promising activity against sEH. Two undescribed protostanes, 3ß-hydroxy-25-anhydro-alisol F (1) and 3ß-hydroxy-alisol G (2) were isolated from Alisma orientale and identified as new sEH inhibitors with IC50 values of 10.06 and 30.45 µM, respectively. Potential lead compound 1 was determined as an uncompetitive inhibitor against sEH, which had a Ki value of 5.13 µM. In-depth molecular docking and molecular dynamics simulations revealed that amino acid residue Ser374 plays an important role in the inhibition of 1, which also provides an idea for the development of sEH inhibitors based on protostane-type triterpenoids.

Amentoflavone from Selaginella tamariscina as a potent inhibitor of gut bacterial ß-glucuronidase: Inhibition kinetics and molecular dynamics stimulation.

Gut bacterial ß-glucuronidase (GUS) plays a pivotal role in the metabolism and reactivation of a vast of glucuronide conjugates of both endogenous and xenobiotic compounds in the gastrointestinal tract of human, which has been implicated in certain drug-induced gastrointestinal tract (GI) toxicity in clinic. Inhibitors of gut microbial GUS exhibited great potentials in relieving the drug-induced GI toxicity. In this study, Selaginella tamariscina and its major biflavonoid amentoflavone (AMF) were evaluated for their inhibitory activity against Escherichia coli GUS. Two selective probe substrates for GUS (a specific fluorescent probe substrate for GUS, DDAOG and a classical drug substrate for GUS, SN38G) were used in parallel for charactering the inhibition behaviors. Both the extract of S. tamariscina and its major biflavonoid AMF displayed evident inhibitory effects on GUS, and the IC50 values of AMF against GUS mediated DDAOG and SN-38G hydrolysis were 0.62 and 0.49 µM, respectively. Inhibition kinetics studies indicated that AMF showed mixed type inhibition for GUS-mediated DDAOG hydrolysis, while displayed competitive type inhibition against GUS-mediated SN-38G hydrolysis, with the Ki values of 0.24 and 1.25 µM, respectively. Molecular docking studies and molecular dynamics stimulation results clarified the role of amino acid residues Leu361, Ile363, and Glu413 in the inhibition of AMF on GUS. These results provided some foundations for the potential clinical utility of S. tamariscina and its major biflavonoid AMF for treating drug-induced enteropathy.

Medicinal Inula Species: Phytochemistry, Biosynthesis, and Bioactivities.

As a genus of the Asteraceae, Inula is widely distributed all over the world, and several of them are being used in traditional medicines. A number of metabolites were isolated from Inula species, and some of these have shown to possess ranges of pharmacological activities. The genus Inula contains abundant sesquiterpenoids, such as eudesmanes, xanthanes, and sesquiterpenoid dimers and trimers. In addition, other types of terpenoids, flavonoids, and lignins also exist in the genus Inula. Since 2010, more than 300 new secondary metabolites, including several known natural products that were isolated for the first time from the genus Inula. Most of them exhibited potential bioactivities in various diseases. The review aimed to summarize the advance of recent researches (2010-2020) on phytochemical constituents, biosynthesis, and pharmacological properties of the genus Inula for providing a scientific basis and supporting its application and exploitation for new drug development.

A highly selective near infrared fluorescent probe for carboxylesterase 2 and its biological applications.

Carboxylesterase 2 (CES 2) is a key enzyme in the activation of the prodrug irinotecan (CPT-11) in the treatment against colorectal cancer and also has some relationship with the side effect of CPT-11 in clinical applications. Herein, a near infrared (NIR) fluorescent probe (DSAB) has been designed for CES 2 which possesses the advantages of prominent selectivity and high sensitivity, and DSAB has been successfully applied for the imaging of endogenous CES 2 in living cells. Moreover, a high-throughput screening method for CES 2 inhibitors has been established using DSAB and discovered four novel CES 2 inhibitors from various herbal medicines. These results fully demonstrated that DSAB is a promising molecular tool for the investigation of the biological functions of CES 2 in living systems and the discovery of novel CES 2 inhibitors for the treatment of CES 2 related physiological diseases.

Alisma genus: Phytochemical constituents, biosynthesis, and biological activities.

The genus Alisma contains 11 species distributed worldwide, of which at least two species (A. orientale [Sam.] Juzep. and A. plantago-aquatica Linn.) have been used as common herbal medicines. Secondary metabolites obtained from the genus Alisma are considered to be the material basis for the various biological functions and medicinal applications. In this review, we mainly focused on the recent investigations of secondary metabolites from plants of the genus Alisma and their biological activities, with the highlighting on the diversity of the chemical structures, the biosynthesis of interesting secondary metabolites, the biological activities, and the relationships between structures and bioactivities.

Investigation of the inhibitory effect of protostanes on human carboxylesterase 2 and their interaction: Inhibition kinetics and molecular stimulations.

Carboxylesterase 2 (CES 2), plays a pivotal role in endobiotic homeostasis and xenobiotic metabolism. Protostanes, the major constituents of the genus Alisma, display a series of pharmacological activities. Despite the extensive studies of pharmacological activities, the investigation on inhibitory effects of protostanes against CES 2 is rarely reported. In this study, the inhibitory activities of a library of protostanes (1-25) against human CES 2 were investigated for the first time, using 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate (DDAB) as the specific fluorescent probe for human CES 2. Compounds 1, 2, 7, 8, 12, 13, 18, 19, and 25 showed strong inhibitory effects towards CES 2. For the most potent compounds 1, 7, 13, and 25, the inhibition kinetics were further investigated, and these four protostanes were all uncompetitive inhibitors against human CES 2 with the inhibition constant (Ki) values ranging from 0.89 µM to 2.83 µM. In addition, molecular docking and molecular dynamics stimulation were employed to analyze the potential interactions between these protostanes and CES 2, and amino acid residue Gln422 was identified to play a crucial role in the strong inhibition of protostanes towards CES 2.

The genus Uncaria: A review on phytochemical metabolites and biological aspects.

The genus Uncaira (Rubiaceae) comprises of 34 species, many of which are usually used as traditional Chinese medicines (TCMs) to treat hypertension, fever, headache, gastrointestinal illness, and fungal infection. Over the past twenty years, Uncaira species have been paid the considerable attentions in phytochemical and biological aspects, and about 100 new secondary metabolites, including alkaloids, triterpenes, and flavonoids, have been elucidated. This review aims to present a comprehensive and up-to date overview of the biological source, structures and their biosynthetic pathways, as well as the pharmacological of the compounds reported in the genus Uncaria for the past two decades. It would provide an insight into the emerging pharmacological applications of the genus Uncaria.

The inhibition effect of uncarialin A on voltage-dependent L-type calcium channel subunit alpha-1C: Inhibition potential and molecular stimulation.

Cardiovascular diseases, such as hypertension and cardiac failure, have become the most major and global cause for threatening human health in recent years. Uncaria rhynchophylla as a traditional Chinese medicine is widely used to treat hypertension for a long history, whereas its medicinal effective components and potential action mechanism are uncertain. Therefore, twenty-four alkaloids (1-24) isolated from U. rhynchophylla were assayed for their relaxant effects against phenylephrine (Phe)-induced contraction of rat mesenteric arteries. Among them, we surprisingly found that uncarialin A (21) exhibited most potent relaxation effect against Phe-induced contraction (IC50 = 0.18 µM) in the manner of independent on endothelium-derived vasorelaxing factors and endothelium. All the experiments including measurement of Ca2+ in vascular smooth muscle cells (VSMCs) by fluorescence microscopy, whole-cell path clamp, molecular docking, and molecular dynamics, demonstrated that uncarialin A (21) could significantly inhibit L-type calcium channel subunit alpha-1C (Cav1.2) via the hydrogen bond interaction with amino acid residue Met1186, allowing the inhibition of Ca2+ inward current. Our results suggested that uncarialin A (21) could be served as a potential L-type Cav1.2 blocker in the effective treatment of cardiovascular diseases.

The study of inhibitory effect of natural flavonoids toward ß-glucuronidase and interaction of flavonoids with ß-glucuronidase.

ß-Glucuronidase plays a vital role in the metabolism of drugs and endogenous substance. Herein, we assayed the inhibitory effects of thirty-six flavonoids (1-36) toward ß-glucuronidase (Escherichia coli) using the probe reaction of DDAO-glu hydrolysis. The results showed that kushenol X (6), (2S)-farrerol (10), 5,7,2'-trihydroxy-8,6'-dimethoxy flavone (20), demethylbellidifolin (31), and gentisin (32) exhibited potent inhibitory activities toward ß-glucuronidase with the IC50 values of 2.07 ± 0.26, 8.95 ± 0.74, 4.97 ± 0.61, 0.91 ± 0.11, and 0.68 ± 0.10 µM, respectively. Furthermore, the inhibition kinetics studies indicated that demethylbellidifolin (31) and gentisin (32) exhibited mixed-type inhibiton toward ß-glucuronidase, the Ki values were caculated to be 4.05 and 2.02 µM, respectively. Additionally, the circular change of dichroism (CD) spectrum verified the interaction between demethylbellidifolin (31) and gentisin (32) with ß-glucuronidase; following by the molecular docking and molecular dynamics further revealed the potential interaction amino acid site in ß-glucuronidase. All our findings not only developed some potent novel ß-glucuronidase inhibitors but also indicated the potential herb drug interaction (HDI) effects of flavonoids with some clinical drugs which had enterohepatic circulation and further revealed the vital pharamcophoric requirement of natural flavonoids for ß-glucuronidase inhibition activity.

Uncarialins A-I, Monoterpenoid Indole Alkaloids from Uncaria rhynchophylla as Natural Agonists of the 5-HT1A Receptor.

Nine new monoterpenoid indole alkaloids, uncarialins A-I (1-9), were isolated from Uncaria rhynchophylla as well as 14 known analogues (10-23). Their structures were determined by HRESIMS, 1D and 2D NMR, and experimental and calculated electronic circular dichroism data. Compounds 5, 7, 15, and 22 displayed significant agonistic effects against the 5-HT1A receptor with EC50 values of 2.2 ± 0.1, 0.1 ± 0.1, 1.6 ± 0.3, and 2.0 ± 0.5 µM, respectively. The mechanisms of action of these four compounds with the 5-HT1A receptor were investigated by molecular docking, and the results suggested that amino acid residues Asp116, Thr196, Asn386, and Tyr390 played critical roles in the observed activity of the above-mentioned compounds.

Highly potent non-steroidal FXR agonists protostane-type triterpenoids: Structure-activity relationship and mechanism.

Farnesoid X receptor (FXR) is a key regulator in charge of bile acid synthesis, transport, and metabolism. Activation of FXR represses bile acid synthesis and increases its excretion and transport, consequently protecting the liver functions. Thus, FXR is considered as a critical therapeutic target of cholestasis and nonalcoholic steatohepatitis. Herein, we isolated and identified fourteen new protostane-type triterpenoids (1-14) and four known analogues (15-18) from Alisma orientale, and finally constructed a small library of protostane-type triterpenoids (1-70) to investigate their structure-activity relationship with FXR, further leading to obtain compound 15 with potent agonistic activity against FXR (EC50 = 90 nM). Extensive in vitro investigation confirmed high efficacy of compound 15 against FXR in living cell, and revealed its underlying mechanism for FXR activation (amino acid residues Arg331 and Ser332) by molecular docking and site-directed mutagenesis technology.

Demethylbellidifolin isolated from Swertia bimaculate against human carboxylesterase 2: Kinetics and interaction mechanism merged with docking simulations.

In this study, forty-nine kinds of traditional Chinese medicines (TCMs) were evaluated for their inhibitory activities against human carboxylesterase 2 (HCE 2) using a human liver microsome (HLM) system. Swertia bimaculata showed significant inhibition on HCE 2 at 10 µg/mL among forty-nine kinds of TCMs. The extract of Swertia bimaculata was separated by preparative HPLC to afford demethylbellidifolin (1) identified by MS, 1H NMR, and 13C NMR spectra. Demethylbellidifolin (1) was assayed for its inhibitory HCE 2 effect by HCE 2-mediated DDAB hydrolysis, and its potential IC50 value was 3.12 ±â€¯0.64 µM. Demethylbellidifolin (1) was assigned as a mixed-type competitive inhibitor with the inhibiton constant Ki value of 6.87 µM by Lineweaver-Burk and slope plots. Living cell imaging was conducted to corroborate its inhibitory HCE 2 activity. Molecular docking indicated potential interactions of demethylbellidifolin (1) with HCE 2 through two hydrogen bonds of the C-3 and C-5 hydroxy groups with amino acid residues Glu227 and Ser228 in the catalytic cavity, respectively.

Oxidative coupling of coumarins catalyzed by laccase.

Laccase (LAC) belongs to the blue multi­copper lignolytic oxidase enzymes, and has been regarded as an important tool to produce some important dimers in the application of biotechnology. In this study, sixteen coumarins 1-16 were screened to investigate the catalytic ability of LAC, and three coumarins 6, 7, and 16 could be catalyzed to produce three coumarin derivative coupling with acetone 6a, 7a, and 16a. The potential interaction mechanisms of three coumarins 6, 7, and 16 with LAC were analyzed by molecular docking. The kinetic analyses of catalytic reactions for coumarins 6, 7, and 16 with LAC were performed by using the transformed products 6a, 7a, and 16a as standard substances. Km values of coumarins 6, 7, and 16 were ranged from 0.87 ±â€¯0.07 µM to 2.74 ±â€¯0.29 µM, respectively. This finding suggested that LAC was a reliable method to catalyze oxidative coupling.