Genetic deletion or pharmacological inhibition of soluble epoxide hydrolase attenuated particulate matter 2.5 exposure mediated lung injury.

Air pollution represented by particulate matter 2.5 (PM2.5) is closely related to diseases of the respiratory system. Although the understanding of its mechanism is limited, pulmonary inflammation is closely correlated with PM2.5-mediated lung injury. Soluble epoxide hydrolase (sEH) and epoxy fatty acids play a vital role in the inflammation. Herein, we attempted to use the metabolomics of oxidized lipids for analyzing the relationship of oxylipins with lung injury in a PM2.5-mediated mouse model, and found that the cytochrome P450 oxidases/sEH mediated metabolic pathway was involved in lung injury. Furthermore, the sEH overexpression was revealed in lung injury mice. Interestingly, sEH genetic deletion or the selective sEH inhibitor TPPU increased levels of epoxyeicosatrienoic acids (EETs) in lung injury mice, and inactivated pulmonary macrophages based on the MAPK/NF-κB pathway, resulting in protection against PM2.5-mediated lung injury. Additionally, a natural sEH inhibitor luteolin from Inula japonica displayed a pulmonary protective effect towards lung injury mediated by PM2.5 as well. Our results are consistent with the sEH message and protein being both a marker and mechanism for PM2.5-induced inflammation, which suggest its potential as a pharmaceutical target for treating diseases of the respiratory system.

Direct targeting of sEH with alisol B alleviated the apoptosis, inflammation, and oxidative stress in cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) is a pathological condition characterized by a rapid decrease in glomerular filtration rate and nitrogenous waste accumulation during hemodynamic regulation. Alisol B, from Alisma orientale, displays anti-tumor, anti-complement, and anti-inflammatory effects. However, its effect and action mechanism on AKI is still unclear. Herein, alisol B significantly attenuated cisplatin (Cis)-induced renal tubular apoptosis through decreasing expressions levels of cleaved-caspase 3 and cleaved-PARP and the ratio of Bax/Bcl-2 depended on the p53 pathway. Alisol B also alleviated Cis-induced inflammatory response (e.g. the increase of ICAM-1, MCP-1, COX-2, iNOS, IL-6, and TNF-α) and oxidative stress (e.g. the decrease of SOD and GSH, the decrease of HO-1, GCLC, GCLM, and NQO-1) through the NF-κB and Nrf2 pathways. In a target fishing experiment, alisol B bound to soluble epoxide hydrolase (sEH) as a direct cellular target through the hydrogen bond with Gln384, which was further supported by inhibition kinetics and surface plasmon resonance (equilibrium dissociation constant, K D = 1.32 µM). Notably, alisol B enhanced levels of epoxyeicosatrienoic acids and decreased levels of dihydroxyeicosatrienoic acids, indicating that alisol B reduced the sEH activity in vivo. In addition, sEH genetic deletion alleviated Cis-induced AKI and abolished the protective effect of alisol B in Cis-induced AKI as well. These findings indicated that alisol B targeted sEH to alleviate Cis-induced AKI via GSK3ß-mediated p53, NF-κB, and Nrf2 signaling pathways and could be used as a potential therapeutic agent in the treatment of AKI.

Total flavonoids of Inula japonica alleviated the inflammatory response and oxidative stress in LPS-induced acute lung injury via inhibiting the sEH activity: Insights from lipid metabolomics.

BACKGROUND: Acute lung injury (ALI) is a severe respiratory disease characterized by diffuse lung interstitial and respiratory distress and pulmonary edema with a mortality rate of 35%-40%. Inula japonica Thunb., known as "Xuan Fu Hua" in Chinese, is a traditional Chinese medicine Inulae Flos to use for relieving cough, eliminating expectorant, and preventing bacterial infections in the clinic, and possesses an anti-pulmonary fibrosis effect. However, the effect and action mechanism of I. japonica on ALI is still unclear. PURPOSE: This study aimed to investigate the protective effect and underlying mechanism of total flavonoids of I. japonica (TFIJ) in the treatment of ALI. STUDY DESIGN AND METHODS: A mouse ALI model was established through administration of LPS by the intratracheal instillation. Protective effects of TFIJ in the inflammation and oxidative stress were studied in LPS-induced ALI mice based on inflammatory and oxidative stress factors, including MDA, MPO, SOD, and TNF-α. Lipid metabolomics, bioinformatics, Western blot, quantitative real-time PCR, and immunohistochemistry were performed to reveal the potential mechanism of TFIJ in the treatment of ALI. RESULTS: TFIJ significantly alleviated the interstitial infiltration of inflammatory cells and the collapse of the alveoli in LPS-induced ALI mice. Lipid metabolomics demonstrated that TFIJ could significantly affect the CYP2J/sEH-mediated arachidonic acid metabolism, such as 11,12-EET, 14,15-EET, 8,9-DHET, 11,12-DHET, and 14,15-DHET, revealing that sEH was the potential target of TFIJ, which was further supported by the recombinant sEH-mediated the substrate hydrolysis in vitro (IC50 = 1.18 µg/ml). Inhibition of sEH by TFIJ alleviated the inflammatory response and oxidative stress via the MAPK, NF-κB, and Nrf2 signaling pathways. CONCLUSION: These results demonstrated that TFIJ could suppress the sEH activity to stabilize the level of EETs, allowing the alleviation of the pathological course of lung injury in LPS-treated mice, which suggested that TFIJ could serve as the potential agents in the treatment of ALI.

Total terpenoids of Inula japonica activated the Nrf2 receptor to alleviate the inflammation and oxidative stress in LPS-induced acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a life-threatening lung disease and characterized by pulmonary edema and atelectasis. Inula japonica Thunb. is a commonly used traditional Chinese medicine for the treatment of lung diseases. However, the potential effect and mechanism of total terpenoids of I. japonica (TTIJ) on ALI remain obscure. PURPOSE: This study focused on the protective effect of TTIJ on lipopolysaccharide (LPS)-induced ALI in mice and its potential mechanism. STUDY DESIGN AND METHODS: A mouse model of ALI was established by intratracheal instillation of LPS to investigate the protective effect of TTIJ. RNA-seq and bioinformatics were then performed to reveal the underlying mechanism. Finally, western blot and real-time qPCR were used to verify the effects of TTIJ on the inflammation and oxidative stress. RESULTS: TTIJ notably attenuated LPS-induced histopathological changes of lung. The RNA-seq result suggested that the protective effect of TTIJ on LPS-induced ALI were associated with the Toll-like receptor 4 (TLR4) and nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathways. Pretreatment with TTIJ significantly reduced the inflammation and oxidative stress via regulating levels of pro-inflammatory and anti-oxidative cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), superoxide dismutase (SOD), and glutathione (GSH), in LPS-induced ALI mice. TTIJ treatment could suppress the cyclooxygenase-2 (COX-2) expression level and the phosphorylation of p65, p38, ERK, and JNK through the inactivation of the MAPK/NF-κB signaling pathway in a TLR4-independent manner. Meanwhile, TTIJ treatment upregulated expression levels of proteins involved in the Nrf2 signaling pathway, such as heme oxygenase-1 (HO-1), NAD(P)H: quinoneoxidoreductase-1 (NQO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glutamate-cysteine ligase modifier subunit (GCLM), via activating the Nrf2 receptor, which was confirmed by the luciferase assay. CONCLUSION: TTIJ could activate the Nrf2 receptor to alleviate the inflammatory response and oxidative stress in LPS-induced ALI mice, which suggested that TTIJ could serve as the potential agent in the treatment of ALI.

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.

Comparative pharmacokinetics of baicalin and geniposide in juvenile and adult rats after oral administration of Qingkailing Granules.

Objective: To explore the effect of age on Qingkailing Granules disposition by comparing the pharmacokinetics of geniposide and baicalin in juvenile and adult rats. Methods: A simple and rapid LC-MS/MS method was developed and validated to simultaneously determine geniposide and baicalin in rat plasma after a simple protein precipitation. The analytes were separated on an Agilent ZORBAX Extend-C18 column. The mobile phase consisted of acetonitrile and water with 0.1% (volume percent) formic acid at a flow rate of 0.6 mL/min. The ionization was conducted using an ESI source in negative ion mode. Multiple reaction monitoring was used for quantification at transitions of m/z 445.0 â†’ m/z 268.9 for baicalin, m/z 433.2 â†’ m/z 225.0 for geniposide, m/z 431.0 â†’ m/z 341.0 for vitexin (IS). Juvenile and adult rats were administrated Qingkailing Granules (3 g/kg) orally. Plasma concentrations of baicalin and geniposide were determined by LC-MS/MS. Results: The linear ranges of the analytes were 1-1000 ng/mL for baicalin and 2-2000 ng/mL for geniposide. The method was successfully applied to compare the pharmacokinetics of the analytes between juvenile and adult rats after oral administration of Qingkailing Granules. AUC was bigger in adult rats, while t 1/2 was longer in juvenile rats. Conclusion: These results suggested that the absorption and elimination of baicalin and geniposide in juvenile rats was lower than that in adult rats. Additional attention should be paid to the pharmacokinetic difference when Qingkailing Granules were used in children.

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.

Evaluation of the stability of tyrosol esters during in vitro gastrointestinal digestion.

Lipophenols such as tea polyphenol palmitate derivatives (palmitoyl esters of tea polyphenols) have been classified as non-toxic food additives due to their better protective effects on lipidic food matrices from oxidation, but their digestion and absorption have remained unexplored. In this study, the digestive stability of tyrosol acyl esters (TYr-Es) with fatty acids of different chain lengths and different degrees of unsaturation such as C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, and C22:6 was evaluated using an in vitro simulated gastrointestinal tract model containing various digestive enzymes (pancreatin, pancreatic lipase and phospholipase A2). HPLC-UV measurements demonstrated that only pancreatin and pancreatic lipase, but not phospholipase A2, could hydrolyze TYr-Es to free TYr. The degree of TYr-E hydrolysis negatively correlated with the chain length but positively correlated with the degree of unsaturation of their lipid moiety. In addition, the fact that TYr in fatty acid ester forms could be absorbed by the intestinal lumen, at least partially in the form of free TYr, may explain a sustained release behavior of TYr-Es to TYr during the time-course following the digestion process.

Uncaria rhynchophylla Ameliorates Parkinson's Disease by Inhibiting HSP90 Expression: Insights from Quantitative Proteomics.

BACKGROUND/AIMS: Uncaria rhynchophylla, known as "Gou-teng", is a traditional Chinese medicine (TCM) used to extinguish wind, clear heat, arrest convulsions, and pacify the liver. Although U. rhynchophylla has a long history of being often used to treat central nervous system (CNS) diseases, its efficacy and potential mechanism are still uncertain. This study investigated neuroprotective effect and the underlying mechanism of U. rhynchophylla extract (URE) in MPP+-induced SH-SY5Y cells and MPTP-induced mice. METHODS: MPP+-induced SH-SY5Y cells and MPTP-induced mice were used to established Parkinson's disease (PD) models. Quantitative proteomics and bioinformatics were used to uncover proteomics changes of URE. Western blotting was used to validate main differentially expressed proteins and test HSP90 client proteins (apoptosis-related, autophagy-related, MAPKs, PI3K, and AKT proteins). Flow cytometry and JC-1 staining assay were further used to confirm the effect of URE on MPP+-induced apoptosis in SH-SY5Y cells. Gait analysis was used to detect the behavioral changes in MPTP-induced mice. The levels of dopamine (DA) and their metabolites were examined in striatum (STR) by HPLC-EC. The positive expression of tyrosine hydroxylase (TH) was detected by immunohischemical staining and Western blotting. RESULTS: URE dose-dependently increased the cell viability in MPP+-induced SH-SY5Y cells. Quantitative proteomics and bioinformatics results confirmed that HSP90 was an important differentially expressed protein of URE. URE inhibited the expression of HSP90, which further reversed MPP+-induced cell apoptosis and autophagy by increasing the expressions of Bcl-2, Cyclin D1, p-ERK, p-PI3K p85, PI3K p110α, p-AKT, and LC3-I and decreasing cleaved caspase 3, Bax, p-JNK, p-p38, and LC3-II. URE also markedly decreased the apoptotic ratio and elevated mitochondrial transmembrane potential (DΨm). Furthermore, URE treatment ameliorated behavioral impairments, increased the contents of DA and its metabolites and elevated the positive expressions of TH in SN and STR as well as the TH protein. CONCLUSIONS: URE possessed the neuroprotective effect in vivo and in vitro, regulated MAPK and PI3K-AKT signal pathways, and inhibited the expression of HSP90. U. rhynchophylla has potentials as therapeutic agent in PD treatment.

Afatinib Decreases P-Glycoprotein Expression to Promote Adriamycin Toxicity of A549T Cells.

We investigated the reversal effect of afatinib (AFT) on activity of adriamycin (ADR) in A549T cells and clarified the related molecular mechanisms. A549T cells overexpressing P-glycoprotein (P-gp) were resistant to anticancer drug ADR. AFT significantly increased the antitumor activity of ADR in A549T cells. AFT increased the intracellular concentration of ADR by inhibiting the function and expression of P-gp at mRNA and protein levels in A549T cells. Additionally, the reversal effect of AFT on P-gp mediated multidrug resistance (MDR) might be related to the inhibition of PI3K/Akt pathway. Cotreatment with AFT and ADR could enhance ADR-induced apoptosis and autophagy in A549T cells. Meanwhile, the co-treatment significantly induced cell apoptosis and autophagy accompanied by increased expression of cleaved caspase-3, PARP, LC3B-II, and beclin 1. Apoptosis inhibitors had no significant effect on cell activity, while autophagy inhibitors decreased cell viability, suggesting that autophagy may be a self protective mechanism of cell survival in the absence of chemotherapy drugs. Interestingly, when combined with AFT and ADR, inhibition of apoptosis and/or autophagy could enhance cell viability. These results indicated that in addition to inhibit P-gp, ADR-induced apoptosis, and autophagy promoted by AFT contributed to the antiproliferation effect of combined AFT and ADR on A549T cells. These findings provide evidence that AFT combined ADR may achieve a better therapeutic effect to lung cancer in clinic. J. Cell. Biochem. 119: 414-423, 2018. © 2017 Wiley Periodicals, Inc.

Diterpenoids from the roots of Euphorbia ebracteolata and their inhibitory effects on human carboxylesterase 2.

A chemical investigation of the roots of Euphorbia ebracteolata identified eighteen diterpenoids and glycosides. On the basis of spectroscopic data, they were determined to be ent-kauranes, ent-atisanes, tigliane derivatives, ingenane, and ent-abietanes, among which were eleven previously undescribed diterpenoids. The inhibitory effects of the isolated compounds against human carboxylesterase 2 (hCE-2) were evaluated in vitro, which revealed moderate inhibitory effects with IC50 values < 50 µM. Next, the inhibitory kinetics were evaluated for the putative hCE-2 inhibitor 4ß,9α,16,20-tetrahydroxy-14(13 → 12)-abeo-12αH-1,6-tigliadiene-3,13-dione (IC50 3.88 µM), and results indicated competitive inhibition with Ki 4.94 µM. Additionally, none of the diterpenoids showed cytotoxic effects against five human tumor cell lines as determined by MTT assays.

Phenolic glycosides and monoterpenoids from the roots of Euphorbia ebracteolata and their bioactivities.

The bioactive substance investigation of Euphorbia ebracteolata obtained 17 compounds by various chromatographic techniques. Their structures were elucidated using widely spectroscopic data, including ESI-MS, HRESI-MS, CD, 1D- and 2D-NMR, which gave 5 new phenolic glucosides and 4 new monoterpenoids. The phenolic glucosides and monoterpenoids showed the inhibitory effect against the human carboxylesterase-2 (hCE-2) using a fluorescence bioassay in vitro, with the strongest inhibitor compound 4 (IC50 7.17µM). The antioxidant effects of these isolated compounds were evaluated using a DPPH scavenging assay. All of the phenolic acids displayed the DPPH scavenging effect, especially that eight compounds have better effect than vitamin C, with the IC50 values ranging from 4.52 to 7.52µM. Additionally, compounds 1-17 showed no cytotoxic effect against five human cancer cell lines by MTT assay.

Alisol B 23-acetate protects against non-alcoholic steatohepatitis in mice via farnesoid X receptor activation.

Alisol B 23-acetate (AB23A) is a natural triterpenoid isolated from the traditional Chinese medicine rhizoma alismatis, which exhibits a number of pharmacological activities, including anti-hepatitis virus, anti-cancer and antibacterial effects. In this study we examined whether AB23A protected against non-alcoholic steatohepatitis (NASH) in mice, and the mechanisms underlying the protective effects. NASH was induced in mice fed a methionine and choline-deficient (MCD) diet for 4 weeks. The mice were simultaneously treated with AB23A (15, 30, and 60 mg·kg-1·d-1, ig) for 4 weeks. On the last day, blood samples and livers were collected. Serum liver functional enzymes, inflammatoru markers were assessed. The livers were histologically examined using H&E, Oil Red O, Masson's trichrome and Sirius Red staining. Mouse primary hepatocytes were used for in vitro experiments. The mechanisms underlying AB23A protection were analyzed using siRNA, qRT-PCR, and Western blot assays. AB23A treatment significantly and dose-dependently decreased the elevated levels of serum ALT and AST in MCD diet-fed mice. Furthermore, AB23A treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration and hepatic fibrosis in the mice. AB23A-induced decreases in serum and hepatic lipids were related to decreased hepatic lipogenesis through decreasing hepatic levels of SREBP-1c, FAS, ACC1 and SCD1 and increased lipid metabolism via inducing PPARα, CPT1α, ACADS and LPL. The reduction in inflammatory cell infiltration corresponded to deceased serum levels of mKC and MCP-1 and decreased hepatic gene expression of MCP-1 and VCAM-1. The reduction in hepatic fibrosis was correlated with decreased hepatic gene expression of fibrosis markers. The protective effects of AB23A were FXR-dependent, because treatment with the FXR agonist CDCA mimicked AB23A-induced hepato-protection in the mice, whereas co-administration of FXR antagonist guggulsterone abrogated AB23A-induced hepato-protection. In mouse primary hepatocytes, FXR gene silencing abrogated AB23A-induced changes in gene expression of Apo C-II, CPT1α, ACADS and LPL. AB23A produces protective effects against NASH in mice via FXR activation.

Identification of the hydroxylated derivatives of bufalin: phase I metabolites in rats.

Bufalin was a typical bioactive bufadienolide, existed in the traditional Chinese medicine Chan Su with the high content of 1-5%. The in vivo metabolites (1-5) of bufalin were prepared by various chromatographic techniques from the bile samples of SD rats, which were administrated with bufalin orally. Their structures were determined on the basis of the widely spectroscopic data, including HRESIMS, 1D-, and 2D NMR. And 1-3, 5 were new compounds. In the in vitro cytotoxicity assay, metabolites (1-5) showed weaker cytotoxic effects than bufalin against human cancer cell lines A549 and H1299, which indicated that the metabolism was a significant pathway for the detoxification of bufalin. Structures analyses indicated that metabolites 1-5 were hydroxylated derivatives of bufalin. This study suggested that Phase I metabolism catalyzed by CYP450 enzymes was one of the metabolic ways of bufalin, which may promote the excretion of bufalin.

Inhibitory Effects of Highly Oxygenated Lanostane Derivatives from the Fungus Ganoderma lucidum on P-Glycoprotein and α-Glucosidase.

Twelve new highly oxygenated lanostane triterpenoids and nine known ganoderic acids were isolated from the fruiting body of Ganoderma lucidum. The new compounds were lanostane nortriterpenoids with 27 carbons (1-5 and 8), lanostane nor-triterpenoids with 25 carbons (6 and 7), and lanostane triterpenoids (9-12) based on multiple spectroscopic data analysis, including HRESIMS, 1D-NMR, 2D-NMR, and CD. Compounds 1-5 were identified as rare nor-lanostanoids that contain a 17ß-pentatomic lactone ring. Compound 13, possessing a lactone ring, had been isolated previously. The P-glycoprotein (P-gp) inhibitory effects of compounds 1-21 were evaluated at a concentration of 20 µM using an adriamycin (ADM)-resistant human breast adenocarcinoma cell line (MCF-7/ADR). Compounds 1, 5, 18, and 20 and verapamil increased the accumulation of ADM in MCF-7/ADR cells approximately 3-fold when compared with the negative control. These data support the significant P-glycoprotein inhibitory activities of compounds 1, 5, 18, and 20. In silico docking analysis suggested these compounds had similar P-gp recognition mechanisms compared with those of verapamil (a classical inhibitor). Furthermore, in an in vitro bioassay, compounds 2, 4, 5, 6, and 18 showed moderate inhibitory effects against α-glucosidase compared with those of the positive control acarbose.

Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis.

Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp) and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes.

Characterization of phase I metabolism of resibufogenin and evaluation of the metabolic effects on its antitumor activity and toxicity.

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has displayed great potential as a chemotherapeutic agent in oncology. However, it is a digoxin-like compound that also exhibits extremely cardiotoxic effects. The present study aimed to characterize the metabolic behaviors of RB in humans as well as to evaluate the metabolic effects on its bioactivity and toxicity. The phase I metabolic profile in human liver microsomes was characterized systemically, and the major metabolite was identified as marinobufagenin (5ß-hydroxylresibufogenin, 5-HRB) by liquid chromatography-mass spectrometry and nuclear magnetic imaging techniques. Both cytochrome P450 (P450) reaction phenotyping and inhibition assays using P450-selective chemical inhibitors demonstrated that CYP3A4 was mainly involved in RB 5ß-hydroxylation with much higher selectivity than CYP3A5. Kinetic characterization demonstrated that RB 5ß-hydroxylation in both human liver microsomes and human recombinant CYP3A4 obeyed biphasic kinetics and displayed similar apparent kinetic parameters. Furthermore, 5-HRB could significantly induce cell growth inhibition and apoptosis in A549 and H1299 by facilitating apoptosome assembly and caspase activation. Meanwhile, 5-HRB displayed very weak cytotoxicity of human embryonic lung fibroblasts, and in mice there was a greater tolerance to acute toxicity. In summary, CYP3A4 dominantly mediated 5ß-hydroxylation and was found to be a major metabolic pathway of RB in the human liver, whereas its major metabolite (5-HRB) displayed better druglikeness than its parent compound RB. Our findings lay a solid foundation for RB metabolism studies in humans and encourage further research on the bioactive metabolite of RB.

Bioactive metabolites of Schisanlactone E transformed by Cunninghamella blakesleana AS 3.970.

Schisanlactone E (SE) is a major triterpene obtained from the plants of genus Kadsura. The aim of this research was to investigate the transformed metabolites of SE by fungi and evaluate the bioactivities of these products. After screening 10 strains of filamentous fungi, Cunninghamella blakesleana AS 3.970 was chosen as a potent organism to be used for the biotransformation of SE. 13 metabolites were obtained and determined to be new compounds through the use of spectroscopic data, including UV, 1D-, 2D-NMR, and HR-ESIMS. Furthermore, in an in vitro bioassay, metabolites 7 and 9 showed moderate inhibitory effects on the nitric oxide production in LPS-induced macrophages with IC50 values of 16.73, 5.91 µM, respectively; 9 could inhibit the proliferation of acetaldehyde-induced HSC-T6 cells, with the IC50 value of 21.4 µM. Preliminary findings on the structure-activity relationships for these metabolites were also discussed.

Microbial transformation of resibufogenin by Curvularia lunata AS 3.4381.

In this paper, the microbial transformation of resibufogenin by Curvularia lunata AS 3.4381 was investigated, and four transformed products were isolated and characterized as 3-epi-resibufogenin (2), 12α-hydroxy-3-epi-resibufogenin (3), 12-oxo-16ß-hydroxy-3-epi-resibufogenin (4), and 12ß,15-epoxy-3-epi-bufalin-14,15-ene (5). Among them, 4 and 5 are new compounds, and isomerization, hydroxylation, and oxidation reactions in microbial transformation process were observed. Additionally, the cytotoxicities of transformed products (2-5) were also investigated.