Pharmacokinetic study of deufruquintinibs in rats by HPLC-MS/MS after oral administration.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry method was established to quantitatively determine the pharmacokinetics of fruquintinib (HMPL-013) and its derivatives [deufruquintinib-3D (HMPL-013-3D), deufruquintinib-6D (HMPL-013-6D) and deufruquintinib-9D (HMPL-013-9D)] in rats. Detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The method established in this assay was successfully applied to a pharmacokinetic study of HMPL-013 and HMPL-013-Ds after oral administration. These results showed that HMPL-013-Ds had longer half-life and larger area under the plasma concentration-time curve than HMPL-013, especially HMPL-013-6D, which provides a significant basis for innovative ideas for drug structure transformation to reduce drug administration frequency and dosage.

Hepatoprotective Effects of Phloridzin against Isoniazid-Rifampicin Induced Liver Injury by Regulating CYP450 and Nrf2/HO-1 Pathway in Mice.

The protective effect of phloridzin (PHL) and its potential mechanism were examined in mice with liver injury induced by isoniazid (INH) and rifampicin (RFP). The mice were randomly divided into normal control group, model group, low (80 mg/kg), medium (160 mg/kg) and high (320 mg/kg) phloridzin-treated groups. After 28 d treatment, blood and liver tissue were collected and analysed. The results revealed that PHL regulated liver function related indicators and reduced the pathological tissue damage, indicating that PHL significantly alleviated the liver injury. Furthermore, the level of CYP450 enzyme, the expression of CYP3A4, CYP2E1, heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and protein were inhibited by PHL. These results indicated that PHL exerts a protecting effect against liver injury induced by combination of RFP and INH. The potential mechanisms may be concerned with the activation of Nrf2/HO-1 signaling pathway containing its key antioxidant enzymes and regulation of CYP3A4 and CYP2E1.

[Studies on absorption and transportation of coumarins in Angelica dahurica 'Yubaizhi' across human intestinal epithelial by using human Caco-2 cell monolayers].

The absorption is the key to the resulted efficacy of orally administered drugs and the small intestine is the main site to absorb the orally administered drug. In this paper, internationally recognized human colon adenocarcinoma cell line(Caco-2) monola-yer model which can simulate small intestinal epithelial cell was used to comparatively study the absorption and transportation diffe-rences of total coumarins and main individual coumarin in Angelica dahurica 'Yubaizhi' by separately using 6-and 12-well plates. It was found that apparent permeability coefficient(P_(app)) values of oxypeucedanin hydrate, byakangelicin and phellopterin were at the quantitative degree of 1 × 10~(-5) cm·s~(-1) when the individual administration was conducted independently, indicating that they were well-absorbed compounds. P_(app) ratio of their bi-directional transportation was close to 1, indicating that they can be absorbed across Caco-2 monolayer by passive diffusion mechanism without carrier mediation during the transportation. The similar trend of transportation was also observed for imperatorin, isoimperatorin and bergapten. The P_(app) values of oxypeucedanin hydrate, byakangelicin and bergapten were at quantitative degree of 1 × 10~(-5) cm·s~(-1) when the administration of total coumarins in Angelica dahurica 'Yubaizhi' was conducted, indicating that they were well-absorbed compounds. The results were consistent with those of independent administration of individual coumarins. Whereas, the P_(app) values of imperatorin, phellopterin and isoimperatorin in the total coumarins decreased, indicating that the interaction between compounds may exist although the P_(app) value ratio of bi-directional transportation was between 0.5 and 1.5. The results laid the foundation for intestinal absorption study of Angelica dahurica 'Yubaizhi' coumarins in compound Chinese medicine.

[Chemical constituents from lipophilic parts in roots of Angelica dahurica cv.Yubaizhi].

The chemical constituents from lipophilic parts in the roots of Angelica dahurica cv. Yubaizhi were studied in this paper. The compounds were separated and purified by repeated column chromatographic methods on silica gel and HPLC, and the chemical structures of compounds were determined by spectral data analyses. Thirty-three compounds were obtained and identified as isoimperatorin (1), imperatorin (2), stigmasterol (3), isooxypeucedanin (4), pabulenol (5), psoralen (6), bergapten (7), isodemethylfuropinarine (8), phellopterin (9), osthenol (10), alloimperatorin (11), xanthotoxin (12), xanthotoxol (13), isopimpinellin (14), alloisoimperatorin (15), ß-sitosterol (16), oxyalloimperatorin (17), pabularinone (18), 5-hydroxy-8-methoxypsoralen (19), columbianetin (20), heracol (21), isogosferol (22), 2″R-neobyakangelicol (23), byakangelicin ethoxide (24), byakangelicin (25), oxypeucedanin hydrate (26), uracil (27), umbelliferone (28), bergaptol (29), demethylfuropinarine (30), isobyakangelicol (31), oxypeucedanin ethanolate (32), heraclenol (33). Among them, compounds 8, 10, 17, 21, and 30 were obtained from the roots of title plant for the first time.

[Chemical constituents from polarity part in roots of Angelica dahurica var. formosana cv. Chuanbaizhi].

The chemical constituents from polarity part in the roots of Angelica dahurica var. formosana cv. Chuanbaizhi were studied in this paper. The compounds were separated and purified by repeated column chromatographic methods on silica gel and HPLC, and the chemical structures of compounds were determined by spectral data analyses. Fourteen compounds were obtained and identified as tert-O-ß-D-glucopyranosyl-(R)-byakangelicin (1), (2"S) -3"-O-ß-D-glucopyranosyl-oxypeucedanin hydrate (2), marmesinin (3), sec-O-ß-D-glucopyranosyl-byakangelicin (4), isofraxidin-7-O-ß-D-glucopyranoside (5), benzyl-O-ß-D-glucopyranoside (6), 8-O-ß-D-glycopyranosylxanthotoxol (7), prenyl-O-ß-D-glucopyranoside (8), scopolin (9), (2' R) -5'-hydroxymarmesin-5'-O-ß-D-glucopyranoside (10), (2'S,3'R) -3'-hydroxymarmesinin (11), skimmin (12), benzyl-O-ß-D-apiofuranosyl-(1"--> 6')-ß-D-glucopyranoside (13), and decuroside IV (14). Among them, compounds 2, 5, 6, 8, and 10-13 were obtained from the roots of title plant for the first time.

[Chemical constituents from lipophilic parts in roots of Angelica dahurica var. formosana cv. Chuanbaizhi].

The chemical constituents from lipophilic parts in the roots of Angelica dahurica var. formosana cv. Chuanbaizhi were studied in this paper. The compounds were separated and purified by repeated column chromatographic methods on silica gel and HPLC, and the chemical structures of compounds were determined by spectral data analyses. Twenty-nine compounds were obtained and identified as isoimperatorin (1), ß-sitosterol (2), imperatorin (3), bergapten (4), osthenol (5), xanthotoxin (6), isoimpinellin (7), dehydrogeijerin (8), phellopterin (9), isodemethylfuropinarine (10), 7-demethylsuberosin (11), alloimperatorin (12), xanthotoxol (13), isooxypeucedanin (14), alloisoimperatorin (15), demethylfuropinarine (16), 5-hydroxy-8-methoxypsoralen (17), oxypeucedanin methanolate (18), pabulenol (19), byakangelicin (20), marmesin (21), (+) -decursinol (22), heraclenol (23), oxypeucedanin hydrate (24), marmesinin (25), ulopterol (26), erythro-guaiacylglycerol-ß-ferulic acid ether (27), threo-guaiacylglycerol-ß-ferulic acid ether (28), and uracil (29). Compounds 5, 8, 11, 18, 21-23, and 26-28 were obtained from the roots of title plant for the first time.

Hepatoprotective effects of phloridzin on hepatic fibrosis induced by carbon tetrachloride against oxidative stress-triggered damage and fibrosis in rats.

The present study was to study the hepatoprotective effects of phloridzin (PHL) on hepatic fibrosis induced by carbon tetrachloride (CCl4) in rats, on the basis of this investigation, the possible mechanism of PHL was elucidated. Male Sprague Dawley (SD) rats were randomly divided into six groups: control, model, PHL-L, PHL-M, PHL-H and colchine. All rats except control group were intraperitoneally injected with CCl4, and control rats were injected with olive oil, twice a week for eight weeks. At the same time, the rats were orally given homologue drugs once a day, respectively. Hepatoprotective effects of PHL were evaluated by liver weight indexes, biochemical values, total antioxidant capacity and total-superoxide dismutase, histopathological observations, hepatic fibrosis, and the hepatic fibrosis relative gene and protein expressions. PHL significantly improved hepatic function; remarkably decreased serum hyaluronic acid (HA), transforming growth factor-ß1 (TGF-ß1), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and liver tissues hydroxyproline, malondialdehyde (MDA) levels, increased glutathione peroxidase (GSH-Px), total-antioxygen capacity (T-AOC) and total-superoxide dismutase (T-SOD) contents of liver tissues; Real-time polymerase chain reaction (PCR) and immunohisto-chemical results showed PHL might markedly reverse the up-regulated mRNA and protein expressions of the α-smooth muscle actin (SMA), TGF-ß1 and tissue inhibitor of metalloproteinase-1 (TIMP1), up-regulate the matrix metalloproteinase-1 (MMP1) mRNA and protein expressions. Histopathological observations provided supportive evidence for biochemical analyses and the hepatic fibrosis relative gene and protein expressions, and with the dose of PHL increasing, the aforesaid improvement became more and more strong. The studies demonstrated that PHL exerted beneficially hepatoprotective effects on hepatic fibrosis induced by CCl4, mainly enhancing antioxidant capacity of liver organizations, reduce the level of lipid peroxidation induced by CCl4, and protect hepatocyte membranes from damage, and alleviate hepatic fibrosis.

AMPK/Sirt1-mediated inflammation is positively correlated with myocardial fibrosis during ageing.

OBJECTIVES: Cardiovascular disease is the leading cause of death in the world, and it increases dramatically with ageing. The objective of this study was to elucidate age-dependent molecular changes of inflammation and its correlation with the progression of myocardial fibrosis. METHODS: Methods: Male SD rats aged 3, 6, 9 and 24 months were used in this study. H&E staining was used to assessed histo-morphological changes in different ages. Masson's trichrome staining was used to evaluate myocardial fibrosis. Immunofluorescence as well as western blot was carried out to detect the expression of vimentin. Real-time PCR was used to detect the level of pro-inflammatory chemokines MCP-1, IL1ß, TNFα and IL-6. Western blotting was also carried out to detect p-AMPK, Sirt1, AC-NF-κB expression. RESULTS: Myocardial pathological changes and fibrosis are positively correlated with age. Ageing rats showed an enhanced expression of inflammatory factors and the activation of cardiac fibroblasts increases. Meanwhile, the expression of p-AMPK, Sirt1 and downstream AC-NF-κB increased significantly during ageing. Furthermore, the 15-24 months of age in rats is the fastest changing stage of increased inflammation and decreased Sirt1 activity. CONCLUSIONS: Ageing is an independent risk factor for the occurrence and development of myocardial fibrosis. During ageing, myocardial fibroblasts are activated, accompanied by an increase in extracellular matrix deposition. The inflammation mediated by AMPK/Sirt1/NF-κB signalling pathway is closely positively correlated with the activation of myocardial fibroblasts and the progression of myocardial fibrosis.

Estrogenic activities of compound GL-1, isolated from Ganoderma lucidum.

In this study, the estrogenic effect of GL-1, a component of the Ganoderma lucidum, was studied, and the possible mechanism was discussed preliminarily. The binding ability of GL-1 to estrogen receptor was calculated by computer aided simulation. The effects of GL-1 on the proliferation of estrogen sensitive estrogen receptor (ER) (+) MCF-7 cells and estrogen insensitive ER (-) MDA-MB-231 cells were detected by MTT method. The effects of GL-1 on the proliferation of estrogen-induced MCF-7 cells, and the effects of the estrogen receptor inhibitor ICI182780 on the proliferation of GL-1-induced MCF-7 cells were detected by MTT assay. The expression of ERα and ERß monoclonal antibody were detected by Western blot. The results showed that GL-1 has a good binding ability to estrogen receptor ß, and has estrogen-like effect, which might be related to secretion of estrogen and expression of ERß by binding to ERs.

Effects of phloridzin on blood glucose and key enzyme G-6-Pase of gluconeogenesis in mice.

The effects of phloridzin (PHL), main component of Malus hupehensis (MH) tea leaves, on blood glucose (BG) and glucose-6-phosphatase (G-6-Pase) were investigated to provide a basis for finding a scheme of stabilizing BG. Glucose uptake of insulin resistant HepG2 cells was measured by glucose oxidase method. Glucose tolerance, fasting BG (FBG) and postprandial BG (PBG) were determined by BG test strips. The expression of G-6-Pase was detected by Western blot. The results showed that glucose uptake was enhanced and the expression of G-6-Pase was inhibited by PHL in insulin resistant HepG2 cells. Glucose tolerance was enhanced, FBG level was increased and PBG level was decreased by PHL in mice. The expression of G-6-Pase in the liver was enhanced under fasting state, and was inhibited by the low and medium dose under postprandial state. It indicated that PHL has a positive effect on stabilizing BG in mice, which is related to bidirectional regulation of G-6-Pase activity. PRACTICAL APPLICATIONS: Malus hupehensis, edible and medicinal plant, which has been proved by long-term application and experiments that it has a good effect on stabilizing blood glucose, preventing diabetes and adjuvant treatment. Its effect is closely related to its main component PHL. Thus, MH can be used as a dietary regulating drink for daily life to maintain blood glucose. Its main ingredient is PHL, which can be developed as a candidate drug for diabetes treatment.

Trametenolic Acid B Triggers HSP90AA4P and Autophagy in HepG2/2.2.15 Cells by Proteomic Analysis.

Our previous studies have demonstrated that trametenolic acid B (TAB) extracted from the Laetiporus sulphureus (Fr.) Murrill owned effective anti-proliferation of HepG2/2.215 cells and induced autophagy activity. The present aim was to further investigate its mechanisms involved by proteomic analysis. The iTRAQ of TAB on HepG2/2.215 was carried out and the western blot was used to verify the results of the proteomics analysis. According to the peptide segment quantitative standard (FDR ≤ 1%), a total of 5324 proteins were identified in HepG2/2.215 by proteomic analysis. The results identified that the major up-regulated proteins were HSP90AA4P, MYB, SERPINE1, and down-regulated proteins were Rho C, SERPINA1, and PIK3R4, which were related to PI3K/Akt signaling pathway, cell metastasis, and autophagy. HSP90AA4P and Rho C's proteomics analysis were further confirmed by the western blot. The proteomic results demonstrated that the anti-hematoma effect of TAB was closely related to the increase of HSP90AA4P protein expressions and autophagy, which may be a critical target of TAB, which was expected to be a candidate drug for the treatment liver cancer.

Tissue distribution study of Angelica dahurica cv. Yubaizhi in rat by ultra-performance liquid chromatography with tandem mass spectrometry.

A sensitive and specific ultra-performance liquid chromatographic-tandem mass (UPLC-MS/MS) spectrometric method was established to investigate tissue distribution of fourteen coumarins of Angelica Dahurica cv. Yubaizhi roots (ADYR) in rat tissues, including isoimperatorin (1), imperatorin (2), isooxypeucedanin (3), byakangelicin (4), oxypeucedanin hydrate (5), bergapten (6), 2"R-neobyakangelicol (7), phellopterin (8), xanthotoxin (9), isopimpinellin (10), oxypeucedanin ethanolate (11), isobyakangelicol (12), columbianetin (13), (-)-marmesin (14). Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction-mode (MRM). The method established in this assay was successfully applied to tissue distribution study of the selected 14 coumarins after oral administration of the extract of ADYR in rat tissues, including heart, liver, spleen, lung, kidney, stomach, small intestine, muscle, testis, and brain. Tissue distribution characteristics of the fourteen coumarins were clearly elucidated, and the results of this study indicated that the fourteen coumarins were distributed to rat tissues rapidly and could be detected in all of the selected tissues after oral administration. Concentrations of the coumarins were obviously higher in kidney, liver and stomach tissues, and lower in testis, brain and muscle tissues. As an important part of ADMET/Act. study on ADYR, the tissue distribution of multiple coumarins of ADYR in rats provides a significant basis for better evaluation of the metabolism and disposition process in vivo of the herb medicine. The information provided in this research is very useful for further understanding of the metabolic mechanism of ADYR in vivo.

Anti-inflammatory coumarins with short- and long-chain hydrophobic groups from roots of Angelica dahurica cv. Hangbaizhi.

The (1)H NMR-guided fractionation of a cyclohexane soluble portion of the 75% ethanolic extract of the roots of Angelica dahurica cv. Hangbaizhi led to the isolation of two coumarins, namely, 5-(3"-hydroxy-3"-methylbutyl)-8-hydroxyfuranocoumarin, and isobyakangelicin hydrate-3"-ethyl ether, and ten coumarins with short- or long-chain hydrophobic groups, namely, andafocoumarins A-J. Their structures were elucidated by extensive spectroscopic analyses. The absolute configurations of the C-2" secondary alcohols in ten of these compounds were deduced via the circular dichroism data of the in situ formed [Rh2(OCOCF3)4] complex, and oxidation reactions were utilized to determine location of the double bonds in the lipid chain of andafocoumarins H and I, respectively. The long-chain hydrophobic group of andafocoumarin J was determined by the method of chemical degradation and GC-MS analysis. It was the first time that coumarins with short- or long-chain hydrophobic groups in this plant had been comprehensively investigated. All isolates were assayed for their inhibitory effect against nitric oxide (NO) production in a lipopolysaccharide (LPS)-activated RAW264.7 macrophage cell line, among which andafocoumarins A and B exhibited a potent inhibition on LPS-activated NO production with IC50 values of 19.7 and 13.9 µM, respectively, indicating their stronger inhibitory activity than l-N(6)-(1-iminoethyl)-lysine (IC50=23.7 µM), a selective inhibitor of inducible nitric oxide synthase.

New coumarins from the roots of Angelica dahurica var. formosana cv. Chuanbaizhi and their inhibition on NO production in LPS-activated RAW264.7 cells.

A new linear pyranocoumarin named (-)-hydroxydecursinol (1) and a new biscoumarin named (±)-dahuribiscoumarin (2), together with six known compounds isoimperatorin (3), imperatorin (4), phellopterin (5), isodemethylfuropinarine (6), demethylfuropinarine (7), and (+)-decursinol (8) were isolated from the 75% ethanolic extract of the roots of Angelica dahurica var. formosana cv. Chuanbaizhi. Their structures were elucidated by extensive spectroscopic techniques, including 2D NMR spectroscopy and mass spectrometry, and the structure of 2 was confirmed by single-crystal X-ray diffraction. All of the isolated compounds were evaluated for the inhibition against nitric oxide (NO) production in the lipopolysaccharide (LPS)-activated RAW264.7 macrophage cell line, and exhibited the inhibitory activity on NO production in a concentration-dependent manner. Furthermore, real-time PCR analysis revealed that compounds 2, 5-8 could significantly suppress the expression levels of inducible nitric oxide synthase mRNA in a concentration-dependent manner. And their primary structure-activity relationships of NO inhibitory effects were also briefly discussed. These compounds are potential candidates for further bioassay studies to determine their suitability as drug leads.