Spirocitromycetin, a Fungal Polyketide with an Antiosteoporotic Pharmacophore.

Spirocitromycetin, an antiosteoporotic polyketide bearing a unique spirocycle, was characterized from a human mucus sputum-derived Penicillium velutinum. Its structure and absolute configuration were elucidated spectrally, with its biosynthetic pathway likely mediated via polivione, a reported heptaketide. Spirocitromycetin was shown to be antiosteoporotic at 0.1 µM in the prednisolone-induced osteoporotic zebrafish model. A combination of spirocitromycetin variant synthesis and bioassay has identified 5'-methyl-3'H-spiro[chromane-3,2'-furan]-3',4-dione as an unreported antiosteroporotic pharmacophore. Collectively, this work offers new starting (sub)structures that may be of significance for antiosteoporotic drug discovery.

Holistic quality evaluation of commercial Agastache rugosa by multiple chromatographic and chemometric analysis.

In present study, a comprehensive strategy integrating multiple chromatographic and chemometric methods to simultaneously characterize the volatile and non-volatile components was developed for the holistic quality evaluation of commercial Agastache rugosa (AR), a common edible and medicinal herb, collected in China. The volatile components and the non-volatile components were characterized by GC-MS and UPLC-QTOF-MS/MS, respectively. And the data were analyzed either independently or integratively by multivariate statistical analysis (MVS) for the quality assessment of commercial samples. The results revealed that the commercial AR samples were different in both the composition and the content of volatile components. However, the compositions of non-volatile components in commercial AR were generally similar, whereas the contents of some components were different. All the results indicated that the holistic quality of commercial AR was inconsistent, and the commercial samples collected could be classified into two main groups, the volatile components were majorly responsible for the classification. Whether or not the holistic quality variations affect the efficacy of AR deserves further investigation.

[Effect of lupeol on invasion and metastasis of human hepatoma HepG2 and SK-HEP-1 cells and its mechanism].

Epithelial-mesenchymal transformation(EMT) exists in embryonic development and is closely related to cell migration and invasion. The increased EMT level in tumors showed that E-cadherin was replaced by N-cadherin, and the expression of interstitial markers such as α-SMA and vimentin was up-regulated. It has been reported that lupeol can reduce the expression of matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9) and N-cadherin to inhibit the metastasis of osteoma cells. However lupeol has been less studied in liver cancer. Therefore, this paper investigated the effect of lupanol on invasion and metastasis of human hepatoma cell line HepG2 and SK-HEP-1 and its possible mechanism. MTT assay and Annexin V/PI double staining were used to investigate the effect of lupeol on activity and apoptosis of HepG2 cells and SK-HEP-1 cells. Moreover, the effect of lupeol on the invasion of HepG2 cells and SK-HEP-1 cells were evaluated by Transwell assay. The expressions of E-cadherin, N-cadherin, α-SMA, vimentin and MMP-9 were measured by Western blot. The model of subcutaneous transplantation of nude mice and the lung metastasis model of H22 hepatocellular carcinoma cells were established to evaluate the efficacy of lupeol in vivo on tumor growth and lung metastasis by HE staining combined with immunohistochemical assay. The results showed that lupeol inhibited the activity and invasion of HepG2 cells and SK-HEP-1 cells in a dose-dependent manner and induced apoptosis. Western blot showed that the expression of E-cadherin, a landmark protein for EMT, was induced by lupeol, and the expressions of N-cadherin, α-SMA, vimentin and MMP-9 were decreased. In vivo experiments showed that lupeol inhibited tumor growth in mice bearing xenograft. In addition, immunohistochemical experiments confirmed that lupeol could up-regulate the expression of E-cadherin in tumor tissues of nude mice, reduce the expression of N-cadherin, and inhibit the metastasis of liver cancer H22 cells in the lungs of mice. The above results indicated that the mechanism of lupeol inhibiting the invasion and metastasis of HCC cells may be related to the regulation of EMT process.

[Acute toxicity mechanism of Panax notoginseng saponins in larvae zebrafish based on metabonomics].

Based on metabolomics,the metabolites of larvae zebrafish with overdose of Panax notoginseng saponins( PNS) were compared with those in normal group of larvae zebrafish to investigate the possible toxicity mechanism of overdose PNS in larvae zebrafish. An experimental animal model of long-term toxicity induced by PNS overdose was established by administering 1-6 dpf at low,medium and high doses of PNS,respectively. The ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry( UPLC-Q-TOF-MS) technique was combined with principal component analysis( PCA) and orthogonal partial least squares discriminant analysis( OPLS-DA) to screen and identify biomarkers associated with toxicity,and then the MetaboAnalyst database was used to analyze metabolism-related pathways. The results showed that the metabolites of each group could be distinguished distinctly,and they deviated more from the normal group in a time and dose dependent manner. Twenty-nine potential biomarkers related to toxicity( VIP>1,P<0. 05) were identified preliminarily,mainly involving six metabolic pathways. From the metabonomics point of view,the toxicity mechanism of overdose PNS may be related to the disorders of lipid metabolism,amino acid metabolism and energy metabolism.

Design and synthesis of novel celastrol derivative and its antitumor activity in hepatoma cells and antiangiogenic activity in zebrafish.

Two series of celastrol derivatives were designed and synthesized by modifying carboxylic acid at the 28th position with amino acid, and their intermediates with isobutyrate at the third position. All compounds were evaluated for their antiproliferation activity by four human cancer cell lines (SCG7901, HGC27, HepG2, and Bel7402) and one normal cell LO2. The most promising compound, compound 8, showed superior bioactivity and lower toxicity than others including celastrol. Further underlying tests illustrated that compound 8 induced apoptosis and cell arrest at G2/M and inhibited proliferation and mobility of human hepatoma cells by suppressing the signal transducer and activator of transcription-3 signaling pathway. Besides these, a highly accurate and reproducible high performance liquid chromatography protocol was established to determine celastrol and compound 8 absorption in zebrafish, and results demonstrated that their concentration increased rapidly within 4 hr in a time-dependent manner and the concentration of compound 8 was higher than that of celastrol. In addition, without detection at 12 hr, compound 8 was rapidly metabolized in vivo. These findings are very helpful for the structural modification of celastrol and other bioactive compounds to improve their bioactivity, toxicity, and absorption.

Citrofulvicin, an Antiosteoporotic Polyketide from Penicillium velutinum.

Citrofulvicin (1), along with its early shunt product fulvionol (2), was characterized as a skeletally unprecedented antiosteoporotic agent from a human sputum-derived fungus Penicillium velutinum. The unique citrofulvicin framework is likely formed by a nonenzymatic intermolecular Diels-Alder cycloaddition between heptaketide-based intermediates. Citrofulvicin and fulvionol were demonstrated to be osteogenic at 0.1 µM in the prednisolone-induced osteoporotic zebrafish.

Acaulins A and B, Trimeric Macrodiolides from Acaulium sp. H-JQSF.

Acaulin A (1) and its macrolactone ring-opened congener acaulin B (2) were characterized from the culture of Acaulium sp. H-JQSF (an isopod-associated fungus) as architecturally undescribed trimeric macrodiolides, with the former being antiosteoporotic at 0.4 µM in the prednisolone-induced osteoporotic zebrafish. Identification of acaudiolic acid (3) as the monomeric macrodiolide precursor facilitated the proposal of the acaulin biosynthetic pathway.

Acaulide, an Osteogenic Macrodiolide from Acaulium sp. H-JQSF, an Isopod-Associated Fungus.

Acaulide (1), a macrodiolide with an unprecedented framework, was characterized along with its shunt products-acaulones A (2) and B (3)-from the culture of Acaulium sp. H-JQSF associated with the isopod Armadillidium vulgare. The spiro-linked 14-, 14-, and 6-membered cycles of 1 arise likely from iterative intermolecular Michael addition reactions. Biological evaluation in the prednisolone-induced osteoporotic zebrafish demonstrated that 1 is antiosteoporotic at 0.4 and 2.0 µM.

Biotransformation of ginsenosides F4 and Rg6 in zebrafish.

Ginsenosides F4 and Rg6 (GF4 and GRg6), two main active components of steamed notoginseng or red ginseng, are dehydrated disaccharide saponins. In this work, biotransformation of ginsenosides F4 and Rg6 in zebrafish was investigated by qualitatively identifying their metabolites and then proposing their possible metabolic pathways. The prediction of possible metabolism of ginsenosides F4 and Rg6 using zebrafish model which can effectively simulate existing mammals model was early and quickly performed. Metabolites of ginsenosides F4 and Rg6 after exposing to zebrafish for 24 h were identified by Ultraperformance Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometry. A total of 8 and 6 metabolites of ginsenosides F4 and Rg6 were identified in zebrafish, respectively. Of these, 7 and 5, including M1, M3-M5, M7-M9 and N1 (N5), N2, N4 (N9), N7-N8 were reported for the first time as far as we know. The mechanisms of their biotransformation involved were further deduced to be desugarization, glucuronidation, sulfation, dehydroxylation, loss of C-17 and/or C-23 residue pathways. It was concluded that loss of rhamnose at position C-6 and glucuronidation at position C-3 in zebrafish were considered as the main physiologic and metabolic processes of ginsenosides F4 and ginsenosides Rg6, respectively.

Metabolism of five diterpenoid lactones from Dioscorea bulbifera tubers in zebrafish.

Diterpenoid lactones (DLs) have been reported to be the main hepatotoxic constituents in Dioscorea bulbifera tubers (DBT), a traditional Chinese medicinal herb. The acquisition of early information regarding its metabolism is critical for evaluating the potential hepatotoxicity of DLs. We investigated, for the first time, the main metabolites of diosbulbin A (DIOA), diosbulbin C (DIOC), diosbulbin (DIOG), diosbulbin (DIOM) and diosbulbin (DIOF) in adult zebrafish. By using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS), 6, 2, 7, 5 and 4 metabolites of DIOA, DIOC, DIOF, DIOM and DIOG were identified in the zebrafish body and the aqueous solution, respectively. Both phase-I and phase-II metabolites were observed in the metabolic profiles and the metabolic pathways involved in hydroxyl reduction, glucuronidation, glutathione conjugation and sulfation. The above results indicated that hepatocytic metabolism might be the major route of clearance for DLs. This study provided important information for the understanding of the metabolism of DLs in DBT.

[Ideas and methods on efficient screening of traditional medicines for anti-osteoporosis activity based on M-Act/Tox integrated evaluation using zebrafish].

The increasingly apparent liver injury problems of bone strengthening Chinese medicines have brought challenges for clinical application, and it is necessary to consider both effectiveness and safety in screening anti-osteoporosis Chinese medicines. Metabolic transformation is closely related to drug efficacy and toxicity, so it is significant to comprehensively consider metabolism-action/toxicity(M-Act/Tox) for screening anti-osteoporosis Chinese medicines. The current evaluation models and the number of compounds(including metabolites) severely restrict efficient screening in vivo. By referring to previous relevant research and domestic and abroad literature, zebrafish M-Act/Tox integrative method was put forward for efficiently screening anti-osteoporosis herb medicines, which has organically integrated zebrafish metabolism model, osteoporosis model and toxicity evaluation method. This method can break through the bottleneck and blind spots that trace compositions can't achieve efficient and integrated in vivo evaluation, and realize both efficient and comprehensive screening on anti-osteoporosis traditional medicines based on in vivo process taking both safety and effectiveness into account, which is significant to accelerate discovery of effective and safe innovative traditional Chinese medicines for osteoporosis.

[Research and application of hepatotoxicity evaluation technique of traditional Chinese medicine].

The safety of traditional Chinese medicine (TCM) has received the widespread attention in recent years. Hepatotoxicity of TCM is one of the key problems of the safety of TCM. This article summarized research progress and application prospect in the mechanism of TCM hepatotoxicity, biomarkers, toxic omics database, prevention of hepatotoxicity of the liver cell lines, subcellular fraction, three-dimensional cultivation models, the model animals, aiming to provide theoretical basis for TCM toxicity evaluation and technical guidelines, thus promoting the development of TCM toxicity studies. Hope for Chinese medicine liver toxicity evaluation method provides the theoretical foundation and technical guidelines, promote the development and improvement of TCM liver toxicity research system.

Rapid identification of herbal compounds derived metabolites using zebrafish larvae as the biotransformation system.

Metabolites derived from herbal compounds are becoming promising sources for discovering new drugs. However, the rapid identification of metabolites from biological matrixes is limited by massive endogenous interference and low abundance of metabolites. Thus, by using zebrafish larvae as the biotransformation system, we herein proposed and validated an integrated strategy for rapid identification of metabolites derived from herbal compounds. Two pivotal steps involved in this strategy are to differentiate metabolites from herbal compounds and match metabolites with their parent compounds. The differentiation step was achieved by cross orthogonal partial least-squares discriminant analysis. Automatic matching analysis was performed on R Project based on a self-developed program, of which the number of matched ionic clusters and its corresponding percentage between metabolite and parent compound were taken into account to assess their similarity. Using this strategy, 46 metabolites screened from incubation water samples of zebrafish treated with total Epimedium flavonoids (EFs) could be matched with their corresponding parent compounds, 37 of them were identified and validated by the known metabolic pathways and fragmentation patterns. Finally, 75% of the identified EFs metabolites were successfully detected in urine samples of rats treated with EFs. These experimental results indicate that the proposed strategy using zebrafish larvae as the biotransformation system will facilitate the rapid identification of metabolites derived from herbal compounds, which shows promising perspectives in providing additional resources for pharmaceutical developments from natural products.

Hepatic inflammation-fibrosis-cancer axis in the rat hepatocellular carcinoma induced by diethylnitrosamine.

PURPOSE: Hepatocellular carcinoma (HCC) cases are closely associated with chronic inflammation and fibrosis which is known as hepatic inflammation-fibrosis-cancer (IFC) axis. The aim of this study is to elucidate the development characteristics of the rat HCC model based on IFC axis. METHODS: The diethylnitrosamine (DEN)-induced rat HCC, which presents a stepwise histopathological progression that is similar to human HCC, was used to analyze the features of the different stages (inflammation, fibrosis, cancer). Rats were injected DEN at a dose of 30 mg/kg body weight twice a week for 11 weeks and the animals were observed until week 20. Time series sera and organ samples from the DEN animal model were collected to evaluate the dynamic changes. RESULTS: It was found that serum biochemical indicators (AST, ALT, ALP, TP, T-BIL, IL-6, TNF-α) from DEN-treated group were higher than that from control group. Fibrosis-related index in serum and live tissue were increased, respectively, from week 4 after DEN treatment. The expression of TGF-ß1 and α-SMA in DEN-treated group was higher than that in control group. JAK2/STAT3 signaling was significantly up-regulated in DEN-treated group compared to that in control group. The histological examination confirmed that the hepatocarcinogenesis model was successfully established, and 100% of the animals in the DEN-exposed group developed liver tumors at 20 weeks. According to the pathological changes, the model characterized resulted in three stages: the inflammation stage (week 2-6), the fibrosis stage (week 8-12), and the HCC stage (week 14-20). CONCLUSIONS: The results suggested that the HCC development was associated with IFC axis. The serial progression of hepatocarcinogenesis was according to the sequence of hepatic inflammation, fibrosis and then hepatic tumor.

Absence of myocyte regeneration by mobilization of bone marrow stem cells after myocardial infarction.

The study aimed to test the potential for bone marrow stem cells (BMSC) mobilized by granulocyte macrophage colony stimulating factor (GM-CSF) to promote neovascularization and cardiomyocytes regeneration in a rat model of myocardial infarction. The myocardial infracted rats were randomly assigned to receive GM-CSF injection as GM-CSF group or received saline injection as control group. Evaluation of CD34+ stem cells was performed by flow cytometry. Cardiac functions were monitored using a multiple channel recorder via cardiac catheterization. Immunobiological staining including factor VIII and Ki67, and phosphotungstic acid-hematoxylin (PTAH) staining, were performed to assess angiogenesis and myogenesis and calculated myocardial infarction size. The CD34+ stem cells in blood and bone marrow of GM-CSF group increased significantlyon day 7 and day 14 comparing with control group, and declined on day 28. Immunobiological staining showed neovasculature formation and more Ki67 expression in the infracted regions of theGM-CSF group.Ki67 and PTAH double staining showed Ki67 positive signals were overlap with lymphocytes, fibroblasts and endothelial cells but not myocytes. No significant decrease of infracted size occurred in the GM-CSF group. These results suggested BMSC could be mobilized effectively by GM-CSF after myocardial infarction, which could only promote neovascularization without myogenesis.

Gualou Xiebai decoction inhibits cardiac dysfunction and inflammation in cardiac fibrosis rats.

BACKGROUND: Gualou Xiebai Decoction (GXD) is a well-known traditional Chinese recipe. It has been used to treat cardiovascular disorders for nearly two thousand years. But there is a lack of reports on cardiac fibrosis and underlying mechanism. METHODS: Myocardial infarction was performed by ligation of left anterior descending coronary artery (LAD) in male Wistar rats. Rats with myocardial infarction were treated with GXD (1.14 g/kg, 4.53 g/kg) daily for 4 weeks. Cardiac function was evaluated by echocardiography. Hemodynamic parameters and infarct size were measured in each group. Myocardial enzymes were examined by biochemical tests. Inflammatory cytokines were assessed by ELISA, and interrelated proteins were detected by western blot. RESULTS: Cardiac function was significantly improved in GXD-treatment rats after myocardial infarction (MI), which was accompanied with decreased infarct size. Administration of GXD to myocardial fibrosis rats significantly ameliorated the activities of AST, LDH and CK-MB in serum. The increase in inflammatory factors (TNF-α, IL-1ß) were markedly reduced upon GXD treatment. Furthermore, the inflammatory mediators (NF-κB p65, TNF-α, MCP-1) were down-regulated by GXD in the myocardial fibrosis rats. CONCLUSIONS: Treatment with GXD improved cardiac function induced by myocardial fibrosis by inhibiting expression of inflammatory mediators associated with NF-κB.

[Screening on antiosteoporotic active parts of dipsacus radix based on zebrafish model].

OBJECTIVE: Prednisolone-induced osteoporosis model using zebrafish was used to screen the antiosteoporotic active parts of Dipsacus Radix, in order to investigate the applicability and rationality of the zebrafish model of osteoporosis. METHODS: Zebrafish larvae at 5 days post fertilization (dpf) were exposed with 25 micromol/L prednisolone and 0.5% DMSO for 48 h, then except one group of 25 micromol/L prednisolone, other groups of 25 micromol/L prednisolone were treated with a range of concentration (0.025, 0.25, 2.5, 25 microg crude drug/mL) of extract of Dipsacus Radix and its different concentration ethanol elution parts of macroporous resin with 25 micromol/L prednisolone. All groups were incubated in 24-well plates (28.5 degrees C) until 10 dpf. Zebrafish skeleton at 10 dpf were anesthetized and fixed for staining with alizarin red. Quantitative analysis of the stained area was performed by microscopic inspection and digital imaging methods to reflect the amount of zebrafish head skeleton mineralization. RESULTS: The results indicated that head skeleton mineral area and integrated optical density (IOD) of 25 micromol/L prednisolone model group were significantly decreased when compared with vehicle control group, and the extract of Dipsacus Radix and its 30%, 50%, 70% and 90% ethanol elution parts of macroporous resin rescued the further bone loss of zebrafish induced by prednisolone when compared with the model group. HPLC analysis indicated that components of 30%, 50%, 70% and 90% ethanol elution parts of macroporous resin containing saponins and nonsaponins components. CONCLUSION: Both saponins and nonsaponins can prevent bone loss of zebrafish induced by prednisolone. This novel osteoporosis zebrafish model was successfully used to screen antiosteoporotic active parts of Dipsacus Radix, which had advantages of simple, high efficiency and easy to perform.

[Two-dimensional zebrafish model combined with hyphenated chromatographic techniques for evaluation anti-osteoporosis activity of epimendin A and its metabolite baohuoside I].

This article firstly established a new efficient method for screening anti-osteoporosis ingredients, which used two-dimensional zebrafish model combined with hyphenated chromatographic techniques to evaluate anti-osteoporosis activities of epimedin A and its metabolite baohuoside I. Adult zebrafish was used for metabolism of epimedin A in 0.5% DMSO, and LC-MS was used for analysis of the metabolite, which was captured by HPLC, and prednisolone-induced osteoporosis model of zebrafish was used to evaluate the anti-osteoporotic activities of trace amounts of epimedin A and baohuoside I. The results indicated that epimedin A and baohuoside I can prevent prednisolone-induced osteoporosis in zebrafish. The developed method in this paper enables the separation, enrichment and analysis of micro-amount metabolite of epimedin A, and anti-osteoporosis activities in vivo of epimedin A and baohuoside I was simple and efficient screening resorting to zebrafish osteoporosis mode. This paper would provide new ideas and methods for a rapid and early discovery of anti-osteoporosis activities of micro-ingredients and its metabolite of traditional Chinese medicine.

[Ideas and methods of two-dimensional zebrafish model combined with chromatographic techniques in high-throughput screening of active anti-osteoporosis components of traditional Chinese medicines].

OBJECTIVE: To break through the restrictions of the evaluation model and the quantity of compounds by using the two-dimensional zebrafish model combined with chromatographic techniques, and establish a new method for the high-throughput screening of active anti-osteoporosis components. METHOD: According to the research group-related studies and relevant foreign literatures, on the basis of the fact that the zebrafish osteoporosis model could efficiently evaluate the activity, the zebrafish metabolism model could efficiently enrich metabolites and the chromatographic techniques could efficiently separate and analyze components of traditional Chinese medicines, we proposed that the inherent combination of the three methods is expected to efficiently decode in vivo and in vitro efficacious anti-osteoporosis materials of traditional Chinese medicines. RESULT AND CONCLUSION: The method makes it simple and efficient in the enrichment, separation and analysis on components of traditional Chinese medicines, particularly micro-components and metabolites and the screening anti-osteoporosis activity, fully reflects that efficacious materials of traditional Chinese medicines contain original components and metabolites, with characteristic of "multi-components, multi-targets and integral effect", which provides new ideas and methods for the early and rapid discovery of active anti-osteoporosis components of traditional Chinese medicines.

[Construction of research system for processing mechanism of traditional Chinese medicine based on chemical composition transformation combined with intestinal absorption barrier].

Based on practice of Epimedium processing mechanism for many years and integrated multidisciplinary theory and technology, this paper initially constructs the research system for processing mechanism of traditional Chinese medicine based on chemical composition transformation combined with intestinal absorption barrier, which to form an innovative research mode of the " chemical composition changes-biological transformation-metabolism in vitro and in vivo-intestinal absorption-pharmacokinetic combined pharmacodynamic-pharmacodynamic mechanism". Combined with specific examples of Epimedium and other Chinese herbal medicine processing mechanism, this paper also discusses the academic thoughts, research methods and key technologies of this research system, which will be conducive to systematically reveal the modem scientific connotation of traditional Chinese medicine processing, and enrich the theory of Chinese herbal medicine processing.