Function-oriented mechanism discovery of coumarins from Psoralea corylifolia L. in the treatment of ovariectomy-induced osteoporosis based on multi-omics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus (Bu Gu Zhi) is the fruit of Psoralea corylifolia L. (PCL) and has been used for centuries in traditional Chinese medicine formulas to treat osteoporosis (OP). A new drug called "BX" has been developed from PCL, but its mechanism for treating OP is not yet fully understood. AIM OF THE STUDY: To explore the mechanism of action of BX in the treatment of ovariectomy-induced OP based function-oriented multi-omics analysis of gut microbiota (GM) and metabolites. MATERIALS AND METHODS: C57BL/6 mice were bilaterally ovariectomized to replicate the OP model. The therapeutic efficacy of BX was evaluated by bone parameters (BMD, BV/TV, Tb.N, Tb.Sp), hematoxylin and eosin (H&E) staining results, and determination of bone formation markers procollagen type Ⅰ amino-terminal peptide (PⅠNP) and bone-specific alkaline phosphatase (BALP). Serum and fecal metabolomics and high-throughput 16S rDNA sequencing were performed to evaluate effects on endogenous metabolites and GM. In addition, an enzyme-based functional correlation algorithm (EBFC) algorithm was used to investigate functional correlations between GM and metabolites. RESULTS: BX improved OP in OVX mice by increasing BMD, BV/TV, serum PⅠNP, BALP, and improving Tb.N and Tb.Sp. A total of 59 differential metabolites were identified, and 9 metabolic pathways, including arachidonic acid metabolism, glycerophospholipid metabolism, purine metabolism, and tryptophan metabolism, were found to be involved in the progression of OP. EBFC analysis results revealed that the enzymes related to purine and tryptophan metabolism, which are from Lachnospiraceae_NK4A136_group, Blautia, Rs-E47_termite_group, UCG-009, and Clostridia_UCG-014, were identified as the intrinsic link between GM and metabolites. CONCLUSIONS: The regulation of GM and restoration of metabolic disorders may be the mechanisms of action of BX in alleviating OP. This research provides insights into the function-oriented mechanism discovery of traditional Chinese medicine in the treatment of OP.

Study on the intervention effect of Epimedium before and after suet-oil-processed on kidney yang deficiency rats based on intestinal flora and fecal metabolomics.

Epimedium is a Chinese herbal medicine commonly used in clinical practice to reinforce yang. Previous studies have shown that Epimedium fried with suet oil based has the best effect on warming kidney and promoting yang. Evidence suggests a relationship between kidney yang deficiency syndrome (KYDS) and metabolic disorders of the intestinal microflora. However, the specific interaction between KYDS and the intestinal microbiome, as well as the internal regulatory mechanism of the KYDS intestinal microbiome regulated by Epimedium fried with suet oil, remain unclear. The purpose of this study was to investigate the regulatory effects of different processed products of Epimedium on intestinal microflora and metabolites in rats with kidney yang deficiency, and to reveal the processing mechanism of Epimedium fried with suet oil warming kidney and helping yang. 16 S rRNA and LC-MS/MS technology were used to detect fecal samples. Combined with multivariate statistical analysis, differential intestinal flora and metabolites were screened. Then the content of differential bacteria was then quantified using quantitative real-time fluorescence PCR. Furthermore, the correlation between differential bacterial flora and metabolites was analyzed using Spearman's method. The study found that the composition of intestinal flora in rats with kidney yang deficiency changed compared to healthy rats. Epimedium fried with suet oil could increase the levels of beneficial bacteria, while significantly reducing the levels of harmful bacteria. Real-time quantitative PCR results were consistent with 16 S rRNA gene sequencing analysis. Fecal metabolomics revealed that KYDS was associated with 30 different metabolites, involving metabolic pathways steroid hormone biosynthesis etc. Moreover, differential bacteria were closely correlated with potential biomarkers. Epimedium could improve metabolic disorders associated with KYDS by acting on the intestinal flora, with Epimedium fried with suet oil demonstrating the most effective regulatory effect. Its potential mechanism may involve the regulation of abnormal metabolism and the impact on the diversity and structure of the intestinal flora.

Preparation of sustainable mineral oil-free offset printing ink with vegetable oil esters.

Mineral oils are used in substantial quantities for the production of varnishes and inks due to their abundance and versatility. However, as part of the production process, some of mineral oil components are separated as waste material, whereupon they can mix with air, water, or soil and become potentially harmful to the environment. Almost all these waste materials are volatile organic compounds (VOCs), chemicals that can easily evaporate at room temperature and have toxic effect. Therefore, a novel green, mineral oil-free offset printing ink was produced using vegetable oil esters as bio-renewable raw materials. Accompanying varnishes were prepared with linseed oil, methyl oleate, octyl stearate, and four types of resin (A, B, C, and D). The application of these varnishes to magenta color offset ink was subsequently studied to screen out the best combination of resin and ester in terms of setting time. Meanwhile, dyeing force tests were conducted to evaluate the ink's printability, while rheological analysis was done via viscosity and flowability tests. The setting time of the magenta color offset ink made by varnish A was observed to be considerably shorter than that of the ink samples prepared using varnishes B, C, and D. Furthermore, varnish A proved to be a good alternative varnish for the production of yellow, cyan, and black color offset printing inks. Samples of these inks were printed on coated paper, and their printability was contrasted against that of vegetable oil-based (pure vegetable oil), mineral oil-based, and other mineral oil-free offset printing inks. Results determined that the varnishes produced with linseed oil, methyl oleate, and octyl stearate can replace mineral oil-based varnishes for the production of offset printing ink.

Integrating strategies of metabolomics, network pharmacology, and experiment validation to investigate the processing mechanism of Epimedium fried with suet oil to warm kidney and enhance yang.

Introduction: Epimedium, a traditional Chinese medicine (TCM) commonly used in ancient and modern China, is one of the traditional Chinese medicines clinically used to treat kidney yang deficiency syndrome (KYDS). There are differences in the efficacy of Epimedium before and after processing, and the effect of warming the kidney and enhancing yang is significantly enhanced after heating with suet oil. However, the active compounds, corresponding targets, metabolic pathways, and synergistic mechanism of frying Epimedium in suet oil to promote yang, remain unclear. Methods: Herein, a strategy based on comprehensive GC-TOF/MS metabolomics and network pharmacology analysis was used to construct an "active compounds-targets-metabolic pathways" network to identify the active compounds, targets and metabolic pathways involved. Subsequently, the targets in kidney tissue were further validated by real-time quantitative polymerase chain reaction (RT-qPCR). Histopathological analysis with physical and biochemical parameters were performed. Results: Fifteen biomarkers from urine and plasma, involving five known metabolic pathways related to kidney yang deficiency were screened. The network pharmacology results showed 37 active compounds (13 from Epimedium and 24 from suet oil), 159 targets, and 267 pathways with significant correlation. Importantly, integrated metabolomics and network pharmacologic analysis revealed 13 active compounds (nine from Epimedium and four from suet oil), 7 corresponding targets (ALDH2, ARG2, GSTA3, GSTM1, GSTM2, HPGDS, and NOS2), two metabolic pathways (glutathione metabolism, arginine and proline metabolism), and two biomarkers (Ornithine and 5-Oxoproline) associated with improved kidney yang deficiency by Epimedium fried with suet oil. Discussion: These finds may elucidate the underlying mechanism of yang enhancement via kidney warming effects. Our study indicated that the mechanism of action mainly involved oxidative stress and amino acid metabolism. Here, we demonstrated the novel strategies of integrating metabolomics and network pharmacology in exploring of the mechanisms of traditional Chinese medicines.

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.

The Toxicity and Metabolism Properties of Herba Epimedii Flavonoids on Laval and Adult Zebrafish.

Zebrafish is being increasingly used for metabolism and toxicity assessment. The drugs consumed in zebrafish metabolism studies are far less than those used in rat studies. In our study, zebrafish embryos were exposed to icariin, Baohuoside I (BI), Epimedin A (EA), Epimedin B (EB), Epimedin C (EC), Sagittatoside A (SA), Sagittatoside B (SB), and 2''-O-rhamnosylicariside II (SC), respectively, to examine the toxicity and metabolic profiles of these flavonoids. The order of toxicity was SC, SB > EC, SA > BI, icariin, EA, EB. After 24 h exposure to SB and SC, the mortality of zebrafish larvae reached 100% and yolk sac swollen was obvious. Both SC and SB caused severe hepatocellular vacuolization and liver cells degeneration in adult zebrafish after 15 consecutive days' treatment. The metabolic profiles of these flavonoids with trace amount were also monitored in larvae. BI was the common metabolite shared by icariin, EA, EB, SA, and SB, via deglycosylation. Both BI and SC remained as the prototype in the medium, suggesting that it is hard for BI and SC to cleave the rhamnose residue. EC was metabolized into SC and BI in zebrafish, inferring that SC might be responsible for the toxicity observed in EC group. The metabolites of icariin, EA, EB, EC, and BI in zebrafish larvae coincided with results from rats and intestinal flora. These data support the use of this system as a surrogate in predicting metabolites and hepatotoxicity risk, especially for TCM compound with trace amount.

Total C-21 steroidal glycosides, isolated from the root tuber of Cynanchum auriculatum Royle ex Wight, attenuate hydrogen peroxide-induced oxidative injury and inflammation in L02 cells.

Oxidative stress plays an important role in the pathology of liver disorders. Total C­21 steroidal glycosides (TCSGs), isolated from the root tuber of Cynanchum auriculatum Royle ex Wight, have been reported to exert numerous effects, including liver protective and antioxidant effects. In order to investigate the potential mechanisms underlying the protective effects of TCSGs on liver function, the present study used the human normal liver cell line, L02, to evaluate the effects of TCSGs on hydrogen peroxide (H2O2)­induced oxidative injury and inflammatory responses. The L02 cells were pretreated with various concentrations of TCSGs, followed by exposure to 1.5 mM H2O2. Cell viability was determined by a 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and nitric oxide (NO) were measured using colorimetric assays. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH­Px) and the production of malondialdehyde (MDA) were also determined. Intracellular reactive oxygen species (ROS) levels were detected using a fluorescent probe. H2O2­induced oxidative toxicity was attenuated following treatment with TCSGs, as indicated by the increase in cell viability, the decreased levels of ALT, AST, LDH, NO, MDA and ROS, and the increased activities of SOD, CAT and GSH­Px. To further explore the possible mechanisms of action of TCSGs, the nuclear factor erythroid 2­related factor 2 (Nrf2) and nuclear factor­κB (NF)­κB pathways were examined. The results revealed that treatment with TCSGs markedly induced Nrf2 nuclear translocation and upregulated the expression of heme oxygenase­1 (HO­1) in the L02 cells damaged by H2O2. In addition, pretreatment with TCSGs inhibited the NF­κB signaling pathway by blocking the degradation of the inhibitor of nuclear factor κBα (IκBα), thereby reducing the expression and nuclear translocation of NF­κB, as well as reducing the expression of tumor necrosis factor­α (TNF­α), interleukin-6 (IL­6), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX­2). On the whole, the findings of this study demonstrate that TCSGs can protect L02 cells against H2O2­induced oxidative toxicity and inflammatory injury by increasing the expression of Nrf2 and HO­1, mediated by the NF­κB signaling pathway.

Assessment of Toxicity and Absorption of the Novel AA Derivative AA-Pme in SGC7901 Cancer Cells In Vitro and in Zebrafish In Vivo.

BACKGROUND Asiatic acid (AA; 2α,3ß,23-trihydroxyurs-12-ene-28-oic acid) is an active compound derived from Centella asiatica, a traditional medicinal plant used widely in many Asian countries, particularly for the treatment of cancer. However, the modified AA derivative N-(2α,3ß,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester (AA-PMe) has shown markedly better anti-tumor activity than AA. MATERIAL AND METHODS We evaluated the toxicity of AA and AA-PMe on zebrafish morphology, mortality, and hatching rate and determined the effect on SGC7901 cancer cells by acute toxicity assay. AA-PMe absorption in vitro in SGC7901 cells and in vivo in zebrafish was determined by establishing a highly accurate and reproducible HPLC protocol. RESULTS In zebrafish, the toxicity of AA-PMe was lower than AA, with an acute toxic dose of AA-PMe above 25 µM, compared to acute toxicity at doses above 10 µM for AA. However, chronic toxicity of AA-PMe began occurring at doses below 25 µM but became apparent for AA at doses below 10 µM. Although low doses of AA-PMe were tolerated acutely, it became chronically toxic during zebrafish development, resulting in morphological abnormalities, including peripheral and abdominal edema, hemorrhage, abnormal body shape, enlarged yolk sac, and reduced motility. At low concentrations, absorption of AA-PMe by cells and zebrafish embryos occurred in a dose-dependent manner, but this stabilized as the concentration increased. CONCLUSIONS This pharmacokinetic study outlines the cellular and organismal effects of AA-PMe and suggests a theoretical basis that may underlie its mechanism of action.

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.

The potential of Epimedium koreanum Nakai for herb-drug interaction.

OBJECTIVES: This study aims to investigate potential herb-drug interactions (HDI) of Epimedium koreanum Nakai. METHODS: Human liver microsomes (HLMs) were used to determine the enzyme kinetics of the major human cytochrome P450s (CYPs). Inducible potential of E. koreanum on CYP1A2, 2B6, 2C19 and 3A4 activities of human primary hepatocytes was also examined. KEY FINDINGS: Ethanol extract of E. koreanum showed direct inhibitory potency for CYP1A2 (IC50  = 121.8 µg/ml, Ki  = 110.7 ± 36.8 µg/ml) and CYP2B6 (IC50  = 59.5 µg/ml, Ki  = 18.1 ± 2.9 µg/ml). For CYP2C9, 2C19, 2D6, 2E1 and 3A4, only negligible effect was observed. Time-dependent (irreversible) inhibition by E. koreanum was observed for CYP1A2 (KI  = 32.9 ± 18.4 µg/ml, kinact  = 0.031 ± 0.006 min-1 ). However, ethanol extract of E. koreanum (1.5-150 µg/ml) did not change the activity or mRNA expressions for CYP3A4, 1A2, 2C19 and 2B6. CONCLUSIONS: The ethanol extract of E. koreanum is not likely to cause HDI via inducing the major human CYPs. But the potential for interactions between E. koreanum extract and substrates of CYP1A2 or 2B6 cannot be overlooked.

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.

TPGS/Phospholipids Mixed Micelles for Delivery of Icariside II to Multidrug-Resistant Breast Cancer.

The biggest challenge for the treatment of multidrug resistant cancer is to deliver a high concentration of anticancer drugs to cancer cells. Icariside II is a flavonoid from Epimedium koreanum Nakai with remarkable anticancer properties, but poor solubility and significant efflux from cancer cells limited its clinical use. In our previous study, a self-assembled mixture of micelles (TPGS-Icariside II-phospholipid complex) was successfully constructed, which could substantially increase the solubility of Icariside II and inhibit the efflux on Caco-2 cells. In this study, we evaluate the anticancer effect of the mixed micelles encapsulating Icariside II (Icar-MC) on MCF-7/ADR, a multidrug-resistant breast cancer cell line. The cellular uptake of the micelles was confirmed by fluorescent coumarin-6-loaded micelles. The IC50 of Icar-MC in MCF-7/ADR was 2-fold less than the free drug. The in vitro study showed Icar-MC induced more apoptosis and lactate dehydrogenase release. Intravenous injection of Icar-MC into nude mice bearing MCF-7/ADR xenograft resulted in a better antitumor efficacy compared with the administration of free drug, without causing significant body weight changes in mice. The antitumor effect was further verified by magnetic resonance imaging and immunohistochemical assays for Ki-67, a proliferative indicator. Moreover, Icar-MC treatment also elevated Bax/Bcl-2 ratio and the expressions of cleaved caspase-3, -8, -9 and AIFM1 in tumors. This study suggests that phospholipid/TPGS mixed micelles might be a suitable drug delivery system for Icariside II to treat multidrug resistant breast cancer.

Metabolism of ginsenosides Rk3 and Rh4 from steamed notoginseng in zebrafish by ultraperformance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

In this work, the metabolite profiles of ginsenosides Rk3 and Rh4 from steamed notoginseng in zebrafish were qualitatively investigated, and their possible metabolic pathways were subsequently proposed. Metabolites of ginsenosides Rk3 and Rh4 after exposing to zebrafish for 24 h were identified by Ultraperformance Liquid Chromatography/Quadrupole -Time-of-Flight Mass Spectrometry (UPLC-TOF/MS). The separation was performed with a UPLC BEH C18 column using a binary gradient elution of 0.1 % formic acetonitrile -0.1 % formic acid water. The quasi-molecular ions of compounds were detected in negative mode. According to the quasi-molecular ions and MS2 spectra, after comparison with reference standards, molecular mass information and the potential structures about metabolites of ginsenosides Rk3 and Rh4 were obtained. In all, 5 and 6 metabolites of ginsenoside Rk3 and Rh4 including M1(M6), M2(M7), M3-M5 and N1, N2(N7), N3-N6 were identified in zebrafish respectively, which were to our knowledge reported for the first time. The results indicated that ginsenosides Rk3 and Rh4 were metabolized via desugarization, glucuronidation, sulfation, dehydroxylation, and loss of C-17 and C-23 residue pathways in zebrafish. Loss of glucose at position C-6 and glucuronidation at position C-3 or C-6 in zebrafish were considered as the main physiologic processes of ginsenosides Rk3 and Rh4.

[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.