Identification of key pharmacodynamic markers of American ginseng against heart failure based on metabolomics and zebrafish model.

Background: American ginseng (Panax quinquefolium L., AG) is a traditional Chinese medicine with multiple cardiovascular protective properties. Many bioactive components have been discovered in AG over these years. However, the understanding of these key pharmacodynamic components of activity against heart failure is insufficient. Methods: A heart failure model was established using AB line wild-type zebrafish (Danio rerio) to evaluate the anti-heart failure activity of AG. Untargeted metabolomics analysis based on ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap-mass spectrometry technology (UHPLC-QE-Orbitrap-MS) was performed to screen differential components from AG samples. The potential active components were verified using the zebrafish model. Simultaneously, network pharmacology and molecular docking techniques were used to predict the possible mechanism. Finally, the key targets of six key pharmacodynamic components were verified in zebrafish using quantitative real-time-polymerase chain reaction (Q-PCR) techniques. Results: The heart failure model was successfully established in 48 h of post-fertilization (hpf) zebrafish larvae by treating with verapamil hydrochloride. The zebrafish assay showed that the anti-heart failure effects of AG varied with producing regions. The result of the herbal metabolomic analysis based on UHPLC-QE-Orbitrap-MS indicated that ginsenoside Rg3, ginsenoside Rg5, ginsenoside Rg6, malic acid, quinic acid, L-argininosuccinic acid, 3-methyl-3-butenyl-apinosyl (1→6) glucoside, pseudoginsenoside F11, and annonaine were differential components, which might be responsible for variation in efficacy. Further analysis using zebrafish models, network pharmacology, and Q-PCR techniques showed that ginsenoside Rg3, ginsenoside Rg5, ginsenoside Rg6, malic acid, quinic acid, and pseudoginsenoside F11 were the pharmacodynamic markers (P-markers) responsible for anti-heart failure. Conclusion: We have rapidly identified the P-markers against heart failure in AG using the zebrafish model and metabolomics technology. These P-markers may provide new reference standards for quality control and new drug development of AG.

An Integrated Strategy for Rapid Discovery and Identification of Quality Markers in Gardenia Fructus Using an Omics Discrimination-Grey Correlation-Biological Verification Method.

Background: Gardenia Fructus (GF), a traditional Chinese medicine of Gardenia Ellis in Rubiaceae family, has the potential to clear heat and purge fire and has been widely used to treat multiple infection-related diseases. However, the quality markers (Q-Markers) of GF have not been revealed comprehensively. Methods: In this experiment, the transgenic zebrafish lines, Tg (l-fabp:EGFP) and Tg (lyz:EGFP), were used to evaluate two main kinds of traditional efficacies of GF, hepatoprotective and anti-inflammatory effects. All the GF samples from different production areas were tested their anti-liver injury and anti-inflammantory activities. High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry method (HPLC-Q-TOF/MS) was employed for herbal metabonomic analysis of GF samples. Gray correlation analysis (GCA) was utilized to screen out the components closely associated with the activities. Finally, the zebrafish model was applied to verify the bioactivity of the crucial components to determine the Q-Markers of GF. Results: The zebrafish models were established by inducing with hydrogen peroxide or copper sulfate and applied to quickly evaluate the hepatoprotective effect and inflammation of GF samples. 27 potentially active components for liver protection and 21 potentially active components with anti-inflammatory properties were identified by herbal metabolomic analysis based on HPLC-Q-TOF/MS. The GCA result showed that five of the 27 components were highly correlated with liver protection, 15 of the 21 components were highly correlated with anti-inflammatory activity. Among them, geniposide and crocin-1 were confirmed their bioactivities on zebrafish experiment to be responsible for the protective effects of GF against liver injury, and genipin-1-ß-D-gentiobioside, quinic acid, gardenoside, d-glucuronic acid, l-malic acid, mannitol, rutin, and chlorogenic acid were confirmed to be responsible for the anti-inflammatory effects. Finally, according to the screening principles of Q-Markers, genipin-1-ß-D-gentiobioside, geniposide, and gardenoside were preliminarily identified to be the Q-Markers of GF. Conclusion: This study established an effective research strategy of "Omics Discrimination-Grey Correlation-Biological Verification," which enabled the rapid identification of key pharmacological components of GF. These markers have provided a scientific basis for constructing a modern quality evaluation system for GF.

Formulation and evaluation of a α-linolenic acid and vitamin E succinate microemulsion with low surfactant content and free of co-surfactant for use as a nutritional supplement.

Herein, we have designed an alcohol-free and low-surfactant microemulsion to safely and effectively supply α-linolenic acid (ALA) and vitamin E (VE). Ternary phase diagrams show that the use of medium- or short-chain alcohols as the co-surfactant (CoS) was unfavorable for the formation of the ALA microemulsion due to the competitive hydrogen bonding effect and vitamin E succinate (VES) significantly increased the ALA microemulsion region by improving the hydrophilicity of the oil phase. The optimal microemulsion formulation (Mav) was 6.86% ALA, 1.14% VES, 12% surfactant and 80% water, with uniformly dispersed spherical particles with diameters of ~ 25.41 nm and viscosity of 35.17 mPa·s. The Mav was stable to high temperature, ionic strength and pH, and exhibited good physical and anti-oxidation stability. The Mav facilitated the release and hydrolysis of VES, indicating that the CoS-free microemulsion with low surfactant content is promising for the safe and effective supply of ALA and VE.

Discovery and identification of antithrombotic chemical markers in Gardenia Fructus by herbal metabolomics and zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Gardenia Fructus (GF), a traditional Chinese medicine for clearing heat and purging fire, has been reported to use to treat thrombotic related diseases, but the antithrombotic components are not clear. AIM OF THE STUDY: To develop efficient research methods for discovering some representative antithrombotic compounds of GF. MATERIALS AND METHODS: AB line zebrafish induced by arachidonic acid (AA) was used as a fast and trace-sample-required valuation model for antithrombptic effect of GF samples. Among nine samples of GF from different production areas, two samples with the largest difference in bioactivity were selected for downstream analysis. High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS) was applied to detect compounds in the GF samples. And herbal metabolomics and grey correlation analysis (GCA) were used to identify crucial compounds with potential antithrombotic activity. Then the bioactivity of those important compounds was verified on the zebrafish model. Network pharmacology was used to explore the protein targets and signaling pathways of these compounds. RESULTS: Among the GF samples, S1 (Huoshan City, Anhui Province), and S6 (Jichun City, Hubei Province), significantly differed in thrombus inhibiting bioactivity. HPLC-Q-TOF/MS identified a total of 614 compounds in each GF sample. 19 compounds were selected as important potential variables from metabolomics data by orthogonal partial least squares discriminant analysis (OPLS-DA). And 10 compounds among them were further found to be positively correlated with the antithrombotic bioactivity of GF by GCA. Finally, 3 compounds in them, geniposide, citric acid, and quinic acid, were confirmed as representative antithrombotic chemical markers of GF. Using network pharmacology analysis, some key protein targets, such as proto-oncogene tyrosine-protein kinase Src (SRC) and cyclin-dependent kinase 2 (CDK2), and some signaling pathways were found to supply powerful evidence about antithrombotic mechanisms of three compounds and GF. CONCLUSIONS: This research have succeeded to discover and identify three representative antithrombotic compounds of GF using an efficient integrated research strategy we established, an Omics Discriminant-Grey Correlation-Biological Activity strategy. The antithrombotic chemical makers we found could also contribute to provided more accurate index components for comprehensive quality control of GF.

Xiaoaiping Induces Developmental Toxicity in Zebrafish Embryos Through Activation of ER Stress, Apoptosis and the Wnt Pathway.

The aim of the study was to determine the developmental toxicity of the traditional Chinese medicine Xiaoaiping (XAP) and to investigate its underlying mechanism of action. Zebrafish embryos were incubated with 0.4, 0.8, 1.2, and 1.6 mg/mL XAP. Endpoints such as mortality, hatching rate, malformation, body length, morphology score, swimming behavior, histological changes, reactive oxygen species (ROS) production, total superoxide dismutase (T-SOD) activity, and the mRNA expression of genes related to oxidative stress, endoplasmic reticulum (ER) stress, apoptosis, and the Wnt pathway were evaluated. Our results demonstrated that XAP exposure increased mortality and malformation and reduced the hatching rate. XAP resulted in severe malformation, including swim bladder deficiency, yolk retention, pericardial edema, and tail curvature. Histopathological analysis showed that XAP induced liver, heart and muscle injury. High doses (≥1.2 mg/mL) of XAP notably decreased the locomotor capacity of zebrafish. ROS generation was remarkably increased and T-SOD activity was decreased, confirming that oxidative stress was induced by XAP. The mRNA expression levels of ER stress-related genes (chop, hspa5, hsp90b1, and perk), apoptosis-related genes (caspase-3, bax, and p53) and wnt11 were significantly upregulated by XAP exposure. The expression levels of the oxidative stress-related genes (cat, sod1, and gstp2), Wnt pathway-related genes (ß-catenin, wnt3a, and wnt8a) and bcl-2 initially increased and then decreased as the XAP exposure dose increased. In conclusion, we provide evidence for the first time that XAP can induce dose-related developmental toxicity, and ER stress, apoptosis and the Wnt pathway participate in the toxicity regulation.

Preclinical pharmacokinetics of the novel PI3K inhibitor GDC-0941 and prediction of its pharmacokinetics and efficacy in human.

The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation is associated with the development of many cancers. GDC-0941, a potent and selective inhibitor of PI3K, was characterised preclinically in in vitro and in vivo studies. Plasma protein binding was extensive, with free fraction less than 7%, and blood-to-plasma ratio ranged from 0.6 to 1.2 among the species tested. GDC-0941 human hepatic clearance was predicted to be moderate by liver microsomal incubations. GDC-0941 had high permeability in Madin-Darby canine kidney cells. The clearance of GDC-0941 was high in mouse (63.7 mL/min/kg), rat (49.3 mL/min/kg) and cynomolgus monkey (58.6 mL/min/kg), and moderate in dog (11.9 mL/min/kg). The volume of distribution ranged from 2.52 L/kg in rat to 2.94 L/kg in monkey. Oral bioavailability ranged from 18.6% in monkey to 77.9% in mouse. Predicted human clearance and volume of distribution using allometry were 6 mL/min/kg and 2.9 L/kg, respectively. The human efficacious doses were predicted based on results from preclinical pharmacokinetic studies and xenograft models. GDC-0941 preclinical characterisation and predictions of its properties in human supported its progression towards clinical development. GDC-0941 is currently in phase II clinical trials.