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Phytolacca acinosa Roxb (PAR), a traditional Chinese medicine, has been widely used as a diuretic drug for a long period of time for the treatment edema, swelling, and sores. However, it has been reported that PAR might induce hepatotoxicity, while the mechanisms of its toxic effect are still unclear. In this study, network toxicology and metabolomic technique were applied to explore PAR-induced hepatotoxicity on zebrafish larvae. We evaluated the effect of PAR on the ultrastructure and the function of the liver, predictive targets, and pathways in network toxicology, apoptosis of liver cells by PCR and western blot, and metabolic profile by GC-MS. PAR causes liver injury, abnormal liver function, and apoptosis in zebrafish. The level of arachidonic acid in endogenous metabolites treated with PAR was significantly increased, leading to oxidative stress in vivo. Excessive ROS further activated the p53 signal pathway and caspase family, which were obtained from KEGG enrichment analysis of network toxicology. The gene levels of caspase-3, caspase-8, and caspase-9 were significantly increased by RT-PCR, and the level of Caps3 protein was also significantly up-regulated through western blot. PAR exposure results in the liver function abnormal amino acid metabolism disturbance and motivates hepatocyte apoptosis, furthermore leading to liver injury.

In this study, data of amino acids of Cordyceps samples from Qinghai and Tibet was analyzed with self-organizing map neural network. A model of XY-Fused network was established with the content of 8 major amino acids and total amino acids for the identification of geographical origins of Cordyceps from Qinghai and Tibet. It had the prediction accuracy of 83.3% for the test set. In addition, data mining indicated that methionine was a special kind of amino acid in Cordyceps which could serve as a marker to identify its geographical origins. On this basis, the content ratio of methionine to total amino acids was proposed to be a quantifiable indicator to distinguish Cordyceps from Qinghai and Tibet.

Metallomics is a frontier interdisciplinary subject at its vigorous development stage. Its goal is to systematically study the content, distribution, chemical species, structural characteristics and functions of metal elements in biological system. It is also a comprehensive discipline to study the existing state and function of free or complex metal elements in life. Metallomics is an ideal tool to study the biological behavior of inorganic elements, which can be used to solve many problems in the research of mineral Chinese medicine(MCM). It provides a strong theoretical basis and technical support for the research of MCM. Its theory and methods provide re-ference and enlightenment for the in-depth study of MCM, and also provide new ideas and open up new ways for the research of MCM. The application of metallomics theory and methods in the research of MCM is of great significance to reveal the material basis and mec-hanism of MCM, promote the process of basic research on MCM, fully exploit and utilize medicinal mineral resources and carry forward the traditional MCM treasure in China. In this paper, we introduced the concept, academic development, research content and research methods of metallomics, and discussed the application prospects of metallomics in the analysis of inorganic element composition characteristics and quality control, material basis and mechanism of MCM, so as to provide reference for further researches on MCM.

With the increasing incidence of hepatobiliary diseases, it is particularly important to understand the role of molecular, cellular and physiological factors in the clinical diagnosis and treatment with traditional Chinese medicine(TCM) in the development of liver disease. Appropriate animal models can help us identify the possible mechanisms of relevant diseases. Danio rerio(zebrafish) model was traditionally used to study embryonic development, and has been gradually used in screening and evaluation of liver diseases and relevant drug in recent years. Zebrafish embryos develop rapidly and the digestive organs of 5-day-old juvenile fish are all mature. At this stage, they may develop hepatobiliary diseases induced by developmental defects or compounds. Zebrafish liver is similar to human liver in cell composition, function, signal transduction, response to injury and cell process mediating liver disease. Furthermore, due to the high conservation of genes and proteins between humans and zebrafish, zebrafish becomes an alternative system for studying basic mechanisms of liver disease. Therefore, genetic screening could be performed to identify new genes involving specific disease processes, and chemical screening could be made for drugs in specific processes. This paper briefly introduced the experimental properties of zebrafish as model system, emphasized the study progress of zebrafish models for pathological mechanism of liver diseases, especially fatty liver, and drug screening and evaluation, so as to provide ideas and techniques for the future liver toxicity assessment of TCM.

Ginseng has been widely applied in clinical practice, but the cultivation age cannot be ignored as it influences the quality of ginseng and its products. In this work, different cultivation ages of fresh ginseng (FG) from four to seven years were analysed by UPLC-Q-TOF-MS/MS. Principal component analysis and supervised orthogonal partial least squared discrimination analysis, which belong to the normal method of multivariate statistical analysis, were applied to discover the characteristic components of FG at different cultivation ages. The components of new type of red ginseng (NRG) derived from FG at different cultivation ages were compared by HPLC analysis. The pharmacological anti-inflammatory activity was evaluated by ELISA and qPCR. The result showed that the characteristic components of both 6- and 7-year-old ginseng were ginsenoside Rb1, mal-ginsenoside Rb1, ginsenoside Rc, mal-ginsenoside Rc, mal-ginsenoside Rb1 isomer, and mal-ginsenoside Rb2. Moreover, the characteristic components of both 4- and 5-year-old ginseng were ADP-glucose and 3-hydroxyhexanoyl CoA. In addition, 6-year-old NRG has higher rare ginsenosides than 4-year-old NRG, which possesses great anti-inflammatory activity in vitro. The results reveal the ginsenoside transformation law of NRG processing and suggest that the cultivation age of FG influences the content of ginsenosides in NRG. Therefore, 6-year-old ginseng is more suitable for red ginseng processing and clinical use.

Clinical reports on hepatotoxicity that arise from Rhizoma Paridis have recently received widespread attention. Because the hepatotoxicity mechanism is little understood, this research strived to investigate the hepatotoxicity mechanism of Rhizoma Paridis extracts based on iTRAQ quantitative proteomics and metabonomics. The extraction solutions were administrated to rats for 7 days by gavage, and the hepatotoxicity was assessed through quantification of biochemical indexes and Oil red O staining. Additionally, the mechanism of hepatotoxicity was investigated by metabonomics based upon GC-MS and iTRAQ quantitative proteomics. The biochemical and histopathological analysis stood out that Rhizoma Paridis extract could induce liver injury, which was proved by the formation of fat droplets, the changes of mitochondrial structure, and biochemical parameters. The iTRAQ proteomics and metabonomics revealed that Rhizoma Paridis-induced hepatotoxicity was chiefly connected with the abnormal activity of mitochondrion function, which brought about oxidative stress injuries and inflammation, finally causing cell apoptosis. Collectively, we have provided previously uncharacterized hepatotoxic mechanism induced by Rhizoma Paridis and a reference to ensure its safe use in the future.

In this paper, some quality problems of mineral medicine Calamina and calcined Calamina have been discussed after determination and analysis of the quality parameters of a large number of market samples, and the countermeasures are put forward. According to the XRD results, as well as the results of tests included in Chinese Pharmacopoeia(2015 edition), the authenticity of Calamina and calcined Calamina samples were identified. The content of zinc oxide in samples were determined by the method of determination in Chinese Pharmacopoeia. Individually, inductively coupled plasma mass spectrometry(ICP-MS), inductively coupled plasma atomic emission spectrometry(ICP-AES) and atomic fluorescence spectrometry(AFS) methods were used for the determination of impurity elements and harmful elements in Calamina and calcined Calamina samples. Four kinds of impurity elements of magnesium(Mg), iron(Fe), aluminum(Al), calcium(Ca) and five harmful elements such as lead(Pb), cadmium(Cd), arsenic(As), copper(Cu), mercury(Hg) were measured. The study showed that: ① Fake Calamina products on the market were overflowing; ② The mineral origin of the mainstream Calamina in the market is inconsistent with that stipulated in Chinese Pharmacopoeia(2015 edition); ③ The contents of harmful elements Pb and Cd in Calamina and calcined Calamina are generally higher, while the contents of harmful elements As and Cu in some inferior Calaminae are higher; ④ Parts of calcined Calamina were improperly or inadequately processed. In view of these quality problems, the countermeasures are put forward as follows: ① It is suggested that hydrozincite should be approved as the mineral source of Calamina, and be included by Chinese Pharmacopoeia; ② Strengthen the research on the specificity of Calamina identification methods to improve the quality control level; ③ Strengthen the research on the processing of Calamina, and formulate the limit standards for the content of Pb and Cd in Calamina; ④ Carry out research on the artificial synthesis of Calamina and calcined Calamina, in order to cope with the current shortage of Calamina resources and ensure the sustainable development of Calamina medicinal materials.

Zebrafish larvae were used to further understand the liver toxicity of nux vomica. The histopathology, protein expression, and gene expression were assessed to confirm apoptosis in the liver, and then, profiles of the metabolites in zebrafish were investigated by untargeted metabolomic assessment to understand the potential toxicity mechanism of nux vomica. Histopathological observations showed that nux vomica caused damage to liver cells. Western blot results indicated increased expression of activated caspase3, and the result of real-time polymerase chain reaction showed a significant increase in the expression level of caspase-3, caspase-8, and caspase-9 genes (p 0.05) compared with the control group. The liver injury from nux vomica was linked to the downregulation of amino acid (e.g., proline and alanine) and fatty acid (e.g., palmitoleic acid) metabolism and upregulation of some other fatty acid (e.g., arachidic acid) and purine (e.g., xanthine and uric acid) metabolism. The hepatotoxicity of nux vomica resulted from metabolic pathway disturbances, including small molecules involved in energy, purine, lipids, and amino acid metabolism.

Background PPVI 1 and PPⅦ 2 were a group of Pennogenin compounds extracted from the Paris polyphylla and caused hepatotoxicity in human, while the potential underlying mechanism was unclear. Purpose To evaluated the adverse effects of PP VI and PP Ⅶ on the liver in the zebrafish. Method In this study, 4dpf zebrafish were used for acute toxicity test, LC0 was calculated, and 1/2LC0 and 3/5LC0 were selected for pathological section and liver area measurement to verify the hepatotoxicity of PPVI and PPⅦ. Etabonomics study was then conducted to further explore the mechanism of hepatotoxicity of PPVI and PPⅦ. Lovastatin was used as an inhibitor, and PCR was used to verify the results. Result The result showed that under the condition of sub-lethal concentration exposure, hepatotoxicity-included changes in liver phenotype (liver area), hepatocyte swelling and degeneration, liver cell apoptosis and disturbed biochemical index were observed in zebrafish treated with PPVI and PPVII. Furthermore, the transcriptome was conducted to confirm the toxicity mechanism shared with PPVI and PPVII, and we found that steroid biosynthesis process and the related target genes were mainly affected. While, lovastatin treatment effectively ameliorated PPVII-induced zebrafish liver injury by improving the liver tissue structure and regulate the expression of associated genes including HMGCRA, SREBP, LSS, CYP2R1, PIK3R3A, GDPD1 and PFKFB-2. Conclusion This study was the first investigation to provide the direct evidence of hepatotoxicity of PP Ⅵ and PP Ⅶ in vivo zebrafish model, which were related to the steroid biosynthesis. furthermore, in lovastatin played an important role in protection against hepatotoxicity induced by PPVI and PPⅦ by regulating the cholesterol metabolism.

Ethnopharmacological relevance Forsythiae Fructus (called Lianqiao in Chinese), the fruit of Forsythia suspensa (Thunb.) Vahl, is utilized as a common traditional medicine in China, Japan and Korea. It is traditionally used to treat pyrexia, inflammation, gonorrhea, carbuncle and erysipelas. Depending on the different harvest time, Forsythiae Fructus can be classified into two forms, namely Qingqiao and Laoqiao. The greenish fruits that start to ripen are collected as Qingqiao, while the yellow fruits that are fully ripe are collected as Laoqiao. Both are applied to medical use. This review aims to provide a systematic summary of F. suspensa (Forsythia suspensa (Thunb.) Vahl) and to reveal the correlation between the traditional uses and pharmacological activities so as to offer inspiration for future research. Materials and methods All corresponding information about F. suspensa was searched by Scifinder and obtained from scientific databases including Springer, Science Direct, Wiley, Pubmed and China Knowledge Resource Integrated (CNKI). Local dissertations and books were searched as well. Results According to classical Chinese herbal texts and Chinese Pharmacopoeia, Forsythiae Fructus dominantly displays heat-clearing and detoxifying effects in TCM prescriptions. In modern research, more than 230 compounds were separated and identified from F. suspensa. 211 Of them were isolated from fruits. Lignans and phenylethanoid glycosides are considered as the characteristic and active constituents of this herb, such as forsythiaside, phillyrin, rutin and phillygenin. They exhibited anti-inflammatory, antioxidant, antibacterial, anti-virus, anti-cancer and anti-allergy effects, etc. Currently, there is no report on the toxicity of Forsythiae Fructus, despite slight toxicity of forsythiaside reported in local publications. Compared to Laoqiao, Qingqiao contains higher levels of forsythiaside, forsythoside C, cornoside, rutin, phillyrin, gallic acid and chlorogenic acid and lower levels of rengyol, β-glucose and S-suspensaside methyl ether. Conclusion Heat-clearing actions of Forsythiae Fructus are based on the anti-inflammatory and antioxidant properties of lignans and phenylethanoid glycosides. Detoxifying effects attribute to the antibacterial, antiviral and anti-cancer activities of Forsythiae Fructus. And traditional Chinese medicine (TCM) characteristics of Forsythiae Fructus (bitter flavor, slightly cold nature and lung meridian) supported its strong anti-inflammatory effects. In addition, the remarkable anti-inflammatory and antioxidant capacities of Forsythiae Fructus contribute to its anti-cancer and neuroprotective activities. The higher proportion of lignans and phenylethanoid glycosides in Qingqiao than Laoqiao might explain the better antioxidant ability of Qingqiao and more frequent uses of Qingqiao in TCM prescriptions. For future research, more in vivo experiments and clinical studies are encouraged to further clarify the relation between traditional uses and modern applications. Regarding to Qingqiao and Laoqiao, they remain to be differentiated by all-round quality control methods, and the chemical compositions and clinical effects between them should be compared.

Euphorbia pekinensis Rupr. (EP) is a Euphorbia species of Euphorbiaceae, which is widely used in traditional Chinese medicine. It has been reported to exhibit therapeutic effects on solid tumors, leukemias, and malignant ascites although underlying molecular mechanisms are poorly delineated. Anti-angiogenic therapy is a recognized strategy for treating cancer-based solid tumors, and is also associated with malignant ascites treatment.To study the anti-angiogenic properties of the water extract of EP vinegar preparation (WEVEP).Following WEVEP treatment, intersegmental blood vessels were assessed during the development of transgenic Tg (flk: mCherry) zebrafish as was the proliferation, migration and network formation of HUVECs in vitro. mRNA expression of specific angiogenic-related genes including VEGF family members, Met, and NRP2 was also measured using quantitative real-time PCR (Q-PCR).Data demonstrated that angiogenesis was inhibited by the WEVEP in zebrafish (from 100µg/mL to 250µg/mL, p0.0001) and in the HUVEC model (from 100µg/mL to 400µg/mL, p0.0001). In the zebrafish model, the mean vessel numbers of administered groups were 26.00 ± 1.29 (100µg/mL), 24.54 ± 2.20 (150µg/mL), 22.66 ± 2.68 (200µg/mL), 20.80 ± 1.75 (250µg/mL), compared to 27.67 ± 0.96 of control group. Relative quantitative gene expression in zebrafish treated with WEVEP demonstrated that only VEGFR3 was significantly increased and other 23 genes including Met, VEGFA, Flt-1 were significantly decreased.WEVEP can positively modulate angiogenesis via multiple targeting mechanisms. Our novel results contribute towards the discovery of a possible mechanism(s) of the traditional use of EP in the treatment of cancer and malignant ascites.

Glutamate-induced excitotoxicity is a key pathological mechanism in many neurological disease states. Ecdysterones derived from Rhaponticum carthamoides (Willd.) Iljin (RCI) have been shown to alleviate glutamate-induced neuronal damage; although their mechanism of action is unclear, some data suggest that they enhance signaling in the mechanistic target of rapamycin (mTOR) signaling pathway. This study sought to elucidate the mechanisms underlying ecdysterone-mediated neuroprotection. We used in silico target prediction and simulation methods to identify putative ecdysterone binding targets, and to specifically identify those that represent nodes where several neurodegenerative diseases converge. We then used histological analyses in a rat hippocampal excitotoxicity model to test the effectiveness of ecdysterones in vivo. We found that RCI-derived ecdysterones should bind to glutamatergic NMDA-type receptors (NMDARs); specifically, in vivo modeling showed binding to the GRIN2B subunit of NMDARs, which was found also to be a node of convergence in several neurodegenerative disease pathways. Computerized network construction by using pathway information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed putative links between GRIN2B and mTOR pathway elements including phosphoinositide-3kinase (PI3K), mTOR, and protein kinase C (PKC); these elements are associated with neuronal survival. Brain tissue western blots of ecdysterone-treated rats showed upregulated PI3K, Akt, mTOR, and phosphorylated Akt and mTOR, and down regulated GRIN2B and the apoptotic enzyme cleaved caspase-3. Ecdysterone treatment also prevented glutamate-induced rat hippocampal cell loss. In summary, RCI-derived ecdysterones appear to prevent glutamatergic excitotoxicity by increasing mTOR/Akt/PI3K signaling activity.

The decline of male fertility and its consequences on human populations are important public-health issues. However, there are limited choices for treatment of male infertility. In an attempt to identify a compound that could promote male fertility, we identified and characterized a library of small molecules from an ancient formulation Wuzi Yanzong-Pill, which was used as a folk medicine since the Tang dynasty of China. We found that SB enabled evident repairs in oligoasthenospermia-associated testicular tissue abnormality and in spermatogenesis disruption, resulting in significant improvements of sperm count, mobility, and reproductive ability in oligoasthenospermia mice. Furthermore, SB could alter substantial testicular genes (2033), among which, upregulation of Fst while downregulation of Inhba involved in reproductive signaling pathway could explain its role in enhancing spermatogenesis. The encouraging preclinical data with pharmacokinetics warranted a rapid development of this new class of therapeutic agent. Our finding provides a strong potent drug for treatment of male infertility.

Ethnopharmacological relevance Ranunculus japonicus Thunb. (short for R. japonicus) is a topically applied herb with the activities of removing jaundice, nebula and edema, preventing malaria, stopping asthma, promoting diuresis and relieving pain. It was firstly recorded in Zhouhou Beiji Fang and has been used for the treatment of malaria, ulcers, carbuncle, jaundice, migraine, stomachache, toothache and arthritis for over 1800 years. Aim of the study This study aimed to uncover the potentially effective components of R. japonicus and the pharmacological mechanisms against rheumatoid arthritis (RA) by combing LC-MS and network pharmacology. Materials and methods Firstly, the chemical constituents of R. japonicus were qualitatively identified by UPLC-ESI-LTQ-Orbitrap MS. Then we performed target prediction by PharmMapper, protein-protein interaction (PPI) analysis via String, GO and KEGG pathway enrichment analysis by DAVID and constructed the compound-target-pathway network using Cytoscape. Thirdly, crucial compounds in the network were quantitatively analyzed to achieve quality control of R. japonicus. Finally, the pharmacological activities of R. japonicus and two potentially bioactive ingredients were validated in RA-FLSs (Rheumatoid Arthritis Fibroblast-like Synoviocytes) in vitro. Results Overall fifty-four ingredients of R. japonicus were identified and forty-five components were firstly discovered in R. japonicus. Among them, twenty-seven validated compounds were predicted to act on twenty-five RA-related targets and they might exhibit therapeutic effects against RA via positive regulation of cell migration, etc. Nine potentially bioactive components of R. japonicus which played important roles in the compound-target-pathway network were simultaneously quantified by an optimized UPLC-ESI-Triple Quad method. In vitro, compared to control group, R. japonicus extract, berberine and yangonin significantly inhibited the migration capacity of RA-FLSs after 24 h treatment. Conclusion This study clarified that R. japonicus and the bioactive ingredients berberine and yangonin might exert therapeutic actions for RA via suppressing the aggressive phenotypes of RA-FLSs through combined LC-MS technology and network pharmacology tools for the first time. The present research provided deeper understanding into the chemical profiling, pharmacological activities and quality control of R. japonicus and offered reference for further scientific research and clinical use of R. japonicus in treating RA.

Realgar is a mineral traditional medicine with definite efficacy. The function of realgar is detoxicating, insecticiding, eliminating dampness and phlegm, etc. It is widely applied in clinical practice by compatibility medicines. However, the safety and scientificalness of clinical application are questioned because of the toxic effect caused by arsenic compounds. At present, there are still many problems in the research of realgar, which are mainly manifested in three areas: the expression of main components and effective substances are inconsistent; the anti-tumor mechanism is difficult to explain at the molecular level; the mechanism of compatibility is not clear. As a result, realgar and realgar-containing Chinese patent medicines are frequently prohibited from entering the international market, and the reputation of traditional Chinese medicine is also damaged. This paper would analyze the research status of realgar at home and abroad as well as its problems from its main components, effective substances, anti-tumor mechanism and compatibility mechanism. In view of these difficulties, quantum chemical calculation method is proposed to solve them, so as to make up for the shortcomings and limitations of experimental technology and experimental conditions, reduce the cost of realgar research and improve research efficiency. Moreover, it provides inspiration for research of other mineral medicine.

Objective To explore the developmental toxicity and the potential toxicological mechanism of safflower ( Carthamus tinctorius L.) on zebrafish embryos/larvae. Methods Mortality, malformations and increased apoptosis induced by safflower were assessed in zebrafish embryos from 6 to 96 hours post-fertilization. Enzymes and genes in the anti-oxidative and apoptotic pathways were also assayed. Results The lethal concentration 50 of safflower to zebrafish embryos was 345.6 mg/L. Hatching inhibition, abnormal spontaneous movement, depressed heart rate, pericardial edema, yolk sac edema, abnormal head-trunk angle, inhibition of melanin release, enlarged yolk, and short body length were observed in safflower-treated zebrafish. Additional apoptotic cells mainly appeared around the heart. Safflower exposure changed the activities of defense enzymes (SOD↑, CAT↑, MDA↑, GPX without trend), increased MDA content, decreased caspase-3 activity, and altered mRNA levels of related genes ( ogg1 ↓, p53 ↓, Cu/Zn-sod ↑, Mn-sod ↓, cat ↓, gpx ↑). Conclusion Safflower exhibits developmental toxicity for zebrafish embryos/larvae. The developing heart was speculated as the target organ of toxicity. Oxidative stress and increased apoptosis have roles in the developmental toxicity of safflower. This article provides a novel method to research the teratogenicity and possible mechanisms of toxicity of traditional Chinese medicines that are prohibited or contraindicated in pregnant women.

As a classical traditional Chinese medicine, Wuzi-Yanzong-Wan (WZYZW) has been widely applied for several centuries to treat non-obstructive oligoasthenozoospermia (NOA), although its pharmacological mechanisms remain largely unknown. In this study, both plasma and urine metabolomics profiling was first analysed to explore the therapeutic mechanisms of WZYZW in NOA rats induced by removal of the unilateral testicle. Then, 106 identified compounds comprising WZYZW (our previous work), for which putative targets were discovered using systems pharmacology, were systematically analysed via mRNA microarrays to validate their putative targets. Finally, metabolomics-tested WZYZW-regulated metabolites were connected with validated targets using Spearman correlation analysis to further confirm the targets from a biological perspective. The results suggested that WZYZW plays key roles in modulating the concentrations of 18 metabolites in the metabolism of amino acids, lipids and so on, normalizing the metabolic phenotype and regulating metabolic disorders. Moreover, 27 targets of WZYZW (23 compounds) against NOA were validated, and metabolomics-tested metabolites were also found to be significantly related to these identified targets, suggesting that these targets and compounds are worthy of further research. This work offers the first systematic investigation of the efficacy of WZYZW against NOA and illustrates a practicable approach for explaining the molecular mechanisms of multicomponent drugs.

Background Euphorbia kansui is effective in treating various diseases, such as ascites and edema, but its liver toxicity is a major obstacle in its wide use in the clinic. However, further investigations have suggested that Euphorbia kansui can cause liver injury. Hypothesis The study aims to investigate the effect of Euphorbia kansui exposure on zebrafish, and explain the underlying toxicity mechanisms from a comprehensive perspective. Study design The 4dpf zebrafish larvae were exposed to Euphorbia kansui at a sub-lethal concentration. Methods We evaluated the effect of Euphorbia kansui on the ultrastructure and function of the liver, apoptosis of liver cells by PCR and western blot, and metabolic profile by GC-MS based on sub-lethal concentrations. Results Our results suggested Euphorbia kansui could lead to liver injury and significant alteration of the metabolomics of the zebrafish larvae in sub-lethal concentration conditions. It could also induce alterations in liver microstructure, hepatic function, gene expression and protein associated with the apoptosis process, as well as endogenous metabolism. KEGG pathway analysis identified some biological processes on the basis of different metabolisms and their associated processes especially for amino acid metabolism. Conclusion The results bring us closer to an in-depth understanding of the toxic effects of Euphorbia kansui on zebrafish liver, which will be significantly helpful in effectively guiding safer clinical application of this herb in the clinic. Furthermore, our results also showed the zebrafish model is reliable for evaluation of Euphorbia kansui extract hepatotoxicity and as a methodological reference for the evaluation of Traditional Chinese Medicine with underlying liver toxicity.

Licorice is one of the most common herbs in traditional Chinese medicine, and classified as top grade in Shen Nong Ben Cao Jing. There are three different original plants of licorice stipulated in Chinese Pharmacopeia, Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L., and Glycyrrhiza inflata Bat. However, previous investigation showed that the pharmacodynamic effects of the three licorices were quite different. It is very difficult to identify them by the classical identification methods. In order to establish a fast and effective identification method, we collected 240 licorice plants from 21 populations of 7 provinces, and amplified their ITS and psbA-trnH sequences. ITS sequences with a full length of 616 bp and psbA-trnH sequences with a full length of 389 bp were obtained separately. Using DNAMAN to analyze these sequences, 4 variable sites were found in ITS sequences and 2 ITS haplotypes were determined, and 3 variable sites were found in psbA-trnH sequences and 4 psbA-trnH haplotypes were determined. With the combination analysis of ITS and psbA-trnH sequences, the molecular identification method of original licorice was established. Using this method, 40 samples of licorice slices collected from 4 main herbal material markets in China were identified successfully. Furthermore, the contents of 2 triterpenes, 18α-glycyrrhizic acid and 18β-glycyrrhizic acid, and 4 flavonoids, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin in these licorice pieces were examined by HPLC and the results were analyzed using SPSS 21.0. This study provides a new method in identification of licorice, which may serve as a guideline for quality control of licorice slices.

Bupleuri Radix is one of the most frequently used herbal medicines in China with a 2 000-year medicinal history. However, the use of Bupleuri Radix is very confused. Twenty-five species and eight varieties of Bupleurum have been used as Bupleuri Radix in different regions of China. It is very difficult to identify these Bupleurum species using traditional morphological method. In order to establish a fast and effective method to identify these Bupleurum species, we collected 168 Bupleurum medicinal plants from 14 populations of 9 provinces, and amplified their ITS sequences. 168 ITS sequences with a full length of 600-606 bp were obtained. DNAMAN analyzing results showed that 86 variable sites were present in these sequences and 19 haplotypes (TH1-TH19) were determined. After calculating K2P distance and analyzing an NJ tree, we established a molecular identification method based on ITS sequence. Using this method, 52 samples of Bupleuri Radix were identified successfully. Furthermore, we tested saikosaponin a, c, d content in these Bupleuri Radix by HPLC and analyzed the results by ANOVA and LSD T test to evaluate the quality of Bupleuri Radix. This method is significant for effective identification of Bupleurum medicinal plants, and quality control of Bupleuri Radix in the market.

Euphorbia lathyris ( E. lathyris ), a traditional Chinese medicine, rich in diterpenoids, has been widely used for the treatment of hydropsy, especially the ascites in China, with its mechanism is still unclear. For exploring possible diuresis mechanism of E. lathyris , in this study, a potential diuresis related target, urea transporter B (UT-B), and its potential ligands were discovered by using bioinformatics method of “Inverse Docking”. Then, homology modeling, molecular docking and ligand-based pharmacophore methods were applied to research the possible mechanism of diuresis. Bioinformatics analysis showed that most of diterpenoids in E. lathyris were optimal ligands for the UT-B protein and these predicted compounds also exhibited stable affinity to UT-B binding site, a hydrophobic region in urea passage. The HPLC-TOF-MS method was used to characterize the main diterpenoids content of E. lathyris extracts and three out of eight proposed diterpenoids were detected and assigned. In vitro UT-B inhibition experiment of erythrocyte lysis assay was then applied to confirm the diuretic effects of the diterpenoids concentrated extracts of E. lathyris . The half erythrocyte lysis rate of 23.89 µg/ml of diterpenoids concentrated extracts showed moderate UT-B inhibition efficacy. All these performances might also raise an alternative approach for the design of new diuretic drugs.

An accurate and sensitive high-performance liquid chromatography method coupled with ultralviolet detection and precolumn derivatization was developed for the simultaneous quantification of the major bile acids in Artificial Calculus bovis, including cholic acid, hyodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid. The extraction, derivatization, chromatographic separation, and detection parameters were fully optimized. The samples were extracted with methanol by ultrasonic extraction. Then, 2-bromine-4'-nitroacetophenone and 18-crown ether-6 were used for derivatization. The chromatographic separation was performed on an Agilent SB-C18 column (250 × 4.6 mm id, 5 μm) at a column temperature of 30°C and liquid flow rate of 1.0 mL/min using water and methanol as the mobile phase with a gradient elution. The detection wavelength was 263 nm. The method was extensively validated by evaluating the linearity (r(2) ≥ 0.9980), recovery (94.24-98.91%), limits of detection (0.25-0.31 ng) and limits of quantification (0.83-1.02 ng). Seventeen samples were analyzed using the developed and validated method. Then, the amounts of bile acids were analyzed by hierarchical agglomerative clustering analysis and principal component analysis. The results of the chemometric analysis showed that the contents of these compounds reflect the intrinsic quality of artificial Calculus bovis, and two compounds (hyodeoxycholic acid and chenodeoxycholic acid) were the most important markers for quality evaluating.

Lipidomics is a lipid-targeted metabolomics approach focusing on comprehensive analysis of all lipids with which they interact in biology systems. Recent technological advances in MS and chromatography have greatly enhanced the developments and applications of metabolic profiling of diverse lipids in complex biological samples. Lipidomics will not only provide insights into the specific functions of lipid species in health and disease, but will also identify potential biomarkers for establishing preventive or therapeutic programs for human disease. In this review, recent applications of lipidomics to understand animal models of disease such as metabolic syndromes, neurodegenerative diseases, cancer and infectious diseases are considered. We also discuss the lipidomics for the future perspectives and their potential problems.

Salvianolic acid Y (TSL 1), a new phenolic acid with the same planar structure as salvianolic acid B, was isolated from Salvia officinalis. The structural elucidation and stereochemistry determination were achieved by spectroscopic and chemical methods, including 1D, 2D-NMR (1H-1H COSY, HMQC and HMBC) and circular dichroism (CD) experiments. The biosynthesis pathway of salvianolic acid B and salvianolic acid Y (TSL 1) was proposed based on structural analysis. The protection of PC12 cells from injury induced by H2O2 was assessed in vitro using a cell viability assay. Salvianolic acid Y (TSL 1) protected cells from injury by 54.2%, which was significantly higher than salvianolic acid B (35.2%).

Rhizoma Paridis, as a Traditional Chinese Medicine (TCM), has been used in clinic for thousands of years. Recently, the hepatic toxicity was reported in some published articles while its hepatotoxicity mechanisms have not been well established. Therefore, the present study was performed to determine the effect of Rhizoma Paridis treatment on the lipid deposition and metabolism, oxidative stress and mitochondrial dysfunction, and explore the underlying molecular mechanism through L02 cell, rat and zebrafish larvae. Rhizoma Paridis could diminish cell activity and cell proliferation, brought on cell apoptosis and elevated the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared with the control group, as evaluated in cell cultures. Rhizoma Paridis could result in the change of the liver structure and the liver function in the rat model and zebrafish larvae. Our results showed that Rhizoma Paridis could increase hepatic lipid accumulation, which was similar to the previous study and probably exerted toxic effect through intensive fatty acid lipogenesis, inhibition of fat degradation. Meanwhile, this experiment highlighted the importance of the oxidative stress, mitochondrial dysfunction, ER function, and the inflammation response in Rhizoma Paridis-induced disorder of hepatic lipid metabolism, which proposed a novel mechanism for interpretation of Rhizoma Paridis exposure inducing the disorder of lipid metabolism in vertebrates. Furthermore, the result of this experiment suggested that the toxicity response of zebrafish larvae was similar to the conventional model with a significant advantage.

Rhizoma Paridis hepatotoxicity is a risk factor limiting its extensive use in clinic, there is limited information available regarding the mechanism by which typical environmental levels of exposure can contribute to the onset of this disease. The adult zebrafish were exposed to Rhizoma Paridis at a sub-lethal concentration. The alterations in protein expression profiles and metabolite levels in the adult zebrafish liver, a popular model for toxicity assessment, exposed to the Rhizoma Paridis were observed. The result showed that Rhizoma Paridis exposure treatment caused an obvious toxic effect on the zebrafish liver, resulting in a significant change of the liver organization structure and various biochemical parameters. The hepatotoxicity of adult zebrafish liver induced by Rhizoma Paridis was mainly associated with lipid metabolism and energy metabolism disorder. Furthermore, oxidative stress injury, inflammation, and endoplasmic reticulum stress might also be involved in the hepatotoxicity. Our study facilitated the understanding of molecular signatures of toxic effects of Rhizoma Paridis causing liver injury to move away from the risk assessment based on in vivo animal experiments.

Ten lathyrane-type diterpenoids named Euphorbia Factor L12-L21 (1-10) and twelve known diterpenoids (11-22) were isolated from seeds of Euphorbia lathyris. The structures of these compounds were determined by extensive spectroscopic (UV, IR, HRESIMS, 1D and 2D NMR) analyses. In addition, the configuration of Euphorbia Factor L12 (1) was further confirmed by X-ray crystallographic and circular dichroism (CD) analyses. A putative biogenetic relationship to these compounds was proposed. Cytotoxicity of the isolated compounds against C6 and MCF-7 cell lines were evaluated. Compounds 1, 5, 7, 12 and 17 exhibited considerable cytotoxic activities (IC50 12.4-36.2 μM).

Two new compounds beauvericins M1 (1) and S1 (2) were isolated from Bombyx batryticatus. Their structures were established as (3α,6α)-3-benzyl-6-secbutyl-4-methylmorpholine-2,5-dione (1) and (5α,8α)-epidioxyergosterol-24-one-6,22-dien-3β-ol (2) by various spectroscopic techniques including 1D NMR ((1)H NMR and (13)C NMR), 2D NMR (HSQC, HMBC, (1)H-(1)H COSY, NOESY), and HR-ESI-TOF-MS.

In the last decade, proteomics and metabolomics have contributed substantially to our understanding of different diseases. Proteomics and metabolomics aims to comprehensively identify proteins and metabolites to gain insight into the cellular signaling pathways underlying disease and to discover novel biomarkers for screening, early detection and diagnosis, as well as for determining prognoses and predicting responses to specific treatments. For comprehensive analysis of cellular proteins and metabolites, analytical methods of wider dynamic range higher resolution and good sensitivity are required. Ultra performance liquid chromatography-mass spectrometry(Elevated Energy) (UPLC-MS(E)) is currently one of the most versatile techniques. UPLC-MS(E) is an established technology in proteomics studies and is now expanding into metabolite research. MS(E) was used for simultaneous acquisition of precursor ion information and fragment ion data at low and high collision energy in one analytical run, providing similar information to conventional MS(2). In this review, UPLC-MS(E) application in proteomics and metabolomics was highlighted to assess protein and metabolite changes in different diseases, including cancer, neuropsychiatric pharmacology studies from clinical trials and animal models. In addition, the future prospects for complete proteomics and metabolomics are discussed.

A chemical study on the constituents of Danshen injection (lyophilized powder) led to the isolation of 15 compounds, including 13 phenolic acids, (R)-salvianolic acid G methyl ester (1), (R)-salvianolic acid G (2), tournefolic acid B (3), isosalvianolic acid C methyl ester (4), rosmarinic acid (5), and 8 simple phenolics (6–13), together with 5-hydroxymethyl-2-furaldehyde (14) and 5-oxopyrrolidine-2-carboxylic acid (15). Among them, compound 1 was new, and 10 compounds showed potent antioxidant activities by in vitro DPPH· and ABTS ·+ scavenging methods with microplate assays.

In this study, The fingerprints of Cuscutae Semen and its processed products such as qing chao pin (QCP), jiu zhi pin (JZP), and yan zhi pin (YZP) were performed using high performance liquid chromatography. Ten chromatographic peaks were selected as characteristic peaks, the relative peak areas (RPA) of which were calculated for quantitative expression of the HPLC fingerprints. The RPA of 10 chromatographic peaks were subjected to principal component analysis (PCA) in order to classify raw Cuscutae Semen and its processed products. Principal component analysis (PCA) of the analytical data showed that raw Cuscutae Semen and its processed products could be clustered reasonably into four different groups corresponding to different processed procedure. The chemical markers of different samples given by the loadings plot of PCA were identified tentatively by electrospray ionization iontrap mass spectrometry and partially verified by the available reference standards. The results from this study indicate that ...

A new tetrahydrofuran-type lignan, episesaminone (1), was isolated from Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. Its structure was established by spectroscopic techniques (HR-MS, 1D NMR, 2D NMR, and circular dichroism). The anti-inflammatory activity in RAW 264.7 macrophages was carried out on 1 and other eight known compounds, the epimer of 1 (2) and seven known furofurans-type lignan (3-9) obtained from A. heterotropoides Fr. Schmidt var. mandshuricum. Compounds 1, 2, 3, 4, 5, 7, and 9 showed significant anti-inflammatory activity, particularly 50 μM compound 3 inhibited 69.2% NO production compared with the lipopolysaccharide group.

Despite the advances in science and technology and wide use of chemical drugs, dietary intervention (or food therapy) remains useful in preventing or treating many human diseases. A huge body of evidence shows that the dietary pattern or habit is also an important contributing factor to the development of chronic diseases such as hypertension, type 2 diabetes, hyperlipidemia, and cancers. In recent years, over-the-counter health foods, nutraceuticals, and plant-derived medicinal products have been gaining popularity all over the world, particularly in developed countries. Unfortunately, owing to the contamination with various harmful substances in foods and the presence of toxic food components, food-borne diseases have also become increasingly problematic. Incidents of food poisonings or tainted food have been increasing worldwide, particularly in China and other developing countries. Therefore, the government should put in a greater effort in enforcing food safety by improving the surveillance mechanism and exerting highest standards of quality control for foods.

Ethnopharmacological relevance BX is a traditional Chinese medicine (TCM) from the plant Pinellia ternata (Thunb.) Berit. It has been traditionally used to treat cough, vomiting, infection and inflammation. Despite of its potentially clinical utility, it also has many side effects and toxicity. Aim of the study We propose here an ultra performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC Q-TOF/MS) metabonomics approach to elucidate the toxicity in rats induced by orally administered BX in multiple organs including the kidney, liver, and heart. Materials and methods Serum samples were collected from Sprague-Dawley male rats exposed to BX (6 g/kg/day, n =10) and healthy controls ( n =10) at the 48 h, 144 h, 240 h, and 336 h postdose for LC/MS analyses. Through principal component analysis and orthogonal partial least squares-discriminant analysis of the integrated serum MS data, we distinguished the BX group from the healthy control group and identified the differential metabolites and pertinent altered biological pathways in response to the herbal toxin. The liver, kidney, heart were assessed using conventional histopathological examinations at the end point of the experiment. The serum samples at the 336 h postdose were assessed using biochemistry test. Results Significant differences in the serum levels of phospholipids, amino acids, l -carnitine and l -acetylcarnitine were observed in BX-induced rats, indicating the perturbations of phospholipid metabolism, amino acid metabolism and carnitine metabolism in BX-induced rats. Conclusions In the paper, we used metabonomics approach to study the toxicity of BX for the first time. With blood biochemistry, histopathological examinations and metabonomics methods, we validated that oral administration of crude BX caused no obvious liver and kidney toxicity in SD rats. BX may possess certain cardio toxicity in SD rats. The metabolism changes suggested that metabonomic approach was a promising tool to study and diagnose TCM-induced toxicity.

A new eudesmanolide sesquiterpene, atractylenolide IV (1), together with seven known compounds were isolated from the 70% aqueous acetonic extract of the whole plant of Sarcandra glabra (Chloranthaceae). Their structures were established by spectral analysis, mainly UV, IR, HRESI-MS, 1D and 2D-NMR experiments (HSQC, HMBC and NOESY). Compounds 1-4 showed no remarkable cytotoxic activity against Hela, HCT-8 and MCF-7 cancer cell lines with IC50 > 50 µg mL(-1).

Ethnopharmacological relevance Poria cocos Wolf (Polyporaceae) is a well-known medicinal fungus. The epidermis of the sclerotia (“Fu-Ling-Pi” in Chinese) is used as a diuretic and traditionally used for promoting urination and reduce edema. Aim of the study Traditional Chinese medicines (TCM) treat many diseases through multi-components, multi-ways and multi-targets. However, the molecular mechanisms of TCM are not yet well understood. In the present work, ultra performance liquid chromatography-based metabonomics analysis was applied to investigate the urinary metabolite profiling of the renoprotective effect of FLP on adenine-induced chronic kidney disease (CKD) rat model and involved possible mechanism. Material and methods A metabonomic approach based on ultra performance liquid chromatography coupled with quadrupole time-of-flight high-sensitivity mass spectrometry and a novel mass spectrometryElevated Energy data collection technique was developed. The resulting dataset was analyzed by principal component analysis and partial least squares discriminant analysis. The identification of all potential biomarkers was performed using reference standard by comparing their mass spectra, MSE fragments information, isotopic pattern and MassLynx i-FIT algorithm. Results By partial least squares-discriminate analysis, 15 biomarkers in rat urine were identified and 11 of them were related to the pathway of adenine metabolism and amino acid metabolism. Among these biomarkers, eight biomarkers like adenine, l -acetylcarnitine, 8-hydroxyadenine, hypoxanthine, creatine, methionine, phytosphingosine and phenylalanine were reversed to the control level in FLP-treated group and six biomarkers like 2,8-dihydroxyadenine, indole-3-carboxylic acid, 3-methyldioxyindole, ethyl-N2-acetyl- l -argininate, 3-O-methyldopa and xanthurenic acid were reversed to high control group by FLP, which indicates that the urinary metabolic pattern significantly changed after FLP treatment. Conclusions Our study indicates that FLP treatment can ameliorate CKD by intervening in some dominating metabolic pathways, such as adenine metabolism and amino acid metabolism. The metabonomic results not only supplied a systematic view of the development and progression of CKD and mechanism studies of FLP but also provided the theoretical basis for the prevention or treatment of CKD.

Carpesii Fructus, the dried fruit of Carpesium abrotanoides L., has been used as a traditional Chinese medicine for centuries to kill intestinal parasites in children. It has been recorded as a mildly toxic medicine in the Chinese pharmacopoeia. However, little proof of its toxicology has been reported in modern pharmacology. This study investigated for the first time its developmental toxicity on zebrafish embryos/larvae from 6 to 96 h post-fertilization (hpf). In addition, the enzymes and genes associated with oxidative stress and apoptosis were tested to investigate the potential toxicologic mechanism preliminarily. The observation of toxicologic endpoints showed the developmental toxicity of Carpesii Fructus. Pericardial edema, yolk sac edema, bleeding tendency, and enlarged yolk were the most commonly occurring morphological changes observed in our study. According to the results of acridine orange staining and morphological observation, the developing heart was speculated to be the target organ of toxicity. Furthermore, Carpesii Fructus exposure changed the activities of defense enzymes, increased malondialdehyde (MDA) content, decreased caspase-3 activity, and altered mRNA levels of related genes (ogg1, p53, Cu/Zn-Sod, Mn-Sod, and Cat↓; Gpx↑) in zebrafish larvae, indicating that oxidative stress and additional apoptosis should have roles in the developmental toxicity of Carpesii Fructus. This is the first study that provides proof of modern pharmacology on the teratogenicity and possible toxicologic mechanism of Carpesii Fructus.

The ethnic drug Melastoma dodecandrum Lour. (MDL) is widely distributed throughout South China, and is the major component of Gong Yan Ping Tablets/Capsules and Zi Di Ning Xue San. Although the pharmacological effects of MDL have been well documented, its chemical profile has not been fully determined. In this study, we have developed a rapid and sensitive UPLC-ESI-Q-Exactive Focus-MS/MS method to characterize the chemical constituents of MDL in the positive and negative ionization modes. A comparison of the chromatographic and spectrometric data obtained using this method with data from databases, the literature and reference standards allowed us to identify or tentatively characterize 109 compounds, including 26 fatty acids, 26 organic acids, 33 flavonoids, six tannins, 10 triterpenoids, two steroids and six other compounds. Notably, 55 of the compounds characterized in this study have never been detected before in this plant. The information obtained in this study therefore enriches our understanding of the chemical composition of MDL and could be used in quality control, pharmacological research and the development of drugs based on MDL. In addition, this study represents the first reported comprehensive analysis of the chemical constituents of MDL.

2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), a natural product purified from the Chinese medical herb Polygoni multiflori Radix, has been demonstrated to possess many pharmacological activities, including antioxidant, anti-inflammatory, hepatoprotective, and antitumor properties. Ultra-performance liquid chromatography–time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and high-performance liquid chromatography-ultraviolet detection (HPLC-UV) methods were developed for the pharmacokinetics, bioavailability, absorption, and metabolism studies of THSG in rats following a single intravenous or oral administration. The metabolites (M1 and M2) were identified in plasma by UPLC-Q-TOF/MS. The concentrations of the THSG in rat plasma, and bile, urine, or feces samples were determined by HPLC-UV. The results show THSG was rapidly absorbed within 30 min in plasma. Absolute bioavailability of THSG was 40%. Total recovery of unchanged THSG within 24 hr were low (0.041% in bile, 0.06% in feces), whereas the a...

2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti-inflammatory and hepatoprotective activities. Ultra-performance LC-quadrupole TOF-MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MS(n) methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC-MS(n). The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra-performance LC-MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long-term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.