Three new pterosins from Pteris semipinnata.

Three new pterosins, named as semipterosin A (1), B (2) and C (3), together with 11 known pterosins (4-14), were isolated from the aerial parts of Pteris semipinnata. Their structures were elucidated by HRESI-MS, NMR spectral data, CD and literature comparisons. Three new pterosins were assessed for their anti-inflammatory activity. Compounds 1-3 inhibited the NF-kB induction by 40.7%, 61.9% and 34.0%, respectively. This is the first report of the isolation of compounds 6-14 from this plant.

Three new flavonoid glycosides isolated from Pteridium aquilinum.

Three new flavonoid glycosides, (S)-4',6,8-trihydroxyflavanone-7-C-glucoside (1), (R)-4',6,8- trihydroxyflavanone-7-C-glucoside (2) and distenin-7-O-ß-D-glucoside (3), along with nine known flavonoids (4-12) were isolated from the aerial of Pteridium acquilinum. Their structures were elucidated by the analysis of spectroscopy data and their comparison with the reported values. The two C-glycosyl flavanones (1 and 2), were isolated from this specie, which might be chemotaxonomic markers of this specie. In addition, three new flavonoids were preliminarily examined for their anti-inflammatory activity. Compounds 1-3 inhibited the NF-κB induction by 46.3%, 59.6% and 29.2%, respectively.

UPLC-Q-TOF-MS/MS-based urine metabolomics studies on the toxicity and detoxication of Tripterygium wilfordii Hook. f. after roasting.

Tripterygium wilfordii (TW), a well-known traditional Chinese medicine, was widely used in the treatment of autoimmune disorders and inflammatory diseases. However, the clinical use of TW was limited by severe toxicities, such as hepatotoxicity and nephrotoxicity. Our previous studies indicated that roasting was an effective approach for reducing TW-induced toxicity. After roasting, celastrol was completely decomposed, partially converted into 1-hydroxy-2,5,8-trimethyl-9-fluorenone and the total alkaloids content were significantly reduced. However, the detoxication mechanisms of roasting on TW were poorly unknown. This study aimed to explore the toxicity and detoxification mechanisms of TW after roasting based on urine metabolomics. Promising biomarkers were evaluated by multiple comparison analyses. Sixteen toxicity biomarkers were identified between control group and total extract group. Twelve toxicity biomarkers were identified between control group and total alkaloids group. Eight toxicity biomarkers were identified between control group and celastrol group. These metabolites were mainly involved in seven metabolic pathways, summarized as pentose and glucuronate interconversions, lipid metabolism (sphingolipid metabolism, glycerophospholipid metabolisms, fatty acid biosynthesis and steroid hormone biosynthesis) and amino acid metabolism (taurine and hypotaurine metabolism, tryptophan metabolism). After roasting, the toxicities of total extract, total alkaloids and celastrol were relieved by ameliorative serum parameters and pathological changes in hepatic and renal tissues which revealed that the reduction of celastrol and total alkaloids played important roles in the detoxification of roasting on TW. Furthermore, roasting regulated the levels of fourteen potential biomarkers in the total extract group, ten potential biomarkers in the total alkaloids group and seven candidate biomarkers in the celastrol group to normal levels. Biological pathway analysis revealed that roasting may ameliorate TW-induced metabolic disorders in pentose and glucuronate interconversions, lipid metabolism and amino acid metabolism. This study provided evidence for the application of roasting in TW.

The Effect of W Content on the Microstructure, Mechanics and Electrical Performance of an FeCrCo Alloy.

In this paper, FeCrCoW alloys with different W contents (0.4, 2.1 and 3.4 at%) are designed and studied in order to overcome the existing shortcomings of resistance materials. These resistance materials have high resistivity and a low temperature coefficient of resistivity. It is observed that the addition of W has a remarkable effect on the phase structure of the alloy. In particular, when the W content is 3.4 at%, the single BCC phase of the alloy can be transformed into the BCC and FCC phase. Meanwhile, when analyzed by transmission electron microscopy, there are stacking faults and martensite in FeCrCoW alloy with W content of 3.4 at%. These features are related to excessive W content. In addition, the strength of the alloy can be improved, and the ultimate tensile strength and yield strength are both very high, which are considered as grain-boundary strengthening and solid solution strengthening, caused by the addition of W. The electrical resistivity of the FeCrCoW alloys decreases when the content of W is more than 2.1 at%. The maximum resistivity of the alloy is 170 ± 1.5 µΩ·cm. Moreover, the unique properties of the transition metal allow the alloy to have a low temperature coefficient of resistivity in the temperature range of 298~393 K. The temperature coefficient of resistivity values of the W0.4, W2.1 and W3.4 alloys are -0.0073, -0.0052 and -0.0051 ppm/K. Therefore, this work provides a vision for resistance alloys, which can achieve highly stable resistivity and high strengths in a certain temperature range.

Targeting tryptophan metabolism reveals Clematichinenoside AR alleviates triptolide-induced hepatotoxicity.

Liver toxicity induced by Triptolide (TP) has limited its clinical application on rheumatoid arthritis (RA). Saponins have been proved as an efficacious remedy to mitigate hepatotoxicity. However, the mechanism of reducing hepatotoxicity by saponins intervention remains incompletely characterized. Tryptophan (Trp) metabolites activate transcriptional regulators to mediate host detoxification responses. Our study aimed to investigate whether Clematichinenoside AR (C-AR) could attenuate TP-induced liver damage by regulating Trp metabolism. We used targeted metabolomics to quantify Trp metabolites in the serum and liver samples of collagen-induced arthritis rats treated by TP. Multiple comparison analyses helped the evaluation of promising biomarkers. The pronounced changed levels of Trp, indole acetic acid, and indole-3-carboxaldehyde in the serum and indole acetic acid, indole, and tryptamine in the liver are relevant to TP-induced liver injury. Intervention with C-AR could relieve TP-induced hepatotoxicity evidenced by ameliorative serum parameters and hepatic histology. In addition, C-AR regulated the levels of these indoles biomarker candidates to normal. Therapeutic modulation with natural compounds might be a useful clinical strategy to ameliorate toxicity induced by TP. Deciphering Trp metabolism will facilitate a better understanding of the pathogenesis of diseases and drug responding.

Orthogonal label and label-free dual pretreatment for targeted profiling of neurotransmitters in enteric nervous system.

Neurotransmitter (NT) abnormalities in the enteric nervous system have been reported as crucial roles to regulate the intestinal inflammation and gut immune homeostasis. Capturing quantitative changes at the NT metabolome provides an opportunity to develop an understanding of neuroimmune-mediated inflammation. Given the wide diversity of chemical characterizations in the NTs, only partial coverage of the NT metabolome can be simultaneously quantified in a single-run analysis. Herein, we summarized the distribution of functional groups of compound entries in the NT metabolome. Based on this information, an orthogonal dansyl-labeling and label-free dual pretreatment approach was separately designed to target phenol and amine NTs and tertiary amine and choline NTs. By combining the dansyl-labeled and unlabeled NTs within a single vial, a comprehensive and practical approach was optimized for quantifying high coverage of NT metabolome in a single-run analysis on the reversed-phase C18 column. Method validation indicated good linearity with correlation coefficients (R2) > 0.99, intra- and interday accuracy with relative error < ±20%, and precision with relative standard deviations of ≤15%. With this method, we could simultaneously monitor the alterations of cholines, amines, amino acids, tryptophan and phenylalanine biological pathways in dextran sulphate sodium-induced colitis mice. The measured levels of NT metabolome ranged from 0.0007 to 3.540 µg/mg in intestinal contents and 0.013-154.54 µg/mL in serum samples. The NT metabolism was disrupted by colitis, characterized by the changed NT levels in serum and excessive amino acid NTs accumulation in the intestinal contents. We envisage that the orthogonal approach is of great significance for the comprehensive determination of targeted metabolomics. NTs have the potential to be biomarkers for clinical metabolomics.

Three novel pterosin dimers form Pteris obtusiloba.

Three novel pterosin dimmers, named as obtupterosin A (1), B (2) and C (3), together with eight known pterosins (4-11) were isolated from Pteris obtusiloba. Their structures were elucidated on the basis of ESI-MS, 1D and 2D NMR spectral data, CD, X-ray and literature comparisons. Compounds 1 and 2 were a pair of isomers. Compounds 1 and 3 were the novel type of pterosin dimer. The new compounds (1-3) were assessed for their cytotoxic activities and their α-glucosidase inhibition activity. Compounds 1-3 exhibited cytotoxic activity against HCT-116 cells with IC50 value of 27.5 µM, 30.6 µM and 12.8 µM, respectively. However, all were found to be inactive at 200 µM for α-glucosidase inhibition.

Effects of the Isomerized Thiophene-Fused Ending Groups on the Performances of Twisted Non-Fullerene Acceptor-Based Polymer Solar Cells.

Recently, benefiting from the merits of small-molecule acceptors (NFAs), polymer solar cells (PSCs) have achieved tremendous advances. From the perspective of the structural characteristics of the π-conjugated acceptor-donor-acceptor (A-D-A) type of organic molecules, the backbone's planarity and the terminal groups and their substituents have strong influences on the performances of the constructed NFAs. Through enlarging the dihedral angle of the conjugated main chain of NFAs, a certain degree of enhancement of photovoltaic parameters has been achieved. To further probe the influences of ending groups on the performances of nonplanar NFAs, we synthesized two new NFAs i-cc23 and i-cc34 with isomerized thiophene-fused ending groups and a twisted π-conjugated main chain. Compared to i-cc23 containing the 2-(6-oxo-5,6-dihydro-4H-cyclopenta[b]thiophen-4-ylidene)malononitrile ending group, the acceptor i-cc34 containing 2-(6-oxo-5,6-dihydro-4H-cyclopenta[c]thiophen-4-ylidene)malononitrile has a relatively higher molar extinction coefficient, bathochromic-shifted absorption spectrum, and deepened energy levels. When mixed with PBDB-T in solar cells, the i-cc23-based device achieved an excellent open-circuit voltage (VOC) of 1.10 V and a moderate power conversion efficiency of 7.34%. Although the VOC of the i-cc34-related device was decreased to 0.96 V, the short-circuit current density and fill factor were improved, giving rise to an enhanced efficiency of 9.51%. Apart from the distinct photovoltaic performances, the two isomer-based devices exhibit a high radiative efficiency of 8 × 10-4, leading to a very small nonradiative loss of 0.19 V. Our results emphasize the importance of the isomerized thiophene-fused ending groups on the performances of nonplanar NFA-based PSCs.

Effects of Monofluorinated Positions at the End-Capping Groups on the Performances of Twisted Non-Fullerene Acceptor-Based Polymer Solar Cells.

Recently, main-chain twisted small molecules are attractive as electron-acceptors in polymer solar cells (PSCs) for their upshifted molecular energy levels, enhanced extinction coefficients, and better charge extraction properties along with longer carrier lifetimes and lower recombination rates relative to their planar analogues, which are conducive to the power conversion efficiency (PCE) promotion of PSCs. To further probe the "structure-performance" correlation of main-chain twisted acceptors, in particular the monofluorine-substituted sites on the performances of the resultant acceptors, two new main-chain twisted small molecules were synthesized, in which a fluorine atom was introduced at different sites on the end-capping group 2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile (INCN). Although fine structural modification was adopted, quite different performances were obtained for the two acceptors. Compared to the 3-fluorinated analogue (i-IEICO-F3), a mixture of 4-fluorinated and 5-fluorinated isomers (i-IEICO-2F) exhibited a higher dipole moment, enlarged molar extinction coefficient with a bathochromic-shifted absorption region, suppressed charge recombinations with balanced charge mobilities, and slightly enhanced crystallinity. In combination with a fluorobenzotriazole-based medium-band gap polymer (J52), a high efficiency of 12.86% was resultantly achieved in an i-IEICO-2F-based device, which is superior to the result (7.65%) of the i-IEICO-F3 device, revealing the importance of monofluorinated positions on the performances of main-chain twisted non-fullerene acceptors.

Saponins from Clematis mandshurica Rupr. regulates gut microbiota and its metabolites during alleviation of collagen-induced arthritis in rats.

Gut microbiota and their metabolites (short-chain fatty acids, SCFAs) are associated with the pathogenesis of rheumatoid arthritis (RA). Total Clematis triterpenoid saponins (CTSs) prepared from Clematis mandshurica Rupr. possess therapeutic benefits for arthritic diseases. However, the poor pharmacokinetic properties of CTSs have obstructed the translation of these natural agents to drugs. Here, we examined the effects of CTSs on arthritis symptoms, gut microbiota and SCFAs in rats with collagen-induced arthritis (CIA). Our results showed that the arthritis index scores of CIA rats treated with CTSs were significantly lower than those of the model group. Most importantly, CTSs moderated gut microbial dysbiosis and significantly downregulated the total SCFA concentration in CIA rats. Compared to the control group, CTSs treatment have no significant side effects on the gut microbiota and SCFA metabolism in normal rats. Two differential analyses (LEfSe and DESeq2) were combined to study the details of the changes in gut microbiome, and twenty-four marker taxa at the genus level were identified via a comparison among control, model and CIA rats treated with high doses of CTSs. In particular, the mostly significantly increased gram-negative (G-) and decreased gram-positive (G+) genera in CIA rats were well restored by CTSs. The observed SCFA concentrations demonstrated that CTSs tend to maintain the balance of the gut microbiota. The data presented herein suggest that CTSs could ameliorate arthritis-associated gut microbial dysbiosis and may be potential adjuvant drugs that could provide relief from the gastrointestinal damage caused as a side effect of commonly used drugs.

Ammonium fluoride-induced stabilization for anion attachment mass spectrometry: Facilitating the pseudotargeted profiling of bile acids submetabolome.

Mass spectrometry-based approaches enable us to capture changes in the metabolome in biological systems with high sensitivity and resolution. But global MS-based profiling of the bile acids (BAs) submetabolome is still a challenging task. Particularly for unconjugated BAs, the collision-induced dissociation (CID) fragment ions showed low ion intensities which were insufficient for analysis. This study is aimed at the development of an anion attachment MS-based approach for pseudotargeted profiling of the BAs submetabolome. We demonstrated that anion attachment MS with the combination use of ammonia fluoride (NH4F) and formate could provide stable anionic adduct ([M + HCOO]-) with good MS responses for unconjugated BAs. A mechanistic study revealed that the underlying rationale is due to the NH4F-induced approximate matching of attractions between BAs and anion for the 24-carboxyl hydrogen. This 24-carboxyl hydrogen regioselectivity is useful to screen for potential unconjugated BAs from the biological matrix. The stability and regioselectivity of anion attachment allowed the establishment of SRM transitions for unconjugated BAs for the first time. To profile conjugated BAs that come from the conjugation of glycine or taurine at 24-carboxyl hydrogen, specific precursor/fragment ion transitions were used for the detection. Finally, SRM-based UPLC-MS/MS method was developed for the pseudotargeted profiling of the BAs submetabolome with good linearity (r2 > 0.995) and high sensitivity (0.20-1.37 ng mL-1 for LLOQ). With this method, a total of 83 BAs, covering 45 unconjugated BAs and 38 conjugated BAs, were successfully determined in different biosamples from experimental colitis mice. The BAs metabolism homeostasis was disrupted by colitis, characterized by the decreased BAs levels in serum and excessive BAs accumuation in the gall bladder and colon. Overall, the present anion attachment MS-based approach is sufficiently sensitive and robust to comprehensively measure various BAs.

Four New Pterosins from Pteris cretica and Their Cytotoxic Activities.

Phytochemical investigation of the aerial parts of Pteris cretica led to the isolation and elucidation of nine pterosins, including four new pterosins, creticolacton A (1), 13-hydroxy-2(R),3(R)-pterosin L (2), creticoside A (3), and spelosin 3-O-ß-d-glucopyranoside (4), together with five known pterosins 5-9. Their structures were identified mainly on the basis of 1D and 2D NMR spectral data, ESI-MS and literature comparisons. Compounds 1 and 3 were new type of petrosins with a six membered ring between C-14 and C-15. The new compounds were tested in vitro for their cytotoxic activities against four human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116, Lovo). Results showed that compounds 1 and 2 exhibited cytotoxic activity against HCT-116 cells with IC50 value of 22.4 µM and 15.8 µM, respectively.

Development and validation of a systematic platform for broad-scale profiling of microbial metabolites.

Liquid chromatography-mass spectrometry based profiling of microbial metabolites has been a challenging task due to their diverse physicochemical properties and wide concentration ranges. This study is aimed to develop a systematic platform for the broad-scale profiling of microbial metabolites by integrating aqueous-lipophilic biphasic extractions and chemical derivatizations with a data-dependent automatable metabolite annotation algorithm. This complementary strategy of detection will not only largely expand the metabolite coverage, but also facilitate the drawing out of interested submetabolome using designed chemical derivatizations. Then, the data-dependent metabolite annotation algorithm is able to automatically match the raw MS/MS data with those of compounds in the self-collected databases. The performance of this platform is illustrated through the analysis of two representative bacteria (Escherichia coli and Pseudomonas aeruginosa) and intestinal contents samples from experimental colitis mice. As a result, 292 metabolites corresponding to 875 annotated features distributing over 25 chemical families were putatively annotated in a short time. Of these metabolites, 197 and 218 are respectively from the bacteria and intestinal contents, and 107 are identified in all three biological samples. This systematic platform could be used to accomplete high-coverage detection and high-quality data processing of microbial metabolites. At the same time, chemical derivatization design and the establishment of self-collected databases will facilitate self-driven untargeted analysis.

Efficient Polymer Solar Cells Having High Open-Circuit Voltage and Low Energy Loss Enabled by a Main-Chain Twisted Small Molecular Acceptor.

A new main-chain twisted small molecular acceptor with nonhalogenated end groups (i-IEICO) is designed and synthesized. In contrast to its planar analogue IECIO, i-IEICO possesses an obviously twisted backbone, leading to significant hypsochromic shift in film absorption, slight enhancement in solution extinction coefficient, and significantly elevated molecular energy level. Benefited from these features, i-IEICO is matched well with two wide band gap polymer donor materials (J52 and PBDB-T) both in absorption spectra and molecular energy levels. Relative to the planar-molecule IEICO-based devices, the open-circuit voltage ( VOC), short-circuit current density, and fill factor of the i-IEICO-based devices are simultaneously improved, giving rising to a 10.48% (with J52) and 8.79% (with PBDB-T) power conversion efficiency, respectively. Moreover, J52:i-IEICO device exhibits a high VOC of 0.96 V accompanied by a small energy loss of 0.64 eV, which can be further improved to 1.01 V and 0.59 eV for the PBDB-T-based device. The obtained VOC of i-IEICO-based devices are among one of the highest values of either J52 or PBDB-T-based binary devices, suggesting the effectiveness of main-chain twisted strategy coupled with end-group modification to achieve highly efficient nonfullerene acceptors with low energy loss and high VOC.

Structures and biological evaluation of phenylpropanoid derivatives from Murraya koenigii.

Four new phenylpropanoid derivatives (1-4), together with eleven known analogues (5-15) were isolated and identified by comparison with their references and extensive spectroscopic methods from Murraya koenigii for the first time. Compounds (1-15) were assayed for their inhibitory activities by measuring IL-6-induced STAT3 promoter activities in HepG2 cells, and found compounds 1, 2, 6, and 15 showed inhibitory effects with IC50 values of 11.5, 18.7, 8.9, and 22.7 µM, respectively. The inhibitory activities of compounds (1-15) were screened against NO production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells, and found compounds 3, 4, 9, 11, and 14 exhibited inhibitions against LPS-induced NO production in RAW264.7 macrophages, with IC50 values of 32.7, 7.9, 42.1, 58.9, and 62.4 µM, respectively.

Immunogenicity and Safety of an F-Genotype Attenuated Mumps Vaccine in Healthy 8- to 24-Month-Old Children.

Background: Mumps vaccine immunizations have reduced the incidence of this disease. With the variation of mumps circulating strain, novel vaccine strains are always important. Methods: A 2-center parallel, randomized, double-blind noninferiority trial was performed to compare an F-genotype attenuated mumps vaccine (SP strain) to the A-genotype vaccine (S-79, Jeryl-Lynn strain) in 1080 healthy children aged 8-24 months in Hubei, China. Results: Participants were randomly assigned to receive a high or low dose of the SP or S79 vaccine and then assessed clinically at 30 minutes and 1-28 days postinoculation. No differences in local or systemic reactivity were observed. A similar incidence of severe adverse events associated with the vaccine was observed in the high-dose group and the positive control group. Based on throat swab collections, no viral shedding was present at the 4th and 10th days in any group. Neutralizing and hemagglutination-inhibiting antibody assays with the F- or A-genotype strains showed similar trends in geometric mean titers in the high-dose SP and S79 groups. Increased cytotoxic T lymphocyte responses were observed in all groups. Conclusions: The F-genotype attenuated mumps vaccine is safe, offers immunogenicity against a homologous virus, and is noninferior to the A-genotype vaccine in 8- to 24-month-old children.

A chemical derivatization based UHPLC-LTQ-Orbitrap mass spectrometry method for accurate quantification of short-chain fatty acids in bronchoalveolar lavage fluid of asthma mice.

Recent studies have demonstrated the important role of short-chain fatty acids (SCFAs) in the maintenance of homeostasis of respiratory immunity. However, there is still no report focus on the determination of SCFAs level in bronchoalveolar lavage fluid (BALF), the most common sample used for screening biomarkers of the pulmonary diseases. Herein, an ultra-high-performance liquid chromatography with LTQ-Orbitrap mass spectrometer (UHPLC-LTQ-Orbitrap) oriented 3-nitrophenylhydrazine (3-NPH)-based derivatization method was developed for the quantification of SCFAs in BALF. To achieve accurate quantitation, d4-acetate was used as internal standard to compensate for the matrix effects. Method validation showed a good linearity (R2 > 0.9992) with wide concentration range, and the intra-day and inter-day precision for determination of eight SCFAs in BALF samples was ≤ 14.79%. The quantitation accuracy, assessed by relative recoveries, ranged from 90% to 110% for target SCFAs at three concentration levels. Matrix effects ranged from 85% to 115%, and the lower limits of quantification of these targeted SCFAs were varied from 3 to 24 nmol/L. The SCFAs-targeted method was then applied to determine the changed levels in BALF samples from OVA-induced asthma mice and normal mice. In addition, the universality of our developed method was also demonstrated by determining the SCFAs concentrations in feces, serum and lung tissue samples from asthma and normal mice. These results indicate that 3-NPH derivatization based UHPLC-LTQ-Orbitrap provides accurate view of global SCFAs alternation in different samples, giving a support to deduce the origin of SCFAs in lung. The present study is of great importance for understanding the role of SCFAs in modulation of host metabolism and immunity.

Succinate induces synovial angiogenesis in rheumatoid arthritis through metabolic remodeling and HIF-1α/VEGF axis.

BACKGROUND AND PURPOSE: In response to hypoxic succinate accumulates in arthritis synovium, however, the implication is little known. This study aims to investigate whether succinate could act as a metabolic signal linking metabolic alternation with angiogenesis in arthritis synovium. EXPERIMENTAL APPROACH: The interaction between elevated succinate and VEGF production was examined in endothelial cells. Succinate production, HIF-1α induction and angiogenesis in the hypoxic synovium of collagen-induced arthritis rats were also investigated. KEY RESULTS: Intracellular succinate promoted VEGF production and induced angiogenic response dependent on HIF-1α induction in endothelial cells. Luciferase reporter assay showed that succinate increased VEGF expression through gene promoter activation dependent on HIF-1α induction. Intracellular succinate released into intercellular space, where extracellular succinate activated succinate receptor G-protein-coupled receptor 91 (GPR91) and induced VEGF production, further exacerbating angiogenesis. In addition, TGF-ß1 treatment increased succinate production due to the reversal of succinate dehydrogenase (SDH) activation, and consistently, SDH inhibitor dimethyl malonate reduced angiogenesis in the arthritis synovium. CONCLUSION AND IMPLICATIONS: More than an intermediate, succinate functioned as a signaling molecule to link metabolic reprograming with angiogenesis. Intracellular succinate induced angiogenesis through HIF-1α induction, while extracellular succinate acted on GPR91 activation, working together to disturb energy metabolism and exacerbate inflammation and angiogenesis in arthritis synovium. Our work suggested that suppression of SDH could prevent succinate accumulation and inhibit angiogenesis via blocking HIF-1α/VEGF axis. This finding not only provides a novel insight into angiogenesis, but also reveals a potential therapeutical strategy to attenuate revascularization in arthritis.

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.

Comparison of three officinal species of Callicarpa based on a biochemome profiling strategy with UHPLC-IT-MS and chemometrics analysis.

Traditional Chinese medicine (TCM) materials with closely related species are frequently fungible in clinical use. Therefore, holistic comparison of the composition in bioactive compounds is essential to evaluate whether they are equivalent in efficacy. Taking three officinal species of Callicarpa as a case, we proposed and validated a standardized strategy for the discrimination of closely related TCM materials, which focused on the extraction, profiling and multivariate statistical analysis of their biochemome. Firstly, serial liquid-liquid extractions were utilized to prepare different batches of Callicarpa biochemome, and the preparation yields were utilized for the normalization of sampling quantity prior to UHPLC-IT-MS analysis. Secondly, 34 compounds, including 19 phenylethanoid glycosides, 10 flavonoids and 5 terpenoids, were identified based on an untargeted UHPLC-IT-MS method. Thirdly, method validation of linearity, precision and stability showed that the UHPLC-IT-MS system was qualified (R2>0.995, RSD<15%) for subsequent biochemome profiling. After PCA and PLS-DA analysis, 30 marker compounds were screened and demonstrated to be of good predictability using genetic algorithm optimized support vector machines. Finally, a heatmap visualization was employed for clarifying the distribution of marker compounds, which could be helpful to determine whether the three Callicarpa species are, in fact, equivalent substitutes. This study provides a standardized biochemome profiling strategy for systemic comparison analysis of closely related TCM materials, which shows promising perspectives in tracking the supply chain of pharmaceutical suppliers.