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.

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.

UPLC/ESI-QTOF-MS-based metabolomics survey on the toxicity of triptolide and detoxication of licorice.

Triptolide (TP) from Tripterygium wilfordii has been demonstrated to possess anti-inflammatory, immunosuppressive, and anticancer activities. TP is specially used for the treatment of awkward rheumatoid arthritis, but its clinical application is confined by intense side effects. It is reported that licorice can obviously reduce the toxicity of TP, but the detailed mechanisms involved have not been comprehensively investigated. The current study aimed to explore metabolomics characteristics of the toxic reaction induced by TP and the intervention effect of licorice water extraction (LWE) against such toxicity. Obtained urine samples from control, TP and TP + LWE treated rats were analyzed by UPLC/ESI-QTOF-MS. The metabolic profiles of the control and the TP group were well differentiated by the principal component analysis and orthogonal partial least squares-discriminant analysis. The toxicity of TP was demonstrated to be evolving along with the exposure time of TP. Eight potential biomarkers related to TP toxicity were successfully identified in urine samples. Furthermore, LWE treatment could attenuate the change in six of the eight identified biomarkers. Functional pathway analysis revealed that the alterations in these metabolites were associated with tryptophan, pantothenic acid, and porphyrin metabolism. Therefore, it was concluded that LWE demonstrated interventional effects on TP toxicity through regulation of tryptophan, pantothenic acid, and porphyrin metabolism pathways, which provided novel insights into the possible mechanisms of TP toxicity as well as the potential therapeutic effects of LWE against such toxicity.

Dose-response characteristics of Clematis triterpenoid saponins and clematichinenoside AR in rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine metabolomics.

Clematidis Radix et Rhizoma is a traditional Chinese medicine widely used for treating arthritic disease. Clematis triterpenoid saponins (TS) and clematichinenoside AR (C-AR) have been considered to be responsible for its antiarthritic effects. However, the underling mechanism is still unclear because of their low bioavailability. To address of this issue, metabolomics tools were performed to determine metabolic variations associated with rheumatoid arthritis (RA) and responses to Clematis TS, C-AR and positive drug (Triptolide, TP) treatments. This metabolomics investigation of RA was conducted in collagen-induced arthritis (CIA) rats. Liquid chromatography/mass spectrometry and multivariate statistical tools were used to identify the alteration of serum and urine metabolites associated with RA and responses to drug treatment. As a result, 45 potential metabolites associated with RA were identified. After treatment, a total of 24 biomarkers were regulated to normal like levels. Among these, PC(18:0/20:4), 9,11-octadecadienoic acid, arachidonic acid, 1-methyladenosine, valine, hippuric acid and pantothenic acid etc, were reversed in Clematis TS and C-AR groups. Tetrahydrocortisol was regulated to normal levels in Clematis TS and TP groups, while 3,7,12-trihydroxycholan-24-oic acid was regulated in C-AR and TP groups. Biomarkers like citric acid, p-cresol glucuronide, creatinine, cortolone were reversed in TP group.

A novel phloroglucinol and two new phenolic glycosides from Psidium littorale.

One novel phloroglucinol, psidosone A (1), and two new phenolic glycosides, psidoside A (2), and psidoside B (3), together with nine known phenol compounds (4-12), were isolated from the fruits of Psidium littorale Raddi. Their structures were elucidated using data obtained from MS, 1H and 13C NMR spectra, and correlation experiments (HMQC and HMBC), as well as by comparison with published data.

Succinate/NLRP3 Inflammasome Induces Synovial Fibroblast Activation: Therapeutical Effects of Clematichinenoside AR on Arthritis.

Clematichinenoside AR (C-AR) is a triterpene saponin isolated from the root of Clematis manshurica Rupr., which is a herbal medicine used in traditional Chinese medicine for the treatment of arthritis. C-AR exerts anti-inflammatory and immunosuppressive properties, but little is known about its action in the suppression of fibroblast activation. Low oxygen tension and transforming growth factor-ß (TGF-ß1) induction in the synovium contribute to fibrosis in arthritis. This study was designed to investigate the effect of C-AR on synovial fibrosis from the aspects of hypoxic TGF-ß1 and hypoxia-inducible transcription factor-1α (HIF-1α) induction. In the synovium of rheumatoid arthritis (RA) rats, hypoxic TGF-ß1 induction increased succinate accumulation due to the reversal of succinate dehydrogenase (SDH) activation and induced NLRP3 inflammasome activation in a manner dependent on HIF-1α induction. In response to NLRP3 inflammasome activation, the released IL-1ß further increased TGF-ß1 induction, suggesting the forward cycle between inflammation and fibrosis in myofibroblast activation. In the synovium of RA rats, C-AR inhibited hypoxic TGF-ß1 induction and suppressed succinate-associated NLRP3 inflammasome activation by inhibiting SDH activity, and thereby prevented myofibroblast activation by blocking the cross-talk between inflammation and fibrosis. Taken together, these results showed that succinate worked as a metabolic signaling, linking inflammation with fibrosis through NLRP3 inflammasome activation. These findings suggested that synovial succinate accumulation and HIF-1α induction might be therapeutical targets for the prevention of fibrosis in arthritis.

Structural characterization and discrimination of Chinese medicinal materials with multiple botanical origins based on metabolite profiling and chemometrics analysis: Clematidis Radix et Rhizoma as a case study.

Traditional Chinese medicines (TCMs)-based products are becoming more and more popular over the world. To ensure the safety and efficacy, authentication of Chinese medicinal materials has been an important issue, especially for that with multiple botanical origins (one-to-multiple). Taking Clematidis Radix et Rhizoma (CRR) as a case study, we herein developed an integrated platform based on metabolite profiling and chemometrics analysis to characterize, classify, and predict the "one-to-multiple" herbs. Firstly, the predominant constituents, triterpenoid saponins, in three Clematis CRR were rapid characterized by a novel UPLC-QTOF/MS-based strategy, and a total of 49 triterpenoid saponins were identified. Secondly, metabolite profiling was performed by UPLC-QTOF/MS, and 4623 variables were extracted and aligned as dataset. Thirdly, by using pattern recognition analysis, a clear separation of the three Clematis CRR was achieved as well as a total number of 28 variables were screened as the valuable variables for discrimination. By matching with identified saponins, these 28 variables were corresponding to 10 saponins which were identified as marker compounds. Fourthly, based on the relative intensity of the marker compounds-related variables, genetic algorithm optimized support vector machines (GA-SVM) was employed to predict the species of CRR samples. The obtained model showed excellent prediction performance with a prediction accuracy of 100%. Finally, a heatmap visualization was employed for clarifying the distribution of identified saponins, which could be useful for phytochemotaxonomy study of Clematis herbs. These results indicated that our proposed platform was a powerful tool for chemical profiling and discrimination of herbs with multiple botanical origins, providing promising perspectives in tracking the formulation processes of TCMs products.

[Quality standard study on Pteris multifida].

The quality control method and standard were established to control the quality of Pteris multifida in this paper. The tests of water content, total ash, acid-unsoluble ash and ethanol-soluble extractives of P. multifida were carried out according to the methods recorded in appendix of Chinese Pharmacopeia (2010 edition, volume 1) . The TLC method was established by using rhoifolin as references, and a mixture of CHCl3 -MeOH-HAc (6: 1: 1) as the developing solvent system on GF254 thin layer plate. The contents of rhoifolin was determined by HPLC on a Diamonsil C18 (4.6 mm x 250 mm, 5 µm) column, using acetonitrile-water (containing 0.15% formic acid) (16: 84) as mobile phase at a flow rate of 1.0 mL x min(-1). The column temperature was 30 degrees C and the detection wave-length was 350 nm. As a result, pterosin C 3-O-ß-D-glucosidede and the other constituents were well separated on TLC detected under the UV light at 254 nm . The methodology validation for the assay of rhoifolin presented that it was in good linear correlation in the ranges of 0.025 5-5.1 µg with the regression equations of Y = 1 092.4X + 9.503 5 (r = 0.999 8), and the average recoveries were 100.3% (RSD 1.3%). The content range of rhoifolin from 16 different batches of Pteris multifida was 0.08-5.06 mg x g(-1). The water content, total ash, acid-unsoluble ash and ethanol-soluble extractives of 16 samples varied in the ranges of 7.35% - 12.96%, 6.90% - 16.33%, 2.07% -11.38% and 13.29% -23.87%, respectively. The suggesting limes in the quality standard for water content, total ash, acid-unsoluble ash, ethanol-soluble extractives and rhoifolin content were ≤ 12% , ≤ 15% , ≤ 8.5% , ≥ 14% and ≥ 0.040%, respectively. The result proved that the established quality of control method was specific and accurate, which can be used for the quality control of P. multifida.

A new alkaloid from the fruit of Nandina domestica Thunb.

A new steroidal alkaloid, (20S,22R,24R)-24-ethyl-3-oxocholest-4-en-22-amino, named as nandsterine (1), together with 10 known alkaloids, palmatine (2), O-methylbulbocapnine (3), nantenine (4), dehydronantenine (5), glaucine (6), didehydroglaucine (7), dehydrocorydaline (8), jatrorrhizine (9), magnoflorine (10) and berberine (11), was isolated from the fruit of Nandina domestica Thunb. Their structures were elucidated by using spectroscopic methods as well as by comparing with the published data. Compound 1 was a new class of steroidal alkaloid isolated from the family Berberidaceae, meanwhile compounds 2, 3, 6-8 and 10 were obtained from N. domestica for the first time. Compound 1 exhibited cytotoxicity against HL-60 cells (human leukaemia) with IC50 values of 52.1 µM.

[Study on the diterpenoids of Tripterygium wilfordii by electrospray ionization tandem and electron impact mass spectrometry].

OBJECTIVE: To study the diterpenoids of Tripterygium wilfordii by electrospray ionization tandem (ESI) and electron impact (EI) mass spectrometry and establish the methods for quickly and on line identification of these diterpenoids. METHODS: The diterpenoids were analyzed by ESI-MS and EI-MS under positive ion model. RESULTS: In ESI-Trap-MS (+) model, the quasi-molecular ions [M + H] + of the diterpenoids took place bond cleavage and generally lost neutral molecules such as H2O, CH2CHCH3, CO and CH2CO, then generated characteristic fragment ion series (m/z 197, 183, 169). In ESI-TOF-MS (+) model, the quasi-molecular ions [M + H] + of the diterpenoids took place bond cleavage and generally lost neutral molecules such as H2O, CO, then generated characteristic fragment ion series (m/z 277, 185, 93). In EI-MS model, the molecular ions of the diterpenoids took place bond cleavage and generally lost neutral molecules such as H2O, CO, CH4, and isopropyl radical, then generated characteristic fragment ion series (m/z 149, 105, 91, 71, 55, 43). CONCLUSION: The ESI-MS and EI-MS characteristics of diterpenoids of Tripterygium wilfordii are reported for the first time. Based on these characteristics, the methods for quickly and on line identification of diterpenoids are established.

A new triterpenoid from Verbena officinalis L.

To investigate the chemical constituents of Verbena officinalis L., five triterpenoid constituents were isolated and elucidated as 3α,19,23-trihydroxyurs-12-en-28-oic acid, namely, 4-epi-barbinervic acid (1), 2α,3ß-dihydroxyurs-12-en-28-oic acid (2), 3α,24-dihydroxyurs-12-en- 28-oic acid (3), 3α,24-dihydroxy-olean-12-en-28-oic acid (4), ursolic acid (5), using spectroscopic methods and comparing with published data. Compounds 2 and 4 were obtained from V. officinalis for the first time, and compound 1 is a new triterpenoid compound, which exhibits significantly higher antitumor activity against human hepatoma cell line Bel 7402 in vitro than the blank control.

[Studies on chemical constituents from Grewia biloba].

OBJECTIVE: To investigate the chemical constituents of Grewia biloba and find its bioactive compounds. METHODS: Compounds were isolated by silica gel, MCI, Sephadex LH-20 and Rp-18 column chromatography, and purified by recrystallization. Their structures were elucidated by spectral analysis and other methods. RESULTS: Six compounds were isolated and identified as friedelin (1) epi-friedelan-3-ol (2), heneicosanoic acid (3), beta-sitosterol (4), Propyl palmitate (5), eatechin (6), respectively. CONCLUSION: Compounds 1-3, 5, 6 are isolated from Grewia genus for the first time and compounds 4 is isolated from Grewia biloba for the first time.

[Terpenoid compounds isolated from Ligularia sagitta].

OBJECTIVE: To study the chemical constituents of radix and rhizome of Ligularia sagitta. METHOD: Isolation and purification were carried out on the column chromatography of silica gel and sephadex LH -20. The structures were identified by spectral analysis. RESULT: Seven terpenoid compounds were isolated from the radix and rhizome of L. sagitta. Five of them are sesquiterpenoids, the structures were determined as 7alpha-hydroxy-9(10) -ene-1, 8-dioxo-6, 7-dihydrofuranoeremophilane (1), 1p, 10P3-epoxy-6beta, 8 beta-dihydroxy-eremophil7 (11) -en-12, 8alpha-olide (2) , 1-oxo-9-desoxycacalol (3), benzofuranoeremophil-l-ene (4) and bakkenolide (5); other two compounds belong to triterpenoid. 3p, 16P3-dihydroxy-12-oleanen-28-al (6) and lupeol (7). CONCLUSION: Compoundsl-3 and 6 were: isolated from L. sagitta for the first time, and compounds 3 and 6 were obtained from Ligularia for the first time.