Dissecting the Regulation of Arachidonic Acid Metabolites by Uncaria rhynchophylla (Miq). Miq. in Spontaneously Hypertensive Rats and the Predictive Target sEH in the Anti-Hypertensive Effect Based on Metabolomics and Molecular Docking.

Uncaria rhynchophylla (Miq). Miq. (UR), as a traditional Chinese medicine, was employed in treating hypertension as a safe and effective therapy. The pharmacological properties of UR have characteristics of multiple biological targets and multiple functional pathways. Hypertension is related to impaired metabolic homeostasis and is especially associated with the abnormal regulation of arachidonic acid metabolites, the classical cardiovascular active compounds. This study aimed to examine the anti-hypertensive effect of UR extract (URE) and its regulating role in differential metabolic pathways. The results showed that daily administration of URE at a dose of 4 g crude drug/kg orally could exert hypotensive effects on spontaneously hypertensive rats (SHRs) for 8 weeks. Non-targeted metabolomics analysis of the plasma samples suggested that the anti-hypertension effect of URE in SHRs was associated with the reorganization of the perturbed metabolic network, such as the pathways of glycerophospholipid metabolism, linoleic acid metabolism, and arachidonic acid metabolism. For the targeted metabolomics, twenty-eight arachidonic acid metabolites in SHRs were quantitatively analyzed for the first time based on ultra-high performance liquid chromatography-tandem mass spectrometry method after URE administration. URE restored the functions of these cardiovascular active compounds and rebalanced the dynamics of arachidonic acid metabolic flux. Among them, the inhibition of soluble epoxide hydrolase (sEH) enzyme activity and up-regulation of vasodilators epoxyeicosatrienoic acids (EETs) were identified as contributors to the anti-hypertension effect of URE on SHRs, and sEH represented an attractive and promising drug-binding target of URE. With the molecular docking approach, 13 potential anti-hypertension ingredients as well as sEH inhibitors were discovered, which were worthy of further investigation and verification in future studies.

Quantitative imaging of natural products in fine brain regions using desorption electrospray ionization mass spectrometry imaging (DESI-MSI): Uncaria alkaloids as a case study.

Uncaria species (Rubiaceae) are used as traditional Chinese medicines (TCMs) to treat central nervous system (CNS) diseases, and monoterpene indole alkaloids are the main bioactive constituents. Localization and quantification of CNS drugs in fine brain regions are important to provide insights into their pharmacodynamics, for which quantitative mass spectrometry imaging (MSI) has emerged as a powerful technique. A systematic study of the quantitative imaging of seven Uncaria alkaloids in rat brains using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was presented. The distribution of the alkaloids in thirteen brain regions was quantified successfully using the calibration curves generated by a modified on-tissue approach. The distribution trend of different Uncaria alkaloids in the rat brain was listed as monoterpene indole alkaloids > monoterpene oxindole alkaloids, R-configuration epimers > S-configuration epimers. Particularly, Uncaria alkaloids were detected directly in the pineal gland for the first time and their enrichment phenomenon in this region had an instructive significance in future pharmacodynamic studies.

Restoring perturbed oxylipins with Danqi Tongmai Tablet attenuates acute myocardial infarction.

Salvianolic acids have a special synergic effect on panax notoginsenosides in acute myocardial infarction (AMI) and have been developed into a new drug as Danqi Tongmai Tablet (DQTT). To explore candidate targets and mechanisms of DQTT on AMI, a network pharmacology-based analysis was performed on absorbed prototype compounds of DQTT in rat plasma. Target prediction from network analysis indicated that the arachidonic acid pathway might contribute to the therapeutic effects of DQTT on AMI, and the regulatory effects on cyclooxygenase (COX) and lipoxygenase (LOX) were validated using an oxygen-glucose deprivation/reoxygenation model established on H9c2 cardiomyocytes. To further explore the action mechanisms of DQTT, 38 oxylipins were quantitatively analyzed among high, medium, and low doses of DQTT using a rat AMI model with an ultra high performance liquid chromatograph coupled with a triple quadrupole mass spectrometry (UHPLC-QqQ/MS) detection system. As attenuation was observed in AMI with DQTT treatment, the perturbed arachidonic acid metabolome was partly restored in a dose-dependent fashion with a significant elevation of anti-inflammatory metabolites, while pro-inflammatory lipids were decreased. Cytokine array analysis also supported the anti-inflammatory effects of DQTT, as significant down-regulation of pro-inflammatory cytokines was observed. The analysis of ischemic heart tissues demonstrated that COX and LOX, the inflammation-induced catalytic enzymes of arachidonic acid metabolism, were inhibited on both gene expression and protein level. These results confirmed that DQTT could restore the arachidonic acid metabolome to maintain an anti-inflammatory profile against the ischemic tissue injury and support that DQTT can be a promising medicinal therapy against AMI.

Fast determination of 16 circulating neurotransmitters and their metabolites in plasma samples of spontaneously hypertensive rats intervened with five different Uncaria.

This study aimed to establish a sensitive, reproducible, and rapid liquid chromatography method with tandem mass spectrometry detection to perform simultaneous quantitative analysis of 16 neurotransmitters and their metabolites in rat plasma, including levodopa, dopamine, norepinephrine, epinephrine, L-tryptophan, kynurenic acid, serotonin, melatonin, choline, acetylcholine, histamine, phenylethylamine, as well as excitatory (L-glutamic acid and L-aspartic acid) and inhibitory (γ-aminobutyric acid and L-glycine) neurotransmitters. These analytes were measured by ultra-high performance chromatography coupled with triple quadrupole mass spectrometry using a hydrophilic interaction chromatographic column (ethylene-bridged hybrid amide column). The internal standards of stable isotope labeling were used to improve the reliability of the results. Our method provided high linearity for all neurotransmitters (for all coefficients measured > 0.99), with inter- and intra-day accuracy from -14.82% to 17.49% and precision was between 0.89% and 17.70%. The method was subsequently verified in an animal study, where the intervention of five different Uncarias, the traditional Chinese medicine with hypotensive effects, was applied to the spontaneously hypertensive rats (SHRs). SHRs showed dysregulated plasma kynurenic acid, acetylcholine, and norepinephrine levels, and these neuroactive analytes were significantly restored by Uncaria treatment compared with the model group (SHR group). Compared with captopril, included as a positive control for its hypotensive effect, Uncaria had more effects on perturbing the levels of plasma neurotransmitters, which might indicate Uncaria's potential in treating symptoms related to the nervous system. These results suggested that the changes in the neurotransmitters and their metabolites in plasma may be related to the pathogenesis of hypertension. It also provided valuable information about the action mechanisms of Uncaria on its hypotensive effects.

Exploring the effective materials of flavonoids-enriched extract from Scutellaria baicalensis roots based on the metabolic activation in influenza A virus induced acute lung injury.

Flavonoids-enriched extract from Scutellaria baicalensis roots (FESR) ameliorated influenza A virus (IAV) induced acute lung injury (ALI) in mice by inhibiting the excessive activation of complement system in vivo. However, FESR had no anti-complementary activity in vitro. In order to reveal the effective materials of FESR for the treatment of IAV-induced ALI, the present research explored the metabolic process of FESR both in nomal and IAV infected mice by the method of UHPLC-ESI-LTQ/MS, as well as the metabolic activating mechanism. The results showed that the inactive flavonoid glycosides of FESR were partly metabolized into anti-complementary aglycones in vivo, mainly including 5,7,4'-trihydroxy-8-methoxy-flavone, norwogonin, baicalein, wogonin, oroxylin A and chrysin. Moreover, compared with the normal mice, IAV-induced ALI mice exhibited more efficient on producing and absorbing these active metabolites, with AUC0-t and Cmax in plasma and concentrations in lungs and intestines markedly elevated in the IAV treated groups (P <  0.05). Interestingly, the intestinal bacteria from IAV-induced ALI mice showed stronger ß-glucuronidase activity and also had higher efficiency on transforming FESR to the flavonoid aglycones. These findings suggested that the anti-complementary aglycones produced by metabolic activation in vivo should be the potential effective materials of FESR against IAV infections, and intestinal bacteria might play an important role on the higher bioavailability of FESR in IAV infected mice. Additionally, the animals under the pathological state are more suitable for the metabolic study of traditional Chinese medicine.

Exploring the protective effects of Danqi Tongmai tablet on acute myocardial ischemia rats by comprehensive metabolomics profiling.

BACKGROUND: Danqi Tongmai tablet (DQTM), a combination of salvianolic acids (SA) and panax notoginsenosides (PNS), is now in phase II clinical trial developed for the treatment of cardiovascular diseases. However, the mechanisms of its protective effects through regulating endogenous metabolites remain unclear. PURPOSE: The purpose of this study was to explore the protective effects of DQTM on acute myocardial ischemia rats by comprehensive metabolomics profiling. STUDY DESIGN: The rats were divided into three groups: sham-operating, acute myocardial ischemia (AMI) and DQTM groups. The plasma and heart were collected and profiled by LC-MS based metabolomics and lipidomics. Based on the identified differential metabolites, the pathway analysis results were obtained and further validated using the network pharmacology approach. METHODS: The AMI model was induced by ligating the left anterior descending coronary artery. The metabolomics and lipidomics profiling were based on two established LC-QTOF/MS analysis methods. The raw data were processed using XCMS Online, then the differential metabolites with nonparametric t-test p value less than 0.05 were selected and identified using HMDB and METLIN. The pathway analysis was conducted using MetaboAnalyst and validated with the predicted network results obtained by BATMAN-TCM. RESULTS: The metabolomics and lipidomics profiles of plasma and heart in response to AMI and DQTM were significantly different. The AMI operation had a serious influence on metabolites in heart ischemia region, while DQTM had a greater impact on lipids in heart non-ischemia region. A total of 151 differential metabolites were identified, including mainly amino acids and fatty acids. Multiple metabolic pathways were disturbed after AMI and could be restored by DQTM, of which arachidonic acid metabolism was further validated with the predicted results of network pharmacology. CONCLUSION: The protective effects of DQTM on acute myocardial ischemia rats could be achieved through the regulation of multiple metabolic pathways.

Dissecting the Metabolic Phenotype of the Antihypertensive Effects of Five Uncaria Species on Spontaneously Hypertensive Rats.

The sourcing of plants from multiple botanical origins is a common phenomenon in traditional Chinese medicines. Uncaria Stem with Hooks (UHs) are approved for using five botanical origins in the Chinese Pharmacopoeia (2015 Edition). All five UHs are commonly used for treating hypertension even though the plants have different chromatographic fingerprints based on our previous study. However, their hypotensive effects and metabolic phenotypes have not been fully studied. In the present study, spontaneously hypertensive rats (SHRs) were orally administered five aqueous extracts (4 g crude drug/kg) prepared from the different UHs over a 6-week period. Systolic blood pressure (SBP) was measured every week, and urine was collected after SBP measurement. Untargeted metabonomics was performed using ultra performance liquid chromatography (UPLC) coupled with an LTQ-Orbitrap mass spectrometer. Bidirectional orthogonal projection to latent structures discriminant analysis (O2PLS-DA), Student's t test, and correlation analysis were used for pattern recognition and the selection of significant metabolites. A similar and prolonged reduction in SBP was observed in each of the groups given the five different UHs, while the metabolic profiles were perturbed slightly compared with that of SHR. Further analysis has shown that only a few common, different components were observed within the five groups, which showed the similar antihypertensive effect in spite of the distinct metabolic pathways due to their different alkaloid composition. These results help in understanding the mechanisms of the phenomenon "different species, same effect" of UHs.

Study on the herb-herb interaction of Danqi Tongmai Tablet based on the pharmacokinetics of twelve notoginsenoides in acute myocardial ischemia and sham rats.

Danqi Tongmai tablet (DQTT), an innovative TCM formula under clinical trials, is composed of salvianolic acids (SA) and panax notoginsenosides (PNE) for the treatment of coronary heart disease and angina pectoris. However, the in vivo herb-herb interaction of DQTT remains unclear. In the present research, a rapid, reliable and sensitive method for quantitative analysis of multi-notoginsenoside in rat plasma based on ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-TQ/MS) was established and then applied to explore the herb-herb interaction mechanism of DQTT based on the pharmacokinetics in acute myocardial ischemia (AMI) and sham rats after oral administration of DQTT and PNE. Compared with sham rats after oral administration of PNE, the values of AUC0-t for Rf and Rb2 were significantly higher in DQTT group. Compared with AMI rats after oral PNE, AUC0-t for NR1, Rg1, Re, Rb1, Rd, Rg2, Rb2, NR2, Rh1, F1 and F2 were significantly increased after oral administration of DQTT. These results hinted that SA could improve the bioavailability of notoginsenosides in AMI rats, which provides scientific information for better understanding the herb-herb interaction mechanism and offers a reference for clinical administration of DQTT. Additionally, the presently developed methodology was simple, robust, accurate, precise, and would be useful for the pharmacokinetic studies for all kinds of notoginsenosides and other herbal saponins.

In vivo effect of quantified flavonoids-enriched extract of Scutellaria baicalensis root on acute lung injury induced by influenza A virus.

BACKGROUND: Scutellaria baicalensis root is traditionally used for the treatment of common cold, fever and influenza. Flavonoids are the major chemical components of S. baicalensis root. PURPOSE: To evaluate the therapeutic effects and action mechanism of flavonoids-enriched extract from S. baicalensis root (FESR) on acute lung injury (ALI) induced by influenza A virus (IAV) in mice. METHODS: The anti-influenza, anti-inflammatory and anti-complementary properties of FESR and the main flavonoids were evaluated in vitro. Mice were challenged intranasally with influenza virus H1N1 (A/FM/1/47) 2  h before treatment. FESR (50, 100 and 200  mg/kg) was administrated intragastrically. Baicalin (BG), the most abundant compound in FESR was given as reference control. Survival rates, life spans and lung indexes of IAV-infected mice were measured. Histopathological changes, virus levels, inflammatory markers and complement deposition in lungs were analyzed. RESULT: Compared with the main compound BG, FESR and lower content aglycones (baicalein, oroxylin A, wogonin and chrysin) in FESR significantly inhibited H1N1 activity in virus-infected Madin-Darby canine kidney (MDCK) cells and markedly decreased nitric oxide (NO) production from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In vitro assays showed that FESR and BG had no anti-complementary activity whereas baicalein, oroxylin A, wogonin and chrysin exhibited obvious anti-complementary activity. Oral administration of FESR effectively protected the IAV-infected mice, increased the survival rate (FESR: 67%; BG: 33%), decreased the lung index (FESR: 0.90; BG: 1.00) and improved the lung morphology in comparing with BG group. FESR efficiently decreased lung virus titers, reduced haemagglutinin (HA) titers and inhibited neuraminidase (NA) activities in lungs of IAV-infected mice. FESR modulated the inflammatory responses by decreasing the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), and increasing the levels of interferon-γ (IFN-γ) and interleukin-10 (IL-10) in lung tissues. Although showing no anti-complementary activity in vitro, FESR obviously reduced complement deposition and decreased complement activation product level in the lung . CONCLUSION: FESR has a great potential for the treatment of ALI induced by IAV and the underlying action mechanism might be closely associated with antiviral, anti-inflammatory and anti-complementary properties. Furthermore, FESR resulted in more potent therapeutic effect than BG in the treatment of IAV-induced ALI.

Comparative in vivo constituents and pharmacokinetic study in rats after oral administration of ultrafine granular powder and traditional decoction slices of Chinese Salvia.

Salvia miltiorrhiza, one of the most well-known herbal medicines, is commonly used for the treatment of coronary heart diseases in China. Besides traditional decoction slices (TDS), another relatively new product of S. miltiorrhiza, ultrafine granular powder (UGP; D90 < 45 µm), is also increasingly being used. In this paper, a UHPLC-LTQ-Orbitrap MS technique was developed for a metabolite profile study after oral administration of UGP and TDS of S. miltiorrhiza. The results showed that the number of in vivo absorbed compounds from UGP was much greater than that from TDS, and different types of products from S. miltiorrhiza will have different metabolic processes in vivo. Furthermore, a UHPLC-Q-Trap MS/MS method for simultaneously determining four tanshinones (tanshinone IIA, dihydrotanshinone I, tanshinone I and cryptotanshinone) was established and applied to assess the pharmacokinetics of the two types of products. All of the analytes displayed significant higher area under the concentration-time curve and peak concentration after oral administration of UGP than after TDS, indicating that ultrafine powder product could improve the bioavailability and absorption of cryptotanshinon,tanshinone II A,dihydrotanshinonE I and tanshinone I in vivo. The present study provides scientific information for further exploration of the pharmacology of these two types of S. miltiorrhiza and offers a reference for clinical administration of S. miltiorrhiza.

[Chemical comparison of different Farfarae Flos by NMR-based metabolomic approaches].

1H NMR-based metabolomic approach combined with multivariate statistical analysis was used to evaluate the quality of 21 Farfarae Flos (FF) samples from different growth regions. Principal component analysis showed that wild and cultivated FF could be separated clearly, suggesting a big chemical difference existed between them. Supervised PLS-DA analysis indicated that the wild samples showed higher levels of secondary metabolites, such as bauer-7-ene-3ß, 16α-diol, chlorogenic acid, rutin, 7-(3'-ethylcrotonoyloxy)-1α-(2'-methyl-butyryloxy)-3, 14-dehydro-Z-notonipetranone (EMDNT), tussilagone, ß-sitosterol and sitosterone. This is consistent with traditional experience that the quality of wild samples are better than that of cultivated ones. The content of pyrrolizidine alkaloids senkirkine also differed greatly among samples from different habitats. The Pearson correlation analysis showed that senkirkine is positively correlated with 4, 5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, rutin, kampferol analogues, to a statistically significant extent. The correlation between the toxic compounds and the bioactive components in FF should be further studied.

Metabolomic profiling of the antitussive and expectorant plant Tussilago farfara L. by nuclear magnetic resonance spectroscopy and multivariate data analysis.

This study aims to find metabolites responsible for antitussive and expectorant activities of Tussilago farfara L. by metabolomic approach. Different parts (roots, flower buds, and leaves) of the title plant were analyzed systematically. The in vivo study revealed that the leaves and flower buds had strong antitussive and expectorant effects. Then ¹H NMR spectrometry together with principal component analysis (PCA) and partial least squares discriminant (PLS-DA) analysis were used to investigate the compounds responsible for the bioactivities. PCA was used to find the differential metabolites, while PLS-DA confirmed a strong correlation between the observed effects and the metabolic profiles of the plant. The result revealed that chlorogenic acid, 3,5-dicaffeoylquinic acid, and rutin may be closely related with the antitussive and expectorant activities. The overall results of this study confirm the benefits of using metabolic profiling for screening active principles in medicinal plants.

Metabolic fingerprinting of Tussilago farfara L. using ¹H-NMR spectroscopy and multivariate data analysis.

INTRODUCTION: The flower bud of Tussilago farfara L. is widely used for the treatment of coughs, bronchitis and asthmatic disorders in traditional Chinese medicine. In Europe, the plant has been used as herbal remedies for virtually the same applications, but the leaves are preferred over flowers. OBJECTIVE: To systematically evaluate the chemical profiles of Tusssilago farfara leaves and flowers along with the identification of the polar and non-polar metabolites. METHODOLOGY: Metabolic profiling carried out by means of ¹H-NMR spectroscopy and multivariate data analysis was applied to crude extracts from flowers and leaves. Metabolites were identified directly from the crude extracts through one-dimensional and two-dimensional NMR spectra. RESULTS: A broad range of metabolites were detected without any chromatographic separation. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) of ¹H-NMR data provided a clear separation between the samples. The corresponding loadings plot indicated that higher levels of phenylpropanoids, amino acids, organic acids and fatty acids, as well as lower levels of sugars, terpenoids and sterols were present in the leaves, as compared with flowers. For the flowers, more phenylpropanoids were present in fully open flowers, while more sugars and fatty acids were present in flower buds. CONCLUSION: NMR spectra (one- and two-dimensional) are useful for identifying metabolites, especially for the overlapped signals. The NMR-based metabolomics approach has great potential for chemical comparison study of the metabolome of herbal drugs.

Metabolomic profiling of the flower bud and rachis of Tussilago farfara with antitussive and expectorant effects on mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Flower bud of Tussilago farfara L. is widely used for the treatment of cough, bronchitis and asthmatic disorders in the Traditional Chinese Medicine. However, due to the increasing demands, adulteration with rachis is frequently encountered in the marketplace. No report demonstrated the chemical and pharmacological differences between flower bud and rachis before. MATERIALS AND METHODS: The water extracts were orally administrated to mice. Ammonia induced mice coughing model was used to evaluate the antitussive activity. The expectorant activity was evaluated by volume of phenol red in mice's tracheas. Metabolites were identified directly from the crude extracts through 1D- and 2D-NMR spectra. A metabolic profiling carried out by (1)H NMR spectroscopy and multivariate data analysis was applied to crude extracts from flower bud and rachis. RESULTS: Flower bud significantly lengthened the latent period of cough, decreased cough frequency caused by ammonia and enhanced tracheal phenol red output in expectorant evaluation. Principal component analysis (PCA) yielded good separation between flower bud and rachis, and corresponding loading plot showed that the phenolic compounds, organic acid, sugar, amino acid, terpene and sterol contributed to the discrimination. CONCLUSIONS: These findings provide pharmacological and chemical evidence that only flower bud can be used as the antitussive and expectorant herbal drug. The high concentration of chlorogenic acid, 3,5-dicaffeoylquinic acid, rutin in flower buds may be related with the antitussive and expectorant effects of Flos Farfara. To guarantee the clinical effect, rachis should be picked out before use.

Colorimetric detection of thiols using a chromene molecule.

A new thiol-containing colorimetric probe has been developed by using a chromene derivative, 7-nitro-2,3-dihydro-1H-cyclopenta[b]chromen-1-one (1). The molecule exhibited high selectivity and sensitivity for detecting thiol species as cysteine, homocysteine, and glutathione in aqueous solution through a rapid visual color change from colorless to yellow.