Beihua Bao

Integrated plasma and urine metabolomics coupled with HPLC/QTOF-MS and chemometric analysis on potential biomarkers in liver injury and hepatoprotective effects of Er-Zhi-Wan

AbstractMetabolomics techniques are the comprehensive assessment of endogenous metabolites in a biological system and may provide additional insight into the molecular mechanisms. Er-Zhi-Wan (EZW) is a traditional Chinese medicine formula, which contains Fructus Ligustri Lucidi (FLL) and Herba Ecliptae (HE). EZW is widely used to prevent and treat various liver injuries through the nourishment of the liver. However, the precise molecular mechanism of hepatoprotective effects has not been comprehensively explored. Here, an integrated metabolomics strategy was designed to assess the effects and possible mechanisms of EZW against carbon tetrachloride-induced liver injury, a commonly used model of both acute and chronic liver intoxication. High-performance chromatography/quadrupole time-of-flight mass spectrometry (HPLC/QTOF-MS) combined with chemometric approaches including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to discover differentiating metabolites in metabolomics data of rat plasma and urine. Results indicate six differentiating metabolites, tryptophan, sphinganine, tetrahydrocorticosterone, pipecolic acid, l-2-amino-3-oxobutanoic acid and phosphoribosyl pyrophosphate, in the positive mode. Functional pathway analysis revealed that the alterations in these metabolites were associated with tryptophan metabolism, sphingolipid metabolism, steroid hormone biosynthesis, lysine degradation, glycine, serine and threonine metabolism, and pentose phosphate pathway. Of note, EZW has a potential pharmacological effect, which might be through regulating multiple perturbed pathways to the normal state. Our findings also showed that the robust integrated metabolomics techniques are promising for identifying more biomarkers and pathways and helping to clarify the function mechanisms of traditional Chinese medicine. Graphical AbstractOverview of the integrated metabolomics strategy

Anti-inflammatory effects of Huangqin tang extract in mice on ulcerative colitis

ETHNOPHARMACOLOGICAL RELEVANCE HuangqinTang (HQT) is a traditional Chinese formula which is composed of Scutellaria baicalensis Georgi, Paeonia lactiflora Pall, Glycyrrhiza uralensis Fisch, and Ziziphus jujube Mill. HQT has been used in China for a wide range of disorders, especially in gastrointestinal inflammation with symptoms of nausea, vomiting, diarrhea, abdominal cramps and so on. AIM OF THE STUDY To investigate the protective effects of HQT extract on 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) induced colitis in mice. MATERIALS AND METHODS Different doses of HQT extract (1, 2 and 4 g/kg/day) and salicylazosulfapyridine (SASP, 500 mg/kg/day) were administered by gavage for 7 days after the induction of colitis with TNBS. The effects were studied by macroscopic score, histological analysis, immunohistochemical study of Cyclo-oxygenase-2 protein expression, as well as by determination of inflammation markers such as myeloperoxidase (MPO) and mRNA expression levels of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6. RESULTS In TNBS induced group, mice body weight decreased gradually and did not recover at the end of the experiment, as compared with that of control group (p<0.01). Edema and redness were also discovered in the colons profoundly and scores representing inflammation were all high in this group (p<0.01). The level of colonic MPO activity and the tissue levels of TNF-α, IL-1β and IL-6 were markedly increased (p<0.01). The mice treated with HQT extract and SASP recovered significantly compared with the TNBS group (p<0.01). CONCLUSION Our results suggested that the efficacy of HQT extract, especially at the higher dose, was analogous to that of SASP, which implicated its potential application as a natural alternative medicine in colitis treatment.

Quality assessment of Fructus Ligustri Lucidi by the simultaneous determination of six compounds and chemometric analysis

A comprehensive strategy was designed for the quality assessment of Fructus Ligustri Lucidi, a well-known and commonly used herbal medicine in clinical practice in China. First, a simple and stable method of high-performance liquid chromatography was developed for the simultaneous quantitative analysis of six compounds, namely, salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid in Fructus Ligustri Lucidi. The separation of analytes was conducted on a C18 column (200 mm × 4.6 mm, 5 μm) at 30°C, and the wavelength of UV detector was set at 210 nm. In quantitative analysis, all of the calibration curves showed good linear regression (R(2) > 0.9994) within the tested ranges, and the mean recoveries of three different concentrations ranged from 95.21-102.34%. The described method was applied to determine 11 batches of samples collected from different stores in China. Then multiple chemometrics analysis including hierarchical cluster analysis and principal component analysis were performed to classify samples and search significant compounds. Three notable compounds, specnuezhenide, oleanic acid, and acetyl oleanic acid, were discovered for better quality control compared with those stated in the China pharmacopeia. The results demonstrated that this strategy could be readily utilized for the comprehensive quality control of Fructus Ligustri Lucidi.

Effects of Schizonepetin on Activity and mRNA Expression of Cytochrome P450 Enzymes in Rats

The aim of this study was to find out whether Schizonepetin influences the pharmacokinetics of the main substrates drugs of CYP1A2, CYP3A1/2, CYP2E1, CYP2C19 and CYP2D6 in rats; the influence on the levels of CYP mRNA was also studied. Phenacetin, dapsone, chlorzoxazone, omeprazole and metoprolol were selected as probe substrates for CYP1A2, CYP3A1/2, CYP2E1, CYP2C19 and CYP2D6 respectively. HPLC methods were employed for the determination of these substrates in plasma and the pharmacokinetic parameters were calculated. Real-time RT-PCR was used to determine the effects of Schizonepetin on the mRNA expression of CYP3A1, CYP1A2 and CYP2E1 in the rat liver. After the rats were orally administrated with Schizonepetin once a day for seven consecutive days, there were significant differences in plasma concentration of phenacetin, dapsone, chlorzoxazone and metoprolol, but not omeprazole, as compared with pre-administration. In addition, Schizonepetin induced the expression of CYP3A1, CYP1A and CYP2E1 at dosages of 24 and 48 mg/kg. Our results indicated that Schizonepetin had significant induction effects on CYP3A1/2 and inhibition effects on CYP1A2, CYP2E1 or CYP2D6 as oriented from the pharmacokinetic profiles of the substrates. Moreover, in the mRNA expression levels, Schizonepetin could induce the mRNA expression of CYP3A1, CYP1A and CYP2E1. In conclusion, co-administration of some CYP substrates with Schizonepetin may lead to an undesirable herb-drug interaction.

Application of UHPLC-ESI-Q-TOF-MS to Identify Multiple Constituents in Processed Products of the Herbal Medicine Ligustri Lucidi Fructus

Ligustri Lucidi Fructus (LLF), the fruit of Ligustrum lucidum Ait. (Oleaceae), has been used as a common herbal medicine in clinical practice in China for nearly 2000 years. In most cases, LLF is prescribed in decoctions in the form of processed products rather than crude drugs. In this study, an ultra-high performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UHPLC-ESI-Q-TOF-MS) method was established for rapid separation and identification of multiple constituents in the 80% methanol extract of processed-LLF. A total of 50 compounds (one phenylethanoid, seven phenylethanoid glycosides, seven flavonoids, 25 iridoids, nine triterpenoids and one cyclohexanecarboxylic acid) were either unambiguously identified or tentatively characterized with the aid of authentic standards or published data. Luteolin-7-O-rutinoside, oleoside and secologanoside were detected in LLF for the first time. This study enriches the chemical profiling of processed-LLF and could provide valuable information for the quality control and further investigation of processed-LLF and crude LLF.

Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism.

Pekinenin C is a casbane diterpenoid separated from the root of the traditional Chinese medicine, Euphorbia pekinensis Rupr., which is used as drug for the treatment of edema, ascites, and hydrothorax. Whereas pekinenin C exhibits severe cytotoxicity, the exact toxicity mechanism is unclear. In this study, the effects of pekinenin C on cell inhibition, cell cycle, and cell apoptosis were examined to explain its toxic mechanism. The proliferation of IEC-6 cells was accessed via MTT colorimetric assay after incubated with different concentrations of pekinenin C. Pekinenin C-treated IEC-6 cells labeled with RNase/PI and Annexin V/PI were analyzed by flow cytometric analyses for evaluation of cell cycle distribution and cell apoptosis, respectively. The apoptosis mechanism of pekinenin C on IEC-6 was investigated through assaying the activities of caspase-3, 8, 9 by enzyme-linked immunosorbent assay (ELISA), protein expression of Bax, Bcl-2, apoptosis-inducing factor (AIF), Apaf-1, Fas-associated death domain (FADD) and type 1-associated death domain (TRADD) by Western-blot, mRNA expression of Fas receptor (FasR), Fas ligand (FasL), tumor necrosis factor receptor (TNFR1) and NF-κB by RT-PCR. The results showed that pekinenin C has exhibited obvious IEC-6 cells toxicity and the IC50 value was 2.1 μg·mL−1. Typical apoptosis characteristics were observed under a transmission electron microscopy, and it was found that pekinenin C could cause G0/G1 phase arrest in IEC-6 cells in a dose-dependent manner and induce apoptosis of IEC-6 cells. Additionally, pekinenin C could increase the expressions of Bax, AIF, Apaf-1, FasR, FasL, TNFR1 and NF-κB, suppress the expression of Bcl-2, FADD and TRADD, then activate caspase-3, 8, 9 cascades, and at last result in apoptosis. These results demonstrated that pekinenin C effectively promoted cell apoptosis, and induced IEC-6 cells apoptosis through both the mitochondrial and death receptor pathways.

Antioxidant capacity of Typha angustifolia extracts and two active flavonoids

Abstract Context: The pollen of Typha angustifolia L. (Typhaceae) has been used as a traditional Chinese medicine for improving the microcirculation and promoting wound healing. Flavonoids are the main constituent in the plant, but little is known about the antioxidant activity of the principal constituent of the pollen in detail. Objectives: To assess the antioxidant activities of ethanol and water extracts and two constituents of the pollen. Materials and methods: Plant material (1 g) was extracted by 95% ethanol and water (10 mL × 2, 1 h each), respectively. The extracted activities (0.8–2.6 mg/mL) were measured by DPPH and the reducing activity of ferric chloride (1.7–2.6 mg/mL). Typhaneoside and isorhamnetin-3-O-neohesperidoside (I3ON) (2.8–70 μmol/L) were investigated on the relationship between NO, MDA and SOD in HUVECs treated with 100 μg/mL of LPS for 24 h. Results: Nine compounds were identified by UPLC-MS. Ethanol extract showed IC50 values in DPPH (39.51 ± 0.72) and Fe3+ reducing activity (82.76 ± 13.38), higher than the water extract (50.85 ± 0.74) and (106.33 ± 6.35), respectively. Typhaneoside and I3ON promoted cell proliferation at the respective concentration range of 2.8 to 70 μmol/L (p < 0.01). This two compounds decreased MDA (1.91 ± 0.10, 1.80 ± 0.34, p < 0.05) and NO levels (14.64 ± 0.08, 13.10 ± 0.88, p < 0.01), respectively, and increased SOD level (22.94 ± 2.48, 23.57 ± 2.38, p < 0.01) at the concentration of 70 μmol/L compared with LPS group. Conclusions: The constituents from Typha angustifolia could be a novel therapeutic strategy for LPS-induced inflammation.

Anti-thrombotic and pro-angiogenic effects of Rubia cordifolia extract in zebrafish

ETHNOPHARMACOLOGICAL RELEVANCE Rubia cordifolia is a common traditional Chinese medicine that promotes blood circulation and eliminates blood stasis, and has been used to cure diseases related to blood stasis syndrome (BSS) clinically for many years. It has been previously demonstrated that anti-thrombosis and pro-angiogenesis can improve BSS. However, the anti-thrombotic and pro-angiogenic activities of Rubia cordifolia have not been well investigated. AIM OF STUDY To determine the potential anti-thrombotic and pro-angiogenic activities of Rubia cordifolia and to elucidate the underlying mechanisms. In addition, the major chemical constituents of Rubia cordifolia extract (QC) were qualitatively analysed by UPLC-Q-TOF/MS to explore the association between pharmacological activity and chemical constituents. MATERIAL AND METHODS The QC samples were composed of a 95% ethanol extract and an aqueous extract following extraction using 95% ethanol. UPLC-Q-TOF/MS was used to analyse the major chemical constituents of QC. For the anti-thrombotic experiment of QC, a phenylhydrazine (PHZ)-induced AB strain zebrafish thrombosis model was used. The zebrafish larvae were stained using O-dianisidine, and the heart and caudal vein of the zebrafish were observed and imaged with a fluorescence microscope. The staining intensity of erythrocytes in the heart (SI) of each group and the morphology of thrombus in the caudal vein were used to assess the anti-thrombotic effect of QC. For the pro-angiogenic assay of QC, the intersegmental blood vessel (ISV) insufficiency model of Tg(fli-1: EGFP)y1 transgenic zebrafish (Flik zebrafish), which was induced by the VEGF receptor tyrosine kinase inhibitor II (VRI), was used. The morphology of the intact ISVs and defective ISVs was observed to evaluate the pro-angiogenic activity of QC. The mechanism involved in promoting angiogenesis was studied with real-time PCR. RESULTS A total of 12 components in QC were identified based on standard compounds and references, including nine anthraquinones and three naphthoquinones. After treatment with QC, the PHZ-induced thrombosis in AB strain zebrafish larvae decreased to a certain degree, which we believe was related to its dosages, and the therapeutic effect within the 50-200 µg/mL QC treatment groups was especially prominent (P < 0.01, P < 0.001) compared to that in the PHZ model group. Similarly, QC also recovered the loss of the ISVs, which was induced by VRI in Flik zebrafish larvae, which have a certain dose-effect relationship. The pro-angiogenic activity of QC was also conspicuous (P < 0.01, P < 0.001) compared to that of the VRI model group. The following real-time PCR assay proved that QC significantly restored the VRI-induced downregulation of vWF, VEGF-A, kdrl, and flt-1 in Flik zebrafish (P < 0.05, P < 0.01, P < 0.001). CONCLUSIONS A total of 12 compounds from QC were analysed by UPLC-Q-TOF/MS. The data of the pharmacological experiments demonstrated that QC presented anti-thrombotic and pro-angiogenic activities in zebrafish, and the principal active components were likely anthraquinones and naphthoquinones. Thus, the current study provided a theoretical basis for the clinical use of Rubia cordifolia as a traditional Chinese medicine in promoting blood circulation and eliminating stasis.

Simultaneous quantitation and comparison of eight components in Jiao‐ai decoction and Si‐wu decoction by ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry

An ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry method has been established to evaluate the variations of multiple components of Chinese herbal preparations, Jiao-ai decoction and Si-wu decoction, through the simultaneous determination of eight major active compounds with a huge difference in the level of content. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with a mobile phase consisting of acetonitrile (0.1% formic acid) and water (0.1% formic acid) under gradient elution. A triple quadrupole tandem mass spectrometer was operated in positive and negative ionization modes, respectively, with multiple reaction monitoring for the detection of the eight compounds. All calibration curves showed excellent linear regressions (r > 0.99) within the test range. The precision, repeatability, and stability of the eight compounds were below 5.0% in terms of relative standard deviation. The recoveries were 97.0-102.4% with a relative standard deviation of 1.21-3.65% for all samples. In conclusion, a rapid, sensitive, precise, accurate, and reliable method has been developed for the simultaneous detection of eight active compounds in the pharmaceutical samples of Jiao-ai decoction and Si-wu decoction, which can be applied for the multicomponent comparison and further quality control.

Cellular Metabolomics Revealed the Cytoprotection of Amentoflavone, a Natural Compound, in Lipopolysaccharide-Induced Injury of Human Umbilical Vein Endothelial Cells

Amentoflavone is one of the important bioactive flavonoids in the ethylacetate extract of “Cebaiye”, which is a blood cooling and hematostatic herb in traditional Chinese medicine. The previous work in our group has demonstrated that the ethylacetate extract of Cebaiye has a notable antagonistic effect on the injury induced by lipopolysaccharide (LPS) to human umbilical vein endothelial cells (HUVECs). The present investigation was designed to assess the effects and possible mechanism of cytoprotection of amentoflavone via metabolomics. Ultra-performance liquid chromatography/quadrupole time of flight-mass spectrometry (UPLC/QTOF-MS) coupled with multivariate data analysis was used to characterize the variations in the metabolites of HUVECs in response to exposure to LPS and amentoflavone treatment. Seven putative metabolites (glycine, argininosuccinic acid, putrescine, ornithine, spermidine, 5-oxoproline and dihydrouracil) were discovered in cells incubated with LPS and/or amentoflavone. Functional pathway analysis uncovered that the changes of these metabolites related to various significant metabolic pathways (glutathione metabolism, arginine and proline metabolism, β-alanine metabolism and glycine, serine and threonine metabolism), which may explain the potential cytoprotection function of amentoflavone. These findings also demonstrate that cellular metabolomics through UPLC/QTOF-MS is a powerful tool for detecting variations in a range of intracellular compounds upon toxin and/or drug exposure.