Impact of 14 types of genetic polymorphisms on antihypertensive efficacy of felodipine in healthy Chinese subjects
.

OBJECTIVE: This study evaluated different influences of 14 single nucleotide polymorphisms (SNPs) and demographic factors leading to individual differences in the antihypertensive efficacy of felodipine in healthy Chinese subjects. MATERIALS AND METHODS: 24 subjects were sequenced for candidate SNPs. Plasma samples were obtained as clinical trial protocol, and were determined by a HPLC-MS/MS method. Pharmacokinetic parameters were calculated by WinNonlin 6.0. Statistical analysis was mainly performed by SPSS 22.0. A multiple linear regression model provided different weight coefficients of different demographic and genetic factors. RESULTS: The trend of Cmax is almost consistent with AUCss increase, but tmax of individuals is different; the antihypertensive effect of felodipine is individually different. A significant association was observed between systolic blood pressure decrease (ΔSBP) and SNPs of CACNA1C, CACNA1D, GNB3 respectively, while CACNA1C and CACNA1 were associated with diastolic blood pressure decrease (ΔDBP). CYP3A5 rs766746 and CYP3A4 rs2242480 were linked with Cmax and AUCss, and ABCB1 rs1045642 was associated with T1/2. Significant relationships were shown between AUCss and ΔSBP (p = 0.022) as well as Cmax and ΔSBP (p = 0.015). CONCLUSION: The efficacy of felodipine is individually different, influenced especially by CACNA1C rs1051375 and ABCB1 rs1045642. ΔDBP is associated with ΔSBP in multiple-dosing of felodipine in healthy Chinese subjects.

Toxic effects of triptolide on adrenal steroidogenesis in H295R cells and female rats.

Triptolide (TP), a major active ingredient of Tripterygium wilfordii, exerts potent immunosuppressive effects in the treatment of rheumatoid arthritis but is not widely used in clinical practice due to its multiorgan toxicity, particularly hepatotoxicity, nephrotoxicity, and reproductive toxicity. An LC-MS/MS approach was employed to explore the endocrine-disrupting effects of TP. The endocrine-disrupting effects of various concentrations (0-100 nM) of TP for 48 hour were firstly investigated using an in vitro model (H295R cell line). It was found that TP did not decrease cell viability. The transcriptional levels of steroidogenic enzymes in H295R cells were assessed by quantificational real-time polymerase chain reaction. The possible adrenal and endocrine effects of oral administration of TP (0, 50, and 500 µg/kg) for 28 days on both normal and collagen-induced arthritis (CIA) rats were also explored. The serum and adrenal tissue hormone levels (corticosterone and progesterone) and adrenal histopathology were analyzed, with the results that TP significantly decreased the level of cortisol in H295R cells and the level of plasma corticosterone in both normal and CIA rats. Histological alterations in adrenal cortex were observed at the dose of 500 µg/kg. Exposure to TP for 48 hour had an obvious inhibitory effect on the messenger RNA transcript levels of HSD3B2, CYP21A2, CYP17A1, and CYP11B1, which is essential for the synthesis of corticosteroids. In a word, TP leads to the disorder of corticosteroid synthesis and secretion, and corticosteroid may be a potential biomarker for the treatment of multiorgan toxicity of TP.

Analysis of dihydroindole-type alkaloids in Strychnos nux-vomica unprocessed and processed seeds by high-performance liquid chromatography coupled with diode array detection and mass spectrometry.

The ripened seeds of Strychnos nux-vomica L. have been extensively used as herbal medicines in Asian countries. Dihydroindole-type alkaloids are not only the active constituents but also the toxicants in Strychnos. However, the simultaneous determination of these alkaloids in both crude and processed Semen Strychni is still lacking. The present study represents the first quantitation and relative quantitation assay of 12 dihydroindole-type alkaloids in Strychnos nux-vomica unprocessed and sand-processed seeds using high-performance liquid chromatography coupled with diode array detection and mass spectrometry. The relative concentration of ten alkaloids was calculated by semi-quantification using the internal standard and their amounts in unprocessed and detoxified Semen Strychni were compared. We report here for the first time the significant increase of the two alkaloids, 19-N-methyl-strychnine, and 2,3-dimethoxy-19-N-methyl-strychnine, during the processing of Semen Strychni. Our study provides new insight into the true complexity of seed processing procedure and valuable information for assessing the efficacy and safety for clinical applications of Semen Strychni-containing drugs.

Characterisation and identification of dihydroindole-type alkaloids from processed semen strychni by high-performance liquid chromatography coupled with electrospray ionisation ion trap time-of-flight mass spectrometry.

INTRODUCTION: For centuries, Semen Strychni (the ripened seeds of Strychnos nux-vomica) has been used extensively as a herbal medicine in Asian countries. However, the chemical composition of the dihydroindole-type alkaloids contained in processed Semen Strychni is not fully understood. OBJECTIVE: To develop an improved strategy using mass defect filtering (MDF) in combination with MS(n) analysis and theoretical calculations for identification and structural characterisation of dihydroindole-type alkaloids in processed Semen Strychni extracts. METHODS: The experimental work was conducted using a high-performance liquid chromatography coupled with electrospray ionisation ion trap time-of-flight mass spectrometry (HPLC-ESI/IT-TOF/MS) system. Upon acquisition of full-scan MS data, the potential dihydroindole-type alkaloids were screened using a well-defined mass defect range of 50 mDa. With the assistance of MS(n) analysis, the diagnostic fragment ions (DFIs) were used as primary screening references for targeting the characteristic analogues. For better discrimination of the isomers, quantum chemical calculations were utilised to provide additional structural information. RESULTS: Twenty-four dihydroindole-type alkaloids, including four that were previously not described, were tentatively identified. CONCLUSION: A new, rapid and sensitive method was developed for the discovery and characterisation of dihydroindole-type alkaloids in extracts of processed Semen Strychni. The successful application of this method indicates a potential for adaptation to other classes of natural product from other sources.

In vitro metabolism study of Strychnos alkaloids using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry.

In this report, the in vitro metabolism of Strychnos alkaloids was investigated using liquid chromatography/high-resolution mass spectrometry for the first time. Strychnine and brucine were selected as model compounds to determine the universal biotransformations of the Strychnos alkaloids in rat liver microsomes. The incubation mixtures were separated by a bidentate-C18 column, and then analyzed by on-line ion trap/time-of-flight mass spectrometry. With the assistance of mass defect filtering technique, full-scan accurate mass datasets were processed for the discovery of the related metabolites. The structural elucidations of these metabolites were achieved by comparing the changes in accurate molecular masses, calculating chemical component using Formula Predictor software and defining sites of biotransformation based upon accurate MS(n) spectral information. As a result, 31 metabolites were identified, of which 26 metabolites were reported for the first time. These biotransformations included hydroxylation, N-oxidation, epoxidation, methylation, dehydrogenation, de-methoxylation, O-demethylation, as well as hydrolysis reactions.

Analysis of saikosaponins in rat plasma by anionic adducts-based liquid chromatography tandem mass spectrometry method.

Saikosaponins (SSs) are a class of triterpene saponins with a wide spectrum of bioactivities. A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of saikosaponin a, saikosaponin c, saikosaponin d and saikosaponin b2 in rat plasma. Plasma samples were prepared by liquid-liquid extraction. The analytes and the internal standard (IS) digoxin were well separated on an octadecyl column using gradient elution and analyzed by monitoring the fragmentation transition pair of anionic adducts to deprotonated molecules in negative-mode electrospray. By neutral loss of HCOOH, the transition pairs of m/z 825 → 779 for SSa, SSd, SSb2 and the IS, and m/z 971 → 925 for SSc were sensitive for MS/MS detection with the lower limits of quantification in the range of 0.20-0.40 ng/mL. Method validation experiments were performed, including selectivity, precision, accuracy, linearity, matrix effect, recovery and stability. The validated method was further applied to determine the pharmacokinetics parameters of SSa, c and d in rats following a single oral administration of the extract of chaihu (the dried roots of Bupleurum chinense DC).

A four-component combination derived from Huang-Qin Decoction significantly enhances anticancer activity of irinotecan.

Huang-Qin Decoction (HQD) is a classic prescription for diarrhea in Chinese medicine treatment. Recent studies have demonstrated that HQD and its modified formulation PHY906 could ameliorate irinotecan (CPT-11) induced gastrointestinal (GI) toxicity and enhance its anticancer therapeutic efficacy. Nevertheless, which constituents in HQD are effective is still unclear so far. The study aims to screen out the key bioactive components combination from HQD that could enhance the anticancer effect of CPT-11. First, the potential bioactive constituents were obtained through system pharmacology strategy. Then the bioactivity of each constituent was investigated synthetically from the aspects of NCM460 cell migration, TNF-α release of THP-1-derived macrophage and MTT assay in HCT116 cell. The contribution of each constituent in HQD was evaluated using the bioactive index Ei, which taken the content and bioactivity into comprehensive consideration. And then, the most contributing constituents were selected out to form a key-component combination. At last, the bioefficacy of the key-component combination was validated in vitro and in vivo. As a result, a key-component combination (HB4) consisting of four compounds baicalin, baicalein, glycyrrhizic acid and wogonin was screened out. In vitro assessment indicated that HB4 could enhance the effect of CPT-11 on inhibiting cell proliferation and inducing apoptosis in HCT116. Furthermore, the in vivo study confirmed that HB4 and HQD have similar pharmacological activity and could both enhance the antitumor effect of CPT-11 in HCT116 xenograft model. Meanwhile, HB4 could also reduce the CPT-11 induced GI toxicity.

Ardeemins and cytochalasins from Aspergillus terreus residing in Artemisia annua.

Three new alkaloids, 15b-dehydro-5- N-acetylardeemin ( 3), 10-phenyl-[12]-cytochalasins Z16 ( 6) and Z17 ( 7), were characterized from the liquid culture of the endophytic fungus ASPERGILLUS TERREUS IFB-E030 along with six known derivatives, 5- N-acetylardeemin ( 1), 15b- ß-hydroxyl-5- N-acetylardeemin ( 2), cytochalasin E ( 4), rosellichalasin ( 5), cytochalasins Z11 ( 8), and Z13 ( 9). The structures of the new metabolites were established mainly by a combination of their 1D- and 2D-NMR spectra, single crystal X-ray diffraction, and the modified Mosher reaction. Biological assays indicated that cytochalasin Z17 ( 7) had moderate cytotoxicity against human nasopharyngeal epidermoid tumor KB cell line with an IC (50) value of 26.2 µM.

Systems Pharmacology Based Strategy for Q-Markers Discovery of HuangQin Decoction to Attenuate Intestinal Damage.

The quality control research of traditional Chinese medicine (TCM) is lagged far behind the space of progress in modernization and globalization. Thus the concept of quality marker (Q-marker) was proposed recently to guide the quality investigations of TCM. However, how to discover and validate the Q-marker is still a challenge. In this paper, a system pharmacology based strategy was proposed to discover Q-marker of HuangQin decoction (HQD) to attenuate Intestinal Damage. Using this strategy, nine measurable compounds including paeoniflorin, baicalin, scutellarein, liquiritigenin, norwogonin, baicalein, glycyrrhizic acid, wogonin, and oroxylin A were screened out as potential markers. Standard references of these nine compounds were pooled together as components combination according to their corresponding concentration in HQD. The bioactive equivalence between components combination and HQD was validated using wound healing test and inflammatory factor determination experiment. The comprehensive results indicated that components combination is almost bioactive equivalent to HQD and could serve as the Q-markers. In conclusion, our study put forward a promising strategy for Q-markers discovery.

Metabolomics Reveals the Efficacy of Caspase Inhibition for Saikosaponin D-Induced Hepatotoxicity.

Saikosaponin d (SSd) is a major hepatoprotective component of saikosaponins derived from Radix Bupleuri, which was also linked to hepatotoxicity. Previous studies have demonstrated that caspases play a key role in SSd-induced liver cell death. Our in vitro and in vivo studies also showed that treatment with caspase inhibitor z-VAD-fmk could significantly reduce the L02 hepatocyte cells death and lessen the degree of liver damage in mice caused by SSd. In order to further reveal the underlying mechanisms of caspase inhibition in SSd-induced hepatotoxicity, mass spectrometry based untargeted metabolomics was conducted. Significant alterations in metabolic profiling were observed in SSd-treated group, which could be restored by caspase inhibition. Bile acids and phospholipids were screened out to be most significant by spearman correlation analysis, heatmap analysis and S-Plot analysis. These findings were further confirmed by absolute quantitation of bile acids via targeted metabolomics approach. Furthermore, cytokine profiles were analyzed to identify potential associations between inflammation and metabolites. The study could provide deeper insight into the hepatotoxicity of SSd and the efficacy of caspase inhibition.

HuangQin Decoction Attenuates CPT-11-Induced Gastrointestinal Toxicity by Regulating Bile Acids Metabolism Homeostasis.

Irinotecan (CPT-11) is a potent chemotherapeutic agent, however, its clinical usage is often limited by the induction of severe gastrointestinal (GI) toxicity, especially late-onset diarrhea. HuangQin Decoction (HQD), commonly used for the treatment of GI ailments, has been proved could significantly ameliorate the intestinal toxicity of CPT-11. To reveal the mechanisms of CPT-11-induced toxicity and the modulation effects of HQD, a previous untargeted metabolomics study was performed and the results indicated that HQD may protect the GI tract by altering the metabolism of bile acids (BAs). Nevertheless, the untargeted assays are often less sensitive and/or efficient. In order to further confirm our previous findings, here in this paper, serum and tissues metabolic profiles of 17 BAs were analyzed using liquid chromatography-tandem mass spectrometry based targeted metabolomics. The results indicated that serum and tissues levels of most BAs were significantly decreased after CPT-11 administration, except some hydrophobic BAs. Co-treatment with HQD could markedly attenuate CPT-11-induced GI toxicity and reverse the alterations of hydrophobic BAs. Despite the fact that the BAs pool size remained unchanged, the balance of BAs had shifted leading to decreased toxicity after HQD treatment. The present study demonstrated for the first time that the precise interaction between HQD, CPT-11-induced intestinal toxicity and BAs' homeostasis.

Renal Medulla is More Sensitive to Cisplatin than Cortex Revealed by Untargeted Mass Spectrometry-Based Metabolomics in Rats.

Nephrotoxicity has long been the most severe and life-threatening side-effect of cisplatin, whose anticancer effect is therefore restricted. Previous pathological studies have shown that both renal cortex and medulla could be injured by cisplatin. Our TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) assay results further uncovered that medulla subjected more severe injury than cortex. In order to depict the underlying metabolic mechanism of spatial difference in response to cisplatin, in the present study, mass spectrometry-based untargeted metabolomics approach was applied to profile renal cortex and medulla metabolites of rat after receiving a single dose of cisplatin (2.5, 5 or 10 mg/kg). Eventually, 53 and 55 differential metabolites in cortex and medulla were screened out, respectively. Random forest, orthogonal partial least squares-discriminant analysis and metabolic cumulative fold change analysis revealed that metabolic changes in medulla were more obviously dose-dependent than those in cortex, which confirmed the conclusion that medulla was more sensitive to cisplatin exposure. Furthermore, 29 intermediates were recognized as the most contributive metabolites for the sensitivity difference. Metabolic pathways interrupted by cisplatin mainly included amino acid, energy, lipid, pyrimidine, purine, and creatine metabolism. Our findings provide new insight into the mechanism study of cisplatin-induced nephrotoxicity.

Combination of LC/MS and GC/MS based metabolomics to study the hepatotoxic effect of realgar nanoparticles in rats.

Realgar nanoparticles (NPs) are increasingly used as therapeutic agents for their enhanced anti-proliferation effect and cytotoxicity on cancer cells. However, the alteration of particle size may enhance biological reactivity as well as toxicity. A LC/MS and GC/MS based metabolomics approach was employed to explore the mechanism of realgar NPs-induced hepatotoxicity and identify potential biomarkers. Male Sprague-Dawley rats were administrated intragastrically with realgar or realgar NPs at a dose of 1.0 g·kg-1·d-1 for 28 days and toxic effects of realgar NPs on liver tissues were examined by biochemical indicator analysis and histopathologic examination. Increased levels of serum enzymes and high hepatic steatosis were discovered in the realgar NPs treated group. Multivariate data analysis revealed that rats with realgar NPs-induced hepatotoxicity could be distinctively differentiated from the animals in the control and realgar treated groups. In addition, 21 and 32 endogenous metabolites were apparently changed in the serum and live extracts, respectively. Realgar NPs might induce free fatty acid and triglyceride accumulation, resulting in hepatotoxicity. In conclusion, the present study represents the first comprehensive LC/MS- and GC/MS-based metabolomics analysis of realgar NPs-induced hepatotoxicity, which may help further research of nanotoxicity.

Liver metabolomics study reveals protective function of Phyllanthus urinaria against CCl4-induced liver injury.

Phyllanthus Urinaria L. (PUL) is a traditional Chinese medicine used to treat hepatic and renal disorders. However, the mechanism of its hepatoprotective action is not fully understood. In the present study, blood biochemical indexes and liver histopathological changes were used to estimate the extent of hepatic injury. GC/MS and LC/MS-based untargeted metabolomics were used in combination to characterize the potential biomarkers associated with the protective activity of PUL against CCl4-induced liver injury in rats. PUL treatment could reverse the increase in ALT, AST and ALP induced by CCl4 and attenuate the pathological changes in rat liver. Significant changes in liver metabolic profiling were observed in PUL-treated group compared with liver injury model group. Seventeen biomarkers related to the hepatoprotective effects of PUL against CCl4-induced liver injury were screened out using nonparametric test and Pearson's correlation analysis (OPLS-DA). The results suggested that the potential hepatoprotective effects of PUL in attenuating CCl4-induced hepatotoxicity could be partially attributed to regulating L-carnitine, taurocholic acid, and amino acids metabolism, which may become promising targets for treatment of liver toxicity. In conclusion, this study provides new insights into the mechanism of the hepatoprotection of Phyllanthus Urinaria.

Quantitative Evaluation of the Compatibility Effects of Huangqin Decoction on the Treatment of Irinotecan-Induced Gastrointestinal Toxicity Using Untargeted Metabolomics.

Huangqin decoction (HQD), a traditional Chinese medicine (TCM), has been widely used to treat gastrointestinal syndrome in China for thousands of years. Chemotherapy drug irinotecan (CPT-11) is used clinically to treat various kinds of cancers but limited by its side effects, especially delayed diarrhea. Nowadays, HQD has been proved to be effective in attenuating the intestinal toxicity induced by CPT-11. HQD consists of four medicinal herbs including Scutellaria baicalensis Georgi, Glycyrrhiza uralensis Fisch, Paeonia lactiflora Pall, and Ziziphus jujuba Mill. Due to its complexity, the role of each herb and the multi-herb synergistic effects of the formula are poorly understood. In order to quantitatively assess the compatibility effects of HQD, mass spectrometry-based untargeted metabolomics studies were performed. The serum metabolic profiles of rats administered with HQD, single S. baicalensis decoction, S. baicalensis-free decoction and baicalin/baicalein combination were compared. A time-dependent trajectory upon principal component analysis was firstly used to visualize the overall differences. Then metabolites deregulation score and relative area under the curve were calculated and used as parameters to quantitatively evaluate the compatibility effects of HQD from the aspect of global metabolic profile and the specifically altered metabolites, respectively. The collective results indicated that S. baicalensis played a crucial role in the therapeutic effect of HQD on irinotecan-induced diarrhea. Both HQD and SS decoction regulated glycine, serine and threonine pathway. This study demonstrated that metabolomics was a promising tool to elucidate the compatibility effects of TCM or combinatorial drugs.

Comparative analysis of volatile oils in the stems and roots of Ephedra sinica via GC-MS-based plant metabolomics.

With a great difference in therapeutic effects of Mahuang (MH, the stems of Ephedra sinica) and Mahuanggen (MHG, the roots of Ephedra sinica), chemical differences between MH and MHG should be investigated. In the present study, gas chromatography-mass spectrometry (GC-MS)-based plant metabolomics was employed to compare volatile oil profiles of MH and MHG. The antioxidant activities of volatile oils from MH and MHG were also compared. 32 differential chemical markers were identified according to the variable importance in the projection (VIP) value of orthogonal partial least squares discriminant analysis (OPLS-DA) and P value of Mann-Whitney test. Among them, chemical markers of tetramethylpyrazine (TMP) and α-terpineol were quantified. Their contents were much higher in most MH samples compared with MHG. The antioxidant assay demonstrated that MH had significantly higher free radical-scavenging activity than MHG. Although MH and MHG derived from the same medicinal plant, there was much difference in their volatile oil profiles. MH samples had significantly higher content of two reported pharmacologically important chemical markers of TMP and α-terpineol, which may account for their different antioxidant activities.

Quantification of iodiconazole by LC-MS/MS, a new topical transdermal cream, in dermatophytosis patients' plasma.

BACKGROUND: A rapid and sensitive LC-MS/MS method was established to measure iodiconazole (ADKZ) in dermatophytosis patients treated topically with 2% ADKZ cream. METHODOLOGY: ADKZ was extracted by liquid-liquid extraction (LLE) and separated by an Agilent Zorbax SB-C18 column (2.1 × 150 mm, 3.5 µm) using methanol - 0.01% formic acid (70:30, v/v) as the mobile phase. All the validation assays met the acceptable criteria and the linearity ranged from 10 to 1000 pg/ml. CONCLUSION: The method has been validated to be simple, sensitive and successfully applied to the study. The average amount of ADKZ absorbed into blood was approximately 0.51 µg daily, and ADKZ blood concentrations were consistent during the four-week treatment course. The cumulation of ADKZ in vivo was almost negligible.

A new 1,4-phenanthraquinone from Menispermum dauricum.

A new 1,4-phenanthraquinone was isolated from the roots of Menispermum dauricum. The structure was elucidated as 7-hydroxy-3,6-dimethoxy-1,4-phenanthraquinone on the basis of spectral evidence.

[Determination of verbascoside in herba of Galeobdolon Chinense by RP-HPLC].

OBJECTIVE: To determine Verbascoside in Galeobdolon chinense. METHOD: HPLC method was used, with the column packed with Kromasil C18(4.6 mm x 150 mm, 5 microns). The mobile phase was a mixture of acetonitrile and 1% acetic acid in a ratio of 13:87, and the ultraviolet wavelength was set at 350 nm. RESULT: The average recovery was 97.0%, the RSD being 1.69%. CONCLUSION: This method is sensitive and reliable. It can be used to determine being Verbascoside in Galeobdolon chinense.