Shouhong Gao

A simple and sensitive HPLC method for the simultaneous determination of eight bioactive components and fingerprint analysis of Schisandra sphenanthera

A simple and sensitive high performance liquid chromatography method with photodiode array detection (HPLC-DAD) was developed for simultaneous determination of eight bioactive constituents (schisandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyshisandrin, schisandrin B and schisandrin C) in the ripe fruit of Schisandra sphenanthera and its traditional Chinese herbal preparations Wuzhi-capsule by optimizing the extraction, separation and analytical conditions of HPLC-DAD. The chemical fingerprint of S. sphenanthera was established using raw materials of 15 different origins in China. The chromatographic separations were obtained by an Agilent Eclipse XDB-C18 reserved-phase column (250 mm x 4.6 mm i.d., 5 microm) using gradient elution with water-formic acid (100:0.1, v/v) and acetonitrile, at a flow rate of 1.0 mL min(-1), an operating temperature of 35 degrees C, and a wavelength of 230 nm. The constituents were confirmed by (+) electrospray ionization LC-MS. The new method was validated and was successfully applied to simultaneous determination of components in 13 batches of Wuzhi-capsule. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for quantitative analysis and quality control of S. sphenanthera.

Simultaneous determination of active xanthone glycosides, timosaponins and alkaloids in rat plasma after oral administration of Zi-Shen Pill extract for the pharmacokinetic study by liquid chromatography–tandem mass spectrometry

A sensitive and reliable liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for simultaneous determination of active components, i.e., xanthone glycosides (neomangiferin and mangiferin), timosaponins (timosaponin E1, timosaponin B-II and timosaponin B) and alkaloids (palmatine and berberine) in rat plasma after oral administration of Zi-Shen Pill extract. Plasma samples were pretreated by protein precipitation with acetonitrile containing the internal standards ginsenoside Re (for xanthone glycosides and timosaponins) and tetrahydroberberine (for alkaloids). LC separation was achieved on a Zorbax SB-C(18) column (150 mm x 2.1 mm I.D., 3.5 microm) with gradient elution using a mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.25 mL/min. The detection was carried out by a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between negative (for xanthone glycosides and timosaponins) and positive (for alkaloids) ionization mode. Linear calibration curves were obtained over the concentration range of 5-1000 ng/mL for mangiferin, 0.5-100 ng/mL for neomangiferin, timosaponin E1, timosaponin B-II and timosaponin B, and 0.05-10 ng/mL for palmatine and berberine. The mean recovery of all the analytes ranged from 64.7 to 93.8%. The intra- and inter-day precision (% R.S.D.) was within 11.7% and accuracy (% bias) ranged from -9.0 to 10.9%. This fully validated method was successfully applied to pharmacokinetic study of the above seven compounds in rats.

A rapid and sensitive liquid chromatography-tandem mass spectrometric method for the determination of timosaponin B-II in blood plasma and a study of the pharmacokinetics of saponin in the rat.

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of Timosaponin B-II (TB-II), a pharmacologically active constituent isolated from Anemarrhena asphodeloides. This method was used to examine the pharmacokinetics and bioavailability of TB-II in rats using ginsenoside Re as an internal standard. After simple protein precipitation of the plasma samples with acetonitrile, the analytes were separated on an ODS column (150 mm x 2.1 mm i.d., 5 microm) with the mobile phase of acetonitrile-water (35:65, v/v) containing 0.05% formic acid and detected by electrospray ionization mass spectrometry in the negative multiple reaction monitoring (MRM) mode with a chromatographic run time of 3.0 min. The calibration curves were linear over the range of 5-15,000 ng/ml and the lower limit of quantification (LLOQ) was 5 ng/ml in rat plasma. In this range, relative standard deviations (R.S.D.) were <7.4% for intra-day precision and <9.0% for inter-day precision. The accuracy was within the range of 97.7-107.3%. The method was successfully applied to assess the pharmacokinetics and oral bioavailability of TB-II after intravenous and oral administration in rats, with the oral bioavailability being only 1.1%.

Biosynthesis of the active compounds of Isatis indigotica based on transcriptome sequencing and metabolites profiling

BackgroudIsatis indigotica is a widely used herb for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine (TCM). Various structural classes of compounds have been identified as effective ingredients. However, little is known at genetics level about these active metabolites. In the present study, we performed de novo transcriptome sequencing for the first time to produce a comprehensive dataset of I. indigotica.ResultsA database of 36,367 unigenes (average length = 1,115.67 bases) was generated by performing transcriptome sequencing. Based on the gene annotation of the transcriptome, 104 unigenes were identified covering most of the catalytic steps in the general biosynthetic pathways of indole, terpenoid, and phenylpropanoid. Subsequently, the organ-specific expression patterns of the genes involved in these pathways, and their responses to methyl jasmonate (MeJA) induction, were investigated. Metabolites profile of effective phenylpropanoid showed accumulation pattern of secondary metabolites were mostly correlated with the transcription of their biosynthetic genes. According to the analysis of UDP-dependent glycosyltransferases (UGT) family, several flavonoids were indicated to exist in I. indigotica and further identified by metabolic profile using UPLC/Q-TOF. Moreover, applying transcriptome co-expression analysis, nine new, putative UGTs were suggested as flavonol glycosyltransferases and lignan glycosyltransferases.ConclusionsThis database provides a pool of candidate genes involved in biosynthesis of effective metabolites in I. indigotica. Furthermore, the comprehensive analysis and characterization of the significant pathways are expected to give a better insight regarding the diversity of chemical composition, synthetic characteristics, and the regulatory mechanism which operate in this medical herb.

SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge, which contains tanshinones and phenolic acids as major classes of bioactive components, is one of the most widely used herbs in traditional Chinese medicine. Production of tanshinones and phenolic acids is enhanced by methyl jasmonate (MeJA). Transcription factor MYC2 is the switch of jasmontes signaling in plants. Here, we focused on two novel JA-inducible genes in S. miltiorrhiza, designated as SmMYC2a and SmMYC2b, which were localized in the nucleus. SmMYC2a and SmMYC2b were also discovered to interact with SmJAZ1 and SmJAZ2, implying that the two MYC2s might function as direct targets of JAZ proteins. Ectopic RNA interference (RNAi)-mediated knockdown experiments suggested that SmMYC2a/b affected multiple genes in tanshinone and phenolic acid biosynthetic pathway. Besides, the accumulation of tanshinones and phenolic acids was impaired by the loss of function in SmMYC2a/b. Meanwhile, SmMYC2a could bind with an E-box motif within SmHCT6 and SmCYP98A14 promoters, while SmMYC2b bound with an E-box motif within SmCYP98A14 promoter, through which the regulation of phenolic acid biosynthetic pathway might achieve. Together, these results suggest that SmMYC2a and SmMYC2b are JAZ-interacting transcription factors that positively regulate the biosynthesis of tanshinones and Sal B with similar but irreplaceable effects.

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C₈ column (3.5 μm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.

Liquid chromatography–tandem mass spectrometry method for simultaneous determination of seven commonly used anticancer drugs in human plasma

This paper describes the development and validation of a novel, general liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of cyclophosphamide, ifosfamide, irinotecan, etoposide, gemcitabine, carboplatin and pemetrexed concentrations in human plasma. Samples were prepared by two kinds of extraction method and analyzed using a gradient separation over an Atlantis T3-C18 column (2.1 mm×100 mm, 3 μm, Waters). Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.1% formic acid and 10mM ammonium acetate) at a flow rate of 0.25 mL/min. Linear coefficients of correlation were >0.992 for all analytes. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 9.2%, while the accuracy was within ± 10.5%. The mean recovery of all the analytes ranged from 50.0 to 81.0%. This method was successfully applied to clinical samples from cancer patients.

X-3, a mangiferin derivative, stimulates AMP-activated protein kinase and reduces hyperglycemia and obesity in db/db mice

Diabetes mellitus is a major health concern, affecting nearly 10% of the population. Here we describe a potential novel therapeutic agent for this disease, X-3, a derivative of mangiferin. Therapeutic administration of X-3 significantly and dose-dependently reduced plasma glucose and triglycerides in db/db mice following 8 week-treatments. Treatment with X-3 dose-dependently increased the number of insulin-positive β-cell mass. Importantly, X-3 did not cause any death or signs of toxicity in acute toxicity studies. Study of mechanism of action revealed that X-3 increased glucose uptake in parallel with increased phosphorylation of AMP-activated protein kinase (AMPK) in 3T3-L1 cells. It activates AMPK in both LKB1-dependent and -independent manner. Furthermore, administration of X-3 resulted in activation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC) in the hypothalamus, liver, muscle and adipose tissues of C57BL/6 mice. An 80 mg/kg X-3 was more potent than metformin at 500 mg/kg in the hypothalamus, and interscapular fat tissues, potent than MF at the same dose in the liver. Thus, we conclude that X-3 is a promising new class of AMPK activating drug, and can potentially be used in the treatment of type 2 diabetes.

An liquid chromatography-tandem mass spectrometry assay for determination of trace amount of new antifungal drug iodiconazole in human plasma.

A simple, rapid and sensitive LC-MS/MS method for determination of trace amount of new antifungal drug iodiconazole in human plasma was developed (1-(1H-1,2,4-triazole)-2-(2,4-diflurophenyl)-3-[N-methyl-N-(3-chlor-benzyl)amino]-2-propanol), was used as internal standard (IS). The analytes were extracted by liquid-liquid extraction with n-hexane after internal standard spiked. The separation was performed by a ZORBAX SB-C(18) column (3.5 microm, 2.1 mm x 100 mm) with an isocratic mobile phase consisting of methanol-water-formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The lower limit of quantification (LLOQ) was 0.10 ng/mL. This method was successfully used to determine the concentration of iodiconazole in human plasma following dermal administration.

Metabolite profiling of Zi-Shen pill in rat biological specimens by UPLC-Q-TOF/MS.

This study aimed to profile the chemical constituents of Zi-Shen pill (ZSP) and its metabolites in plasma, urine, and prostate tissue, after administration into rats. Based on the chromatographic retention behavior, fragmentation patterns of chemical components, published literatures, and literature databases, an UPLC-Q-TOF/MS (LC-TOF/MS) method was established to identify the components of ZSP and its metabolites in biological samples. A total of 101 compounds were identified and tentatively characterized from the ZSP, including alkaloids, xanthones, and timosaponins. Except for 33 prototype components, 22 metabolites were detected in the plasma, urine, and prostate, and mainly came from Phellodendri Amurensis Cortex and Anemarrhenae Rhizoma. It was found that glucuronidation and sulfation were the major metabolic processes of xanthones, while oxidation, demethylation, and glucuronidation were the major metabolic pathways of alkaloids. In summary, the present study provided important chemical information on the metabolism of ZSP, indicating that alkaloids might be able to be absorbed into the prostate. The results provided a basis for further studies of the mechanisms of action for ZSP.