Association of genetic variants in soy isoflavones metabolism-related genes with decreased lung cancer risk.

BACKGROUND: Soy isoflavones have been reported to exhibit anti-tumor effects. We hypothesize that genetic variants in soy isoflavone metabolism-related genes are associated with the risk of lung cancer. METHODS: A two-stage case-control study design was conducted in this study. The discovery stage included 300 lung cancer cases and 600 healthy controls to evaluate the association of candidate genetic variants with lung cancer risk. The validation stage involved 1200 cases and 1200 controls to validate the associations found. Furthermore, qPCR was performed to assess the mRNA expression levels of different genotypes of the SNP. ELISA was used to explore the association between genotype and soy isoflavone levels, as well as the association between soy isoflavone levels and lung cancer risk. RESULTS: A nonlinear association was observed between plasma soy isoflavone levels and lung cancer risk, with higher soy isoflavone levels associated with lower lung cancer risk (P < 0.001). The two-stage case-control study identified that UGT1A1 rs3755319 A > C was associated with decreased lung cancer risk (Recessive model: adjusted OR = 0.69, 95 %CI = 0.57-0.84, P < 0.001). Moreover, eQTL analysis showed that the expression level of UGT1A1 in the rs3755319 CC genotype was lower than in the AA + AC genotype (P < 0.05). The plasma concentration of soy isoflavones in the rs3755319 CC genotype was higher than in the AA + AC genotype (P = 0.008). CONCLUSIONS: We identified a potentially functional SNP, UGT1A1 rs3755319 A > C, as being associated with decreased lung cancer risk. Further experiments will be needed to explore the mechanisms underlying the observed associations.

Effects of Soy Isoflavones, Resistant Starch and Antibiotics on Polycystic Ovary Syndrome (PCOS)-Like Features in Letrozole-Treated Rats.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-aged women. Recently, various dietary interventions have been used extensively as a novel therapy against PCOS. In the present study, we show that soy isoflavone metabolites and resistant starch, together with gut microbiota modulations, were successful in decreasing the severity of PCOS-like reproductive features while increasing the expression of gut barrier markers and butyric acid in the gut. In the letrozole-induced PCOS model rats, the intake of both 0.05% soy isoflavones and 11% resistant starch, even with letrozole treatment, reduced the severity of menstrual irregularity and polycystic ovaries with a high concentration of soy isoflavones and equol in plasma. Antibiotic cocktail treatment suppressed soy isoflavone metabolism in the gut and showed no considerable effects on reducing the PCOS-like symptoms. The mRNA expression level of occludin significantly increased with soy isoflavone and resistant starch combined treatment. Bacterial genera such as Blautia, Dorea and Clostridium were positively correlated with menstrual irregularity under resistant starch intake. Moreover, the concentration of butyric acid was elevated by resistant starch intake. In conclusion, we propose that both dietary interventions and gut microbiota modulations could be effectively used in reducing the severity of PCOS reproductive features.

A rapid ultra high performance liquid chromatography-tandem mass spectrometry method for the quantification of daidzein, its valine carbamate prodrug, and glucuronide in rat plasma samples: Comparison of the pharmacokinetic behavior of daidzine valine carbamate prodrugs.

Two valine carbamate prodrugs of daidzein were designed to improve its bioavailability. To compare the pharmacokinetic behavior of these prodrugs with different protected phenolic hydroxyl groups of daidzein, a rapid and sensitive method for simultaneous quantification of daidzein, its valine carbamate prodrug, and daidzein-7-O-glucuronide in rat plasma was developed and validated in this study. The samples were processed using a fast one-step protein precipitation method with methanol added to 50 µL of plasma and were analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry. To improve the selectivity, peak shape, and peak elution, several key factors, especially stationary phase and the composition of the mobile phase, were tested, and the analysis was performed using the Kinetex® C18 column (100 × 2.1 mm, 2.6 µm) within only 2.6 min under optimal conditions. The established method exhibited good linearity over the concentration range of 2.0-1000 ng/mL for daidzein, and 8.0-4000 ng/mL for the prodrug and daidzein-7-O-glucuronide. The accuracy of the quality control samples was between 95.5 and 110.2% with satisfactory intra- and interday precision (relative standard deviation values < 10.85%), respectively. This sensitive, rapid, low-cost, and high-throughput method was successfully applied to compare the pharmacokinetic behavior of different daidzein carbamate prodrugs.

Characterization of the metabolites of dichotomitin in rat, monkey and human by ultra-high-performance liquid chromatography/high-resolution mass spectrometry.

RATIONALE: Dichotomitin is one of the bioactive constituents isolated from Belamcanda chinensis. The goal of this study was to identify the metabolites of dichotomitin produced by liver microsomes and hepatocytes. METHODS: Using ultra-high-performance liquid chromatography and high-resolution mass spectrometry (UPLC/HRMS), the metabolites were profiled and identified. The exact masses, elemental compositions and product ions of the metabolites were used to characterize their structures. RESULTS: A total of ten metabolites were found and identified. The main metabolites identified in the incubation samples were M6 (3',5,6,7-tetrahydroxy-4',5'-dimethoxyisoflavone) and M8 (8-hydroxydichotomitin). Opening of 1,3-benzodioxole, demethylation, hydroxylation, glucuronidation and sulfation were among the metabolic modifications for dichotomitin. A human recombinant cytochrome P450 enzyme study revealed that CYP 1A2, 2C19, and 2D6 facilitated the formation of M6, whereas CYP 1A2 catalyzed the formation of M8 exclusively. CONCLUSIONS: For the first time, data on the in vitro metabolic fates of dichotomitin were revealed in this work which would be helpful for us to understand the disposition of this bioactive constituent.

Isoflavone biomarkers are inversely associated with atherosclerosis progression in adults: a prospective study.

BACKGROUND: Many studies have examined associations between dietary isoflavones and atherosclerosis, but few used objective biomarkers. OBJECTIVES: We examined the associations of isoflavone biomarkers (primary analyses) and equol production (secondary analyses) with the progression of carotid intima-media thickness (cIMT), and whether inflammation, systolic blood pressure (SBP), blood lipids, and sex hormone-binding globulin (SHBG) mediated these associations, in Chinese adults. METHODS: This 8.8-y prospective study included 2572 subjects (40-75 y old) from the GNHS (Guangzhou Nutrition and Health Study; 2008-2019). The concentrations of daidzein, genistein, and equol were assayed by an HPLC-tandem MS in serum (n = 2572) at baseline and in urine (n = 2220) at 3-y intervals. The cIMT of the common carotid artery (CCA) and bifurcation segment were measured by B-mode ultrasound every 3 y, and the progressions of cIMT ( ∆cIMT) were estimated using the regression method. RESULTS: Multivariable linear mixed-effects models (LMEMs) and ANCOVA revealed that subjects with higher serum isoflavones tended to have lower increases of CCA-cIMT. The mean ± SEM differences in 8.8-y ∆CCA-cIMT between extreme tertiles of serum isoflavones were -17.1 ± 8.4, -20.6 ± 8.3, and -23.3 ± 10.4 µm for daidzein, total isoflavone, and equol (P-trends < 0.05), respectively. LMEMs showed that the estimated yearly changes (95% CIs) (µm/y) in CCA-IMT were -2.0 (-3.8, -0.3), -1.9 (-3.6, -0.1), and -2.1 (-3.8, -0.3) in the highest (compared with the lowest) tertile of daidzein, genistein, and total isoflavones, respectively (P-interaction < 0.05). Path analyses indicated that the serum equol-atherosclerosis association was mediated by increased SHBG and decreased SBP. Similar beneficial associations were observed in the secondary analyses. CONCLUSIONS: Serum isoflavones and equol exposure were associated with reduced cIMT progression, mediated by SHBG and SBP.

Metabolic tracking of isoflavones in soybean products and biosamples from healthy adults after fermented soybean consumption.

Fermentation may enhance the nutritional properties of foods by increasing metabolite bioactivity or bioavailability. This study explored the effect of fermentation on isoflavone bioavailability and metabolism. Isoflavone metabolites were tracked in foods and biospecimens of healthy adults after fermented soybean (FS) or non-fermented soybean (NFS) consumption in a randomized, controlled, crossover intervention study. The change in soybean isoflavones caused by fermentation resulted in faster absorption and higher bioavailability after consumption of FS. Although the urinary level of total isoflavone metabolites was similar after the consumption of the two diets, urinary genistein 7-O-sulfate was derived as a discriminant metabolite for the FS diet by partial least squares discriminant analysis. This study suggests that an isoflavone conjugate profile might be a more appropriate marker than total isoflavone levels for discriminating between the consumption of FS and NFS diets.

Simultaneous quantification and rat pharmacokinetics of formononetin-7-O-ß-d-glucoside and its metabolite formononetin by high-performance liquid chromatography-tandem mass spectrometry.

Formononetin-7-O-ß-d-glucoside has been proved to have significant anti-inflammatory effect. To evaluate its rat pharmacokinetics, a rapid, sensitive, and specific liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of formononetin-7-O-ß-d-glucoside and its main metabolite formononetin in rat plasma. Samples were pretreated using a simple protein precipitation and the chromatographic separation was performed on a C18 column by a gradient elution using a mobile phase consisting of water and acetonitrile both containing 0.1% formic acid. Both analytes were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. The assay showed wide linear dynamic ranges of both 0.10-100 ng/mL, with acceptable intra- and inter-batch accuracy and precision. The lower limits of quantification were both 0.10 ng/mL using 50 µL of rat plasma for two analytes. The method has been successfully used to investigate the oral pharmacokinetic profiles of both analytes in rats. After oral administration of formononetin-7-O-ß-d-glucoside at the dose of 50 mg/kg, it was rapidly absorbed in vivo and metabolized to its metabolite formononetin. The plasma concentration-time profiles both showed double-peak phenomena, which would be attributed to the strong enterohepatic circulation of formononetin-7-O-ß-d-glucoside.

Effects of a low-fat dietary regimen enriched with soy in children affected with heterozygous familial hypercholesterolemia.

INTRODUCTION: Familial hypercholesterolemia (FH) is an inheritable, autosomal dominant disorder leading to pathologically increased levels of low-density-lipoprotein cholesterol (LDL-C). Dietary treatment remains an important tool in the management of affected children even after the decision for the initiation of pharmacotherapy is made. However, little evidence is available regarding the optimal dietary regimen for the treatment of children affected with FH. METHODS: We present results from a randomized controlled trial in paediatric patients affected with heterozygous FH, assessing the effect of a soy-enriched fat modified diet (soy group) compared to fat modified diet (Control group) alone on LDL-C over a period of 13 weeks. Furthermore, we monitored isoflavone levels in plasma and urine as markers of adherence to the dietary treatments. RESULTS: LDL-C decrease was statistically significantly greater in the soy group compared to the control group at week 7 (Control group 176.3 ± 27.8 mg/dl, soy group 154.7 ± 29.2 mg/dl, p = 0.038), and showed a trend towards significant at week 13 (Control group 179.9 ± 41.8 mg/dl, soy group 155.0 ± 30.2 mg/dl, p = 0.089). Relative LDL-C decrease correlated significantly with the following plasma isoflavone concentrations measured in week 7: daidzein (p < 0.004, r = 0.576) and genistein (p < 0.017, r = 0.490). CONCLUSIONS: We provide evidence from a small randomized-controlled trial for the effectiveness and safety of a dietary treatment with soy in paediatric patients affected with heterozygous FH. The decrease in LDL-C was highly correlated with isoflavone levels, further highlighting a direct effect of soy ingestion. This study was registered under ClinicalTrials.gov Identifier No. NCT03563547.

Pharmacokinetics, Tissue Distribution, and Druggability Prediction of the Natural Anticancer Active Compound Cytisine N-Isoflavones Combined with Computer Simulation.

Cytisine N-methylene-(5,7-dihydroxy-4'-methoxy)-isoflavone (CNF2) is a new compound isolated from the Chinese herbal medicine Sophora alopecuroides. Preliminary pharmacodynamic studies demonstrated its activity in inhibiting breast cancer cell metastasis. This study examined the pharmacokinetics, absolute bioavailability, and tissue distribution of CNF2 in rats, and combined computer-aided technology to predict the druggability of CNF2. The binding site of CNF2 and the breast cancer target human epidermal growth factor receptor-2 (HER2) were examined with molecular docking technology. Next, ACD/Percepta software was used to predict the druggability of CNF2 based on the quantitative structure-activity relationship (QSAR). Finally, a simple and effective HPLC method was used to determine plasma pharmacokinetics and tissue distribution of CNF2 in rats. Prediction and experimental results show that compared with the positive control HER2 inhibitor SYR127063, CNF2 has a stronger binding affinity with HER2, suggesting that its efficacy is stronger; and the structure of CNF2 complies with the Lipinski's Rule of Five and has good drug-likeness. The residence time of CNF2 in rats is less than 4 h, and the metabolic rate is relatively fast; But the absolute bioavailability of CNF2 in rats was 6.6%, mainly distributed in the stomach, intestine, and lung tissues, where the CNF2 contents were 401.20, 144.01, and 245.82 µg/g, respectively. This study constructed rapid screening and preliminary evaluation of active compounds, which provided important references for the development and further research of such compounds.

Simultaneous quantitation of glycyrrhetic acid and puerarin in plasma using ultra flow liquid chromatography tandem mass spectrometry and application in a pharmacokinetics study in healthy and alcoholic liver injury rats.

A rapid, sensitive, and accurate ultra flow liquid chromatography tandem mass spectrometry (UFLC-MS/MS ) method was developed and validated for simultaneous quantitation of glycyrrhetic acid and puerarin in plasma derived from healthy and alcoholic liver injury rats. Plasma samples from healthy and model rats were deproteinated with methanol using liquiritin as an internal standard. Chromatography separation was performed by a Waters BEH (ethylene-bridged hybrid) C18 column (2.1 × 50 mm; 1.7 µm) using a gradient elution from acetonitrile and water (containing 0.1% formic acid) and at a flow rate of 0.4 mL/min. Quantitation was performed on a Triple Quad 4500 tandem mass spectrometer coupled with an electrospray ionization source in negative multiple reaction monitoring mode. Specificity, carryover, dilution integrity, recovery, linearity, precision and accuracy, matrix effect, and stability were within acceptable limits. The newly established method was successfully applied to a pharmacokinetics study to investigate glycyrrhetic acid and puerarin in healthy and alcoholic liver injury rats.

Use of Physiologically Based Kinetic Modeling to Predict Rat Gut Microbial Metabolism of the Isoflavone Daidzein to S-Equol and Its Consequences for ERα Activation.

SCOPE: To predict gut microbial metabolism of xenobiotics and the resulting plasma concentrations of metabolites formed, an in vitro-in silico-based testing strategy is developed using the isoflavone daidzein and its gut microbial metabolite S-equol as model compounds. METHODS AND RESULTS: Anaerobic rat fecal incubations are optimized and performed to derive the apparent maximum velocities (Vmax ) and Michaelis-Menten constants (Km ) for gut microbial conversion of daidzein to dihydrodaidzein, S-equol, and O-desmethylangolensin, which are input as parameters for a physiologically based kinetic (PBK) model. The inclusion of gut microbiota in the PBK model allows prediction of S-equol concentrations and slightly reduced predicted maximal daidzein concentrations from 2.19 to 2.16 µm. The resulting predicted concentrations of daidzein and S-equol are comparable to in vivo concentrations reported. CONCLUSION: The optimized in vitro approach to quantify kinetics for gut microbial conversions, and the newly developed PBK model for rats that includes gut microbial metabolism, provide a unique tool to predict the in vivo consequences of daidzein microbial metabolism for systemic exposure of the host to daidzein and its metabolite S-equol. The predictions reveal a dominant role for daidzein in ERα-mediated estrogenicity despite the higher estrogenic potency of its microbial metabolite S-equol.

A novel molecularly imprinted sensor based on PtCu bimetallic nanoparticle deposited on PSS functionalized graphene with peroxidase-like activity for selective determination of puerarin.

In this work, PtCu bimetallic nanoparticle was deposited on poly (styrene sulfonate) (PSS) functionalized graphene (Gr) to form a nanocomposite PtCu/PSS-Gr and its enzyme-like activity was investigated. Benefiting from the synergistic effect from Pt and Cu monometal as well as the superior properties of PSS-Gr, such as large surface area, good dispersity, strong adsorption of substrate and additional peroxidase-like activity, the PtCu/PSS-Gr nanocomposite was demonstrated as an excellent peroxidase mimic to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Combined with molecularly imprinted polymer (MIP), a new colorimetric approach for puerarin detection was proposed with the linear range of 2 × 10-5-6 × 10-4 mol L-1 and LOD of 1 × 10-5 mol L-1. The combination of MIP with PtCu/PSS-Gr nanocomposite not only endowed the determination of puerarin with high selectivity, but also realized the detection of small molecules which are neither substrate of the nanozyme nor substances with strong oxidizing or reducing activity by using peroxidase-like catalytic activity of nanozyme, expanding the application of nanozyme.

Untargeted metabolomics reveals alterations in metabolites of lipid metabolism and immune pathways in the serum of rats after long-term oral administration of Amalaki rasayana.

Amalaki rasayana, a traditional preparation, is widely used by Ayurvedic physicians for the treatment of inflammatory conditions, cardiovascular diseases, and cancer. Metabolic alterations induced by Amalaki rasayana intervention are unknown. We investigated the modulations in serum metabolomic profiles in Wistar rats following long-term oral administration of Amalaki rasayana. Global metabolic profiling was performed of the serum of rats administered with either Amalaki rasayana (AR) or ghee + honey (GH) for 18 months and control animals which were left untreated. Amalaki rasayana components were confirmed from AR extract using HR-LCMS analysis. Significant reductions in prostaglandin J2, 11-dehydrothromboxane B2, and higher levels of reduced glutathione and glycitein metabolites were observed in the serum of AR administered rats compared to the control groups. Eleven different metabolites classified as phospholipids, glycerophospholipids, glucoside derivatives, organic acids, and glycosphingolipid were exclusively observed in the AR administered rats. Pathway analysis suggests that altered metabolites in AR administered rats are those associated with different biochemical pathways of arachidonic acid metabolism, fatty acid metabolism, leukotriene metabolism, G-protein mediated events, phospholipid metabolism, and the immune system. Targeted metabolomics confirmed the presence of gallic acid, ellagic acid, and arachidonic acid components in the AR extract. The known activities of these components can be correlated with the altered metabolic profile following long-term AR administration. AR also activates IGF1R-Akt-Foxo3 signaling axis in heart tissues of rats administered with AR. Our study identifies AR components that induce alterations in lipid metabolism and immune pathways in animals which consume AR for an extended period.

LC-MS/MS analysis of puerarin and 18ß-glycyrrhetinic acid in human plasma after oral administration of Samso-eum and its application to pharmacokinetic study.

The aim of this study was to confirm pharmacokinetic screening of multiple components in healthy Korean subjects after oral administration of Samso-eum and perform quantitation of active components in the human plasma. Thirteen potential bioactive components [puerarin (PRR), daidzin, nodakenin, ginsenoside Rb1, 18ß-glycyrrhetinic acid (18ß-GTA), 6-shogaol, naringin, glycyrrhizin, hesperidin, platycodin D, naringenin, hesperetin, and 6-gingerol] were screened based on literature. The results showed that three analytes (daidzin, naringenin, and hesperetin) were detected in trace amounts. In addition, PRR and 18ß-GTA were detected in human plasma after the oral administration of Samso-eum. In this study, a liquid chromatography-electrospray ionization-tandem mass spectrometry method was validated for the simultaneous determination of PRR and 18ß-GTA in human plasma. This was the first study to evaluate pharmacokinetics of PRR and 18ß-GTA after the usual oral dose of Samso-eum (30 g containing 102.48 mg PRR, 48.18 mg glycyrrhizin) in human subjects.

Comprehensive investigation of mechanism and effective ingredients of Fangji Huangqi Tang by serum pharmacochemistry and network pharmacology.

Fangji Huangqi Tang (FHT), has been reported to show effects on nephrotic syndrome, but its mechanism of action and bioactive components have not yet been determined. In this study, a method using UPLC-HRMS/MS was established for the detection and identification of the chemical constituents and metabolites absorbed into the blood. Absorbed components in serum were then used for the network pharmacology analysis to deduce the mechanism and effective components. A total of 86 compounds were identified or tentatively characterized. Based on the same instrumental conditions, 85 compounds were found in rat serum after oral administration of FHT, including 22 prototypes and 63 metabolites. Network pharmacology analysis showed that absorbed components, such as (3R)-2',3',4',7-tetrahydroxyisoflavan, astrapterocarpan, cycloastragenol, 7,2'-dihydroxy-3',4'-dimethoxyisoflavan, astragaloside IV, astrapterocarpan glucoside and glycyrrhetinic acid, could be responsible for the pharmacological activity of nephrotic syndrome by regulating the VEGF signaling pathway, focal adhesion and MAPK signaling pathway. Furthermore, the pathway-target network showed that the MAPK1, AKT2 and CDC42 were involved in the signal pathways above. This study provides a scientific basis for the mechanism and effective ingredients of FHT.

The profiling and identification of the absorbed constituents and metabolites of Naoshuantong capsule in mice biofluids and brain by ultra- fast liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry.

Naoshuantong capsule (NSTC) is an oral traditional Chinese medicine formula used widely in the clinic for ischemic stroke. The absorbed ingredients and metabolites of NSTC have never been reported before. In this study, a method incorporating rapid resolution liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to identify absorbed ingredients and metabolites after oral administration of NSTC. A total of 15 constituents were detected and identified as prototypes of NSTC. 109 metabolites related to catechin, gallic acid, paeoniflorin, chlorogenic acid, protocatechuate, typhaneoside, ß-elemene, calycosin were identified in serum, urine and brain. 19 metabolites of typhaneoside, 3 metabolites of ß-elemene, 12 metabolites of calycosin were reported for the first time. This is the first time to explore the absorption and metabolism of NSTC. The work will provide helpful information for further research of the mechanism and application of NSTC.

A network pharmacology integrated pharmacokinetics strategy for uncovering pharmacological mechanism of compounds absorbed into the blood of Dan-Lou tablet on coronary heart disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Dan-Lou tablet (DLT) is developed from the traditional Chinese medicine (TCM) formula Gualou Xiebai Baijiu Tang which has been used for at least 2000 years in China. DLT has been widely used in clinical practice to treat cardiovascular diseases. AIM OF THE STUDY: This study aimed to uncover the pharmacological mechanism of the compounds absorbed into the blood of Dan-Lou tablet (DLT) on coronary heart disease (CHD) using a network pharmacology integrated pharmacokinetics strategy. MATERIALS AND METHODS: A rapid and sensitive method was developed for the simultaneous determination of the six compounds (puerarin, formononetin, calycosin, paeoniflorin, cryptotanshinone and tanshinone IIA) in rat plasma by liquid chromatography tandem mass spectrometry (LC-MS/MS). Then, the pharmacology network was established based on the relationship between five compounds absorbed into the blood targets (puerarin, formononetin, calycosin, cryptotanshinone and tanshinone IIA) and CHD targets. RESULTS: The intra-and inter-day precision were less than 11% and the accuracy ranged from 88.2% to 112%, which demonstrated that the LC-MS/MS method could be used to evaluate the pharmacokinetic feature of the six compounds in rats after oral administration of DLT. The pathway enrichment analysis revealed that the significant bioprocess networks of DLT on CHD were positive regulation of estradiol secretion, negative regulation of transcription from RNA polymerase II promoter, lipopolysaccharide-mediated signaling pathway and cytokine activity. CONCLUSION: The proposed network pharmacology integrated pharmacokinetics strategy provides a combination method to explore the therapeutic mechanism of the compounds absorbed into the blood of multi-component drugs on a systematic level.

Simultaneous determination of eight constituents in rat plasma by HPLC-MS/MS and its application to a pharmacokinetic study after oral administration of Shejin-liyan Granule.

Shejin-liyan Granule (SJLY) is an effective traditional Chinese prescription medicine for the treatment of acute pharyngitis. In this study, a selective and convenient HPLC-MS/MS method was developed and validated for the simultaneous determination of the following eight constituents in the plasma: galuteolin, tectoridin, tectorigenin, iridin, irigenin, irisflorentin, arctiin and arctigenin. The plasma samples were prepared by a protein precipitation method using acetonitrile, and analysis was carried out on a C18 column using a gradient elution at a flow rate of 0.3 mL/min. The concentration of these analytes was quantified in the positive ion and multiple reaction monitoring modes. The method was validated for selectivity, linearity, accuracy, precision, recovery, matrix effect and sample stability. The obtained results were well within the acceptable limits. The established method was then successfully applied to study the pharmacokinetic profiles of the multiple constituents of Shejin-liyan Granule. According to the area under the curve and maximum concentration data, tectorigenin exhibited the highest exposure followed by arctigenin, irigenin, arctiin and irisflorentin. The concentrations of galuteolin, tectoridin and iridin were low, and a complete concentration-time curve could not be plotted. This research provides useful information for understanding the pharmacokinetics of Shejin-liyan Granule.

Simultaneous determination of five isoflavones in rat plasma by LC-MS/MS: Comparative pharmacokinetic characteristics of Puerariae lobatae radix in normal and type 2 diabetic rats.

A rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed and validated for simultaneous determination of puerarin, daidzin, daidzein, 3'-hydroxy puerarin, and genistein in rat plasma after oral administration of Puerariae lobatae radix extract. The method of protein precipitation with acetonitrile was used for sample preparation. Chromatographic separation was achieved on a C18 column with the mobile phases of acetonitrile/water containing 0.1% formic acid. The analytes were detected by mass spectrometer with an electrospray ionization source operating in the negative ion mode. The linearity, precision, accuracy, dilution reliability, recovery, matrix effects, and stability of the method were within acceptable ranges. The developed method was successfully used to compare the pharmacokinetic characteristics of five analytes in normal and type 2 diabetics rats after oral administration of Puerariae lobatae radix extract. Several pharmacokinetic alterations were observed and this might be caused by the pathological state of type 2 diabetes.

Validated LC-MS/MS method for simultaneous quantification of seven components of Naodesheng in rat serum after oral administration and its application to a pharmacokinetic study.

A simple, precise and reliable LC-MS/MS method was developed and validated for simultaneous quantification of vitexin, notoginsenoside R1, hydroxysafflor yellow A, ginsenoside Rd, puerarin, daidzein and senkyunolide I as components of Naodesheng (NDS) in rat serum. The Linearity ranges in rat serum were 0.045-4.5 µg/mL for vitexin, 0.0476-4.76 µg/mL for notoginsenoside R1, 0.0422-4.22 µg/mL for hydroxysafflor yellow A, 0.0426-4.26 µg/mL for ginsenoside Rd, 0.0436-4.36 µg/mL for puerarin, 0.026-2.6 µg/mL for daidzein, and 0.05-5 µg/mL for senkyunolide I, with the correlation coefficients greater than 0.99. The established method was validated in terms of intra- and inter-day precision and accuracy, recovery, matrix effect and stability. Furthermore, the method was successfully applied for pharmacokinetic study of these seven components in rat serum after oral administration of NDS.