3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

Qualitative, quantitative, and pharmacokinetic study on the absorbed components of Ardisia japonica (Thunb.) Blume in rat plasma based on molecular networking combined with quadrupole time-of-flight LC/MS and triple quadrupole LC/MS.

Isolation and screening of different compounds from plant extracts are always the key for natural drug research, and the absorbed prototype components have been considered as potential active ingredients. UHPLC combined with quadrupole time-of-flight mass spectrometry (Q-TOF-LC/MS) has been widely used in the research of natural drugs; however, we still need a more effective tool to compare and treat from a raw data. In this study, we provided a fast analytical method to measure the absorbed prototype components and their metabolites both qualitatively and quantitatively based on molecular networking (MN). For example, in Ardisia japonica (Thunb.) Blume, a total of eight absorbed prototype components in rat plasma were identified. Furthermore, pharmacokinetic study was also successfully performed on the eight absorbed prototype components in rat plasma. Our findings have provided important information on the investigation of A. japonica in vivo. More importantly, the MS network analysis pattern serves as an integral solution for qualitative and quantitative determination of phytochemical compounds in natural drugs.

Serum Proteomic Analysis of Cannabis Use Disorder in Male Patients.

Cannabis use has been growing recently and it is legally consumed in many countries. Cannabis has a variety of phytochemicals including cannabinoids, which might impair the peripheral systems responses affecting inflammatory and immunological pathways. However, the exact signaling pathways that induce these effects need further understanding. The objective of this study is to investigate the serum proteomic profiling in patients diagnosed with cannabis use disorder (CUD) as compared with healthy control subjects. The novelty of our study is to highlight the differentially changes proteins in the serum of CUD patients. Certain proteins can be targeted in the future to attenuate the toxicological effects of cannabis. Blood samples were collected from 20 male individuals: 10 healthy controls and 10 CUD patients. An untargeted proteomic technique employing two-dimensional difference in gel electrophoresis coupled with mass spectrometry was employed in this study to assess the differentially expressed proteins. The proteomic analysis identified a total of 121 proteins that showed significant changes in protein expression between CUD patients (experimental group) and healthy individuals (control group). For instance, the serum expression of inactive tyrosine protein kinase PEAK1 and tumor necrosis factor alpha-induced protein 3 were increased in CUD group. In contrast, the serum expression of transthyretin and serotransferrin were reduced in CUD group. Among these proteins, 55 proteins were significantly upregulated and 66 proteins significantly downregulated in CUD patients as compared with healthy control group. Ingenuity pathway analysis (IPA) found that these differentially expressed proteins are linked to p38MAPK, interleukin 12 complex, nuclear factor-κB, and other signaling pathways. Our work indicates that the differentially expressed serum proteins between CUD and control groups are correlated to liver X receptor/retinoid X receptor (RXR), farnesoid X receptor/RXR activation, and acute phase response signaling.

UHPLC-MS/MS method for pharmacokinetic and bioavailability determination of five bioactive components in raw and various processed products of Polygala tenuifolia in rat plasma.

CONTEXT: Sibiricose A5 (A5), sibiricose A6 (A6), 3,6'-disinapoyl sucrose (DSS), tenuifoliside A (TFSA) and 3,4,5-trimethoxycinnamic acid (TMCA) are the main active components of Polygala tenuifolia Willd. (Polygalaceae) (PT) that are active against Alzheimer's disease. OBJECTIVE: To compare the pharmacokinetics and bioavailability of five active components in the roots of raw PT (RPT), liquorice-boiled PT (LPT) and honey-stir-baked PT (HPT). MATERIALS AND METHODS: The median lethal dose (LD50) was evaluated through acute toxicity test. The pharmacokinetics of five components after oral administration of extracts of RPT, LPT, HPT (all equivalent to 1.9 g/kg of RPT extract for one dose) and 0.5% CMC-Na solution (control group) were investigated, respectively, in Sprague-Dawley rats (four groups, n = 6) using UHPLC-MS/MS. In addition, the absolute bioavailability of A5, A6, DSS, TFSA and TMCA after oral administration (7.40, 11.60, 16.00, 50.00 and 3.11 mg/kg, respectively) and intravenous injection (1/10 of the corresponding oral dose) in rats (n = 6) was studied. RESULTS: The LD50 of RPT, LPT and HPT was 7.79, 14.55 and 15.99 g/kg, respectively. AUC 0- t of RPT, LPT and HPT were as follows: A5 (433.18 ± 65.48, 680.40 ± 89.21, 552.02 ± 31.10 ng h/mL), A6 (314.55 ± 62.73, 545.76 ± 123.16, 570.06 ± 178.93 ng h/mL) and DSS (100.30 ± 62.44, 232.00 ± 66.08, 197.58 ± 57.37 ng h/mL). The absolute bioavailability of A5, A6, DSS, TFSA and TMCA was 3.25, 2.95, 2.36, 1.17 and 42.91%, respectively. DISCUSSION AND CONCLUSIONS: The pharmacokinetic and bioavailability parameters of each compound can facilitate future clinical studies.

[Effects of gut microbiota on five absorbed components of Berberis kansuensis in rat serum by HPLC-QqQ-MS].

To elucidate the absorption and metabolism of alkaloids in Berberis kansuensis in vivo, a high performance liquid chromatography-triple quadrupole mass spectrometry(HPLC-QqQ-MS) method was developed to qualitatively and quantitatively analyze the absorption components in rat serum in multiple-reaction monitoring mode. The mobile phase consisted of 0.1% formic acid and acetonitrile with a gradient elution mode. In addition, to investigate the effects of gut microbiota on five absorbed components of B. kansuensis in rat serum, diabetic rat and pseudo germ-free diabetic rat models were established, and partial least squares discriminant analysis and One-way ANOVA were used to study the content differences of five components among different groups. In this study, a HPLC-QqQ-MS method for quantitative analysis of five components in rat serum after oral administration of B. kansuensis was established for the first time. It was found that there were differences in the five constituents in rat serum between different groups. By comparing the normal group with the diabetic model group, we found that the absorption and metabolism capacities of berberine and magnoflorine were different under the health and pathological conditions. It was also found that the serum levels of berberine, magnoflorine and jatrorrhizine in pseudo germ-free diabetic rats were significantly lower than those in diabetic rats, indicating that gut microbiota plays an important role in the metabolism of alkaloids of B. kansuensis in vivo. These results provide a good reference for clarifying the active ingredients of B. kansuensis in the treatment of diabetes.

A Novel Tomato-Soy Juice Induces a Dose-Response Increase in Urinary and Plasma Phytochemical Biomarkers in Men with Prostate Cancer.

Background: Tomato and soy intake is associated with reduced prostate cancer risk or severity in epidemiologic and experimental studies. Objective: On the basis of the principle that multiple bioactives in tomato and soy may act on diverse anticancer pathways, we developed and characterized a tomato-soy juice for clinical trials. In this phase 2 dose-escalating study, we examined plasma, prostate, and urine biomarkers of carotenoid and isoflavone exposure. Methods: Men scheduled for prostatectomy were recruited to consume 0, 1, or 2 cans of tomato-soy juice/d before surgery (mean ± SD duration: 24 ± 4.6 d). The juice provided 20.6 mg lycopene and 66 mg isoflavone aglycone equivalents/177-mL can. Plasma carotenoids and urinary isoflavone metabolites were quantified by HPLC-photometric diode array and prostate carotenoids and isoflavones by HPLC-tandem mass spectrometry. Results: We documented significant dose-response increases (P < 0.05) in plasma concentrations of tomato carotenoids. Plasma concentrations were 1.86-, 1.69-, 1.73-, and 1.69-fold higher for lycopene, ß-carotene, phytoene, and phytofluene, respectively, for the 1-can/d group and 2.34-, 3.43-, 2.54-, and 2.29-fold higher, respectively, for the 2-cans/d group compared with 0 cans/d. Urinary isoflavones daidzein, genistein, and glycitein increased in a dose-dependent manner. Prostate carotenoid and isoflavone concentrations were not dose-dependent in this short intervention; yet, correlations between plasma carotenoid and urinary isoflavones with respective prostate concentrations were documented (R2 = 0.78 for lycopene, P < 0.001; R2 = 0.59 for dihydrodaidzein, P < 0.001). Secondary clustering analyses showed urinary isoflavone metabolite phenotypes. To our knowledge, this is the first demonstration of the phytoene and phytofluene in prostate tissue after a dietary intervention. Secondary analysis showed that the 2-cans/d group experienced a nonsignificant decrease in prostate-specific antigen slope compared with 0 cans/d (P = 0.078). Conclusion: These findings provide the foundation for evaluating a well-characterized tomato-soy juice in human clinical trials to define the impact on human prostate carcinogenesis. This trial is registered at clinicaltrials.gov as NCT01009736.

Comparative Study on Pharmacokinetics of Four Active Compounds in Rat Plasma after Oral Administration of Raw and Wine Processed Chuanxiong Rhizoma.

In Chinese medicine, the effect of promoting blood circulation and removing stasis could be enhanced after Chuanxiong Rhizoma is processed by wine. However, the relevant mechanism remains unclear. In this manuscript, a rapid and sensitive quantification method employing ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established and validated to simultaneously determine butylidenephthalide, ligustilide, senkyunolide A and ferulic acid in rat plasma after oral administration of raw Chuanxiong Rhizoma (RCR) and wine-processed Chuanxiong Rhizoma (WCR) respectively. All analytes were extracted from plasma by proteins precipitation with methanol. Chromatographic separation was carried out on a Hypersil GOLD C18 column by using a gradient mobile phase system of acetonitrile and water with 0.01% formic acid, the flow rate was 0.3 mL/min. For exact mass detecting, quick switching mode was used, positive and negative ions could be detected in one injection. The pharmacokinetic profiles of four components in the two groups were evaluated and compared. The results showed that, compared to the RCR group, the Vd and AUC0→t values of four active compounds were increased and decreased respectively in WCR group, which revealed the effect of wine processing to Chuanxiong Rhizoma: the stronger the effect, the wider the distribution.

[UPLC-Q-TOF-MS/MS analysis on prototypes components and metabolites of effective fractions of Polygonum orientale flower in rat serum and urine].

Ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOF-MS/MS) method was applied to analyze the prototypes and metabolites of the effective components of Polygonum orientale in SD rat serum and urine. The separation was performed on Agilent Eclipse Plus C_(18) column( 2. 1 mm×100 mm,1. 8 µm),with 0. 1% formic acid solution( A)-acetonitrile( B) as the mobile phase for gradient elution. Mass spectrometry data of biological samples were obtained under positive and negative electrospray ion mode. By comparing chromatogram differences between blank samples and drug treatment samples,prototype components and metabolites of the effective components of P. orientale extract were identified. The results showed that 12 metabolites were detected in serum and 26 metabolites in urine( including cross-components) of rats. The main metabolic pathways included hydrogenation,hydroxylation,glucuronidation,sulfation reaction,and methylation-glucuronidation,etc. The method established in this study was reliable and effective for studying the metabolic characteristics of the effective components of P. orientale in rats,and it can provide a reference for further studies on therapeutic material basis of this herb.

Identification of methylated metabolites of oat avenanthramides in human plasma using UHPLC QToF-MS.

Oat avenanthramides (AVAs) are a group of phenolic alkaloids, consisting of an anthranilic acid and a hydroxycinnamic acid linked by a pseudo-peptide bond. Bioavailability of AVA is poor in humans, suggesting transformations for rapid excretion. Thus, we aim to identify metabolites of AVA isomers in plasma of humans after consuming AVA-enriched oats. After lipid removal, AVA and their metabolites in plasma were extracted with ethyl acetate and analysed using an Agilent UHPLC-QToF-MS. Pharmacokinetics of AVA-O showed a bimodal distribution with Cmax1 and 2 for AVA-O at 5.9 ± 5.2 and 7.9 ± 7.0 ng/mL and Tmax1 and 2 at 1.7 ± 0.7 and 3.1 ± 1.2 h, respectively. Only the methyl-AVA-O showed a single Cmax at 14 ± 9.9 ng/mL AVA-O equivalents and a Tmax of 2.4 ± 2.7 h. This analysis is the first to identify methylated metabolites of AVAs and AVA aglycones in human blood after acute AVA consumption.

Acute Consumption of Flavan-3-ol-Enriched Dark Chocolate Affects Human Endogenous Metabolism.

Flavan-3-ols and methylxanthines have potential beneficial effects on human health including reducing cardiovascular risk. We performed a randomized controlled crossover intervention trial to assess the acute effects of consumption of flavan-3-ol-enriched dark chocolate, compared with standard dark chocolate and white chocolate, on the human metabolome. We assessed the metabolome in urine and blood plasma samples collected before and at 2 and 6 h after consumption of chocolates in 42 healthy volunteers using a nontargeted metabolomics approach. Plasma samples were assessed and showed differentiation between time points with no further separation among the three chocolate treatments. Multivariate statistics applied to urine samples could readily separate the postprandial time points and distinguish between the treatments. Most of the markers responsible for the multivariate discrimination between the chocolates were of dietary origin. Interestingly, small but significant level changes were also observed for a subset of endogenous metabolites. 1H NMR revealed that flavan-3-ol-enriched dark chocolate and standard dark chocolate reduced urinary levels of creatinine, lactate, some amino acids, and related degradation products and increased the levels of pyruvate and 4-hydroxyphenylacetate, a phenolic compound of bacterial origin. This study demonstrates that an acute chocolate intervention can significantly affect human metabolism.

Plant miRNAs found in human circulating system provide evidences of cross kingdom RNAi.

BACKGROUND: Emerging evidence indicates that plant miRNAs can present within human circulating system through dietary intake and regulate human gene expression. Hence we deduced that comestible plants miRNAs can be identified in the public available small RNA sequencing data sets. RESULTS: In this study, we identified abundant plant miRNAs sequences from 410 human plasma small RNA sequencing data sets. One particular plant miRNA miR2910, conserved in fruits and vegetables, was found to present in high relative amount in the plasma samples. This miRNA, with same 6mer and 7mer-A1 target seed sequences as hsa-miR-4259 and hsa-miR-4715-5p, was predicted to target human JAK-STAT signaling pathway gene SPRY4 and transcription regulation genes. CONCLUSIONS: Through analysis of public available plasma small RNA sequencing data, we found the supporting evidence for the plant miRNAs cross kingdom RNAi within human circulating system.

Quantitative analysis of absorption, metabolism, and excretion of benzoxazinoids in humans after the consumption of high- and low-benzoxazinoid diets with similar contents of cereal dietary fibres: a crossover study.

PURPOSE: Benzoxazinoids (BXs) are a group of wholegrain phytochemicals with potential pharmacological properties; however, limited information exists on their absorption, metabolism, and excretion in humans. The aim of this study was to investigate the dose-dependent uptake and excretion of dietary BXs in a healthy population. METHODS: Blood and urine were collected from 19 healthy participants from a crossover study after a washout, a LOW BX diet or HIGH BX diet, and analysed for 12 BXs and 4 phenoxazinone derivatives. RESULTS: We found that the plasma BX level peaked approximately 3 h after food intake, whereas BXs in urine were present even at 36 h after consuming a meal. No phenoxazinone derivatives could be detected in either plasma or urine. The dominant BX metabolite in both plasma and urine was 2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc), even though 2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc) was the major component in the diet. CONCLUSION: The dietary BX treatment correlated well with the plasma and urine levels, illustrating strong dose-dependent BX absorption, which also had a rapid washout, especially from the plasma compartment.

Phytochemical uptake following human consumption of Montmorency tart cherry (L. Prunus cerasus) and influence of phenolic acids on vascular smooth muscle cells in vitro.

PURPOSE: To investigate the phytochemical uptake following human consumption of Montmorency tart cherry (L. Prunus cerasus) and influence of selected phenolic acids on vascular smooth muscle cells in vitro. METHODS: In a randomised, double-blinded, crossover design, 12 healthy males consumed either 30 or 60 mL of Montmorency tart cherry concentrate. Following analysis of the juice composition, venous blood samples were taken before and 1, 2, 3, 5 and 8 h post-consumption of the beverage. In addition to examining some aspects of the concentrate contents, plasma concentrations of protocatechuic acid (PCA), vanillic acid (VA) and chlorogenic (CHL) acid were analysed by reversed-phase high-performance liquid chromatography (HPLC) with diode array for quantitation and mass spectrometry detection (LCMS) for qualitative purposes. Vascular smooth muscle cell migration and proliferation were also assessed in vitro. RESULTS: Both the 30 and 60 mL doses of Montmorency cherry concentrate contained high amounts of total phenolics (71.37 ± 0.11; 142.73 ± 0.22 mg/L) and total anthocyanins (62.47 ± 0.31; 31.24 ± 0.16 mg/L), as well as large quantities of CHL (0.205 ± 0.24; 0.410 ± 0.48 mg/L) and VA (0.253 ± 0.84; 0.506 ± 1.68 mg/L). HPLC/LCMS identified two dihydroxybenzoic acids (PCA and VA) in plasma following MC concentrate consumption. Both compounds were most abundant 1-2 h post-initial ingestion with traces detectable at 8 h post-ingestion. Cell migration was significantly influenced by the combination of PCA and VA, but not in isolation. There was no effect of the compounds on cell proliferation. CONCLUSIONS: These data show new information that phenolic compounds thought to exert vasoactive properties are bioavailable in vivo following MC consumption and subsequently can influence cell behaviour. These data may be useful for the design and interpretation of intervention studies investigating the health effects of Montmorency cherries.

Rapid discovery of absorbed constituents and metabolites in rat plasma after the oral administration of Zi Shen Wan using high-throughput UHPLC-MS with a multivariate analysis approach.

Zi Shen Wan is a typical formula consisting of three herbs, Phellodendri Amurensis Cortex, Rhizoma Anemarrhenae, and Cortex Cinnamomi, and has been widely used for treating prostatitis and infection diseases. However, it lacks in-depth research of the constituents of Zi Shen Wan in vivo and in vitro. In this work, ultra high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and MassLynx software was established to characterize the chemical compositions of Zi Shen Wan in vivo and in vitro. In total, 92 peaks were characterized in vitro and 33 peaks were characterized in vivo based on mass spectrometry and tandem mass spectrometry data. Among the 33 compounds characterized in rat plasma, 22 prototype components absorbed in rat serum and 11 metabolites were identified in vivo. This work was fully reports the chemical constituents of traditional Chinese formula of Zi Shen Wan, it demonstrated that ultra high performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry coupled to MassLynx software and multivariate data processing approach could be successfully applied for rapid screening and comprehensive analysis of chemical constituents in vitro and prototype components or metabolites in vivo of traditional Chinese medicine.

Sample preparation for liquid chromatographic analysis of phytochemicals in biological fluids.

INTRODUCTION: Natural products have been used traditionally for the treatment and prevention of diseases for thousands of years and are nowadays consumed as dietary supplements and herbal medicine. To ensure the safe and effective use of these herbal products, information about bioavailability of active compounds in plasma or target tissues should be provided via validated analytical methods combined with appropriate sampling methods. OBJECTIVE: To provide comprehensive and abridged information about sample preparation methods for the quantification of phytochemicals in biological samples using liquid chromatography analysis. METHODS: Sample pre-treatment procedures used in analytical methods for in vivo pharmacokinetic studies of natural compounds or herbal medicines were reviewed. These were categorised according to the biological matrices (plasma, bile, urine, faeces and tissues) and sample clean-up processes (protein precipitation, liquid-liquid extraction and solid-phase extraction). RESULTS: Although various kinds of sample pre-treatment methods have been developed, liquid-liquid extraction is still widely used and solid-phase extraction is becoming increasingly popular because of its efficiency for extensive clean up of complex matrix samples. However, protein precipitation is still favoured due to its simplicity. CONCLUSION: Sample treatment for phytochemical analysis in biological fluids is an indispensable and critical step to obtain high quality results. This step could dominate the overall analytical process because both the duration of the process as well as the reliability of the data depend in large part on its efficiency. Thus, special attention should be given to the choice of a proper sample treatment method that targets analytes and their biomatrix.

A sensitive UPLC/ESI/MS/MS method for concomitant quantification of active plant constituent combinations in rat plasma after single oral administration.

Ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC/ESI/MS/MS) for the concomitant quantification of active plant constituents, namely quercetin and piperine, in rat plasma was developed and validated to assess pharmacokinetics after a single oral administration. Liquid-liquid extraction technique with ethyl acetate and n-hexane (1 : 1) was used, and fisetin was added as an internal standard (IS). Effective chromatographic separation of quercetin, piperine and IS was executed on a Waters Acquity BEH C18 column (50.0 mm × 2.1 mm, 1.7 µm) using formic acid both (0.1% w/v) in water (A) and acetonitrile (B) as the mobile phase in gradient mode. For detection purposes, positive electrospray ionization (ESI) mode was used with multiple reaction monitoring (MRM) mode for estimation using [M + H]+ fragment ions m/z 303.04 → 152.9 for quercetin, 286.12 → 201.04 for piperine and 287.01 → 136.93 for IS. The method was linear over the calibration range of 0.1-200 ng mL-1. The lower limit of quantification (LLOQ) of quercetin and piperine was obtained as 0.1 ng mL-1 in rat plasma, along with negligible matrix effect and acceptable stability. Furthermore, the bioanalytical method was successfully implemented to determine the pharmacokinetic profiles of quercetin-and piperine-enriched nanostructured lipid carriers (NLCs) in rat plasma after oral administration. The enhancement in the oral bioavailability of quercetin and piperine was 20.72 and 4.67 fold, respectively, compared to their native pristine dispersions. Future exploration of the concentrations of these active constituents in human plasma and organs is feasible using this sensitive, validated UPLC/ESI/MS/MS method.

Screening of blood-activating active components from Curcuma wenyujin Y.H. Chen et C. Ling rhizome based on spectrum-effect relationship analysis and network pharmacology.

Curcuma wenyujin Y.H. Chen et C. Ling rhizome (also called EZhu in China) has long been used as plant medicine for its traditional effect on promoting blood circulation and remove blood stasis. However, the active components of EZhu are still unclear at present. This research is managed to investigate the pharmacodynamics material basis on removing blood stasis of EZhu by exploring the spectrum-effect relationship between UPLC-Q/TOF-MS fingerprints and pharmacologic actions. Hemorheology and related functional parameters were detected to evaluate the pharmacologic actions of EZhu. Relative content Changes of components in rat plasma were detected by UPLC-Q/TOF-MS. A compound-target-pathway network was built to predict the pharmacological activity of components in plasma. Then, bivariate correlation analysis (BCA) was used to explore the correlation degree between components in plasma and pharmacologic actions of EZhu. In UPLC-Q/TOF-MS fingerprints of rat plasma, 10 prototype components were identified. BCA results show that 8 components were concerned with the pharmacological activity for treating blood stasis syndrome (BSS) in varying degrees (R > 0.5, P < 0.05). Among them, zedoarofuran and curzerenone have shown correlation with more pharmacological indicators. The network predicted that 80 targets were closely related to 10 components, in which 48 targets were connected with 159 metabolic pathways including arachidonic acid metabolism, sphingolipid signaling pathway, and linoleic acid metabolism. Overall, this study provided a scientific basis for TCM quality control to ensure its safety and efficacy.

UPLC-MS/MS Method for the Simultaneous Quantification of Eight Compounds in Rat Plasma and Its Application to a Pharmacokinetic Study after Oral Administration of Veratrum (Veratrum nigrum L.) Extract.

Veratrum nigrum L. is a well-known traditional Chinese medicine with a lot of pharmacological activities including antihypertensive, anticancer, and antifungal effects. In the current experiment, a rapid and sensitive UPLC-MS/MS method that takes only 7 min run time has been established and validated for simultaneous determination of eight bioactive compounds including cyclopamine, jervine, veratramine, polydatin, quercetin, apigenin, resveratrol, and veratrosine in rat plasma. The chromatographic separation of analytes and internal standard was performed on a Phenyl-Hexyl column (2.1 × 100 mm, 1.7 µm) with the mobile phase consisting of water (0.1% formic acid) and acetonitrile at a flow rate of 0.3 mL/min. An electrospray ionization (ESI) source was used to detect the samples in both positive and negative ion modes. The intra- and interday precisions of the compounds were less than 9.5% and the accuracy ranged from -10.8% to 10.4%. The extraction recoveries of the compounds were in the range of 85.1 ± 1.5% to 102.6 ± 8.0%, and the matrix effect ranged from 91.2 ± 4.5% to 113.8 ± 1.5%. According to the results of the stability test, the eight compounds have good stability under various conditions and the relative standard deviation (RSD) less than 13.2%. The pharmacokinetic parameters of the eight compounds in rat plasma after oral administration of Veratrum nigrum L. extract were successfully determined by the established UPLC-MS/MS method.

[Simultaneous rapid determination of 84 toxic plant constituents in plasma and urine by ultra-performance liquid chromatography- triple quadrupole/linear ion trap mass spectrometry].

An ultra-performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry (UPLC-Qtrap MS) method was developed for the determination of 84 toxic plant constiuents in plasma and urine. Plasma was precipitated by acetonitrile to remove proteins and then passed through a Prime HLB SPE column to remove phospholipids, while urine was diluted with methanol. Chromatographic separation of the analytes was achieved on an Acquity BEH C18 column (100 mm×2.1 mm, 1.7 µm) by gradient elution using the mobile phase of 0.1% (v/v) formic acid and 2 mmol/L ammonium formate both in 97% (v/v) acetonitrile aqueous solution and water. Electrospray ionization mass spectrometry was carried out in the positive ion mode with multiple reaction monitoring-information dependent acquisition-enhanced product ion scan mode (MRM-IDA-EPI). The 84 analytes were quantified by the matrix working standard curve internal standard method, and a good linear relationship was observed, with correlation coefficients of ≥ 0.9911. The limits of detection (LODs) in plasma and urine were 0.01-1 µg/L and 0.03-2 µg/L, respectively. The intra- and inter-day precisions of these analytes were 0.7%-18.4% and 1.1%-18.5%, and the accuracy of all analytes ranged from 70.6% to 124.5%. This method is simple, sensitive, and accurate for the measurement of these analytes in plasma and urine for both clinical and forensic applications.

Hyphenated chromatography detection and compound-target-disease investigation on herb-pair Chuanxiong Rhizoma - Xiangfu Rhizoma.

ETHNOPHARMACOLOGY RELEVANCE: The combination of Chuanxiong Rhizoma (Ligusticum chuanxiong Hort., umbelliferae) with Xiangfu Rhizoma (the rhizoma of Cyperus rotundus L., Cyperaceae), is deemed as CR-XR herb-pair (Yaodui) in China. Their compatible mechanism needs a further research using modern analytical techniques and bioinformatic tool. METHODS: Head Space- Solid Phase Micro Extraction coupled with Gas Chromatography/Mass Spectrometer detection (HS-SPME-GC/MS) and Liquid Chromatography coupled to quadrupole Time of Flight - Mass Spectrometry (LC-qTOF-MS) were applied in an accurate identification of the absorbed phytochemicals in mice serum; Their potential targets were available after compound-protein interaction (CPI) prediction and molecular docking verification; Then the corresponding disease types, as well as the relevant Traditional Chinese Medicine (Zhongyi) syndromes (Zheng), were matched from databases and references. RESULTS: Resolution from hyphenated chromatographic datasets, thirty-eight phytochemicals were detected in serum samples from mice. Seventy potential target proteins were thereby found through a bioinformatic calculation, which mainly focused on circulatory, endocrine and nervous diseases in Western medicine, also related with Qizhi and Xueyu Zheng from the perspective of Zhongyi. Part of the relationships among compound-Target-Disease have been confirmed by literatures. These virtual data were sketched out as 'The active Compound - potential Target' network, 'Target - Disease' network and 'Target - Zhongyi Disease' network, in which the network topology was used to analyze them. CONCLUSIONS: Our work successfully explained the compatible mechanism of CR-XR Yaodui, which exert 'multi-components, multi targets' in treating Qizhi and Xueyu Zheng.