Quality evaluation of Lonicerae Japonicae Flos and Lonicerae Flos based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis.

INTRODUCTION: Lonicerae Japonicae Flos (LJF) and Lonicerae Flos (LF) belong to different genera of Caprifoliaceae. They have been historically utilised as herbal medicine to treat various diseases. However, the comprehensive assessment of them still remains a challenge. OBJECTIVE: To develop a comprehensive method of ultra-fast liquid chromatography-tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) coupled with multivariate statistical analysis for the quality evaluation and reveal differential components of LJF and LF. METHODOLOGY: A validated UFLC-QTRAP-MS/MS method was established for simultaneous determination of 50 constituents, including 12 organic acids, 12 flavonoids, 6 iridoids, 3 saponins, 13 amino acids and 4 nucleosides. The obtained data were employed to multivariate statistical analysis. Principal component anlysis (PCA) and partial least squares determinant analysis (PLS-DA) were performed to classify and reveal differential components of samples; grey relational analysis (GRA) was introduced to assess the samples according to the contents of 50 constituents by calculating the relative correlation degree of each sample. RESULTS: Fifty constituents were simultaneously determined of LJF and LF. Based on obtained data, PCA and PLS-DA were easy to distinguish samples and the classification of the samples was related to 11 chemical constituents. GRA implied the quality of LJF was better, and that the flower buds were superior to the flowers. Moreover, organic acids are the main components of samples. CONCLUSION: This study not only established a method of simultaneous determination of multiple bioactive constituents in LJF and LF, but provided comprehensive information on the quality control of them. The developed method is conducive to distinguish orthologues or paralogues of them, and supply the support for "heterologous effects".

Anti-depressant effect of Zhi-zi-chi decoction on CUMS mice and elucidation of its signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhi-zi-chi decoction (ZZCD) is used for treating depression as an effectively traditional Chinese medicine. Until now, studies on pharmacological research of ZZCD have mostly been centered in pharmacokinetic level. Little was known about its pharmacological mechanism of relieving depression. AIM OF THE STUDY: This study was to evaluate the effect of ZZCD on relieving depression via behavioral tests, serum metabolomics and signaling target expression analysis on chronic unpredictable mild stress (CUMS) model mice. MATERIALS AND METHODS: The CUMS exposure lasted 7 consecutive weeks. The mice were administrated with ZZCD for the last 3 weeks. Behavioral tests were applied and a serum metabolomics method based on UFLC/Q-TOF-MS with multivariate statistical and global metabolic network analysis was performed to identify relevant metabolites and pathways. Finally, the protein expressions in mouse hippocampi were determined by western blot to verify the metabolomics deduction. RESULTS: Behavioral parameters were visibly changed after modeling, while high and medium dosage groups showed status improvement compared to the model group. Seventy six metabolites were identified as potential biomarkers from the metabolomics profiles in C18 and HILIC systems. In addition, 9 significant pathways related to changed biomarkers were conducted. The pathways were closely connected by some key targets, which were significantly reduced in the model group compared with those in control group, while ZZCD treated groups showed corrections after 3-week administration. The results revealed that the anti-depression efficacy of ZZCD might be associated with PKA-CREB-BDNF-TrkB-PSD-95 pathway influenced by metabolic changes, verifying the pathway annotation speculation. CONCLUSION: This study demonstrated that ZZCD had a positive treatment effect on CUMS depression model mice. Metabolomics results revealed the holistic and interconnected metabolic changes of ZZCD in CUMS mice. The metabolic pathway annotation suggested that the anti-depression mechanism of ZZCD might be related to signaling pathway in brain. PKA-CREB-BDNF-TrkB-PSD-95 signaling expression was a verification and complement to the metabolomics results.

Comprehensive Comparison of Two Color Varieties of Perillae Folium Using Rapid Resolution Liquid Chromatography Coupled with Quadruple-Time-of-Flight Mass Spectrometry (RRLC-Q/TOF-MS)-Based Metabolic Profile and in Vivo/in Vitro Anti-Oxidative Activity.

Perillae Folium (PF), which is extensively used as a dietary vegetable and medicinal herb, contains two varietal forms corresponding to purple perilla leaf (Perilla frutescens var. crispa) and green perilla leaf (Perilla frutescens var. frutescens). However, the components and efficacy of different PF varieties remain underexplored so far. In the present work, a nontargeted rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q/TOF-MS)-based metabolomics approach was developed to investigate the difference in the chemical compositions between green PF and purple PF. A total of 71 compounds were identified or tentatively identified, among which 7 phenolic acids, 10 flavonoids, and 9 anthocyanins were characterized as differential metabolites. In addition, heatmap visualization and ultraperformance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-TQ-MS/MS)-based quantitative analysis revealed that flavonoids and anthocyanins especially had higher contents in purple PF. Furthermore, the anti-oxidative activities of two varietal PFs were evaluated in vivo zebrafish and in vitro human umbilical vein endothelial cells (HUVECs). The results showed that the purple PF had more pronounced anti-oxidative activities than did the green PF, which may be due to the presence of anthocyanins and a higher concentration of flavonoids in its phytochemical profile. The outcome of the present study is expected to provide useful insight on the comprehensive utilization of a PF resource.

Distribution Patterns for Bioactive Constituents in Pericarp, Stalk and Seed of Forsythiae Fructus.

Forsythiae Fructus (FF) is a widely used folk medicine in China, Japan, and Korea. The distribution of bioactive constituents throughout the fruit segments has rarely been addressed, although mounting evidence suggests that plant secondary metabolites are synthesized and distributed regularly. The phytochemical profiles of three segments of FF (pericarp, stalk and seed) were firstly revealed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitative analysis of twenty-one bioactive constituents, including three phenylethanoid glycosides, five lignans, eight flavonoids, and five phenolic acids to explore the spatial distribution of bioactive constituents. Furthermore, the hierarchical clustering analysis (HCA) and one-way analysis of variance (one-way ANOVA) were conducted to visualize and verify the distribution regularity of twenty-one analytes among three segments. The results showed that phytochemical profiles of the three segments were similar, i.e., phenylethanoid glycosides covering the most part were the predominant compounds, followed by lignans, flavonoids and phenolic acids. Nevertheless, the abundance of twenty-one bioactive constituents among three segments was different. Specifically, phenylethanoid glycosides were highly expressed in the seed; lignans were primarily enriched in the stalk; flavonoids were largely concentrated in the pericarp, while the contents of phenolic acids showed no much difference among various segments. The research improves our understanding of distribution patterns for bioactive constituents in FF, and also complements some scientific data for further exploring the quality formation mechanism of FF.

Beta-elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation.

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta-elemene (ß-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated that ß-elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. ß-Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that ß-elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of ß-elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose-1,6-bisphosphate (FBP) and L-cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and ß-elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti-metastatic agent from natural compounds.

LC-Q-TOF/MS-oriented systemic metabolism study of pedunculoside with in vitro and in vivo biotransformation.

As a triterpene saponin, pedunculoside is one of the most abundant, representative and active components in plants of genus Ilex (Aquifoliaceae). Pedunculoside has been used to treat myocardial ischemia, ameliorate hyperlipidemia and prevent liver injury. In this paper, a systemic in vitro liver microsomes / S9 and intestinal bacteria incubation, and in vivo animal experiment were performed, using LC-Q-TOF/MS analysis and a three-step data processing protocol. As a result, Bifidobacterium adolescentis and Bifidobacterium breve were identified to potentially metabolize pedunculoside among the intestinal bacteria tested. A total of 11 metabolites were found and tentatively identified, with 6 in both microsomal and bacterial incubation systems, and 9 after rats orally administered with pedunculoside. The metabolites detected involving both phase I and phase II metabolism, mainly through deglycosylation (hydrolyzation), dehydrogenation, hydroxylation and conjugation, and some of them underwent more than one-step metabolic reactions. Most of the metabolites have not been reported before. In vitro, liver microsome and intestinal bacteria prefer to metabolize pedunculoside in totally different ways; while in vivo, intestinal tract is the most important site for the metabolism and excretion of pedunculoside, where both intestinal bacteria and the host metabolic enzymes participate in its metabolism and disposition. The importance of intestinal bacteria should be highlighted. This study would contribute to a better understanding of pedunculoside metabolism, which can provide scientific evidence for its pharmacodynamic mechanism research and prove its clinical application.

A Method to Study the Distribution Patterns for Metabolites in Xylem and Phloem of Spatholobi Caulis.

Spatholobi Caulis (SC), the vine stem of Spatholobus suberectus Dunn, is a widely used traditional Chinese medicine (TCM) for the treatment of blood stasis syndrome and related diseases. Xylem and phloem are the main structures of SC and the color of xylem in SC is red brown or brown while the phloem with resin secretions is reddish brown to dark brown. They are alternately arranged in a plurality of concentric or eccentric rings. In order to investigate the distribution patterns of metabolites in xylem and phloem of SC, an analytical method based on UFLC-QTRAP-MS/MS was established for simultaneous determination of 22 constituents including four flavanols, nine isoflavones, two flavonols, two dihydroflavones, one flavanonol, one chalcone, one pterocarpan, one anthocyanidin and one phenolic acid in the samples (xylem and phloem) from Laos. Furthermore, according to the contents of 22 constituents, heat map, principal components analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and t-test were used to evaluate the samples and discover the differences between xylem and phloem of SC. The results indicated that the measured ingredients in xylem and phloem were significantly different. To be specific, the contents of flavonoids in xylem were higher than that in phloem, while the content of protocatechuic acid showed a contrary tendency. This study will not only reveal the distribution patterns of metabolites in xylem and phloem of SC but also facilitate further study on their quality formation.

Simultaneous Determination of Quercitrin, Afzelin, Amentoflavone, Hinokiflavone in Rat Plasma by UFLC-MS-MS and Its Application to the Pharmacokinetics of Platycladus orientalis Leaves Extract.

Leaves of Platycladus orientalis have been used as blood cooling and homeostatic therapy for thousands of years in traditional Chinese medicine. Emerging evidences of modern pharmacology have proved flavonoids as the key elements responsible for the efficacies. However, there has been no report on pharmacokinetic study of the flavonoids from Platycladus orientalis leaves extract. In this study, a sensitive and rapid ultra-flow liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous determination of amentoflavone, afzelin, hinokiflavone and quercitrin in rat plasma. The four flavonoids and luteolin (internal standard, IS) were recovered from rat plasma by methanol-ethyl acetate (v:v, 50:50). Chromatographic separation was performed on a C18 column with gradient elution. Our results showed that the recoveries from spiked control samples were more than 85% for all analytes and IS. The relative standard deviations of intra-day and inter-day precision were within 15% while the REs ranged from -6.6% to 8.0%. The validated method in this study was successfully applied to pharmacokinetic study in healthy rats after oral administration of P. orientalis leaves extract.

Comparison of Multiple Bioactive Constituents in Different Parts of Eucommia ulmoides Based on UFLC-QTRAP-MS/MS Combined with PCA.

Eucommia ulmoides Oilv. (EU), also called Du-zhong, is a classical traditional Chinese medicine. Its bark, leaf, and male flower are all used for medicinal purposes, called Eucommiae Cortex (EC), Eucommiae Folium (EF), and Eucommiae Flos Male (EFM). In order to study the difference in synthesis and the accumulation of metabolites in different parts of EU, a reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was developed for the simultaneous determination of a total of 21 constituents, including two lignans, 6 iridoids, 6 penylpropanoids, 6 flavonoids, and one phenol in the samples (EC, EF, and EFM). Furthermore, principal component analysis (PCA) was performed to evaluate and classify the samples according to the contents of these 21 constituents. All of the results demonstrated that the chemical compositions in EC, EF, and EFM were significantly different and the differential constituents (i.e., aucubin, geniposidic acid, chlorogenic acid, pinoresinol-di-O-ß-d-glucopyranoside, geniposide, cryptochlorogenic acid, rutin, and quercetin) were remarkably associated with sample classifications. The research will provide the basic information for revealing the laws of metabolite accumulation in EC, EF, and EFM from the same origin.

Quality Evaluation of Apocyni Veneti Folium from Different Habitats and Commercial Herbs Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Apocyni Veneti Folium (AVF) is a kind of staple traditional Chinese medicine with vast clinical consumption because of its positive effects. However, due to the habitats and adulterants, its quality is uneven. To control the quality of this medicinal herb, in this study, the quality of AVF was evaluated based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis. A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was developed for the simultaneous determination of a total of 43 constituents, including 15 flavonoids, 6 organic acids, 13 amino acids, and 9 nucleosides in 41 Luobumaye samples from different habitats and commercial herbs. Furthermore, according to the contents of these 43 constituents, principal component analysis (PCA) was employed to classify and distinguish between AVF and its adulterants, leaves of Poacynum hendersonii (PHF), and gray relational analysis (GRA) was performed to evaluate the quality of the samples. The proposed method was successfully applied to the comprehensive quality evaluation of AVF, and all results demonstrated that the quality of AVF was higher than the PHF. This study will provide comprehensive information necessary for the quality control of AVF.

A comprehensive in vitro and in vivo metabolism study of hydroxysafflor yellow A.

As the most important marker component in Carthamus tinctorius L., hydroxysafflor yellow A (HSYA) was widely used in the prevention and treatment of cardiovascular diseases, due to its effect of improving blood supply, suppressing oxidative stress, and protecting against ischemia/reperfusion. In this paper, both an in vitro microsomal incubation and an in vivo animal experiment were conducted, along with an LC-Q-TOF/MS instrument and a 3-step protocol, to further explore the metabolism of HSYA. As a result, a total of 10 metabolites were searched and tentatively identified in plasma, urine, and feces after intravenous administration of HSYA to male rats, although no obvious biotransformation was found in the simulated rat liver microsomal system. The metabolites detected involving both phase I and phase II metabolism including dehydration, deglycosylation, methylation, and glucuronic acid conjugation. A few of the metabolites underwent more than one-step metabolic reactions, and some have not been reported before. The study would contribute to a further understanding of the metabolism of HSYA and provide scientific evidence for its pharmacodynamic mechanism research and clinical use.

Quality evaluation of Eucommiae Cortex processed by different methods and "sweating" conditions based on simultaneous determination of multiple bioactive constituents combined with gray relational analysis.

Eucommiae Cortex is a classical traditional Chinese medicine, which needs to be processed by "sweating" methods. To select the suitable processing method and "sweating" processing condition for Eucommiae Cortex, in this study, the quality of Eucommiae Cortex was evaluated based on simultaneous determination of multiple bioactive constituents combined with gray relational analysis. The contents of lignans, iridoids, penylpropanoids, flavonoids, and phenols in samples were simultaneously determined using ultra-fast performance liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry. The chromatographic separation was performed on a Synergiۛ Hydro-RP 100 Å column (100 mm × 2.0 mm, 2.5 µm) at 30°C with a gradient elution of acetonitrile with 0.1% formic acid/0.1% aqueous formic acid as the mobile phase. Furthermore, gray relational analysis was performed to evaluate and sort the samples according to the contents of 14 constituents by calculating the relative correlation degree of each sample. The results demonstrated that the quality of Eucommiae Cortex "sweating" at source area was better and the better "sweating" condition was to scrape off the cork layer before "sweating" with straw covering and sun drying. The developed method could provide the foundation and support for "sweating" processing method of Eucommiae Cortex in normalization and standardization.

[Difference of chemical constituents in Eucommiae Cortex from different habitats by LC-QTOF MS/MS].

In order to study the influence of ecological environment regarding the synthesis and accumulation of metabolites in Eucommiae Cortex, LC-QTOF MS/MS method combined with multivariate statistical analysis was used to analyze the differences of chemical constituents in Eucommiae Cortex from different habitats. Through the analysis of the multistage tandem mass spectrometry, the characteristic peaks were extracted with mass spectrometry data peak matching, peak alignment, and noise filtering. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for data processing. The chemical constituents were identified or tentative presumed according to MS accurate mass and MS/MS spectrometry fragmentation information, combined with the software of database search, comparison with reference standards and literature. The results show the differences among samples of Eucommiae Cortex from different habitats are distinguishable. A total of 23 chemical constituents in Eucommiae Cortex were identified or tentative presumed. Among of them, 14 kinds of common differential chemical constituents (aucubin, geniposidic acid, neochlorogenic acid, syringin, olivil-4',4'-di-O-ß-D-glucopyranoside, chlorogenic acid, cryptochlorogenic acid, 1-hydroxypinoresinol- 4',4'-di-O-ß-D-glucopyranoside, caffeic acid, pinoresinol-di-O-ß-D-glucopyranoside, syringaresionl-di-O-ß-D-glucopyranoside, pinoresinol-4'-O-ß-D-glucopyranoside, eucommiol, isochlorogenic acid C and asiatic acid) presented different changing laws. This study provides basic information for revealing the influence law of ecological environment on the biosynthesis of metabolites in Eucommiae Cortex.

Untargeted serum metabolomics reveals Fu-Zhu-Jiang-Tang tablet and its optimal combination improve an impaired glucose and lipid metabolism in type II diabetic rats.

Fu-Zhu-Jiang-Tang tablet, a six-herb preparation, was proved to show beneficial effects on type II diabetes patients in clinical. This study aims to optimize the component proportion of the six-herb preparation and explore the serum metabolic signatures of type II diabetes rats after treatment with Fu-Zhu-Jiang-Tang tablet and its optimal combination. The component proportion of the preparation was optimized using uniform experimental design and machine learning techniques. Untargeted GC-MS metabolomic experiments were carried out with serum samples from model group and treatment groups. Data were normalized, multivariate and univariate statistical analysis performed and metabolites of interest putatively identified. 23 metabolites were significantly changed by Fu-Zhu-Jiang-Tang tablet treatment and the majority of these were decreased, including various carbohydrates (glucose, mannose, fructose, allose and gluconic acid), unsaturated fatty acids (palmitic acid, 9-octadecenoic acid, oleic acid, arachidonic acid), alanine, valine, propanoic acid, 3-hydroxybutyrate, along with pyrimidine and cholesterol. Increased concentrations of oxalic acid, leucine, glycine, serine, threonine, proline, lysine and citrate were observed. In the optimal combination-fed group, 21 metabolites were significantly affected and strikingly, the magnitudes of changes here were generally much greater than that of Fu-Zhu-Jiang-Tang tablet treated rats. 18 metabolites affected in both groups included various carbohydrates (mannose, glucose, allose, fructose and gluconic acid), unsaturated fatty acids (palmitic acid, 9-octadecenoic acid, oleic acid and arachidonic acid), short-chain fatty acids (oxalic acid, 3-hydroxybutyrate), and amino acids (alanine, valine, leucine, glycine, proline and lysine), as well as pyrimidine. Metabolites exclusively affected in optimal combination treated rat included succinic acid, cysteine and phenylalanine, whilst four metabolites (propanoic acid, citrate, serine and threonine) were only altered in Fu-Zhu-Jiang-Tang tablet treated rat. Our investigation demonstrated Fu-Zhu-Jiang-Tang tablet and its optimal combination treatments were able to ameliorate impaired glucose and lipid metabolism, down- regulate the high level of glucose to a lower level and reverse abnormal levels of metabolites in serum of type II diabetes rats. However, the optimal combination treatment was able to maximize the magnitudes of changes in some metabolites. These findings may be helpful in clarifying the anti-diabetic mechanism of FZJT tablet and its optimal combination.

Quality Evaluation of Pseudostellariae Radix Based on Simultaneous Determination of Multiple Bioactive Components Combined with Grey Relational Analysis.

Pseudostellariae Radix (PR) is an important traditional Chinese herbal medicine (TCM) with vast clinical consumption because of its positive effects. However, little attention has been devoted to simultaneous analysis of its bioactive components for quality control of PR based on its different harvesting times, different growing habitats, and different processing methods. In this research, the quality of PR was evaluated based on simultaneous determination of multiple bioactive components combined with grey relational analysis (GRA). A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established to simultaneously determine the contents of 30 components in PR, including two cyclopeptides, 12 nucleosides, and 16 amino acids. Furthermore, grey relational analysis was performed to evaluate the quality of PR samples according to the contents of these 30 components. The results showed that the quality of PR harvested in 6 August 2013, cultivated in Jurong, Jiangsu, and treated by oven drying 60 °C was better than that of other PR samples. The proposed method is useful for the overall assessment on the quality of PR, and this study provides valuable information for revealing the dynamic change laws of metabolite accumulation in PR and choosing the most suitable harvesting time and reasonable processing method of PR to obtain the best quality.

Peperomin E reactivates silenced tumor suppressor genes in lung cancer cells by inhibition of DNA methyltransferase.

Advanced lung cancer has poor prognosis owing to its low sensitivity to current chemotherapy agents. Therefore, discovery of new therapeutic agents is urgently needed. In this study, we investigated the antitumor effects of peperomin E, a secolignan isolated from Peperomia dindygulensis, a frequently used Chinese folk medicine for lung cancer treatment. The results indicate that peperomin E has antiproliferative effects, promoting apoptosis and cell cycle arrest in non-small-cell lung cancer (NSCLC) cell lines in a dose-dependent manner, while showing lower toxicity against normal human lung epidermal cells. Peperomin E inhibited tumor growth in A549 xenograft BALB/c nude mice without significant secondary adverse effects, indicating that it may be safely used to treat NSCLC. Furthermore, the mechanisms underlying the anticancer effects of peperomin E have been investigated. Using an in silico target fishing method, we observed that peperomin E directly interacts with the active domain of DNA methyltransferase 1 (DNMT1), potentially affecting its genome methylation activity. Subsequent experiments verified that peperomin E decreased DNMT1 activity and expression, thereby decreasing global methylation and reactivating the epigenetically silenced tumor suppressor genes including RASSF1A, APC, RUNX3, and p16INK4, which in turn activates their mediated pro-apoptotic and cell cycle regulatory signaling pathways in lung cancer cells. The observations herein report for the first time that peperomin E is a potential chemotherapeutic agent for NSCLC. The anticancer effects of peperomin E may be partly attributable to its ability to demethylate and reactivate methylation-silenced tumor suppressor genes through direct inhibition of the activity and expression of DNMT1.

Simultaneous determination of ten alkaloids of crude and wine-processed Rhizoma Coptidis aqueous extracts in rat plasma by UHPLC-ESI-MS/MS and its application to a comparative pharmacokinetic study.

Rhizoma coptidis (R.C.), a widely used traditional Chinese medicine, has been used for centuries in the treatment of hypertension, inflammation, dysentery and liver diseases, etc. Wine-processing is a specialized technology by sautéing crude herbal medicine using Chinese rice wine. This paper was designed to establish a simultaneous quantitative method of ten alkaloids (berberine, coptisine, palmatine, jatrorrhizine, epiberberine, magnoflorine, columbamine, noroxyhydrastinine, oxyberberine and 8-oxocoptisine) in rat plasma. Furthermore, the pharmacokinetics of those alkaloids after administration of crude and wine-processed R.C. aqueous extracts was compared. As a result, a ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method was developed and validated for the first time. Chromatographic separation was achieved on a C18 column using gradient elution with the mobile phase consisting of acetonitrile and water (containing 0.2% formic acid) at a flow rate of 0.2 ml/min. The validated method showed good linearity over a wide concentration range (r>0.99), and lower limits of quantification less than 5.46 ng/ml for the each analyte. The intra- and inter-day assay variability was below 9.9% and 10.5% for all analytes, respectively. The extraction recovery of those alkaloids and I.S. ranged from 65.3% to 90.7%. The validated method has been successfully applied to pharmacokinetic comparison after administration of crude and wine-processed R.C. aqueous extracts. Pharmacokinetic comparative study showed that Cmax of coptisine and 8-oxocoptisine and AUC0-t of coptisine, palmatine and 8-oxocoptisine were increased significantly (p<0.05) after wine-processing, while other compounds didn't show significant difference, which suggested that wine-processing exerted limited effects on the absorption of alkaloids. These results might be helpful for R.C.' clinical reasonable application and further studies on its wine-processing mechanism.

Effects of food and gender on the pharmacokinetics of ginkgolides A, B, C and bilobalide in rats after oral dosing with ginkgo terpene lactones extract.

The ginkgo terpene lactones (GTL), mainly including bilobalide (BB), ginkgolide A (GA), ginkgolide B (GB) and ginkgolide C (GC) possess different biological activities such as peripheral vasoregulation, platelet-activating factor (PAF) receptor antagonism, neuroprotective properties and prevention of membrane damage caused by free radicals. To investigate the effects of food and gender on the bioavailability of BB, GA, GB and GC after oral administration of GTL extract, a rapid UPLC-MS/MS method was developed and validated. A reversed phase C18 column (100mm×2.1mm, i.d., 1.7µm) and a mobile phase consisted of methanol and 1mM ammonium acetate (70/30, v/v) were employed. Compared with the fasted group, the t1/2 values for BB, GA, GB and GC in fed were all increased (p<0.05), AUC0-t and AUC0-∞ values of BB, GA, GB and GC were all significantly increased (p<0.05), but the Cmax values of BB, GA, GB and GC were significantly decreased (p<0.05). In comparison with the male group, all of the t1/2 values and AUC0-t values for BB, GA, GB and GC in female were higher (p<0.05), but no statistical difference in Tmax values for BB, GA, GB and GC between these two groups. Food and gender factor showed significant effects on the pharmacokinetics of BB, GA, GB, and GC. The results suggested that oral doses of GTL should be lowered for fasted and female subjects, compared with the fed and male subjects, respectively.

The effects of wine-processing on ascending and descending: The distribution of flavonoids in rat tissues after oral administration of crude and wine-processed Radix scutellariae.

ETHNOPHARMACOLOGICAL RELEVANCE: Ascending and descending theory is a core principle of traditional Chinese medicine (TCM) theories. It plays an essential role in TCM clinical applications. Some TCM medicine has specific properties, which could alter the inclination and direction of their actions. The properties of the ascending and floating process of one herbal medicine are affected by means of herb processing. Wine-processing, which is sautéing with rice wine, is one of the most popular technologies of herb processing. Wine-processing increases the inclination and direction of its actions, thereby producing or strengthening their efficacy in cleaning the upper-energizer heat. Radix scutellariae, the dried roots of Scutellaria baicalensis Georgi, is a well-known TCM used for the treatment of inflammation, pyrexia, jaundice, etc. Recently, wine-processed Radix scutellariae was normally applied in clinical studies for the treatment of upper-energizer syndrome. In order to investigate the effects of wine-processing on ascending and descending of Radix scutellariae, the comparative study of distribution of flavonoids in rat tissues of triple energizers (SanJiao-upper, middle, lower jiao) after oral administration of crude and wine-processed Radix scutellariae aqueous extracts was carried out. MATERIALS AND METHODS: The rats were randomly assigned to two groups and orally administered with crude and wine-processed Radix scutellariae aqueous extracts, respectively. At different pre-determined time points after administration, the concentrations of compounds in rat tissue homogenate were determined, and the main tissue pharmacokinetic parameters were investigated. Tissue pharmacokinetic parameters including AUC0-t, t1/2, Tmax and Cmax were calculated using DAS 2.0. An unpaired Student t-test was used to compare the differences in tissue pharmacokinetic parameters between the two groups. All the results were expressed as arithmetic mean±S.D. RESULTS: The parameters of Cmax and AUC0-t of some flavonoids in wine-processed Radix scutellariae were remarkably increased (p<0.05, p<0.01, p<0.001) in the rat upper-energizer tissues (lung and heart) compared with those of the crude group. However, in the rat middle- and lower-energizer tissues (spleen, liver and kidney), the Cmax and AUC0-t of some flavonoids were significantly decreased (p<0.05, p<0.01) compared with the crude group. The main explanation for these differences seems to the effects of wine-processing on ascending and descending theory. CONCLUSIONS: All of these differences in the distribution of triple energizers after oral administration of crude and wine-processed Radix scutellariae aqueous extracts may lead to the increase of efficacy on the upper-energizer tissues and were in compliance with the ascending and descending theory. Therefore, wine-processing was recommended when Radix scutellariae was used for cleaning the upper-energizer heat and humidity. The obtained knowledge can be used to evaluate the impact of these differences on the efficacy of both the drugs in clinical applications and might be helpful in explaining the effects of wine-processing on ascending and descending theory.

Effects of storage period and heat treatment on phenolic compound composition in dried Citrus peels (Chenpi) and discrimination of Chenpi with different storage periods through targeted metabolomic study using HPLC-DAD analysis.

Dried Citrus peels, known as Chenpi in Chinese medicine, are a traditional medicine for the treatment of indigestion and inflammatory syndromes. In this study, we evaluated the effect of storage periods (1-year vs. 3-year) and heat treatment (90min vs. 3h at 120°C) on the total phenolic content (TPC) and bioactivity (anti-oxidant activity) of Chenpi. It was found that the long-term stored Chenpi had a higher TPC and superior 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity levels compared to the regular stored Chenpi, and that heat treatment increased both TPC and bioactivity. Subsequently, we developed and validated a high performance liquid chromatography with diode-array detection (HPLC-DAD) method to determine individual phenolic acids. Eleven phenolic compounds were determined in different Chenpi samples. Concentrations of total phenolic compounds were higher in long-term stored Chenpi and heat treatment raised the levels of those in regular stored Chenpi. In further study, a targeted metabolomic approach was applied to discriminate Chenpi with different storage periods. Two different phenolic acid fractions (free and ester) from the regular and long-term stored Chenpi were analyzed using the developed HPLC-DAD and the data were used in principal component analysis (PCA) on the HPLC-DAD peak areas of the 11 phenolic acids. Two principal components (PC1 and PC2) accounted for 87.1% of the variation between the regular and long-term stored Chenpi. In a two-dimensional plot of PC1 and PC2, the samples divided into four groups: two storage periods and two fractions.