Quantitative Determination of Quercitrin Levels in Rat Plasma Using UHPLC-MS/MS and its Application in a Pharmacokinetic Study after the Oral Administration of Polygoni cuspidati Folium Capsules.

BACKGROUND: Quercitrin is widely found in herbal medicines, and it is particularly important in the design of new therapeutic agents. Because of its wide range of biological activities, methods for detecting quercitrin and its pharmacokinetics in biological samples must be investigated. OBJECTIVES: To develop and validate a sensitive and reliable ultra-high-performance liquid chromatography- tandem mass spectrometry (UHPLC-MS/MS) method for the quantitative determination of quercitrin levels in rat plasma, and test its application in a pharmacokinetic investigation after the oral administration of Polygoni cuspidati folium capsules (HC). METHODS: First, a rapid analytical method implementing UHPLC-MS/MS for the quantification of quercitrin levels in rat plasma was developed and validated. The analyte and internal standard (IS) tinidazole were extracted from rat plasma via protein precipitation with 800 µL of methanol and 50 µL of 1% formic acid solution. Chromatographic separation was performed using an Agilent ZORBAX C18 column within 4 min. Mass spectrometry was performed for quantification using a triple-quadrupole mass spectrometer employing electrospray ionization in the negative ion mode. The MRM transitions for quercitrin and IS were m/z 447.2→229.9 and m/z 246.0→125.8, respectively. The UHPLC-MS/MS method for the quantitative determination of quercitrin levels in rat plasma was then applied to investigate its pharmacokinetics after the oral administration of HC in rats. RESULTS: The developed UHPLC-MS/MS method for detecting quercitrin in rat plasma was linear over the range of 0.1-160 ng/mL. The linear regression equation was Y = (0.7373 ± 0.0023)X - (0.0087 ± 0.0021) (r2 = 0.9978). The intra- and interday precision values were within 7.8%, and the recoveries of quercitrin and IS exceeding 67.3%. The UHPLC-MS/MS method was successfully applied to characterize the pharmacokinetic profile of quercitrin in eight rats after the oral administration of HC. The experimentally obtained values were fit to a one-compartment, first-order pharmacokinetic model, and they appeared to fit the concentration-time curve. CONCLUSION: Quercitrin was proven to be stable during sample storage, preparation, and the analytical procedures. The pharmacokinetic parameters suggested that quercitrin may be present in the peripheral tissues of rats.

Accelerated crystallization of magnetic 4A-zeolite synthesized from red mud for application in removal of mixed heavy metal ions.

To cope with the increasing environmental issues of red mud, an integrated technological route for its comprehensive utilization was developed through the extraction of valuable components and the synthesis of magnetic 4A-zeolite. To accelerate the crystallization process of the synthesized 4A-zeolite, sodium chloride (NaCl) was innovatively employed under hydrothermal treatment. The effects of various parameters, including mass ratio of red mud/NaOH, alkali fusion temperature, alkali fusion time and molar ratio of NaCl/Al2O3, were systematically investigated. The results showed that approximately 81.0% Al, 76.1% Si and 95.8% Fe were utilized from red mud using alkali fusion and acid leaching methods. The optimal conditions of the alkali fusion process were determined as: mass ratio of red mud/NaOH = 1/2, alkali fusion temperature of 800 °C, and time of 90 min. Furthermore, when the molar ratio of NaCl/Al2O3 was kept at 1.5, the crystallization time reduced from 240 min to 150 min, and particle size distributions narrowed from 20-100 µm to 1-10 µm. The practical applications in removal of mixed heavy metal ions (Zn2+, Cu2+, Cd2+, Ni2+, and Pb2+) from wastewater indicated that the as-synthesized magnetic 4A-zeolite is a promising candidate for heavy metals adsorption.

[Synergistic Effects of Clopidogrel and Xuesaitong Dispersible Tablet by Modulating Plasma Protein Binding].

Objective: The ginsenoside Rb1,which account for platelet aggregation of Xuesaitong dispersible tablet, was selected to investigate the synergistic effects of clopidogrel( CPG) and Xuesaitong dispersible tablet drug by modulating plasma protein binding rate aspect. Methods: The HPLC and equilibrium dialysis were employed to determine the concentration of Rb1 both in dialysate( PBS) and blank plasma from healthy volunteer blood donors. The differences in protein-binding rate between Xuesaitong dispersible tablet alone( the concentration of ginsenoside Rb1 were 5. 0,1. 0,0. 4 µg / m L,respectively) and combined with CPG( each add CPG 2 µg / m L) were then compared. The three-dimensional spatial structure of the blank plasma albumin( HSA) in the subjects was construct by rabbit plasma albumin( PDB ID 3V09) template and evaluated by PRO-CHECK and ERRAT methods. Molecular simulation technique was used to display the competition mechanism with human plasma protein. Results: The protein binding rate of Xuesaitong dispersible tablet alone group in plasma PBS and human plasma at high( the concentration of ginsenoside Rb1 were 5. 0 µg / m L),middle( the concentred of ginsenoside Rb1 were 1. 0 µg / m L) and low( the concentration of ginsenoside Rb1 were 0. 4 µg / m L) concentrations were( 58. 17 ±3. 82) %,( 57. 43 ± 3. 21) %,( 55. 63 ± 3. 42) % respectively. When combined with CPG( each add CPG 2 µg / m L),the protein binding rate value were decline to( 46. 54 ± 3. 35) %,( 49. 25 ± 3. 56) %,( 48. 15 ± 3. 76) %,respectively. The molecular simulation results suggested that the two compounds have competitive synergistic effects with human plasma protein. Conclusion: The present investigation suggestes that there are synergistic effects of CPG and Xuesaitong dispersible tablet by modulating plasma protein binding rate of ginsenoside Rb1.

[Tissue Distribution of Alkaloids from Coptidis Rhizoma in Rats Determined by UPLC-MS].

Objective: To develop an UPLC-MS method for simultaneous determination of alkaloids from Coptidis Rhizoma in rat tissues, and to study the tissue distribution of alkaloids from Coptidis Rhizoma in rats. Methods: The samples were extracted with ethyl acetate, and analyzed by UPLC-MS with acetonitrile-0. 2% formic acid solution in a gradient elution mobile phase, the flow rate was 0. 2m L/min. Tetrahydropalmatine was used as an internal standard. The mass spectrometer was operated in selected reaction monitoring( SIM) mode with positive electrospray ionization, the transition were m/z 191. 904 /118. 973( noroxyhydrastinine), m/z 335. 877 /308. 072( 8-ocoptisine),m/z 351. 94 /294. 554( palmatine chloride),m/z 335. 94 /262. 112( epiberberine), m/z 337. 94 /322. 422( columbamine), m/z 319. 904 /292. 037( coptisine), m/z 355. 977 /192. 036( tetrahydropalmatine),m/z 335. 94 /320. 036( berberine hydrochloride),m/z 351. 94 /321. 995( oxyberberin), m/z 337. 94 /322. 949( jatrorrhizine respectively). Results: Excellent linearity was observed in all alkaloids in their linear range( r & 0. 9901). The RSD of precision of the developed method was less than 15%,and the accuracy and stability were less than ± 15%,the extraction recovery was 72. 1% ~ 82. 9% with RSD less than 15%. Coptisine,epiberberine,berberine,jatrorrhizine,columbamine,palmatine were widely distributed in rat tissues. Noroxyhydrastinine,8-ocoptisine,oxyberberin could only be determined in liver and heart or kidney. Conclusion: The established method is simple and accurate. Satisfactory results are obtained with applying this method to the tissue distribution study of alkaloids from Coptidis Rhizoma.

[The relationship between catechol O-methyltransferase and diseases].

Catechol O-methyltransferase (COMT), one of the endogenous phase II metabolizing enzymes, expressed by chromosome 22. COMT catalyzes the transfer of a methyl group from common methyl donor S-adenosyl-L-methionine(Ado Met or SAM) to one of the catechol hydroxyls. COMT participates in the metabolism of many catechols in vivo, e.g. dopamine, epinephrine, noradrenaline, estradiol. Furthermore COMT also plays important roles in the metabolism of xenobiotic catechols from food and drug. COMT play a critical role in the management of catechols. Metabolism disorders of COMT can cause many diseases or an increased risk of diseases, e.g. Pakinson diseases, schizophrenia, and breast cancer. In this review, we explains the relationship of COMT and related-diseases through expounding disease caused by the COMT metabolic disorders. Finally, we hope that there will be more effective treatments for the COMT metabolism related diseases.

[Computational Pharmacological Study on Clopidogrel Metabolism Enzymes Influenced by Fufang Danshen Dripping Pill].

OBJECTIVE: To investigate the effect of Fufang Danshen Dripping Pill on Clopidogrel metabolism enzymes target such as human liver carboxylesterasel (CES1), cytochrome P450 3A4, CYP450 2C19, CYP450 1A2, and CYP450 2B6, and to interpret the interaction effects. METHODS: The CES1, cytochrome P450 3A4, CYP450 2C19, CYP450 1A2 and CYP450 2B6 which involved in Clopidogrel metabolism were selected at first, the chemical ligand database were created then, and finally the interaction effects between the ligand database and Clopidogrel metabolism target were explored. RESULT: 1 MX1 (CES1), 3NXU (CYP450 3A4), 4GQS (CYP450 2C19), 2HI4 (CYP450 1A2) and 3IBD(CYP450 2B6) as well as THA, RIT, OXU, Chlorzoxazone and CPZ were used as receptors and cutoff for each target respectively. The number of hits with potentially positive activities with metabolism enzymes target from the bioactive compounds in the preparation was 29, 8, 31, 51 and 44, respectively. These computational pharmacological docking studies were in accordance with the referenced cocktail experiment results. CONCLUSION: It is suggested that Fufang Danshen Dripping Pill has inhibitory effects on Clopidogrel metabolism enzymes target such as CES1, Cytochrome P450 3A4, CYP450 2C19, CYP450 1A2 and CYP450 2B6.

[Studies on effects of Achyranthes bidentata on tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in vivo pharmacokinetics].

To study on the effects of Achyranthes bidentata on Tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in rats in vivo pharmacokinetic behaviors, a method for the simultaneous determination of chlorogenic acid, isoliquiritin, harpagoside and liquiritigenin in rat plasma was established by UPLC-MS/MS. The analysis was performed on a waters Acquity BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with the mixture of acetonitrile and 0.1% formic acid/water as mobile phase, and the gradient elution at a flow rate of 0.3 mL x min(-1). The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. It turned out that the analytes of Tongsaimai pellets groups C(max) and AUC(Q-infinity) values were higher than that with A. bidentata group, and the C(max) values of chlorogenic acid had significantly difference (P < 0.05), the AUC(0-infinity) values of chlorogenic acid and glycyrrhizin had significantly difference (P < 0.05); The T(max) and CL values of two groups had no significantly difference. Results showed that the established method was specific, rapid, accurate and sensitive for the studies of Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic, and A. bidentata have varying degrees of effects on Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic behaviors.

[Application of oral micro-carrier drug delivery system in studies on traditional Chinese medicine].

Drug-loading micro-particles are a targeted, positioned and controlled-release drug delivery carrier with a wide application prospect. Various micro-carrier drug delivery systems have their own advantages in promoting absorption, improving stability, targeting and controlled release. Accordingly, it is of far-reaching significance for the studies on micro carrier drug delivery systems to build oral traditional Chinese medicine (TCM) compound micro-carrier drug delivery systems with effective TCM components and effective fractions. This article introduces several features and advantages of oral micro-carrier drug delivery systems, and summarizes their application in the field of TCMs.

In vitro metabolism in Sprague-Dawley rat liver microsomes of forsythoside A in different compositions of Shuang-Huang-Lian.

Shuang-Huang-Lian (SHL), a traditional Chinese formula containing Lonicerae japonicae flos (LJF), Scutellariae radix (SR) and Forsythiae fructus (FF), is commonly used to treat acute upper respiratory tract infection, acute bronchitis and light pneumonia. Forsythoside A is one of the main active ingredients in Forsythiae fructus, a key herb in SHL. In the present study, effects of different compositions in SHL on the in vitro metabolism in Sprague-Dawley rat liver microsomes of forsythoside A were investigated. The observations from Sprague-Dawley rat liver microsomes in the presence of ß-NADPH or UDPGA that forsythoside A may be the substrates of CYP3A4, CYP2C9, CYP1A2, UGT1A6, UGT1A3, UGT1A1 and UGT1A9; Chlorogenic acid may be the substrates of CYP3A4, CYP2C9, CYP1A2, CYP2C19, UGT1A6, UGT1A3 and UGT1A1; Baicalin may be the substrates of CYP3A4, CYP2C19, CYP1A2, UGT1A9, UGT1A1 and UGT1A3; Baicalein may be the substrates of CYP3A4, CYP2E1 and UGT1A6. It was also found that the residue of forsythoside A in SHL, FF+LJF and FF+SR was greatly increased compared with that in FF in Sprague-Dawley rat liver microsomes in the presence of ß-NADPH or UDPGA, which indicated that the metabolism of forsythoside A in SHL may be influenced by chlorogenic acid in LJF acting on the CYP3A4, CYP2C9, CYP1A2, UGT1A6, UGT1A3 and UGT1A1; baicalin in SR acting on the CYP3A4, CYP1A2, UGT1A9, UGT1A1 and UGT1A3; baicalein acting on the CYP3A4 and UGT1A6 respectively.

Intestinal absorption of forsythoside A in different compositions of Shuang-Huang-Lian.

Shuang-Huang-Lian (SHL), a traditional Chinese formula containing Lonicerae japonicae flos (LJF), Scutellariae radix (SR) and Forsythiae fructus (FF), is commonly used to treat acute upper respiratory tract infection, acute bronchitis and light pneumonia. Forsythoside A is one of the main active ingredients in Forsythiae fructus, a key herb in SHL. In the present study, effects of different compositions in SHL on the intestinal absorption of forsythoside A were investigated. The observations from in situ intestinal circulation model showed that A/%(h(-1)) of forsythoside A in FF+LSF, FF+SR and SHL were all reduced greatly compared with that in FF. However, in pharmacokinetics study, C(max) and AUC(0→1440) of forsythoside A all increased and T(1/2) prolonged in SHL, FF+LJF and FF+SR compared with FF. The results indicated that the different compositions of SHL decreased absorption but increased bioavailability of forsythoside A, which may be related to its metabolism inhibited in intestine or liver.

[Intestinal absorption of forsythoside A by rat circulation in situ].

This study is to investigate the effects of concentration, intestinal section, pH, paracellular route, substrate/inhibitor of enzyme (CYP3A) and proteins (P-gp, MRP2, SGL1) on the absorption of forsythoside A. The absorption of three concentrations (2.6, 5.2, and 10.4 microg x mL(-1)) of forsythoside A in different intestinal segments was studied with phenol red as the marker by rat circulation in situ. The results showed that the residue of forsythoside A with different concentrations had little significant difference from that obtained after perfusing via duodenum, jejunum, ileum and colon, which indicated that the absorption of forsythoside A was passive diffusion and had no difference in different segments of rat intestine. The residue of forsythoside A increased to 466.160 and 463.429 microg respectively when cyclosporine (4 microg x mL(-1)) or midazolam (50 micromol x L(-1)) was added to the circulation fluid, which showed significant difference compared to the control group (P < 0.05). Moreover, the residue of forsythoside A showed a tendency of increase with the increase of cyclosporine or midazolam. When digoxin (50 micromol x L(-1)) or EDTA (10 microg x mL(-1)) was added to the circulation fluid, the residue of forsythoside A decreased to 325.110 and 369.888 microg respectively, which showed significant difference as compared to the control group (P < 0.05). Besides, the residue of forsythoside A showed a tendency of reduction with the increase of digoxin or EDTA. However, there is no significant change in the absorption of forsythoside A when the different concentrations of mannitol were added to the circulation fluid. The results above indicated that the absorption of forsythoside A was mainly passive diffusion and involved paracellular route at the same time. In addition, the substrates of P-gp or CYP3A had dose-dependent effect on the absorption of forsythoside A.

[Research on the quality changes in pre- and post-processed pieces of Eucommia ulmoides].

OBJECTIVE: To study the quality changes in pre- and post-processed pieces of Eucommia ulmoides Oliv. METHODS: The changes of the content of pinoresinol diglucoside, extract and fingerprint were studied. RESULTS: Pinoresinol diglucoside contents in post-processed pieces were lower than those in pre-processed and alcohol extract had different changes because of its different habitats. Eucommia ulmoides consists of 11 common peaks, the one processed by salt-water consists of 8 Peaks. CONCLUSION: Processing can reduce the content of pinoresinol diglucoside. Alcohol extract has different changes. Eucommia ulmoides common peaks of its fingerprint reduce and mostly components descend after processed by salt-water.

[Screening and application of enhancer-like sequences from vaccinia virus].

OBJECTIVE: To screen enhancer-like sequences from vaccinia virus genome, to construct an expression vector harboring prokaryotic enhancer-like sequence and study the effect of interferon gene expression. METHODS: Enhancer-like element from vaccinia virus genome was obtained by using the chloramphenicol acetyl-transferase cat gene as reporter gene. An expression vector harboring prokaryotic enhancer-like sequence VV1 from vaccinia virus was constructed. Interferon was expressed and assayed. RESULTS: Eighteen enhancing sequences were found. From them two enhancer-like sequences with distance and orientation independence property were screened and named VV1 and VV16 respectively. Quantification test showed that the direct and reverse orientation of VV1 could increase the activity of beta-galactosidase with 10.9 and 3.8 times and those of VV16 could increase by 9.0 times and 4.1 times respectively. The enhancing activity of the element was on transcription level. An expression vector harboring prokaryotic enhancer-like sequence VV1 was constructed. Using this vector the antiviral activity of interferon alpha-2b was increased by 2.6 times in comparison with the original expression plasmid. CONCLUSION: Two enhancer-like sequences were screened from vaccinia virus genome. Interferon gene was highly expressed by using an expression vector harboring enhancer-like sequences.