The regioselective glucuronidation of morphine by dimerized human UGT2B7, 1A1, 1A9 and their allelic variants.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7 is expressed mostly in the human liver, lung and kidney and can transfer endogenous glucuronide group into its substrate and impact the pharmacological effects of several drugs such as estriol, AZT and morphine. UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGT1A1 and UGT1A9, as well as their allelic variants, and then change their enzymatic activities in the process of substrate catalysis. The current study was designed to identify this mechanism using morphine as the substrate of UGT2B7. Single-recombinant allozymes, including UGT2B7*1 (wild type), UGT2B7*71S (A71S, 211G>T), UGT2B7*2 (H268Y, 802C>T), UGT2B7*5 (D398N, 1192G>A), and double-recombinant allozymes formed by the dimerization of UGT1A9*1 (wild type), UGT1A9*2 (C3Y, 8G>A), UGT1A9*3 (M33T, 98T>C), UGT1A9*5 (D256N, 766G>A), UGT1A1 (wild type) with its splice variant UGT1A1b were established and incubated with morphine in vitro. Each sample was analyzed with HPLC-MS/MS. All enzyme kinetic parameters were then measured and analyzed. From the results, the production ratio of its aberrant metabolism and subsequent metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), changes regioselectively. Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant alloyzymes. Compared to UGT2B7*1, UGT2B7*2 singles or doubles have lower Km values for M3G and M6G, whereas UGT2B7*5 allozymes perform opposite effects. The double allozymes of UGT1A9*2 or UGT1A9*5 with UGT2B7 tend to produce M6G. Interestingly, the majority of single or double allozymes significantly reduce the ratio of M3G to M6G. The UGT1A9*2-UGT2B7*1 double enzyme has the lowest M3G:M6G ratio, reflecting that more M6G would form in morphine glucuronide metabolism. This study demonstrates that UGT2B7 common SNPs and their dimers with UGT1A1 and UGT1A9 and their allelic variants can regioselectively affect the generation of two metabolites of morphine via altering the CLint ratios of M3G to M6G. These results may predict the effectiveness of morphine antinociception in individualized opioid treatment.

[Expression and regulation of drug transporters in placenta].

Placenta, an important organ, mediates the exchange of nutrients and metabolites between mother and fetus. The transporters, including ATP-binding cassette (ABC) transporters and solute carrier (SLC), expressed in the syncytiotrophoblast play a vital role in substance exchange. Some transporters, such as organic cation transporters (OCTs) and organic anion transporters (OATs), mediate the uptake of endogenous substances and drugs. Some transporters, such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins(MRPs), can excrete their substrates from the syncytiotrophoblast to the maternal circulation. However, the expression and activity of these transporters are not uniform throughout the gestation period, since they can be affected by physiological and pathological changes during pregnancy or drugs. Thus, an understanding of the role of placental transporters and the variation in their expression and activity in response to physiological and pathological changes is essential for efficient and safe therapy during pregnancy, and it also has important value in the development of drug treatment in pregnancy.

[Establishment and application of cell models with stable expression of hOCTN1/2].

Human carnitine/organic cation transporter 1 and 2（hOCTN1 and hOCTN2） mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1 (+) plasmid vector containing hOCTN1 or hOCTN2（pcDNA3.1(+)-hOCTN1/2), several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, K(m) and V(max) of ergothioneine, mediated by MDCK-hOCTN1, were 8.19 ± 0.61 µmol·L-1 and 1 427 ± 49 pmol·mg(-1)(protein)·min(-1); while K(m) and V(max) of mildronate by MDCK- hOCTN2 were 52.3 ± 4.3 µmol·L(-1) and 2 454 ± 64 pmol·mg(-1)(protein)·min(-1). Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCK- hOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.

[Establishment of MDCK cell models expressing human MATE1 or co-expressing with human OCT1 or OCT2].

To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.

[In vitro interaction of deferiprone with cellular membrane transporters of hOCTs and hOAT1].

OBJECTIVE: To develop a LC-MS/MS method for determination of deferiprone in cell lysate and to study the potential interaction between deferiprone and hOCTs or hOAT1 transporters in vitro. METHODS: The determination was performed on an Agilent Eclipse Plus C18 column(3.5 µm, 2.1 mm×50 mm).The gradient mobile phase was composed of solvent A:0.1% formic acid in water, and B:0.1% formic acid in acetonitrile. The mass spectrometer with an electrospray interface was operated in positive ion mode with multiple reaction monitoring (MRM) scan mode monitored the ion pair of deferiprone at m/z 140→96, or phenacetin at m/z 180→110. The effects of deferiprone on the accumulation of typical substrates of hOCTs and hOAT1 were evaluated by MDCK-hOCTs and MDCK-hOAT1 cells respectively. The accumulation of deferiprone was also investigated in MDCK-hOCTs cells and mock cells with or without typical inhibitors. RESULTS: The standard curve was linear over the range of 5-300 nmol/L. The assay recovery of deferiprone was above 94%, and the intra-day precision (RSD) was less than 2.0%. The accumulation of MPP(+) in MDCK-hOCTs cells with 300 µmol/L deferiprone were 73.5%, 87.1% and 70.4%, respectively. The uptake of deferiprone in MDCK-hOCTs and mock cells did not show significant difference. Deferiprone of 100 µmol/L did not significantly affect the accumulation of 6-CF in MDCK-hOAT1 cell. CONCLUSION: The method is sensitivity and suitable for the determination of deferiprone in cell lysate. Deferiprone can significantly inhibit hOCT1 and hOCT3, but has no effects on hOCT2 and hOAT1. hOCTs may not play a major role in the transport of deferiprone.

Stereoselective binding of chiral drugs to plasma proteins.

Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed.

Stereoselective interaction between tetrahydropalmatine enantiomers and CYP enzymes in human liver microsomes.

Tetrahydropalmatine (THP), with one chiral center, is an alkaloid that possesses analgesic and many other pharmacological actives. The aim of the present study is to investigate stereoselective metabolism of THP enantiomers in human liver microsomes (HLM) and elucidate which cytochrome P450 (CYP) isoforms contribute to the stereoselective metabolism in HLM. Additionally, the inhibitions of THP enantiomers on activity of CYP enzymes are also investigated. The results demonstrated that (+)-THP was preferentially metabolized by HLM. Ketoconazole (inhibitor of CYP3A4/5) inhibited metabolism of (-)-THP or (+)-THP at same degree, whereas the inhibition of fluvoxamine (inhibitor of CYP1A2) on metabolism of (+)-THP was greater than that of (-)-THP; moreover, the metabolic rate of (+)-THP was 5.3-fold of (-)-THP in recombinant human CYP1A2. Meanwhile, THP enantiomers did not show obvious inhibitory effect on the activity of various CYP isoforms (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2E1, and 3A4/5), whereas (-)-THP, but not (+)-THP, significantly inhibited the activity of CYP2D6 with the Ki value of 6.42 ± 0.38 µM. The results suggested that THP enantiomers were predominantly metabolized by CYP3A4/5 and CYP1A2 in HLM, and (+)-THP was preferentially metabolized by CYP1A2, whereas CYP3A4/5 contributed equally to metabolism of (-)-THP or (+)-THP. Besides, the inhibition of CYP2D6 by (-)-THP may cause drug-drug interaction, which should be considered.

Stereoselective binding of mexiletine and ketoprofen enantiomers with human serum albumin domains.

AIM: To investigate the stereoselective binding of mexiletine or ketoprofen enantiomers with different recombinant domains of human serum albumin (HSA). METHODS: Three domains (HSA DOM I, II and III) were expressed in Pichia pastoris GS115 cells. Blue Sepharose 6 Fast Flow was employed to purify the recombinant HSA domains. The binding properties of the standard ligands, digitoxin, phenylbutazone and diazepam, and the chiral drugs to HSA domains were investigated using ultrafiltration. The concentrations of the standard ligands, ketoprofen and mexiletine were analyzed with HPLC. RESULTS: The recombinant HSA domains were highly purified as shown by SDS-PAGE and Western blotting analyses. The standard HSA ligands digitoxin, phenylbutazone and diazepam selectively binds to DOM I, DOM II and DOM III, respectively. For the chiral drugs, R-ketoprofen showed a higher binding affinity toward DOM III than S-ketoprofen, whereas S-mexiletine bound to DOM II with a greater affinity than R-mexiletine. CONCLUSION: The results demonstrate that HSA DOM III possesses the chiral recognition ability for the ketoprofen enantiomers, whereas HSA DOM II possesses that for the mexiletine enantiomers.

Stereoselective metabolism of tetrahydropalmatine enantiomers in rat liver microsomes.

Tetrahydropalmatine (THP), with one chiral center, is an active alkaloid ingredient in Rhizoma Corydalis. The aim of the present paper is to study whether THP enantiomers are metabolized stereoselectively in rat, mouse, dog, and monkey liver microsomes, and then, to elucidate which Cytochrome P450 (CYP) isoforms are predominately responsible for the stereoselective metabolism of THP enantiomers in rat liver microsomes (RLM). The results demonstrated that (+)-THP was preferentially metabolized by liver microsomes from rats, mice, dogs, and monkeys, and the intrinsic clearance (Cl(int)) ratios of (+)-THP to (-)-THP were 2.66, 2.85, 4.24, and 1.67, respectively. Compared with the metabolism in untreated RLM, the metabolism of (-)-THP and (+)-THP was significantly increased in dexamethasone (Dex)-induced and ß-naphthoflavone (ß-NF)-induced RLM; meanwhile, the Cl(int) ratios of (+)-THP to (-)-THP in Dex-induced and ß-NF-induced RLM were 5.74 and 0.81, respectively. Ketoconazole had stronger inhibitory effect on (+)-THP than (-)-THP, whereas fluvoxamine had stronger effect on (-)-THP in untreated and Dex-induced or ß-NF-induced RLM. The results suggested that THP enantiomers were predominately metabolized by CYP3A1/2 and CYP1A2 in RLM, and CYP3A1/2 preferred to metabolize (+)-THP, whereas CYP1A2 preferred (-)-THP.

[The role of ADME evaluation in translation research of innovative drug].

New Chemical Entities (NCEs) development is a systematic long-term project that involves multiple disciplines. The translation research will help to build an advanced R&D system from the basic laboratory research, preclinical studies and clinical evaluation to clinical application of drug, for the purpose of shortening the R&D cycle and accelerate the launch of new drugs. In new drug R&D and its clinical application, drug disposition (absorption, distribution, metabolism, excretion, ADME) properties are important criteria for assessing drug-likeness of candidates. ADME evaluation of NCEs plays an important role in the translation research throughout innovative drug R&D process. Therefore, ADME evaluation at the early stage of drug design and development will be helpful to improve the success rate and reduce costs, and further access to safe, effective drugs.

[Inhibitory and inductive effects of (-)- and (+)-tetrahydropalmatine on CYP450 in mice].

OBJECTIVE: To investigate the inhibitive and inductive effect of (-)-tetrahydropalmatine (THP) and (+)-THP on main CYP450 isoforms in mouse liver microsomes. METHODS: The in vitro inhibitory effect was evaluated by incubating (-)-THP or(+)-THP with the probe substrates of main phase I metabolic enzymes in mouse liver microsomes, and the remaining substrates were determined by HPLC or LC-MS/MS method. Mice were administered with (-)-THP or(+)-THP at dosage of 240 mg/kg or 60 mg/kg by gastric lavage for successive 7 days, then the cocktail-LC-MS method was applied to assess the activities of main CYP450 isoforms in mouse liver microsomes. RESULT: The IC(50) values of both (-)-THP and (+)-THP on isoforms studied were higher than 100 µmol/L except that IC(50) value of (+)-THP on CYP2C was 43.89 µmol/L, indicating weak inhibition of (-)-THP and (+)-THP on CYP1A2, CYP2D22, CYP2E1 and CYP3A11 in vitro. Compared with the vehicle group, the activities of CYP2D22, CYP2E1 and CYP3A11 were not increased significantly in (-)-THP and (+)-THP treatment groups, while the activities of CYP1A2 in 60 mg/kg and 240 mg/kg (-)-THP groups were 68.7% and 73.0% higher, than that of the vehicle group (P < 0.05, P < 0.01, respectively), the activity of CYP2C37 in 240 mg/kg (-)-THP treatment group was 80.4%, higher than that of the vehicle group (P < 0.05). CONCLUSION: There is negligible or weak inhibition on main CYP450 in mouse liver microsomes by (-)-THP and (+)-THP in vitro. (+)-THP does not induce main CYP450 in mouse liver microsomes while (-)-THP weakly induces CYP1A2 and CYP2C37.

[Determination of quercetin metabolism in UGT1A3 cDNA-expressing cells by RP-HPLC].

OBJECTIVE: To develop a RP-HPLC method for the determination of quercetin in UGT1A3 cDNA-transfected cells. METHODS: The lysate of cells transfected with human recombinant uridine 5-diphosphate glucuronosyltransferases UGT1A3 cDNA was co-incubated with quercetin, the reaction was terminated with acetonitrile, and luteolin was used as internal standard. The determination was performed on a C(1) reversed phase column with a mobile phase of methanol-0.1% formic acid (V/V) at a flow rate of 1.0 ml/min. The gradient elution was as follows: 0 - 25 min (30:70-80:20, methanol:0.1% formic acid), > 25-25.5 min (80:20), >25.5-27 min (80:20-30:70), > 27-30 min (30:70). A UV-VIS detector was operated at 368 nm. RESULT: The standard curve was linear over the concentration range of 5-200 µmol/L (r = 0.9999). The limit of detection was 1.25 µmol/L(S/N ≥ 3), and the limit of quantification was 5 µmol/L (S/N >10, RSD = 6.99%). The method afforded recoveries of 99.1%-103.5%, and precisions for inter- and intra-assay were < 2.5% and < 8%, respectively. In addition, kinetic analysis indicated that the K(m), V(max) and CL(int) (V(max)/K(m)) values for quercetin glucuronide were (62.95 ± 13.16) µ mol/L, (284.50 ± 24.35)nmol*min⁻¹*g⁻¹ and 4.52 ml*min⁻¹*g⁻¹, respectively. CONCLUSION: The method established is accurate and simple and suitable for the determination of quercetin in UGT1A3 cDNA-expressed cells.

[Chiral separation of fluvastatin enantiomers with in vitro cellular method].

OBJECTIVE: To establish a chiral separation method for determination of fluvastatin enantiomer with in vitro cellular model. METHODS: The determination was performed on Chiralpak AD column (4.6 mm × 250 mm); and the phase consisted of hexane-isopropanol-trifluoroacetic acid (90:10:0.1) at a flow rate of 0.5 ml/min with UV detection of 239 nm. RESULT: The standard curve was linear over the concentration range of 20 µmol/L-300 µmol/L (r² = 0.9993, r² = 0.9997). The recovery for this assay was (99.4 ± 0.8)%, precision for inter-assay and intra-assay was <10 %. CONCLUSION: The normal-phase HPLC chiral separation method was accurate and suitable for study on the stereoselectivity of fluvastatin with in vitro cellular model.

Stereoselective protein binding of tetrahydropalmatine enantiomers in human plasma, HSA, and AGP, but not in rat plasma.

Tetrahydropalmatine (THP) is one of the active alkaloid ingredients of Rhizoma Corydalis. THP has a chiral center, and the stereoselective pharmacokinetics and tissue distribution have been reported. The aim of the present article is to study the stereoselective protein binding of THP using equilibrium dialysis followed by HPLC-UV analysis. The results showed that THP stereoselectively binds to human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), and proteins in human plasma. The fraction binding of (+)-THP was significantly higher than that of (-)-THP, whereas such stereoselectivity was not found in rat plasma. The affinity of HSA and AGP to (+)-THP, expressed as nK(A), were 9.0 x 10(3) M(-1) and 2.34 x 10(5) M(-1), respectively, which were notablely higher than to (-)-THP, with the nK(A) of 3.4 x 10(3) M(-1) and 1.44 x 10(5) M(-1), respectively. The binding site of HSA for (-)-THP was Site I, whereas for (+)-THP was both Site I and Site II. The F1/S variants of AGP were proved to be the key variants (-)- and (+)-THP binding to both. Finally, the AGP binding drugs, such as mifepristone, were demonstrated to reduce the fraction binding of (-)- and (+)-THP with pure AGP (1 mg/ml) but did not affect the fraction binding of both (-)- and (+)-THP with proteins in human plasma. It can be concluded that protein binding of THP is species dependent and stereoselective, both HSA and AGP contribute to the stereoselective binding to THP enatiomers, and AGP binding drugs may not cause the drug-drug interaction on THP in healthy human plasma.

[Antiarrhythmic effect of ethyl acetate extract from Chrysanthemum Morifolium Ramat on rats].

OBJECTIVE: To investigate the effect of ethyl acetate extract from Chrysanthemum Morifolium Ramat (CME) on experimental arrhythmia induced by ischemia/reperfusion or aconitine in rats and to explore its underlying mechanisms. METHODS: Arrhythmia model in intact rat was induced by aconitine (30 microg/kg body weight, i.v.). In isolated Langendorff perfused rat hearts, regional ischemia and reperfusion was induced by ligation and release of left anterior descending artery. The ventricular fibrillation threshold (VFT), effective refractory period (ERP), and diastolic excitation threshold (DET) in the isolated heart were measured. The action potentials of papillary muscle in rat right ventricle were recorded by conventional glass microelectrode technique. RESULTS: Compared with control group CME significantly decreased the number and duration of ventricular tachycardia (VT); delayed the occurrence of ventricular premature beats (VPB) and VT induced by aconitine. Arrhythmia score of the CME group was lower than that in aconitine-treated group. CME markedly prolonged the ERP and increased the VFT in the isolated perfused rat hearts during ischemia and reperfusion. CME prolonged action potential duration at 50% and 90% repolarization of the right ventricular papillary muscles and decreased the maximal rate of rise of the action potential upstroke, but did not affect the resting potential, amplitude of action potential. CONCLUSION: CME can reduce myocardial vulnerability and exerts its antiarrhythmic effects induced by aconitine or ischemia/reperfusion, which may be related to its prolongation of action potential duration and effective refractory period that enhance the electrophysiological stability of myocardiaium.

Inhibition of superoxide anion-mediated impairment of endothelium by treatment with luteolin and apigenin in rat mesenteric artery.

This study was designed (i) to test the hypothesis that the endothelium-derived hyperpolarizing factor (EDHF) component of ACh-induced vasorelaxation and hyperpolarization of smooth muscle cells (SMCs) are impaired following exposure to superoxide anion, and (ii) to further investigate whether luteolin and apigenin induce vasoprotection at the vasoactive concentrations in rat mesenteric artery. Rat mesenteric arterial rings were isolated for isometric force recording and electrophysiological studies. Perfusion pressure of mesenteric arterial bed was measured and visualization of superoxide production was detected with fluorescent dye. 300 microM pyrogallol significantly decreased the relaxation and hyperpolarization to ACh. Luteolin and apigenin both induced vasoprotection against loss of the EDHF component of ACh-induced relaxation and attenuated the impairment of hyperpolarization to ACh. Oxidative fluorescent microtopography showed that either luteolin or apigenin significantly reduced the superoxide levels. The results suggest that superoxide anion impairs ACh-induced relaxation and hyperpolarization of SMC in resistance arteries through the impairment of EDHF mediated responses. Luteolin and apigenin protect resistance arteries from injury, implying that they may be effective in therapy for vascular diseases associated with oxidative stress.

Intestinal absorption of luteolin from peanut hull extract is more efficient than that from individual pure luteolin.

Luteoin is one of the main flavones and the crucial effective component of peanut hull extract (PHE). The present paper aims to elucidate the absorption mechanism of luteolin and clarify whether its absorption occurs primarily at a specific site of the intestine by an in situ single-pass intestinal perfusion (SPIP) model. Moreover, the paper investigates the difference in absorption of luteolin when it is administered in PHE form and as pure luteolin by the SPIP model and in vivo pharmacokinetics studies. Results showed that the effective permeability ( P eff) and absorption rate constant ( k a) of pure luteolin(5.0 microg/mL) in duodenum and jejunum were not significantly different, but markedly higher than that in the colon and ileum. The P eff and k a of luteolin in jejunum were concentration-independent, and the ATP inhibitor (DNP) did not influence P eff and k a of pure luteolin. However, the P eff and k a of luteolin in PHE were significantly greater than that of pure luteolin. The pharmacokinetics study showed that following oral administration of a single dose of pure luteolin (14.3 mg/kg) or PHE (= 14.3 mg/kg of luteolin) in rats, the peak concentration of luteolin in plasma ( C max) and the area under the concentration curve (AUC) for pure luteolin were 1.97 +/- 0.15 microg/mL and 10.7 +/- 2.2 microg/mL.h, respectively. These parameters were significantly lower than those of the PHE group ( P < 0.05), C max = 8.34 +/- 0.98 microg/mL and AUC = 20.3 +/- 1.3 microg/mL.h, respectively. It can be concluded that luteolin is absorbed passively in the intestine of rats and that its absorption is more efficient in the jejunum and duodenum than in the colon and ileum. The bioavailability of luteolin in PHE form is significantly greater than that of pure luteolin.

Simultaneous determination of three flavonoid aglycones in Elsholtzia blanda by adopting orthogonal test and high-performance liquid chromatography.

A new HCl hydrolysis/HPLC method, by adopting L9(34) orthogonal test to optimize hydrolysis condition, has been developed for simultaneous determination of three flavonoid aglycones in Elsholtzia blanda benth. The HCl concentration, methanol concentration, hydrolysis temperature, hydrolysis time are taking as four inspecting foctors, and the contents of luteolin, apigenin, and 5-hydroxy-6,7-dimethoxyflavone in hydrolytic solution are used as the evaluation indexes. Agilent Zorbax SB-C18 is used as analytical column. The mobile phase is a mixture of methanol-0.2% phosphoric acid (70:30, v/v), and UV detector is set at 350 nm. The flow rate is 1.0 mL/min, the temperature of column is maintained at 30 degrees C. The optimal hydrolysis conditions are 3.0M HCl, 70% methanol, 85 degrees C hydrolytic temperature and 3 h hydrolytic time. Standard curves are linear over the concentration range 8.54-85.4 microg/mL, 1.2-12 microg/mL, 9.2-92 microg/mL, and their average recoveries are 96.8%, 98.0%, and 100.5% for luteolin, apigenin, 5-hydroxy-6, 7-dimethoxyflavone, respectively. Thus, the optimum hydrolysis condition is relatively gentle, and the HPLC method is proved to be simple, accurate, and sensitive, so it will be able be applied to quality control of medicinal plant of Elsholtzia blanda.

[Determination of bis (p-fluorobenzyl) trisulfide and bis (p-fluoro-benzyl) disulfide in lungs of rat by HPLC].

OBJECTIVE: To establish an HPLC method for analysis of bis(p-fluorobenzyl) trisulfide(BFTS) and bis(p-fluorobenzyl)disulfide(BFDS) in the lungs of rat. METHODS: 5.0 ml extract solvent (n-hexane: isopropyl alcohol=95:5, v/v) and 20 microl of 11.50 microg/ml dibenzyl disulfide (internal standard) were added to 0.2 g lung sample followed by homogenization. After centrifugation, 4.0 ml of supernatant was separated and vaporized to dryness, and the residue was reconstituted in mobile phase for HPLC analysis. The HPLC analysis was performed on an SB C18 column using acetonitrile and water (65:35, v/v) as mobile phase with a flow rate of 1.0 ml/min with UV detection at 220 nm. RESULT: The calibration curves for BFTS and BFDS in sample were linear over the concentration ranges of 0.04712-14.78 microg/g(r=0.999) and 0.04831-23.96 microg/g(r=0.999), respectively. The limits of quantification were 0.04712 microg/g and 0.04831 microg/g for BFTS and BFDS, respectively. The assay recoveries for BFTS and BFDS ranged from 95.71%-107.2% and 90.00%-110.5%, respectively. The precisions were obtained with RSD of <10%. The developed method was successfully applied to study the content of BFTS and BFDS in the lungs of rats after intravenous injection of 12.5 mg/kg BFTS. CONCLUSION: The method developed is simple, selective, repeatable and accurate, which can be applied to study the tissue distribution of BFTS and BFDS.

Absorption and excretion of luteolin and apigenin in rats after oral administration of Chrysanthemum morifolium extract.

Chrysanthemum morifolium extract (CME) has the protective effect on cardiovascular diseases. Luteolin and apigenin are two major bioactive components in vivo when CME is orally administrated to experimental animal. The present paper shows the study of the absorption and excretion of luteolin and apigenin in rats after a single oral dose of CME (200 mg/kg). The levels of luteolin and apigenin in plasma, urine, feces, and bile were measured by HPLC after deconjugation with hydrochloric acid or beta-glucuronidase/sulfatase. The results showed that the plasma concentrations of luteolin and apigenin reached the highest peak level at 1.1 and 3.9 h after dosing, respectively. The area under the concentration-time curves (AUC) for luteolin and apigenin were 23.03 and 237.6 microg h mL-1, respectively. The total recovery of the dose was 37.9% (6.6% in urine; 31.3% in feces) for luteolin and 45.2% (16.6% in urine; 28.6% in feces) for apigenin. The cumulative luteolin and apigenin excreted in the bile was 2.05% and 6.34% of the dose, respectively. All of the results suggest apigenin may be absorbed more efficiently than luteolin in CME in rats, and both luteolin and apigenin have a slow elimination phase, with a quick absorption, so a possible accumulation of the two flavonoids in the body can be hypothesized.