Chemical profiling of the tuber of Stephania cambodica Gagnep. (Menispermaceae) and analytical control by UHPLC-DAD.

A new aporphine glycoside (1), named 'angkorwatine', and eight known alkaloids: oblongine (2), stepharine (3), asimilobine-ß-d-glucopyranoside (4), isocorydine (5), tetrahydropalmatine (THP) (6), jatrorrhizine (7), palmatine (PAL) (8), and roemerine (ROE) (9) were simultaneously isolated from the tuber of Stephania cambodica. The development and validation of UHPLC-DAD method was carried out for the quantification of marker compounds (PAL, ROE, THP) of S. cambodica. In addition to good selectivity and linearity (r2 > 0.997), trueness, precision, and accuracy of the method did not exceed the acceptance limit of ±10% for ROE, THP and ±20% for PAL. Consequently, this method is able to provide accurate results between 1.39-4.18 µg/mL, 2.01-30.72 µg/mL, and 4.29-64.42 µg/mL for PAL, ROE, and THP, respectively. This study shows that the validated UHPLC method is a rapid, innovative and effective analytical approach to control quality of tubers of S. cambodica and to regulate the usage of this plant in traditional medicine.

HPLC analysis of Stephania rotunda extracts and correlation with antiplasmodial activity.

Stephania rotunda (Menispermaceae), a creeper commonly found in the mountainous areas of Cambodia, has been mainly used for the treatment of fever and malaria. Thus, the aim of this study is to investigate the chemical composition and antiplasmodial activity of different samples of S. rotunda and compare their antiplasmodial activity with their alkaloid content. Sixteen samples from different parts (roots, stem, and tuber) of S. rotunda were collected from four regions of Cambodia (Battambang, Pailin, Siem Reap, and Kampot). Reversed-phase HPLC was used to determine the content of three bioactive alkaloids (cepharanthine, tetrahydropalmatine, and xylopinine). These three alkaloids have been found in all samples from Battambang and Pailin (samples I-IX), whereas only tetrahydropalmatine was present in samples from Siem Reap and Kampot (samples X-XVI). The analyzed extracts were evaluated for their antiplasmodial activity on W2 strain of Plasmodium falciparum. Among them, 13 extracts were significantly active with inhibitory concentration 50 (IC(50) ) from 1.2 to 3.7 µg/mL and 2 extracts were moderately active (IC(50) = 6.1 and 10 µg/mL, respectively), whereas sample XI was not active (IC(50) = 19.6 µg/mL). A comparison between antiplasmodial activity and concentration of the three bioactive alkaloids in S. rotunda extracts has been realized.

Use of various models for in vitro percutaneous absorption studies of ultraviolet filters.

BACKGROUND: The percutaneous absorption test aims to estimate the passage of a substance across the skin. The absorption process can be described in three steps: (a) penetration of a substance into the skin layer, followed by (b) penetration from one layer into another (permeation) and finally (c) resorption into the vascular system. In vivo and in vitro models are available but owing to ethical reasons as well as the latter providing greater feasibility, in vitro models are preferred. AIMS: This present study reviews the natural membranes (human skin and animal models: pig, rabbit, rat, hairless mouse, guinea-pig and mouse), artificial skin equivalents and synthetic membranes that are currently being used for in vitro percutaneous absorption studies of UV filters, in order to provide the researcher with a greater insight when selecting membrane models for given experimental conditions.

Cytotoxic activity of alkaloids isolated from Stephania rotunda [corrected].

Three major alkaloids: cepharanthine (1), tetrahydropalmatine (2) and xylopinine (3) isolated from Stephania rotunda tuber were investigated for their cytotoxic activity in a panel of human cancer cells (HT29, LS174T, SW620 and HepG2) using MTT assay. In the present study, cepharanthine (1) exerted potent cytotoxicity against colon and hepatoma cancer cell lines with IC(50) values between 2.4 and 5.3 microM while tetrahydropalmatine (2) and xylopinine (3) displayed weak cytotoxicity. In addition, the mutagenic activity of cepharanthine (1) was investigated using a modified liquid incubation technique of the Salmonella/microsomal assay. This alkaloid (1) was found to be non-mutagenic for doses up to 8.2 microM.

Validation of a simple HPLC method for assay of haplamine and its metabolites in plasma suitable for pharmacokinetic application in rats.

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation.

A new small volume holding chamber for asthmatic children: comparison with Babyhaler spacer.

When a new holding chamber for administrating inhaled medication is to be marketed, it needs to be compared with existing chambers with two questions in mind: is this chamber well accepted by patients and is there an in vitro equivalence? We compared the new small volume non-electrostatic valved holding chamber, usable with all pressurized metered-dose inhalers and equipped with a funny facemask, Vortex (Pari GmbH, Germany), to the most frequently prescribed holding chamber in France, Babyhaler (GlaxoSmithKline Laboratories). Preferences were studied for 75 families with a child no more than 4 yr old, using standard questionnaires. An in vitro study assessed the delivered dose and the particle size distribution of two HFA beclomethasone dipropionate pressurized metered dose inhalers (Becotide 250 microg per dose and Nexxair 100 microg per dose) by dose uniformity sample apparatus and cascade impactor according to the European Pharmacopoeia. Vortex was preferred by 95% of the families because of its small size, its duck facemask, and its robust appearance. Among children able to give their opinion, 86% preferred Vortex to Babyhaler. In vitro, both holding chambers reduced the delivered dose of beclomethasone dipropionate and increased the quantity of particles smaller than 5 microm in diameter with both medications. A higher proportion of fine particles was obtained with Nexxair than with Becotide (p < 0.05) and with Vortex than with Babyhaler (p < 0.05). As expected, throat deposition is dramatically reduced for both drugs with both holding chambers. The in vitro difference in the particle size distribution of beclomethasone dipropionate with both holding chambers probably has no clinical influence.

Inter-species variability of haplamine metabolism and identification of its phase I metabolites from liver microsomes.

Haplamine, a pyranoquinoline alkaloid, was isolated from the genus Haplophyllum. The inter-species variability of haplamine metabolism was determined by reversed phase high performance liquid chromatography (HPLC) with UV detection. Microsomes from the liver of rats, mice, rabbits, guinea-pigs and humans were incubated with haplamine. After incubation, samples were extracted with a mixture of ethyl acetate and isopropyl alcohol (90 : 10; v/v). Haplamine and its metabolites were separated by HPLC using Nucleosil C18 Nautilus (5 microm) connected with a precolumn of the same type. The HPLC mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85 : 15; v/v) (B) used in a gradient mode (17 to 27 % B for 10 min, 27 to 90 % B for 37 min, 90 to 17 % B for 3 min, and finally 17 % B for 3 min) at 1 mL/min. Quantitative and qualitative results showed significant inter-species differences in haplamine metabolism. Qualitative similarities were found between guinea-pigs, rabbits, and humans. The metabolites were isolated by HPLC and identified by GC/MS after silylation. The phase I metabolites identified in human liver microsomes were TRANS/CIS-3,4-dihydroxy-9-O-desmethylhaplamine, TRANS/CIS-3,4-dihydroxyhaplamine and 9-O-desmethylhaplamine.

Effect of several compounds on biliary excretion of paclitaxel and its metabolites in guinea-pigs.

The objective of this study was to evaluate the in vivo metabolic profile of paclitaxel and to examine the effect of potential co-administered drugs on the biliary secretion of paclitaxel and its metabolites in guinea-pigs. We first investigated in vitro paclitaxel metabolism using liver microsomes obtained from various species to identify the most suitable animal model with a similar metabolism to humans. Then, in vivo paclitaxel metabolism was investigated in male guinea-pigs. The levels of paclitaxel and its metabolites were measured by high-performance liquid chromatography in bile samples from guinea-pigs after paclitaxel i.v. injection (6 mg/kg). We further evaluated the effects of various drugs (quercetin, ketoconazole, dexamethasone, cotrimoxazole) on the biliary secretion of paclitaxel and its metabolites in guinea-pigs. This work demonstrated significant in vitro interspecies differences in paclitaxel metabolism. Our findings showed both in vitro and in vivo similarities between human and guinea-pig biotransformation of paclitaxel. 6alpha-Hydroxypaclitaxel, the main human metabolite of paclitaxel, was found in guinea-pig bile. After paclitaxel combination with ketoconazole or quercetin in guinea-pigs, the cumulative biliary excretion of paclitaxel and its metabolites up to 6 h was significantly decreased by 62 and 76%, respectively. The co-administration of cotrimoxazole or pretreatment with dexamethasone did not alter significantly cumulative biliary excretion. The guinea-pig is a suitable model to study metabolism and biliary excretion of paclitaxel, and to investigate in vivo drug interactions.

Interspecies variability and drug interactions of loxapine metabolism in liver microsomes.

Loxapine is a dibenzoxazepine neuroleptic that is metabolized by the liver in humans. In the present study, we investigated first in vitro loxapine metabolism in liver microsomes from various species including rats, mice, guinea pigs, dogs, rabbits, monkeys and humans. This enables us to choose between species to further validate drug-drug interaction studies. We observed the formation of desmethyl- and hydroxy- metabolites of loxapine after incubation of the different species liver microsomes. Hydroxylation pathway was major in all species. Wide interspecies variability of loxapine metabolism was observed. Loxapine metabolism was similar in human, guinea pig and dog microsomes. We screened in vitro effects of 67 molecules, representative of 8 therapeutic classes, on loxapine metabolism. Loxapine (100 microM) was incubated with guinea pig liver microsomes (1 mg/ml) 30 min at 37 degrees C with and without the presence of interacting drug. We found that most of psychotropics (alimemazine, cyamemazine and levomepromazine), antifungal (ketoconazole), anticancer drugs (daunorubicin, pirarubicin) and analgesic (nefopam) inhibited more than 50% of hydroxyloxapine formation in vitro. Complementary clinical and pharmacokinetic studies should be performed to confirm these results.