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Analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis.

The ability of capillary zone electrophoresis in the development of analytical methods devoted to the quality control of the thiol drug penicillamine is shown. Using 50 mM phosphate running buffer (pH 2.5), good quantitations of underivatized penicillamine and its disulfide were achieved; detection at 200 nm allowed checking the presence of the disulfide impurity in pharmaceuticals. The use of 1,1-[ethenylidenebis(sulfonyl)]bis-benzene as a thiol specific reagent resulted in an increased sensitivity for the quantitation of D-penicillamine (limit of detection at 200 nm wavelength was 1.5 microM). Introducing beta-cyclodextrin as chiral selector in the running buffer, enantioseparation of D-L-penicillamine was obtained; for this purpose (+)-camphor-10-sulfonic acid, a chiral ion-pairing reagent, was found to be an essential additive in obtaining a baseline separation. The resulting enantioseparative system was validated in order to evaluate the presence of the toxic L-penicillamine enantiomer in pharmaceutical samples.

Disulfides↗

Determination of sunscreen agents in cosmetic products by supercritical fluid extraction and high-performance liquid chromatography.

A rapid supercritical fluid extraction (SFE) procedure for the isolation of five of the most common sunscreen agents (2-ethylhexyl-p-dimethylaminobenzoate, 2-hydroxy-4-methoxybenzophenone, 2-ethylhexyl-p-methoxycinnamate, 4-methylbenzylidene camphor and 4-tert.-butyl-4'-methoxydibenzoylmethane) from cosmetic products is described. Investigation of the factors affecting the extraction efficiency in SFE indicated that sunscreen recoveries were affected mainly by the supercritical CO2 pressure and by the trapping method. The sunscreens were analyzed by reversed-phase high-performance liquid chromatography after a 10-min extraction of the cosmetic product with CO2 at 250 bar and 40 degrees C, using sequential glass surface and C18 sorbent as collection system. A quantitative comparison of SFE with a liquid extraction procedure was performed on commercial cosmetics. The SFE method yielded recoveries higher than 94.8% compared with conventional liquid extraction and exhibited a precision better than 5.3% relative standard deviation. Moreover, SFE minimized sample handling, reduced the consumption of harmful solvents and afforded a more effective purification of the cosmetic matrices.

Chromatography, High Pressure Liquid↗

Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system.

Escherichia coli K12 assay-system is designed in order to detect bioantimutagens, agents preventing mutagenesis by modulation of DNA repair and replication. The assay is composed of four tests aimed at the detection of inhibition of spontaneous and induced mutations (Tests A and B) and at the estimation whether the anti-mutagenic agent acts by increasing the fidelity of DNA replication (Test B), by inhibition of SOS error prone repair (Test C), or by favoring error-free recombinational repair (Test D). In Test A, repair proficient strain and its uvrA counterpart are used for detection of spontaneous and UV-induced mutations, while in Test B mismatch repair deficient strains (mutH, mutS, mutL and uvrD) are used for amplified detection of spontaneous mutations caused by replication errors. In Test C, repair proficient strain carrying sfiA::lacZ fusion is used for measuring the level of SOS induction by monitoring the level of beta-galactosidase. In Test D, the strains carrying different recA alleles (recA+, recA730 and DeltarecA) are used for measuring intrachromosomal recombination between nonoverlapping deletions in duplicated lac operon, by monitoring Lac+ recombinants. The assay-system is validated with model bioantimutagens and used for detection of anti-mutagenic potential of different terpenoid fractions from sage (Salvia officinalis L.). Extract E1/3 of cultivated sage, distinguished from others by its high content of monoterpenoid camphor, reduces UV-induced mutagenesis in Test A, while it has no effect in Tests B and C. In Test D, it enhances intrachromosomal recombination in untreated and UV-irradiated recA+ and recA730 strains. The results suggest that the protective effect is due to stimulation of recombinational repair, similarly to coumarin. We speculate that monoterpenoids from sage enhance genetic recombination by intervening in a formation of RecA-DNA complex and channeling it into recombination reaction.

Antimutagenic Agents↗

Antifungal activity of the essential oil of flowerheads of garland chrysanthemum (Chrysanthemum coronarium) against agricultural pathogens.

The antifungal activity of Chrysamthemum coronarium was evaluated against 12 agricultural pathogens. Flowerhead oil was active both in contact and headspace in vitro assays producing hyphal growth inhibition, although there was less activity on faster growing fungi. The main compounds identified in the oil were camphor (29.2%), alpha-pinene (14.8%), beta-pinene (9.5%) and lyratyl acetate (9.8%). The blue color of the oil was due to the presence of chamazulene (0.5%).

Antifungal Agents↗

Estrone sulfonates as inhibitors of estrone sulfatase.

In our continuing quest to design efficient inhibitors of estrone sulfatase activity and to assess the recognition of estrone sulfate surrogates by estrone sulfatase, we synthesized and evaluated several sulfonate derivatives of 5,6,7,8-tetrahydronaphth-2-ol and estrone. 5,6,7,8-Tetrahydronaphth-2-methanesulfonate (11), and 5,6,7,8-tetrahydronaphth-2-(p-toluene)sulfonate (12) were found not to inhibit estrone sulfatase activity; estrone-3-methane-sulfonate (5), estrone-3-ethanesulfonate (6), estrone-3-butanesulfonate (7), and estrone-3-[(+)10-camphor]sulfonate (8) all weakly inhibited estrone sulfatase, and the best inhibitor, from this class of compounds, was estrone-3-(p-toluene)sulfonate (9). At 10 microM, it inhibited estrone sulfatase activity by 91%. These results emphasize some of the requirements needed for high-affinity binding to the enzyme.

Chromatography, Thin Layer↗

Emergency pulpotomy: pain relieving effect with and without the use of sedative dressings.

Seventy-three teeth with acute irreversible pulpitis were subjected to emergency pulpotomies. After removal of the coronal portion of the pulp, a sterile cotton pellet or zinc oxide-eugenol cement was placed against the remaining pulp tissue. The cotton pellet was either dry or moistened with camphorated phenol, cresatin, eugenol, or isotonic saline. This gave six different groups of treatment following the pulpotomy. All teeth were sealed with zinc oxide-eugenol cement. By means of questionnaires, symptoms were recorded after the anesthetic effect was gone and also at 1, 7, and 30 days after treatment. A total of 70 patients (96%) reported pain relief. Three patients did not experience relief and returned for pulpectomy. After the first postoperative day, no pain was reported by any of the patients. The frequency of discomfort decreased from 11% 1 day postoperatively to 1% 30 days after the emergency treatment. There was no difference in recorded symptoms among the six treatment groups. Thus, removal of caries, pulpotomy, and sealing of the cavity apparently was a reliable means to relieve pain. The use of the various dressings did not contribute to the relief of pain.

Analgesics↗

Intracanal pH changes of calcium hydroxide pastes exposed to carbon dioxide in vitro.

It has been suggested that the main benefit of using calcium hydroxide as an intracanal medicament lies in its bactericidal effect, provided that the pH of the paste is above 12.5. The purpose of this study was to measure changes in the pH of several calcium hydroxide pastes sealed in root canals for 30 days. Sixty-two extracted, single-rooted human teeth were endodontically prepared using K-files up to size 60. The teeth were separated at random into six equal groups to be filled with either Calxyl, Hydrocalcium, or a paste made by mixing calcium hydroxide powder with either distilled water, camphorated p-monochlorophenol, local anesthetic solution, or Solvidont. Cavidentin was used to seal the coronal orifice of the teeth that were placed individually in vials containing 10 mL distilled water. Five vials of each group were exposed to air at room temperature, whereas the other five vials were exposed to carbon dioxide in a closed container. The pH of the paste in the root canal was measured after 30 days. There was no significant (p > 0.01) change in the pH (mean 13.11) of the pastes placed in teeth before and after exposure to air, whereas the pH of the pastes in teeth exposed to carbon dioxide was significantly (p < 0.01) reduced (mean 12.54). There was no significant difference in pH between the six preparations. After 30 days of exposure to carbon dioxide, they still maintained a purportedly bactericidal pH within the root canal.

Analysis of Variance↗

Chiral recognition of terpenoids in some pharmaceuticals derived from natural sources.

Capillary gas chromatography was applied to explore the enantiomeric composition of some terpenoids in pharmaceuticals of natural origin. The drugs under investigation were produced in Germany (Rowachol, Rowatinex), Poland (Terpichol, Terpinex, Rub arom, Herbolen and Oleum Camphoratum), Slovenia (Uroterp, Mentoklar) and United Kingdom (Olbas oil, Vicks Vapo Rub). The model compounds tested were: menthone, isomenthone, menthol, fenchone, borneol and camphor. It has been found that depending on the manufacturer, pharmaceuticals possessing similar chemical composition may differ considerably in enantiomer composition. Exceptionally large discrepancies have been found for the content of borneol and fenchone enantiomers in pharmaceuticals applied in liver and kidney diseases. It seems that the changes in enantiomeric composition are the main reason of the lack of general acceptance of natural medicines by clinicians. The study of enantiomeric composition may sometimes lead to information concerning the origin of preparation, i.e. natural or synthetic.

Biological Factors↗

Bacterial photomutagenicity testing: distinction between direct, enzyme-mediated and light-induced events.

A bacterial photomutagenicity test system has been established according to a predetermined protocol using a light source emitting multiple wave lengths, including UV-A and UV-B, and the tester strains used for standard bacterial mutagenicity testing. 8-Methoxypsoralen (8-MOP) was used as a photomutagenic positive control showing no mutagenicity in the absence of light and borderline (Salmonella typhimurium TA1537) or clear (Salmonella typhimurium TA102 and Escherichia coli WP2) mutagenic effects in the presence of light. Using the same experimental conditions, the UV filters p-aminobenzoic acid (PABA), octyl dimethyl PABA (Eusolex 6007), and 4-methylbenzylidene camphor (Eusolex 6300) were non-mutagenic (in the absence of light) and non-photomutagenic (in the presence of light). Dihydroxyacetone (DHA), used as an active ingredient in self-tanning lotions, slightly increased the number of revertants in Salmonella typhimurium TA100 and TA102 in the absence of light. However, the relevance of these effects is equivocal, since they occurred at very high, cytotoxic concentrations (5000 micrograms/plate). Furthermore, these increases were not potentiated by light, thus demonstrating the non-photomutagenicity of DHA. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA), which is non-mutagenic per se and is activated by external metabolizing systems (e.g., S9-mix) in the absence of light, was clearly mutagenic in Salmonella typhimurium TA98 and TA1537 in the presence of light (and the absence of S9-mix). Although the photomutagenic potency of DMBA, on a molar basis, was certainly lower than that of 8-MOP, the absolute mutagenic effects of DMBA in the respective bacterial strains were in a similar range under either S9-mix or photoactivation conditions. The strain specificity of the mutagenic effects were, however, different for either enzyme- or light-mediated mutagenicity. This indicates that different reactive intermediates are responsible for the mutagenicity in the tests using the two different activation systems. These results further suggest to use DMBA as a positive photomutagenic control compound alternatively to 8-MOP, since the latter is non- or only weakly photomutagenic in Salmonella typhimurium TA98 and TA1537. Furthermore, the usefulness and application of this photomutagenicity test system could be demonstrated for the testing of different photoabsorbing chemicals and cosmetic ingredients.

9,10-Dimethyl-1,2-benzanthracene↗

Protein engineering of cytochromes P-450.

The cytochromes P-450 are an immensely important superfamily of heme-containing enzymes. They catalyze the monooxygenation of an enormous range of substrates. In bacteria, cytochromes P-450 are known to catalyze the hydroxylation of environmentally significant substrates such as camphor, phenolic compounds and many herbicides. In eukaryotes, these enzymes perform key roles in the synthesis and interconversion of steroids, while in mammals hepatic cytochromes P-450 are vital for the detoxification of many drugs. As such, the cytochromes P-450 are of considerable interest in medicine and biotechnology and are obvious targets for protein engineering. The purpose of this article is to illustrate the ways in which protein engineering has been used to investigate and modify the properties of cytochromes P-450. Illustrative examples include: the manipulation of substrate selectivity and regiospecificity, the alteration of membrane binding properties, and probing the route of electron transfer.

Animals↗

In situ biodegradation determined by carbon isotope fractionation of aromatic hydrocarbons in an anaerobic landfill leachate plume (Vejen, Denmark).

Concentrations and isotopic compositions (13C/12C) of aromatic hydrocarbons were determined in eight samples obtained from the strongly anoxic part of the leachate plume downgradient from the Vejen Landfill (Denmark), where methanogenic, sulfate-reducing and iron-reducing conditions were observed. Despite the heterogeneous distribution of the compounds in the plume, the isotope fractionation proved that ethylbenzene and m/p-xylene were subject to significant biodegradation within the strongly anoxic plume. The isotope fractionation factors (alphaC) for the degradation of the m/p-xylene (1.0015) and ethylbenzene (1.0021) obtained from the field observations were similar to factors previously determined for the anaerobic degradation of toluene and o-xylene in laboratory experiments, and suggest that in situ biodegradation is one major process controlling the fate of these contaminants in this aquifer. The isotope fractionation determined for 1,2,4-trimethylbenzene and 2-ethyltoluene suggested in situ biodegradation; however, the isotopic composition did not correlate well with the respective concentration as expressed by the Rayleigh equation. Some other compounds (1,2,3-trimethylbenzene, o-xylene, naphthalene and fenchone) did not show significant enrichments in delta13C values along the flow path. The compound concentrations were too low for accurate isotope analyses of benzene, toluene, 1- and 2-methylnaphthalene, while interferences in the chromatography made it impossible to evaluate the isotopic composition for 4-ethyltoluene, 1,3,5-trimethylbenzene and camphor. In addition to demonstrating the potential of assessing isotopic fractionation as a means for documenting the in situ biodegradation of complex mixtures of aromatic hydrocarbons in leachate plumes, this study also illustrates the difficulties for data interpretation in complex plumes and high analytical uncertainties for isotope analysis of organic compounds in low concentration ranges.

Bacteria, Anaerobic↗

Effect of a proprietary rubefacient "Tiger Balm" on rabbit skin.

The effect of continuous exposure to proprietary rubefacients (Tiger Balm Red and Tiger Balm White), which contain menthol and camphor as well as clove, cajuput and cassia oils, was tested in rabbits. Dermal irritancy was determined by a 21-day continuous-application patch test (0.5 g/patch) and was assessed according to the Draize method of scoring. At the end of the 21-day test period, the skin was also examined histopathologically. In these tests, Tiger Balm Red (which contained 5% cassia oil plus 5% clove oil) caused irritation consisting of erythema, eschar formation and some oedema, to which a degree of tolerance developed. This irritation resulted in hyperkeratosis and sometimes inflammatory changes but no major damage to the skin. Tiger Balm White (no cassia oil and 2% clove oil) was better tolerated and produced less irritation and histological change than either TIger Balm Red or a mixture of commercial waxes similar in composition to the wax base for Tiger Balm Red. None of the treatments produced any signs of systemic toxicity.

Animals↗

Antibacterial and antioxidant activities of Artemisia annua essential oil.

The essential oil of Artemisia annua aerial parts, consisting of camphor (44%), germacrene D (16%), trans-pinocarveol (11%), beta-selinene (9%), beta-caryophyllene (9%) and artemisia ketone (3%), was screened for its antimicrobial activity. The essential oil remarkably inhibited the growth of tested Gram-positive bacteria Enterococcus hirae and both tested fungi. This oil has shown an antioxidant activity equivalent to 18% of the reference compound (alpha-tocopherol).

Anti-Bacterial Agents↗

In vitro inhibition of CYP2B1 monooxygenase by beta-myrcene and other monoterpenoid compounds.

beta-myrcene (MYR) is an acyclic monoterpene found in the essential oils of several useful plants such as lemongrass (Cymbopogon citratus), hop, bay, verbena and others. Recently it has been reported that MYR as well as lemongrass oil blocked the metabolic activation of some promutagens (e.g., cyclophosphamide and aflatoxin B1) in in vitro genotoxicity assays. The present study was performed to evaluate the inhibitory effects of MYR and some other monoterpenoid compounds on microsomal enzymes involved in the activation of genotoxic substances. The effects of MYR and other monoterpenes on the activity of pentoxyresorufin-O-depenthylase (PROD), a selective marker for CYP2B1, was determined in a pool of liver microsomes prepared from phenobarbital-treated rats. The effect of MYR on the activity of ethoxyresorufin-O-deethylase (EROD), a marker for CYP4501A1, was investigated in liver microsomes of untreated rats. Results revealed that MYR had almost no effect on EROD (IC50 > 50 microM), but produced a concentration-dependent inhibition of PROD activity (IC50 =0.14 microM). The analysis of alterations produced by MYR on PROD kinetic parameters (Lineweaver-Burk plot) suggested that inhibition is competitive (Ki = 0.14 microM). The inhibitory effects of seven other monoterpenes on PROD activity (pentoxyresorufin 5 microM) were also studied and the IC50 were as follows: (-)-alpha-pinene, 0.087 microM; (+)-alpha-pinene, 0.089 microM; d-limonene, 0.19 microM; alpha-terpinene, 0.76 microM; citral, 1.19 microM; citronellal, 1.56 microM, and (+/-) camphor, 7.89 microM. The potent inhibitory effects on CYP4502B1 suggest that MYR, and other monoterpenes, interfere with the metabolism of xenobiotics which are substrates for this isoenzyme.

Acyclic Monoterpenes↗

Compositions and the in vitro antimicrobial activities of the essential oils of Achillea setacea and Achillea teretifolia (Compositae).

GC-MS analysis of the isolated essential oils from air-dried aerial parts of Achillea setacea and Achillea teretifolia, an endemic taxon, resulted in the identification of 51 constituents (79.8% of the total oil) and 42 constituents (87.1% of the total oil), respectively. Eucalyptol (1,8-cineole) was the major constituent of both oils studied (18.5 and 19.9%, respectively). The antimicrobial activities of the essential oils were individually evaluated against 14 microorganisms. Both oils exhibited inhibitory effects on Clostridium perfringens, Acinetobacter lwoffii and Candida albicans with a range of minimum inhibitory concentration values extended from 0.28 to 2.25 mg/ml. Camphor and their derivatives, borneol, terpinen-4-ol and eucalyptol (1,8-cineol) can be considered as the main antimicrobial constituents of the oils studied.

Achillea↗

Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae).

The in vitro antimicrobial and antioxidant activities of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae) were investigated. GC-MS analysis of the essential oil resulted in the identification of 36 compounds constituting 90.8% of the total oil. Eucalyptol, camphor, alpha-terpineol, beta-pinene, and borneol were the principal components comprising 60.7% of the oil. The oil strongly reduced the diphenylpicrylhydrazyl radical (IC(50)=1.56 micro g/ml) and exhibited hydroxyl radical scavenging effect in the Fe(3+)-EDTA-H(2)O(2) deoxyribose system (IC(50)=2.7 micro g/ml). It also inhibited the nonenzymatic lipid peroxidation of rat liver homogenate (IC(50)=13.5 micro g/ml). The polar phase of the extract showed antioxidant activity. The oil showed antimicrobial activity against Streptococcus pneumoniae, Clostridium perfringens, Candida albicans, Mycobacterium smegmatis, Acinetobacter lwoffii and Candida krusei while water-insoluble parts of the methanolic extracts exhibited slight or no activity. This study confirms that the essential oil of Achillea millefolium possesses antioxidant and antimicrobial properties in vitro.

Achillea↗

A preliminary study of the effect of essential oils on skeletal and smooth muscle in vitro.

The pharmacological activity of nine commercial essential oils was studied on the rat isolated phrenic nerve diaphragm preparation and compared with activity on field-stimulated guinea-pig ileum preparations. The essential oils at final bath concentrations of 2 x 10(-5) and 2 x 10(-4) g/ml produced four different effects on skeletal muscle, whilst only a contracture with or without a decrease in response to field stimulation in smooth muscle. The first type of effect on skeletal muscle involved a contracture and inhibition of the twitch response to nerve stimulation shown by a sample of clary sage, dill, fennel, frankincense and nutmeg; a second, shown by thyme produced a contracture without a change in the twitch response; a third, shown by lavender reduced the twitch response alone and the fourth, shown by camphor, increased the size of the twitch response. Angelica root oil at the highest concentration studied showed no response on skeletal muscle.

Animals↗

ION-pair liquid chromatography technique for the estimation of metformin in its multicomponent dosage forms.

A simple, precise and accurate high performance liquid chromatography (HPLC) method was developed for the simultaneous estimation of metformin with gliclazide and glipizide present in multicomponent dosage forms. The method was carried out on Inertsil C(18) column. A mobile phase composed of acetonitrile-water containing camphor sulphonic acid (adjusted to pH 7 using 0.1 N sodium hydroxide; 75 mM) at a flow rate of 1 ml min(-1) was used for the separation. Detection was carried out at 225 nm. Tolbutamide was used as internal standard. Validation of the developed HPLC method was carried out.

Chemistry, Pharmaceutical↗