Vanni Cavrini

β-Amyloid aggregation induced by human acetylcholinesterase: inhibition studies

The aggregation of beta-amyloid (1-40) (Abeta) induced by human recombinant acetylcholinesterase (HuAChE) was studied by means of circular dichroism (CD) and by thioflavin T fluorescence spectroscopy. Abeta was incubated alone and with HuAChE. The kinetic of fibrils formation was followed for 48 hr. The increasing beta-conformation content induced by HuAChE, preliminary to the formation of Abeta fibrils, was determined by circular dichroism. This phenomenon was found to be related to the thioflavin T emission of fluorescence at 490 nm. Incubation experiments were performed in the presence of known AChE inhibitors (physostigmine, edrophonium, decamethonium, propidium) and drugs used for Alzheimers disease (AD) (tacrine, donepezil), to test their capability of preventing the HuAChE-induced Abeta aggregation. The non-competitive or mixed mode of AChE inhibition was confirmed to be an essential feature. At 100 microM propidium, decamethonium, donepezil and physostigmine were found to inhibit the HuAChE-induced Abeta aggregation by 82, 25, 22 and 30%, respectively.

GC/MS evaluation of thyme (Thymus vulgaris L.) oil composition and variations during the vegetative cycle.

Capillary GC/MS analysis based on polar and non-polar columns has been applied to evaluation of the volatile oils hydrodistilled from thyme (Thymus vulgaris L.) plants. The adopted methodology has been used to monitor seasonal variations in the composition of the oil obtained from thyme herbs harvested at different periods during the plant vegetative and life cycles. Oils from thyme plants of young (2 years) and old (5 years) cultivations have been evaluated from four and two collections, respectively, effected throughout May/December growth period. Generally, the oil was found to be rich in the active monoterpene phenols (thymol and carvacrol) and their corresponding monoterpene hydrocarbon (HC) precursors (p-cymene and gamma-terpinene), which collectively showed synchronized patterns of variation during the different collection periods and in different seasons. The oil from old plant collected in May/June period (0.15% v/w) was characterized by significantly lower levels of monoterpene HCs (mainly gamma-terpinene) and the highest levels of the oxygenated monoterpenes (linalool and borneol), monoterpene phenols (mainly thymol) and their derivatives (mainly carvacrol methyl ether), sesquiterpenes (mainly beta-caryophyllene) and their oxygenated derivatives (e.g. caryophyllene oxide) in comparison with all other samples. A characteristic presence of camphor and thymodihydroquinone was also observed in the old plant oils. On the other hand, the young plant, collected in June/July just before the end of the vegetative cycle, provided the best oil yield (1.2%) with also the highest % content of the monoterpene phenols (thymol: 51.2% and carvacrol: 4%). This latter growth period can represent the best harvest time of young thyme plants in order to obtain an essential oil with better quality and quantity.

Analysis of ACE inhibitors in pharmaceutical dosage forms by derivative UV spectroscopy and liquid chromatography (HPLC)

Derivative UV spectroscopy and high performance liquid chromatography (HPLC) were applied to the determination of angiotensin-converting enzyme (ACE) inhibitors in their pharmaceutical dosage forms. For spectrophotometric determinations, the more appropriate derivative order was selected for each drug: ramipril (third-order), benazepril (second-order), enalapril maleate (second-order), lisinopril (first- and second-order) and quinapril (first-order). Reverse phase HPLC procedures (ODS column) were developed able to provide a single, symmetric peak for each drug; mixtures A-B, where A is 20 mM sodium heptansulphonate (pH 2.5) and B is acetonitrile-THF (95:5 v/v), proved to be suitable mobile phases to obtain selective separations of the cited ACE inhibitors. At ambient temperature, a low pH value (2.5) was found to be critical to avoid peak splitting and band broadening.

Acetylcholinesterase inhibitors for potential use in Alzheimer's disease: molecular modeling, synthesis and kinetic evaluation of 11H-indeno-[1,2-b]-quinolin-10-ylamine derivatives.

Continuing our work on tetracyclic tacrine analogues, we synthesized a series of acetylcholinesterase (AChE) inhibitors of 11H-indeno-[1,2-b]-quinolin-10-ylaminic structure. Selected substituents were placed in synthetically accessible positions of the tetracyclic nucleus, in order to explore the structure-activity relationships (SAR) and the mode of action of this class of anticholinesterases. A molecular modeling investigation of the binding interaction of the lead compound (1a) with the AChE active site was performed, from which it resulted that, despite the rather wide and rigid structure of 1a, there may still be the possibility to introduce some small substituent in some positions of the tetracycle. However, from the examination of the experimental IC50 values, it derived that the indenoquinoline nucleus probably represents the maximum allowable molecular size for rigid compounds binding to AChE. In fact, only a fluorine atom in position 2 maintains the AChE inhibitory potency of the parent compound, and, actually, increases the AChE-selectivity with respect to the butyrylcholinesterase inhibition. By studying the kinetics of AChE inhibition for two representative compounds of the series, it resulted that the lead compound (1a) shows an inhibition of mixed type, binding to both the active and the peripheral sites, while the more sterically hindered analogue 2n seems to interact only at the external binding site of the enzyme. This finding seems particularly important in the context of Alzheimers disease research in the light of recent observations showing that peripheral AChE inhibitors might decrease the aggregating effects of the enzyme on the beta-amyloid peptide (betaA).

Analysis of phenolic acids by micellar electrokinetic chromatography: application to Echinacea purpurea plant extracts.

A micellar electrokinetic chromatographic (MEKC) method was developed for the separation of ten phenolic acids including cichoric acid and caftaric acids, specific marker phytochemicals of Echinacea purpurea. The MEKC method involved the use of 70 mM sodium deoxycholate (SDC) in 40 mM borate (pH 9.2) buffer and UV detection at 300 nm. The bile acid was used as biosurfactant able to provided a micellar system with different and more selective properties than sodium dodecyl sulfate. The effects of SDC and borate concentration and buffer pH on the analyte resolution were evaluated. The validated method was applied to the determination of cichoric acid and related compounds in E. purpurea root extracts, and in commercial E. purpurea based dried extracts and tablets.

Application of high-performance liquid chromatography with diode-array detection and on-line post-column photochemical derivatization to the determination of analgesics

HPLC analyses of pharmaceutical dosage forms containing analgesics and related compounds (acetylsalicyclic acid, paracetamol, propyphenazone, caffeine and chlorpheniramine) were performed on C18 and cyano columns under reversed-phase conditions. The performance of the methods was enhanced by introducing postcolumn on-line photochemical derivatization in combination with a diode-array detection. The column effluents were subjected on-line to UV irradiation (254 nm) and the characteristic photo-induced spectral modifications were useful for the unambiguous identification of the various analgesic compounds. The proposed HPLC methods were successfully applied to the analysis of commercially available analgesic dosage forms.

Design of experiments for capillary electrophoretic enantioresolution of salbutamol using dermatan sulfate.

Statistical experimental design was used for the optimization and for robustness evaluation of a capillary electrophoretic method developed for the enantioresolution of salbutamol. Dermatan sulfate was used as chiral selector. The goal of the study was to obtain an efficient and fast separation. An eight-run Plackett-Burman matrix was used during the optimization process for the screening of the factors and to adjust the experimental domain under study. Response surface methodology was adopted after the screening phase to obtain information about how the factors percentage of chiral selector, pH and voltage affected the considered responses resolution and analysis time. The Derringer desirability function, which makes it possible to combine results obtained for properties measured on different scales, was used to simultaneously optimize the two responses. Robustness testing was carried out using a Plackett-Burman matrix. The method was found robust as regards the response resolution while voltage and chiral selector were found to be critical factors for the robustness of analysis time response. The proposed CE method permitted the complete enantioseparation of racemic salbutamol and was applied to its chiral resolution in spiked urine samples.

On-line post-column photochemical derivatization in liquid chromatographic—diode-array detection analysis of binary drug mixtures☆

HPLC methods were developed for the analysis of pharmaceutical creams containing binary drug mixtures (betamethasone valerate-chlorocresol; hydrocortisone-miconazole nitrate; desonide pivalate-chlorhexidine; dexamethasone-clotrimazole; triamcinolone acetonide-econazole nitrate). The chromatographic separations were performed on C-18 and cyano columns under reversed-phase conditions. A post-column on-line photochemical reactor (irradiation at 254 nm) was arranged between the analytical column and the diode-array detector to enhance the performance of the method. Two UV spectra (photoreactor on and off) were obtained for each analyte and these additional sources of information proved to be useful for the unambiguous identification of the various analytes. The method was applied to the quality control of commercial creams using a solid-phase extraction procedure for the sample clean-up.

HPLC analysis of imidazole antimycotic drugs in pharmaceutical formulations

Reversed-phase HPLC on different column packing materials (Hypersil C-18, Spherisorb-CN, Chromspher-B) is used to obtain selective separations of imidazole antimycotic drugs, such as ketoconazole, clotrimazole, tioconazole, bifonazole, isoconazole, econazole, miconazole and fenticonazole. The use of a post-column on-line photochemical reactor is shown to be useful for the enhancement of the sensitivity of the HPLC analysis with UV detection. The proposed HPLC methods are applied to the analysis of commercial dosage forms (creams) with solid-phase extraction (SPE) procedure, using a diol sorbent, being found to be a convenient technique for the sample preparation giving quantitative drug recovery.

Analysis of catechins in extracts of Cistus species by microemulsion electrokinetic chromatography.

A microemulsion electrokinetic chromatographic (MEEKC) method was developed for the separation of six catechins, specific marker phytochemicals of Cistus species. The MEEKC method involved the use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent and butan-1-ol as co-solvent. In order to have a better stability of the studied catechins, the separation was performed under acidic conditions (pH 2.5 phosphate buffer). The effects of SDS concentration and of the amount of organic solvent and co-solvent on the analyte resolution were evaluated. The optimized conditions (heptane 1.36% (w/v), SDS 2.31% (w/v), butan-1-ol 9.72% (w/v) and 50 mM sodium phosphate buffer (pH 2.5) 86.61% (w/v)) allowed a useful and reproducible separation of the studied analytes to be achieved. These conditions provided a different separation profile compared to that obtained under conventional micellar electrokinetic chromatography (MECK) using SDS. The method was validated and applied to the determination of catechin and gallocatechin in lyophilized extracts of Cistus incanus and Cistus monspeliensis.