Giovanni Mazzi

Evaluation of chemical stability of St. John's wort commercial extract and some preparations

Thermal and photostability of a commercial dried extract and capsules of St. Johns wort (Hypericum perforatum L.) were evaluated under the ICH test conditions. The extract was considered as drug substance and its preparations as drug products. In addition, capsules of different colours corresponding to different opaficient and pigment contents were also evaluated as primary package of drug product and the tests in the secondary pack were performed with amber containers, as well. A selective high-performance liquid chromatography (HPLC) for determination of stability of all the characteristic constituents, namely flavonols, hyperforins and hypericins, was carried out. Photostability testing showed all the constituents to be photosensitive in the tested conditions. However, different opaficients and pigments present in the capsules influenced the stability of the different classes of constituents. Amber containers suggested as secondary packages influenced only in part the photostability of the investigated constituents. Long-term thermal stability testing showed a very low (less than 4 months) hyperforins and hypericins t(90), even if ascorbic and citric acids were added to the formulation. From the results we have obtained it is clear that for St. Johns wort preparations, a mere translation of the ICH guidelines to the field of herbal products, as suggested by the WPHMP of the EMEA, cannot be accepted. A revision and adaptation of the storage conditions should be elaborated.

Stability of the constituents of Calendula, Milk-thistle and Passionflower tinctures by LC-DAD and LC-MS

As a part of our investigations on the stability of tinctures, we evaluated 40 and 60% v/v tinctures of Calendula flower, Milk-thistle fruit and Passionflower. These preparations are widely employed in phytotherapy, thus Calendula is used externally for anti-inflammatory properties, Milk-thistle and Passionflower are employed for hepatic injuries and in tenseness with difficulty in falling asleep, respectively. Aim of this work was to assess the chemical stability of their active or marker constituents from accelerated and long-term testing by using HPLC. For Calendula flower and Passionflower active constituents are not known, however, flavonoids seem to have a crucial importance for the activity, and thus are considered the markers of Calendula and of Passionflower. Active constituents of Milk-thistle are represented by silymarin that is a phytocomplex mainly constituted by three flavolignans: silybin, silychristin and silydianin. Our investigation showed a very low thermal stability of the constituents from accelerated and long-term testing and determined by HPLC-DAD and -MS analyses and was related both to the class of flavonoids and water content of the investigated tinctures. Thus, shelf-lives at 25 degrees C of the most stable tincture (Passionflower 60% v/v) was about 6 months and only about 3 months the stability of Milk-thistle tinctures.

1H-NMR NOE and molecular modelling to characterize thymol and carvacrol β-cyclodextrin complexes

Abstract In this study the structural conformations of the complexes between β-cyclodextrin and the geometrical isomers, thymol and carvacrol, were investigated using 1 H-NMR and molecular modelling techniques. The NOE experiments were performed in aqueous solutions and showed the presence of only one complex with a well-defined orientation either for thymol or for carvacrol. Molecular modelling investigations were useful to clarify the structural conformation of the supramolecular edifice. To develop graphic simulations closer to the experimental conditions, the studies were performed in dynamic at 300 K with and without evaluating the dielectric constant, D. The best agreement of 1 H-NMR NOE findings with the theoretical data was obtained after the evaluation of the dielectric constant parameter and these results confirmed the inclusion of thymol and carvacrol inside the βCD hydrophobic cavity.

Molecular modelling and NMR NOE experiments: Complementary tools for the investigation of complex ibuproxam-β-cyclodextrin topology

Abstract The aim of this paper is to study the use of computer molecular modelling and 1 H-NMR NOE data for gaining insight into the mode of inclusion of the ibuproxam (IBX) molecule in the hydrophobic cavity of β-cyclodextrin (βCyD) in solution. In the IBX-βCyD complex, the guest molecule shows very favourable structural features for a spectroscopic investigation of this type. The interatomic distances relative to some atoms of the IBX-βCyD complex, were calculated with molecular graphic systems. Through the comparison between the experimental values of nuclear Overhauser enhancements and the theoretical data obtained with molecular modelling studies, it was possible to establish the real structure of the IBX-βCyD complex in solution.

NMR spectroscopy: a useful tool for characterisation of plant extracts, the case of supercritical CO2 arnica extract.

The efficiency of two-dimensional homonuclear (1)H-(1)H correlated spectroscopy (COSY) and two-dimensional reverse heteronuclear shift correlation spectroscopy (i.e. heteronuclear multiple quantum correlation, HMQC) in characterising the content of the constituents of innovative extracts is demonstrated. These experiments were performed directly on a supercritical carbon dioxide (CO(2)) commercial extract of arnica and were able to fully characterise the active constituents, sesquiterpenes, and other metabolites extracted with the supercritical CO(2), namely polyketides. Identification of constituents was performed by combining literature data and information obtained by 2D-NMR experiments.

Analysis and stability of the constituents of artichoke and St. John's wort tinctures by HPLC-DAD and HPLC-MS.

ABSTRACT In continuing our investigations on tinctures, which represent both herbal drug preparations and herbal medicinal products, 40% and 60% v/v tinctures of artichoke and St. Johns wort were investigated. Artichoke is largely used in hepatic disorders, while St. Johns wort is an anti-inflammatory, antidepressant, and healing agent. Both herbal drugs contain various constituents, although the compounds responsible for the main effects have not yet been completely identified. However, caffeoylquinic acids and flavones seem to be of crucial importance for the activity of artichoke, as well as flavonoids, naphthodianthrones, and phloroglucinol derivatives for St. Johns wort, and they are used as marker constituents. Thus, quantification of all these constituents was performed using high-performance liquid chromatography–diode array detection (HPLC–DAD) and HPLC–mass spectrometry (MS) analyses with rutin as external standard. In addition the stability of the constituents of these tinctures from accelerated and long-term testing was also evaluated. From the results it was evidenced that constituent content depends on the solvent used for the extraction. The stability was also shown to be very different and seems to be related to the water content of the tinctures.

Letter: Membrane inlet mass spectrometry in the study of the release kinetics of volatile active principles in tisanes of vegetable drugs

Dear Sir Obtaining tisanes from vegetable drugs involves a process of infusion or decoction, whose duration is related to the extraction of active principles. Several studies have been performed to assess the optimum time to obtain an exhaustive extraction. In the case of volatile active principles, e.g. essential oils, quantitative determination in tisanes requires both long process time and extremely delicate extractive procedures 1,2 and consequently alternative, effective and simpler analytical methodologies are of great interest. In this area a new methodology has become available, resulting in the highly effective detection of organics in aqueous solutions. The membrane inlet mass spectrometry (MIMS) technique exploits the selective partitioning properties of a plastic membrane to act as an interface between water solutions of organic analytes and the evacuated environment of the mass spectrometer ion source. 3–8 Such an approach has been successfully employed in many different fields, such as in the monitoring of volatiles in fermentation broths, 9 the analysis of organic compounds in water solutions 10 and of solution equilibria 11 and the detection of drugs in blood and urine. 12,13 More recently, the capabilities of MIMS have been demonstrated in the online detection of volatile organic compounds in air at ppt levels, in the on-line monitoring of biological reactions and in on-site environmental analysis. 16

Stabilization of tinctures with cyclodextrins: a tincture from the fruits of Oenanthe aquatica L.

Abstract An original system of stabilization of the active principles of tinctures by means of cyclodextrins (α, β, γ-CyD) is proposed. In the particular case of a tincture from the fruits of Oenanthe aquatica L. the microinclusion of the components was found to be incomplete in all 3 cyclodextrins. However, β-CyD was the most efficient since a solid form was derived from it in which the most unstable active principles, polyacetylenes and terpenes, are included in the cyclic olygosaccharide and the others, lignans and steroids, are present as a physical mixture. GC checks confirmed the full correspondence of the components of this new form to those of the tincture from which it derives. The advantages of its use lie in the stabilization of the most unstable microincluded active principles within it, which was verified by means of artificial ageing studies, and in the implication that simple and complex solid dosage forms may be more simply prepared.