Pharmacological in vivo test to evaluate the bioavailability of some St John's Wort innovative oral preparations.

In this study, the optimisation of biopharmaceutical properties of a dried commercial extract of St John's Wort were evaluated employing the in vivo forced swimming test (FST). Three new dosage forms containing beta-cyclodextrin and surfactants (SDS, ASC8) were compared in the FST with the commercial extract. The commercial extract showed antidepressant activity in mice after 60 min at a dosage of 100 mg/kg. The same antidepressant activity appeared in 30 min with a micellar solution of SDS containing the same quantity of extract (100 mg/kg), while with micelles of ASC8 the effect appeared at 15 min and with a dosage of 30 mg/kg. In the case of beta-cyclodextrin the best results were obtained at 30 min, administering 60 mg/kg of the extract. Finally, the influence of the formulations on the water solubility of the constituents of the extract is reported. The tensides dramatically enhanced solubility, in particular that of the more lipophilic compounds, in the case of beta-cyclodextrin this effect was very pronounced for flavonoids and biapigenin, lower for hypericins and practically insignificant for hyperforins.

HPLC-DAD-ESI-MS analysis of the constituents of aqueous preparations of verbena and lemon verbena and evaluation of the antioxidant activity.

Verbena and lemon verbena aqueous preparations were investigated for their content of constituents, especially polyphenols by HPLC/DAD/ESI/MS analysis because they are used worldwide as herbal teas. The main class of compounds of these plants were phenylpropanoids (from 16 to 120 mg/g of dried extract), being verbascoside the most abundant in all the preparations up to 97% of the total phenylpropanoids. Also iridoids, hastatoside and verbenalin together with flavonoids, mono- and di-glucuronidic derivatives of luteolin and apigenin were found. These simple preparations, especially that obtained from infusion of lemon verbena, could be lyophilized to obtain a powder having interesting technological properties to be used as ingredients of cosmetics, food supplements and herbal medicinal products do to the many biological properties of verbascoside. In addition, the antioxidant property of the lemon verbena infusion was evaluated by the DPPH test using Trolox as the reference compound.

Liposomes as carriers for verbascoside: stability and skin permeation studies.

In this study the influence of liposomal incorporation on both the stability and the in vitro (trans) dermal delivery of verbascoside was evaluated. The effect of drug entrapment into vesicles on its radical scavenging activity was also studied. Liposomes were obtained from soy phosphatidylcholine and cholesterol according to the film hydration method. Stability of verbascoside-loaded vesicles was studied over 6 months. Results showed that verbascoside can be incorporated in liposomes (E% = 57-66%), preventing its degradation. Stability studies (dynamic lager light scattering [DLLS] measurements and transmission electron microscopy [TEM] visualization) pointed out that vesicles were stable for 90 days and neither verbascoside leakage nor vesicle size alteration occurred during this period. The effects of vesicular incorporation on verbascoside diffusion through skin were investigated in vitro using newborn pig skin. Results showed that liposomes promoted drug accumulation into the stratum corneum but they did not give rise to any significant transdermal verbascoside delivery. Finally, results obtained from a 1, 1-diphenyl-2-pierylhydrazyl (DPPH) radical assay demonstrated that liposomes did not interfere with the radical scavenging activity of verbascoside.

Supercritical extraction of carotenoids from Rosa canina L. hips and their formulation with beta-cyclodextrin.

The purpose of this research was to preliminary assess the suitability of a new method for the preparation of a solid formulation in form of powder composed by beta-cyclodextrin and the supercritical extract of Rosa canina hips. The method implies the extraction of carotenoids, in particular beta-carotene, from freeze dried fruits of R. canina with supercritical CO2 at 70 degrees C and 300 bar, in the presence of varying quantity of ethanol as entrainer. The obtained supercritical solution is then expanded at ambient conditions into an aqueous solution of beta-cyclodextrin to favour the interaction between beta-cyclodextrin and the lipophilic components of the extract. beta-carotene solubility (mole fraction) in supercritical CO2 or in supercritical CO2/ethanol mixtures were in the order of 1 10(-7). The beta-carotene extracted from R. canina fruits (nearly 10 microg/g of dry matrix), interacts almost quantitatively with beta-cyclodextrin affording a solid phase, which presents a low apparent solubility in water. Finally the interaction with beta-cyclodextrin results in a higher concentration of the beta-carotene trans- form relative to the cis- form in the extracted product when collected in an aqueous solution of beta-cyclodextrin with respect to the extract in n-hexane.

Analysis and stability of the constituents of St. John's wort oils prepared with different methods.

St. John's wort is a medicinal plant with a long history of use in traditional medicine all over Europe. Traditional preparations and in particular the infused oil from SJW flowers remains one of the most popular and curative topical remedy against ulcerations and burns. The presence of the characteristic polyprenylated acylphloroglucinol derivatives, namely hyperforin and analogs are instead related to the oil's therapeutic activity. Indeed, it is well known that hyperforin has a potent antibacterial activity. In this study we tried to rationalize the production system of the oily preparation in order to obtain the highest concentration and stability of phloroglucinols. Five different samples of SJW oils were evaluated by HPLC-DAD-MS analysis to verify the variability and stability of the constituents according to the following factors: different harvesting time, fresh or dried plant material, use of sunlight or heating systems during extraction. The stability of these oils during 1 year was also tested.

Analysis of condensed and hydrolysable tannins from commercial plant extracts.

High performance liquid chromatography (HPLC)/DAD and MS qualitative and quantitative analyses of polyphenols, hydrolysable and condensed tannins from Pinus maritima L. and tannic acid (TA) extracts were performed using normal and reverse phase. Normal-phase HPLC was more suitable for pine bark (PBE) and tannic acid extracts analysis. The chromatographic profile revealed that P. maritima L. extract was mainly composed by polymeric flavanols (containing from two to seven units) and tannic acid (characterized by a mixture of glucose gallates containing from three to seven units of gallic acid). Concerning their antimycotic properties, P. maritima L. extract exhibited a broad activity towards yeast strains of the genera Candida, Cryptococcus, Filobasidiella, Issatchenkia, Saccharomyces: MICs from 200 to 4000 microg/ml (corresponding to 140-2800 microg/ml of active polyphenols) were determined. Conversely, no activity of tannic acid was observed over the same target microorganisms. Taken into consideration the above results of HPLC analysis and on the basis of the current literature, we may conclude that only 70.2% of polyphenols (recognized as condensed tannins) occurring in P. maritima L. extract can be apparently considered responsible for its antimycotic activity.

Evaluation of skin permeability of sesquiterpenes of an innovative supercritical carbon dioxide Arnica extract by HPLC/DAD/MS.

Recently, a supercritical carbon dioxide dried extract of Amica flower, with a very high sesquiterpene content was developed. In view of using this extract in formulations for cutaneous application, the ability of sesquiterpenes to permeate the skin was evaluated by HPLC/DAD/MS using the following permeation enhancers: oleic acid (OA), dimethylsulfoxide (DMSO), lauroglycol, isopropyl myristate and Tween 80. A skin permeation study was performed using a modified Franz diffusion cell and the human stratum corneum and epidermis as membrane. Solutions of the enhancers were directly analysed after dilution with methanol or DMSO. A simple RP-HPLC-DAD-MS method for the quantification of the sesquiterpenes was developed and the method showed no interference with the other substances extracted from the skin and the permeation enhancers. The study evidenced that among the selected skin permeation enhancers, DMSO and OA canbe considered as good candidates to be used in preparations for cutaneous application.

Anti-inflammatory and immunologically active polysaccharides of Sedum telephium.

Two major polysaccharides, rhamnogalacturonans with mean M(r)s of 13,500 and 13,000, were isolated from dried leaves of Sedum telephium by column chromatography on DEAE-Sepharose CL-6B and gel filtration on Fractogel TSK HW-50 (S) and Sephacryl 200 HR. The structures were determined mainly by NMR spectroscopy, methylation analysis and GC-MS of the partially methylated alditol acetates, carboxyl reduction and by analysis of acidic and enzymatic degradation products. Both polysaccharides exert an anticomplementary effect in vitro, induce TNF-alpha-production, enhance phagocytosis in vitro and in vivo, and exhibit anti-inflammatory activity.

Inclusion complex formation of 1,8-dihydroxyanthraquinone with cyclodextrins in aqueous solution and in solid state.

Complex formation between cyclodextrins and 1,8-dihydroxyanthraquinone in buffer solution has been investigated using absorption, its second derivative (D2), and fluorescence spectroscopy. The results showed that whereas the self-association process was found for 1,8-dihydroxyanthraquinone alone, the monomeric form is microincluded in beta- and gamma-cyclodextrins. The interaction is more favored as the cavity size of cyclodextrins is larger, the molecule being more tightly bound with gamma- than with beta-cyclodextrin. The complex formation inhibits the excited-state intramolecular proton transfer process that has already been reported for 1,8-dihydroxyanthraquinone alone.

Flavone photoreactivity. UV-induced reactions in organic solvents and micellar systems.

Flavonoid photochemistry is a subject of interest in studies dealing with the role of phenolic compounds as screening pigments in plants. In order to contribute to the understanding of the processes involved in the interaction between flavonoids and UV radiation, we have studied UV-induced flavone photodegradation in both organic solvents and micellar systems. The results obtained show that flavone photosensitivity depends on the characteristics of the reaction environment and is influenced by the medium polarity and the charges on the micellar surface. Qualitative and quantitative differences in the photodegradation products were demonstrated by high performance liquid chromatography (HPLC) analysis.

Analysis of plant complex matrices by use of nuclear magnetic resonance spectroscopy: St. John's wort extract.

The efficiency of two-dimensional homonuclear (1)H--(1)H correlated spectroscopy and two-dimensional reverse heteronuclear shift correlation spectroscopy (i.e., heteronuclear multiple quantum correlation) in characterizing and evaluating the relative content of herbal extract constituents is demonstrated. These experiments are able to fully assign the proton and carbon resonances of all three classes of constituents present in dried commercial extract of St. John's wort, that is, flavonols, phloroglucinols, and naphthodianthrones, with particular regard to the very unstable phloroglucinols. In addition, shikimic and chlorogenic acids, sucrose, lipids, polyphenols, and traces of solvents of the extractive process (methanol) were also identified. These experiments can be considered to be a very simple and fast analytical method for determining the quality and stability of the titled commercial extract. They represent a generally applicable technique for a rapid screening and a specific measurement of other commercial phytochemicals or, in selected cases, an alternative to the classical analytical techniques such as high-performance thin-layer chromatography, high-performance liquid chromatography, capillary gas chromatography, and electrophoresis.

Polyphenolic content in five tuscany cultivars of Olea europaea L.

In this study polyphenolic compounds extracted from olive fruits of five registered cultivars were analyzed. A solid-liquid extraction (LSE) procedure with Extrelut cartridge (diatomaceous earth) using different eluents was developed to obtain polyphenolic compounds. HPLC-DAD and HPLC-MS methods were applied for the quali-quantitative analysis of each fraction obtained from LSE. The results of this work show that the LSE procedure with diatomaceous earth cartridge supplies a rapid and reproducible fractioning method able to obtain a quantitative recovery of all compounds and to collect fractions directly analyzed by HPLC. A comparison among different cultivars shows significant quantitative differences in some polyphenols, such as verbascoside, anthocyanic compounds, and oleuropein derivatives.

Comparison among differential pulse voltammetry, amperometric biosensor, and HPLC/DAD analysis for polyphenol determination.

Polyphenols are widespread in vegetables and fruits. They can play an important role in human diet and health, and they influence the sensorial properties of many foods, and act as natural antioxidants. This study was conducted using HPLC/DAD, tyrosinase biosensor, and differential pulse voltammetry (DPV) analyses to detect polyphenolic compounds in natural complex matrices. The analyses were applied to a series of both standards and natural extracts derived from grape, olives, and green tea. The pure compounds include phenolic acids, flavones, flavonols, catechins, tannins, and oleuropein. HPLC/DAD, DPV, and the biosensor approach were used as independent analytical techniques. Bare graphite screen-printed electrodes were employed in DPV and in the biosensor analysis. The most accurate data were obtained by HPLC/DAD analysis, while the DPV approach using screen-printed electrodes could represent a quick screening method for the determination of polyphenols in natural extracts. Use of the biosensor for the analysis of complex matrices needs further study in order to improve its performance.

Chemical composition of some traditional herbal drug preparations: essential oil and aromatic water of costmary (Balsamita suaveolens Pers.).

The compositions of the essential oil and the aromatic water of costmary (Balsamita suaveolens Pers.) cultivated in Tuscany were investigated. They represent the main ingredients of some traditional preparations sold commercially. The essential oil as such and the n-hexane extract of the aromatic water were analyzed by GC and GC-MS. Both samples were found to be rich in monoterpenes. Eighty-five compounds were identified, accounting for 95.1 and 95.4% of the essential oil and n-hexane extract of aromatic water, respectively. Carvone was the main compound (43.5% in the essential oil and 74.9% in the n-hexane extract of aromatic water). In addition, solid phase microextraction was used to sample the volatile organic compounds emitted from the fresh plant and from the aromatic water, and carvone was again the main component, amounting to 46.2 and 41.3%, respectively.

Identification by HPLC-DAD and HPLC-MS analyses and quantification of constituents of fennel teas and decoctions.

Qualitative and quantitative differences among the constituents in various fennel (Foeniculum vulgare Mill., family Apiaceae) teas prepared by classical infusion, microwave decoction, and dissolution are reported. Different commercial starting materials, such as fruit (unbroken and crushed), four herbal teas, and two instant herbal teas were evaluated. Chlorogenic acid (1), quercetin-3-O-beta-D-glucuronide (2), p-anisaldehyde (3), and trans-anethole (4) were identified by HPLC-DAD and HPLC-MS as constituents of fennel teas. No coumarins, which are characteristic constituents of plants of Apiaceae family, were found. Trans-anethole (4), the main constituent of the essential oil, was present in all teas. In addition p-anisaldehyde (3), a degradation product of trans-anethole, was also identified in all teas with the exception of two samples. Chlorogenic acid (1) and quercetin-3-O-beta-D-glucuronide (2) were also present in all teas. In addition, minor unidentified flavonol constituents were found in two teas. Quality, activity, and safety of the content of the investigated preparations are also discussed.

HPLC analysis of flavonoids and secoiridoids in leaves of Ligustrum vulgare L. (Oleaceae).

Identification and quantification of flavonol glycosides and secoiridoids was carried out on leaves of Ligustrum vulgare L. (Oleaceae) by means of HPLC-DAD and HPLC-MS analysis. In addition to previously reported secoiridoids (oleuropein, ligustaloside A, ligustaloside B, and ligstroside) four kaempferol glycosides (kaempferol 3-O-glucoside 7-O-rhamnoside, kaempferol 3, 7-O-dirhamnoside, kaempferol 3-O-rhamnoside, and kaempferol 3-O-glucoside) and two quercetin glycosides (quercetin 3-O-glucoside 7-O-rhamnoside and quercetin 3,7-O-dirhamnoside) were present in leaves of L. vulgare L. Although secoiridoids accounted for nearly the 76% of the total leaf polyphenols content (with ligustaloside A as the main component), kaempferol glycosides were also accumulated in the leaves of L. vulgare L. to a relatively high extent (23%). Contribution of quercetin derivatives was minor under our experimental conditions. Our findings suggest that flavonol glycosides may have a central role in both the ecology and the biology of L. vulgare L.

Polyphenolic content in olive oil waste waters and related olive samples.

The production of olive oil yields a considerable amount of waste water, which is a powerful pollutant and is currently discarded. Polyphenols and other natural antioxidants, extracted from olives during oil extraction process, partially end up in the waste waters. Experimental and commercial olive oil waste waters from four Mediterranean countries were analyzed for a possible recovering of these biologically interesting constituents. Identification and quantitation of the main polyphenols were carried out by applying HPLC-DAD and HPLC-MS methods. Representative samples of ripe olives were also analyzed at the same time to correlate, if possible, their polyphenolic profiles with those of the corresponding olive oil waste waters. The results demonstrate that Italian commercial olive oil waste waters were the richest in total polyphenolic compounds with amounts between 150 and 400 mg/100 mL of waste waters. These raw, as yet unused, matrices could represent an interesting and alternative source of biologically active polyphenols.

Antioxidant and other biological activities of olive mill waste waters.

During olive oil production, large volumes of water are generated and subsequently discarded. Olives contain a variety of bioactive components, and some of them, according to their partition coefficients, end up in the water phase. The current investigation aimed at comparing different methods for the extraction of biologically active components of the olive mill waste waters (OMWW) and evaluating the in vitro antioxidant and anti-inflammatory activities of the resulting extracts. The results indicate that OMWW extracts are able to inhibit human LDL oxidation (a process involved in the pathogenesis of atherosclerosis) and to scavenge superoxide anions and hypochlorous acid at concentrations as low as 20 ppm. Finally, two of the three extracts also inhibited the production of leukotrienes by human neutrophils. The potency of the extracts depended on their degree of refinement: extracts containing only low molecular weight phenols were the most effective.

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. John's 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. John's 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.

Stabilisation of natural anthocyanins by micellar systems.

In this paper, we discuss the influence of different micellar systems on the degradation of natural anthocyans, either glycosides and aglycones, at pH values ranging from 2.8 to 6.0. The interaction of anthocyanins, in suitable dispersed systems such as negative micelles of sodium dodecylsulphate (SDS), consistently increased their chemical stability in aqueous solutions. The results of these experiments point out how both the number of available negative charges and the presence of an organised distribution of the negative charges on the micellar surface appear to be necessary conditions to achieve the anthocyanins' stability and colour retention. The sodium dodecylbenzensulphonate (SDBS), containing an aromatic ring near the negative surface of the micelle, seems to increase the rate of decomposition. Preliminary findings of circular dicroism (CD) investigation allowed us to hypothesise that these pigments undergo an intermolecular self-association process induced by the SDS micelles and this phenomenon presumably contribute to increase stability.