HPLC-DAD and HPLC-ESI-MS analyses of Tiliae flos and its preparations.

In the present study extensive HPLC-DAD, HPLC-ESI-MS and NMR analyses were undertaken in the aqueous preparations (decoctions, infusions) and tinctures of Tilia platyphyllos Scop inflorescences. The aim of this work was to examine in depth the qualitative and quantitative profile of the investigated preparations, which find until today wide applications in pharmaceutical and cosmetic industry, and to propose a validated method for their quality control. An HPLC-DAD-ESI-MS method was developed and optimised for the quantitative determination of the constituents. Marker constituents of Tiliae flos are the flavonoids, while the volatile content is also used for the quality control. However, the analyses of the non-volatile fraction gave complex chromatographic fingerprints containing simple phenolics and low molecular weight procyanidins. The use of different HPLC columns permitted a good separation of the constituents and enabled their quantitation, while HPLC-MS analyses permitted the detection of procyanidin oligomers. Overall, 31 constituents were detected and identified. Extensive preparative chromatographic investigations and 2D-NMR analyses allowed the characterisation of procyanidins as epicatechin derivatives. Finally, the HPLC method was validated and complied with ICH guidelines. This is the first report of detailed analysis of the chemical composition of Tiliae flos.

Cyclodextrins as carriers for kavalactones in aqueous media: spectroscopic characterization of (S)-7,8-dihydrokavain and beta-cyclodextrin inclusion complex.

Kavalactones represent the active constituents of kava-kava (Piper methysticum G. Forster), endowed with sedative and anaesthetic properties. Kavalactones are polar constituents, but poorly soluble in water with a low bioavailability. In this study, the formation of inclusion complexes of one of the most representative kavalactone isolated from kava-kava extract, (S)-7,8-dihydrokavain (DHK), with beta-cyclodextrin (beta-CyD) was investigated mainly by spectroscopic methods. NMR experiments were extensively used for the complete characterization of the complex and included (1)H NMR complexation shifts analysis, (1)H NMR diffusion measurements (DOSY), and ROESY experiments. In particular DOSY experiments demonstrated that in the presence of beta-CyD the translational diffusion of kavalactone is sizably slowed down (2.5x10(-10)m(2)/s) with respect to the free drug (4.4x10(-10)m(2)/s) according to the inclusion of DHK in the cavity of (beta-CyD). ROESY experiments confirmed the inclusion of DHK in the hydrophobic pocket of beta-CyD through the primary hydroxyl rim, being the most relevant interactions between the H3' of beta-CyD and the ortho protons on the phenyl ring of the DHK, and between H5' of beta-CyD and the meta/para protons of DHK phenyl ring. The inclusion of the phenyl ring of DHK, leaving the lactone moiety outside of CyD was also confirmed by the induced CD effects. The binary solution DHK/beta-CyD shows a 50% intensity increase of the negative band of the pi-pi* transitions of the phenyl ring with respect to the absorption observed with DHK alone. Molecular dynamics simulations results corroborated and further clarify observed spectroscopic data. It was found that the phenylethyl substituent at C6 has a preferential equatorial position in the free state, and an axial one in the complex, justifying the large downfield shift experienced by H6 of DHK upon binding. Finally the influence of beta-CyD on water solubility of DHK was investigated by phase-solubility studies. In the range 2-4mM of host, solubility of DHK was increased only two-fold, but being beta-CyD also a penetration enhancer, in vivo studies will be performed to clarify a possible role of the complex on the bioavailability of DHK.

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.

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.

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.

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.

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.

Simultaneous analysis of artemisinin and flavonoids of several extracts of Artemisia annua L. obtained from a commercial sample and a selected cultivar.

Artemisia annua L. (Qinghao) is a promising and potent antimalarial herbal drug. This activity has been ascribed to its component artemisinin, a sesquiterpene lactone that is very effective against drug-resistant Plasmodium species with a low toxicity. Our studies indicate that several flavonoids of A. annua can promote and enhance the reaction of artemisinin with hemin. These data are in good agreement with previous investigations on the in vitro potentiation of antimalarial activity of artemisinin by such flavonoids. As a consequence, in view of a possible use of the phytocomplex rather than pure artemisinin, an HPLC/DAD/MS method is proposed for the simultaneous detection and quantification of both flavonoids and artemisinin. Different extracts, obtained from two different herbal drugs, a commercial sample and a selected cultivar, were analyzed in order to determine which solvents provide the best yields of both artemisinin and flavonoids. Qualitative and quantitative results obtained using an HPLC method are described, which will be useful for developing highly effective herbal drug preparations.

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.

An optical DNA-based biosensor for the analysis of bioactive constituents with application in drug and herbal drug screening.

The efficient and rapid detection of bioactive compounds in complex matrices of different origins (natural or synthetic) is a key step in the discovery of molecules with potential application in therapy. Among them, molecules able to interact with nucleic acids can represent important targets. In this study, an optical DNA biosensor, based on surface plasmon resonance (SPR) transduction, has been studied in its potential application as new analytical device for drug screening. This device was applied to the analysis of pure synthetic or natural molecules and also to some fractions obtained by chromatographic separation of an extract of Chelidonium majus L. (great celandine), a plant containing benzo[c]phenanthridinium alkaloids having intercalating properties. The ability of these molecules to interact with the double stranded nucleic acid (dsDNA) immobilised on the sensor surface has been investigated. The optical sensing relies on the SPR-based bench instrument Biacore Xtrade mark and represents an example of multiuse sensor. The results obtained demonstrate the potential application of this device for the rapid screening of bioeffective compounds. The characteristics of the biosensor offer the possibility to be coupled to chemical analysis as in hyphenated technologies.

Commercial and laboratory extracts from artichoke leaves: estimation of caffeoyl esters and flavonoidic compounds content.

Artichoke leaf extracts are widely used alone or in association with other herbs for embittering alcoholic and soft drinks and to prepare herbal teas or herbal medicinal products. Despite this wide diffusion, the European Pharmacopoeia does not report an official method for the determination of the active principles of artichoke leaf extracts. This work reports a quali-quantitative determination by HPLC/DAD and HPLC/MS techniques of both cynnamic acids and flavonoids present in some artichoke leaf commercial extracts (Com) compared with two different laboratory extracts (Lab). Most of the commercial extracts showed a similar quali-quantitative pattern with a single exception having five-six times higher value. The quantitative data from the Italian Pharmacopoeia(IP) official method does not evaluate the flavonoidic fraction and showed an overestimation of the caffeoyl esters with respect to the HPLC/DAD results. The proposed HPLC/DAD method was able to completely characterize and quantify this matrix and represents a contribution to better quality control of these herbal extracts.

Disposable electrochemical sensor for rapid determination of heavy metals in herbal drugs.

Analysis of herbal drugs and extracts need rapid and affordable methods to assure the quality of products. The application of the electrochemical sensors in the field of quality control of herbal drugs, herbal drug preparations and herbal medicinal products appears very promising, advantageous and alternative to conventional methods due to their inherent specificity, simplicity and for the fast response obtained. This paper presents a proposal about the application of disposable electrochemical sensors associated with electroanalytical instrumentation for the detection of heavy metal analysis in herbal drugs. In particular samples of St. John's wort were analysed applying anodic stripping voltammetry. The content of Cd and Pb were evaluated. The ICP spectroscopy was used as reference method.

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

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. John's wort were investigated. Artichoke is largely used in hepatic disorders, while St. John's 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. John's 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.

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.

Variability in the content of the constituents of Hypericum perforatum L. and some commercial extracts.

Seven samples of Hypericum perforatum L. (St. John's wort) were collected throughout Tuscany; the dried extracts were assayed to determine the concentration of the constituents. Total flavonol content ranged from 4.58% to 15.90%; hypericins ranged from 0.05% to 0.11%; and hyperforins ranged from 1.37% to 20.80%. In addition, four commercially dried extracts were analyzed using the same high-performance liquid chromatographic (HPLC) method; their flavonol contents varied from 10.64% to 15.01%, hypericins varied from 0.03% to 0.20%, and hyperforins varied from 1.18% to 6.54%. The aim of this investigation was to evaluate the contents of the different constituents depending on environmental factors and drying and storage conditions of the wild samples. In addition, the contents of the constituents of the products available to the consumer that were related to quality and the relation of this to safety and efficacy were also evaluated.

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.

Identification of volatile constituents of Tambourissa leptophylla.

The volatile fraction of Tambourissa leptophylla fruit skin was extracted by petrol ether, purified by adsorption chromatography (LPC) and analysed by gas chromatographic-spectroscopic methods. 27 non-oxygenated terpene hydrocarbons and 10 oxygenated derivatives were identified. The most abundant components were: limonene (24.0%), cis-alpha-bergamotene (23.2%), delta-3-carene (8.2%), alpha-curcumene (6.0%), trans-alpha-bergamotene (5.1%), alpha-copaene (4.1%), alpha-pinene (4.0%), p-cymene (4.0%) and bicyclogermacrene (3.3%). The crude volatile fraction was tested in vitro against Cladosporium cucumerinum in direct bioautography on TLC plates on the basis of the antifungal use of fruit skin. Activity of petrol ether extract against this micro-organism was demonstrated.

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.

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.