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In the present study an HPLC-DAD method was developed for the determination of the main carotenoid, zeaxanthin dipalmitate, in the fruits of Lycium barbarum. The aim was to develop and optimize an extraction protocol to allow fast, exhaustive, and repeatable extraction, suitable for labile carotenoid content. Use of liquid N2 allowed the grinding of the fruit. A step of ultrasonication with water removed efficiently the polysaccharides and enabled the exhaustive extraction of carotenoids by hexane/acetone 50:50. The assay was fast and simple and permitted the quality control of a large number of commercial samples including fruits, juices, and a jam. The HPLC method was validated according to ICH guidelines and satisfied the requirements. Finally, the overall method was validated for precision (% RSD ranging between 3.81 and 4.13) and accuracy at three concentration levels. The recovery was between 94 and 107% with RSD values2%, within the acceptable limits, especially if the difficulty of the matrix is taken into consideration.

This study focused on herbalists, herbal shops and people currently using traditional Arabic medicine. Informants were asked to list plants and methods for obtaining plant herbal extracts used to manage a range of illnesses. A total of 109 plants were identified, of these, principle plant elements included, leaves (47.3%), fruits (18.5%) and seeds (18.0%) were most commonly utilized. Extraction methods included decoction (boiling) 51%, and infusion 17% and prepared as creams, powders, syrups, food or cooked. Many plant species are used by herbalists for treating a range of ailments. This study suggests that a lack of methodological standardization during herbal extraction could compromise herbal stability. There is also a need to monitor for potential adverse drug interactions when used concurrently with prescribed medications.

Objectives This study reports on the rapid isolation of verbascoside from Lippia citriodora H.B.K. (Verbenaceae), an inexpensive and widespread source, and the evaluation of its antihyperalgesic activity. Methods Isolation of verbascoside was achieved by size exclusion chromatography with Sephadex LH-20 eluting with 50% EtOH, which is proposed as a fast and efficient method of separation. Key findings The antihyperalgesic activity of verbascoside was tested by in-vivo assay using the paw-pressure test in two animal models of neuropathic pain: a peripheral mononeuropathy produced either by a chronic constriction injury of the sciatic nerve (CCI) or by an intra-articular injection of sodium monoiodoacetate (MIA). Conclusions Verbascoside administered intraperitoneally at a dose of 100 mg/kg reverted the mechanical hyperalgesia in both CCI and MIA treated rats, as evaluated in the paw-pressure test. Verbascoside was also effective against mechanical hyperalgesia after oral administration at doses of 300 and 600 mg/kg.

In the present study, a method based on liquid chromatography with diode array detection (HPLC-DAD) coupled to an electrospray ionisation (ESI) interface was developed for the determination of the constituents in the aqueous preparations of Viola odorata L. flowering tops. The developed assay was fast, simple and effective and permitted the quality control of the preparations. The aim of this work was to assess the qualitative and quantitative profile of the investigated preparations, which find until today wide applications in food and cosmetic industry, and to propose a validated method for their quality control. Characteristic constituents of V. odorata flowers are considered to be the anthocyanins; however, a detailed literature research showed that data concerning their chemical content are scarce. HPLC-DAD-ESI-MS analyses supported by extensive preparative chromatographic investigations and 2D NMR analyses revealed the predominance of complex flavonol glycosides and permitted the complete characterisation of the content of V. odorata preparations. This is the first report of detailed analysis of the chemical composition of V. odorata flowers.

In the present study a method based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of phenolic constituents in three aqueous preparations of the herbal medicinal drug Stachys recta. The developed assay was simple and effective and permitted the quality control of S. recta decoctions and infusion. Overall, 30 constituents were detected and identified, belonging mainly to three classes of compounds: caffeoylquinic acids, phenylethanol glycosides and flavonoids. 15 of them were quantified having a lower limit not less than 0.02% of the lyophilized extracts. Only seven of them were previously reported in this species, while 23 were identified for the first time as constituents of S. recta. HPLC-DAD-ESI-MS analysis provided evidence for the certain identification of the main constituents and in some cases of their isomers. Eight constituents were isolated and their structure elucidated by HPLC-ESI-MS and 1D- and 2D-NMR spectroscopy. Among the investigated preparations, the infusion seems to be the best method to extract the native constituents of the plant, while decoction is a more aggressive treatment and causes partial degradation of some acylated flavonoids.

Background A number of scientific reports have investigated the possible implications of refined seed oils in allergic reactions, resulting in conflicting points of view. Also the total amount of residual proteins after refinement is still a matter of debate. Nevertheless, seed oils are now blamed as possible cause of allergic reactions. Objective To determine the true amount of proteins after oil refinement and to shed new lights on allergenic properties of refined seed oils. Methods We optimized a protein extraction procedure on several commercial refined seed oils. Both colorimetric and amino acid analysis were used to measure residual protein content. SDS–PAGE was also used for characterizations of protein band patterns. Sensitized child patients sera were tested by Western blot on PAGE-resolved proteins. Results Our extraction method proved to be effective and reproducible. Amino acid analysis resulted more accurate in determining the protein content with respect to colorimetric methods, indicating a higher protein content than that previously reported. IgE responsive residual proteins were found in peanut oil extracts. Conclusions Our preliminary data suggest that fully refined seed oils should be taken into account in the context of allergic reactions and would benefit of further toxicological studies.

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.

The flavonoidic fraction composition of the hydroalcoholic extract of Chrysobalanus icaco L. (Chrysobalanaceae) leaves, which are largely used in the traditional medicine in Northern Brazil to control the glycaemia of diabetic patients, was characterised. Myricetin 3-O-glucuronide (miricitrin) and quercitrin, among other minor myricetin derivatives, were evidenced by HPLC/DAD and HPLC/MS analysis. A fração flavonoídica do extrato hidroalcoólico de folhas de Chrysobalanus icaco L. (Chrysobalanaceae), que são largamente utilizadas na medicina tradicional do Norte do Brasil para controlar a glicemia de pacientes diabéticos, foi caracterizada. Miricetina-3-O-glucuronídeo (miricitrina) e quercitrina, entre outros derivados de miricetina minoritários, foram evidenciados por análises com sistemas CLAE/DAD e CLAE/EM.

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.

The reactions with hemoglobin of artemisinin and of its parent compounds, sodium artesunate and dihydroartemisinin, were investigated by visible absorption spectroscopy under standard solution conditions (50 mM phosphate buffer, pH 7, 37 degrees C). Notably, these antimalarial drugs were found to react with hemoglobin (i.e., ferrous heme), but not with methemoglobin (i.e., ferric heme). The reaction selectively occurs at the heme sites and consists of the progressive, slow decay of the Soret band, as a consequence of heme alkylation and subsequent loss of pi electron delocalization. For the various tested compounds the process reaches completion within approximately 30-70 h. Additional experiments were carried out upon adopting the solution conditions described by Meunier et al. and by Kannan et al. in their recent studies. Some reactivity of artemisinin with methemoglobin was indeed detected after addition of 50% v/v acetonitrile, most likely as a consequence of extensive protein unfolding. A unified description for the reactions of artemisinins with hemoglobin is given.

We describe chemical hydrolytic procedures to evaluate the total amount of tyrosol and hydroxy-tyrosol free and/or linked to secoiridoidic molecules (acid hydrolysis). At the same time a rapid determination of the lignans in complex minor polar compound (MPC) extracts is proposed (alkaline hydrolysis). High-performance liquid chromatography/diode array detection (HPLC/DAD) and HPLC/MS were applied as reference methods to evaluate the quantitative results from the hydrolysis experiments. The optimized acid hydrolysis procedures were first applied to an oleuropein standard and then to MPC fractions extracted from several commercial extra virgin olive oils. The results confirm the applicability of the method, consisting in the acid hydrolysis of complex mixtures of secoiridoidic derivatives, to determine the antioxidant potential in terms of MPC. These data can contribute to forecasting the potential ageing resistance of an extra virgin olive oil in terms of antioxidant potency. Finally, alkaline hydrolysis allows confirmation and easy determination of the amount of lignans, especially in those MPC fractions which are particularly complex. Copyright © 2006 Society of Chemical Industry

The antiradical activity, polyphenols, flavonoids and total condensed tannins contents have been determined in the case of seven local edible Brassicaceae , i.e. Italian kale, broccoli, Savoy and white cabbage, cauliflower, green cauliflower and Brussels sprouts. Rapid spectrophotometric methods were applied. The results achieved were compared with the quali–quantitative information obtained by HPLC/DAD and HPLC/MS. The polyphenolic compounds detected were: kaempferol and quercetin glycosides and hydroxycinnamic esters. The EC 50 values ranged from 81.45 to 917.81 mg sample/mg DPPH and the total phenolic content from 4.30 to 13.80 gallic acid equivalents (mg gallic acid/g sample). The peculiar characteristics of these vegetables can be evaluated and can increase their value as functional food.

Solid olive residues (SOR) are byproducts of the olive-milling process, but they have an increasing importance in the pharmaceutical industry due to their rich content of biophenols. Such compounds are studied widely for their antioxidant and antimicrobial activities, but there is a lack of information about their quantitative recovery. This research highlighted the key role played both by the selection of the cultivar and by the degree of olive fruit ripening on the phenolic content on the SOR. The extraction methods were selected to reach the best quantitative results mainly using a safe food solvent. In light of the results the Soxhlet extraction with ethanol could be proposed as preferential for a higher recovery of verbascoside and its analogues.

A rapid and inexpensive HPLC method has been developed for simultaneous separation of the three main classes of polyphenol in the leaves of Cistus salvifolius L. Time devoted to extraction of polyphenols, which was performed using small volume of solvent, did not exceed 120 min. We identified three ellagitannins (punicalagin and related compounds), a total of ten glycosyl derivatives of quercetin and myricetin, and two coumaroyl glucosyl kaempferols by use of both diode-array detection (DAD) and mass spectrometry. The polyphenol composition of C. salvifolius leaves, which may contribute to the metabolic plasticity of the species, may explain its distribution in infertile soils of the Mediterranean area, and may also indicate this shrub is an important source of metabolites of potential use in human health care.

Fresh aerial parts of different chicory varieties: green chicory (c.v. "Catalogna"), two red chicory varieties ("radicchio rosso di Chioggia" and "radicchio rosso di Treviso"), and Witloof or Belgian endive were analyzed by HPLC/DAD/MS. The chromatographic fingerprint was diagnostic for each variety. A monocaffeoyl tartaric acid, chlorogenic acid, and chicoric acid were detected in all the varieties, while cyanidin 3-O-glucoside, delphinidin 3-O-(6'' malonyl) glucoside, and cyanidin 3-O-(6'' malonyl) glucoside were the main phenolic compounds in the red varieties. The flavonoidic compounds, quercetin 3-O-glucuronide and luteolin 7-O-glucuronide, were absent in the Witloof sample. The phenolic compounds from total leaves were the same as those obtained from only the colored parts; nevertheless, the total amount was remarkably lower with a decrease of up to 80% for Belgian endive. Chemical stability at high temperature was observed for the phenolic fraction from the green variety after decoction at 100 degrees C for 30 min.

This paper focuses on comparing the main chemical characteristics of 16 fresh commercial samples of extra virgin olive oil obtained from four harvest years (1999–2002) and derived from both stoned and whole fruits. The qualitative and quantitative contents of minor polar compounds (MPCs) together with other reference analytical parameters (acidity, peroxides, UV absorption values and Rancimat test) were evaluated. An investigation of the MPCs and oil composition obtained from only stoned olives was also carried out. The acidity values of the oils from stoned fruits were always similar to or lower than those of the corresponding oils from whole fruits. For almost all the samples from stoned olives a better resistance to oxidation was revealed in comparison with the corresponding traditional oils. Five pairs obtained from the 2000 and 2001 harvests showed higher concentrations of both MPCs and hydroxytyrosol derivatives in the oils from stoned fruits, in agreement with their higher Rancimat values. Overall, our findings with regard to acidity values, % hydrolysis, the Rancimat test and the qualitative and quantitative distribution of MPCs suggest a higher antioxidant capacity of the oils from stoned olives. Copyright © 2004 Society of Chemical Industry

The mechanism of action of trioxane antimalarial drugs is still largely controversial and warrants further investigation. We report here on the direct reaction of artemisinin with hemin, carried out in DMSO, in the absence of reducing agents. The reaction was analysed, independently, by visible spectroscopy, HPLC-ESI/MS and 1H NMR. Two isomeric artemisinin–hemin long-lived adducts are unambiguously detected. Eventual degradation of the porphyrin ring and loss of the Soret band are observed as well. Implications of the present results for the mechanism of action of artemisinin-based antimalarials are discussed.

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.

Virgin olive oil is a typical component of the Mediterranean diet, consumed unrefined and rich in important molecules, such as minor polar compounds (hydroxytyrosol, tyrosol, secoiridoids and flavonoids) and fatty acids. These molecules not only influence the sensorial properties of both olives and virgin oil but they are also important markers for typicity, biodiversity and quality determination of this product. The aim of this study was to evaluate the minor polar compound and fatty acid contents of 10 monocultivar virgin olive oils, typical of Tuscany, in order to have better knowledge about the quali-quantitative profiles of these compounds in samples obtained from both the same collecting season and same processing technique. Quali-quantitative analysis (performed by HPLC/DAD, HPLC/MS and GC) could be a useful tool to better correlate the typicity of the virgin olive oil with its minor polar compound and fatty acid pattern. Further studies are in progress to isolate the unknown compounds and to further investigate the quality index of this food product.

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.

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.

The efficiency of one- and two-dimensional NMR experiments in characterizing the content of the constituents of both herbal drugs and herbal drug preparations is demonstrated for kava-kava. These experiments directly detect active constituents represented by kavalactones in both a finely powdered herbal drug and a commercial extract. In addition, NMR spectroscopy can detect all other compounds present in the extract. As previously evidenced, NMR experiments can represent a generally applicable technique for rapid screening and are a complement to the classical analytical techniques such as high-performance thin-layer chromatography, high-performance liquid chromatography, capillary gas chromatography, and electrophoresis. These experiments can be considered a very simple and fast analytical method to obtain a fingerprint of the herbal drugs and their preparations, and to quantify the content of the active principles of the extract.

St. John's wort (Hypericum perforatum L.) is a medicinal plant traditionally used, both externally and internally, in all Europe for many pathologies. Paracelsus named it "arnica of the nerves" because of its empirical use in nervous diseases. In the last two decades many studies have proved the efficacy of some St. John's wort extracts in mild to moderate depression and it has been successful as an antidepressant both in Europe and the US. Its high efficacy and tolerability is unquestionable and from the clinical studies the activity is comparable to other antidepressants while lacking major side effects, making it a safe antidepressant.However, recently its potential to induce the metabolism of co-administered medications has been reported because it may potentate certain enzymes of the cytochrome P450 enzyme system. This resulted in a lowering of serum concentration of a number of concomitant drugs, including warfarin, digoxin, theophylline, cyclosporin, and indinavir. Many drugs and also several common foods and drinks can influence this enzyme system. So, even if its safety has been well established, physicians should be aware that St. John's wort administration might significantly affect other prescribed medicines.

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.

Malaria is a major health problem in many countries and according to an estimate of the WHO, more than 500 million infections occur per year. Artemisinin, a sesquiterpene from Artemisia annua L., has received considerable attention as a promising and potent antimalarial drug for its stage speciticity, its rather low toxicity, effectiveness against drug-resistant Plasmodium species and activity against cerebral malaria. From recent studies it seems that hemin is primarily involved in the antimalarial activity of the constituents of Artemisia annua L. Thus, the interaction of a compound with hemin may represent a crucial screening test to define its efficacy. In this study the interaction between artemisinin and hemin was investigated by UltraViolet/Visible (UV/Vis) spectrophotometry and High Performance Liquid Chromatography/Diode Array Detector/Mass Spectrometry (HPLC/DAD/MS). In addition, some flavonols isolated from Artemisia annua L. were also tested to investigate their possible role in the interaction between artemisinin and hemin. These two simple physico-chemical methods can be useful as rapid and widespread screening methods for the search of other alkylating antimalarial constituents from natural sources or for the evaluation of the activity of semisynthetic analogues of artemisinin.

Infusions and decoctions of three chemotypes of Lippia alba (Mill.) N. E. Brown (Verbenaceae) were investigated for their quantitative profiles by HPLC–DAD–ESI-MS analyses. An RP-HPLC method was developed which permitted the quality control of the preparations. The correct choice of the column allowed the detailed characterization of the constituents in a total analysis time of 35 min. The HPLC method was accordingly validated for linearity range, LOD, LOQ, accuracy and precision. For the quantitative analysis the three major phytochemical groups were taken into consideration, namely iridoids, phenylpropanoids and flavonoids. Comparative quantitative analyses revealed significant differences among the chemotypes that should be taken into account in the uses of the herbal teas. The developed HPLC-UV assay proved to be an efficient and alternative method for the discrimination of the three chemotypes. This is the first report of detailed analysis of the chemical composition of the constituents of L. alba chemotypes’ teas.

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.

The efficiency of two-dimensional homonuclear 1H−1H 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 comme...

Vitamin C behaves as a very powerful radical scavenger in aqueous media and can be successfully used to prevent peroxidation of important unsaturated organic molecules. Its hydrophobic derivatives (i.e. alkyl-esters and ethers) perform the same antioxidant activity in self-assembled structures such as micelles and microemulsion systems. In this work the study on the micellar properties obtained from these hydrophobic derivatives by viscosity, light-scattering, and small-angle neutron-scattering (SANS) experiments, and a comparison between the antioxidant activity of the vitamin C derivatives and the most common natural antioxidants are reported.

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.

Matricaria recutita L. is a spontaneous herbaceous perennial plant and its drug is largely used, as an infusion, for its anti-inflammatory properties, especially for respiratory and gastroenteric tracts. Granular soluble extracts of this drug are also used in children’s diets.

A liquid-solid extraction and purification procedure (LSE) was developed to identify and quantify polyphenols in the leaf tissue ofMyrtus communis L. Identification and quantitation of individual compounds was performed using HPTLC, HPLC-DAD and HPLC-MS analysis. Leaves ofMyrtus communis L. contain small amounts of phenolic acids (caffeic, ellagic and gallic acids) and quercetin derivatives (quercetin 3-O-galactoside and quercetin 3-O-rhamnoside), whereas catechin derivatives (epigallocatechin, epigallocatechin 3-O-gallate, epicatechin 3-O-gallate) and myricetin derivatives (myricetin 3-O-galactoside, myricetin 3-O-rhamnoside) are present in large amounts. This is the first report on the occurrence of galloyl-derivatives of catechin and gallo-catechin inMyrtus communis L. leaves.

The effects of gamma irradiation on cascara bark were studied. Induced degradation products and modification in cascaroside ratio were investigated by a dedicated HPLC method. The presence of free radicals was evaluated by EPR technique. Both HPLC and EPR measurements were performed at different times up to four months after the irradiation process.

Establishment of molecular modelling able to simulate the behaviour of the β CyD-complexes in aqueous solution was studied by applying the fundamentals of classic mechanics. The existence of stable complexes was experimentally determined by the findings of 1 H NMR NOE (Nuclear Overhauser Effect) experiments in aqueous solution. A `training set' of molecules was chosen to test the ability of the mathematic models to describe the behaviour of β CyD-complexes. A `test set' group of molecules was defined to better confirm the validity of the proposed models. Based on the results, molecular modelling seems to be a useful tool for forecasting the behaviour of β CyD-complexes in water solution since the results of the mathematic models are close to the experimental findings obtained from NOE experiments.

Liquid-liquid (LLE) and liquid-solid extraction (LSE) procedures were developed to identify polyphenols in phillyrea (Phillyrea angustifolia L.) leaves. The liquidsolid extraction, carried out by using two serial Bond-Elut® CH and Bond-Elut® SAX cartridges allowed the collection of both polyphenols and soluble carbohydrates during a single extraction of leaf. This method seems to be suitable for both phytochemical and physiological study of the species, allowing the identification of two classes of organic compounds which have a central role in the metabolism of phillyrea plants. A method of isolation and concentration of polyphenols for further molecular characterization, using centrifuge chromatography, was also developed. Molecular characterization, carried out by UV-Vis spectrophotometry and mass spectrometry, demonstrated the presence of compounds with interesting biological activity, i.e. flavonoids and oleuropein derivatives. The soluble carbohydrate composition of phillyrea leaves did not substantially differ from that ofOlea europaea L. leaves, except for a higher mannitol/glucose ratio. This carbohydrate distribution may be linked to the evolution pattern of this species which usually grows in severely stressed environments.

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.

Four new diterpenoids, helioscopinolides F (2), H (4), I (5), and L (6), were isolated from three different cultured cell lines of Euphorbia calyptrata var. involucrata. Helioscopinolide B (1) and jolkinolide E (3), previously found in Euphorbia helioscopia and Euphorbia jolkini, respectively, were also isolated from the same cultures. Structures were identified by spectroscopic methods.

Aqueous alcoholic mallow flower extracts were analyzed both by HPTLC-densitometry in the reflectance mode at 530 nm and by reversed-phase HPLC with gradient elution. For the mallow flower anthocyanins the best chromatographic resolution was obtained by HPLC, which revealed only two main compounds, confirmed by FAB-MS: malvidin 3,5-O-diglucoside (malvin) and malvidin 3-O-(6"-O-malonylglucoside)-5-O-glucoside. The HPTLC densitometric method on cellulose plates provides accuracy, reproducibility and selectivity for the quantitative analysis of the anthocyanins and this method was shown to be much more sensitive than the HPLC-DAD system, at 530 nm. Both methods give comparable quantitative results for total anthocyanins when applied to mallow flowers from two different sources: Italy and Albania.

An HPLC/MS application to anthocyanic compounds of Vitis vinifera L. is described. A series of liquid-liquid and liquid-solid extractions yielded an extract containing anthocyanic compounds only. This extract was analyzed by HPLC/DAD and HPLC/MS, and UV-vis and MS spectra of each compound were obtained. An API ion-spray interface allowed coupling between the chromatographic system and a mass spectrometer. This interface allowed application of the chromatographic conditions normally used in the HPLC analysis of anthocyanic compounds, which led to the identification of the 3-glucosides, the 3-acetylglucosides, and the 3-p-coumaroylglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin, already known in the literature. Two 3-caffeoylglucoside derivatives were identified too, and it has been possible to identify, for the first time, some 3,5-diglucosides that are known to be present in Vitis sp. but not in Vitis vinifera L. The investigated cultivars showed the same anthocyanic profile but dramatic quantitative differences.

Negative-ion fast-atom bombardment mass spectrometry together with precursor and product-ion spectroscopies has been validly employed in the identification of six flavonol glycosides present in the juice of Sedum telephium leaves. The proposed technique proved to be a valuable alternative to the combination of high performance liquid chromatography and mass spectrometry in the analysis of the complex polyphenol mixtures present in some medicinal plants.

The technical standardization of an intermediary of a phytomedicine is a decisive step for the quality standard that the product will show. After the validation of the alleged use, popular form of use, posology and agronomic certification of the plant material, for which phytochemical and pharmacognostic pattern will be developed to monitor the physical, chemical and physico-chemical characteristics of the plant, ensuring homogeneity of samples and the similarity to specimens tested in experimental stage. The plant material is then extracted and the obtained extract is used in the development of the formulation. This extract must be in accordance to the Brazilian Pharmacopoeia, respecting as far as possible the characteristics of the popular form of use. Before the next step, the pharmacotechnical handling, the extract will be phytochemically analysed, in order to determine the metabolic substance classes present in the sample. Then it must be pharmacologically investigated to assure that its activity, is similar to that originally alleged and experimentally tested; its physical and physico-chemical characteristics will be determined, and, subsequently, the extract undergoes chemical and chromatographic analysis to have identified substances that could characterize the plant and serve as chemical and, preferably, pharmacological quality markers. Substances which are chemically stable, responsible for the activity to be presented by the phytomedicine and able to be detectable and quantifiable by usual analytical methods, such as chromatography and ultraviolet spectroscopy or mass spectrometry, are potential candidates to be used as markers.

Qualitative and quantitative analyses of carbohydrates in olive (Olea europaea L.) tissues have been carried out by HPLC-RI. Sample purification was by two successive solid-liquid extractions to remove completely plant phenolics and pigments. Five carbohydrate peaks; sucrose (stachyose + raffinose + sucrose), glucose, galactose, fructose (fructose + myo-inositol) and mannitol were detected when plant extracts were run on a Sugar SC 1011 column operating at 75°C, using water as eluent 0.5 ml min−1. The use of two serial Sugar SC 1011 columns operating at 90°C and eluting the plant extracts with H2O−CH3CN (95/5 v/v) enables identification and quantification of nine carbohydrates, including tetra and tri-saccharides.

The use of Traditional Arabic Medicine (TAM) for various diseases has been popular but scarcely studied in Syria. In the present study, we carried out ethnobotanical and ethnopharmacological research on the plants traditionally used to cure various diseases in northern Syria. The information was collected from the city and villages of the Aleppo governorate “Mohaafazah” in the north of Syria, collecting data directly on the basis of a detailed survey of inhabitants and herbalists. In this survey, we found that hundreds of plant species are still in use in TAM for the treatment of various diseases. We selected the most common 100 species, used in the treatment of more than 25 diseases. Among these plants, 53 are used for treating gastrointestinal disorders, 38 for respiratory system diseases, including asthma, bronchitis and cough, 34 for skin diseases, 21 for diabetes, 17 for kidney and urinary disorders, 16 for cardiac disorders, 14 for infertility and sexual impotency, 13 for treating liver diseases, 13 for several types of cancer, 9 for enhancing breast milk excretion, 8 for weight loss, 5 for reducing cholesterol, and three for weight gain. Plants were collected and identified: scientific Latin names, local names, the used parts of the plant, the herbal preparations and the local medical uses are described. Scientific literature concerning the activity of the investigated species is also reported and discussed according to their traditional uses.

The concerted applications of a variety of one- and two-dimensional NMR techniques (COSY, NOESY, HET-COR, COLOC, etc.) to the structural elucidation of the natural 3,4-bis[(3,4-dimethoxyphenyl)methyl] dihydro(3R-trans)-2(3H)-Furanone, isolated from Oenanthe aquatica fruits, in CDCl 3 solution are reported. The magnitude of the 1 H, 1 H coupling constants obtained from the simulated spectrum together with a series of homonuclear NOE difference spectra were used to determine the full stereochemistry of the system

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.

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.

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.