Beneficial effects of polyphenol-rich olive oil in patients with early atherosclerosis.

PURPOSE: Diets rich in plant-derived polyphenols such as olive oil (OO) and/or catechins such as epigallocatechin 3-gallate (EGCG) have been shown to reduce the incidence of cardiovascular diseases, potentially by improving endothelial function, an important surrogate for atherosclerosis. The possible augmentation of endothelial function with the combined efforts of OO and EGCG is intriguing, yet unknown. METHODS: Eighty-two patients with early atherosclerosis (presence of endothelial dysfunction) were enrolled in this double-blind, randomized trial with 52 completing the study. The aim of the study was to compare the effect of a daily intake of 30 ml simple OO, with 30 ml of EGCG-supplemented OO, on endothelial function as well as on inflammation and oxidative stress after a period of 4 months. Endothelial function was assessed noninvasively via peripheral arterial tonometry (Endo-PAT®). RESULTS: After 4 months, when OO and EGCG-supplemented OO groups were combined, OO significantly improved endothelial function (RHI, 1.59 ± 0.25-1.75 ± 0.45; p < 0.05). However, there were no significant differences in results between the two olive oil groups. Interestingly, with OO supplementation there was a significant reduction in inflammatory parameters: sICAM (196 to 183 ng/mL, p = < 0.001); white blood cells (WBCs) (6.0 × 109/L-5.8 × 109/L, p < 0.05); monocytes (0.48 × 109/L to 0.44 × 109/L, p = 0.05); lymphocytes (1.85 × 109/L to 1.6 × 109/L, p = 0.01); and platelets (242-229 × 109/L, p = 0.047). CONCLUSIONS: Improvement in endothelial dysfunction in patients with early atherosclerosis in association with significant reduction in leukocytes may suggest an important role of early cellular inflammatory mediators on endothelial function. The current study supports one potential mechanism for the role of olive oil, independent of EGCG, modestly supplemented to a healthy cardiovascular diet.

Honey extracts inhibit PTP1B, upregulate insulin receptor expression, and enhance glucose uptake in human HepG2 cells.

Honey is a food known for its medical properties. In this work, we have studied the impact of different types of honey on insulin signalling pathway. We found that honey extracts inhibit the enzyme PTP1B, one of the main negative regulators of insulin receptor signalling. HPLC-MS analysis allowed us to confirm the presence of several natural PTP1B inhibitors in the honey extracts analysed. Statistical analysis methods show a correlation between specific 1H-NMR resonance frequencies/HPLC peaks and the inhibitory power of the samples. This finding will allow the prediction of the biological properties of honey samples applying relative simple analytical methods. Finally, we demonstrated that the treatment of HepG2 cells with honey extracts enhances the expression of insulin receptor, and stimulates glucose uptake. For the first time, our results demonstrate that bioactive components of honey could improve glycaemic control by both inhibiting PTP1B and stimulating the expression of insulin receptor in liver cells.

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.

Triterpenoids and ellagic acid derivatives from in vitro cultures of Camptotheca acuminata Decaisne.

The metabolic profile of secondary products in calli and cell suspension cultures of Camptotheca acuminata Decaisne was investigated and compared to that of the leaves and roots taken from the plant. Neither in vitro system produced the anticancer quinoline alkaloid camptothecin (CPT); they accumulated discrete quantities of polyhydroxylated triterpenoids, different from those found in the plant organs, and ellagic acid derivatives. Nine ellagic acid derivatives (1a-1d and 2a-2e) and eight triterpenoid acids (3a-3e and 4a-4c) were isolated and characterised. All the identified triterpenes were related to ursane- or oleanane-type skeletons and their concentrations rose to 4.5% in suspended cells.

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.

An in vitro evaluation of the antibacterial activity of the non-volatile phenolic fraction from rosemary leaves.

In this study, the aim was to evaluate the antimicrobial action of the non-volatile phenols of rosemary leaves against two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis). Three extracts with different phenolic compositions were tested. By the agar disc diffusion method, Gram-positive bacteria were more sensitive to the extracts, and S. epidermidis showed the highest inhibition zones. Overall, all the extracts tested by the broth dilution method showed higher activity than results from the agar disc diffusion method. The minimal bactericidal concentration values indicated that E. coli was the most susceptible strain. This study demonstrated that the flavonoidic fraction of rosemary leaves does not play a crucial role as antimicrobial agents against these microorganisms. The most active extract was characterised by the highest amount of non-volatile terpenoidic compounds.

Flavonoids accumulate in leaves and glandular trichomes of Phillyrea latifolia exposed to excess solar radiation.

Experiments were conducted on Phillyrea latifolia plants grown under a dense overstorey of Pinus pinea (shade plants) or on seashore dunes (sun plants) in a coastal area of Tuscany (42° 46' N, 10° 53' E). Total integrated photon flux densities averaged 1.67 and 61.4 m mol m-2 d-1 for shade and sun sites, respectively. A leaf morphological-structural analysis, a qualitative and quantitative analysis of phenylpropanoids of leaf tissue and leaf surface, and a histochemical localization of flavonoids were conducted. The area of sun leaves reached 57% of that of shade leaves, whereas leaf angle (ß), sclerophylly index (ratio of leaf d. wt:leaf area), and trichome frequency (trichome number mm-2 ) were markedly greater in leaves exposed to full solar radiation than in leaves acclimated to shade. The total thickness of sun leaves was 78% higher than that of shade leaves, mostly owing to a greater development of both palisade parenchyma and spongy mesophyll. The concentration, but not the composition, of leaf tissue phenylpropanoids varied significantly between sun and shade leaves, with a marked increase in flavonoid glycosides in sun leaves. Flavonoids occurred almost exclusively in the upper epidermal cells of shade leaves. By contrast, flavonoids largely accumulated in the upper and lower epidermis, as well as in the mesophyll tissue of leaves that were acclimated to full sunlight. Flavonoid glycosides were found exclusively in the secretory products of glandular trichomes of P. latifolia leaves exposed to high levels of light; luteolin 7-O- glucoside and quercetin 3-O-rutinoside were the major constituents. By contrast, verbascoside and an unidentified caffeic acid derivative constituted 72% of total phenylpropanoids secreted by glandular trichomes of shade leaves, whereas they were not detected in glandular trichomes of sun leaves. These findings suggest that the light-induced synthesis of flavonoids in glandular trichomes of P. latifolia probably occurs in situ and concomitantly inactivates other branch pathways of the general phenylpropanoid metabolism. This is the first report of the key role of glandular trichomes and of flavonoid glycosides in the integrated mechanisms of acclimation of P. latifolia to excess light.

Hydroxytyrosol, as a component of olive mill waste water, is dose- dependently absorbed and increases the antioxidant capacity of rat plasma.

Hydroxytyrosol is the most potent phenolic antioxidant of olive oil and olive mill waste water (OMWW) and its biological activities have stimulated research on its potential role in cardiovascular protection. However, evidence of the absorption of OMWW phenolics and on their possible in vivo activity has, until now, never been provided. Three groups male Sprague-Dawley rats were administered 1, 5, or 10 mg/Kg of the OMWW extract, respectively, providing 41.4, 207, and 414 microg/Kg of hydroxytyrosol, respectively. Urine was collected for 24 h and the urinary levels of hydroxytyrosol were quantified by mass spectrometry. Hydroxytyrosol was dose-dependently (R(2) = 0.95) absorbed and excreted in the urines mostly as a glucuronide conjugate. Further, the administration of an hydroxytyrosol-rich OMWW extract (10 mg/kg) to the rats was also associated with an increase of their plasma antioxidant capacity. Future experiments will eventually further clarify its metabolic fate and its in vivo actions.

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.

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.

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.

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.

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.

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.

HPTLC and reflectance mode densitometry of anthocyanins in Malva silvestris L.: a comparison with gradient-elution reversed-phase HPLC.

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.

Sedum telephium L. polysaccharide content affects MRC5 cell adhesion to laminin and fibronectin.

In traditional medicine the fresh leaves and juice of Sedum telephium L. are used as wound-healing promoters. Cell adhesion represents a primary event in wound repair and in tissue homeostasis, and therefore we have investigated the effect of Sedum juice and its main fractions, polysaccharides and flavonols, on human fibroblast (MRC5) adhesion to fibronectin and laminin. Our findings revealed that total Sedum juice strongly inhibited cell adhesion to laminin and fibronectin (EC50 1.03+/-0.12 mg mL(-1)). This anti-adhesive feature was concentrated mainly in the two polysaccharide fractions (EC50 values comprised between 0.09 and 0.44 mg mL(-1)). The flavonol fractions did not seem to contribute to this effect. A first attempt to elucidate the polysaccharide-related anti-adhesive feature of Sedum juice was also performed. The results confirmed that natural polysaccharides, with chemical structures different from heparin, were able to interfere with integrin-mediated cell behaviour and they contributed to the outstanding effects of Sedum juice and to the role of polysaccharides in cell-matrix interaction.

In-vitro antioxidant and in-vivo photoprotective effect of three lyophilized extracts of Sedum telephium L. leaves.

Sedum telephium L. is a medicinal plant used in antiquity to cure many types of inflammatory skin diseases. The leaves (without the external cuticle), are used to promote healing and reduce skin inflammation and pain, and contain various components. We found two major components: flavonol glycosides and polysaccharides, with molecular weight between 13,000 and 13,500 Da. We evaluated the in-vitro antioxidant and in-vivo skin photoprotective effects of three lyophilized extracts obtained from the juice of S. telephium L. leaves: a total lyophilized juice, a lyophilized flavonolic fraction, and a lyophilized polysaccharidic fraction. Two in-vitro models were used: the bleaching of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH*) radical, and the protective effect against UV-induced peroxidation on phosphatidylcholine multilamellar vesicles, as model membranes. The antioxidant/radical scavenging activity of each lyophilized extract was also assessed in-vivo by determining their ability to reduce UVB-induced skin erythema (monitored by reflectance spectrophotometry) in healthy human volunteers. The findings of the in-vitro experiments clearly demonstrated that, unlike the lyophilized polysaccharidic fraction, the lyophilized flavonolic fraction and total lyophilized juice possess strong antioxidant/free radical scavenging properties, which are likely due to phenolic compounds. Consistent with these findings, gel formulations of both the total lyophilized juice and, to a greater degree, the lyophilized flavonolic fraction appeared to possess a strong protective effect against UV-induced skin erythema in-vivo, whereas the lyophilized polysaccharidic fraction was completely ineffective. The in-vitro and in-vivo results suggest that, both the total lyophilized juice and, in particular, the lyophilized flavonolic fraction, but not the lyophilized polysaccharidic fraction of S. telephium L. leaves, have photoprotective effects against UVB-induced skin damage.