Antonella Saija

Flavonoids as antioxidant agents: Importance of their interaction with biomembranes

Flavonoids, a group of phenolic compounds widely occurring in the plant kingdom, have been reported to possess strong antioxidant activity. In the present study, four flavonoids (quercetin, hesperetin, naringenin, rutin), chosen according to their structural characteristics, were tested in two different in vitro experimental models: (1) Fe(2+)-induced linoleate peroxidation (Fe(2+)-ILP), by detection of conjugated dienes; and (2) autooxidation of rat cerebral membranes (ARCM), by using thiobarbituric acid for assay of free malondialdehyde production. The results obtained were also interpreted in the light of flavonoid interactions, studied by differential scanning calorimetry, with dipalmitoylphosphatidylcholine (DPPC) vesicles as a biological membrane model. The antilipoperoxidant activity of the flavonoids tested can be classified as follows: rutin > hesperetin > quercetin > naringenin in the Fe(2+)-ILP test: quercetin > rutin > hesperetin > naringenin in the ARCM test. Quercetin, hesperetin, and naringenin interacted with DPPC liposomes causing different shifts, toward lower values, of the main transition peak temperature (Tm) typical for DPPC liposomes; however, no change in Tm of DPPC dispersion was observed in the presence of rutin. The hypothesis will be discussed that flavonoid capacity to modify membrane-dependent processes, such as free-radical-induced membrane lipoperoxidation, is related not only to their structural characteristics but also to their ability to interact with and penetrate the lipid bilayers.

Mechanisms of Antibacterial Action of Three Monoterpenes

ABSTRACT In the present paper, we report the antimicrobial efficacy of three monoterpenes [linalyl acetate, (+)menthol, and thymol] against the gram-positive bacterium Staphylococcus aureus and the gram-negative bacterium Escherichia coli. For a better understanding of their mechanisms of action, the capability of these three monoterpenes to damage biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein from unilamellar vesicles with different lipidic compositions (phosphatidylcholine, phosphatidylcholine/phosphatidylserine [9:1], phosphatidylcholine/stearylamine [9:1], and phosphatidylglycerol/cardiolipin [9:1]). Furthermore, the interaction of the terpenes tested with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry. Finally, the results were related to the relative lipophilicity and water solubility of the compounds examined. Taken together, our findings lead us to speculate that the antimicrobial effect of (+)menthol, thymol, and linalyl acetate may result, at least partially, from a perturbation of the lipid fraction of microorganism plasma membrane, resulting in alterations of membrane permeability and in leakage of intracellular materials. Besides being related to physicochemical characteristics of the drugs (such as lipophilicity and water solubility), this effect seems to be dependent on lipid composition and net surface charge of microbial membranes. Furthermore, the drugs might cross the cell membranes, penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.

On the in-vitro antimicrobial activity of oleuropein and hydroxytyrosol.

Secoiridoides (oleuropein and derivatives), one of the major classes of polyphenol contained in olives and olive oil, have recently been shown to inhibit or delay the rate of growth of a range of bacteria and microfungi but there are no data in the literature concerning the possible employment of these secoiridoides as antimicrobial agents against pathogenic bacteria in man.

In vitro and in vivo evaluation of caffeic and ferulic acids as topical photoprotective agents

Topically-applied antioxidant drugs represent a successful strategy for protecting the skin against UV-mediated oxidative damage. However, they can afford to the skin a satisfactory photoprotection only if able to permeate through the stratum corneum and thus to reach deeper cutaneous layers. Caffeic and ferulic acids, dissolved in saturated aqueous solutions at pH 3 or 7.2, have been tested for their capability to permeate through excised human skin mounted in Franz cells. At both pH values, ferulic and, at a lower degree, caffeic acids appeared able to permeate through the stratum corneum. The known higher lipophilicity of ferulic acid may explain the fact that it permeates through the stratum corneum better than caffeic acid. However, vehicle pH values proved to have no influence on biophenol skin permeation profile; this observed lack of pH effect may reflect the drug higher concentration attainable in saturated solutions at high pH. On the basis of the findings obtained in these in vitro experiments, we designed the schedule of a series of in vivo experiments, carried out to evaluate the ability of caffeic and ferulic acids to reduce, in healthy human volunteers, UVB-induced skin erythema, monitored by means of reflectance spectrophotometry. Caffeic and ferulic acids, dissolved in saturated aqueous solution pH 7.2, proved to afford a significant protection to the skin against UVB-induced erythema. To conclude, we have confirmed, by means of in vitro and in vivo experiments, that caffeic and ferulic acids may be successfully employed as topical protective agents against UV radiation-induced skin damage; however their skin absorption is not influenced by the pH of the formulation.

Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry

Aims:  To evaluate the antimicrobial properties of flavonoid‐rich fractions derived from bergamot peel, a byproduct from the Citrus fruit processing industry and the influence of enzymatic deglycosylation on their activity against different bacteria and yeast.

Flavonoids as potential protective agents against photo-oxidative skin damage

Abstract Flavonoids, a group of phenolic compounds widely occurring in the plant kingdom, have been reported to possess strong antioxidant activity. This preliminary study was designed to estimate the potential utility of topically applied flavonoids to prevent photooxidative stress in the skin. With this aim we have evaluated the protective effect of three flavonoids (quercetin, hesperetin and naringenin), chosen according to their structural characteristics, against UV radiation-induced peroxidation on phosphatidylcholine (PC) vesicles as a model membrane. Furthermore ‘in vitro’ human skin permeation of these flavonoids was measured, given that a suitable percutaneous absorption is an essential requirement for satisfactory topically applied photoprotective agents. The flavonoids tested in our study proved to protect efficiently PC liposomes from UV radiation-induced peroxidation, probably by scavenging oxygen free radicals generated by UV irradiations; their antilipoperoxidative activity can be classified as follows: quercetin > hesperetin > naringenin. In addition, naringenin, hesperetin and, at a very lower degree, quercetin were able to permeate through the stratum corneum (which is the main barrier against the penetration of exogenous substances through the skin) and, so, to penetrate into deeper skin layers. Taken together, these findings suggest that topically applied flavonoids could be excellent candidates for successful employment as protective agents in certain skin diseases caused, initiated or exacerbated by sunlight irradiation.

`In vitro' evaluation of the antioxidant activity and biomembrane interaction of the plant phenols oleuropein and hydroxytyrosol

Abstract Oleuropein and hydroxytyrosol, two phenolic compounds contained in olives and olive oil, are known to possess several biological properties, many of which may be related, partially at least, to their antioxidant and free radical-scavenger ability. Hence, together with their scavenging activity against the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test), we have investigated the antioxidative effect of oleuropein and hydroxytyrosol in a model system consisting of dipalmitoylphosphatidylcholine/linoleic acid unilamellar vesicles (DPPC/LA LUVs) and a water-soluble azo compound as a free radical generator (LP–LUV test). The results obtained were also interpreted in the light of biophenol interactions, studied by differential scanning calorimetry (DSC), with dimyristoylphosphatidylcholine (DMPC) vesicles as a biological membrane model. Our results obtained in the DPPH and LP–LUV tests confirm the good scavenger activity and antioxidant effect of oleuropein and hydroxytyrosol. However, while both compounds exhibit comparable effectiveness in the DPPH test (hydroxytyrosol being slightly more active than oleuropein), oleuropein seems, in the LP–LUV test, a better antioxidant than hydroxytyrosol. Besides oleuropein shows a better antioxidant activity in the membranous system than in homogenous solution. Furthermore, oleuropein, but not hydroxytyrosol, interacts with DMPC vesicles, causing shifts, toward lower values, of the calorimetric peak temperature ( T m ), associated to the gel to liquid-crystal phase transition, typical for DMPC multilayers. The hypothesis will be discussed that hydroxytyrosol can serve as scavenger of aqueous peroxyl radicals near the membrane surface, while oleuropein acts also as a scavenger of chain-propagating lipid peroxyl radicals within the membranes.

Ferulic and caffeic acids as potential protective agents against photooxidative skin damage

The biological properties and, particularly, the antioxidant activity of plant hydroxycinnamic acids, such as caffeic and ferulic acids, are well recognised. This preliminary study was designed to estimate the potential utility of caffeic and ferulic acids to prevent, when topically applied, photooxidative stress in the skin. With this aim we have evaluated the antioxidant activity of ferulic and caffeic acids in two experimental models: (1) the UV radiation-induced peroxidation in phosphatidylcholine (PC) liposomal membranes; (2) the scavenging activity against nitric oxide (a radical involved in oxidative reactions). In addition, given that a suitable percutaneous absorption is an essential requirement for successful topical photoprotective agents, we measured their in vitro permeation through excised human skin. Caffeic and ferulic acids efficiently protected PC liposomes from UV radiation-induced peroxidation and reacted with nitrogen oxides. In addition, caffeic and ferulic acids were able to permeate through the stratum corneum (the main barrier against the penetration of exogenous substances through the skin). Taken together, these findings suggest that caffeic and ferulic acids should be good canditates for successful employment as topical protective agents against UV radiation-induced skin damage. © 1999 Society of Chemical Industry

Antioxidant activity and phenolic profile of pistachio (Pistacia vera L., variety Bronte) seeds and skins

Pistachio (Pistacia vera L.; Anacardiaceae) is native of aride zones of Central and West Asia and distributed throughout the Mediterranean basin. In Italy, a pistachio cultivar of high quality is typical of Bronte (Sicily), an area around the Etna volcano, where the lava land and climate allow the production of a nut with intense green colour and aromatic taste, very appreciated in international markets. Pistachio nuts are a rich source of phenolic compounds, and have recently been ranked among the first 50 food products highest in antioxidant potential. Pistachio nuts are often used after removing the skin, which thus represents a significant by-product of pistachio industrial processing. The present study was carried out to better characterize the phenolic composition and the antioxidant activity of Bronte pistachios, with the particular aim to evaluate the differences between pistachio seeds and skins. The total content of phenolic compounds in pistachios was shown to be significantly higher in skins than in seeds. By HPLC analysis, gallic acid, catechin, eriodictyol-7-O-glucoside, naringenin-7-O-neohesperidoside, quercetin-3-O-rutinoside and eriodictyol were found both in pistachio seeds than in skins; furthermore, genistein-7-O-glucoside, genistein, daidzein and apigenin appeared to be present only in pistachio seeds, while epicatechin, quercetin, naringenin, luteolin, kaempferol, cyanidin-3-O-galactoside and cyanidin-3-O-glucoside are contained only in pistachio skins. The antioxidant activity of pistachio seeds and skins were determined by means of four different assays (DPPH assay, Folin-Ciocalteau colorimetric method and TEAC assay, SOD-mimetic assay). As expected on the basis of the chemical analyses, pistachio skins have shown to possess a better activity with respect to seeds in all tests. The excellent antioxidant activity of pistachio skins can be explained by its higher content of antioxidant phenolic compounds. By HPLC-TLC analysis, gallic acid, catechin, cyanidin-3-O-galactoside, eriodictyol-7-O-glucoside and epicatechin appeared to be responsible for the antioxidant activity of pistachio skin, together with other unidentified compounds. In conclusion, our work has contributed to clarify some particular characteristics of Bronte pistachios and the specific antioxidant power of pistachio skins. Introduction of pistachios in daily diet may be of undoubted utility to protect human health and well-being against cancer, inflammatory diseases, cardiovascular pathologies and, more generally, pathological conditions related to free radical overproduction. On the other hand, pistachio skins could be successfully employed in food, cosmetic and pharmaceutical industry.

In vitro and in vivo evaluation of polyoxyethylene esters as dermal prodrugs of ketoprofen, naproxen and diclofenac.

Novel polyoxyethylene esters of ketoprofen (1(a-e)), naproxen (2(a-e)) and diclofenac (3(a-e)) were synthesized and evaluated as potential dermal prodrugs of naproxen, ketoprofen and diclofenac. These esters were obtained by coupling these drugs with polyoxyethylene glycols by a succinic acid spacer. The aqueous solubilities, lipophilicities and hydrolysis rates of esters 1(a-e), 2(a-e) and 3(a-e) were determined in a buffered solution and in porcine esterase. The permeation of these prodrugs through excised human skin was studied in vitro. Furthermore we investigated the in vivo topical anti-inflammatory activity of esters 1(d), 2(e) and 3(e), which showed the best in vitro profile, evaluating the ability of these compounds to inhibit methyl nicotinate (MN)-induced skin erythema on healthy human volunteers. Esters 1(a-e), 2(a-e) and 3(a-e) showed good water stability and rapid enzymatic cleavage and their hydrolysis rates, both chemical and enzymatic, were not significantly affected by the length of the polyoxyethylenic chain used as promoiety. Concerning in vitro percutaneous absorption studies, only esters 1(d-e), 2(d-e) and 3(c-e) showed an increased flux through stratum corneum and epidermis membranes compared to their respective parent drugs. In vivo results showed an interesting delayed and sustained activity of esters 1(d) and 3(e) compared to the parent drugs. In conclusion polyoxyethylene glycols could prove to be suitable promoieties for ketoprofen, naproxen and diclofenac design since esters 1(d-e), 2(d-e) and 3(c-e) showed some requirements (chemical stability, enzymatic lability and an increased skin permeation) needed to obtain successful dermal prodrugs. Furthermore, was observed an appreciable and sustained in vivo topical anti-inflammatory activity of esters 1(d) and 3(e), compared to the parent drugs, using MN-induced erythema in human volunteers as inflammation model.