Francesco Bonina

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.

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.

Lipid nanoparticles for prolonged topical delivery: An in vitro and in vivo investigation

Dermal therapy is still a challenge due to the difficulties in controlling the active pharmaceutical ingredient (API) fate within the skin. Recently, lipid nanoparticles have shown a great potential as vehicle for topical administration of active substances, principally owing to the possible targeting effect and controlled release in different skin strata. Ketoprofen and naproxen loaded lipid nanoparticles were prepared, using hot high pressure homogenization and ultrasonication techniques, and characterized by means of photo correlation spectroscopy and differential scanning calorimetry. Nanoparticle behavior on human skin was assessed, in vitro, to determine drug percutaneous absorption (Franz cell method) and, in vivo, to establish the active localization (tape-stripping technique) and the controlled release abilities (UVB-induced erythema model). Results demonstrated that the particles were able to reduce drug penetration increasing, simultaneously, the permeation and the accumulation in the horny layer. A prolonged anti-inflammatory effect was observed in the case of drug loaded nanoparticles with respect to the drug solution. Direct as well as indirect evidences corroborate the early reports on the usefulness of lipid nanoparticles as carriers for topical administration, stimulating new and deeper investigations in the field.

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

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.

Influence of different penetration enhancers on in vitro skin permeation and in vivo photoprotective effect of flavonoids

In the present study the permeation of two flavonoids (naringenin and hesperetin) through excised human skin mounted in Franz diffusion cells and its possible optimization following skin pretreatment with two penetration enhancers (D-limonene and lecithin) were determined. Hesperetin and naringenin were able to permeate through excised human skin; moreover, skin pretreatment with D-limonene and lecithin increased, to different degrees, their cutaneous permeation. On the basis of findings obtained in these in vitro experiments, we designed a schedule for a series of in vivo experiments, in which the protective effect of topically applied naringenin and hesperetin against UV-B-induced skin damage was assessed monitoring the extent of erythema in human volunteers by means of reflectance spectrophotometry. Hesperetin and naringenin from formulations containing the flavonoid alone were completely ineffective in decreasing UV-B-induced erythema. Furthermore, both D-limonene and lecithin have enhanced, to a significant extent, the photoprotective activity of naringenin and hesperetin. Taken together, these data demonstrate that hesperetin and naringenin may be successfully employed as topical photoprotective agents. However their topical activity needs to be optimized by using suitable penetration enhancers.

Effects of phospholipid based formulations on in vitro and in vivo percutaneous absorption of methyl nicotinate

In this paper we evaluate the influence of phospholipid based formulations (PBFs) on skin absorption. In particular we describe the production and characterization of different PBFs, namely liposomes and w/o microemulsion gels, and their influence on in vitro and in vivo absorption of methyl nicotinate (MN) used as model compound. In order to compare the influence of various vehicles on skin absorption, Franz cell and MN induced erythema were used as in vitro and in vivo experimental models respectively. The formulations tested were: (a) unilamellar liposomes consisting of soybean lecithin/ cholesterol (9:1 w/w) suspended in water or incorporated into hydrophilic gels (Carbomer and carboxymethyl cellulose based gels) and (b) soybean lecithin based gels. The results indicate that vehicles containing phospholipids in liposomal form provided enhanced in vivo MN skin permeation compared to the corresponding vehicles without phospholipids. Lecithin gel showed a different behaviour characterized by a short and intense persistence of MN induced erythema.

Evaluation of alternative strategies to optimize ketorolac transdermal delivery

In the present study, 2 alternative strategies to optimize ketorolac transdermal delivery, namely, prodrugs (polyoxyethylene glycol ester derivatives, I–IV) and nanostructured lipid carriers (NLC) were investigated. The synthesized prodrugs were chemically stable and easily degraded to the parent drug in human plasma. Ketorolac-loaded NLC with high drug content could be successfully prepared. The obtained products formulated into gels showed a different trend of drug permeation through human stratum corneum and epidermis. Particularly, skin permeation of ester prodrugs was significantly enhanced, apart from ester IV, compared with ketorolac, while the results of drug release from NLC outlined that these carriers were ineffective in increasing ketorolac percutaneous absorption owing to a higher degree of mutual interaction between the drug and carrier lipid matrix. Polyoxyethylene glycol esterification confirmed to be a suitable approach to enhance ketorolac transdermal delivery, while NLC seemed more appropriate for sustained release owing to the possible formation of a drug reservoir into the skin.