New Technologies in Rice Derivatives.

In this research the incapsulation of ferulic acid (FA) in advanced systems of protection with the aim of improving its stability and photostability was studied. Lipoparticles and polymeric microparticles as incapsulation systems were prepared and characterized. Lipoparticles were completely of natural origin, while microparticles were obtained using chitosan as natural polymer. In both systems FA stability was greatly increased and its organoleptic properties in the emulsions did not change.

Improving photoprotection: 4-methylbenzylidene camphor microspheres.

We propose a new approach for photoprotection. 4-Methylbenzylidene camphor (4-MBC), one of the most widely used UV filters, was encapsulated in microspheres, with a view to overcoming problems (percutaneous absorption, photodegradation and lack of lasting effect) arising with organic sunscreens, and to achieve safe photoprotection. We focused on this filter in the light of the Cosmetics Europe opinion concerning its possible effects on the thyroid gland. Microspheres were prepared by emulsification-solvent evaporation, using different amounts of 4-MBC and characterized for morphology, encapsulation efficiency and particle size. The particles were then mixed in O/W emulsions. The in vitro sun protection factors, in vitro release and photostability were investigated and compared with emulsions containing the free sunscreen. The new microspheres offer good morphology and loading (up to 40%), and the same photoprotection as the free filter while at the same time protecting it from photodegradation. The systems also give a slower release from the emulsions.

Scale-up of polyamide and polyester Parsol® MCX nanocapsules by interfacial polycondensation and solvent diffusion method.

The scale-up of oil-containing polyamide nanocapsules produced by simultaneous interfacial polycondensation and solvent diffusion was successfully achieved. Up to 1,500 mL were produced by using a Y-shaped mixer device. The sizes of nanocapsules containing olive oil were modulated from 646 to 211 nm by changing process parameters without modification of the formulation composition. All the results of nanocapsule diameters (dsc) expressed as a function of the Reynolds number (Re) showed the existence of a typical power-law relationship. It was demonstrated that the high turbulences created upon nanocapsule formation are the most important parameter allowing to nanocapsule size to be controlled without modifying the formulation composition. Finally, the power-law relationship was used to predict the size of nanocapsules composed of polyamide or polyester and loaded with Parsol(®) MCX. The physico-chemical properties of both polyamide and polyester nanocapsules at the laboratory scale were compared to the ones obtained at the pilot scale. The encapsulation efficiency was higher than 98% in both types of nanocapsules at the laboratory and the pilot scales. The in vitro releases of Parsol(®) MCX from polyester nanocapsules were reproducible at both scales. This is the first time such a power-law was described for the preparation of nanocapsules by interfacial polycondensation and solvent diffusion.

GC-MS profiling of the phytochemical constituents of the oleoresin from Copaifera langsdorffii Desf. and a preliminary in vivo evaluation of its antipsoriatic effect.

Copaiba is the oleoresin (OR) obtained from Copaifera (Fabaceae), a neotropical tree which grows in Amazon regions. The balsam, constituted by an essential oil and a resinous fraction is used as folkloristic remedy in the treatment of several inflammatory diseases and for its antioxidant and antibacterial properties. Aim of this work was (a) to carry out a characterization by GC-MS of the volatile and nonvolatile constituents of Copaifera langsdorffii Desf. oleoresin (OR); (b) to investigate the mechanism of its anti-inflammatory activity; (c) to evaluate its antipsoriatic effect after oral intake/topical application. The volatile fraction (yield: 22.51%, w/w) shows: α-bergamotene (48.38%), α-himachalene (11.17%), ß-selinene (5.00%) and ß-caryophyllene (5.47%). The OR residue (77.49%, w/w), after derivatization, showed as main constituents the following compounds: copalic, abietic, daniellic, lambertinic, labd-7-en-15-oic, pimaric, isopimaric acids and kaur16-en18-oic acid. Preincubation of LPS-stimulated human THP-1 monocytes with increasing concentrations of the OR purified fraction (OR-PF), containing diterpene acids, diterpenes and sesquiterpenes, reduced the release of pro-inflammatory cytokines (IL-1ß, IL-6, TNFα) in a dose-range of 0.1-10 µM. In addition, in cell culture system of human THP-1 monocytes, 1 µM OR-PF counteracts LPS-driven NF-κB nuclear translocation. In a preliminary clinical trial three patients affected by chronic psoriasis, treated with oral intake or topical application of the OR, exhibited a significant improvement of the typical signs of this disease, i.e. erythema, skin thickness, and scaliness. In conclusion, the results of this work, beside an extensive analytical characterization of the OR chemical composition, provide strong evidences that its anti-inflammatory activity is related to the inhibition of the NF-κB nuclear translocation, and consequently of proinflammatory cytokines secretion.

Scale-up of nanoemulsion produced by emulsification and solvent diffusion.

The scale-up of nanoemulsions (NEs) produced by emulsification and solvent diffusion process was successfully achieved in the present work. Up to 1500 mL of NEs were produced with olive oil, castor oil, almond oil, or Arlamol™ E by using a Y-shaped mixer device. NE droplet sizes were significantly modulated from 290 to 185 nm by changing the process parameters without modification of the formulation composition. Smaller NE droplet sizes were obtained by (1) decreasing the internal diameter of the Y-mixer from 5 to 0.8 mm, (2) increasing the flow rates of the organic and the aqueous phases upon mixing, and (3) increasing the temperature of the experiment from 5°C to 40°C. All the results of NE diameters (d(sc) ) expressed as a function of the Reynolds number (Re) and the shear rate inside the Y-mixer (\documentclass{article}\usepackage{amssymb}\begin{document}\pagestyle{empty}$\dot \gamma$\end{document}) showed the existence of typical power-law relationships: d(sc) = 10(2.82) Re(- 0.14) and \documentclass{article}\usepackage{amssymb}\begin{document}\pagestyle{empty}$d_{{\rm sc}} = 10^{2.60} \dot \gamma ^{- 0.06}$\end{document}, respectively. The existence of these power-laws for NE formation by emulsification and solvent diffusion process has never been reported in the literature yet and constitutes a new finding in this work. We definitely proved that the high turbulences created upon NE formation are the most important parameter allowing to decrease droplet size.

Polyamide nanocapsules and nano-emulsions containing Parsol® MCX and Parsol® 1789: in vitro release, ex vivo skin penetration and photo-stability studies.

PURPOSE: To prepare polyamide nanocapsules for skin photo-protection, encapsulating α-tocopherol, Parsol®MCX (ethylhexyl methoxycinnamate) and/or Parsol®1789 (butyl methoxydibenzoylmethane). METHODS: Nanocapsules were obtained by combining spontaneous emulsification and interfacial polycondensation reaction between sebacoyl chloride and diethylenetriamine. Nano-emulsions used as control were obtained by the same process without monomers. The influence of carrier on release rate was studied in vitro with a membrane-free model. Epidermal penetration of encapsulated sunscreens was ex vivo evaluated using Franz diffusion cells. Ability of encapsulated sunscreens to improve photo-stability was verified by comparing percentage of degradation after UV radiation exposure. RESULTS: Sunscreen-containing nanocapsules (260-400 nm) were successfully prepared; yield of encapsulation was >98%. Parsol®MCX and Parsol®1789 encapsulation led to decreased release rate by up to 60% in comparison with nano-emulsion and allowed minimum penetration through pig ear epidermis. Presence of polyamide shell protected encapsulated sunscreen filters from photo-degradation without affecting their activity. CONCLUSIONS: Encapsulation of Parsol®MCX and Parsol®1789 into oil-core of polyamide nanocapsules allowed protection from photo-degradation, controlled release from nanocapsules, and limited penetration through pig ear epidermis.

New multifunctional surfactants from natural phenolic acids.

Several new multifunctional molecules derived from natural sources such as amino acids and hydroxycinnamic acids were synthesized. They exhibit various activities such as emulsifying, UV-protecting, and radical scavenging, thereby conforming to the latest requirements for cosmetic ingredients. The synthesis comprises only a few steps: (i) the amino acid, the acid groups of which are protected by esterification, is coupled with ferulic or caffeic acid; (ii) the p-hydroxyl group of the cinnamic derivative reacts with dodecyl bromide in the presence of potassium carbonate (the resulting compounds are highly lipophilic and tested as water/oil (W/O) emulsifiers); (iii) these molecules, by deprotonating the acid groups of the amino acids, with successive salification, are more hydrophilic, with stronger O/W emulsifying properties. The new multifunctional surfactants might prove useful for the treatment of multiple skin conditions, including loss of cellular antioxidants, damage from free radicals, damage from UV, and others.

Lutein nanocrystals as antioxidant formulation for oral and dermal delivery.

Lutein is a well known antioxidant and anti-free radical used in cosmetic, nutraceutical industry with potential application in pharmaceutics as supportive antioxidant in treatments. As lipophilic molecule it is poorly soluble in water and has a low bioavailability. Lutein nanosuspension was prepared to enhance dissolution velocity, saturation solubility (C(s)), which are major factors determining oral bioavailability and penetration into the skin. High pressure homogenization (HPH) was used to prepare lutein nanosuspension. Particle size was determined by photon correlation spectroscopy (PCS) and laser diffractometry (LD). The lowest PCS diameter obtained was about 429 nm, the LD diameter 90% of 1.2 µm. The zeta potential was about -40 mV in water and -17 mV in the original dispersion medium. The 3 month storage study at different temperatures (4°C, 25°C, 40°C) confirmed physical stability despite the low zeta potential of -17 mV in original surfactant solution. A pronounced increase in saturation solubility by 26.3 fold was obtained for lutein nanocrystals compared to coarse powder. The lutein nanosuspension was converted into pellets and filled into hard gelatin capsules for nutraceutical use, showed a superior in vitro release (factor of 3-4). Lyophilized nanosuspension was prepared for subsequent incorporation into creams and gels. The lyophilized nanosuspension was very well re-dispersible (435 nm). Using cellulose nitrate membranes as in vitro model, permeation through this barrier was 14× higher for lutein nanocrystals compared to coarse powder. However, pig ear skin did not allow lutein to permeate but supported localization of the lutein in the skin where it should act anti-oxidatively.

The human olfactory receptor 17-40: requisites for fitting into the binding pocket.

To gain structural insight on the interactions between odorants and the human olfactory receptor, we did homology modelling of the receptor structure, followed by molecular docking simulation with ligands. Molecular dynamics simulation on the structures resulting from docking served to estimate the binding free energy of the various odorant families. A correlation with the odorous properties of the ligands is proposed. We also investigated which residues were involved in the binding of a set of properly synthesised ligands and which were required for fitting inside the binding pocket. Olfactive stimulation of the olfactory receptor with odorous molecules was also investigated, using calcium imaging or electrophysiological recordings.

Lipid nanocarriers for dermal delivery of lutein: preparation, characterization, stability and performance.

Topical application of lutein as an innovative antioxidant, anti-stress and blue light filter, which is able to protect skin from photo damage, has got a special cosmetic and pharmaceutical interest in the last decade. Lutein is poorly soluble, and was therefore incorporated into nanocarriers for dermal delivery: solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and a nanoemulsion (NE). Nanocarriers were produced by high pressure homogenization. The mean particle size was in the range of about 150 nm to maximum 350 nm, it decreased with increasing oil content of the carriers. The zeta potential in water was in the range -40 to -63 mV, being in agreement with the good short term stability at room temperature monitored for one month. In vitro release was studied using a membrane free model. Highest release in 24h was observed for the nanoemulsion (19.5%), lowest release (0.4%) for the SLN. Release profiles were biphasic (lipid nanoparticles) or triphasic (NE). In vitro penetration study with a cellulose membrane showed in agreement highest values for the NE (60% in 24h), distinctly lower values for the solid nanocarriers SLN and NLC (8-19%), lowest values for lutein powder (5%). Permeation studies with fresh pig ear skin showed that no (SLN, NLC) or very little lutein (0.4% after 24h) permeated, that means the active remains in the skin and is not systemically absorbed. The nanocarriers were able to protect lutein against UV degradation. In SLN, only 0.06% degradation was observed after irradiation with 10 MED (Minimal Erythema Dose), in NLC 6-8%, compared to 14% in the NE, and to 50% as lutein powder suspended in corn oil. Based on size, stability and release/permeation data, and considering the chemical protection of the lutein prior to its absorption into the skin, the lipid nanoparticles are potential dermal nanocarriers for lutein.

Permeation and distribution of ferulic acid and its α-cyclodextrin complex from different formulations in hairless rat skin.

Ferulic acid (FA) is a natural product that occurs in seeds of many plants where it is generally located in the bran. This compound is a multifunctional ingredient endowed with antioxidative, radical scavenging, sunscreening and antibacterial actions. The aim of this study was to analyse the ferulic acid cutaneous permeation and distribution, through and into the skin layers, from different cosmetic vehicles, an O/W emulsion (pH 6.0) and two gel-type formulations at different pH levels (6.0 and 7.4), containing FA alone or an inclusion complex with α-cyclodextrin (CD-FA). In vitro permeation studies were performed in vertical diffusion cells using hairless rat excised skin. At appropriate intervals of time, the amount of permeated sunscreen/radical scavenger was evaluated by high-performance liquid chromatography (HPLC). At the end of experiments, treated skin samples were sectioned with a cryomicrotome and the FA content of the individual slices was analysed by HPLC. FA-containing formulations, O/W emulsion, gels A and B, originated FA fluxes of 8.48 ± 2.31, 8.38 ± 0.89 and 5.72 ± 0.50 µg/cm(2) h, respectively, thus suggesting the pH influence on FA percutaneous permeation. The use of the inclusion complex, CD-FA, determined in all cases a decrease of FA transdermal permeation while no influence of pH was observed. Gel-type formulations containing FA ensured higher sunscreen storage in the superficial layers if compared with O/W emulsion. When FA was included in α-cyclodextrin, FA amount retained into skin layers decreased markedly.

Non-covalent inclusion of ferulic acid with alpha-cyclodextrin improves photo-stability and delivery: NMR and modeling studies.

Ferulic acid (FA) is a highly effective antioxidant and photo-protective agent, already approved in Japan as a sunscreen, but it is poorly suited for cosmetic application because of its low physicochemical stability. We prepared the inclusion complex of FA with alpha-cyclodextrin by co-precipitation from an aqueous solution, and used (1)H NMR and molecular dynamics to investigate the most probable structure of the inclusion complex. In rotating frame nuclear Overhouser effect spectroscopy (ROESY) experiments FA penetrated the alpha-CD hydrophobic cavity with the alpha,beta-unsaturated part of the molecule and some of its aromatic skeleton. In proton chemical shift measurements of FA and alpha-cyclodextrins we determined the stoichiometry of the association complex (1:1) by Job's method, and its stability constant (K(1:1) 1162+/-140 M(-1)) and described the molecular dynamics of the complex on the basis of theoretical studies. Encapsulation with alpha-cyclodextrin improves (i) the chemical stability of FA against UVB stress (10 MED [Minimal Erythemal Dose: 1 MED=25 mJ/cm(2) for skin phototype II: 30]), since no degradation products are formed after irradiation, and (ii) the bioavailability of FA on the skin, slowing its delivery (Strainer cell model).

Analytical characterization of a ferulic acid/gamma-cyclodextrin inclusion complex.

Ferulic acid (FA) is a well-known antioxidant of natural source with promising properties as photoprotective agent (approved in Japan as sunscreen) and its derivatives (alkyl ferulates) are under screening for the prevention of photoinduced skin tumours. In the present work we describe the preparation of a solid inclusion complex between ferulic acid and gamma-cyclodextrin (gamma-CD) and its characterization by different analytical techniques: differential scanning calorimetry (DSC), X-ray diffractometry (XRD), nuclear magnetic resonance spectroscopy (1H NMR) and by supporting information of molecular modelling. All these approaches indicate that ferulic acid is able to form an association complex with gamma-CD but only 1H NMR and molecular modelling studies give an unequivocal evidence that the antioxidant molecule is embedded into the gamma-CD cavity to form an inclusion complex. In detail it is entrapped inside the hydrophobic core of gamma-CD with the lipophilic aromatic ring and the ethylenic moieties, leaving the more polar functional groups close to wider rim or outside the cavity.

Interaction of ferulic acid derivatives with human erythrocytes monitored by pulse field gradient NMR diffusion and NMR relaxation studies.

Ferulic acid (Fer), a natural anti-oxidant and chemo-protector, is able to suppress experimental carcinogenesis in the forestomach, lungs, skin, tongue and colon. Several Fer derivatives have been suggested as promising candidates for cancer prevention, being the biological activity related also to the capacity of partitioning between aqueous and lipid phases. In the present work, pulsed field gradient (PFG) NMR diffusion measurement and NMR relaxation rates have been adopted for investigating the interaction of three Fer derivatives (Fer-C11, Fer-C12 and Fer-C13) with human erythrocytes. Binding to the erythrocyte membrane has been shown for all derivatives, which displayed a similar interaction mode such that the aromatic moiety and the terminal part of the alkyl chain were the most affected. Quantitative analysis of the diffusion coefficients was used to show that Fer-C12 and Fer-C13 display higher affinity for the cell membrane when compared with Fer-C11. These findings agree with the higher anti-oxidant activity of the two derivatives.

Antioxidant activity of ferulic acid alkyl esters in a heterophasic system: a mechanistic insight.

The antioxidant activity of some esters of ferulic acid with the linear fatty alcohols C7, C8 (branched and linear), C9, C11, C12, C13, C15, C16, and C18 has been studied in homogeneous and heterogeneous phases. Whereas in homogeneous phase all of the alkyl ferulates possessed similar radical-scavenging abilities, in rat liver microsomes they showed striking differences, the more effective being C12 (7) (IC50 = 11.03 M), linear C8 (3) (IC50 = 12.40 microM), C13 (8) (IC50 = 18.60 microM), and C9 (5) (IC50 = 19.74 microM), followed by C7 (2), C15 (9), C11 (6), branched C8 (4), C16 (10), and C18 (11) (ferulic acid was the less active, IC50 = 243.84 microM). All of the molecules showed similar partition coefficients in an octanol-buffer system. Three-dimensional studies (NMR in solution, modeling in vacuo) indicate that this behavior might be due to a different anchorage of the molecules with the ester side chain to the microsomal phospholipid bilayer and to a consequent different orientation/positioning of the scavenging phenoxy group outside the membrane surface against the flux of oxy radicals.

Conformational analysis: a tool for the elucidation of the antioxidant properties of ferulic acid derivatives in membrane models.

With the aim to search and design more effective and safe antioxidant molecules to be used as functional ingredients in cosmetic formulations for UV protection, we evaluated the antioxidant/radical scavenging activities of ferulic acid and of some alkyl ferulates in both acellular and cellular systems. Ferulic acid esters, equipotent as antioxidant in homogeneous phase, showed when tested in membranous systems (rat liver microsomes, rat erythrocytes) marked differences in antioxidant potency. The n-C(12) derivative was the most potent, followed by n-C(8), n-C(16) and branched C(8), and then by ferulic acid. A conformational study carried out by NMR and modelling, indicates that the different antioxidant activity of ferulates in membrane models is due to the different spatial conformation and arrangement of the side chain of the molecule, which governs the access and binding to the phospholipid bilayer, the modality of orientation of the scavenging/quenching nucleus (phenol moiety), and hence the overall antioxidant potency of the derivative. These results emphasize the need of analytical studies (NMR and molecular modelling) addressed to the knowledge of the conformational parameters in combination with conventional antioxidant testings for understanding the antioxidant behaviour of a molecule in a biological membrane/system.

Involvement of oxidative stress in apoptosis induced by a mixture of isothiazolinones in normal human keratinocytes.

A 3:1 combination of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) is widely used to preserve cosmetic products. We show here that CMI/MI induced apoptosis in normal human keratinocytes (NHK) as at low concentrations (0.001-0.05% documented by subdiploid DNA content and phosphatidylserine exposure, while at the highest concentration (0.1% as supplied, 15 p.p.m.) the response was necrosis. Various molecular events accompanied the cytotoxic effects of CMI/MI. Generation of ROS and hyperpolarization of mitochondrial transmembrane potential (DeltaPsim) were early events, followed by increased Fas expression and activation of caspase-8, and then activation of caspase-3 and -9. The drop in DeltaPsim occurred only later in the cell death pathway, when NHK showed signs of apoptosis. Pretreatment of cells for 2 h with the redox-active agent N-acetyl-L-cysteine conferred complete protection against the CMI/MI-induced cytotoxic effects, DeltaPsim loss, and apoptosis. The pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F blocked the CMI/MI-induced apoptosis without preventing ROS generation and the drop in DeltaPsim. These results indicate that the generation of ROS plays an important part in mediating apoptosis and necrosis associated with CMI/MI treatment. This new aspect of the in vitro toxicity of CMI/MI may provide important information about the relationship between the preservative's in vitro apoptotic activity and its in vivo toxicity.

In vitro induction of apoptosis vs. necrosis by widely used preservatives: 2-phenoxyethanol, a mixture of isothiazolinones, imidazolidinyl urea and 1,2-pentanediol.

Preservatives are added to many final products, such as detergents, cosmetics, pharmaceuticals and vaccines. We conducted an in vitro investigation of the apoptosis- and necrosis-inducing potential of brief applications (10 min) of four common preservatives: ethylene glycol monophenyl ether, 2-phenoxyethanol (EGPE), imidazolidinyl urea (IMU), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMI/MI), and 1,2-pentanediol, a "preservative-non-preservative" best known as pentylene glycol. Using HL60 cells, we monitored the kinetics of cell toxicity with the MTT test and analysed extranuclear end points of apoptosis, i.e. phosphatidylserine exposure and nuclear fragmentation. Preservative treatment resulted in a dose-dependent decrease of cell viability. The mode of cell death was dose-dependent: necrosis occurred at high concentrations while apoptosis, shown by DNA laddering, DNA sub-diploid peak and caspase-3 activation, occurred at lower concentrations 0-24hr after exposure to a single dose: CMI/MI induced apoptosis at low concentrations (0.001-0.01%) and necrosis at high concentrations (0.5-0.1%); IMU and EGPE required higher concentrations to induce apoptosis (IMU 0.01-0.1% and EGPE 0.01-0.5%) or necrosis (IMU 0.5-1% and EGPE only at 1%). PG induced apoptosis only at 5%. Externalization of PS, a hallmark of apoptosis, occurred early in HL60 treated with low concentrations of CMI/MI and EGPE and was concomitant with the subdiploid peak in HL60 treated with PG. However, it did not occur in HL60 treated with IMU. In conclusion, at appropriate concentrations, each of the four preservatives modulates the apoptotic machinery by a caspase-dependent mechanism. Thus, apoptosis could be a good parameter to evaluate the cytoxicity of these chemical compounds.