Potential of Olive Oil Mill Wastewater as a Source of Polyphenols for the Treatment of Skin Disorders: A Review.

Current trends toward naturally occurring compounds of therapeutic interest have contributed to an increasing number of studies on olive oil phenolics in the treatment of diseases with oxidative and inflammatory origins. Recent focus has been on olive oil wastewater, which is richer in phenolic compounds than olive oil itself. In this review, we present findings demonstrating the potential use of olive mill wastewater in dermatology. Particular attention is given to compounds with proven benefits in topical pharmacology: caffeic and ferulic acids, tyrosol and hydroxytyrosol, verbascoside, and oleuropein. The review is divided into different sections: inflammatory skin diseases, microbial effects, wound healing in addition to the antimelanoma properties of olive mill waste phenolics, and their potential in sun protection agents. There is strong evidence to support further studies into the valorization of this abundant and sustainable source of phenolic compounds for use in dermatology and dermo-cosmetic preparations.

Varietal Effect on the Concentration and Anti-Inflammatory Activity of Hydroxytyrosol in French Olive Oils.

Numerous studies have been carried out on the bioactive properties of hydroxytyrosol (HT) in olive oils (OOs), although there are few reports comparing anti-inflammatory activity among different olive varieties or regions of production. The purpose of this study was to investigate the in vitro inflammatory action of HT in extracts of four OO varieties in the Languedoc region of the French Mediterranean. Factors other than cultivar were eliminated, which enabled unambiguous varietal differences to be identified. Purified extracts of OO were obtained using an optimized solid-phase extraction procedure by which only polar compounds were recovered. High performance liquid chromatography-photodiode array detection-tandem mass spectrometry was used to identify and quantify HT and oleacein in the extracts. The total polyphenol concentration ranged from 93.00 mg gallic acid equivalent/kg OO for Picholine to 27 mg gallic acid equivalent for Verdale OOs. The concentrations of HT in Picholine, Olivère, and Lucques varieties were 25.3, 18.8 and 12.1 mg/kg, respectively, whereas the concentration of HT in Verdale OOs was less, 1 mg/kg. The in vitro anti-inflammatory response of purified OO extracts, evaluated by the inhibition of nitric oxide release in lipopolysaccharide-induced interferon-γ activated J774.A1 macrophages, strongly correlated with total polyphenol content (R2 > 0.995). The effect increased asymptotically between the equivalent of 2 and 37 mg of OO, reaching, at the maximum tested concentration, 90%, 75%, 62%, and 30% activity for Picholine, Olivière, Lucques, and Verdale, respectively. The results presented here clearly show that, by comparison with authentic standards, the activity of HT in OO extracts was enhanced in a concentration-dependent manner, varying from 3-fold at the highest extract concentration to over 6.5-fold at the lowest extract concentration. Therefore, the anti-inflammatory activity of OOs should be rationalized on the basis of whole extracts rather than solely on the concentration of HT or other bioactive compounds in OO.

High Accumulation and In Vivo Recycling of the New Antimalarial Albitiazolium Lead to Rapid Parasite Death.

Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei-infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei-infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs.

Neuroprotective effect of Picholine virgin olive oil and its hydroxycinnamic acids component against ß-amyloid-induced toxicity in SH-SY5Y neurotypic cells.

The health benefits of Mediterranean diet has long been reported and attributed to the consumption of virgin olive oil (VOO). Here, we evaluated the neuroprotective effect of VOO against Alzheimer's disease by determining its effect on ß-amyloid (Aß)-induced cytotoxicity and oxidative stress, and explored the possibility that its hydroxycinnamic acids (Hc acids) content contribute significantly to this effect. SH-SY5Y cells treated with or without Aß and with VOO or Hc acids (mixture of p-coumaric acid, ferulic acid, vanillic acid, and caffeic acid) were subjected to MTT assay and the results showed that both samples alleviated Aß-induced cytotoxicity. Furthermore, both VOO and Hc acids decreased the reactive oxygen species level. Using western blot to determine the effect of these samples on Aß-induced activation of pERK1/2, p38, and JNK MAPKs, results revealed that both VOO and Hc acids inhibited the activation of pERK1/2 and p-p38 MAPK, but not JNK. Moreover, VOO upregulated the glycolytic enzymes genes hexokinase (HK1), and phosphofructokinase (PFKM) expression which means that VOO enhanced the energy metabolism of the neurotypic cells, and therefore suggests another mechanism by which VOO could provide protection against Aß-induced cytotoxicity. The findings in this study suggest that VOO has a neuroprotective effect, attributable to its hydroxycinnamic acids component, against Aß-induced cytotoxicity and oxidative stress through the inhibition of the activation of MAPKs ERK and p38 and by enhancing the energy metabolism of the neurotypic cells.

Antiallergic effect of Picholine olive oil-in-water emulsions through ß-hexosaminidase release inhibition and characterization of their physicochemical properties.

The inhibitory effect of Picholine olive oil from Montpellier in Southern France on the chemical mediator release in type I allergy, using rat basophilic leukemia (RBL-2H3) cells, was investigated. Oil-in-water (O/W) emulsions prepared using Picholine olive oil showed an inhibitory effect on the chemical mediator release and decreased expressions of genes related to type I allergy in RBL-2H3 cells. We then measured the phenolic compounds present in Picholine olive oil using high-performance liquid chromatography and investigated some physical properties, such as droplet size, size distribution, viscosity, and surface tension of the resulting olive O/W emulsions. Our findings indicate that Picholine olive oil has high flavonoids content, especially apigenin, and the prepared emulsion of Picholine olive oil resulted in a considerable small size distribution, with an average droplet size of 170 nm.

Pharmacokinetic properties and metabolism of a new potent antimalarial N-alkylamidine compound, M64, and its corresponding bioprecursors.

Antimalarial activities and pharmacokinetics of the bis-alkylamidine, M64, and its amidoxime, M64-AH, and O-methylsulfonate, M64-S-Me, derivatives were investigated. M64 and M64-S-Me had the most potent activity against the Plasmodium falciparum growth (IC(50)<12nM). The three compounds can clear the Plasmodium vinckei infection in mice (ED(50)<10mg/kg). A liquid chromatography-mass spectrometry method was validated to simultaneously quantify M64 and M64-AH in human and rat plasma. M64 is partially metabolized to M64-monoamidoxime and M64-monoacetamide by rat and mouse liver microsomes. The amidoxime M64-AH undergoes extensive metabolism forming M64, M64-monoacetamide, M64-diacetamide and M64-monoamidoxime. Strong interspecies differences were observed. The pharmacokinetic profiles of M64, M64-AH and M64-S-Me were studied in rat after intravenous and oral administrations. M64 is partially metabolized to M64-AH; while M64-S-Me is rapidly and totally converted to M64 and M64-AH. M64-AH is mostly oxidized to the inactive M64-diacetamine while its N-reduction to the efficient M64 is a minor metabolic pathway. Oral dose of M64-AH was well absorbed (38%) and converted to M64 and M64-diacetamide. This study generated substantial information about the properties of this class of antimalarial drugs. Other routes of synthesis will be explored to prevent oxidative transformation of the amidoxime and to favour the N-reduction.

Pharmacology of EAPB0203, a novel imidazo[1,2-a]quinoxaline derivative with anti-tumoral activity on melanoma.

In spite of the development of new anticancer drugs by the pharmaceutical industry, melanoma and T lymphomas are diseases for which medical advances remain limited. Thus, there was an urgent need of new therapeutics with an original mechanism of action. Since several years, our group develops quinoxalinic compounds. In this paper, the first preclinical results concerning one lead compound, EAPB0203, are presented. This compound exhibits in vitro cytotoxic activity on A375 and M4Be human melanoma cell lines superior to that of imiquimod and fotemustine. A liquid chromatography-mass spectrometry method was first validated to simultaneously quantify EAPB0203 and its metabolite, EAPB0202, in rat plasma. Thereafter, the pharmacokinetic profiles of EAPB0203 were studied in rat after intravenous and intraperitoneal administrations. After intraperitoneal administration the absolute bioavailability remains limited (22.7%). In xenografted mouse, after intraperitoneal administration of 5 and 20mg/kg, EAPB0203 is more potent than fotemustine. The survival time was increased up to 4 and 2 weeks compared to control mice and mice treated by fotemustine, respectively. The results of this study demonstrate the relationship between the dose of EAPB0203 and its effects on tumor growth. Thus, promising efficacy, tolerance and pharmacokinetic data of EAPB0203 encourage the development towards patient benefit.

Quantitation of SAR97276 in mouse tissues by rapid resolution liquid chromatography-mass spectrometry.

1,12-Bis[5-(2-hydroxyethyl)-4-methyl-1,3-thiazol-3-ium]dodecane dibromide (SAR97276, T3) is a new antimalarial drug, which is currently being evaluated in clinical trials for severe malaria. Drug accumulation inside the parasite and a dual mechanism of action are a major strength of this compound, as it could help delay the development of resistance. The purpose of this article was to develop a rapid resolution LC-MS method for quantifying SAR97276 in mouse tissues. The LC system consisted of Zorbax Eclipse XDB C8 (1.8 microm, 50 x 4.6 mm, 60 degrees C) column. Elution with a gradient mobile phase consisting of ACN-trimethylamine-formate buffer (pH 3) at a flow rate of 1 mL/min yielded sharp, utmost-resolved peaks within 2 min. Tissue samples were powdered under liquid nitrogen. After protein precipitation with citric acid, SPE using WCX cartridges was used for sample preparation. There was no influence of the matrix on the detection of either SAR97276 or the IS. Assay precision was <13% and accuracy was 90-107%. The lower LOQs were 3.3 microg/kg in brain and 33 microg/kg in liver and heart. This newly developed method was used to study the tissue distribution of SAR97276 in mouse as part of the ongoing development of SAR97276.

Quantitation of imidazo[1,2-a]quinoxaline derivatives in human and rat plasma using LC/ESI-MS.

Since several years, our group developed quinoxalinic compounds. Among the synthesized compounds, in the imidazo[1,2-a]quinoxaline series, EAPB0203 has shown interesting activities both on melanoma and lymphoma. The structure of EAPB0203 has been modulated and a new compound, EAPB0503, exhibits an in vitro cytotoxic activity on melanoma cancer cell line 7-9 times higher than EAPB0203. We validated an LC/ESI-MS method to simultaneously quantify EAPB0503 and its metabolite EAPB0603 in human and rat plasma. Chromatography was performed on a C8 Zorbax eclipse XDB column with a mobile phase consisting of acetronitrile and formate buffer gradient elution. LC-MS data were acquired in SIM mode at m/z 305, 291, and 303 for EAPB0503, EAPB0603, and the internal standard, respectively. The drug/internal standard peak area ratios were linked via quadratic relationships to concentrations (low range: 5-300 microg/L, high range: 100-1000 microg/L). The method is precise (precision, < or = 14%) and accurate (recovery, 92-113%). Mean extraction efficiencies, > 72% for each analyte, were obtained. The lower LOQs were 5 microg/L. This highly specific and sensitive method was successfully used to investigate plasma concentrations of EAPB0503 and EAPB0603 in a pharmacokinetic study carried out in rat and would also be useful in clinical trials at a later stage.

Pharmacological properties of a new antimalarial bisthiazolium salt, T3, and a corresponding prodrug, TE3.

A new approach to malarial chemotherapy based on quaternary ammonium that targets membrane biogenesis during intraerythrocytic Plasmodium falciparum development has recently been developed. To increase the bioavailability, nonionic chemically modified prodrugs were synthesized. In this paper, the pharmacological properties of a bisthiazolium salt (T3) and its bioprecursor (TE3) were studied. Their antimalarial activities were determined in vitro against the growth of P. falciparum and in vivo against the growth of P. vinckei in mice. Pharmacokinetic evaluations were performed after T3 (1.3 and 3 mg/kg of body weight administered intravenously; 6.4 mg/kg administered intraperitoneally) and TE3 (1.5 and 3 mg/kg administered intravenously; 12 mg/kg administered orally) administrations to rats. After intraperitoneal administration, very low doses offer protection in a murine model of malaria (50% efficient dose [ED50] of 0.2 to 0.25 mg/kg). After oral administration, the ED50 values were 13 and 5 mg/kg for T3 and TE3, respectively. Both compounds exerted antimalarial activity in the low nanomolar range. After TE3 administration, rapid prodrug-drug conversion occurred; the mean values of the pharmacokinetic parameters for T3 were as follows: total clearance, 1 liter/h/kg; steady-state volume of distribution, 14.8 liters/kg; and elimination half-life, 12 h. After intravenous administration, T3 plasma concentrations increased in proportion to the dose. The absolute bioavailability was 72% after intraperitoneal administration (T3); it was 15% after oral administration (TE3). T3 plasma concentrations (8 nM) 24 h following oral administration of TE3 were higher than the 50% inhibitory concentrations for the most chloroquine-resistant strains of P. falciparum (6.3 nM).

Quantitation of ibogaine and 12-hydroxyibogamine in human plasma by liquid chromatography with fluorimetric detection.

A high-performance liquid chromatographic (HPLC) method with fluorimetric detection was developed for the simultaneous determination of ibogaine and noribogaine in human plasma using fluorescein as internal standard. This method involved a solid phase extraction of the compounds from plasma using N-vinylpyrrolidone-divinybenzene copolymer cartridges. Separation of the three analytes was performed on a reversed-phase Supelcosil C18 analytical column (75 mm x 4.6mm i.d., 3 microm particle size). The excitation wavelength was set at 230 nm for the first 15.8min and then at 440 nm for the following 14.2 min; the emission wavelength was set at 336 nm for the first 15.8 min and then at 514 nm for the following 14.2 min. Obtained from the method validation, inter-assay precision was 6.0-12.5% and accuracy was 95.4-104%. The extraction efficiencies of the assay were higher than 94% and were constant across the calibration range. The lower limits of quantitation were 0.89 ng/ml for ibogaine and 1 ng/ml for noribogaine; at these levels, precision was < or =17% and accuracy was 95-105%. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. Special attention must be paid to sample handling to avoid light degradation of the compounds.

Liquid chromatography-electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells.

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.