Claude-Alain Calliste

Antioxidant, anti-inflammatory and antiproliferative properties of sixteen water plant extracts used in the Limousin countryside as herbal teas

The present paper presents the antioxidant, anti-inflammatory and antiproliferative capabilities of 16 plants. These plants can be found in the Limousin countryside and most of them are used in popular medicine as herbal tea. The biological properties of the water-soluble fractions were measured. Antioxidant properties were evaluated by the ESR method in order to visualize the inhibition of the DPPH, superoxide and hydroxyl radicals. Some extracts were good antioxidants by comparison with reference molecules, e.g. vitamin E and quercetin. Antioxidant effects were correlated with the total amount of phenolic compounds contained in the extracts. Also measured were the anti-inflammatory activities of the 16 water-soluble fractions, by evaluating inhibition of lipoxygenase activity. Finally the effects of these plants on the proliferation of melanoma B16 cells were studied.

Eight flavonoids and their potential as inhibitors of human cytomegalovirus replication

The drugs currently available for treatment of severe human cytomegalovirus (HCMV) infections suffer from many drawbacks, particularly toxicity, and potential teratogenicity contraindicating their use in target populations such as pregnant women. The emergence of drug-resistant strains is still a problem for disease management, particularly in immunosuppressed populations where antivirals are used for extended periods of time. The flavonoid family of drugs contains promising candidates as they have low toxicity and inhibit different targets to currently available antivirals. We report here that, unlike their chalcon homologs, four flavonoids (baicalein, quercetin, quercetagetin and naringenin) inhibit various stages of HCMV replication, the most active anti-HCMV compound being baicalein and the less active and less selective being quercetagetin. These drugs could provide potential inhibitors of virus replication alone or in combination, without increased toxicity.

Redox Reactions Obtained by γ Irradiation of Quercetin Methanol Solution are Similar to In Vivo Metabolism

Abstract Marfak, A., Trouillas, P., Allais, D. P., Calliste, C. A. and Duroux, J. L. Redox Reactions Obtained by γ Irradiation of Quercetin Methanol Solution are Similar to In Vivo Metabolism. Radiat. Res. 159, 218–227 (2003). The flavonol quercetin is one of the most well-known antioxidant flavonoids. Its antioxidant potential has been studied extensively during the last 10 years, but little is known about the metabolites formed in vivo that lead to the formation of depside and small molecules such as benzoic acids. In this study, γ irradiation of a quercetin methanol solution was used as a model of certain oxidative reactions that occur in vivo. Qercetin at concentrations ranging from 5 × 10−5 M to 5 × 10−3 M, was irradiated with γ rays at doses of 2–14 kGy. Quercetin degradation was evaluated by HPLC analysis. The major radiolytic metabolite was identified as a depside by NMR and LC-MS. Formation of 3,4-dihydroxybenzoic acid was also observed. The presence of CH3O· formed during methanol radiolysis is invoked to explain depside formation. Transformation of the 8-methoxy substituted depside (Q1) to the 8-hydroxyl substituted depside (Q2) is discussed. The antioxidant properties of quercetin metabolites are evaluated according to their capacity to decrease the EPR DPPH signal and to inhibit superoxide radical formed by the enzymatic reaction (xanthine + xanthine oxidase). For both assays, the IC50 of Q2 is twice as high as that of quercetin.

Mechanisms of Transformation of the Antioxidant Kaempferol into Depsides. Gamma-Radiolysis Study in Methanol and Ethanol

Abstract Marfak, A., Trouillas, P., Allais, D. P., Calliste, C. A., Cook-Moreau, J. and Duroux, J. L. Mechanisms of Transformation of the Antioxidant Kaempferol into Depsides. Gamma-Radiolysis Study in Methanol and Ethanol. Radiat. Res. 160, 355–365 (2003). In this study, we irradiated the antioxidant kaempferol in ethanol and methanol solutions with γ rays at doses ranging from 0.2–20 kGy. NMR and ES-MS spectroscopy were used to identify radiolysis products. Two depsides, {2-[(4′-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl}(oxo) methyl acetate and {2-[(4′-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl}(oxo) ethyl acetate, were the major compounds of kaempferol degradation in methanol and in ethanol, respectively. Other products formed in low concentrations were identified as [4-hydroxyphenyl](oxo) methyl acetate, [4-hydroxyphenyl](oxo) ethyl acetate, and depside {2-[(4′-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl}(oxo) acetic acid. The formation of the latter was observed in both solvents. We propose degradation mechanisms that suggest that ·CH2OH and CH3·CHOH, produced by solvent radiolysis, react with the 3-OH kaempferol group because of its high H-donor capacity. π-Electron delocalization in the flavonoxy formed after the first H-transfer leads to C-ring opening and consequently to the formation of depsides. G calculation of the degradation products and of ·CH2OH and CH3·CHOH radicals confirmed the proposed mechanism of kaempferol radiolysis. The rate constants for the reaction between kaempferol and these free radicals were also calculated. Formation of depside has also been observed in many studies of the oxidation of flavonoids; those studying human metabolism have suggested similar redox transformation of flavonols. The antioxidant activities of radiolysis products were evaluated and compared to those of kaempferol.

Ex vivo model of congenital cytomegalovirus infection and new combination therapies.

INTRODUCTION Congenital human cytomegalovirus (HCMV) infection is a major public health problem due to severe sequelae in the fetus and newborns. Currently, due to their toxicity anti-CMV treatments cannot be administered to pregnant women. We thus developed an ex vivo model of 1(st) trimester placental CMV infection to observe the route of infection across the placenta and to test the efficacy of various new drugs targeting different stages of viral cycle. METHODS After validation of the viability of floating villi explants by ELISA β-HCG, the kinetics of placental infection were determined by immunochemistry and qPCR in this ex vivo model. Antiviral susceptibility was determined in vitro using focus reduction assay and by qPCR in the ex vivo model. RESULTS The ex vivo model showed viral infection in trophoblasts and mesenchymal space of floating villi. In vitro, antiviral combinations of maribavir with baïcalein or artesunate inhibited viral infection by more than 90%. On the other hand, in ex vivo model, infection was reduced by 40% in presence of maribavir and artesunate. The synergistic effect observed in vitro was not observed ex vivo. DISCUSSION This model allowed us to understand the CMV spread in 1(st) trimester floating villi better and to analyze the anti-CMV efficacy and toxicity of new drugs that could be administered to pregnant women, either alone or in combination. CONCLUSIONS Such an ex vivo model could be applied to other viruses such as rubella or parvovirus B19 and in new drug development.

Partial structural characterization and antioxidant activity of a phenolic–xylan from Castanea sativa hardwood

4-O-Methylglucuronoxylans (MGX) were isolated from chestnut wood sawdust using two different procedures: chlorite delignification followed by the classical alkaline extraction step, and an unusual green chemistry process of delignification using phthalocyanine/H2O2 followed by a simple extraction with hot water. Antioxidant properties of both MGX were evaluated against the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) by electronic spin resonance (ESR). IC50 of water-extracted MGX was found to be less than 225 μg mL(-1), in contrast with alkali-extracted MGX for which no radical scavenging was observed. Characterization of extracts by colorimetric assay, GC, LC-MS and NMR spectroscopy provided some clues to understanding structure-function relationships of MGX in connection with their antioxidant activity.

Comparative antioxidant activity of two Algerian Reseda species

The Reseda genus is found in the Mediterranean and the Southwestern Asian areas [1]. In Algeria, there are twenty two species and subspecies [2, 3]. Eight flavones [4–9], sixteen flavonols [7, 8, 10–16], and one isoflavone [13] have been reported from the genus Reseda. R. villosa (Resedaceae) is an endemic species [1] collected from Ghardaia (Septentrional Algerian Sahara) in March 2002 and authenticated by Prof. Gerard De Belair (Annaba, Algeria). R. duriaeana (Resedaceae) is an endemic species [2] collected from Djebel El-Ouach-Constantine (Eastern Algeria) in May 2004 and authenticated by Prof. Gerard De Belair (Annaba, Algeria). Voucher specimen were deposited in the herbarium of the Laboratory of Therapeutic Substances (LOST) at Mentouri University (LOST/ZKAK Rv/03/02 and LOST/ZKAK Rd/05/04). Air-dried and powdered aerial parts (1 kg) of R. duriaeana and R. villosa (1 kg) were macerated in a methanolic solution (70%). The extract of each plant was successively concentrated to dryness (under low pressure); the residue was dissolved in boiling water and extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol successively. The butanolic extract was column chromatographed on polyamid SC6 and eluted with toluene–methanol with increasing polarity. Whatman 3MM paper chromatography using 15% AcOH and BAW (n-BuOH–AcOH–H2O, 4:1:5 upper phase) and TLC on polyamid DC6, eluted with H2O–MeOH–methyl–ethyl ketone–acetylacetone (13:3:3:1) followed by column flash chromatography over Sephadex LH-20 in MeOH led to five pure flavonoid glycosides from R. duriaeana (1–5) and six flavonoid glycosides (5–10) from R. villosa, which were identified by the usual physicochemical techniques: Rf, fluorescence, UV, NMR spectroscopies, and acid hydrolysis [17–19]. Acid Hydrolysis. The pure compounds were treated with 2 M HCl at 100 C for 1 h. The hydrolysates were extracted with EtOAc, and the aglycones were identified by their UV spectra in methanol and by comparison of their Rf with authentic samples. Sugars in the aqueous residue were identified by comparison with authentic samples on silica gel TLC impregnated with 0.2 M NaH2PO4, solvent Me2CO–H2O (9:1), and revealed with aniline malonate. Compound 1, C21H19O12; mp 176–177 C; UV (MeOH, max, nm): 264.5, 339; NaOH: 273, 322, 389; AlCl3: 273, 394; AlCl3 + HCl: 274, 393. NaOAc: 277, 350; NaOAc + H3BO3: 278, 345. 1H NMR (250 MHz, CD3OD, , ppm, J/Hz): 7.80 (2H, d, J = 8.8, H-2 , H-6 ), 6.90 (2H, d, J = 8.8, H-3 , H-5 ), 6.40 (1H, d, J = 2.0, H-8), 6.20 (1H, d, J = 2.0, H-6), 5.40 (1H, d, J = 1.7, H-1 , 3-O-Rha), 0.90 (3H, d, J = 6.0, CH3, 3-O-Rha). Identified as kaempferol 3-O-rhamnoside. Compound 2, C21H20O10; mp 194–196 C (methanol); UV (MeOH, max, nm): 265.5, 366; NaOH: 292.5, 434; AlCl3: 273, 420; AlCl3 + HCl: 274, 419. NaOAc: 267, 373; NaOAc + H3BO3: 268, 375. 1H NMR (250 MHz, CD3OD, , ppm, J/Hz): 7.80 (2H, d, J = 8.7, H-2 , H-6 ), 6.95 (2H, d, J = 8.7, H-3 , H-5 ), 6.70 (1H, d, J = 2.0, H-8), 6.47 (1H, d, J = 2.0, H-6), 5.41 (1H, d, J = 6.0, H-1 , 7-O-Glu). Characterized as kaempferol 7-O-glucoside.

Lipocarbazole, an efficient lipid peroxidation inhibitor anchored in the membrane

Lipid peroxidation is a major deleterious effect caused by oxidative stress. It is involved in various diseases such as atherosclerosis, rheumatoid arthritis and neurodegenerative diseases. In order to inhibit lipid peroxidation, antioxidants must efficiently scavenge free radicals and penetrate inside biological membranes. Lipocarbazole has recently been shown to be a powerful antioxidant in solution. Here, we show its powerful capacity as lipid peroxidation inhibitor. Its mechanism of action is rationalized based on molecular dynamics simulations on a biomembrane model, quantum calculations and experimental evaluation. The role of the lipocarbazole side chain is particularly highlighted as a critical chemical feature responsible for its antioxidant activity.

Radiolytic transformation of 2,2',4'-trihydroxychalcone.

Abstract Mokrini, R., Trouillas, P., Kaouadji, M., Champavier, Y., Houée-Lévin, C., Calliste, C. A. and Duroux, J. L. Radiolytic Transformation of 2,2′,4′-Trihydroxychalcone. Radiat. Res. 165, 730–740 (2006). Radiolysis of 2,2′,4′-trihydroxychalcone, a natural antioxidant present in fruit and vegetables, was performed in ethanol in the absence or in the presence of dioxygen. The degradation process of chalcone was followed in deaerated solution by HPLC, NMR, FAB-LSIMS mass spectroscopy and analytical TLC. Under anaerobic conditions, six new products (three couples of diastereoisomers) were identified. Four of them kept the chalcone skeleton with OCH2CH3, CH(OH)CH3 and H substitutions on Cα and Cβ. Thus the target was the α-β double bond on which ethanol radicals were added. The two other compounds were formed in a second stage and exhibited a cyclization between the substituent on Cβ and the carbonyl group. In the presence of dioxygen, these reactions were prevented and chalcone was protected. This study was the first step toward understanding of the behavior chalcone in irradiated fruits and vegetables.

Secondary Metabolites, Evaluation of the DPPH Free-Radical Scavenging Effect by Electron Spin Resonance and Antibacterial Activity of the Endemic Species Stachys circinata

The genus Stachys (Lamiaceae), widely known in folk medicine, contains about 200–300 species in the world [1]. In the Flora of Algeria, this genus is represented by 14 species of which four are endemic [2]. In continuation of our work on Stachys genus [3–6], we report here the chemical constituents of the n-butanolic and dichloromethane extracts of the aerial parts of the Algerian species Stachys circinata L Her. which has not been the subject of any previous study. Fifteen known compounds have been identified. We also report here the antibacterial and antioxidant activity of the n-butanolic extract by using disk diffusion and eletron spin resonance methods (ESR). S. circinata L Her. was collected from Djebel El-Ouahch-Constantine (North Eastern Algerian) in April 2005 during the flowering stage and authenticated by Prof. G. De Belair (University of Annaba, Algeria). A voucher specimen (MBAng2005.09) has been deposited in the Herbarium of the Musee Botanique de la Ville d Angers (France). Air-dried and powdered aerial parts (1 kg) of S. circinata were macerated three times at room temperature with MeOH–H2O (7:3, v/v) for 24 h. After filtration, the filtrate was concentrated and dissolved in water (600 mL). The resulting solution was extracted successively with petroleum ether, CH2Cl2, EtOAc, and n-butanol. Concentration in vacuo at room temperature led to the following extracts: petroleum ether (2.3 g), dichloromethane (9 g), EtOAc (5 g), and n-butanol (25 g). The n-butanolic extract of Stachys circinata (BESC) (10 g) was chromatographed over a polyamide column and eluted with a gradient of toluene–methanol with increasing polarity, then on a silica gel column eluting with a gradient of isopropanol–dichloromethane (4:1), ethyl acetate–dichloromethane (3:2), and ethyl acetate–water–methanol (10:1:1, 10:2:1). Further preparative silica gel TLC eluting with ethyl acetate–water–methanol (10:1:1, 10:2:1, 8:2:1) and dichloromethane– methanol (9:1) and paper chromatography (Whatman 3MM) eluting with acetic acid (15%), followed by flash column chromatographies on Sephadex LH20 eluting with MeOH, led to nine compounds. Structures of compounds 1–9 were established by chemical and spectral analysis, mainly HR-ESI-MS and HR-EI-MS, UV, 1H and 13C NMR, DEPT, and 2D NMR experiments (COSY, HSQC, ROESY, and HMBC), as well as by comparing their spectroscopic data with those reported in the literature. The dichloromethane extract of Stachys circinata (DESC) (9 g) was fractionated by silica gel column chromatography eluting with a step gradient of cyclohexane–ethyl acetate and then with increasing percentages of MeOH to yield 17 fractions. After purification by crystallization in EtOAc and a little amount of CH2Cl2, silica gel column chromatography eluting with hexane–ethyl acetate (1:1), CH2Cl2–MeOH (4:1), and CH2Cl2–MeOH (7:3) and preparative TLC on silica gel eluting with hexane–ethyl acetate (4:1) and CH2Cl2–MeOH (9:1) and double elutions with CH2Cl2–MeOH (7:3) lead to six compounds. Compounds 10–15 were identified by the use of 1H and 13C NMR, DEPT, and two-dimensional NMR experiments, and COSY in addition to IR spectroscopy, melting point tests, and HR-EI-MS.