Jean-Noël Wauters

Evaluation of the antioxidant activity of passion fruit (Passiflora edulis and Passiflora alata) extracts on stimulated neutrophils and myeloperoxidase activity assays.

The antioxidant activity of methanol extracts from Passiflora edulis and Passiflora alata pulp, and P. edulis rinds, healthy or infected with the passion fruit woodiness virus (PWV), was investigated using the oxidant activities of the neutrophil and the neutrophil granule enzyme myeloperoxidase (MPO), both playing key roles in inflammation. The reactive oxygen species produced by stimulated neutrophils were evaluated by lucigenin-enhanced chemiluminescence (CL) and the activity of purified MPO was measured by SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection), a technique for studying the direct interaction of a compound with the enzyme. The rind extracts of P. edulis possessed higher and dose-dependent inhibitory effects on CL response and on the peroxidase activity of MPO than total pulp extracts from both passion fruit species. The quantification of isoorientin in the extracts showed a correlation with their antioxidant activity, suggesting the potential of P. edulis rinds as functional food or as a possible source of natural flavonoids.

Quality assessment of Polygonum cuspidatum and Polygonum multiflorum by 1H NMR metabolite fingerprinting and profiling analysis.

The quality assessment and control of traditional Chinese medicines (TCM) nowadays receives a great deal of attention worldwide and particularly in Europe with its increasing local use. Polygonum cuspidatum Siebold & Zucc. and Polygonum multiflorum Thunb. are two members of the Polygonaceae family, which are widely used as Chinese medicinal plants. The aim of this study was to achieve an overview of the quality of P. cuspidatum and P. multiflorum samples available on the Chinese market and to identify important metabolites for their discrimination, using (1)H NMR-based metabolomics. (1)H NMR and multivariate analysis techniques were applied to almost 60 plant samples collected in different places in China. Using (1)H NMR metabolomics, it was possible, without previous evaporation or separation steps, to obtain metabolic fingerprints to distinguish between the species. The important metabolites for discrimination were stilbene derivatives. Finally, a clear distinction between the two species was possible and the discriminant metabolites were identified.

Analysis of passion fruit rinds (Passiflora edulis): isoorientin quantification by HPTLC and evaluation of antioxidant (radical scavenging) capacity

The content of isoorientin in passion fruit rinds (Passiflora edulis fo. flavicarpa O. Degener) was determined by HPTLC (high performance thin layer chromatography) with densitometric analysis. The results revealed a higher amount of isoorientin in healthy rinds of P. edulis (92.275 ± 0.610 mg L-1) than in rinds with typical symptoms of PWV (Passion fruit Woodiness Virus) infection (28.931 ± 0.346 mg L-1). The HPTLC data, allied to assays of radical scavenging activity, suggest the potential of P. edulis rinds as a natural source of flavonoids or as a possible functional food.

Revealing the anti-tumoral effect of Algerian Glaucium flavum roots against human cancer cells.

Glaucium flavum (G. flavum) is a plant from the Papaveraceae family native to Algeria where it is used in local traditional medicine to treat warts. G. flavum root crude alkaloid extract inhibited breast cancer cell proliferation and induced G2/M phase cycle arrest and apoptosis without affecting normal cells, which is a highly awaited feature of potential anti-cancer agents. G. flavum significantly reduced growth and vascularization of human glioma tumors on chicken chorioallantoic membrane (CAM) in vivo. The chromatographic profile of the dichloromethane extract of G. flavum root showed the presence of different constituents including the isoquinoline alkaloid protopine, as the major compound. We report for the first time that G. flavum extract may represent a new promising agent for cancer chemotherapy.

Identification and Quantification of the Main Active Anticancer Alkaloids from the Root of Glaucium flavum

Glaucium flavum is used in Algerian folk medicine to remove warts (benign tumors). Its local appellations are Cheqiq el-asfar and Qarn el-djedyane. We have recently reported the anti-tumoral activity of Glaucium flavum root alkaloid extract against human cancer cells, in vitro and in vivo. The principal identified alkaloid in the extract was protopine. This study aims to determine which component(s) of Glaucium flavum root extract might possess potent antitumor activity on human cancer cells. Quantitative estimation of Glaucium flavum alkaloids was realized by HPLC-DAD. Glaucium flavum effect on human normal and cancer cell viability was determined using WST-1 assay. Quantification of alkaloids in Glaucium flavum revealed that the dried root part contained 0.84% of protopine and 0.07% of bocconoline (w/w), while the dried aerial part contained only 0.08% of protopine, glaucine as the main alkaloid, and no bocconoline. In vitro evaluation of the growth inhibitory activity on breast cancer and normal cells demonstrated that purified protopine did not reproduce the full cytotoxic activity of the alkaloid root extract on cancer cell lines. On the other hand, bocconoline inhibited strongly the viability of cancer cells with an IC50 of 7.8 μM and only a low cytotoxic effect was observed against normal human cells. Our results showed for the first time that protopine is the major root alkaloid of Glaucium flavum. Finally, we are the first to demonstrate a specific anticancer effect of Glaucium flavum root extract against breast cancer cells, which can be attributed, at least in part, to bocconoline.

Rotenoid content and in vitro acaricidal activity of Tephrosia vogelii leaf extract on the tick Rhipicephalus appendiculatus

This study aimed to determine the rotenoid content of leaf extracts of the white (TVW) and purple (TVP) varieties of Tephrosia vogelii, both collected in North-Kivu Province, Democratic Republic of Congo and to evaluate their in vitro acaricidal efficacy on the tick Rhipicephalus appendiculatus. The high performance liquid chromatography analysis of rotenoid compounds from those extracts revealed that the contents of rotenone and deguelin were respectively higher in the leaves of TVW (0.044% and 1.13%) than in TVP (0.014% and 0.66%). Batches of 20 live adult ticks were immersed for 15 min in six different doses of each plant extract (0.625; 1.25; 2.5; 5; 10 and 20mg/mL of distilled water) and in the solution of Milbitraz(®) (12.5%m/v emulsifiable concentrate of amitraz) as a positive control. Additionally 9.5% ethanol and distilled water control groups were included. Tick mortalities were recorded every 24h for 5 days. The results indicated that there was no significant difference (P>0.05) between the acaricidal effect of Milbitraz(®) and the plant material used at a dose of at least 2.5 or 5mg/mL for TVW and TVP respectively. However, the dose response relationship determined at the fifth day after treatment showed a similar acaricidal effect for the two plant varieties with similar lethal dose 50 (LD(50)) of 0.83 and 0.81 mg/mL for TVW and TVP respectively. It is concluded that T. vogelii leaves may be used for the control of R. appendiculatus in areas where synthetic acaricides are either not available or affordable. However, T. vogelii extract should be sprayed in order to limit the potential risks of ecotoxicity linked to rotenoid compounds.

Densitometric evaluation of spiraeoside after derivatization in flowers of Filipendula ulmaria (L.) maxim

In traditional medicines of Europe, thé water extract of Filipendula ulmaria (L.) Maxim. (Rosaceae) flowers has been used as antiinflammatory, analgésie and diuretic (1) and thé compounds of this plant are known to be flavonoids tannins and salicylic acid derivatives (2). Spiraeoside (quercetin-4 glucoside) is thé major and characteristic flavonoid of Filipendula ulmaria flowers and, for this reason, we determined its amount by HPTLC densitometry. We measured thé fluorescence of spiraeoside after derivatization by diphenylboric acid-2-aminoethylester (3). The measurement was achieved by means of a TLC Scanner programmed to work in reflection fluorescence at 330 nm (Mercury lamp, cut-off filter 450 nm). We respected thé following chromatographic procédure: Layer: HPTLC plates silicagel 60 Merck Mobile phase: ethylacetate-formic acid-water (6:1:1 ,v/v/v) Standard solution (0.6,0.8 and 1 ni). 4 mg of spiraeoside SCR in 10 ml methanol. Sample solution: (0.4 M.!): 0.250 g of Filipendula flowers were extracted by 25 ml methanol 60°C (2 hours). The solution was fittrated, evaporated and dissolved in 25 ml methanol. After linearization, thé concentration of spiraeoside was estimated by measurement of thé différent standard and samples mean areas. In ourfindings, thé spiraeoside content ranged from 3 to 4.3 %. The repeatability, reproducibility and thé good linearity were confirmed by thé validation of thé method. This HPTLC method is rapid and suitable for adoption in thé future for thé détermination of spiraeoside in Filipendula ulmaria flowers and extracts. Références 1) Yeo, H.S., Kim, J., Chung, B.S. (1990) Planta Med. 56, 539. 2) Meier, B., Lehmann, D., Sticher, O, Bettschart, A. (1987) Dtsch Apoth. Ztg, 127, 2401-2407. 3) Billeter, M., Meier, B., Sticher, O. (1990) J. Planar Chromatogr. 3,370-375. h I ,

Fingerprinting and validation of a LC-DAD method for the analysis of biflavanones in Garcinia kola-based antimalarial improved traditional medicines

African populations use traditional medicines in their initial attempt to treat a range of diseases. Nevertheless, accurate knowledge of the composition of these drugs remains a challenge in terms of ensuring the health of population and in order to advance towards improved traditional medicines (ITMs). In this paper chromatographic methods were developed for qualitative and quantitative analyses of a per os antimalarial ITM containing Garcinia kola. The identified analytical markers were used to establish TLC and HPLC fingerprints. G. kola seeds were analysed by HPLC to confirm the identity of the extract used by the Congolese manufacturer in the ITM. The main compounds (GB1, GB2, GB-1a and Kolaflavanone) were isolated by preparative TLC and identified by UPLC-MS and NMR. For the quantification of the major compound GB1, a simple and rapid experimental design was applied to develop an LC method, and then its validation was demonstrated using the total error strategy with the accuracy profile as a decision tool. The accurate results were observed within 0.14-0.45mg/mL range of GB1 expressed as naringenin. The extracts used in several batches of the analysed oral solutions contained GB1 (expressed as naringenin) within 2.04-2.43%. Both the fingerprints and the validated LC-DAD were found suitable for the quality control of G. kola-based raw material and finished products, respectively.

Metabolomics analysis of Galium odoratum (L.) Scop.: impact of the plant population origin and growth conditions.

IntroductionGalium odoratum is a plant used in traditional medicine and to prepare beverages. Objective: To study the impact of plant origin and growth conditions on the metabolite content of the plant. Material and methods: Aerial biomass of Galium odoratum was collected from five natural populations (in situ conditions) and from controlled environment (ex situ conditions). NMR-based fingerprinting method was successfully applied to the discriminating chemical profiling of the in situ and ex situ samples. Results: Quantitative analysis of selected phytochemicals including phenylpropranoids and iridoids showed clear differences between the plants from nature and those of controlled growth conditions as well as internal variation within the group. The metabolomic approach emphasized the decrease of the secondary metabolites pool paralleled by an increase of the carbohydrates in ex situ conditions. The quantitative HPLC-UV points out slight variations of each of the analyzed secondary metabolites between populations in natural environment, variations maintained for three populations in the controlled conditions. Conclusion: Metabolomics approaches using H-NMR and HPLC are worth to consider for studying the impact of climate factors on the regulation of the phytochemical profile in relation to the origin of the plant material.