Mise au point et validation de méthodes analytiques pour le contrôle de qualité de plantes médicinales malgaches et la mise en place de cultures locales

This work focuses on the methods development for the quality control of Spirospermum penduliflorum (Menispermaceae) and Centella asiatica (Apiaceae). S. penduliflorum is an endemic species from Madagascar and is largely used for its antihypertensive activities. A bioguided fractionation allowed us to isolate and identify by MS and NMR two vasorelaxant aporphine alkaloids: dicentrine and neolitsine. A HPLC-UV method for the quantification of dicentrine, the major alkaloid in the leaves was developed using experimental design. The method allows the separation of 13 alkaloids and was validated. C. asiatica is the second medicinal plant exported by Madagascar where it is largely used for its wound healing and antioxidant activities. An improved HPLC-UV method for the quantification of the active molecules: asiaticoside, madecassoside, asiatic acid and madecassic acid was developed and validated. A HPTLC method which is rapid and less expensive than HPLC was also developed and validated using the Bland and Altman comparison method.Ce travail porte sur la mise au point de méthodes pour le contrôle de qualité de Spirospermum penduliflorum (Menispermaceae) et de Centella asiatica (Apiaceae). S. penduliflorum est une plante endémique malgache dont les feuilles sont utilisées dans la médecine traditionnelle comme antihypertenseur. Un fractionnement bioguidé a permis d’isoler et d’identifier par SM et RMN pour la première fois dans la plante ; deux alcaloïdes aporphiniques vasorelaxants : la dicentrine et la néolitsine. Une méthode de dosage de la dicentrine, alcaloïde majoritaire de la plante, par CLHP-UV a été mise au point en appliquant un plan d’expérience. 13 alcaloïdes ont été séparés et la méthode a été validée. Centella asiatica est la seconde plante médicinale exportée par Madagascar où elle est très utilisée pour ses propriétés cicatrisantes et antioxydantes. Une méthode améliorée de dosage par CLHP-UV des principes actifs : asiaticoside, madécassoside, acide asiatique et acide madécassique a été mise au point et validée. Une autre méthode de dosage par CCMHP de ces triterpènes, rapide et moins coûteuse que la CLHP a également été développée et validée en utilisant la comparaison de Bland et Altman.(FARM 3) -- UCL, 201

Vasorelaxant alkaloids from Spirospermum penduliflorum (Menispermaceae), a plant used to treat hypertension in malagasy traditional medicine

Spirospermum penduliflorum Thouars (Menispermaceae) is widely used on the eastern coast of Madagascar to treat hypertension. The aim of the present study was to analyse the vasorelaxant properties of different leaf extracts. The activity of the n-hexane, dichloromethane and methanolic extracts was tested on phenylephrine-contracted aorta. The dichloromethane extract was shown to be the most effective. Further fractionation of this extract led to the isolation of an active fraction relaxing phenylephrine-contracted aorta with an IC50 of 0.18 μg/mL {log IC50 (μg/mL) -0.74 ± 0.03} but was much less effective on KCl induced contractions. Bioassay-guided fractionation of this fraction led to the isolation of two aporphinoid alkaloids, neolitsine and dicentrine, which at concentrations of 0.1 μM and 1 μM displaced to the right the phenylephrine concentration-contraction curve. Our results show that Spirospermum penduliflorum extracts possess vasorelaxant activity in vitro that could be related to the presence of dicentrine in the extracts having an alpha1 antagonist activity. This finding is not in accord with the previous studies by Rasoanaivo et al. where no alkaloids were detected in the leaves of Spirospermum pendulifloru

An improved HPLC-UV method for the simultaneous quantification of triterpenic glycosides and aglycones in leaves of Centella asiatica (L.) Urb (APIACEAE).

The simultaneous quantification of madecassoside, asiaticoside, madecassic acid and asiatic acid in Centella asiatica by HPLC-UV is proposed. Asiaticoside was used as reference for the quantification of heterosides and asiatic acid for aglycones. The evaluation of the extraction efficiency of the four molecules led to use Soxhlet extraction for 8 h. The method was validated and was found to be accurate in the concentration range of 1.0-3.0 mg/ml for asiaticoside and 0.5-2.0 mg/ml for asiatic acid with CV <3% for all investigated compounds. LOD and LOQ were, respectively, 0.0113 and 1.0 mg/ml for asiaticoside and 0.0023 and 0.5 mg/ml for asiatic acid. This method was shown to be convenient for routine analysis of samples of C. asiatica

APPLICATION OF DESIGN OF EXPERIMENTS AND DESIGN SPACE METHODOLOGY FOR THE HPLC-UV SEPARATION OPTIMIZATION OF APORPHINE ALKALOIDS FROM LEAVES OF Spirospermum penduliflorum THOUARS

Spirospermum penduliflorum Thouars (Menispermaceae) is an endemic species of Madagascar traditionally used as vasorelaxant. Recently, two aporphine alkaloids known to possess antihypertensive activity (dicentrine and neolitsine) were isolated and identified from the leaves of this plant. In the present study, a HPLC-UV method allowing the separation of all alkaloids and the quantification of dicentrine in the alkaloidic extract of leaves was developed using design of experiments and design space methodology. Three common chromatographic parameters (i.e. the mobile phase pH, the initial proportion of methanol and the gradient slope) were selected to construct a full factorial design of 36 experimental conditions. The times at the beginning, the apex (i.e. the retention time) and the end of each peak were recorded and modelled by multiple linear equations. The corresponding residuals were normally distributed which confirmed that the models can be used for the prediction of the retention times and to optimize the separation. The optimal separation was predicted at pH 3, with a gradient starting at 32% of methanol and a gradient slope of 0.42%/min. Good agreement was obtained between predicted and experimental chromatograms. The method was also validated using total error concept. Using the accuracy profile approach, validation results gave a LOD and LOQ for dicentrine of 3 µg/ml and 10 µg/ml, respectively. A relative standard deviation for intermediate precision lower than 10% was obtained. This method was found to provide accurate results in the concentration range of 10 µg/ml to 75 µg/ml of dicentrine and is suitable for routine analysis