Résumé
To develop a simple and highly sensitive high performance liquid chromatography with electrospray ionization mass spectrometric (LC-ESI-MS) method for the simultaneous determination of madecassoside and its major metabolite madecassic acid in rat plasma, and compare the pharmacokinetics of the two compounds in normal and collagen-induced arthritis (CIA) rats. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was accomplished on an Inertsil ODS-3 column, using a gradient elution with the mobile phase composed of acetonitrile and water acidified with 0.1% (V/V) formic acid. Detection was achieved by ESI-MS under the negative selected ion monitoring (SIM) mode. In normal and CIA rats, madecassoside (30 mg·kg(-1)) was orally administered for 21 consecutive days from the day of arthritis onset. For madecassoside, the linear range was 10-1 000 ng·mL(-1) with the square regression coefficient (r) of 0.998 9, while for madecassic acid, the linear range was 10-500 ng·mL(-1) with the square regression coefficient (r) of 0.996 1. The lower limit of quantification was 10 ng·mL(-1) for both analytes. The intra- and inter-day precision ranged from 1.78% to 13.42% for madecassoside and 2.30% to 14.90% for madecassic acid, and the accuracy was between -0.95% and 6.30% for madecassoside and between -1.48% and 5.34% for madecassic acid. The average recoveries of madecassoside, madecassic acid and IS from spiked plasma samples were > 81%. The developed method was successfully applied to the pharmacokinetic study of madecassoside and madecassic acid in rats after an oral administration of madecassoside. During initial 7 days of dosing, the cmax and AUC of madecassoside were greatly decreased and Vd/F was markedly increased in CIA rats, and no significant difference was observed on the first day of dosing. In contrast, the T1/2, cmax and AUC of madecassic acid were significantly increased, and Ke of madecassic acid was greatly decreased in CIA rats compared with normal rats. Along with repeated administration of madecassoside, the differences of pharmacokinetic parameters of both madecassoside and madecassic acid between CIA and normal rats gradually subsided. The pharmacokinetic characteristics of both madecassoside and madecassic acid in rats were significantly altered by arthritis status, and the differences of pharmacokinetic parameters between arthritis and normal rats coincide with the severity of arthritis.
Sujets)
Animaux , Femelle , Antirhumatismaux , Sang , Pharmacocinétique , Utilisations thérapeutiques , Aire sous la courbe , Arthrite expérimentale , Traitement médicamenteux , Métabolisme , Polyarthrite rhumatoïde , Traitement médicamenteux , Métabolisme , Centella , Chimie , Chromatographie en phase liquide , Méthodes , Collagène , Phytothérapie , Extraits de plantes , Sang , Pharmacocinétique , Utilisations thérapeutiques , Rat Wistar , Valeurs de référence , Indice de gravité de la maladie , Spectrométrie de masse ESI , Méthodes , Spectrométrie de masse en tandem , Méthodes , Triterpènes , Sang , Pharmacocinétique , Utilisations thérapeutiquesRésumé
The aim of this study was to establish an analytical method to detect the presence of the responsible triterpenoids of the anti-inflammatory activity of the leaves of Ugni molinae (murtilla). Successive leaves extracts of EtOAc (EAE) and ethanol (TEE) were prepared, obtaining for the first time from TEE a triterpenoid-rich sub-fraction (TF). The topical anti-inflammatory activity of TF was assessed (43.3 percent at 1 mg/ear) by means of the TPA-induced mouse ear oedema model, which was compared to EAE (83.1 +/- 3.2 percent) and TEE (78.3 +/- 11.8 percent) activities, both previously evaluated by us. These extracts were characterized in their triterpenoids by HPLC-UV and HPLC-ESI-MS. We demonstrated that TF has triterpenoids responsible in part of the anti-inflammatory activity, among them, madecassic and maslinic acids. These two compounds have been reported for the first time for this species. ED50 for madecassic and alphitolic acids are also here reported.
El objetivo de este trabajo fue establecer un método analítico para determinar la presencia de los triterpenoides responsables de la actividad anti-inflamatoria de las hojas de Ugni molinae (murtilla). Fueron preparados los extractos seriados de EtOAc (EAE) y etanólico (TEE) desde sus hojas, obteniendo desde el TEE por primera vez una sub-fracción rica en triterpenoides (TF). Se demostró la actividad anti-inflamatoria tópica del TF por el modelo de edema de oreja de ratón inducida por TPA (43,3 por ciento a 1 mg/oreja), la cual fue comparada con las de los EAE (83,1 +/- 3,2 por ciento) y TEE (78,3 +/- 11,8 por ciento) determinadas en nuestros estudios previos. Dichos extractos fueron caracterizados en sus triterpenoides por CLAE-UV y CLAE-IES-MS. Demostramos que el TF contiene triterpenoides responsables en parte de la actividad anti-inflamatoria, entre ellos, los ácidos madecásico y maslínico, reportados por primera vez para esta especie. Se informan además las DE50 para los ácidos madecásico y alfitólico.