Estudio químico bioguiado de los extractos con actividad analgésica y antiinflamatoria de Adesmia atacamensis Phil.

Abstract

El género Adesmia (145 especies) en Chile se encuentra distribuido a lo largo de la Cordillera de los Andes y varias de sus especies son empleadas para el tratamiento de la inflamación y del dolor. Recientemente, en una investigación paralela, se ha podido demostrar la actividad analgésica, antiinflamatoria, atrapadora de radicales libres y antimicrobiana de A. verrucosa. Continuando con el estudio de las especies de este género, los resultados preliminares del exudado resinoso de A. atacamensis, han demostrado propiedades antiinflamatorias y analgésicas. Mediante un fraccionamiento bioguiado del exudado resinoso (64,3 g) de A. atacamensis, se obtuvieron las siguientes fracciones: AaResA (2,5 g), AaResB (9 g), AaResC (8 g), AaResD (14,3 g) y AaResE (19,4 g) a las que se les determinó su actividad farmacológica. La actividad analgésica oral se evaluó mediante la inducción del efecto algésico por ácido acético al 0,6%, la actividad antiinflamatoria tópica por inducción del edema de oreja por ácido araquidónico (AA) y por 12-miristatoforbol 13-acetato (TPA). Además, se les evaluó la capacidad atrapadora de radicales libres por el método del DPPH, así como también, su capacidad antioxidante por la inhibición de la enzima XO y por último, la actividad antimicrobiana mediante bioautografías, determinando la concentración mínima inhibitoria de los metabolitos activos. Las fracciones bioactivas AaResB, AaResC, AaResD y AaResE, presentaron una importante actividad antiinflamatoria frente a TPA (todas superior a 70%), siendo la E la más activa. Un efecto bastante menor, se observó al inducir la inflamación con AA. Al evaluar la actividad atrapadora de radical libre (DPPH), todas las fracciones resultaron inactivas y por ende, sólo se determinó la posible inhibición de XO a las fracciones AaResD y AaResE por visualizar en su composición mayor cantidad de derivados fenólicos, resultando levemente positivas (< al 50%). Las fracciones AaResB, AaResC y AaResD presentaron actividad antimicrobiana frente a Bacillus subtilis, Microccocus flavus y Staphyloccocus aureus. El aceite esencial se asemeja a la fracción AaResB en cuanto a los terpenos presentes, pero difiere de ésta, en que no posee flavonoides. El aceite esencial, a diferencia de la fracción AaResB, no presentó actividad antiinflamatoria tópica. De la fracción C se aislaron dos flavonoides AaC1 y AaC2 usando columnas rápidas de gel de sílice, identificados por espectroscopía de RMN de 1H y 13C como 3-O-metilgalangina y 3-O-metil-6-metoxicanferol. Ambos compuestos presentaron actividad antiinflamatoria frente a TPA (81,7% y 73,1% respectivamente), siendo el compuesto AaC2 activo frente a AA (36,7%). Ambos flavonoles, fueron inactivos como atrapadores de radicales libres. El compuesto AaC2 presentó actividad antimicrobiana contra B. subtilis y S. aureus (CMI 150 y 240 g/mL respectivamente), mientras que AaC1 sólo presentó actividad contra B. subtilis (CMI: 200 g/mL). Los resultados hasta ahora encontrados proporcionan un respaldo científico al uso de A. atacamensis, destacando su propiedad antiinflamatoria. Las actividades demostradas por la fracción C se deberían a la presencia de flavonoides, entre otros, lo que merece continuar el estudio químico y farmacológico de las otras fracciones obtenidas de esta especie.

In Chile the genus Adesmia (145 species) is distributed along the Los Andes mountain and are employed in folklore medicine for the treatment of several ailments as pain and inflammation. Recent results have demonstrated anti-inflammatory, antioxidant and antimicrobial properties for A. verrucosa due to the presence of bioactive metabolites. Looking forward to study a different species of this genus, preliminary studies of the resinous exudates (64.3 g) of A. atacamensis demonstrated its anti-inflammatory and analgesic, properties. The above extract was submitted to a bioguided fractionation in column chromatography, yielding the following fractions: AaResA (2.5 g), AaResB (9 g), AaResC (8 g), AaResD (14.3 g) and AaResE (19.4 g) and its pharmacological properties were evaluated. To determine the topical anti-inflammatory activity the ear oedema method in CF-1 mice, induced by two different inflammatory agents: phorbol 12-myristate 13-acetate (TPA) and araquidonic was used. The oral analgesic activity was evaluated by means of abdominal writhing method induced by acetic acid in mice, and the fractions were administerd orally. The free radical scavenger properties were evaluated according to the DPPH method as well as the antioxidant capacity by the inhibition of xanthine oxidase method, finally the antimicrobial activity through a bio-autographic agar overlay and the minimal inhibitory concentration (MIC) for the active samples was determined. Fractions AaResB, AaResC, AaResD and AaResE displayed an important anti-inflammatory effect when inflammation was induced by TPA (higher than 70%) being fraction E the most active, while a much lower anti-inflammatory effect was observed when inflammation was induced by AA. Every fraction together with the isolated compounds resulted to be inactive as radical scavenger and only a very low antioxidant activity was observed for fractions AaResD and AaResE, that were assayed as phenolic compounds were observed in its chemical composition. The fractions AaResB, AaResC and AaResD presented antimicrobial activity against Bacillus subtilis, Microccocus flavus and Staphyloccocus aureus. The essential oil (0.72% v/w) was obtained from the fresh aerial part of A. atacamensis and its chemical composition was compared by thin layer chromatography and gas-mass chomatography with all the fractions obtained from the resinous exudates, showing similar terpenoids constituents to AaResB, differing the last for the presence of flavonoids compounds. The essential oil resulted to be inactive as anti-inflammatory. From the active fraction AaResC, two flavonols (AaC1 and AaC2) were isolated by flash column chromatography and identified by NMR of 1H and 13C as 3-O-methylgalangin and 3-O-methyl-6-methoxykaempferol. Both compounds (AaC1 and AaC2) were active as anti-inflammatory when induced by TPA (81.7% and 73.1% respectively), and only AaC2 showed effect in the ear oedema induced by AA (36.7%). Both compounds showed no radical scavenger activity. Mean while AaC2 presented antimicrobial activity against B. subtilis and S. aureus, (MIC: 150 and 240 ug/ml respectively) and AaC1 was active only against B. subtilis (MIC: 200ug/ml). The results afforded in this research provide a scientific endorsement to the use of A. atacamensis, emphasizing its anti-inflammatory properties. The anti-inflammatory activity found for fraction AaResC would be due in part to the presence, among others, of the flavonols here reported and these results deserves to continue the chemical and pharmacological study of the other active fractions yielded from the resinous exudates.