Fitoquímica e potencial farmacológico de Acosmium diffusissimum (Fabaceae:Papilionoideae)

Abstract

Acosmium diffusissimum (Mohlenbr.) Yakovlev, popularly known as black loin, belongs to the Fabaceae (Papilionaceae) family. It has been reclassified and currently belongs to the tribe Dalbergieae. Only three species are described for the genus Acosmium s. strit., among which A. diffusissimum is endemic to Brazil and the only species that grows in caatinga regions. Due to the lack of studies on the species and the potential of caatinga species, this study aims to contribute to phytochemical and pharmacological knowledge by studying A. diffusissimum. The plant material was collected in the municipality of Boa Vista do Tupim, Bahia, identified and an exsiccate is deposited in the Herbarium of the State University of Feira de Santana (HUEFS) under number 246074. This study is registered in SisGen under the code A39AEA5. The roots and stems were extracted by maceration with 95% ethanol, obtaining the respective extracts, which were then defatted. For the roots, the defatted extract was subjected to a liquid-liquid extraction using hexane, chloroform, ethyl acetate and n-butanol. The chloroform phase was subjected to medium pressure liquid chromatography using silica gel, resulting in 19 fractions. Fraction 08 was subjected to high performance liquid chromatography (HPLC) for isolation and purification. For the stems, the defatted extract was subjected to vacuum liquid chromatography, resulting in 7 fractions. The fractions were analyzed by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and entered into the GNPS platform to generate the molecular network and use it as a dereplication tool. After analyzing the fragmentation profiles, the chloroform (AdC-FC) and ethyl acetate (AdC-FA) fractions were prioritized for isolation. AdC-FC and AdC-FA were subjected to classical chromatography (CC), obtaining 14 fractions and 13 fractions, respectively. The fractions obtained were also monitored by HPLC-MS, from which fractions 8-9 and fractions 10-13 of AdC-FC were selected for isolation and purification of compounds. Fractions 6 and 7 of AdC-FA were pooled and subjected to CC with silica gel, obtaining 6 fractions. Fraction 67-2 was subjected to preparative-scale HPLC. The structures were chemically characterized using 1D and 2D NMR, IR and HRESIMS techniques. Two new triterpenes cis-lupacosmeol (1) isolated and in mixture with trans-lupacosmeol (2), and two known compounds, 16β-hydroxy-3β-trans-caffeoyl-olean-12-ene (3) and 16β-hydroxy-3β-p-coumaroyl-lup-20(29)-ene (4), were isolated from the roots of A. diffusissimum. Due to the cytotoxic potential described in the literature for triterpenes, cytotoxicity was assessed against three different cancer cell lines (MCF-7, HCT-116, SK-MEL-28). Only the mixture of cis and trans compounds showed activity against all the strains tested, with the most pronounced ability to inhibit the growth of SK-MEL-28 (IC50: 25.45 μg/mL). Molecular docking studies suggested that the cytotoxic activity was strongly attributed to interactions between cis-lupacosmeol and trans-lupacosmeol with different molecular targets of importance for the treatment of melanoma. Guided isolation of the stems of A. diffusissimum by HPLC-MS resulted in the isolation of five prenylated isoflavonoids, diffusiflavone A-E (1-4 and 9), two prenylated pterocarpans, diffusicarpan A and B (5,6) and two known compounds 6-prenylorobol (7) and 3-O-methylquercetin (8). The in vitro anti-inflammatory potential of compounds 1-6 and 9 was evaluated in macrophages induced with lipopolysaccharide (LPS). Compounds 2, 3, 5 and 9 showed a reduction in NO levels in at least one of the concentrations tested (1.25, 2.5, 5, 10, 20 μg/mL) and also reduced the cytokines IL-1β and IL-6, especially diffusicarpan A which reduced cytokine levels in all concentrations tested. This is the first work on the chemistry and pharmacology of A. diffusissimum and also highlights the application of molecular networking as a dereplication strategy to isolate new bioactive compounds, thus contributing to the knowledge of endemic species of the Brazilian caatinga.