RESUMO

BACKGROUND: Garcinia brasiliensis is a species native to the Amazon forest. The white mucilaginous pulp is used in folk medicine as a wound healing agent and for peptic ulcer, urinary, and tumor disease treatments. The activity of the proprotein convertases (PCs) Subtilisin/Kex is associated with the development of viral, bacterial and fungal infections, osteoporosis, hyperglycemia, atherosclerosis, cardiovascular, neurodegenerative and neoplastic diseases. METHODS: Morelloflavone (BF1) and semisynthetic biflavonoid (BF2, 3 and 4) from Garcinia brasiliensis were tested as inhibitor of PCs Kex2, PC1/3 and Furin, and determined IC50, Ki, human proinflammatory cytokines secretion in Caco-2 cells, mechanism of inhibition, and performed molecular docking studies. RESULTS: Biflavonoids were more effective in the inhibition of neuroendocrine PC1/3 than mammalian Furin and fungal Kex2. BF1 presented a mixed inhibition mechanism for Kex2 and PC1, and competitive inhibition for Furin. BF4 has no good interaction with Kex2 and Furin since carboxypropyl groups results in steric hindrance to ligand-protein interactions. Carboxypropyl groups of BF4 promote steric hindrance with Kex2 and Furin, but effective in the affinity of PC1/3. BF4 was more efficient at inhibiting PCl/3 (IC50 = 1.13 µM and Ki = 0,59 µM, simple linear competitive mechanism of inhibition) than Kex2, Furin. Also, our results strongly suggested that BF4 also inhibits the endogenous cellular PC1/3 activity in Caco-2 cells, since PC1/3 inhibition by BF4 causes a large increase in IL-8 and IL-1ß secretion in Caco-2 cells. CONCLUSIONS: BF4 is a potent and selective inhibitor of PC1/3. GENERAL SIGNIFICANCE: BF4 is the best candidate for further clinical studies on inhibition of PC1/3.

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RESUMO

The accumulation of oxidized ApoB-100-containing lipoproteins in the vascular intima and its subsequent recognition by macrophages results in foam cell formation and inflammation, key events during atherosclerosis development. Agents targeting this process are considered potentially atheroprotective. Since natural biflavonoids exert antioxidant and anti-inflammatory effects, we evaluated the atheroprotective effect of biflavonoids obtained from the tropical fruit tree Garcinia madruno. To this end, the pure biflavonoid aglycones morelloflavone (Mo) and volkensiflavone (Vo), as well as the morelloflavone's glycoside fukugiside (Fu) were tested in vitro in primary macrophages, whereas a biflavonoid fraction with defined composition (85% Mo, 10% Vo, and 5% Amentoflavone) was tested in vitro and in vivo. All biflavonoid preparations were potent reactive oxygen species (ROS) scavengers in the oxygen radical absorbance capacity assay, and most importantly, protected low-density lipoprotein particle from both lipid and protein oxidation. In biflavonoid-treated macrophages, the surface expression of the oxidized LDL (oxLDL) receptor CD36 was significantly lower than in vehicle-treated macrophages. Uptake of fluorescently labeled oxLDL and cholesterol accumulation were also attenuated in biflavonoid-treated macrophages and followed a pattern that paralleled that of CD36 surface expression. Fu and Vo inhibited oxLDL-induced ROS production and interleukin (IL)-6 secretion, respectively, whereas all aglycones, but not the glucoside Fu, inhibited the secretion of one or more of the cytokines IL-1ß, IL-12p70, and monocyte chemotactic protein-1 (MCP-1) in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, in macrophages primed with low-dose LPS and stimulated with cholesterol crystals, IL-1ß secretion was significantly and comparably inhibited by all biflavonoid preparations. Intraperitoneal administration of the defined biflavonoid fraction into ApoE-/- mice was atheroprotective, as evidenced by the reduction of the atheromatous lesion size and the density of T cells and macrophages infiltrating the aortic root; moreover, this treatment also lowered the circulating levels of cholesterol and the lipid peroxidation product malondialdehyde. These results reveal the potent atheroprotective effects exerted by biflavonoids on key events of the oxLDL-macrophage interphase: (i) atheroligand formation, (ii) atheroreceptor expression, (iii) foam cell transformation, and (iv) prooxidant/proinflammatory macrophage response. Furthermore, our results also evidence the antioxidant, anti-inflammatory, hypolipemiant, and atheroprotective effects of Garcinia madruno's biflavonoids in vivo.