Abstract

This article reports the three biflavonoids isolated from the fruit pericarp of Garcinia brasiliensis Mart. (Clusiaceae): Morelloflavone-4```-glycoside (compound 1), (±)-Fukugiside (compound 2), and Morelloflavone (compound 3). Structural modifications by acylation and alkylation reactions were performed on the natural biflavonoid (±) morelloflavone to obtain three semisynthetic compounds: Morelloflavone-7,4`,7``,3```,4```-penta–O–acetyl (compound 4), Morelloflavone-7,4`,7``,3```,4```-penta-O-methyl (compound 5), and Morelloflavone-7,4`,7``,3```,4```-penta-O-butanoyl (compound 6). The inhibitory effects of these naturally isolated biflavonoid-type compounds and three semisynthetic derivatives on the activity of the cysteine proteases papain and cruzain, and on the serine protease trypsin were investigated. The potential inhibitory IC₅₀ of natural bioflavonoids compounds 1, 2, and 3 were 11.0 ± 3.0, 23.0 ± 4.0, and 10.5 ± 0.3 µM, respectively, for papain;  0.86 ± 0.12, 106 ± 7, and 3.8 ± 0.1, 50 ± 2, 119.5 ± 5,  and 9.6 ± 1.0 µM, respectively, for cruzain. On the other hand, the semisynthetic biflavonoids compounds 4, 5, and 6 were more efficient in the inhibition of enzyme activity with IC₅₀ values 0.60 ± 0.02 µM (papain) for biflavonoids compound 4, 1.64 ± 0.11 µM (trypsin) for biflavonoids compound 5, and 8.1 ± 0.6 µM (cruzain) for biflavonoids compound 6. Compound 4 is more active owing to the carbonyl group in the structure; perhaps, this modification could favor a higher nucleophilic attack by serine and cysteine proteases. However, the semisynthetic compound 5 (IC₅₀ = 15.4 ± 0.7 µM for papain), which has no carbonyl group in structure, was less active in the inhibition. Interestingly, structure–activity relationships (SARs) were confirmed by flexible docking simulations. Likewise, the potential usefulness of natural compound 1 as an antioxidant compound was strengthened by our results concerning the antiproteolytic activity.

Key words: Garcinia brasiliensis, biflavonoids, proteases, antiproteolytic activity.

Abbreviation

r-CPB2.8, Recombinant cysteine protease type B; EOAc, ethyl acetate extract; MeOH, methanol; Z-Phe-Arg-MCA, carbobenzoxicarbonil-Phe-Arg-7-amino-4-methylcoumarin; DTT, dithiothreitol; SARs, structure–activity relationships; FT-NMR, Fourier transform nuclear magnetic resonance; Me₄Si, trimethylsilane; TLCK, N-alpha-tosyl-L-lysinyl-chloromethylketone; E-64, 1-[[(Ltrans-epoxysuccinyl)-L-leucyl]amino]-4-guanidinobutane.