Perfil químico e potenciais antioxidante, antibacteriano e anti-inflamatório de extratos hidroetanólicos das folhas de Kalanchoe brasiliensis Cambess. (Crassulaceae)

Abstract

Kalanchoe brasiliensis Cambess. (Crassulaceae DC.), is a Brazilian native plant popularly known as "saião" and traditionally used in the treatment of inflammatory and infectious diseases. The major secondary metabolites present in this species are phenolic compounds, particularly flavonoids. Based on these data, the present study aimed to investigate the chemical composition and the in vitro antioxidant and antibacterial potentials and the in vivo topical anti-inflammatory activity of the hydroethanolic extracts K. brasiliensis leaves. Fresh leaves collected on January (summer) and September (spring) were extracted with a hydroethanolic solution in concentrations of 30% (HEJ30 and HES30), 50% (HEJ50 and HES50) and 70% (HEJ70 and HES70) of ethanol in water using a mechanical crusher. These extracts were chemically characterized by High Performance Liquid Chromatography coupled with Diode Array Detection (HPLC-DAD). The total phenol and flavonoid contents were determined spectrophotometrically and the antioxidant activity was evaluated by the 1, 1-diphenyl-2-picryl-hydrazyl (DPPH•) free radical sequestration, the reduction of the phosphomolybdenum complex and the β-carotene/linoleic acid co-oxidation system assays. The antibacterial activity was determined by Minimal Inhibitory Concentration (MIC) using the broth microdilution method against eight ATCC® reference strains and ten routine ones. Subsequently, the Minimal Bactericidal Concentration (MBC) was established and the bactericidal or bacteriostatic antibacterial effect was classified. The topical anti-inflammatory activity was evaluated using Croton- oil, phenol and histamine induced ear edema models, histological analysis, and determinations of myeloperoxidase (MPO) and N-acetyl-β-D-glycosaminidase (NAG) enzymes and nitric oxide (NO), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) inflammatory mediators. The UV-spectra obtained by HPLC-DAD suggested the presence of flavonoids, specially flavonol and flavone. Total phenol and flavonoid contents ranged from 25.47 to 30.11 mg/g of tannic acid equivalent/g of extract (EAT) and from 11.84 to 16.95 mg/g of rutin equivalent/g of extract (ER), respectively. EC50 values varied of 58.08 ± 15.87 to 257.70 ± 79.47 µg/mL and 8.491 ± 0.364 to 16.780 ± 0.239 RAA% for the DPPH• free radical sequestration and phosphomolybdenum complex reduction methods, in this order. The percentages of inhibition of lipid peroxidation ranged from 37.51 ± 0.47 to 44.46 ± 0.84%. In relation to the antibacterial activity, all extracts were active against Salmonella Choleraesuis (ATCC® 10708™) and Salmonella Typhimurium (ATCC® 13311™), with MIC values of 5000 µg/mL and bacteriostatic effect. HEJ30, HEJ50, HEJ70 and HES70 were active against Escherichia coli (ATCC® 25922™), with MIC of 5000 µg/mL and bacteriostatic effect. HEJ70 and HES70 inhibited the growth of Staphylococcus aureus subsp. aureus (ATCC® 6538™), with MIC of 5000 µg/mL and bacteriostatic effect. In relation to the routine strains, all extracts were active against Salmonella Enteritidis 1406591, 1418594 and 1628260, and for Salmonella spp. fluoroquinolone-resistant 1266695 and 1507788, with MIC of 5000 µg/mL and bacteriostatic effect. HEJ30, HEJ50, HEJ70 and HES70 inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA) 1485279, 1605677, 1830466 and 1664534, with MIC of 5000 µg/mL and bacteriostatic effect. HES was active against MRSA 1605677 and 1830466, with MIC of 5000 µg/mL and bacteriostatic effect. It was not possible to determine the MBC values within the established concentration gradient for any strain tested. With regard to the in vivo topical anti-inflammatory activity, in Croton oil-induced ear edema the values ranged from 74.63 to 85.07% and 62.03 to 80.34% for the measurement and mass of the edema after 24 h of the challenge, respectively. In phenol induced ear edema, the percentages varied of 96.46 to 96.73% and 93.09 to 95.65% for the measurement and mass of the edema, in this order. Considering the edema induced by histamine, no significant statistical decrease was observed.The inhibition of MPO and NAG was 55.27 to 81.74% and from 69.04 to 80.82%, respectively. For the inflammatory mediators, the inhibition was from 52.24 to 67.43%, from 1.68 to 22.78% and from 3.40 to 23.71% for NO, IL-6 and TNF-α, in this order. The obtained results suggest that K. brasiliensis is a promising natural source of bioactive substances, especially of the flavonoid class, with a high topical anti-inflammatory potential and a exploitable antibacterial activity mainly to treat infections caused by Salmonella strains, highlighting this vegetal species as a target for the discovery of new therapeutic agents.