EDINÉIA BONIN

Título da Tese: EXTRATO: BACCHARIS DRACUNCULIFOLIA E TAMARINDUS INDICA L. UM PROMISSOR SUBSTITUTO DE COMPOSTOS SINTÉTICOS COM ATIVIDADE ANTIMICROBIANA

Orientador: Prof. Dr. Ivanor Nunes do Prado

Data da Defesa: 30/06/2020

RESUMO GERAL

 INTRODUCTION AND AIMS: The continuous increase in bacterial resistance has prompted the food industry to reduce or replace synthetic chemical molecules, and studies aimed at discovering new bioactive natural substances capable of inhibiting antibacterial activity have become an important tool. Many plants and co-products such as seeds, leaves, and oils that have antioxidant and antimicrobial properties are potential sources for the discovery of new food additives that are safe to man. Baccharis dracunculifolia (BD study I) is a shrub with medicinal properties popularly known as “Vassourinha or Alecrim do Campo” found in different regions of Brazil. BD is the source of green propolis and is important in the agricultural and food industry as a source of matter raw materials for the preparation of medicines, cosmetics, bioactive compounds. Tamarindus indica L. belongs to the Fabaceae (Leguminosae) dicotyledonous family, its fruits have interesting nutritional and technological characteristics for the food industry. The seeds are considered waste, have in their composition biological properties, high antioxidant activity, good source of protein, carbohydrate, fatty acids, polysaccharides, being considered a safe and low-cost source. The present study have objective, to approach the actions of the BD and TS extracts, as well as to evaluate the BD extract in different concentrations, its chemical and cytotoxic profile, and antimicrobial action against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus) and Gram-negative (Salmonella enterica serovar Enteritidis), and study II evaluated the crude TS extract and partition with ethyl acetate (AE) and hexane (H) solvents, estimating the chemical profile, antioxidant activity and antibacterial action against Escherichia coli, Salmonella enterica serovar Enteritidis, Pseudomonas aeruginosa, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, Candida albicans and Saccharomyces cerevisiae.

MATERIAL AND METHODS: For the first study, an experimental study, the BD plant was collected in the city of Maringá – Paraná, Brazil. The plants were dried in an oven (40 °C), ground (1mm). To obtain the lyophilized Baccharis dracunculifolia (HBDL), hydroalcoholic extract, 10 g of the powder sample were weighed and the extraction was carried out with ethyl alcohol and distilled water (70:30 v/v), alternating agitation 20 min and resting 15 min for 2 h, Afterwards it was concentrated in a water bath, rotary evaporated and lyophilized, the extract was stored at a temperature of 4 °C until use. The chemical profile of the HBDL extract was analyzed by UHPLC/HRMS, diluted with acetonitrile (1:1 w/v). The antibacterial activity was evaluated against the bacteria: Gram-positive (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus) and Gram-negative (Salmonella enterica serovar Enteritidis) were observed by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) according to the Institute of Clinical and Laboratory Standards. Vero cells (ATCC CCL-81) were used to analyze the cytotoxicity of the extract at various concentrations. The integrity or damage of the membrane was analyzed by flow cytometry (CF) using a kit, scanning electron microscopy (SEM) and transmission electron microscopy (MET). For the second study, Tamarindus indica L. seeds were collected in the city of Nova Redenção – Bahia, Brazil. They were dried in an oven at 45 °C for 72 h and ground (1 mm) and then refrigerated at 4 °C. Weighed 50 g of powder, and subjected to the extraction process with ethanol and distilled water (9: 1 v/v). The extraction procedure was performed with stirring intervals in skaker for 30 min and rest for 20 min, the extract was filtered. A portion of the crude TS extract (12.9 g) was suspended in methanol/water (1:1, 100 mL, v/v) and partitioned successively with n-hexane and ethyl acetate (3 x 20 mL, v/v). The solvents were removed under reduced pressure to obtain fractions EA and H. Chemical profile quantified in the EA fraction by UHPLC-HRMS/MS. The TS extract, and the EA and H fractions, were evaluated for antioxidant activity, FRAP (Ferric Reducing Ability Power) assay, ABTS (2,2`-azino-bis-3-ethylbenzthiazoline-6-sulphonic Acid), DPPH (1-diphenyl-2-picrylhydrazyl) and total phenolic compounds (TPC). Antimicrobial action of the extract and fractions were evaluated in Gram-positive bacteria: Bacillus cereus and Shapylococcus aureus and Gram-negative: Escherichia coli, Salmonella enterica serovar Enteritidis, Pseudomonas aeruginosa, Aeromonas hydrophila and against yeast: Candida albicans and Saccharomyces cerevisiae. The susceptibility of bacteria and yeasts were evaluated by the agar diffusion disc method, and the MIC/MBC broth microdilution technique was applied to evaluate the action of the extracts. The bacteria most sensitive to the extract were subjected to FC and SEM analysis.

RESULTS AND DISCUSSION: The literature confirms the importance of bioactive factors and biological compounds of the Baccharis species for the pharmaceutical and food industries. Thus, we evaluated the properties of the HBDL extract as a possible natural, safe and accessible antimicrobial. Twelve constituents were identified and confirmed in the HBDL extract: germacrene B, spatulenol, naringenin, kaempferol, artepillin C, α-pinene, hydroxycinnamic acid, apigenin, kaempferide, limonene, phenylethanol and β-cariofileno, the hydroalcoholic mixture proved to be a suitable system for extraction of chemical constituents from plants, with low-cost and toxicity. The extract showed promising results for Gram-positive bacteria MIC/MBC (125/250 μg/mL) for S. aureus and B. subtilis, and B. cereus the MIC/MBC was 250/500 μg/mL, but not showed a bactericidal effect on Gram-negative bacteria. Based on the results of the MIC, it was evaluated the concentration of the extract in 2x MIC, 4x MIC, 6x MIC and 8x MIC, performing logarithmic UFC/mL counting. When the bacteria were subjected to treatment with 2x MIC, a reduction of S. aureus (0.870), B. subtilis (0.484), B. cereus (0.902) and Salmonella Enteritidis (0.0) was observed. Count to 4x MIC, reduction in log UFC/mL of 5,583 for S. aureus, 5,017 for B. subtilis and 5,004 for B. cereus. Gram-positive bacteria, when exposed to 6x MIC, showed a total reduction, demonstrating an extract potential against these bacteria, without exceeding the allowed cytotoxic limit. The limited action of the extract in Gram-negative battery can be explained due to the external membrane, which hinders the passage of antimicrobial compounds. The cytotoxicity of the HBDL extract was evaluated in Vero cells by determining the concentration capable of destroying 50% or reducing cell viability by 50%, after 24 hours of incubation. The CC50 was greater than 2000 μg/mL, after 48 h was 628.7 ± 279.2 μg/mL. According to this result, the extract shows weak time-dependent cytotoxicity, and the concentrations tested in the tests were within the cytotoxic limit. The FC technique allows the detection of different microbial subpopulations based on the integrity of the membrane through the retention or exclusion of dyes, Gram-positive bacteria treated with 2x and 4x MIC showed an intact membrane in the control and a damaged membrane after treatment, similar results to reduction in log UFC/mL, where we can observe a reduction in cell viability. SEM and MET analyzes also proved this damage to the cell membrane. The AE fraction revealed the presence of fourteen chemical constituents, such as the flavonoid compounds, (+)-Catechin/(-)-Epicatechin Procyanidin B2, Procyanidin C2, Isoquercetin, Quercetin, Luteolin, Rutin, Taxifolin, Eriodictyol, Isokaempferide, Eriodicolol-glucoside, Protocathechuic acid. The crude hydroalcoholic extract (TS) showed the best results for total phenolic compounds TPC: 883.87 GAE (mg/g), antioxidant activity FRAP: 183.29 GAE (mg/g), ABTS: 39.67% and DPPH: 91.08%, and antibacterial activity, for Gram-positive bacteria: Staphylococcus aureus MIC/MBC (62.5/125 μg/mL) Bacillus cereus MIC/MBC (125/250 μg/mL), and Gram-negative: Aeromonas hydrophila MIC/MBC (125/250 μg/mL), Pseudomonas aeruginosa MIC/MBC (250/500 μg/mL), but no satisfactory results were observed for Escherichia coli, Salmonella and yeasts Candida albicans and Saccharomyces cerevisiae. Bacteria sensitive to the TS extract were evaluated when the membrane integrity by CF, where we can observe membrane damage when exposed to treatment, and loss of the characteristic structure when observed by SEM. The tamarind seed has a unique composition and bioactive compounds that should be better explored, being a promising starting point for a new food preservative.

CONCLUSIONS: The HBDL extract presented chemical constituents with bioactive and bactericidal properties, observed in the bacteria S. aureus, B. subtilis and B. cereus, however it was not effective against Salmonella enterica serovar Enteritidis, further studies are necessary for the extract to be effective within the limit cytotoxic. For the second study, we can conclude that the AE fraction of Tamarindus indica, showed to be rich in flavonoids, observed in most chemical compounds, and the crude TS extract had the best antioxidant activity, total phenolic compounds and antimicrobial activity against bacteria S. aureus, B. cereus, A. hydrophila, P. aeruginosa. In general, extracts and fractions were more active against bacteria than against yeasts, showing a certain selectivity. Results indicated that the extracts of Baccharis and tamarind, are considered a promising source with potential to be explored, of bioactive, antimicrobial, safe and low-cost compounds for the development of a new antimicrobial agent, being able to provide useful information for guidance as possible substitutes for synthetic compotes in the food industry.

Key words: Natural extract; Baccharis dracunculifolia; Tamarindus indica seed; Chemical constituents; Antimicrobial activity.

Artigos Publicados Vinculados a Tese:

 DOI: 10.1016/j.lwt.2019.108920