Antibacterial activity of extract and fractions from branches of Protium spruceanum and cytotoxicity on fibroblasts

Abstract The crude ethanol extract (CEE) and fractions from branches of Protium spruceanum were subjected to antibacterial and cytotoxicity assays. Compounds of the most active fraction were identified by GC-MS and LC-MS. CEE was active against 19 bacteria and the ethyl acetate fraction (EAF) showed the lowest minimum bactericidal concentration (MBC 0.3–80.0 mg/mL). Through time-kill assay was observed that EAF induced rapid bactericidal effect against Staphylococcus saprophyticus. The cytotoxicity tests against L929 fibroblasts showed great potential of EAF on the treatment of infections caused by five bacteria (MBC < IC50). The results provide in vitro scientific support to the possible application of branches of P. spruceanum as antimicrobial agent that may contribute for treatment of infections.


Introduction
The increase in drug resistant micro-organisms difficult the treatment of infectious diseases resulting on high morbidity and mortality. This fact stimulates the search for new antibiotics, mainly from medicinal plants . Protium spruceanum (Benth.) Engler (Burseraceae) occurs in Atlantic, Amazon and Cerrado biomes of Brazil where is commonly known as breu (Vieira et al. 2010). Its traditional use as anti-inflammatory and analgesic was attributed to αand β-amyrins (Rodrigues et al. 2013). Sabinene and β-caryophyllene are the major components found in leaf, resin and thin branch essential oils of P. spruceanum (Machado et al. 2003). However, the antimicrobial activity of substances from its branches has not yet been studied. In this study was analysed the in vitro antimicrobial effect of ethanol extract (CEE) of branches, and its fractions. Constituents of the most active fraction were identified by GC-MS and LC-MS. The cytotoxicity against L929 fibroblasts was established to evaluate at which concentrations it could be safely used.

Results and discussion
The antimicrobial activity of others species of genus Protium was reported, such as P. heptaphyllum (Violante et al. 2012) and P. neglectum (Suárez et al. 2007). The present study showed that the crude ethanol extract (CEE) from branches of P. spruceanum, and its fractions presented activity (inhibition zone ≥ 10 mm) against 19 pathogenic bacteria, opening a potential therapeutic application (Table S1).
Several natural products have chemical properties, for instance insolubility in water, that preclude employing Clinical and Laboratory Standards Institute (CLSI) methods fully, requiring modifications (Nascimento et al. 2007). The results of microbial viability obtained through the modified method with methanol evaporation statistically showed no significant changes related to growth controls (Table S2). Therefore, the modified method proposed represents an efficient alternative to analyse antimicrobial activity of extracts and fractions, whereas it doesn't affect the microbial viability and allows assays with extracts in large concentrations and insoluble in the culture medium.
The ethyl acetate fraction (EAF) presented the lowest minimum bactericidal concentration (MBC) (Table S1) and the total antimicrobial activity (TAA) results showed that after partition of CEE, the most bioactive compounds were concentrated in EAF (greater TAA than CEE and other fractions) (Figure 1(A) and Table S3). Time-kill assay was performed to better understand the antimicrobial activity of the EAF against the most sensitive bacteria Staphylococcus saprophyticcus. EAF presented a concentration-and time-dependent antibacterial activity (Figure 1(B)). The effect was observed in concentrations of 2 × MBC and MBC, with complete kill at 3 and 6 h, respectively. There was still a statistical difference in bacterial viability between the growth control and the concentration of ½ MBC at 24 h (p < 0.05), although it is not considered a bactericidal effect. The MBC of the EAF was considered higher to classical antibiotics. The time-kill results showed a promising activity against Staphylococcus saprophyticus with complete kill at a lower time in comparison to nitrofurantoin. Antibiotic used to treat urinary tract infections caused by this bacterium. Antibiotic concentrations ≥ 8 × MIC (48 μg/mL) displayed complete bacterial kill only after 8 h (Lindgren et al. 2015).
Cytotoxicity assays with fibroblasts and keratinocytes indicated a MIC above the cytotoxic concentration is important to evaluate the safety of substances before suggesting its clinical trials (Lopez et al. 2015). The citotoxicity results for L929 fibroblasts showed that MBC of EAF was below to cytotoxic concentration (IC 50 1.27 mg/mL) for tested bacteria. This fact indicates a great potential for treatment of infections by Staphylococcus aureus and S. aureus MRSA, S. saprophyticus, Enterococcus faecalis and Salmonella enteritidis.

Conclusion
For the first time is reported the antimicrobial effect of CEE from branches of P. spruceanum. The EAF developed a rapid activity against S. saprophyticus. The antibacterial effect and low toxicity on fibroblast (MBC < IC 50 ) support to the application of EAF in the development of antibiotics.