It was slightly before the peak of the water levels so the trees were more than half submerged. Maclura tinctoria First, old fustic is a plant from the Americas, discovered after in the forests of tropical America. Kuntze Maclura affinis Miq. Probably all are msclura somewhere, but developing sustainable production seems essential.
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Abstract Background The Atlantic Forest biome extends along the entire Brazilian coast and is home to approximately 20, plant species, many of which are endemic; it is considered one of the hotspot regions of the planet. Several of these species are sources of natural products with biological activities that are still unknown. In this study, we evaluated the antimicrobial activity of 90 extracts derived from native Atlantic Forest tree species against Staphylococcus aureus, an important human and veterinary pathogen.
Methods Extracts from native Atlantic Forest tree species were evaluated for their antimicrobial activity against S. Phytochemical fractionation of the extract from Maclura tinctoria was performed by liquid-liquid partitioning. Damage of cells and alterations in the permeability of cell membrane were determined by atomic force microscopy AFM and crystal violet uptake assay, respectively. In vivo antimicrobial activity was evaluated using Galleria mellonella larvae infected with S.
Results Among the organic or aqueous extracts tested here, 26 showed biological activity. Antibacterial activity was registered for three species for the first time. An organic extract from Maclura tinctoria leaves showed the lowest MIC 0. Fractionation of this extract by liquid-liquid partitioning led to obtaining fraction 11FO d with a MIC of 0. This fraction showed strong activity against veterinary S. The bacterial surface was not altered by the presence of 11FO d, and no cell membrane damage was detected.
Conclusion A fraction enriched in prenylated isoflavones and flavanones from M. These findings justify the need for further research to elucidate the mechanisms of action of these compounds.
Peer Review reports Background Staphylococcus aureus is responsible for high rates of morbidity and mortality worldwide and is the most common cause of skin infections, although it may also be associated with osteomyelitis, bacteremia and sepsis [ 1 ]. In cattle, it is the primary cause of mastitis, which is currently considered the main disease of dairy herds [ 2 ]. Great adaptations to different niches and hosts are determined in part by the variety of bacterial virulence factors that contribute to the establishment of the disease.
The first penicillin-resistant strains arose in the mids, and as early as the s, reports described the isolation of S. The USA strain, which is associated with infections in hospitals and in the community, was initially reported in the US. However, it has been isolated from different continents, and it has become a global concern due to its hypervirulence, reduced susceptibility to different antimicrobials and great epidemic potential.
Plant resources can also be sources of compounds with antimicrobial properties. Several studies on plant extract libraries have been established, and the results show the importance of this strategy for the discovery and development of new drugs [ 5 , 6 ]. Coumarin derivatives have displayed activity against S. Further reports have described the efficacy of intraperitoneal injections of a synthetic dicoumarin derivative in septicemic mice [ 8 ] and hypodermic injections of lysionotin in mouse pneumonia model [ 9 ], confirming the potential use of natural products as leads for new drugs.
The Atlantic Forest biome is the second-largest block of tropical forest in Brazil and has the second-highest biological diversity, behind only the Amazon.
Considering the great diversity of the Atlantic Forest biome, which hosts plant species still unknown to science, along with the urgency to discover new antimicrobial agents, the present work presents a bioprospection of natural products in the native trees extracts from this ecosystem.
The antimicrobial activity of some of these plants was demonstrated for the first time. Methods Microorganisms and culture conditions S. Four field isolates of S. The Brazilian Flora Project was used to check the species names and the botanical families. Each sample was first extracted with a mixture of lower-polarity solvents dichloromethane and methanol, 1: 1 followed by water, and for both extracts, the solvents were completely removed [ 10 ].
The tree species were enumerated randomly, and the extracts were identified by the abbreviations FO organic extract of leaves , FA aqueous extract of leaves , GO organic extract of branches and GA aqueous extract of branches. Table 1 Antimicrobial activity of plant extracts against Staphylococcus aureus ATCC Full size table Agar well diffusion assay The antimicrobial activity was initially evaluated according to the well diffusion test.
A suspension of S. The inhibition halos were measured in millimeters. The tests were performed on three independent biological replicates. After 2 h of incubation, the MIC was defined as the lowest concentration of extract for which the purple color formation, which was indicative of cell viability, was not observed. The MIC of ampicillin for S.
The MIC of the active fraction was estimated as described above using the reference strain and the isolates S. The assays were performed using three biological replicates. Fractionation and phytochemical prospecting of the bioactive extract The extract with the lowest MIC according to the preliminary screening was chosen for further assays. The phytochemical composition was investigated by liquid-liquid partitioning in which the bioactive extract 1. A volume of mL of each solvent was used in this step.
The solvents were evaporated to give the 11 FO h 0. All the fractions were subjected to phytochemical analysis and an antibacterial activity assay. A phytochemical screening was performed by thin layer chromatography in glass plates covered with silica gel Whatman, Kent, UK. Analyses were run in the full scan mode — Da. The mobile phase consisted of water 0. The flow rate was 0.
The UV spectra were registered from to nm. Effect of the 11FO d fraction on S. The experiments were performed using two biological replicates. Atomic force microscopy AFM S. The plant extract was then added at the MIC 0. Cells grown in culture media without the fraction were used as the control. Measurement of permeability with crystal violet Alterations in the cell permeability were evaluated by crystal violet assay [ 22 ].
In brief, a culture of S. CTAB is a cationic detergent that increases cell permeability and therefore was used as a positive control. The negative control consisted of cells incubated only in PBS. After further centrifugation, the supernatants were collected and read at an OD of nm. The absorbance of the violet crystal solution was also checked. The diet consisted of a mix of g wheat bran, g wheat germ, g dried beer yeast, 80 mL glycerin, g skim milk powder, and mL liquid honey.
Larvae weighing — mg were divided into six groups. Unless otherwise stated, all the experiments involved groups containing 10 larvae. Group 1 was injected with a suspension of S. Every 24 h, a new dose of the fraction was applied, and the larvae were monitored for 96 h after the last application. A total of four doses of the fraction were applied to each larva in this group.
The experiments were performed using three biological replicates. Statistical analyses Survival data were collected using the Kaplan-Meier method, and the comparison between the control group and the treatment group was performed using the Log-rank test.
Results Screening of extracts for antimicrobial activity Twenty-six organic and aqueous extracts tested on S. The largest inhibition halo was recorded for the organic extract of Miconia petropolitana leaves 24FO. The extracts from Anadenanthera peregrina led to the formation of inhibition zones ranging from 9 to 13 mm. Inhibition halos were observed when the leaf extracts from Maclura tinctoria 11FA and 11FO were tested. Different extracts from Bathysa nicholsonii, Piptadenia gonoacantha, Tabernaemontana hystrix, Xylopia sericea, and Myrciaria glazioviana also showed activity against S.
One extract from Luehea grandiflora, Ceiba speciosa, Siparuna guianensis, and Alchornea glandulosa produced inhibition halos.
MIC assays were performed with all the extracts that resulted in inhibition zones and values ranged from 0. The 11FO organic extract prepared from Maclura tinctoria leaves had the smallest MIC, and therefore, it was chosen for later assays. The MIC obtained for the dichloromethane fraction of 11FO d was reduced to half that obtained for the crude extract Fig. TLC showed that the fraction 11FO d consisted of a mixture of flavonoids. Data analysis along with literature reports on the Moraceae family suggests that compound 1 is luteone.
Spectra obtained for peaks 3 and 4 showed the UV absorption maxima with two bands at nm, which are characteristic of the flavanone derivatives. These protonated molecules refer to isomers of flavanones that are attached to two prenyl substituents on their aglycone. These data indicated that peaks 3 and 4 refer to isomers of flavanones with two prenyl substituents, one in an open chain form and the other in a closed ring form, confirming the presence of euchrestaflavanone C and cudraflavanone A C25H26O6.
The spectral data suggest that compound 5 is a diprenylated flavanone. The compounds were identified as luteone 1 , wightenone 2 , euchrestaflavanone C 3 , cudraflavanone A 4 , and diprenylated flavanone Full size image Effect of fraction 11FO d on S.
To explore the possible antibacterial mechanism of fraction 11FO d, the morphological changes in S. The alterations in cellular permeability were tested by incubating the cells with crystal violet, a hydrophobic dye that is capable of crossing the lipid bilayer Fig. In the presence of increasing concentrations of CTAB, a cationic detergent, there was greater dye entry, which is consistent with the increased permeability of cell membrane.
The same trend was not observed when the cells were incubated in the presence of the 11FO d fraction, suggesting that the membrane properties were not altered by the compounds found in the fraction. Cells treated with the fraction had a lower uptake of crystal violet in relation to the cells without the added fraction control. The culture supernatant was collected for an absorbance reading at nm. Means obtained for three biological replicates. After 48 h, there were still live larvae in the group treated with 11FO d.
Of the 23 species tested, 12 presented antimicrobial activity against S. This is the first report of the antibacterial activity of species such as T.
Chlorophora tinctoria L. Maclura moraGriseb. Morus tinctoriaL. It produces a yellow dye called fustic primarily known for coloring khaki fabric for U. It is bordered to the north by the United States; to the south and west by the Pacific Ocean; to the southeast by Guatemala, Belize, and the Caribbean Sea; and to the east by the Gulf of Mexico.
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