New dimer and trimer of chalcone derivatives from anti-inflammatory and antinociceptive extracts of Schinopsis brasiliensis roots.

ETHNOPHARMACOLOGY RELEVANCE: Schinopsis brasiliensis Engl. is an endemic tree of the Brazilian semi-arid regions belonging to the Anacardiaceae family. It is the main representative of the genus Schinopsis, mostly native to Brazil and popularly known as "braúna" or "baraúna". Different parts of this plant are employed in Brazilian folk medicines to treat inflammation in general, sexual impotence, cough, and influenza. AIM OF THE STUDY: This work describes the antinociceptive (acetic acid-induced writhing and formalin-induced nociception) and anti-inflammatory (paw edema and neutrophil migration) activities of the extract of the root of S. brasiliensis. Besides, the evaluation of total phenolic compounds and antioxidant, antimicrobial (including MRSA bacteria), and acetylcholinesterase inhibition activities were also determined. MATERIAL AND METHODS: The pure compounds were isolated by different chromatographic techniques and their chemical structures have been unambiguously elucidated based on extensive spectroscopic methods, including 1D (1H, 13C, DEPT, and NOEdiff) and 2D (HSQC, HMBC, and NOESY) NMR experiments, MS data, and comparison with the literature data of similar compounds. The antinociceptive and anti-inflammatory activities were evaluated by acid acetic writhing test, formalin paw edema, and by the investigation of neutrophil migration to the peritoneal cavities of mice. For antimicrobial evaluation were determined MIC and MBC, antioxidant activities were obtained by TPC and DPPH tests, and AChE inhibition by Elmann's methodology. RESULTS: The extracts showed antinociceptive and anti-inflammatory activities and two unusual new compounds, a cyclobutanyl chalcone trimer (schinopsone A) and a cyclohexene-containing chalcone dimer (schinopsone B), with six known compounds were isolated from the active extracts. Additionally, the acetylcholinesterase inhibitory activity for isolated compounds was reported for the first time in this study. Molecular docking studies indicated that the isolated compounds are responsible for the interaction with anti-inflammatory targets (COX 1 and 2 and LOX) with variable binding affinities, indicating a possible mechanism of action of these compounds. CONCLUSIONS: These findings indicate for the first time the correlation between the anti-inflammatory activity different enriched polyphenol-organic soluble fractions of S. brasiliensis, and it contributes to the understanding of the anti-inflammatory potential of S. brasiliensis.

Lactiplantibacillus plantarum strains isolated from spontaneously fermented cocoa exhibit potential probiotic properties against Gardnerella vaginalis and Neisseria gonorrhoeae.

BACKGROUND: Probiotics are important tools in therapies against vaginal infections and can assist traditional antibiotic therapies in restoring healthy microbiota. Recent research has shown that microorganisms belonging to the genus Lactobacillus have probiotic potential. Thus, this study evaluated the potential in vitro probiotic properties of three strains of Lactiplantibacillus plantarum, isolated during the fermentation of high-quality cocoa, against Gardnerella vaginalis and Neisseria gonorrhoeae. Strains were evaluated for their physiological, safety, and antimicrobial characteristics. RESULTS: The hydrophobicity of L. plantarum strains varied from 26.67 to 91.67%, and their autoaggregation varied from 18.10 to 30.64%. The co-aggregation of L. plantarum strains with G. vaginalis ranged from 14.73 to 16.31%, and from 29.14 to 45.76% with N. gonorrhoeae. All L. plantarum strains could moderately or strongly produce biofilms. L. plantarum strains did not show haemolytic activity and were generally sensitive to the tested antimicrobials. All lactobacillus strains were tolerant to heat and pH resistance tests. All three strains of L. plantarum showed antimicrobial activity against the tested pathogens. The coincubation of L. plantarum strains with pathogens showed that the culture pH remained below 4.5 after 24 h. All cell-free culture supernatants (CFCS) demonstrated activity against the two pathogens tested, and all L. plantarum strains produced hydrogen peroxide. CFCS characterisation in conjunction with gas chromatography revealed that organic acids, especially lactic acid, were responsible for the antimicrobial activity against the pathogens evaluated. CONCLUSION: The three strains of L. plantarum presented significant probiotic characteristics against the two pathogens of clinical importance. In vitro screening identified strong probiotic candidates for in vivo studies for the treatment of vaginal infections.

Gardnerella vaginalis and Neisseria gonorrhoeae Are Effectively Inhibited by Lactobacilli with Probiotic Properties Isolated from Brazilian Cupuaçu (Theobroma grandiflorum) Fruit.

In recent years, certain Lactobacillus sp. have emerged in health care as an alternative therapy for various diseases. Based on this, this study is aimed at evaluating in vitro the potential probiotics of five lactobacilli strains isolated from pulp of cupuaçu fruit fermentation against Gardnerella vaginalis and Neisseria gonorrhoeae. Our lactobacilli strains were classified as safe for use in humans, and they were tolerant to heat and pH. Our strains were biofilm producers, while hydrophobicity and autoaggregation varied from 13% to 86% and 13% to 25%, respectively. The coaggregation of lactobacilli used in this study with G. vaginalis and N. gonorrhoeae ranged from 15% to 36% and 32% to 52%, respectively. Antimicrobial activity was present in all tested Lactobacillus strains against both pathogens, and the growth of pathogens in coculture was reduced by the presence of our lactobacilli. Also, all tested lactobacilli reduced the pH of the culture, even in incubation with pathogens after 24 hours. The cell-free culture supernatants (CFCS) of all five lactobacilli demonstrated activity against the two pathogens with a halo presence and CFCS characterization assay together with gas chromatography revealed that lactic acid was the most abundant organic acid in the samples (50% to 62%). Our results demonstrated that the organic acid production profile is strain-specific. This study revealed that cupuaçu is a promising source of microorganisms with probiotic properties against genital pathogens. We demonstrated by in vitro tests that our Lactobacillus strains have probiotic properties. However, the absence of in vivo tests is a limitation of our work due to the need to evaluate the interaction of our lactobacilli with pathogens in the vaginal mucosa. We believe that these findings may be useful in developing a product containing our lactobacilli and their supernatants in order to support with vaginal health.

Anticariogenic activities of Libidibia ferrea, gallic acid and ethyl gallate against Streptococcus mutans in biofilm model.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, ethnopharmacological studies show that Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz is commonly used in folk medicine as an antifungal, antimicrobial and anti-inflammatory. In the Amazon region, the dried fruit powder of L. ferrea are widely used empirically by the population in an alcoholic tincture as an antimicrobial mouthwash in oral infections and the infusion is also recommended for healing oral wounds. However, there are few articles that have evaluated the antimicrobial activity against oral pathogens in a biofilm model, identifying active compounds and mechanisms of action. AIM OF THE STUDY: The aim of this study was to evaluate the antimicrobial and anti-adherence activities of the ethanolic extract, fractions and isolated compounds (gallic acid and ethyl gallate) of the fruit and seed of L. ferrea against Streptococcus mutans. The inhibition of acidicity/acidogenicity and the expression of the S. mutans GTF genes in biofilms were also evaluated. MATERIALS AND METHODS: Minimal Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration of Cell Adhesion (MICA) were evaluated with ethanolic extract (EELF), fractions, gallic acid (GA) and ethyl gallate (EG) against S. mutans. Inhibition of biofilm formation, pH drop and proton permeability tests were conducted with EELF, GA and EG, and also evaluated the expression of the GTF genes in biofilms. The compounds of dichloromethane fraction were identified by GC-MS. RESULTS: This is the first report of shikimic, pyroglutamic, malic and protocatechuic acids identified in L. ferrea. EELF, GA and EG showed MIC at 250 µg/mL, and MBC at 1000 µg/mL by EELF. EELF biofilms showed reduced dry weight and acidogenicity of S. mutans in biofilms. GA and EG reduced viable cells, glucans soluble in alkali, acidogenicity, aciduricity and downregulated expression of gtfB, gtfC and gtfD genes in biofilms. SEM images of GA and EG biofilms showed a reduction of biomass, exopolysaccharide and microcolonies of S. mutans. CONCLUSIONS: The ethanolic extract of fruit and seed of L. ferrea, gallic acid and ethyl gallate showed great antimicrobial activity and inhibition of adhesion, reduction of acidogenicity and aciduricity in S. mutans biofilms. The results obtained in vitro validate the use of this plant in ethnopharmacology, and open opportunities for the development of new oral anticariogenic agents, originated by plants that can inhibit pathogenic biofilm that leads to the development of caries.

Antimicrobial activity of Lactobacillus fermentum TcUESC01 against Streptococcus mutans UA159.

Dental caries is a multifactorial chronic-infection disease, which starts with a bacterial biofilm formation caused mainly by Streptococcus mutans. The use of probiotics has shown numerous health benefits, including in the fight against oral diseases. Strains of Lactobacillus fermentum have already shown probiotic potential against S. mutans, but there are still few studies. Thus, the aim of our study was to evaluate the antimicrobial activity of the inoculum and metabolites produced by L. fermentum TcUESC01 against S. mutans UA159. For this, a growth curve of L. fermentum was performed and both the inoculum and the metabolites formed in the fermentation were tested against the growth of S. mutans UA159 in agar diffusion tests, and only its metabolites were tested to determine the minimum inhibitory concentration, minimal bactericidal concentration and inhibition of cell adhesion. Inhibition of biofilm formation, pH drop and proton permeability were also tested with the metabolites. The zone of inhibition began to be formed at 14 h and continued until 16 h. The inoculum containing L. fermentum also showed zone of inhibition. The MIC for the metabolites was 1280 mg/mL and the MBC was obtained with a concentration higher than the MIC equal to 5120 mg/mL. Half of the MIC concentration (640 mg/mL) was required to inhibit S. mutans adhesion to the surface of the microplates. In the biofilm analyzes, the treatment with the metabolites in the tested concentration was not able to reduce biomass, insoluble glucans and alkali soluble compared to the control biofilm (p > 0.05). The metabolites also did not affect acid production and acid tolerance of S. mutans cells in biofilms compared to saline group (p > 0.05). Lactic acid (50.38%) was the most abundant organic acid produced by L. fermentum. This is the first report showing that the metabolites produced by the Lactobacillus fermentum TcUESC01 have a potential to be used as an antimicrobial agent against S. mutans, showing anti-adherence and bactericidal activity against planktonic cells of S. mutans. Thus, further studies should be carried out in order to better understand the antimicrobial activity of metabolites of L. fermentum TCUESC01.

Administration of Lactobacillus plantarum Lp62 to dam rats at the end of delivery and during lactation affects TGF-ß1 level and nutritional milk composition, and body weight of pups.

PURPOSE: Lactobacillus plantarum Lp62 is a lactic acid bacteria strain that has been isolated from cocoa beans and exhibited probiotic potential. The influence of oral administration of L. plantarum Lp62 on the growth of rat's pups; on yield, cytokines and milk composition was studied. METHODS: Lactobacillus plantarum Lp62 is a lactic acid bacteria strain that has been isolated from cocoa beans. It was administered daily by gavage to Wistar rats (n = 8), from the 7th day before delivery and for 20 days during lactation, in a concentration of 1.44 × 109 CFU/rat. The dam and pups were weighed and milk was collected at 12th and 19th day for determination of protein, triglycerides, cholesterol and lactose by colorimetric assays. TGF-ß1 milk levels were analyzed by ELISA. The mammary glands of rats were removed for histological analysis. To detect statistical differences between the groups, tests of mean differences at a significance level of 5% was performed. RESULTS: Supplementation with L. plantarum L62 resulted in significant higher weight of pups (p < 0.05), with similar weight on dams (p > 0.05). The milk yield was not altered by L. plantarum treatment, but the levels of protein, triglycerides and cholesterol were increased (p < 0.05), with no difference in lactose concentration (p > 0.05). Levels of TGF-ß1 were higher in the milk of L. plantarum treatment (p < 0.05). CONCLUSIONS: The treatment of dams at the end of pregnancy and lactation with L. plantarum Lp62 increased nutritional content of milk, probably contributing to the higher weight of the pups. The higher levels of TGF-ß1 in the milk, could promote immune benefits to the pups. Further studies in this field are needed to prove the potential use of L. plantarum Lp62 as a probiotic.

Evaluation of anti-inflammatory, antinociceptive and biological activities of Cenostigma macrophyllum standardized extracts and determination and quantification of the main metabolites.

The stem barks and leaves of Cenostigma macrophyllum are used in Brazilian folk medicines in the treatment of stomach and intestinal diseases. However, there are no reports of chromatographic methods used to evaluate the bioactives of its standardized extracts and for biological evaluation. An analytical method was developed and validated for simultaneous determination and quantification of the bioactive phenolics gallic acid, methyl gallate, ellagic acid and, the biflavonoids agathisflavone and amentoflavone in the leaves and stem bark of C. macrophyllum. HPLC operating conditions were optimized and the parameters such as selectivity, linearity, precision, accuracy, LOD, LOQ and, robustness of the method were also evaluated. Robustness was evaluated using a multivariate optimization technique. Linear relationships within the range of investigated concentrations were observed with their correlation coefficients greater than 0.9991. The method was validated for repeatability (RSD ≤ 2.88%), intermediate precision (RSD ≤ 3.38%) with recovery between 84.12 and 106.64% and the RSD less than 3.40% and proved to be robust. Besides, antioxidant, acetylcholinesterase inhibition, anti-inflammatory and antinociceptive activities of the standardized hydromethanolic extracts of leaves and stem bark of this species were evaluated. The method was successfully applied in the quantification of the gallic acid, methyl gallate, ellagic acid, agathisflavone and amentoflavone of standardized extracts. The results showed the present method developed was simple, sensitive, reproducible, accurate and precise. The standardized hydromethanolic extracts of leaves and stem bark of C. macrophyllum showed antioxidant activity (EC50 69.09 and 83.06 µg mL-1), acetylcholinesterase inhibition (52.23 and 83.36%) and they were able to inhibit the formalin-induced nociception and also reduced the edema formations at 100 mg kg-1 doses. The anti-inflammatory potentials were evaluated by the decrease of the Cg-induced neutrophils migrations at the same doses.