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Abstract β-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum β-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine β‑lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential β-lactamase inhibitor. In the present study, bla CTX-M-15 and bla KPC-2 represented genes encoding for serine β-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64–128 µg/mL) and cephalosporins (MICs; 128–512 µg/mL). The MICs of the tested β-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-β-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64–512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a β-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-β-lactam inhibitor, which interacts with the crucial residues involved in β-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme’s active site. GQA markedly enhanced the potency of β-lactams against carbapenemase and extended-spectrum β-lactamase-producing strains, reducing the MICs of β-lactams to the susceptible range. The β-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent β-lactamase inhibitors.

Helicobacter pylori is associated with gastrointestinal diseases, and its treatment is challenging due to antibiotic-resistant strains, necessitating alternative therapies. Brazilian red propolis (BRP), known for its diverse bioactive compounds with pharmaceutical properties, was investigated for its anti-H. pylori activity, focusing on biofilm formation inhibition and eradication. BRP was tested against H. pylori (ATCC 43526) using several assays: time–kill, nucleotide leakage, biofilm formation inhibition (determining the minimum inhibitory concentration of biofilm of 50%—MICB50, and cell viability), and biofilm eradication (determining the minimum eradication concentration of biofilm of 99.9%—MBEC). Standardization of H. pylori biofilm formation was also conducted. In the time–kill assay, BRP at 50 µg/mL eliminated all H. pylori cells after 24 h. The nucleotide leakage assay showed no significant differences between control groups and BRP-treated groups at 25 µg/mL and 50 µg/mL. H. pylori formed biofilms in vitro at 109 CFU/mL after 72 h. The MICB50 of BRP was 15.6 µg/mL, and at 500, 1000, and 2000 µg/mL, BRP eradicated all bacterial cells. The MBEC was 2000 µg/mL. These findings suggest that BRP has promising anti-H. pylori activity, effectively inhibiting and eradicating biofilms. Further studies are necessary to elucidate BRP’s mechanisms of action against H. pylori.

Artepillin C, drupanin, and plicatin B are prenylated phenylpropanoids that naturally occur in Brazilian green propolis. In this study, these compounds and eleven of their derivatives were synthesized and evaluated for their in vitro antimicrobial activity against a representative panel of oral bacteria in terms of their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. Plicatin B (2) and its hydrogenated derivative 8 (2′,3′,7,8-tetrahydro-plicatin B) were the most active compounds. Plicatin B (2) displayed strong activity against all the bacteria tested, with an MIC of 31.2 μg/mL against Streptococcus mutans, S. sanguinis, and S. mitis. On the other hand, compound 8 displayed strong activity against S. mutans, S. salivarius, S. sobrinus, Lactobacillus paracasei (MIC = 62.5 μg/mL), and S. mitis (MIC = 31.2 μg/mL), as well as moderate activity against Enterococcus faecalis and S. sanguinis (MIC = 125 μg/mL). Compounds 2 and 8 displayed bactericidal effects (MBC: MIC ≤ 4) against all the tested bacteria. In silico studies showed that the complexes formed by compounds 2 and 8 with the S. mitis, S. sanguinis, and S. mutans targets (3LE0, 4N82, and 3AIC, respectively) had energy score values similar to those of the native S. mitis, S. sanguinis, and S. mutans ligands due to the formation of strong hydrogen bonds. Moreover, all the estimated physicochemical parameters satisfied the drug-likeness criteria without violating the Lipinski, Veber, and Egan rules, so these compounds are not expected to cause problems with oral bioavailability and pharmacokinetics. Compounds 2 and 8 also had suitable ADMET parameters, as the online server pkCSM calculates. These results make compounds 2 and 8 good candidates as antibacterial agents against oral bacteria.

ABSTRACT Vaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 μg/mL) as well as minimum fungicidal concentrations (2 to 32 μg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC50) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 μg/mL and 32 μg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1, and ALS1, were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis. IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB, and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

This study aimed at evaluating the potential of Copaifera lucens, specifically its oleoresin (CLO), extract (CECL), and the compound ent-polyalthic acid (PA), in combating caries and toxoplasmosis, while also assessing its toxicity. The study involved multiple assessments, including determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against cariogenic bacteria. CLO and PA exhibited MIC and MBC values ranging from 25 to 50 μg/mL, whereas CECL showed values equal to or exceeding 400 μg/mL. PA also displayed antibiofilm activity with minimum inhibitory concentration of biofilm (MICB50) values spanning from 62.5 to 1000 μg/mL. Moreover, PA effectively hindered the intracellular proliferation of Toxoplasma gondii at 64 μg/mL, even after 24 h without treatment. Toxicological evaluations included in vitro tests on V79 cells, where concentrations ranged from 78.1 to 1250 μg/mL of PA reduced colony formation. Additionally, using the Caenorhabditis elegans model, the lethal concentration (LC50) of PA was determined as 1000 μg/mL after 48 h of incubation. Notably, no significant differences in micronucleus induction and the NDI were observed in cultures treated with 10, 20, or 40 μg/mL of CLO. These findings underscore the safety profile of CLO and PA, highlighting their potential as alternative treatments for caries and toxoplasmosis.

Background: Increased prevalence of high virulent strains of Mycobacterium tuberculosis and M. kansasii require the development of new antimycobacterial drugs safe and more effective. And, in this context, endophytic fungi have been known as prominent sources of bioactive compounds. Purpose: To study the antimycobacterial and anti-inflammatory activities of six fungal metabolites: austdiol (1), austdiol diacetate (2), mycoleptone A (3) and eugenitin (4) isolated from cultures of endophyte Mycoleptodiscus indicus; emodin (5) from endophyte Penicillium citrinum and δ-lactam (6) from soil fungi Humicola grisea. Methods: Antimycobacterial activity against M. bovis BCG, M. tuberculosis and M. kansasii strains was evaluated using MTT method to determine the MIC50. The anti-inflammatory activity and cytotoxicity assay were carried out in LPS-stimulated RAW 264.7 macrophages. Results: Emodin (5) was the most active compound against high virulent M. tuberculosis and M. kansasii strains exhibiting MIC50 of 8.2 ± 1.0 and 23.9 ± 0.9 μM respectively and against intracellular M. tuberculosis H37Rv growth (MIC50 of 6.5 ± 1.5 μM). Emodin inhibitory activity has been previously described only against extracellular M. tuberculosis strains. Azaphilones (1 and 3) also showed inhibitory activity against hypervirulent M. tuberculosis culture (MIC50 ≤ 40 μM) and intracellular growth (MIC50 ≤ 30 μM) whereas compounds 1 and 2 were active against high virulent M. kansassi strains (MIC50 ≤ 40 μM). Anti-inflammatory activity to inhibit NO, TNF-α and IL-1β production on LPS-stimulated macrophages was observed notably for austdiol (1) and emodin (5) with MIC50 ≤ 10 μM and good potential for compounds 2 and 3. Conclusion: Azaphilone compounds (1, 2 and 3) and emodin (5) exhibiting both activities, antimycobacterial and anti-inflammatory, being promising anti-TB agents for further investigations focusing on dual treatment of severe pulmonary TB and NMT infections associated to hyperinflammation.

Helicobacter pylori is a Gram-negative, microaerophilic, curved-rod, flagellated bacterium commonly found in the stomach mucosa and associated with different gastrointestinal diseases. With high levels of prevalence worldwide, it has developed resistance to the antibiotics used in its therapy. Brazilian red propolis has been studied due to its biological properties, and in the literature, it has shown promising antibacterial activities. The aim of this study was to evaluate anti-H. pylori from the crude hydroalcoholic extract of Brazilian red propolis (CHEBRP). For this, in vitro determination of the minimum inhibitory and bactericidal concentration (MIC/MBC) and synergistic activity and in vivo, microbiological, and histopathological analyses using Wistar rats were carried out using CHEBRP against H. pylori strains (ATCC 46523 and clinical isolate). CHEBRP presented MIC/MBC of 50 and 100 μg/mL against H. pylori strains (ATCC 43526 and clinical isolate, respectively) and tetracycline MIC/MBC of 0.74 µg/mL. The association of CHEBRP with tetracycline had an indifferent effect. In the stomach mucosa of rats, all treatments performed significantly decreased the number of H. pylori, and a concentration of 300 mg/kg was able to modulate the inflammatory response in the tissue. Therefore, CHEBRP showed promising anti-H. pylori in in vitro and in vivo assays.

Denture dentifrices must be effective and not deleterious to prosthetic devices. This study formulated and evaluated dentifrices based on oils of Copaifera officinalis, Eucalyptus citriodora, Melaleuca alternifolia, Pinus strobus, and Ricinus communis. Organoleptic characteristics (appearance, color, odor, taste), physicochemical properties (pH, density, consistency, rheological, abrasiveness, weight loss, and surface roughness) and antimicrobial (Hole-Plate Diffusion–HPD)/anti-biofilm (Colony Forming Units–CFU) action against Staphylococcus aureus, Streptococcus mutans, and Candida albicans were evaluated. Formulations were compared with water (negative control) and a commercial dentifrice (positive control). The data were analyzed by Kruskal-Wallis and Dunn tests (α = 0.05). The organoleptic and physicochemical properties were adequate. All dentifrices promoted weight losses, with high values for C. officinalis and R. communis, and an increase in surface roughness, without differing from each other. For antimicrobial action, C. officinalis and E. citriodora dentifrices were similar to positive control showing effectiveness against S. mutans and C. albicans and no dentifrice was effective against S. aureus; regarding the anti-biofilm action, the dentifrices were not effective, showing higher CFU counts than positive control for all microorganisms. The dentifrices presented satisfactory properties; and, although they showed antimicrobial action when evaluated by HPD, they showed no effective anti-biofilm action on multispecies biofilm.

Context: (−)-6,6′-Dinitrohinokinin (DNHK) display remarkable antiparasitic activity and was, therefore, incorporated into a nanoparticle formulation. Objective: Incorporation of DNHK in poly lactic-co-glycolic acid (PLGA) nanoparticles aiming to improve its biological activities. Materials and methods: Synthesis, characterization and incorporation of DNHK into glycolic acid (PLGA) nanoparticles by nanoprecipitation method. The nanoparticles were characterized by ultraviolet-visible spectroscopy, X-ray diffraction, field emission electron microscopic scanning mansoni (FESEM), and dynamic light scattering (DLS). For the in vitro test with Schistosoma mansoni, the DNHK-loaded PLGA was diluted into the medium, and added at concentrations 10–200 µM to the culture medium containing one adult worm pair. The parasites were kept for 120 h and monitored every 24 h to evaluate their general condition, including: pairing, alterations in motor activity and mortality. Results: The loaded PLGA nanoparticles gave an encapsulation efficiency of 42.2% and showed spherical characteristics in monodisperse polymeric matrix. The adult worm pairs were separated after 120 h of incubation for concentrations higher than 50 µM of DNHK-loaded PLGA. The groups incubated with 150 and 200 µM of DNHK-loaded PLGA for 24 and 120 h killed 100% of adult worms, afforded LC50 values of 137.0 ± 2.12 µM and 79.01 ± 1.90 µM, respectively, which was similar to the effect displayed by 10 µM of praziquantel. Discussion and conclusions: The incorporation of DNHK-loaded showed schistosomicidal activity and allowed its sustained release. The loaded PLGA system can be administered intravenously, as well as it may be internalized by endocytosis by the target organisms.

This study evaluates the antibacterial activity of the Copaifera duckei Dwyer oleoresin and two isolated compounds [eperu-8(20)-15,18-dioic acid and polyalthic acid] against bacteria involved in primary endodontic infections and dental caries and assesses the cytotoxic effect of these substances against a normal cell line. MIC and MBC assays pointed out the most promising metabolites for further studies on bactericidal kinetics, antibiofilm activity, and synergistic antibacterial action. The oleoresin and polyalthic acid but not eperu-8(20)-15,18-dioic provided encouraging MIC and MBC results at concentrations lower than 100 μg mL−1. The oleoresin and polyalthic acid activities depended on the evaluated strain. A bactericidal effect on Lactobacillus casei (ATCC 11578 and clinical isolate) emerged before 8 h of incubation. For all the tested bacteria, the oleoresin and polyalthic acid inhibited biofilm formation by at least 50%. The oleoresin and polyalthic acid gave the best activity against Actinomyces naeslundii (ATCC 19039) and L. casei (ATCC 11578), respectively. The synergistic assays combining the oleoresin or polyalthic acid with chlorhexidine did not afford interesting results. We examined the cytotoxicity of C. duckei oleoresin, eperu-8(20)-15,18-dioic acid, and polyalthic acid against Chinese hamster lung fibroblasts. The oleoresin and polyalthic acid were cytotoxic at concentrations above 78.1 μg mL−1, whereas eperu-8(20)-15,18-dioic displayed cytotoxicity at concentrations above 312.5 μg mL−1. In conclusion, the oleoresin and polyalthic acid are potential sources of antibacterial agents against bacteria involved in primary endodontic infections and dental caries in both the sessile and the planktonic modes at concentrations that do not cause cytotoxicity.

We report herein the synthesis of six diterpene derivatives, three of which are new, generated through known organic chemistry reactions that allowed structural modification of the existing natural products kaurenoic acid (1) and copalic acid (2). The new compounds were fully characterized using high resolution mass spectrometry, infrared spectroscopy, 1H- and 13C-NMR experiments. We also report the evaluation of the anti-tuberculosis potential for all compounds, which showed some promising results for Micobacterium tuberculosis inhibition. Moreover, the toxicity for each of the most active compounds was also assessed.

We evaluated the antibacterial activity of three diterpenes isolated from natural sources against a panel of microorganisms responsible for bovine mastitis. ent-Copalic acid (CA) was the most active metabolite, with promising MIC values (from 1.56 to 6.25 µg mL−1) against Staphylococcus aureus (ATCC and clinical isolate), Staphylococcus epidermidis, Streptococcus agalactiae, and Streptococcus dysgalactiae. We conducted time-kill assays of CA against S. aureus, a commensal organism considered to be a ubiquitous etiological agent of bovine mastitis in dairy farms worldwide. In the first 12 h, CA only inhibited the growth of the inoculums (bacteriostatic effect), but its bactericidal effect was clearly noted thereafter (between 12 and 24 h). In conclusion, CA should be considered for the control of several Gram-positive bacteria related to bovine mastitis.

To assess topical delivery studies of glycoalkaloids, an analytical method by HPLC-UV was developed and validated for the determination of solasonine (SN) and solamargine (SM) in different skin layers, as well as in a topical formulation. The method was linear within the ranges 0.86 to 990.00 µg/mL for SN and 1.74 to 1000.00 µg/mL for SM (r = 0.9996). Moreover, the recoveries for both glycoalkaloids were higher than 88.94 and 93.23% from skin samples and topical formulation, respectively. The method developed is reliable and suitable for topical delivery skin studies and for determining the content of SN and SM in topical formulations.

The antimicrobial activity of four labdane-type diterpenes isolated from the oleoresin of Copaifera langsdorffii as well as of two commercially available diterpenes (sclareol and manool) was investigated against a representative panel of microorganisms responsible for periodontitis. Among all the evaluated compounds, (−)-copalic acid (CA) was the most active, displaying a very promising MIC value (3.1 µg mL−1; 10.2 µM) against the key pathogen (Porphyromonas gingivalis) involved in this infectious disease. Moreover, CA did not exhibit cytotoxicity when tested in human fibroblasts. Time-kill curve assays performed with CA against P. gingivalis revealed that this compound only inhibited the growth of the inoculums in the first 12 h (bacteriostatic effect). However, its bactericidal effect was clearly noted thereafter (between 12 and 24 h). It was also possible to verify an additive effect when CA and chlorhexidine dihydrochloride (CHD, positive control) were associated at their MBC values. The time curve profile resulting from this combination showed that this association needed only six hours for the bactericidal effect to be noted. In summary, CA has shown to be an important metabolite for the control of periodontal diseases. Moreover, the use of standardized extracts based on copaiba oleoresin with high CA contents can be an important strategy in the development of novel oral care products.

Blueberries are among the edible fruits that are recognized best for their potential health benefits. The crude extract from Vaccinium corymbosum was assessed in anti-inflammatory and antinociceptive models. The crude hydroalcoholic extract was administered orally at doses of 100, 200 or 300 mg kg −1 for all the assays. In the carrageenan test, the crude extract reduced rat paw oedema by 9.8, 28.5 and 65.9%, respectively. For the histamine assay, the reductions of oedema were 70.1, 71.7 and 81.9%, respectively. In the myeloperoxidase (MPO) assay, 300 mg kg −1 crude extract produced a significant inhibition of the MPO activity, at 6 h and 24 h after injection of carrageenan, by 42.8 and 46.2%, respectively. With the granulomatous tissue assay dexamethasone displayed significant activity, whereas the blueberry extract was inactive. For the abdominal constriction test, inhibitions of 49.0, 54.5, 53.5%, respectively, were observed for the crude extract, and 61.4% for indometacin. In the formalin test, the crude extract (200 and 300 mg kg −1) and indometacin inhibited only the second phase by 36.2, 35.3 and 45.8%, respectively. Considering that the crude extract of blueberry displayed antinociceptive and anti-inflammatory activity, its consumption may be helpful for the treatment of inflammatory disorders. [ABSTRACT FROM AUTHOR]

Baccharis dracunculifolia D.C. (Asteraceae), a shrub which grows wild in Brazil, is the main botanical source of Brazilian green propolis. Since Brazilian propolis shows an immunomodulatory activity, the goal of this work was to evaluate the action of B. dracunculifolia extracts and some of its isolated compounds on reactive oxygen intermediate (H2O2) production by macrophages obtained from male BALB/c mice. The results showed that the leaf (Bd-L) (25, 50, and 100 μg mL−1), leaf rinse (Bd-LR) (25 μg mL−1), and the root (Bd-R) (25 μg mL−1) extracts enhanced H2O2 release by macrophages. A phytochemical study of the root and leaves of B. dracunculifolia was carried out. The chromatographic fractionation of Bd-R, using several techniques, afforded the isolation of baccharis oxide (1), friedelanol (2), viscidone (11), 11-hydroxy-10,11-dihydro-euparin (12), and 6hydroxy-tremetona (13), while Bd-LR gave the following isolated compounds: baccharis oxide (1), friedelanol (2), isosakuranetin (3), aromadendrin-4'-methyl ether (4), dihydrocumaric acid (5), baccharin (6), hautriwaic acid lactone (7), hautriwaic acid acetate (8), drupanin (9), and cumaric acid (10). Among the isolated compounds, baccharis oxide (1) and friedelanol (2) increased H2O2 production at a concentration of 100 μM. This is the first time that the presence of compounds 7, 8, 12, and 13 in B. dracunculifolia has been reported. Based on these results it is suggested that the crude extracts and some isolated compounds from B. dracunculifolia display an immunomodulatory action. [ABSTRACT FROM AUTHOR]