Green and Red Brazilian Propolis: Antimicrobial Potential and Anti-Virulence against ATCC and Clinically Isolated Multidrug-Resistant Bacteria.

Brazilian green and red propolis stand out as commercial products for different medical applications. In this article, we report the antimicrobial activities of the hydroalcoholic extracts of green (EGP) and red (ERP) propolis, as well as guttiferone E plus xanthochymol (8) and oblongifolin B (9) from red propolis, against multidrug-resistant bacteria (MDRB). We undertook the minimal inhibitory (MIC) and bactericidal (MBC) concentrations, inhibition of biofilm formation (MICB50 ), catalase, coagulase, DNase, lipase, and hemolysin assays, along with molecular docking simulations. ERP was more effective by displaying MIC and MBC values <100 µg mL-1 . Compounds 8 and 9 displayed the lowest MIC values (0.98 to 31.25 µg mL-1 ) against all tested Gram-positive MDRB. They also inhibited the biofilm formation of S. aureus (ATCC 43300 and clinical isolate) and S. epidermidis (ATCC 14990 and clinical isolate), with MICB50 values between 1.56 and 6.25 µg mL-1 . The molecular docking results indicated that 8 and 9 might interact with the catalase's amino acids. Compounds 8 and 9 have great antimicrobial potential.

Novel polyprenylated benzophenone derivatives from Clusia burle-marxii.

Three new caged polyprenylated benzophenone derivatives named burlemarxiones D-F (1-3) were isolated from the hexane extract of Clusia burle-marxii trunks. Burlemarxione D (1) contains the tetracyclo[8.3.1.03,11.05,10]tetradecane core skeleton also observed for burlemarxione A, its probable immediate precursor. However, two additional rings are formed to produce an unprecedented complex-caged core skeleton. These additional rings could be formed by a radical cyclization reaction of one prenyl group at C-5 with C-1 and C-33, followed by oxidative dehydrogenation (rearomatization) or by an intramolecular [4 + 2] radical cycloaddition (Diels-Alder reaction), followed by an enolization reaction (rearomatization). Burlemarxiones E and F were isolated after methylation with diazomethane that was necessary to avoid the interconversion of the pair of ß-diketones in tautomeric equilibrium. The proposed biosynthetic pathway for burlemarxiones D-F involves the condensation of either lavandulyl pyrophosphate or 2-(1-methylvinyl)-hexa-5-enyl pyrophosphate with the acylphloroglucinol derivative 6-benzoyl-5-hydroxy-5-cyclohexen-1,3-dione, followed by consecutive prenylation reactions. Therefore, Clusia burle-marxii reinforces the claim that the genus Clusia is an important source of sophisticated caged polyprenylated benzophenone derivatives.

Polyprenylated benzophenone derivatives with a novel tetracyclo[8.3.1.03,11.05,10]tetradecane core skeleton from Clusia burle-marxii exhibited cytotoxicity against GL-15 glioblastoma-derived human cell line.

Three new polyprenylated benzophenone derivatives (1-3) were identified in the hexane extract of Clusia burle-marxii trunks, through the isolation and structural elucidation of their methyl derivatives, along with two known polyprenylated benzophenone derivatives sampsonine N (4) and obdeltifolione C (5). Burlemarxiones A (1) and B (2) show an unprecedent tetracyclo[8.3.1.03,11.05,10]tetradecane core skeleton. These compounds are a pair of ß-diketones in tautomeric equilibrium, whereas isonemorosonol (3) is the respective ß-diketone pair in tautomeric equilibrium with nemorosonol. Burlemarxione A methyl derivative (1a) and sampsonine N exhibited strong in vitro cytotoxic activity against GL-15 glioblastoma-derived human cell line.

Polycyclic Polyprenylated Xanthones from Symphonia globulifera: Isolation and Biomimetic Electrosynthesis.

Two regioisomeric polycyclic xanthones, 3,16-oxyguttiferone A (2) and 1,16-oxyguttiferone A (3), which are polyprenylated acylphloroglucinol-derived analogues, were isolated from the seeds of Symphonia globulifera, together with their presumed o-dihydroxybenzoyl precursor, guttiferone A (1). Anodic oxidation of 1 into the corresponding o-quinone species proved to be an efficient biomimetic method to generate xanthones 2 and 3 in high overall yield and to confirm their structures. Both compounds displayed cytotoxicity against the HCT 116 colon carcinoma cell line with IC50 values of 8 and 3 µM, respectively.

7-Epiclusianone, a Benzophenone Extracted from Garcinia brasiliensis (Clusiaceae), Induces Cell Cycle Arrest in G1/S Transition in A549 Cells.

Lung cancer is the leading cause of cancer deaths in the world. Disease stage is the most relevant factor influencing mortality. Unfortunately, most patients are still diagnosed at an advanced stage and their five-year survival rate is only 4%. Thus, it is relevant to identify novel drugs that can improve the treatment options for lung cancer. Natural products have been an important source for the discovery of new compounds with pharmacological potential including antineoplastic agents. We have previously isolated a prenylated benzophenone (7-epiclusianone) from Garcinia brasiliensis (Clusiaceae) that has several biological properties including antiproliferative activity against cancer cell lines. In continuation with our studies, the present work aimed to investigate the mechanisms involved with antiproliferative activity of 7-epiclusianone in A549 cells. Our data showed that 7-epiclusianone reduced the viability of A549 cells in a concentration-dependent manner (IC50 of 16.13 ± 1.12 µM). Cells were arrested in G1/S transition and apoptosis was induced. In addition, we observed morphological changes with cytoskeleton disorganization in consequence of the treatment. Taken together, the results showed that cell cycle arrest in G1/S transition is the main mechanism involved with antiproliferative activity of 7-epiclusianone. Our results are promising and open up the prospect of using this compound in further anticancer in vivo studies.

Potent Schistosomicidal Constituents from Garcinia brasiliensis.

Praziquantel is the drug of choice for the treatment of schistosomiasis. However, several strains of Schistosoma mansoni are resistant to praziquantel, making it necessary to discover new drugs that might be used for its treatment. With this in mind, the properties of a schistosomicidal ethanolic extract of Garcinia brasiliensis Mart. epicarp, the fractions obtained by partitioning this extract, including the hexane fractions, ethyl acetate fraction, and the aqueous fraction, and the isolated compounds 7-epiclusianone, a major component from these fractions, and fukugetin were tested in vitro on adult worms of S. mansoni. Mortality, damage to membranes, and excretory system activity were observed at 100.0, 50.0, 75.0, and 14.0 µg/mL for the ethanolic extract of G. brasiliensis Mart. epicarp, its hexane fractions, the ethyl acetate fraction, and 7-epiclusianone, respectively. For 7-epiclusianone, these data were confirmed by fluorescent probe Hoechst 33 258 and resorufin. Additionally, the biocidal effect of 7-epiclusianone was even higher than the hexane fractions. Moreover, an inhibitory effect of 7-epiclusianone on the egg laying of female adult S. mansoni worms was observed in cercariae and schistossomula. Thus, 7-epiclusianone is a promising schistosomicidal compound; however, more studies are needed to elucidate its mechanism of toxicity and to evaluate the in vivo activity of this compound.

Chemosystematic aspects of polyisoprenylated benzophenones from the genus Clusia.

Benzophenone derivatives are special metabolites that arouse great scientific interest. The Clusiaceae family is known for producing large amounts of benzophenone derivatives with several isoprene residues on their structures, which are responsible for the observed complexity and structural variety in this class of substances, and also contribute to their biological activities. Clusia is an important genus belonging to Clusiaceae, with 55 different polyisoprenylated benzophenones identified so far. These substances were analyzed from biosynthetic and chemosystematic points of view, allowing the determination of characteristics regarding their production, accumulation and distribution within this genus. Polyisoprenylated benzophenones found in Clusia showed a high prenylation degree, with 2 to 5 isoprene units and a greater occurrence in flowers and fruits. Section Cordylandra showed a very similar occurrence of 2,4,6-trihydroxybenzophenone derivatives and bicyclo[3.3.1]nonane-2,4,9-trione derivatives, the majority of them with 4 isoprene units. In section Anandrogyne there is a predominance of simple 2,4,6-trihydroxy-benzophenone derivatives, with 2 isoprene units, and in Chlamydoclusia predominates bicyclo[3.3.1]nonane-2,4,9-trione derivatives with 4 isoprene units. Although highly prenylated, these substances showed low oxidation indexes, which from an evolutionary perspective corroborates the fact that Clusiaceae is a family in transition, with some common aspects with both basal and derived botanical families.

Garcinielliptone FC, a polyisoprenylated benzophenone from Platonia insignis Mart., promotes vasorelaxant effect on rat mesenteric artery.

Polyisoprenylated benzophenones represent a group of chemical compounds commonly identified in Clusiaceae species and are responsible for a large amount of biological activities. In this work, the vasorelaxant effect induced by garcinielliptone FC (GFC) isolated from Platonia insignis Mart. (Clusiaceae), a monotype species from Platonia genus, was investigated. GFC promoted an endothelium-independent vasorelaxation on phenylephrine (PHE, 10(-5) mol L(-1))-induced vasoconstriction, but not on KCl (80 mmol L(-1))-induced vasoconstriction, on rat superior mesenteric artery rings. In addition, a concentration-dependent decrease of PHE- or serotonin-induced cumulative concentration-response curves was observed for GFC, and a slight decrease of pD2 value on CaCl2-induced vasoconstriction. In a Ca(2+)-free medium, GFC interfered in calcium mobilisation from PHE (10(-5) mol L(-1))-sensitive intracellular stores. GFC-induced vasorelaxant effect is probably mediated by a dual effect on mobilisation of calcium intracellular stores and attenuation of transmembrane calcium influx.

Antiproliferative activity of the dimeric phloroglucinol and benzophenone derivatives of Hypericum spp. native to southern Brazil.

A large number of plants are known to possess strong antitumor properties. Previous studies have verified the antiproliferative activity of the extracts and fractions from six species of Hypericum spp. growing in southern Brazil. In the present study, the in-vitro antiproliferative effects of two dimeric phloroglucinols (japonicin A and uliginosin B, isolated from Hypericum myrianthum) and two benzophenones (cariphenone A and cariphenone B, isolated from H. carinatum) were investigated against three tumor cell lines (HT-29 - human colon carcinoma cells; U-251 - human glioma cell line, and OVCAR-3 - human ovarian carcinoma cells). In addition, different doses of these compounds were associated with cytotoxic drugs commonly used as chemotherapy in the clinic. Cariphenone A and cariphenone B showed moderate antiproliferative activity against all tumor cell lines at a dose of 100 µg/ml. Unlike benzophenones, japonicin A and uliginosin B exerted antiproliferative effects only in the OVCAR-3 cell line. Moreover, a very strong synergistic effect was demonstrated by the association of subeffective doses of japonicin A with the chemotherapeutic drug paclitaxel, decreasing cellular proliferation of the OVCAR-3 cell line. These preliminary results provide a scientific basis to further pursue these compounds as potential combined therapy for certain tumor types.

Gastroprotective effects of extracts and guttiferone A isolated from Garcinia achachairu Rusby (Clusiaceae) against experimentally induced gastric lesions in mice.

This study was undertaken to evaluate the gastroprotective properties of seed, leaf, and branch methanolic extracts and guttiferone A obtained from Garcinia achachairu (Clusiaceae). Mice were used in all the models, and treatments were administered orally only in pylorus-ligated model of the extracts, and drugs were administered intraduodenally. Treatment with different extracts (500 mg/kg) significantly reduced the ulcerative lesions in the ethanol/HCl-induced model; however, the seed extract was most active. When tested in different doses (50, 250, or 500 mg/kg), the seed extract of G. achaicharu showed a dose-dependent effect with a percentage of inhibition of gastric lesions of 41, 49, and 85 %, respectively. The seed extract also significantly reduced the ulcerative lesions in the indomethacin/bethanechol-induced ulcer. In this model, the percentage of inhibition of ulcer was 24, 58, and 90 %, respectively. Regarding the model of gastric secretion, a reduction of gastric juice volume and total acidity was observed, as well as an increase in gastric pH. Considering that the seed extract was the most active, it was subjected to silica gel column chromatography, leading to the isolation of guttiferone A. The isolated compound and omeprazole were evaluated in the HCl/ethanol-induced ulcer model. In this assay, both compounds at a dose of 30 mg/kg reduced the ulcerative lesions by about 75 %. These results demonstrate, for the first time, that extracts obtained from G. achachairu and guttiferone A produce gastroprotective effects, corroborating ethnomedicinal use of this plant.

Phytochemical analysis and antinociceptive properties of the seeds of Garcinia achachairu.

In a search for new and effective analgesic substances from the Brazilian biodiversity, the present study evaluates the chemical composition and antinociceptive potential of the methanol extract and a pure compound obtained from the seeds of Garcinia achachairu Rusby (Clusiaceae). The methanolic seed extract was directly subjected to purification by column chromatography and the purification was monitored by thin-layer chromatography. The main isolated compound was identified as Guttiferone A by comparison of conventional spectroscopic data (IR, NMR-(1)H and (13)C) to the literature data which was isolated for the first time from this plant. When evaluated in the acetic acid-induced nociception model in mice, the methanolic seed extract had an ID(50) (Inhibitory dose) of 13.1 (11.23-14.91) mg/kg and a maximal inhibition of 72 ± 4%. In the same model, Guttiferone A had an ID(50) of 4.54 (3.29-6.24) mg/kg and a maximal inhibition of 73 ± 5%. The methanolic seed extract and Guttiferone A were also active in pain models induced by formalin, capsaicin, glutamate and carrageenan. These data suggest that the antinociceptive effect of Guttiferone A partly depends on its interference with the synthesis or activity of the cytokine TNF-α, the keratinocyte-derived chemokine KC, and/or PGE(2). These data support, at least in part, the use of G. achachairu in folk medicine and suggest that this plant is an important source of compounds with a suitable profile for development as new and effective medicinal agents to treat pain processes.

Supercritical extraction of phloroglucinol and benzophenone derivatives from Hypericum carinatum: quantification and mathematical modeling.

The aerial parts of Hypericum carinatum (Guttiferae) were extracted with supercritical carbon dioxide under constant temperature (40, 50 or 60°C) and gradual pressure increase (90, 120, 150 and 200 bar) aiming at the recovery of enriched fractions containing uliginosin B, cariphenone A and cariphenone B, compounds of pharmaceutical interest. The yields of these substances were determined by high-performance liquid chromatography and compared with those obtained with n-hexane maceration. The supercritical-fluid extraction showed higher selectivity than the conventional solvent extraction method. After defining 40°C and 90 bar as the best conditions to obtain the target compounds, a mathematical model was used for the extraction process and a good correlation was achieved with the experimental data.

Effects of 7-epiclusianone on Streptococcus mutans and caries development in rats.

The aim of this study was to evaluate the effects of 7-epiclusianone (7-epi) on specific virulence attributes of Streptococcus mutans in vitro and on development of dental caries in vivo. 7-Epi was obtained and purified from fruits of Rheedia brasiliensis. We investigated its influence on surface-adsorbed glucosyltransferase (Gtf) B activity, acid production, and viability of S. MUTANS in biofilms, as well as on caries development using a rodent model. 7-Epi (100 µg/mL) significantly reduced the activity of surface-adsorbed GtfB (up to 48.0 ± 1.8 of inhibition at 100 µg/mL) and glycolytic pH-drop by S. mutans in biofilms (125 and 250 µg/mL) (vs. vehicle control, p < 0.05). In contrast, the test compound did not significantly affect the bacterial viability when compared to vehicle control (15 % ethanol, p > 0.05). Wistar rats treated topically with 7-epi (twice daily, 60-s exposure) showed significantly smaller number of and less severe smooth- and sulcal-surface carious lesions (p < 0.05), without reducing the S. mutans viable population from the animals' dental biofilms. In conclusion, the natural compound 7-epiclusianone may be a potentially novel pharmacological agent to prevent and control dental caries disease.

Antiplasmodial benzophenone derivatives from the root barks of Symphonia globulifera (Clusiaceae).

In an effort to find antimalarial drugs, a systematic in vitro evaluation on a chloroquine-resistant strain of Plasmodium falciparum (FcB1) was undertaken on sixty plant extracts collected in French Guiana. The ethyl acetate extract obtained from the root barks of Symphonia globulifera exhibited a strong antiplasmodial activity (97% at 10 microg/ml). The phytochemical investigation of this extract led to the isolation of nine polycyclic polyprenylated acylphloroglucinol (PPAPs) compounds and two oxidized derivatives. All compounds showed antiplasmodial activity with IC(50)s ranged from 2.1 to 10.1 microM. A LC/ESI-MS(n) study performed on polyprenylated benzophenones previously isolated from Moronobea coccinea provided a reliable method for their detection in the extract and structural elucidation.

Evaluation of sub-critical water as an extraction fluid for model contaminants from recycled PET for reuse as food packaging material.

Recycling of plastics for food-contact packaging is an important issue and research into meaningful and cost-effective solutions is in progress. In this paper, the use of sub-critical water was evaluated as an alternative way of purifying poly(ethylene terephthalate) (PET) flakes for direct food contact applications. The effects of temperature, pressure and flow rate were assessed on the extraction efficiency of two of the most challenging classes of contaminants (toluene and benzophenone) from PET by sub-critical water using a first-order fractional experimental design. Extraction yield was quantified using GC/FID. The most important parameter was flow rate, indicating that the decrease in sub-critical water polarity with temperature was insufficient to eliminate partition effects. Temperature was also important, but only for the optimization of toluene extraction. These results may be explained by the poor solubility of higher molar mass compounds in sub-critical water compared to lower molar mass compounds under the same conditions, and the small decrease in dielectric constant with temperature under the experimental conditions evaluated. As cleaning efficiency is low and PET is very susceptible to hydrolysis, which limits the use of higher temperatures vis-à-vis physical recycling, the proposed extraction is unsuitable for a standalone super-clean process but may be a step in the process.

Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits.

Infections by protozoans of the genus Leishmania are the major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which exert renal and cardiac toxicity. Thus, there is a strong need for safer and more effective treatments against leishmaniasis. The present study was designated to evaluate, by a bioguided assay, the leishmanicidal activity of extracts (hexane, ethyl-acetate and ethanolic) and molecules both obtained by means of extraction from pericarps of Garcinia brasiliensis fruits. The hexane extract presented the best activity on the extracellular (promastigotes) and intracellular (amastigotes) forms of Leishmania (L.) amazonensis, when compared to the other extracts. Based on these findings, this extract was fractionated by silica gel column chromatography, affording nine fractions then resulting in three purified prenylated benzophenones - 7-epi-clusianone (1), garciniaphenone (2) and guttiferone-a (3). They showed significant activity on Leishmania (L.) amazonensis, and little toxicity for mammalian cells. Structure-activity relationships were evaluated showing that the IC(50) value displayed is dependent of prenyl groups and phenolic hydroxyls number, and inversely proportional to the hydrophobicity. Our results are promising, showing that these compounds are biologically active on Leishmania (L.) amazonensis.

Identification of a bioactive compound isolated from Brazilian propolis type 6.

A prenylated benzophenone, hyperibone A, was isolated from the hexane fraction of Brazilian propolis type 6. Its structure was determined by spectral analysis including 2D NMR. This compound exhibited cytotoxic activity against HeLa tumor cells (IC(50)=0.1756microM), strong antimicrobial activity (MIC range-0.73-6.6microg/mL; MBC range-2.92-106microg/mL) against Streptococcus mutans, Streptococcus sobrinus, Streptococcus oralis, Staphylococcus aureus, and Actinomyces naeslundii, and the results of its cytotoxic and antimicrobial activities were considered good.

Antimicrobial activity of benzophenones and extracts from the fruits of Garcinia brasiliensis.

The pericarp and seeds from fruits of Garcinia brasiliensis were subjected to extraction with hexane and ethanol. The pericarp hexane extract (PHE) and seed ethanol extract (SEE) were purified by silica gel column chromatography, which permitted isolation of the prenylated benzophenones 7-epiclusianone (1) and guttiferone-A (2), respectively. The antimicrobial activity of PHE, SEE, and compounds 1 and 2 were evaluated against Candida albicans, Staphylococcus aureus, Escherichia coli, and Bacillus cereus cultures. The minimum inhibitory concentration and minimum bactericidal concentration were established. The substances presented activity against S. aureus and B. cereus as follows: PHE, 4.0 microg/mL and 2.4 microg/mL; SEE, 10.0 microg/mL and 12.6 microg/mL; 7-epiclusianone, 1.2 microg/mL and 0.6 microg/mL; and guttiferone-A, 2.4 microg/mL and 2.4 microg/mL, respectively. The direct relationship between the lipophilic character of the structure and activity in Gram-positive bacteria was specifically observed. Therefore these extracts and prenylated benzophenones represent an interesting topic for further studies and open possibilities for an alternative control of diseases associated with Gram-positive bacteria.

Flavonoids, benzophenones and a new euphane derivative from Clusia columnaris Engl / Flavonóides, benzofenonas e um novo eufano de Clusia columnaris Engl

The polyisoprenylated benzophenones machuone and clusiachromene A have been isolated from the fruits of Clusia columnaris. The hexane extract of the young branches with leaves afforded a new euphane derivative, whose structure was elucidated by spectroscopic methods. On the contrary, the most polar EtOAc and ButOH extracts were constituted of flavonoid C-glucosides (isovitexin, vitexin and vitexin-2"-xyloside) and seven biflavonoids of the so-called Garcinia group.

Dos frutos de Clusia columnaris foram isoladas as benzofenonas poliisopreniladas machuona e clusiacromeno A. Do extrato em hexano obtido de galhos e folhas novas, um novo triterpeno do tipo eufano foi isolado. Sua estrutura foi elucidada através de métodos espectroscópicos. Por outro lado, dos extratos mais polares - em acetato de etila e em butanol, foram isolados os flavonóides C-glicosilados isovitexina, vitexina e vitexina-2"-xilosídeo, além de sete bisflavonóides conhecidos como bisflavonóides do grupo da Garcinia.

Complete assignment of the 1H and 13C NMR spectra of garciniaphenone and keto-enol equilibrium statements for prenylated benzophenones.

This article reports the structural elucidation by IR, UV and MS spectroscopic data along with 1H and 13C NMR chemical shift assignments of two benzophenones isolated from the fruit pericarp of Garcinia brasiliensis Mart. (Clusiaceae): garciniaphenone, (1R,5S,7S)-3-benzoyl-4-hydroxy-6,6-dimethyl-5,7-di(3-methyl-2-butenyl)bicyclo[3.3.1]non-3-ene-2,9-dione, a novel triprenylated benzophenone; and 7-epi-clusianone, a tetraprenylated benzophenone that has already been extracted from another species of the same family. Furthermore, the keto-enol tautomeric equilibrium at solution-state was described for these compounds by 1D and 2D NMR spectral methods and one attempt to rationalize the different ratios between the noted tautomers was based on stereochemical features.