Synergistic antifungal activity of the lipophilic fraction of Hypericum carinatum and fluconazole

ABSTRACT Hypericum species, Hypericaceae, are recognized as a source of therapeutical agents. Purified fractions and isolated compounds have been shown antimicrobial activity. As the indiscriminate use of antifungals and the increase of infections caused by emerging species are leading to the search of new alternative treatments, the aim of this study was to continue the study with Hypericum carinatum Griseb. lipophilic fraction, rich in phloroglucinol derivatives, investigating the effect of its association with fluconazole against emerging yeasts (Candida krusei, C. famata, C. parapsilosis and Cryptococcus neoformans). The synergistic activity between H. carinatum lipophilic fraction and fluconazole was assessed by two methodologies for multiple dose–response analysis: checkerboard and isobologram. Regarding synergistic experiments, the effect of the association was higher than the effect of fluconazole alone against Candida krusei and C. famata isolates (MIC fluconazole decreased about eight and four folds, respectively), suggesting that, somehow, H. carinatum lipophilic fraction compounds are facilitating the action of this drug. On the other hand, when tested against Cryptococcus neoformans and C. parapsilosis, fluconazole showed better results than the association. Thus, against Candida krusei and C. famata, the lipophilic fraction of H. carinatum was able to reduce the MIC values of fluconazole and could be considered as a potential alternative to be used against emerging yeast species.

Physicochemical and antimicrobial properties of copaiba oil: implications on product quality control.

BACKGROUND: The copaiba oil is a common natural product used in cosmetic industry and as a nutraceutical product. However, lack of quality control and scarce knowledge about its antimicrobial activity is a point of concern. The proposal of this study was to investigate the physicochemical properties and the antimicrobial activity of five commercial brands of copaiba oil. METHODS: Acidity and ester index, refractory index, solubility in alcohol, and thin layer chromatography were performed to verify the physicochemical properties of five commercial copaiba oils sold in local pharmacies. Ultra performance liquid chromatography coupled with diode-array detection and electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-DAD/ESI-Q-TOF-MS) was used to investigate diterpene acids while the volatile compounds were analysed by gas chromatography-mass spectrometry (GC-MS). Antibacterial and antifungal activities were also evaluated by agar diffusion technique; and minimal inhibitory concentration and maximal bactericidal concentration were defined for each sample and bacteria. RESULTS: The physical-chemical analysis revealed heterogeneity between all samples analysed. The A1 sample showed characteristics of copaiba oil and was mainly composed by hydrocarbon sesquiterpenes (29.95% ß-bisabolene, 25.65% Z-α-bergamotene and 10.27% ß-cariophyllene). Among diterpene acids, the UPLCDAD/ESI-Q-TOF-MS data are compatible with presence of copalic and/or kolavenic acid (m/z 305 [M + H]+). Candida albicans was sensitive to almost all samples at high concentration and Saccaromyces. Cerevisiae showed sensitivity to A1 sample at 100 mg/mL. Although variable, all samples showed antibacterial activity. Significant activity was seen for A3 (19.0 ±0 and 15.6 ±0.5 mm), A4 (16.6 ±0.5 and 15.6 ±0 mm), and A5 (17.1 ±0 and 17.1 ±0 mm) on Staphylococcus saprophyticus and S. aureus, respectively. All samples were active against Klebsiella pneumoniae showing ≥15 mm diameter halo inhibition; and only A2 was active against Eschirichia coli. Phytopatogens tested revealed resistance of Ralstonia solanacearum CGH12 to all samples and susceptibility of Xcv 112 strain of Xanthomonas campestris pv campestris to almost all samples. MIC and MMC showed bacteriostatic effect against clinical interest bacteria and bactericidal effect against phytopatogens. CONCLUSIONS: The results from physicochemical analysis reinforce the fact that it is imperative to include simple conventional methods in the analysis of oil products. The analysis of copaiba oil gives safe products and purity which ensure products with quality. Also, since copaiba oil is an over-the-counter product the results indicate that pharmacosurveillance must be improved by the governmental regulation agency to avoid microorganism resistance selection and to achieve better international quality products.

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.

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.