New insights into the antiviral activity of nordihydroguaiaretic acid: Inhibition of dengue virus serotype 1 replication.

BACKGROUND: Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4. PURPOSE: We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity. METHODS: The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes. RESULTS: NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process. CONCLUSION: The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process.

Inactivation of Herpes Simplex Virus by Photosensitizing Anthraquinones Isolated from Heterophyllaea pustulata.

Heterophyllaea pustulata is a phototoxic plant from Argentina. Aerial parts extracts, high in photosensitizing anthraquinones, have shown in vitro antiviral activity. The purpose of this study was to study the antiherpetic activity of the main purified anthraquinones, even evaluating their competence as photodynamic sensitizers to photo-stimulate the antiviral effect. In vitro antiviral activity against Herpes Simplex virus type I and the photo-inactivation of viral particle were studied by the Neutral Red uptake test and observation of the cytopathic effect. Rubiadin 1-methyl ether and 5,5'-bisoranjidiol produced a significant effect (≥ 80% inhibition) with minimal damage to host cells (subtoxic concentration). Anthraquinones with poor antiherpetic activity at its maximum noncytotoxic concentration showed an important photo-stimulated effect, such is the case of soranjidiol and 5,5'-bisoranjidiol (28.0 ± 6.3 vs. 81.8 ± 2.1% and 15.5 ± 0.3 vs. 89.8 ± 1.7%, respectively). The study also proved the decrease of viral particles, necessary to reduce infection. Therefore, photosensitizing anthraquinones from natural resources could be proposed to develop new treatments for localized viral lesions with antimicrobial photodynamic therapy.

The anthraquinones rubiadin and its 1-methyl ether isolated from Heterophyllaea pustulata reduces Candida tropicalis biofilms formation.

BACKGROUND: Candida tropicalis is increasingly becoming among the most commonly isolated pathogens causing fungal infections with an important biofilm-forming capacity. PURPOSE: This study addresses the antifungal effect of rubiadin (AQ1) and rubiadin 1-methyl ether (AQ2), two photosensitizing anthraquinones (AQs) isolated from Heterophyllaea pustulata, against C. tropicalis biofilms, by studying the cellular stress and antioxidant response in two experimental conditions: darkness and irradiation. The combination with Amphotericin B (AmB) was assayed to evaluate the synergic effect. STUDY DESIGN/METHODS: Biofilms of clinical isolates and reference strain of Candida tropicalis were treated with AQs (AQ1 or AQ2) and/or AmB, and the biofilms depletion was studied by crystal violet and confocal scanning laser microscopy (CSLM). The oxidant metabolites production and the response of antioxidant defense system were also evaluated under dark and irradiation conditions, being the light a trigger for photo-activation of the AQs. The Reactive Oxygen Species (ROS) were detected by the reduction of Nitro Blue Tetrazolium test, and Reactive Nitrogen Intermediates (RNI) by the Griess assay. ROS accumulation was also detected inside biofilms by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, which was visualized by CSLM. Superoxide dismutase (SOD) activity and the total antioxidant capacity of biofilms were measured by spectrophotometric methods. The minimun inhibitory concentration for sessile cells (SMIC) was determined for each AQs and AmB. The fractional inhibitory concentration index (FICI) was calculated for the combinations of each AQ with AmB by the checkerboard microdilution method. RESULTS: Biofilm reduction of both strains was more effective with AQ1 than with AQ2. The antifungal effect was mediated by an oxidative and nitrosative stress under irradiation, with a significant accumulation of endogenous ROS detected by CSLM and an increase in the SOD activity. Thus, the prooxidant-antioxidant balance was altered especially by AQ1. The best synergic combination with AmB was also obtained with AQ1 (80.5%) (FICI=0.74). CONCLUSION: Under irradiation, the oxidative stress was the predominant effect, altering the prooxidant-antioxidant balance, which may be the cause of the irreversible cell injury in the biofilm. Our results showed synergism of these natural AQs with AmB. Therefore, the photosensitizing AQ1 could be an alternative for the Candida infections treatment, which deserves further investigation.

Reduction of Candida tropicalis biofilm by photoactivation of a Heterophyllaea pustulata extract.

CONTEXT: Biofilm formation is an important problem, since this growth mode confers resistance to drugs usually used in therapeutics. OBJECTIVE: In vitro antifungal activity of extracts obtained from Heterophyllaea pustulata Hook f. (Rubiaceae) were studied against Candida tropicalis biofilms, evaluating the effect of irradiation and the oxidative and nitrosative stresses as possible mechanisms of action. MATERIALS AND METHODS: Hexane, benzene, ethyl acetate and ethanol extracts were evaluated at three concentrations (0.2, 0.1 and 0.05 mg/mL) over mature biofilm, under darkness and irradiation. After 48 h of incubation, biofilm quantitation was performed by the O'Toole and Kolter method. Reactive oxygen species (ROS) was measured by nitro-blue tetrazolium (NBT) reaction and reactive nitrogen intermediates (RNI) by the Griess reagent. Superoxide dismutase activation (SOD, NBT assay) and total antioxidant system (FRAP test) were studied. RESULTS: Only the benzene extract at 0.2 mg/mL reduced the biofilms formation. The slight decrease achieved in darkness (17.06 ± 2.80% reduction) was increased by light action (39.31 ± 3.50% reduction), clearly observing a photostimulation. This great reduction was confirmed by confocal microscopy. In darkness, biofilm reduction was mediated by an increase in RNI, whereas under irradiation, the ROS action was most important. Although no SOD activation was observed, a strong stimulation of the total antioxidant system was detected. HPLC analysis established a high content of several anthraquinones in this extract. DISCUSSION AND CONCLUSION: Biofilm reduction by benzene extract was mainly mediated by oxidative stress triggered under light action, confirming a photodynamic sensitization, which could be attributed to its high content of photosensitizing anthraquinones.

In vitro antiviral activity of Heterophyllaea pustulata extracts.

The antiviral activity was tested of different polarity extracts, with differing chemical composition, obtained from aerial parts of Heterophyllaea pustulata Hook f. (Rubiaceae) against Herpes Simplex Virus Type I (HSV-1) and Saint Louis Encephalitis Virus (SLEV). The Vero cell line was employed as a host cell for the antiviral assessment of benzene (Ben), ethyl acetate (EtOAc) and ethanol (EtOH) extracts by means of the Neutral Red uptake assay and plaque reduction test. None of the extracts showed antiviral activity against SLEV. Only the extracts (Ben and EtOAc) with a high content of anthraquinones (AQs) inhibited HSV-1 replication, exhibiting Selectivity Index (SI) values of 2.7 and 2.4, respectively. Therefore, these extracts could be good candidates as natural sources for antiviral drug development against HSV-1.