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.

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.

Mass spectrometry studies of lycodine-type Lycopodium alkaloids: sauroxine and N-demethylsauroxine.

RATIONALE: Sauroxine and N-demethylsauroxine are lycodine-type Lycopodium alkaloids. In recent years, Lycopodium alkaloids have gained significant interest due to their unique skeletal characteristics as well as due to their acetylcholinesterase activity. It is known that drugs that inhibit acetylcholinesterase can be used to treat the early stages of Alzheimer's disease. METHODS: Sauroxine and N-demethylsauroxine were isolated from the aerial parts of Huperzia saururus (Lam.) Trevis. Electron ionization mass spectrometry (EI-MS) (low resolution) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) fragmentation was conducted using an ion trap, GCQ Plus mass spectrometer with MS/MS. Electron ionization high-resolution mass spectrometry (EI-HRMS) was performed in a magnetic sector mass spectrometer (Micromass VG). RESULTS: Using GC/EI-CID-MS/MS we obtained different fragmentation routes that connect all the ionic populations. In addition, the use of EI-HRMS allowed us to measure the exact masses of all the fragment ions, and, with all this information gathered, we tried to establish a fragmentation scheme concordant with the ascendant and descendant species. CONCLUSIONS: The mass spectrometry studies presented in this work complete our mass studies of Lycopodium alkaloids. The mass spectrometry work presented has been very useful to confirm the structures as well as to support the biogenetic relationships between the lycodine-type Lycopodium alkaloids: sauroxine and N-demethylsauroxine.

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.

A prenylated flavanone from Dalea elegans inhibits rhodamine 6 G efflux and reverses fluconazole-resistance in Candida albicans.

In previous studies, 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin, a prenylated flavonoid isolated from Dalea elegans roots, showed activity against multiresistant Staphylococcus aureus and Candida albicans, as well as an uncoupling effect on mitochondria and antioxidant activity. The aim of this study was to evaluate the inhibitory effects of 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin and fluconazole on the efflux of rhodamine 6 G in azole-resistant C. albicans 12-99 that expresses multidrug transporters Cdr1p, Cdr2p, and Mdr1p. The effect of fluconazole and 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin on rhodamine 6 G efflux was assessed in both azole-sensitive and azole-resistant C. albicans. Between 1 and 1000 µM, 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin inhibited rhodamine 6 G efflux only in azole-resistant C. albicans 12-99 in a concentration-dependent manner (IC50 = 119 µM); a competitive effect was observed. It also showed selectivity of action in comparison with other flavanones (6-prenylpinocembrin, isolated from aerial parts of D. elegans, pinocembrin, naringenin, and hesperetin, all at 250 µM). To check the possible implications of the inhibition of azole efflux on cell growth, antifungal assays were conducted. Minimal inhibitory concentration values were 150 µM for 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin and higher than 400 µM for fluconazole. The combination of both compounds at either inhibitory or subinhibitory concentrations was significantly more effective than each compound separately. Minimal inhibitory concentration for fluconazole decreased by more than 400 times in the presence of 100 µM 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin, reversing azole resistance and giving values similar to those of azole-sensitive C. albicans. These data are consistent with a dual action of 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin: direct antifungal effect on azole-resistant C. albicans 12-99 and inhibition of azole transporters, which results in reversion of fluconazole resistance.

Investigation of the in vitro and ex vivo acetylcholinesterase and antioxidant activities of traditionally used Lycopodium species from South America on alkaloid extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: The study was aimed at evaluating medicinal and therapeutic potentials of two Lycopodiaceae species, Lycopodium clavatum (L.) and Lycopodium thyoides (Humb. & Bonpl. ex Willd), both used in South American folk medicine for central nervous system conditions. Alkaloid extracts were evaluated for chemical characterization, acetylcholinesterase and antioxidant activities. MATERIALS AND METHODS: The alkaloid extracts obtained by alkaline extraction were determined for each species by GC/MS examination. The evaluation of the anticholinesterase and the antioxidant activities of the extracts were tested by determining in vitro and ex vivo models. Effects on acetylcholinesterase (AChE) were tested in vitro using rat brain homogenates and ex vivo after a single administration (25, 10 and 1mg/kg i.p.) of the alkaloid extracts in mice. The in vitro antioxidant effects were tested for the 2-deoxyribose degradation, nitric oxide (NO) interaction, 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging activity and total reactive antioxidant potential (TRAP). After an acute administration (25 and 10mg/kg i.p.) of the extracts in middle-aged (12 months) mice, the antioxidant effects were estimated through the thiobarbituric acid reactive substances test (TBARS), and the antioxidant enzymes activities for catalase (CAT) and superoxide dismutase (SOD) were measured. RESULTS: AChE activity was inhibited in vitro by the alkaloid-enriched extracts of both Lycopodium species in a dose and time-dependent manner in rat cortex, striatum and hippocampus. A significant inhibition was also observed in areas of the brain after acute administration of extracts, as well as decreased lipid peroxidation and increased CAT activity in the cortex, hippocampus and cerebellum. A moderate antioxidant activity was observed in vitro for the extracts. Chemically, the main alkaloids found for the two species were lycopodine and acetyldihidrolycopodine. CONCLUSION: This study showed that the biological properties of the folk medicinal plants Lycopodium clavatum and Lycopodium thyoides include AChE inhibitory activity and antioxidant effects, two possible mechanisms of action in Alzheimer's related processes.

Sauroine, an alkaloid from Huperzia saururus with activity in wistar rats in electrophysiological and behavioral assays related to memory retention.

The present study describes the effects of sauroine (1), the main alkaloid obtained from Huperzia saururus, on memory retention and learning. To evaluate this, electrophysiological experiments and behavioral tests (step down) were performed on male Wistar rats. The results showed that 1 improved memory retention in the step-down test, significantly increasing hippocampal plasticity. Thus, 1 seems to be a constituent responsible for the activity claimed in folk medicine for H. saururus in Argentina.

Anthraquinone derivatives from Heterophyllaea pustulata.

From the leaves of Heterophyllaea pustulata two new monomeric anthraquinones, heterophylline (1,6-dihydroxy-7-methoxy-2-methylanthraquinone, 1) and pustuline (2-hydroxy-3-methoxy-7-methylanthraquinone, 2), and one new bianthraquinone, (S)-5,5'-bisoranjidiol [(S)-5,5'-bis(1,6-dihydroxy-2-methylanthraquinone), 3], were isolated. Furthermore, the iridoid glycoside asperuloside and three known flavonoids, quercetin, isoquercitrin, and quercetin-3-O-beta-d-glucosyl-6' '-acetate, were obtained. The structures were determined by analysis of their spectroscopic data and chemical evidence.

Anticoagulant effect and action mechanism of sulphated flavonoids from Flaveria bidentis.

Quercetin 3-acetyl-7,3',4'-trisulphate (ATS) and quercetin 3,7,3',4'-tetrasulphate (QTS) obtained from Flaveria bidentis (Asteraceae) were investigated in vitro for anticoagulant activity. Three different concentrations of each flavonoid were assayed at different incubation times, showing at 1 mM significant prolongation on the activated partial thromboplastin time (APTT), less on the prothrombin time (PT), and no effect on the thrombin time (TT). In order to define the action mechanism of the anticoagulant activity, all coagulation factors were evaluated and no important activity decrease was observed, indicating that another mechanism is involved. Thus, thrombin inhibition mediated by antithrombin III (ATIII) and heparin cofactor II (HCII) activation was investigated in comparison to the physiological activators, heparin and dermatan sulphate (DS), respectively. As a conclusion, no activation on ATIII for neither flavonoids was observed. On the contrary, QTS much more than ATS produced an activation on HCII comparable to the one of DS, indicating that these flavonoids act as agonists of this inhibitor. A plausible explanation of the effects of both flavonoids could be due to the different degree of sulphation of these molecules. According to the results obtained, and taking in account the high solubility of these natural products in aqueous media and the nontoxic nature of this family of compounds, further investigation on the antithrombotic effects of these flavonoids are merited.