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.

Photosensitization Induced by Heterophyllaea pustulata in Goats: Sequential Development of Skin Lesions.

Five adult Saanen goats received a single oral dose of Heterophyllaea pustulata containing 42.25 µg/kg rubiadin (anthraquinone) and 3 adult goats were untreated controls. All goats were exposed to sunlight and sequential ear skin biopsies were collected before treatment and at 32 hours, 3 days, 8 days, and 15 days after treatment. Changes at 32 hours after dosing included epidermal spongiosis, single cell death and acantholysis, an increased BAX/BCL-2 protein ratio, and dermal edema. Lesions at day 3 included epidermal and adnexal necrosis, crust formation, and acanthosis. Acanthosis, hyperkeratosis, and dermal fibrosis and neovascularization were present at day 15. The pro-apoptotic (BAX)/anti-apoptotic (BCL-2) protein ratio increased at 32 hours, whereas epidermal and dermal PCNA immunolabeling increased between days 8 and 15 after treatment. The cutaneous lesions were consistent with sunlight-induced damage, and the occurrence in treated but not control goats indicates photosensitization.

Parietin: an efficient photo-screening pigment in vivo with good photosensitizing and photodynamic antibacterial effects in vitro.

The photophysical, photoinduced pro-oxidant and antibacterial properties in vitro of the natural occurring parietin (PTN; 1,8-dihydroxy-3-methoxy-6-methyl-9,10-anthraquinone) were evaluated. PTN was extracted from the lichen identified as Teloschistes flavicans (Sw.) Norm. (Telochistaceae). Results indicate that in chloroform solution, PTN presents spectroscopic features corresponding to an excited-state intramolecular proton-transfer (ESIPT) state with partial keto-enol tautomerization. In argon-saturated solutions, the singlet excited state is poorly fluorescent (ΦF = 0.03), decaying by efficient intersystem crossing to an excited triplet state 3PTN*, as detected by laser-flash photolysis experiments. In the presence of triplet molecular oxygen, the 3PTN* was fully quenched producing singlet molecular oxygen (1O2) with a quantum yield of 0.69. In addition, in buffer solutions, PTN has the ability to also generate a superoxide radical anion (O2˙-) in a human leukocyte model and its production was enhanced under UVA-Vis irradiation. Finally, the in vitro antibacterial capability of PTN in the dark and under UVA-Vis illumination was compared in microbial cultures of both Gram positive and negative bacteria. As a result, PTN showed promising photo-induced antibacterial activity through the efficient photosensitized generation of both 1O2 and O2˙- species. Thus, we have demonstrated that PTN, an efficient photo-screening pigment in lichens, is also a good photosensitizer in solution with promising applications in antibacterial 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.

Towards effective natural anthraquinones to mediate antimicrobial photodynamic therapy of cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) is an important tropical neglected disease with broad geographical dispersion. The lack of effective drugs has raised an urgent need to improve CL treatment, and antimicrobial photodynamic therapy (APDT) has been investigated as a new strategy to face it with positive outcomes. Natural compounds have emerged as promising photosensitizers (PSs), but their use in vivo remains unexplored. PURPOSE: In this work, we investigated the potential of three natural anthraquinones (AQs) on CL induced by Leishmania amazonensis in BALB/c mice. STUDY DESIGN/METHODS: ANIMALS WERE INFECTED AND RANDOMLY DIVIDED INTO FOUR GROUPS: CG (control, non-treated group), G5ClSor-gL (treated with 5-chlorosoranjidiol and green LED, 520±10 nm), GSor-bL and GBisor-bL (treated with soranjidiol and bisoranjidiol, respectively, exposed to violet-blue LED, 410±10 nm). All AQs were assayed at 10 µM and LEDs delivered a radiant exposure of 45 J/cm2 with an irradiance of 50 mW/cm2. We assessed the parasite burden in real time for three consecutive days. Lesion evolution and pain score were assessed over 3 weeks after a single APDT session. RESULTS: G5ClSor-gL was able to sustain low levels of parasite burden over time. Besides, GSor-bL showed a smaller lesion area than the control group, inhibiting the disease progression. CONCLUSION: Taken together, our data demonstrate that monoAQs are promising compounds for pursuing the best protocol for treating CL and helping to face this serious health problem. Studies involving host-pathogen interaction as well as monoAQ-mediated PDT immune response are also encouraged.

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.

First report of antiviral activity of nordihydroguaiaretic acid against Fort Sherman virus (Orthobunyavirus).

The genus Orthobunyavirus are a group of viruses within arbovirus, with a zoonotic cycle, some of which could lead to human infection. A characteristic of these viruses is their lack of antiviral treatment or vaccine for its prevention. The objective of this work was to study the in vitro antiviral activity of nordihydroguaiaretic acid (NDGA), the most important active compound of Larrea divaricata Cav. (Zigophyllaceae), against Fort Sherman virus (FSV) as a model of Orthobunyavirus genus. At the same time, the effect of NDGA as a lipolytic agent on the cell cycle of this viral model was assessed. The method of reducing plaque forming units on LLC-MK2 cells was used to detect the action of NDGA on CbaAr426 and SFCrEq231 isolates of FSV. NDGA did not show virucidal effect, but it had antiviral activity with a similar inhibition in both isolates, which was dose dependent. It was established that the NDGA has a better inhibition 1-h post-internalization (p.i.), showing a different behavior in each isolate, which was dependent upon the time p.i. Since virus multiplication is dependent on host cell lipid metabolism, the antiviral effect of NDGA has been previously related to its ability to disturb the lipid metabolism, probably by interfering with the 5-lipoxigenase (5-LOX) and the sterol regulatory element-binding proteins (SREBP) pathway. We determined by using caffeic acid, a 5-LOX inhibitor, that the inhibition of this enzyme negatively affected the FSV replication; and by means of resveratrol, a SREBP1 inhibitor, it was showed that the negative regulation of this pathway only had action on the SFCrEq231 reduction. In addition, it was proved that the NDGA acts intracellularly, since it showed the ability to incorporate into LLC-MK2 cells. The information provided in this work converts the NDGA into a compound with antiviral activity in vitro against FSV (Orthobunyavirus), which can be subjected to structural modifications in the future to improve the activity.

Natural photosensitizers in photodynamic therapy: In vitro activity against monolayers and spheroids of human colorectal adenocarcinoma SW480 cells.

Photodynamic Therapy (PDT), is a treatment option for cancer.It involves the photochemical interaction of light, photosensitizer (PS) and molecular oxygen to produce radical species as well as singlet oxygen which induce cell death. Anthraquinones (AQs) have been extensively studied with respect to their UV/Vis absorption characteristics and their photosensitizing properties in photodynamic reactions. We study the photoactivity of different natural AQs (Parietin, Soranjidiol and Rubiadin) in treating monolayers and multicellular tumor spheroids (MCTSs). Rubiadin and soranjidiol were isolated and purified from the stem and leaves of Heterophyllae pustulata, and PTN was from the liquen Teloschistes flavicans by using repeated combination of several chromatographic techniques. Monolayer and spheroids of human colorectal adenocarcinoma SW480 cells were incubated with different concentrations of the AQs and then irradiated at room temperature. 24 h post-PDT cell viability, nuclear morphology and type of cell death were analyzed. We observed that Soranjidiol and Rubiadin showed no significant difference in the photosensitizing ability on monoculture of colon cancer cells (LD80 at 50 µM and 10 J/cm2, for both AQs). Nevertheless, for Parietin (PTN) LD80 was achieved at (20 µM using the same light dose (10 J/cm2). The death mechanism induced post-PDT was necrosis by use of Soranjidol and Rubiadin and apoptosis by use of PTN. Furthermore, in MCTSs of 300 and 900 µm, the treatment PTN- PDT produces the greatest cytotoxic effect. The three AQs analyzed could be promising chemotherapeutic candidates as anticancer PDT agents.

Natural anthraquinones as novel photosentizers for antiparasitic photodynamic inactivation.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-borne disease caused by obligate protist parasites from the genus Leishmania. The potential toxicity as well as the increased resistance of standard treatments has encouraged the development of new therapeutical strategies. Photodynamic inactivation (PDI) combines the use of a photosensitizer and light to generate reactive oxygen species and kill cells, including microorganisms. Vegetal kingdom constitutes an important source of bioactive compounds that deserve to be investigated in the search of naturally occurring drugs with leishmanicidal activity. PURPOSE: The purpose of this study was to test the antiparasitic activity of PDI (ApPDI) of five natural anthraquinones (AQs) obtained from Heterophyllaea lycioides (Rusby) Sandwith (Rubiacae). To support our results, effect of AQ mediated-PDI on parasite´s morphology and AQ uptake were studied. Cytotoxicity on fibroblasts was also evaluated. STUDY DESIGN/METHODS: Two monomers, soranjidiol (Sor) and 5-chlorosoranjidiol (5-ClSor) plus three bi-anthraquinones (bi-AQs), bisoranjidiol (Bisor), 7-chlorobisoranjidiol (7-ClBisor) and Lycionine (Lyc) were selected for this study. Recombinant L. amazonensis promastigote strain expressing luciferase was subjected to AQs and LED treatment. Following irradiation with variable light parameters, cell viability was quantified by bioluminescence. Alteration on parasite's morphology was analyzed by scanning electron microscopy (SEM). In addition, we verified the AQ uptake in Leishmania cells by fluorescence and their toxicity on fibroblasts by using MTT assay. RESULTS: Bisor, Sor and 5-ClSor exhibited photodynamic effect on L. amazonensis. SEM showed that promastigotes treated with Bisor-mediated PDI exhibited a significant alteration in shape and size. Sor and 5-ClSor presented higher uptake levels than bi-AQs (Bisor, Lyc and 7-ClBisor). Finally, Sor and Bisor presented the lowest toxic activity against fibroblasts. CONCLUSION: Taking together, our results indicate that Sor presents the highest specificity towards Leishmania cells with no toxicity on fibroblasts.

Phototoxic effects of Heterophyllaea pustulata (Rubiaceae).

Heterophyllaea pustulata (Rubiaceae), a South American genus, is a phototoxic shrub that grows in the Andean mountain range of the northwest of Argentina, popularly known as "cegadera". Animals that ingest the aerial parts of this plant suffer a typical primary photosensitization reaction, clinically revealed by dermatitis and blindness in severe cases. Anthraquinone derivatives (AQs), the main metabolites of this species, are characterized as Type I and/or Type II photosensitizers according to their physicochemical properties. The natural toxicity conditions were reproduced in vivo assays by oral administration of soranjidiol and rubiadin, the main components of the aerial parts. By HPLC analysis, the presence of these AQs was determined in serum and quantified in the skin of experimental animals.

Photosensitizing anthraquinones from Heterophyllaea lycioides (Rubiaceae).

Seven anthraquinones were isolated from aerial parts of Heterophyllaea lycioides (Rusby) Sandwith (Rubiaceae), including three derivatives that have not been described before: a hetero-bianthraquinone identified as (R)-2-hydroxymethyl-2'methyl-1,1',6,6'-tetrahydroxy-5,5' bianthraquinone (lycionine), and two mono-chlorinated derivatives related to soranjidiol. One of them is a homo-bianthraquinone: (R)-7-chloro-2,2'-dimethyl-1,1',6,6'-tetrahydroxy-5,5' bianthraquinone (7-chlorobisoranjidiol), whereas the second halogenated derivative corresponds to a monomeric structure: 5-chloro-1,6-dihydroxy-2-methyl anthraquinone (5-chlorosoranjidiol). The four known compounds were already isolated from another species of this genus, H. pustulata, and they were identified as 5,5'-bisoranjidiol, soranjidiol, pustuline and heterophylline. Structural elucidation was performed by means of an extensive spectroscopic analysis, including 1D and 2D NMR data as well as by HRMS analysis. Chemical structures of 7-chlorobisoranjidiol and 5-chlorosoranjidiol were confirmed by their synthesis from 5,5'-bisoranjidiol and soranjidiol, respectively. Type I photosensitizing properties (superoxide anion radical generation, O2-) were assessed by using the nitroblue tetrazolium assay. When lycionine and chlorinated derivatives were irradiated, they enhanced the O2- production with respect to the control; 7-chlorobisoranjidiol stood out by generating an increase of 20%, whereas the other anthraquinones only produced a slight increase of 7%.

On the mechanism of Candida tropicalis biofilm reduction by the combined action of naturally-occurring anthraquinones and blue light.

The photoprocesses involved in the photo-induced Candida tropicalis biofilm reduction by two natural anthraquinones (AQs), rubiadin (1) and rubiadin-1-methyl ether (2), were examined. Production of singlet oxygen (1O2) and of superoxide radical anion (O2•-) was studied. Although it was not possible to detect the triplet state absorption of any AQs in biofilms, observation of 1O2 phosphorescence incubated with deuterated Phosphate Buffer Solution, indicated that this species is actually formed in biofilms. 2 was accumulated in the biofilm to a greater extent than 1 and produced measurable amounts of O2•- after 3h incubation in biofilms. The effect of reactive oxygen species scavengers on the photo-induced biofilm reduction showed that Tiron (a specific O2•- scavenger) is most effective than sodium azide (a specific 1O2 quencher). This suggests that O2•- formed by electron transfer quenching of the AQs excited states, is the main photosensitizing mechanism involved in the photo-induced antibiofilm activity, whereas 1O2 participation seems of lesser importance.

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation.

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3',4'-tetrasulphate (QTS) and quercetin 3-acetyl-7,3',4'-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 microM, final concentration), and the platelet aggregation was induced by using adenosine 5'-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 microM) and Qc (250 microM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 microM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA2) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B2 (TxB2) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA2, as judged by TxB2 formation, in stimulated WP and a marked interference on the TxA2 receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.

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.

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.