Anti-herpes Activity Research Articles

Potential anti-herpes and cytotoxic action of novel semisynthetic digitoxigenin-derivatives

In recent years, new therapeutic possibilities were proposed for cardiac glycosides traditionally used to treat heart diseases, such as anticancer and antiviral activities. In this sense, this work aimed to synthesize the readily accessible 3β-azido-3-deoxydigitoxigenin (5) from digitoxigenin (1). Two new series of compounds were obtained from derivative (5): (i) O-glycosyl trizols through click chemistry with propargyl glycosides; and (ii) compounds substituted in the alpha carbonyl position with different residues linked via an amino-group. All obtained derivatives have their chemical structures confirmed, and their anti-herpes (against HSV-types 1 and 2 replication) and cytotoxic (against PC3, A549, HCT-8 and LNCaP cell lines) activities evaluated. Compounds 10 and 11 exhibited the most promising results against HSV-1 (KOS and 29-R strains) and HSV-2 (333 strain) replication with SI values > 1000. Both compounds were also the most cytotoxic for the human cancer cell lines tested with IC50 values similar to those of paclitaxel. They also presented reduced toxicity toward non-cancerous cell lines (MRC-5 and HGF cells). Promising compounds were tested in regard to their ability to inhibit Na+/K+-ATPase. The inhibition rate correlates suitably with the bioactivity demonstrated by those both compounds against the different human cancer cells tested as well as against HSV replication. Moreover, the results showed that specific chemical features of compound 10 and 11 influenced the bioactivities tested. In summary, it was possible to obtain novel digitoxigenin-derivatives with remarkable cytotoxic and anti-herpes activities as well as low toxicity and high selectivity. In this way, they could be considered potential molecules for the development of new drugs.

Clerodane Diterpenes from the Marine Sponge Raspailia bouryesnaultae Collected in South Brazil.

The marine sponge Raspailia bouryesnaultae, collected in South Brazil, was selected for detailed investigation considering the results of a screening that pointed to an in vitro antiproliferative effect against non-small cells of human lung cancer (A549) and anti-herpes activity against Herpes Simplex virus type 1 (KOS and 29R strains) of ethanolic extracts. The fractionation and chemical investigation of the sponge’s hexanic fraction led to the isolation and structural elucidation of six clerodane diterpenes. The main component was identified as the already-reported raspailol (1), isolated from a sponge of the same genus collected in New Zealand. The structure of a new diterpene (2) with a rearranged skeleton was established by high-resolution mass spectrometry (HRMS) and 1D and 2D Nuclear magnetic resonance spectroscopy (NMR) experiments, and named here as raspadiene. Furthermore, four diterpenes were elucidated as isomers of clerodane diterpenes previously obtained from plants, namely kerlinic acid (3), kerlinic acid methyl ester (4), annonene (5), and 6-hydroxyannonene (6). They differ in their stereochemistry, since these diterpenes are characterized by a trans ring fusion at the decalin moiety and the relative configuration of the two methyl groups at C-8 and C-9 in a cis relationship (type trans/cis). The Raspailia diterpenes have a cis ring fusion at the decalin moiety, and the two methyl groups at C-8 and C-9 are in a trans relationship (type cis/trans). The isolated compounds were evaluated for their potential antiproliferative effects on human cancer cell line A549, and it was observed that the diterpenes bearing a hydroxyl group at C-6 exhibited moderate cytotoxic activity, with 50% inhibitory concentration (IC50) values lower than 25 μM. The evaluation of the potential anti-herpes activity against Herpes Simplex Virus type 1 (HSV-1, KOS and 29R strains) showed that the more promising results were observed for the new compound 2, since it inhibited HSV-1 (KOS and 29R strains) replication by 83% and 74%, respectively.

Importance of lipophilicity for potent anti-herpes simplex virus-1 activity of α-hydroxytropolones.

We previously reported that troponoid compounds profoundly inhibit replication of herpes simplex virus (HSV)-1 and HSV-2 in cell culture, including acyclovir-resistant mutants. Synthesis of 26 alpha-hydroxylated tropolones (αHTs) led to a preliminary structure-activity relationship highlighting the potency of bi-phenyl side chains. Here, we explore the structure-activity relationship in more detail, with a focus on various biaryl and other lipophilic molecules. Along with our prior structure-function analysis, we present a refined structure-activity relationship that reveals the importance of the lipophilicity and nature of the side chain for potent anti-HSV-1 activity in cells. We expect this new information will help guide future optimization of αHTs as HSV antivirals.

Anti-herpes activity of polysaccharide fractions from Stevia rebaudiana leaves

The antiviral potential of natural polysaccharide compounds has been demonstrated, especially against enveloped viruses and members of the Herpesviridae family. Two polysaccharide fractions obtained from Stevia rebaudiana (Bertoni) leaves, that were active against Herpes simplex virus type 1 (HSV-1) were studied to investigate their mode of action. Both polysaccharides - SFW (crude faction) and SSFK (homogeneous alkaline fraction) - exerted antiviral effects on the initial stages of HSV-1 infection by inhibiting viral adsorption and penetration. When added after virus internalization, both fractions decreased plaque size. The effect of the fractions was confirmed by investigating viral glycoprotein expression. Based on the mode of action of the polysaccharides demonstrated in the present work and on their selectivity index, the polysaccharides obtained from S. rebaudiana could be an alternative treatment of infections caused by HSV-1.

Berries grown in Brazil: anthocyanin profiles and biological properties.

Phytochemical profiles of two Brazilian native fruits-pitanga (red and purple) and araçá (yellow and red)-as well as strawberry cultivars Albion, Aromas and Camarosa, blackberry cultivar Tupy and blueberry cultivar Bluegen cultivated in Brazil were characterized for total phenolic content and total anthocyanin content by liquid chromatography coupled to a photodiode array and a quadrupole time-of-flight mass spectrometer. Radical scavenging, antiherpes and cytotoxic activities of these berry extracts were also evaluated. Blueberry presented the highest total anthocyanin content (1202 mg cyanidin-O-glucoside equivalents kg-1 fresh fruit), while strawberry cultivar Aromas presented the highest total phenolic content (13 550 mg gallic acid equivalents kg-1 fresh fruit). Liquid chromatographic-mass spectrometric analysis resulted in the identification of 21 anthocyanins. To the best of our knowledge this is the first report of cyanidin-O-glucoside in yellow and red Araçá fruit and the first time eight anthocyanins have been reported in pitanga fruits. DPPH and ABTS assays showed that blueberry cultivar Bluegen, blackberry cultivar Tupy and pitanga (red and purple) showed the most promising antiradical activities, respectively. No relevant cytotoxicity against three cancer cell lines or antiherpes activity was detected under the experimental conditions tested. Total anthocyanin content of all fruits had a strong positive correlation with their free radical scavenging activity, suggesting anthocyanins contribute to the antioxidant potential of these fruits. © 2018 Society of Chemical Industry.

In vitro schistosomicidal and antiviral activities of Arctium lappa L. (Asteraceae) against Schistosoma mansoni and Herpes simplex virus-1

Schistosomiasis and herpes diseases represent serious issues to the healthcare systems, infecting a large number of people worldwide, mainly in developing countries. Arctium lappa L. (Asteraceae), known as “bardana” and “burdock”, is a medicinal plant popularly used for several purposes, including as antiseptic. In this study, we evaluated the in vitro schistosomicidal and antiherpes activities of the crude extract of A. lappa, which have not yet been described. Fruits of A. lappa L. were extracted by maceration with ethanol: H2O (96:4 v/v) in order to obtain the hydroalcoholic extract of A. lappa (AL). In vitro schistosomicidal assays were assessed against adult worms of Schistosoma mansoni, while the in vitro antiviral activity of AL was evaluated on replication of Herpes simplex virus type-1 (HSV-1). Cell viability was measured by MTT assay, using Vero cells and chemical composition of AL was determined by qualitative UPLC-ESI-QTOF-MS analysis. UPLC-ESI-QTOF-MS analysis of AL revealed the presence of dibenzylbutyrolactone lignans, such as arctiin and arctigenin. Results showed that AL was not cytotoxic to Vero cells even when tested at 400μg/mL. qPCR results indicated a significant viral load decreased for all tested concentrations of AL (400, 50, and 3.125μg/mL), which showed similar antiviral effect to acyclovir (50μg/mL) when tested at 400μg/mL. Also, AL (400, 200, and 100μg/mL) caused 100% mortality and significantly reduction on motor activity of all adult worms of S. mansoni. Confocal laser scanning microscopy showed tegumental morphological alterations and changes on the numbers of tubercles of S. mansoni worms in a dose-dependent manner after treatment with AL. This report provides the first evidence for the in vitro schistosomicidal and antiherpes activities of AL, opening the route to further schistosomicidal and antiviral studies with AL and their compounds, especially lignans.

Дослідження протигерпетичної активності похідних амінопропанолу-2.

Introduction. Chemotherapy and chemoprophylaxis are some of the main and often the only possible ways to effective control of viral infections. Therefore, the study of antiviral properties of new substances with the known chemical structure is one of the main ways to create new antiviral agents.The aim of the study – to research the antiherpes activity of new aminopropanol-2 derivatives against the herpes simplex virus (HSV) antigenic type 1, strain VC.Research Methods. Antiherpes activity was determined in 8 aminopropanol-2 derivatives: norbornyl containing substance (compound No. 51), substance with cyclic substituents in alkoxi group (compound No. 48), substances with alicyclic substituents in alkoxi group (compounds No. 46, 47, 49, 50, 52 and 53). Evaluation of antiherpes activity of the studied compounds was performed in vitro on cell culture VNK (growing culture of hamster kidney). Ability to reduce of virus infectious titer and chemotherapeutic index (HTI) of the studied compounds was determined.Results and Discussion. It is established that the compound No. 53 inhibits HSV-I reproduction in 2 lg ID50 at a concentration of 1.56 µg/ml. HTI of compound No. 53 is equal to 64, which describes it as an effective inhibitor of HSV-I reproduction. Some antiherpes action in compounds No. 46, 47 and 51 was identified also, their HTI were 8, 4 and 4 respectively. Substances No. 48, 49, 50 and 52 do not show the antiherpes action.Conclusions. Among all tested aminopropanol-2 derivatives the compound No. 53 with clear antiherpes properties was determined. Compound No. 53 belongs to the substances with alicyclic substituents in alkoxi group and has such chemical formula: 1-(2-methyl-3-butinox)-3-(2.2.6.6-tetramethyl piperidine)-2-propanol hydrochloride. Compound No. 53 as alicyclic substituent in alkoxy group contains 2-methyl-3-butene, and amine moiety of this substance contains the radical 2.2.6.6 – tetramethylpiperidine. The obtained results will be useful in establishing the natural relationships "structure-activity", also it can be used to create active compounds with certain characteristics.

In vitro antiherpes effect of C-glycosyl flavonoid enriched fraction of Cecropia glaziovii encapsulated in PLGA nanoparticles

In this work is reported a novel and promising approach for the preparation of C-glycosylflavonoid enriched fraction of Cecropia glaziovii (EFF-Cg) loaded PLGA nanoparticles (NP) with antiherpes properties. The purpose of this study was to evaluate and to compare the effect of two nonionic surfactants (poloxamer 188 (PLU) and polyvinyl alcohol (PVA)), and also an emulsion stabilized by solid particles of cellulose nanocrystal (CNC) in place of surfactants. The characterization of these nanoparticles was in terms of size, polydispersity index, zeta potential, morphology, thermogravimetric analysis (TGA), loading capacity and percent yield. Since TGA analysis revealed thermo stability especially for NP-PLU, this formulation was selected for the evaluation of drug release profile, cytotoxicity and antiherpes activity. The drug delivery profile demonstrated a sustained release through the polymer structure and a significant reduction of the polymer molecular weight at 21-day period. The cytotoxicity of these nanoparticles was determined on Vero cells, and the selected formulation did not exhibit cytotoxicity even at the highest tested concentration. The results demonstrated a potential antiherpetic effect of the EFF-Cg loaded NP at 48h of testing. In summary, EFF-Cg loaded NP exhibited a promising system for the effective drug delivery in the treatment of herpes infections.

Novel agents and strategies to treat herpes simplex virus infections

The quiet pandemic of herpes simplex virus (HSV) infection has plagued humanity since ancient times, causing mucocutaneous infection, such as herpes labialis and herpes genitalis. Disease symptoms often interfere with everyday activities and occasionally HSV infections are the cause of life-threatening or sight-impairing disease, especially in neonates and the immunocompromised patient population. After primary or initial infection the virus persists for life in a latent form in neurons of the host, periodically reactivating and often resulting in significant psychosocial distress for the patient. Currently, no cure is available. In the mid-1950s the first antiviral, idoxuridine, was developed for topical treatment of herpes disease and, in 1978, vidarabine was licensed for systemic use to treat HSV encephalitis. Acyclovir (Zovirax), a potent, specific and tolerable nucleosidic inhibitor of the herpes DNA polymerase, was a milestone in the development of antiviral drugs in the late 1970s. In the mid-1990s, when acyclovir became a generic drug, valacyclovir (Valtrex) and famciclovir (Famvir), prodrugs of the gold standard and penciclovir (Denavir), Vectavir), a close analogue, were launched. Though numerous approaches and strategies were tested and considerable effort was expended in the search of the next generation of an antiherpetic therapy, it proved difficult to outperform acyclovir. Notable in this regard was the award of a Nobel Prize in 1988 for the elucidation of mechanistic principles which resulted in the development of new drugs such as acyclovir. Vaccines, interleukins, interferons, therapeutic proteins, antibodies, immunomodulators and small-molecule drugs with specific or nonspecific modes of action lacked either efficacy or the required safety profile to replace the nucleosidic drugs acyclovir, valacyclovir, penciclovir and famciclovir as the first choice of treatment. Recently though, new inhibitors of the HSV helicase-primase with potent in vitro antiherpes activity, novel mechanisms of action, low resistance rates and superior efficacy against HSV in animal models have been discovered. This review summarises the current therapeutic options, discusses the potential of preclinical or investigational drugs and provides an up-to-date interpretation of the challenge to establish novel treatments for herpes simplex disease.

Chemoenzymatic Synthesis of Modified 2′-Deoxy-2′-fluoro-β-d-arabinofuranosyl Benzimidazoles and Evaluation of Their Activity Against Herpes Simplex Virus Type 1

1-(2′-Deoxy-2′-fluoro-β- d -arabinofuranosyl)benzimidazoles containing 4,6-difluoro-, 4,5,6-trifluoro-, 5-fluoro-6-methoxy-, and 5-methoxy-4,6-difluorobenzimidazole fragments were synthesized by using purine nucleoside phosphorylase-catalyzed chemoenzymatic approach. As expected, enzymatic synthesis of nucleosides proceeds in lower yields of target compounds in comparison with the synthesis of ribo- and 2′-deoxyribobenzimidazoles (40–55% vs 60–90%). The compounds obtained were tested against the herpes simplex virus type 1, by using the Vero E6 cells. 5-Methoxy-4,6-difluoro-1-β- d -(2′-deoxy-2′-fluoroarabinofuranosyl)benzimidazole did not show any antiviral activity, when used in nontoxic concentration. All other nucleosides proved to exhibit a selective antiherpes activity. In contrast, it was shown that benzimidazole-β- d -arabinofuranosides of both di- and trisubstituted derivatives, having substituents in positions 4–6 of the benzene ring, as well as unsubstituted compounds, cannot be synthesized by enzymatic transglycosylation. 1-(β- d -Arabinofuranosyl)benzimidazole was obtained through glycosylation of N -trimethylsilylbenzimidazole with 1-chloro-2,3,5- O -methoxymethyl- d -arabinose. The behavior of this compound, as inhibitor of purine nucleoside phosphorylase (PNP) E. сoli , was investigated . 1-(β- d -Arabinofuranosyl)benzimidazole was found to belong to a mixed type of inhibitors of PNP. This fact explains why all attempts to perform enzymatic arabinosylation of 4,6-di-, 5,6-di-, and 4,5,6-trisubstituted benzimidazoles failed.

Pentyl Gallate Nanoemulsions as Potential Topical Treatment of Herpes Labialis

Previous studies have demonstrated the antiherpes activity of pentyl gallate (PG), suggesting that it could be a promising candidate for the topical treatment of human herpes labialis. PG low aqueous solubility represents a major drawback to its incorporation in topical dosage forms. Hence, the feasibility of incorporating PG into nanoemulsions, the ability to penetrate the skin, to inhibit herpes simplex virus (HSV)-1 replication, and to cause dermal sensitization or toxicity were evaluated. Oil/water nanoemulsions containing 0.5% PG were prepared by spontaneous emulsification. The in vitro PG distribution into porcine ear skin after topical application of nanoemulsions was assessed, and the in vitro antiviral activity against HSV-1 replication was evaluated. Acute dermal toxicity and risk of dermal sensitization were evaluated in rat model. Nanoemulsions presented nanometric particle size (from 124.8 to 143.7 nm), high zeta potential (from −50.1 to −66.1 mV), loading efficiency above 99%, and adequate stability during 12 months. All formulations presented anti-HSV-1 activity. PG was able to reach deeper into the dermis more efficiently from the nanoemulsion F4. This formulation as well as PG were considered safe for topical use. Nanoemulsions seem to be a safe and effective approach for topically delivering PG in the treatment of human herpes labialis infection.

In Vitro Evaluation of Mucosa Permeation/Retention and Antiherpes Activity of Genistein from Cationic Nanoemulsions.

In this report, we described the genistein distribution on excised porcine esophageal mucosa from cationic nanoemulsions, as well as the anti-HSV-1 activity against a viral strain resistant to acyclovir. Genistein-loaded cationic nanoemulsions were prepared by spontaneous emulsification. This procedure yielded monodisperse nanoemulsions exhibiting a mean droplet size of approximately 200-300 nm. Hydroxyethyl cellulose (HEC) was added at the end of the manufacturing process as a thickening agent (at 3%). Such formulations exhibit a non-Newtonian pseudoplastic behavior. The addition of HEC significantly reduces the genistein flux through excised porcine mucosa specimens as compared with values elicited by nanoemulsions before thickening. Furthermore, a significant increase of genistein retention in mucosa was observed as compared to the genistein propylene glycol solution, as illustrated by confocal fluorescence microscopy images. Formulations exhibited antiherpetic activity in vitro against HSV-1 (strain 29R). Taken together, these results suggest that these formulations have promising potential to be used topically for herpes infections.

Anti-herpes simplex virus activities of monogalactosyl diglyceride and digalactosyl diglyceride from Clinacanthus nutans, a traditional Thai herbal medicine

ObjectiveTo evaluate the monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) from Clinacanthus nutans (C. nutans) for their in vitro antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by plaque reduction assay. MethodsMGDG and DGDG were extracted with chloroform from C. nutans leaves. MGDG and DGDG were separated from chloroform crude extract using column chromatography, characterized by thin layer chromatography and quantified by high performance liquid chromatography. The anti HSV-1 and 2 activity against pre-treatment and post-treatment of the compounds was evaluated using plaque reduction assay. The cytotoxicity of the extract and the compounds on Vero cells were performed by MTT assay. ResultsMGDG and DGDG obtained by column chromatography showed identical profiles as standard MGDG and standard DGDG using thin layer chromatography and high performance liquid chromatography. MGDG and DGDG from C. nutans showed 100% inhibition of HSV-1 replication at the post step of infection at noncytotoxic concentration with IC50 values of 36.00 and 40.00 μg/mL, and HSV-2 at 41.00 and 43.20 μg/mL, respectively. Moreover, MGDG and DGDG from C. nutans were demonstrated to have anti-herpes simplex activity at the same level as standard synthetic compounds. In contrast, pre-treatment of Vero cells with MGDG and DGDG before HSV-1 and HSV-2 infection did not show inhibitory effect against these viruses. MGDG and DGDG exhibited antiviral activity against HSV-1 with selectivity index of 26.00 and 23.00 and HSV-2 of 23.30 and 21.30. ConclusionsMGDG and DGDG from C. nutans, a traditional Thai herbal medicine illustrated inhibitory activity against HSV-1 and HSV-2, probably by inhibiting the late stage of multiplication, suggesting their promising use as anti-HSV agents.

Chemoenzymatic Synthesis and Antiherpes Activity of 5-Substituted 4,6-Difluorobenzimidazoles Ribo- and 2′-Deoxyribonucleosides

A series of 5,6-disubstituted benzimidazole nucleosides, obtained earlier, did not show any significant antiviral activity at relatively low cytotoxicity in vitro. In the course of our research we have succeeded in introducing an additional fluorine atom into the benzimidazole ring system. A new series of 4,6-difluorobenzimidazoles, bearing various groups (fluoro-, methoxy-, ethoxy-, morpholino-, and pyrrolidino-) in the 5-position of the benzene ring, have been synthesized. All these compounds proved to be substrates for recombinant E. coli purine nucleoside phosphorylase (PNP) in the transglycosylation reaction. Effective methods for the synthesis of ribo- and 2′-deoxyribonucleosides with high yields (60–90%) have been described, and the formation of regioisomeric N 3-nucleosides of benzimidazoles have been detected. The biological activity of the nucleosides obtained against herpes simplex virus type 1 (HSV-1) has been elucidated. All compounds show a low cytotoxicity in the cell culture Vero E6. 4,5,6-Trifluoro-1-(β- d -ribofuranosyl)benzimidazole and 5-methoxy-4,6-difluoro-1-(β- d -2′-deoxyribofuranosyl)benzimidazole proved to inhibit completely the progression of the virus cytopathic effect (CPE) at a multiplicity of infection (MOI) of 0.01 PFU/cell.

Synthesis and Antimicrobial Activity of N-5-Diaryl-7-Methyl-3-OXO-2,3-Dihydro-5H-[1,3]Thiazolo[3,2-a]Pyrimidine-6-Carboxamide Hydrochlorides

One of the most important problems in pharmaceutical chemistry is the design of highly effective and nontoxic drugs. The solution involves targeted synthesis and discovery of new biologically active compounds. In this respect, tetrahydropyrimidine-2(1H)-thiones, their derivatives, and condensed heterocycles based on them are some of the most promising classes of chemical compounds [1 – 7]. 5H-Thiazolo[3,2-a]pyrimidine derivatives are known to possess anti-inflammatory, antiparkinson, and antiherpes activity [8]. The synthesis of 5H-thiazolo[3,2-a]pyrimidines via the reaction of tetrahydropyrimidine-2(1H)-thione derivatives with -halocarboxylate esters was reported [9]. We investigated the reaction of N,6-diaryl-4-methyl-2thioxo-1,2,3,6-tetrahydropyrimidine-5-carboxamides with ethyl chloroacetate in order to prepare new heterocyclic compounds and study their antimicrobial activity. The reaction occurred upon storing the reagents at 120°C for 15-20 without a solvent and formed N,5-diaryl-7-methyl-3-oxo2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxamides (I-IX) (Scheme 1). Apparently, the first step involved nucleophilic substitution of the Cl atom in ethyl chloroacetate as a result of attack by the mercapto S atom formed via isomerization of the starting compound with subsequent intramolecular cyclization of the intermediate (i) (Scheme 2). Compounds I-IX were yellow crystalline compounds that were soluble in DMF and DMSO and with heating in HOAc and EtOH and insoluble in H 2 O, toluene, and benzene. IR spectra of the thiazolopyrimidine hydrochlorides had a characteristic high-frequency absorption band in the range 1752 – 1768 cm –1 that belonged to thiazoline carbonyl stretching vibrations and bands due to amide stretching vibrations (1648 – 1688) and a C=C bond (1600 – 1624). The high-frequency shift of the lactam carbonyl absorption band was apparently explained by protonation of the neighboring N atom. PMR spectra of I-IX showed resonances for aromatic protons and groups bonded to the aromatic ring in addition to resonances for 7-CH 3 protons at 1.80 – 2.28 ppm, a singlet for H-5 at 5.97 – 6.20, a singlet for the amide NH proton at 9.45 – 9.86, and doublets for methylene protons in the range 4.15 – 4.30. 13 C NMR spectra of IX included chemical shifts of C atoms that confirmed the proposed structure (see Experimental). Thus, the observed reaction allowed 5H-[1,3]thiazolo[3,2-a]pyrimidine hydrochlorides with the N-arylamide Pharmaceutical Chemistry Journal, Vol. 49, No. 8, November, 2015 (Russian Original Vol. 49, No. 8, August, 2015)

Anti-herpes simplex virus activity of polysaccharides from Eucheuma gelatinae

Acyclovir is a commonly-used drug for treating herpes simplex virus (HSV) infections, but with its wide clinical application, more and more resistant strains have been found. Therefore, seeking a drug that can act against acyclovir-resistant virus has become an important goal of drug screening and development. In this study, plaque reduction assay, real-time PCR, Western blot, and immunofluorescence technique were used to investigate the antiviral effect of Eucheuma gelatinae polysaccharide (EGP) on HSV and to preliminarily clarify the in vitro anti-HSV mechanism of EGP. EGP was found to significantly inhibit HSV infection in vitro and displayed a good inhibitory effect on acyclovir-resistant strains. More detailed experiments have shown that EGP prevented early HSV-1 infection through directly inactivating HSV-1 particles and impairing virus attachment, but without effect on viral penetration. EGP also inhibited the RNA synthesis of HSV-1 early gene and late gene as well as viral DNA replication; no effect on immediate-early gene synthesis was observed. Besides, through immunofluorescence and western blot, we found that EGP significantly affected the protein synthesis of HSV-1. Taken together, these results demonstrate that EGP exerts its anti-HSV activity mainly through impeding early HSV-1 infection and inhibiting viral RNA and DNA syntheses. The weak cytotoxicity, strong viral inactivation as well as attachment inhibition activity enable EGP to be a virucide candidate for HSV therapy, especially for drug-resistant strains.

Antiherpes Activity and Skin/Mucosa Distribution of Flavonoids from Achyrocline satureioides Extract Incorporated into Topical Nanoemulsions.

This study investigated the inhibitory effects of Achyrocline satureioides extract (ASE) incorporated into a topical nanoemulsion on Herpes Simplex Virus type 1 (HSV-1/KOS strain) replication, as well as the distribution of the main ASE flavonoids (quercetin, luteolin, and 3-O-methylquercetin) in porcine skin and mucosa. The ASE-loaded nanoemulsion showed more pronounced effects against HSV-1 replication when compared to the ASE or pure quercetin, as determined by the viral plaque number reduction assay. All flavonoids were detected in the skin epidermis (2.2 µg/cm2) and the mucosa upper layers (3.0 µg/cm2) from ASE-loaded nanoemulsion until 8 h after topical application. A higher amount of flavonoids was detected when these tissues were impaired, especially in deeper mucosa layers (up to 7-fold). Flavonoids were detected in the receptor fluid only when the mucosa was injured. Such results were supported by confocal microscopy images. Overall, these findings suggest that the tested ASE-loaded nanoemulsion has potential to be used topically for herpes infections.

Flavonoid glycosides profiling in dwarf elder fruits (Sambucus ebulus L.) and evaluation of their antioxidant and anti-herpes simplex activities

Dwarf elder (Sambucus ebulus L.) is a popular medicinal plant, used for centuries in the folk medicine of the Balkan Peninsula. S. ebulus preparations have shown anti-inflammatory, anti-neoplastic and antimicrobial properties, besides abundant wound healing, antioxidant and anti-ulcerogenic activities. We developed a scheme for isolation of individual compounds utilizing different chromatographic techniques, while the structure elucidation was performed by means of 1D and 2D NMR. Five flavonoid glycosides, e.g. quercetin-3-O-laminaribioside [1], isorhamnetin-3-O-laminaribioside [2], quercetin-3-O-rutinoside [3], isorhamnetin-3-O-rutinoside [4], isorhamnetin-3-O-glucoside [5], were identified accordingly. Compounds 1 and 2 are reported for the first time in the genus Sambucus. Several triterpenes – ursolic, oleanolic and maslinic acid – were tentatively identified by GC–MS. The evaluation of anti-herpes simplex virus type 1 and antioxidant (in ORACFL and HORACFL) properties suggests that the dwarf elder fruits might serve as a powerful source of valuable molecules for various purposes.

Chemical characterization and antiherpes activity of sulfated polysaccharides from Lithothamnion muelleri

We report herein the chemical characterization and antiherpes activity of polysaccharides from the red alga Lithothamnion muelleri (Hapalidiaceae). The polysaccharide-rich fractions B1 and B2 were obtained by extraction with Na2CO3 and were purified by size exclusion chromatography to afford Fra-B1 and Fra-B2. The polysaccharides were characterized by FT-IR and chemical analysis (total contents of carbohydrates, proteins, sulfate and uronic acid), whereas their average molecular weights were estimated by high performance gel permeation chromatography. The monosaccharide analysis detected galactose, glucose, xylose, mannose, rhamnose and arabinose in the four polysaccharide samples. Antiherpetic in vitro assays showed that B1 and B2 inhibited Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) when added simultaneously to viral infection affording selectivity indices (SI=CC50/EC50) higher than 20. Investigation of the mechanism of action indicated that B1 and B2 act on the initial steps of HSV replication, mainly inhibiting viral adsorption but also viral penetration into the cells.

HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC

Antimicrobial, antimycobacterial and antiviral activities of crude extracts, fractions and subfractions of Tabernaemontana catharinensis were evaluated. Best antimicrobial results occurred with the dichloromethane (DCM) and butanolic (NB) fractions against Staphylococcus aureus, Micrococcus sp., Enterococcus faecalis and Bacillus subtilis (minimal inhibitory concentration, MIC=31.25–1000μg/mL). Considering the Gram-negative bacteria, only NB fraction was effective against Proteus mirabilis and Aeromonas sp. (MIC=62.5μg/mL and 250μg/mL, respectively). In addition, the fungi Candida albicans, Candida glabrata, Cryptococcus neoformans, Saccharomyces cerevisiae, Aspergillus flavus and Aspergillus fumigatus were particularly vulnerable for DCM fraction (MIC=31.25–1000μg/mL). The fractions and subfractions were effective against Mycobacterium smegmatis (MIC=19.53–156.25μg/mL). DCM (selectivity index – SI=77.92), ethyl acetate (EA) (SI=40.27) and NB (SI=28.97) fractions from the leaves exhibited a potential antiviral activity toward Herpes Simplex Virus type 1 whereas DCM2 subfraction from leaves (SI=12.28) and alkaloidal fraction (AF) (10.71) maintained this good activity. Steroids, terpenoids and phenolics compounds were identified by high-performance liquid chromatography with diode array detection (HPLC/DAD) and may be partially responsible for the antimicrobial and antiherpes activities observed. The results obtained in this study showed that T. catharinensis has antimicrobial and anti-herpetic activities and that these properties are reported for the first time for this species.