Co-encapsulation of acyclovir and curcumin into microparticles improves the physicochemical characteristics and potentiates in vitro antiviral action: Influence of the polymeric composition.

The present study developed and characterized microparticles formulations containing acyclovir and curcumin co-encapsulated in order to overcome the biopharmaceutical limitations and increase the antiviral effect of both drugs. The microparticles were prepared by a spray drying methodology following the ratio 1:3 (drug:polymer), which were made by hydroxypropylmethylcellulose (HPMC) and/or Eudragit® RS100 (EUD). The MP-1 formulation was composed of HPMC and EUD (1:1), MP-2 formulation was composed only of HPMC and MP-3 formulation was composed only of EUD. All formulations showed yielding around 50% and acceptable powder flowability. Drug content determination around 82.1-96.8% and 81.8-87% for acyclovir and curcumin, respectively. The microparticles had spherical shape, size within 11.5-15.3 µm, unimodal distribution and no chemical interactions among the components of the formulations. Of particular importance, the polymeric composition considerably influenced on the release profile of the drugs. The in vitro release experiment demonstrated that the microencapsulation provided a sustained release of acyclovir as well as increased the solubility of curcumin. Besides, mathematical modeling indicated that the experimental fit biexponential equation. Importantly, drugs microencapsulation promoted superior antiviral effect against BoVH-1 virus in comparison to their free form, which could be attributed to the improvement in the aforementioned physicochemical parameters. Therefore, these formulations could be promising technological drug carriers for acyclovir and curcumin, which highlight the great offering a potential alternative treatment for viral herpes.

Photodynamic inactivation of Bovine herpesvirus type 1 (BoHV-1) by porphyrins.

In this work, the photodynamic efficiency of anionic meso-tetrakis sulfonophenyl (TPPS4), cationic meso-tetrakis methylpyridiniumyl (TMPyP) and their zinc complexes (ZnTPPS4 and ZnTMPyP) in the inactivation of Bovine herpesvirus type 1 (BoHV-1) was evaluated. At a non-cytotoxic concentration, all porphyrins showed significant antiviral activity after irradiation using a halogen lamp. The efficiency of the cationic porphyrins was higher than that of the anionic ones. Porphyrin complexation with zinc increases its lipophilicity and the number of absorbed photons, dramatically reducing the time for complete virus inactivation. The high superposition of the compound optical absorption and light source emission spectra played a key role in the virus inactivation efficiency. The results demonstrated the high effectivity of the photodynamic inactivation of BoHV-1. This method can be used as an auxiliary in the treatment of disorders attributed to BoHV-1 infection, and the porphyrins are promising photosensitizers for this application.

Antiviral activity of lambda-carrageenan prepared from red seaweed (Gigartina skottsbergii) against BoHV-1 and SuHV-1.

The antiviral effect of polysaccharides has been known for many years. Carrageenans are considered a good alternative for the prevention of a wide range of diseases, mainly caused by enveloped viruses. The advantages lie on their high availability, low cost and low induction of resistance. The aim of this study was to evaluate the sensitivity of two viral pathogens of veterinary interest to the presence of lambda-carrageenan. This is the first report of a lambda-carrageenan having antiviral activity against animal viruses belonging to the Alphaherpesvirinae subfamily, BoHV-1 (bovine herpesvirus type 1) strain Cooper and SuHV-1 (suid herpesvirus type 1) strain Bartha. Lambda-carrageenan was able to reduce infectivity of both viruses with a more pronounced effect against BoHV-1. These results proved, as previously shown for human herpes virus type 1, that these compounds could be used as potential antiviral agents in the veterinary field.

Polysaccharide and extracts from Lentinula edodes: structural features and antiviral activity.

BACKGROUND: Lentinula edodes, known as shiitake, has been utilized as food, as well as, in popular medicine, moreover, compounds isolated from its mycelium and fruiting body have shown several therapeutic properties. The aim of this study was to determine the antiviral activity of aqueous (AqE) and ethanol (EtOHE) extracts and polysaccharide (LeP) from Lentinula edodes in the replication of poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BoHV-1). METHODS: The time-of-addition assay was performed at the times -2, -1, 0, 1 and 2 h of the infection. The virucidal activity and the inhibition of viral adsorption were also evaluated. Plaque assay was used to monitor antiviral activity throughout. RESULTS: The AqE and LeP were more effective when added at 0 h of infection, however, EtOHE was more effective at the times 1 h and 2 h of the infection. AqE, EtOHE and LeP showed low virucidal activity, and the inhibition of viral adsorption was not significant. CONCLUSIONS: The results allowed us to conclude that AqE, EtOHE and LeP act on the initial processes of the replication of both strains of virus.

Avaliação in vitro do potencial antiviral de Guettarda angelica contra herpesvírus animais / In vitro evaluation of the antiviral potential of Guettarda angelica against animal herpesviruses

Background: The number of antiviral studies using plant extracts has increased in the last decades, and the results have shown that plants are potential sources of compounds that are able to inhibit and/or decrease viral infections. The selection of these plants by ethnopharmacological criteria increases the probability of fi nding new substances with significant pharmacological and biological activities. Hence, Brazil has an advantage in this area due to its extensive biodiversity and ethnological diversity. Guettarda angelica is a plant from the Brazilian Caatinga region the roots of which are popularly used for various therapeutic purposes, including veterinary use. The aim of this work was to investigate the in vitro antiviral activity of extracts of plant parts from G. angelica against three animal herpesviruses: bovine (BoHV-1), suid (SuHV-1) and equine (EHV-1) herpesviruses type 1. Materials, Methods & Results: The extracts of roots, leaves and seeds of G. angelica were initially screened for in vitro antiviral activity against these herpesviruses using the virus yield reduction assay. The MDBK cells were used in assays with BoHV-1 and SuHV-1, and the Vero cells with EHV-1. For these assays, the cells previously treated with the extracts in non-cytotoxic concentrations were inoculated with logarithmic dilutions of each virus. The viral inhibitory activity of extracts was calculated by difference of virus titer between treated infected cells and non-treated infected cells. Only the aqueous extract from seeds (AEs) showed a significant antiviral activity (P < 0.01, ANOVA followed by Tukey test) against all herpesviruses leading continuous studies. Thus, the selectivity index (SI) of this extract was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay by calculating the ratio CC50 over IC50. The 50% cytotoxic concentration (CC50) was defined as the extract concentration that reduced the cell viability by 50% when compared to untreated controls; the 50% inhibitory concentration (IC50) was defined as the concentration of the extract that inhibited 50% of viral replication when compared to the virus control. The CC50 and IC50 were obtained from nonlinear regression analysis of concentration-effect curves by the GraphPad Prism 5 Demo program and represented the means ± standard deviation of three independent experiments. The CC50 for Vero cells was 400.60 ± 0.20 µg/mL, while the CC50 for MDBK cells was 920.50 ± 0.19 µg/mL. The IC50 values of the AEs on the BoHV-1, SuHV-1 and EHV-1 were 22.79 µg/mL, 91.30 µg/mL and 19.95 µg/mL, respectively. The SI values of this extract for each virus obtained from these data were 40.39, 10.08 and 20.08 for BoHV-1, SuHV-1, and EHV-1, respectively. Discussion: To ensure the antiviral activity of a plant extract and consequently its future use as antiviral agent is crucial the obtainment of its selectivity index or safety index. It is guarantee of a true antiviral effect and not the result of cytotoxicity of the extract on cells, and that could be confused with an antiviral activity. Other important point are the extract IC50 values less than 100 µg/mL. The results of the AEs of G. angelica are in accordance with these considerations indicating that the G. angelica seeds may be a potential source of antiviral compounds insurance and encouraging further investigation of them.

Antiviral properties of polysaccharides from Agaricus brasiliensis in the replication of bovine herpesvirus 1.

Natural products are an inexhaustible source of compounds with promising pharmacological activities, including antiviral action. In the present study, the antiviral potential of polysaccharide-peptide (PLS) and an extracted ß-glucan from Agaricus brasiliensis were investigated in the replication of bovine herpesvirus 1 (BoHV-1) in HEp-2 cell cultures. The cytotoxicity (CC50) was assayed by the MTT method and the antiviral activity (IC50) was estimated by the plaque reduction assay. To study the possible mode of action of PLS and ß-glucan, the following protocols were performed: the virucidal assay, adsorption assay and the time-of-addition assay. The PLS presented a selectivity index (SI) higher than 12.50 and ß-glucan 9.19. The antiviral inhibition (67.9%) in cells treated with PLS during virus infection was higher than that in cells treated prior to or post infection. The ß-glucan presented high inhibition of virus replication by plaque assay (83.2%) and by immunofluorescence assay (63.8%). Although the mechanism has yet to be defined, we suggest that PLS and ß-glucan inhibited BoHV-1 replication by interfering with the early events of viral penetration. Additional studies are required for a better understanding of the mechanism of action of PLS and ß-glucan.

In vitro antiviral activity from Acanthospermum australe on herpesvirus and poliovirus.

CONTEXT: The Asteraceae family has been of interest to researchers due to the presence of polyphenolic compounds, mainly flavonoids, which demonstrated antiviral activity. OBJECTIVE: The hydroethanol extract of the aerial parts of Acanthospermum australe (Loefl.) Kuntze (Asteraceae) and its fractions, were evaluated in vitro for their potential cytotoxic and antiviral activity against bovine herpesvirus and human poliovirus. MATERIALS AND METHODS: The sulforhodamine B colorimetric assay were used to evaluate the capacity of the hydroethanol extract and fractions to inhibit the lytic activity of herpes and poliovirus in infected cell cultures and their influence on the viability of uninfected cell cultures. RESULTS AND DISCUSSION: A progressive increase in the antiviral effect against herpesvirus was observed in the course of the purification process of the extract. The hydroethanol extract had a 50% antiviral effective concentration (EC(50)) at 70 µg/mL and 36 µg/mL for herpes and poliovirus, respectively, and it exhibited no cytotoxicity. The fractions F3 (dichloromethane) and F4 (dichloromethane: ethyl acetate (1:1 v/v)) both showed EC(50) at 6.25 µg/mL against herpesvirus, and these fractions showed cytotoxic concentrations (CC(50)) at 12.7 and 11.7 µg/mL, respectively. These fractions had no effect against poliovirus in the concentrations tested. From the bioactive F3, a diterpene lactone (acanthoaustralide-1-O-acetate) was isolated at a concentration of 0.5% and from F4 two flavonoids (quercetin and chrysosplenol D) were isolated at concentrations of 0.14 and 0.24%, respectively. CONCLUSION: The present study reports for the first time the antiviral activity of extracts and fractions from A. australe aerial parts.

Evaluation of trypsin treatment on the inactivation of bovine herpesvirus type 1 on in vitro produced pre-implantation embryos.

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 microl of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor.

In this paper, we describe the purification of an antiviral peptide from seeds of Sorghum bicolor L. by a procedure that included gel filtration, ion exchange, and high-performance liquid chromatography (HPLC) in a reversed-phase column. Its molecular weight, determined by chromatographic mobility on the Shim-pack DIOL-150 gel permeation column in HPLC, was found to be 2000Da. The peptide designated 2kD peptide strongly inhibited the replication of herpes simplex virus type 1 (HSV-1), dose-dependently, at 40-90% of the control level, after incubation with 10-50 microM of the peptide, with EC(50) and EC(90) values of 6.25 and 15.25 microM, respectively. The IC(50) value of the 2kD peptide against Vero cells was 250 microM. Pre-incubation of HSV-1 with various concentrations of the 2kD peptide showed dose-dependent cytopathic effects (CPE) reduction patterns at concentrations from 6.25 to 50 microM. The presence of the 2kD peptide before HSV-1 infections showed moderate inhibition of virus-induced CPE as compared to during or after infections, with EC(50) values of 12.5, 6.25, and 6.25 microM, respectively. Similar results were observed when the 2kD peptide was assayed against bovine herpes virus (BHV), an enveloped virus like HSV-1. On the other hand, the 2kD peptide showed weak activity against poliovirus type 1, a non-enveloped virus. Taken together, these results indicate that the 2kD peptide was able not only to inhibit the initiation and the spread of infection, but also had an in vitro prophylactic effect against HSV-1 infection.

The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca.

We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal plant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 cells, and the 50% inhibitory concentration (IC50) were 22.01 microg/ml (selectivity index (SI)=2.83) and 21.10 microg/ml (SI=2.95), respectively. At the highest concentration of the drug (40 microg/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains.

Antiviral effect of Guazuma ulmifolia and Stryphnodendron adstringens on poliovirus and bovine herpesvirus.

Crude extract (CE) and aqueous (AqF) and ethyl acetate (EtOAcF) fractions of Guazuma ulmifolia Lam., Sterculiaceae and the corresponding AqF, EtOAcF of Stryphnodendron adstringens (Mart.) Coville, Leguminosae were tested for their antiviral activity against poliovirus 1 (P-1) and bovine herpesvirus 1 (BHV-1) in HEp-2 cultured cells. The antiviral activity was monitored by plaque assay and immunofluorescence assay (IFA) under virucidal and therapeutic protocols. The therapeutic protocol demonstrated statistically significant positive results with both plants and for both virus strains. The highest percentages of viral inhibition were found for G. ulmifolia EtOAcF which inhibited BHV-1 and P-1 replication by 100% and 99%, respectively (p<0.05, Student's t-test). For S. adstringens, AqF was the most efficient, inhibiting BHV-1 and P-1 by 97% and 93%, respectively (p<0.05). In the virucidal protocol, G. ulmifolia CE inhibited the replication of BHV-1 and P-1 by 60% and 26%, respectively (p<0.05), while, for S. adstringens, inhibition of 62% (p<0.05) was demonstrated only with EtOAcF for P-1. IFA demonstrated that the greatest reduction in fluorescent cell number occurred with G. ulmifolia, under the therapeutic protocol for both virus strains. However, AqF and EtOAcF of S. adstringens were most efficient with the virucidal protocol for P-1. In conclusion, we demonstrated that G. ulmifolia and S. adstringens inhibited BHV-1 and P-1 replication, as well as, blocked the synthesis of viral antigens in infected cell cultures.

Evaluation of the antiviral activity of an aqueous extract from Phyllanthus orbicularis.

The antiviral and cytotoxic activity of Phyllanthus orbicularis, a member of the Euphorbiaceae, was evaluated in tissue culture. An aqueous extract made from leaves and stems of this plant exhibited selective antiviral indexes of 12.3 and 26 against bovine herpesvirus type 1 and herpes simplex virus type 2, respectively, showing no selective antiviral activity against adenovirus type 5 and mengovirus. Incubation with this plant extract during cell culture infection, impaired the productive replication of both herpes viruses in an extract concentration-dependent manner, also depending on the multiplicity of infection (MOI) used. The results obtained suggested that the P. orbicularis antiviral activity might be partially due to a direct interaction with virus particles or their entry into the cell, instead of interfering with intracellular virus-specific macromolecular synthesis.

Evaluation of the effects of different trypsin treatments on semen quality after BHV-1 inactivation, and a comparison of the results before and after freezing, assessed by a computer image analyzer.

Semen infected experimentally with infectious bovine rhinotracheitis virus (BHV-1) was treated with trypsin at concentrations of 0.30%, 0.25% and 0.15%, with or without (w or w/o) trypsin inhibitor in order to render the semen virus free. The trypsin treatments (at 0.30% and 0.25% by concentration) inactivating the virus up to 10(4) TCID50/ml, and its effects on semen quality were assessed weekly from the 1st to 20th week after being frozen. The following parameters were determined using a computerized semen analysis system (Hamilton Thorn motility analyzer, HTM): total motility, progressive motility and linearity of sperm cells. The results showed that the total and progressive motility of sperm cells were reduced in frozen/thawed semen, principally in the semen treated with trypsin at concentrations of 0.30%. Moreover, the plasma membranes were damaged by trypsin treatments (0.30% by concentration), as determined by the hypoosmotic swelling test (HOS test). These findings suggest that trypsin treatments were effective against the virus however the effects on semen quality and the possibility of a decrease in semen fertility were clear. Trypsin treatment could be recommended at a maximum concentration of 0.25% (w/o trypsin inhibitor) on semen with a high concentration and high motility values of spermatozoa before freezing.

Antiviral activity of crude extracts of Guarea guidona.

Crude extracts of leaves and fruits of Guarea guidona were tested for antiviral activity against pseudorabies virus and foot-and-mouth disease virus in the IB-RS-2 pig cell line and against bovine herpesvirus-1 (BHV-1) in the GBK bovine cell line. The highest nontoxic doses of extracts from fruits and leaves were 125 micrograms/ml and 500 micrograms/ml. respectively. Crude extracts presented antiviral activity against pseudorabies virus with a decrease in virus titer of 3.0 log units at 500 micrograms/ml. Virucidal activity was not observed at 62.5 micrograms/ml. Preformed cell monolayers showed no cytotoxic effect after 48 h in the presence of 500 micrograms/ml in pig cells. G. guidona leaves did not induce an antiviral state but exhibited antiviral effects during the early stage of viral infection.

Antiviral activity of crude extracts of Guarea guidona

Crude extracts of leaves and fruits of Guarea guidona were tested antiviral activity against pseudorabies virus and foot-and-mouth disease virus in the IB-RS-2 pig cell line and against bovine herpesvirus-1 (BHV-1) in the GBK bovine Cell line. The highest nontoxic doses of extracts from fruits and leaves were 125 mug/ml and 500 mug/ml, respectively. Crude extracts presented antiviral activity against pseudorabies virus with a decrease in virus titer of 3.0 log units at 500 mug/ml. Virucidal activity was not observed at 62.5 mug/ml. Preformed cell monolayers showed no cytotoxic effect after 48 h in the presence of 500 mug/ml in pig cells. G. guidona leaves did not induce an antiviral state but exhibited antiviral effects during the early stage of viral infection.