Excoecarianin, Isolated from Phyllanthus urinaria Linnea, Inhibits Herpes Simplex Virus Type 2 Infection through Inactivation of Viral Particles.

Phyllanthus urinaria Linnea (Euphorbiaceae) is one of the traditional medicinal plants widely used by oriental people to treat various diseases. We have previously demonstrated that the acetone extract of P. urinaria inhibits herpes simplex virus type 2 (HSV-2) but not HSV-1 infection. In a continuing effort to clarify the antiviral mechanisms of P. urinaria, we isolated the pure compound excoecarianin from the whole plant of P. urinaria through acetone extraction, and investigated its anti-HSV-1 and HSV-2 activities. Our results indicated that excoecarianin protected Vero cells from HSV-2 but not HSV-1 infection, and its 50% inhibitory concentration (IC(50)) was 1.4 ± 0.1 µM. The antiviral effective concentration of excoecarianin did not affect the viability or the morphology of Vero cells. Although excoecarianin inhibited HSV-2 infection, the inhibitory effect, however, was most prominent when excoecarianin was concurrently added with the virus. Pretreatment of Vero cells with excoecarianin with removal of the drug prior to infection did not yield any antiviral effects, and the same observation was made for post viral entry treatment. Subsequent studies revealed that excoecarianin inactivated HSV-2 virus particles to prevent viral infection. A synergistic antiviral effect against HSV-2 was also observed when Vero cells were treated with a combination of acyclovir (ACV) and excoecarianin. These results suggested that excoecarianin merits to be further explored as an entry inhibitor against HSV-2 and could potentially be investigated for combinatorial drug treatment with nucleoside analogues such as ACV in therapeutic management of HSV-2 infection.

Yin Chen Hao Tang, a Chinese prescription, inhibits both herpes simplex virus type-1 and type-2 infections in vitro.

Yin Chen Hao Tang (YCHT) is one of the most frequently used prescriptions in the long history of traditional Chinese medicine practice. The prescription contains three Chinese herbs, namely Artemisia capillaries Thunb. (Compositae), Rheum officinale Baillon (Polygonaceae), and Gardenia jasminoids Ellis (Rubiaceae), and has been widely used to treat acute hepatitis with jaundice. In this study, the in vitro anti-HSV-1 and HSV-2 activities of the water extract of YCHT were investigated. Results showed that YCHT water extract inhibited both HSV-1 and HSV-2 infections. However, the inhibition was more effective against HSV-2 than against HSV-1. The IC(50) and IC(90) values of YCHT water extract against HSV-1 infection were in the range of 142.5-150.1 and 191.3-393.9 microg/ml, and against HSV-2 infection they were in the range of 19.6-29.4 and 42.2-97.7 microg/ml, respectively. The water extract of YCHT showed no cytotoxic effect at a concentration of 500 microg/ml or below, and had a CC(50) value of 850.7+/-1.7 microg/ml. The prescription was found to diminish HSV-2 infectivity in a dose-dependent manner, and the activity was influenced by the incubation periods and the incubation temperatures. Concurrent addition of virus with YCHT or pre-treatment of the virus with the prescription extract both protected the cells from infection. In summary, the water extract of YCHT was concluded to inhibit infections by HSV-1 and HSV-2 and this effect was likely mediated through direct inactivation of the virus infectivity.

The in vitro anti-herpes simplex virus type-1 and type-2 activity of Long Dan Xie Gan Tan, a prescription of traditional Chinese medicine.

BACKGROUND: Long Dan Xie Gan Tan (LDXGT), a decoction of radix gentianae for purging the pathogenic inflammation of the liver, is a widely used prescription among many in traditional Chinese medicine. The prescription is primarily used to treat the disorders induced by damp-heat in the liver and the gall bladder. METHODS: In this study, the in vitro anti-herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) activity of the water extract of LDXGT was investigated. RESULTS: LDXGT water extract was shown to exhibit anti-HSV activity. The IC(50) values of LDXGT against HSV-1 and HSV-2 infections were 257.5 +/- 12.2 and 494.6 +/- 1.8 microg/ml, respectively. It had a CC(50) value of 4,077.2 +/- 2.4 microg/ml towards Vero cells and showed no cytotoxic effect at a concentration of 2,000 microg/ml or below. The prescription was also found to inactivate HSV-2 infectivity in a dose-, time- and temperature-dependent manner. CONCLUSIONS: In summary, the water extract of LDXGT was concluded to inhibit HSV-1 and HSV-2 infection at different magnitudes of potency, and our observations also suggested that the effect was likely mediated by directly inactivating the virus infectivity.

The in vitro activity of geraniin and 1,3,4,6-tetra-O-galloyl-beta-D-glucose isolated from Phyllanthus urinaria against herpes simplex virus type 1 and type 2 infection.

Phyllanthus urinaria Linnea (Euphorbiaceae) is a widely used traditional medicinal plant by oriental countries and has been reported to possess various biological activities. Previously, the acetone extract from Phyllanthus urinaria was found to inhibit herpes simplex virus (HSV) infection. In this study, geraniin and 1,3,4,6-tetra-O-galloyl-beta-D-glucose (1346TOGDG), both of which were isolated from the acetone extract of Phyllanthus urinaria, were examined for their activity against HSV-1 and HSV-2 in vitro. Results showed that geraniin actively suppressed HSV-2 infection, whereas 1346TOGDG effectively inhibited HSV-1 infection. The 50% inhibitory concentration (IC(50)) was 18.4+/-2.0 microM for geraniin against HSV-2 infection, and 19.2+/-4.0 microM for 1346TOGDG against HSV-1. No toxic effect towards the host cell was observed at the antiviral concentrations. In conclusion, geraniin and 1346TOGDG were found to inhibit HSV-1 and HSV-2 multiplication at different magnitudes of potency.

ent-Epiafzelechin-(4alpha-->8)-epiafzelechin extracted from Cassia javanica inhibits herpes simplex virus type 2 replication.

Herpes simplex virus (HSV) is a ubiquitous organism that causes infections in human populations throughout the world. It causes a variety of diseases ranging in severity from mild to life-threatening. In this study, ent-epiafzelechin-(4alpha-->8)-epiafzelechin (EEE) extracted from the fresh leaves of Cassia javanica L. agnes de Wit (Leguminosae) was investigated for its in vitro anti-HSV-2 activity using XTT and plaque reduction assays. Results showed that EEE inhibited HSV-2 replication in a dose-dependent manner. The IC50 value was 83.8 +/- 10.9 and 166.8 +/- 12.9 microM for XTT and plaque reduction assays, respectively. EEE did not affect the viability and the proliferation of cells at antiviral concentrations. Mechanistic studies demonstrated that EEE prevented HSV-2 from penetrating the cell and also interfered with HSV-2 replication at the late stage of its life cycle. It also disturbed virus attachment but the inhibitory effect was minor. In summary, the conclusion of this study was that EEE exhibits various modes of action in suppressing HSV-2 multiplication.

Acetone, ethanol and methanol extracts of Phyllanthus urinaria inhibit HSV-2 infection in vitro.

Phyllanthus urinaria Linnea (Euphorbiaceae) is one of the traditional medicinal plants that are widely applied by oriental people, especially by Chinese and Indian, to ameliorate various kinds of ailments. Many biological activities, including anti-hepatitis B virus, anti-Epstein-Barr virus and anti-retroviral reverse transcriptase, of P. urinaria have been reported, but not against herpes simplex virus (HSV). In this study, the anti-HSV-1 and HSV-2 activities of different solvents extracted from P. urinaria were investigated in vitro by plaque reduction assay. Results showed that acetone, ethanol and methanol extracts of P. urinaria inhibited HSV-2 but not HSV-1 infection. The 50% inhibitory concentration against HSV-2 infection (IC50) of acetone, ethanol and methanol extracts was 4.3 +/- 0.5, 5.0 +/ -0.4 and 4.0 +/- 0.9 mcg/ml, respectively. All three extracts showed no cytotoxic effect against Vero cells at concentrations of 10.0 mcg/ml or below. The time-of-addition study demonstrated that these three extracts were only effective when added during the HSV-2 infection which, therefore, suggested that they disturb the initial stage of HSV-2 infection. Furthermore, they can diminish virus infectivity without significantly affecting incubation time and temperature. Therefore, the acetone, ethanol and methanol extracts of P. urinaria were concluded to likely inhibit HSV-2 infection through disturbing the early stage of virus infection and through diminishing the virus infectivity.

Mechanism of action of the suppression of herpes simplex virus type 2 replication by pterocarnin A.

The purpose of this study was to investigate the in vitro antiviral properties of pterocarnin A, extracted from the bark of Pterocarya stenoptera (Juylandaceae). Results showed that pterocarnin A exhibited anti-herpes simplex virus (HSV) activity. It had a low selectivity index (SI) value and only possessed some level of cell cytotoxic effect at high antiviral concentrations. Mechanism studies demonstrated that pterocarnin A inhibited herpes simplex virus type 2 (HSV-2) from attaching and penetrating into cells. It also actively suppressed HSV-2 multiplication in Vero cells even when added 12 h after infection. This observation indicated that pterocarnin A affected the late stage(s) of HSV-2 infection cycle. Pterocarnin A also significantly reduced viral infectivity at high concentrations. From these observations, it was concluded that pterocarnin A suppressed both early and late in the replication cycle of HSV-2. The various modes of action of pterocarnin A in interfering with certain steps of viral infection thus merit further investigation.

Hippomanin A from acetone extract of Phyllanthus urinaria inhibited HSV-2 but not HSV-1 infection in vitro.

Phyllanthus urinaria Linnea (Euphorbiaceae) is a commonly used traditional medicinal plant in oriental countries and has been reported to possess various biological activities. Previously, the acetone extract and some pure compounds from P. urinaria were found to suppress herpes simplex virus (HSV). In this study, another two pure compounds were isolated from acetone extract of P. urinaria and were tested for their in vitro anti-HSV-1 and HSV-2 activities. The results showed that hippomanin A impeded HSV-2 but not HSV-1 infection. Corilagin, however, inhibited neither HSV-1 nor HSV-2 replication. The similarity between corilagin and hippomanin A in structure, but difference in antiviral activity, therefore, merit further investigation.

Euphorbia thymifolia suppresses herpes simplex virus-2 infection by directly inactivating virus infectivity.

1. The ethyl acetate (EtOAc) extract and 3-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-d-glucose (3OG46HG) of Euphorbia thymifolia Linnea have been shown to exhibit anti-herpes simplex virus (HSV)-2 activity in vitro. In the present study, we investigated the mode of action of these two compounds in suppressing HSV-2 multiplication. 2. The results demonstrated that the EtOAc extract and 3OG46HG affected virus infectivity in a dose-dependent manner. The EtOAc extract significantly reduced virus infectivity at a concentration of 4.0 microg/mL, whereas 3OG46HG obviously diminished virus infectivity at concentration of a 0.5 microg/mL. The virucidal ability of the EtOAc extract was affected by the incubation period, but not by the incubation temperature. In the case of the action of 3OG46HG against HSV-2, the effects of incubation time and temperature were negligible. 3. In summary, the EtOAc extract and 3OG46HG of E. thymifolia are concluded to inhibit HSV-2 multiplication by reducing virus infectivity.

Antioxidant and antiviral activities of Euphorbia thymifolia L.

The antioxidant and antiviral activities of Euphorbia thymifolia L. (Euphorbiaceae) were investigated in this study. The results showed that all of the fractions (MeOH, CHCl(3), EtOAc, n-butanol and water) and pure compounds (3-O-galloyl-4,6-(S)-HHDP-D-glucose, rugosin B and 1,3,4,6-tetra-O-galloyl-K-beta-D-glucose)tested possessed antioxidant activities, with the exception of the organic aqueous fraction in the anti-lipid and anti-superoxide formation assays. The range of IC(50) of anti-lipid formation, anti-superoxide formation and free radical scavenging assays for all fractions and pure compounds were 2.81-7.63, 0.03-2.18 and 0.013-2.878 mg/ml, respectively. Electron spin resonance studies showed that water extract and pure compounds of E. thymifolia exhibited superoxide radical and hydroxyl radical scavenging activities. Besides antioxidant activities, 3-O-galloyl-4,6-(S)-HHDP-D-glucose and EtOAc fraction also showed anti-HSV-2 activity. Thus, E. thymifolia was concluded to possess antioxidant and anti-HSV-2 activities.

In vitro antiviral activity of prodelphinidin B-2 3,3'-di-O-gallate from Myrica rubra.

In this study, the in vitro antiviral properties of prodelphinidin B-2 3,3'-di- O-gallate (PB233'OG) isolated from the bark of Myrica rubra (Myricaceae) was investigated. Results showed that PB233'OG exhibited anti-herpes simplex virus type 2 (HSV-2) activity with IC (50) values of 5.3 +/- 0.1 and 0.4 +/- 0.04 microM for XTT and plaque reduction assays, respectively. The IC (50) value increased with increasing MOI (multiplicity of infection). PB233'OG did not show a cellular cytotoxic effect at concentrations that possessed antiviral activity. Mechanistic studies demonstrated that PB233'OG inhibited HSV-2 attachment to the Vero cell, interfered with the penetration of HSV-2 into the Vero cell, affected the late stage(s) of the HSV-2 infection cycle, and also reduced the viral infectivity at high concentrations. It is concluded that PB233'OG exhibits various modes of action in its anti-HSV-2 effects.

Putranjivain A from Euphorbia jolkini inhibits both virus entry and late stage replication of herpes simplex virus type 2 in vitro.

AIMS: To investigate the in vitro antiviral properties of putranjivain A, isolated from the whole plant of Euphorbia jolkini Bioss (Euphorbiaceae). METHODS AND RESULTS: Herpes simplex virus (HSV)-2 strain 196-infected Vero cells were used. It was shown that putranjivain A exhibited antiviral activity with an IC50 of 7.9 +/- 1.2 micro M using the XTT assay, with the IC50 value increasing with increasing multiplicity of infection. Using the plaque reduction assay, the IC50 and IC90 were 6.3 +/- 0.8 and 14.5 +/- 3.1 micro M, respectively. Putranjivain A showed no cytotoxic effect on cell multiplication at concentrations that achieved antiviral activity. The 50% cell cytotoxic concentration (CC50) was 80.3 +/- 14.7 microM, and the selectivity index (SI) (ratio of CC50 to IC50) for the XTT and plaque reduction assays was 10.2 and 12.7, respectively. When tested for virucidal activity, putranjivain A significantly reduced viral infectivity at concentrations of 75 and 100 micro M, but not at 50 microM or below. The results of the time-of-addition studies suggested that putranjivain A affected the late stage of HSV-2 replication at 25 microM. Interestingly, putranjivain A also showed inhibition of viral attachment and cell penetration. The combination of putranjivain A and aciclovir produced no interaction. CONCLUSIONS: Putranjivain A possesses antiviral activity, inhibiting viral attachment and penetration, and also interfering with the late stage of viral replication.

Antioxidant and free radical scavenging activities of Terminalia chebula.

Free radicals react with biological molecules and destroy the structure of cells, which eventually causes free-radical induced disease such as cancer, renal failure, aging, etc. In this study, 6 extracts and 4 pure compounds of Terminalia chebula RETZ. were investigated for anti-lipid peroxidation, anti-superoxide radical formation and free radical scavenging activities. The superoxide radical scavenging of the 4 pure compounds was further evaluated using electron spin resonance (ESR) spectrometry. The results showed that all tested extracts and pure compounds of T. chebula exhibited antioxidant activity at different magnitudes of potency. The antioxidant activity of each pure compound was derived from different pathways and was suggested to be specific.