Antiherpetic activities of various protein bound polysaccharides isolated from Ganoderma lucidum

To investigate antiherpetic substances from Ganoderma lucidum, various protein bound polysaccharides, GLhw, GLhw-01, GLhw-02, GLhw-03, were isolated by activity-guided isolation from water soluble substances of the carpophores. These substances were examined for their antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by plaque reduction assay in vitro. Among them, the acidic protein bound polysaccharide, GLhw-02 of a brownish substance, exhibited the most potent antherpetic activity with 50% effective concentrations (EC50) of 300 approximately 520 microg/ml in Vero and HEp-2 cells, and its selectivity indices (SI) were more than 20. GLhw-02 was identified to consist mainly of polysaccharide (approximately 40.6%) and protein (approximately 7.80%) by anthrone test and Lowry-Folin test, and showed the usual molar ratio (C:H:O = 1:2:1) of carbohydrates by elemental analysis. These results suggest that GLhw-02 possesses the possibility of being developed from a new antiherpetic agent.     

Antiherpetic activities of acidic protein bound polysacchride isolated from Ganoderma lucidum alone and in combinations with acyclovir and vidarabine

To investigate antiherpetic activity, an acidic protein bound polysaccharide (APBP) was isolated from carpophores of Ganoderma lucidum. This brownish APBP was isolated from water soluble substances of the carpophores by activity-guided isolation method. APBP was tested for its antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by plaque reduction assay in tissue culture. APBP showed potent antiviral activity against HSV-1 and HSV-2 in Vero cells at its 50% effective concentration (EC(50)) of 300 and 440 microg/ml, respectively. APBP had no cytotoxicity on Vero cells at a concentration of 1 x 10(4) microg/ml. APBP exhibited a potent antiviral activity with selectivity index (SI) of more than 22.73. The combined antiherpetic effects of APBP with nucleoside antiherpetic agents, acyclovir (ACV) and vidarabine (ara-A), were examined on the multiplication of these two strains of herpesviruses in Vero cells by the combination assay. The results of combination assay were evaluated by the combination index (CI) that was calculated by the multiple drug effect analysis. CI values were in the range 0.47-0.51 for a combination of APBP with ACV, and in the range of 1.02-1.18 for a combination of APBP with ara-A. The combinations of APBP with ACV on HSV-1 and HSV-2 showed potent synergistic effects, and these results suggest that the possibility of developing APBP as a new antiherpetic agent.     

Antiherpetic activities of acidic protein bound polysacchride isolated from Ganoderma lucidum alone and in combinations with interferons

To investigate antiherpetic activity, an acidic protein bound polysaccharide (APBP) was isolated from carpophores of Ganoderma lucidum. This brownish APBP was isolated from water soluble substances of the carpophores by activity-guided isolation method. APBP was tested for its antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by plaque reduction assay in tissue culture. APBP showed potent antiviral activity against HSV-1 and HSV-2 in Vero cells at its 50% effective concentration (EC(50)) of 300 and 440 microg/ml, respectively. APBP had no cytotoxicity on Vero cells at a concentration of 1x10(4) microg/ml. APBP exhibited a potent antiviral activity with selectivity index (SI) of more than 22.73. The combined antiherpetic effects of APBP with protein antiviral agents, interferon alpha (IFN alpha) and interferon gamma (IFN gamma), were examined on the multiplication of these two strains of herpesviruses in Vero cells by the combination assay. The results of combination assay were evaluated by the combination index (CI) that was calculated by the multiple drug effect analysis. The combinations of APBP with IFN alpha on HSV-1 and HSV-2 showed more potent synergistic effects with CI values of 0.30-0.62 for 50-90% effective levels than those of APBP with IFN gamma with CI values of 0.65-1.10. These results suggest the possibility of developing APBP as a new antiherpetic agent.     

Possible mode of antiviral activity of acidic protein bound polysaccharide isolated from Ganoderma lucidum on herpes simplex viruses

Two protein bound polysaccharides, a neutral protein bound polysaccharide (NPBP) and an acidic protein bound polysaccharide (APBP), were isolated from water soluble substances of Ganoderma lucidum by EtOH precipitation and DEAE-cellulose column chromatography. Their antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were then investigated by plaque reduction assay. APBP exhibited more potent HSV-1 and HSV-2 antiviral activity than NPBP with 50% effective concentration (EC(50)) of 300-520 microg/ml. In order to examine the possible mode of the antiviral activity of APBP its virucidal effect, antiviral activity in preincubation, attachment and penetration assay were tested with HSV-1 and HSV-2. APBP was found to have a direct virucidal effect on HSV-1 and HSV-2. APBP did not induce IFN or IFN-like materials in vitro and is not expected to induce a change from a normal state to an antiviral state. APBP in concentrations of 100 and 90 microg/ml inhibited up to 50% of the attachment of HSV-1 and HSV-2 to Vero cells and was also found to prevent penetration of both types of HSV into Vero cells. These results show that the antiherpetic activity of APBP seems to be related to its binding with HSV-specific glycoproteins responsible for the attachment and penetration, and APBP impedes the complex interactions of viruses with cell plasma membranes.     

Search for antiviral activity in higher plant extracts

In the course of our search for plant natural products as antiviral agents, extracts of ten plants from the Iberian Peninsula were tested for antiviral activity against herpes simplex type I (HSV-1), vesicular stomatitis virus (VSV) and poliovirus type 1. Aqueous extracts of five of these medicinal plants, namely Nepeta nepetella (150-500 microg/mL), Nepeta coerulea (150-500 microg/mL), Nepeta tuberosa (150-500 microg/mL), Dittrichia viscosa (50-125 microg/mL) and Sanguisorba minor magnolii (50-125 microg/mL), showed a clear antiviral activity against two different DNA and RNA viruses, i.e. HSV-1 and VSV. Only the medicinal plant Dittrichia viscosa was active against an additional virus, poliovirus type 1.     

Antiviral activity of some South American medicinal plants

Folk medicinal plants are potential sources of useful therapeutic compounds including some with antiviral activities. Extracts prepared from 10 South American medicinal plants (Baccharis trinervis, Baccharis teindalensis, Eupatorium articulatum, Eupatorium glutinosum, Tagetes pusilla, Neurolaena lobata, Conyza floribunda, Phytolacca bogotensis, Phytolacca rivinoides and Heisteria acuminata) were screened for in vitro antiviral activity against herpes simplex type I (HSV-1), vesicular stomatitis virus (VSV) and poliovirus type 1. The most potent inhibition was observed with an aqueous extract of B. trinervis, which inhibited HSV-1 replication by 100% at 50-200 micrograms/mL, without showing cytotoxic effects. Good activities were also found with the ethanol extract of H. acuminata and the aqueous extract of E. articulatum, which exhibited antiviral effects against both DNA and RNA viruses (HSV-1 and VSV, respectively) at 125-250 micrograms/mL. The aqueous extracts of T. pusilla (100-250 micrograms/mL), B. teindalensis (50-125 micrograms/mL) and E. glutinosum (50-125 micrograms/mL) also inhibited the replication of VSV, but none of the extracts tested had any effect on poliovirus replication.     

Anti-Herpes simplex virus activity of Bidens pilosa and Houttuynia cordata

The present study evaluated the antiviral activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb., using cytotoxicity test with XTT-based colorimetric assay. BCC-1/KMC cells were infected with herpes simplex virus (HSV) and then were cultured with hot water extract of B. pilosa (HWBP) or H. cordata (HWHC). Results showed that HWBP significantly inhibited the replication of HSV at a concentration of 100 microg/ml (11.9% for HSV-1, p < 0.01; 19.2% for HSV-2, p < 0.005), whereas HWHC had the same effect at a concentration of 250 microg/ml (10.2% for HSV-1, p < 0.05; 32.9% for HSV-2, p < 0.005). The ED50 of HSV type 1 (HSV-1) and HSV type 2 (HSV-2) for HWBP was 655.4 microg/ml and 960 microg/ml respectively, for HWHC it was 822.4 microg/ml and 362.5 microg/ml respectively. Both drugs had selective indexes above 1.04. H. cordata had better effect against HSV-2 than HSV-1, and had a low ED50 against HSV-2. We suggest that H. cordata might be a useful medicinal plant against infection of HSV-2.     

Evaluation of antiviral activity of South American plant extracts against herpes simplex virus type 1 and rabies virus

This paper describes the screening of different South American plant extracts and fractions. Aqueous and organic extracts were prepared and tested for antiherpetic (HSV-1, KOS and 29R strains) and antirabies (PV strain) activities. The evaluation of the potential antiviral activity of these extracts was performed by using an MTT assay for HSV-1, and by a viral cytopathic effect (CPE) inhibitory method for rabies virus (RV). The results were expressed as 50% cytotoxicity (CC(50)) for MTT assay and 50% effective (EC(50)) concentrations for CPE, and with them it was possible to calculate the selectivity indices (SI = CC(50)/EC(50)) of each tested material. From the 18 extracts/fractions tested, six extracts and four fractions showed antiviral action. Ilex paraguariensis, Lafoensia pacari, Passiflora edulis, Rubus imperialis and Slonea guianensis showed values of SI > 7 against HSV-1 KOS and 29-R strains and Alamanda schottii showed a SI of 5.6 against RV, PV strain.     

Anti-herpes simplex virus activities of Eugenia caryophyllus (Spreng.) Bullock & S. G. Harrison and essential oil, eugenol

In this study, an extract from the flower buds of Eugenia caryophyllus (Spreng.) Bullock & S. G. Harrison and the essential oil, eugenol, were evaluated for their anti-herpes simplex virus properties on standard HSV-1(F), standard HSV-2(G) and ten HSV isolates. The plaque reduction assay showed that HSV-1(F), HSV-2(G), two HSV-1 isolates (2, 30) and four HSV-2 isolates (1, 2, 3, 21) were inhibited by E. caryophyllus. Only HSV-1 isolates 1 and 30 were inhibited by eugenol. Thus, strains or isolates of viruses may affect the range of inhibition. Moreover, particles of HSV standard strains were directly inactivated by E. caryophyllus and eugenol. The total virus yield of HSV standard strains and isolates at 30 h also declined after treatment with E. caryophyllus and eugenol. The E. caryophyllus extract exerted higher antiviral replication on HSV-2(G) than on HSV-1(F). The inhibition of the viral yield of HSV-1 isolates was higher than standard HSV-1(F) and standard HSV-2(G) was also inhibited more than most of the HSV-2 isolates. The anti-HSV activity of eugenol against HSV-1(F) and HSV isolates was stronger than with the E. caryophyllus crude extract. However, the percentage inhibition was more pronounced on HSV-1(F) than on HSV-2(G). Moreover, HSV-1(1) and HSV-2(1, 32) could not replicate when eugenol was included in the assay.     

Possible mode of action of antiherpetic activities of a proteoglycan isolated from the mycelia of Ganoderma lucidum in vitro

A bioactive fraction (GLPG) was extracted and purified from the mycelia of Ganoderma lucidum by EtOH precipitation and DEAE-cellulose column chromatography. GLPG was a proteoglycan and had a carbohydrate:protein ratio of 10.4:1. Its antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were investigated by the cytopathic effect (CPE) inhibition assay in cell culture. This kind of polysaccharide inhibited the development of the cytopathic effect in dose-dependent manner in HSV-infected cells, moreover did not show any cytotoxic effects on cells even when a concentration was as high as 2000 microg/ml. In order to study the possible mode of action of the antiviral activity of GLPG, cells were treated with GLPG before, during and after infection, and the viral titers in the supernatant of cell culture 48 h post-infection were tested by TCID(50) assay. The antiviral effects in pre-treated and treated during virus infection with GLPG were more remarkable than the treatment of post-infection. Although the precise mechanism has yet to be defined, our work suggested that GLPG inhibits viral replication by interfering with the early events of viral adsorption and entry into target cells. Thus, this proteoglycan seems to be a potential candidate for anti-HSV agents.     

藏药甘青乌头抗单纯疱疹病毒Ⅱ型体内外作用研究

 目的探讨藏药甘青乌头抗单纯疱疹病毒Ⅱ型(HSV-2)的作用和机制。方法MTT法测定甘青乌头对Vero细胞的毒性,采用细胞病变法和蚀斑法测定甘青乌头体外抗病毒活性,以半数抑制浓度和治疗指数为评价指标;定量荧光PCR法和蚀斑法考察甘青乌头体外抗病毒作用机制。用HSV-2建立小鼠脑炎模型,对甘青乌头体内抗单纯疱疹病毒的抗病毒活性进行评价。结果甘青乌头对Vero细胞的半数中毒浓度(CC₅₀)为5.57 g·L^-1。细胞病变法和蚀斑法测得甘青乌头的半数有效浓度(EC₅₀)分别为2.25,1.68 g·L^-1,治疗指数TI分别为2.47,3.32。LD₅₀值为0.85 g·kg^-1。甘青乌头延长了小鼠的平均存活时间,对小鼠的死亡显示出10%的保护率。甘青乌头能直接灭活HSV-2,能够显著抑制HSV-2的感染性;对病毒吸附到细胞没有抑制作用,能抑制HSV-2大分子的增值。抑制HSV-2 DNA合成的抑制倍数达102。结论甘青乌头体外具有较强的抗HSV-2的活性,抗病毒作用是通过抑制病毒复制循环的各个环节起作用的。     

In vitro antiviral activity of Phyllanthus orbicularis extracts against herpes simplex virus type 1

The antiviral activity of butanol- and acetic acid-soluble fractions, prepared from the leaves and stems of Phyllanthus orbicularis H.B.K., has been investigated against acyclovir-sensitive or -resistant herpes simplex virus type 1 (HSV-1) strains, using human foreskin fibroblast (HFF) and green ape kidney (Vero) cell lines. Both fractions showed antiviral selectivity indexes (SI) from 10.3 to 22.8, while their extracellular virucidal activities reached SI values ranging from 371 to 1,040. Time-addition experiments suggested that the active compounds present in the studied fractions acted on early steps of the virus replication cycle.     

Effects of Ilex paraguariensis A. St. Hil. (yerba mate) on herpes simplex virus types 1 and 2 replication

The antiherpes effects of the crude extract obtained from Ilex paraguariensis leaves (yerba mate) and their purified fractions were investigated. The most active fraction was selected and assayed to determine the viral multiplication steps upon which it acted. In order to detect the major components of this fraction, thin layer chromatography (TLC) analysis was performed. The antiviral activity was evaluated against HSV-1 and HSV-2 by a viral plaque number reduction assay (IC(50) ) and the cytotoxicity by a MTT assay (CC(50) ). According to the obtained results, all tested samples showed antiherpes activity at noncytotoxic concentrations, and the ethyl acetate fraction was the most active (SI = CC(50) /IC(50) = 188.7 and 264.7 for HSV-1 and HSV-2, respectively). The results also demonstrated that this fraction exerts antiviral activity by the reduction of viral infectivity, the inhibition of virus entry into cells and cell-to-cell virus spread, as well as by the impaired levels of ICP27, ICP4, gD and gE proteins of HSV-1. The TLC analysis showed that this fraction contains monodesmosidic triterpenoid saponins, matesaponin-1 (a bidesmosidic one), caffeic and chlorogenic acids and rutin, which suggests that they could act synergistically and be responsible for the detected antiherpes activity.     

Evaluation of the antiherpetic activity of standardized extracts of Achyrocline satureioides

Traditionally, Achyrocline satureioides or 'marcela' has been used in South America for the treatment of several disorders. For the present study, three spray-dried extracts (N1, N2 and N3) were used, all of them prepared with 50% of an hydroethanolic extract rich in flavonoid compounds and 50% of blends of different adjuvants. The cytotoxic concentration which causes destruction in 50% monolayer cells (CC50) was 62.5 microg/ml for the three extracts. The antiviral activity was evaluated by using two different strains of herpes simplex virus (HSV-1) and the best results were obtained with KOS strain and N2 extract. Studies concerning the mechanism of the antiherpetic activity demonstrated that N2 extracts showed no virucidal effect or activity on cellular receptors. HSV-1 DNA synthesis was not inhibited. The antiherpetic activity occurred between the second and ninth hour of the virus replication cycle, probably indicating a perturbation on late stages of this cycle.     

Antiherpes virus activity of extracts from the medicinal plant Geranium sanguineum L

The herpes virus inhibitory effect of five extracts from the Bulgarian medicinal plant Geranium sanguineum L. (Geraniaceae) was investigated. The water extract (WE) from the aerial roots of the plant was the least toxic for cell cultures and inhibited significantly the replication of herpes simplex virus type 1 and type 2 (EC50 = 3.6-6.2 microg/ml) as shown by the reduction of virus induced cytopathogenic effect (CPE) and the protection of cells in MTT assay. The inhibition was dose-related, strain-specific and depended on virus inoculum. In higher concentrations (MIC90 = 120 microg/ml) the preparation exhibited strong extracellular virus inactivating activity. The presence of WE throughout the whole replicative cycle was necessary for the full expression of the antiviral effect. In a preliminary experiment in albino guinea pigs the extract delayed the development of herpetic vesicles following primary infection with HSV-1, strain Kupka. Phytochemical investigation of the plant preparation revealed the presence of flavonoids, catechins, a polyphenolic acid and condensed tannins. The inhibitory effect of the extract on herpes simplex virus replication is related to the rich content of polyphenol compounds.     

Antiviral Chinese medicinal herbs against respiratory syncytial virus.

Forty-four medicinal herbs were tested for antiviral activities against respiratory syncytial virus (RSV) by means of the cytopathologic effect (CPE) assay. Twenty-seven of the 44 medicinal herbs showed potent or moderate antiviral activities against RSV with 50% inhibition concentration (IC(50)) ranging from 6.3 to 52.1 microg/ml, and with selectivity index (SI) ranging from 2.0 to 32.1. Further purification of the active extracts from Sophora flavescens Ait. and Scutellaria baicalensis Georgi led to the identification of anagyrine (2), oxymatrine (7), sophoranol (10), wogonin (12), and oroxylin A (13) as the potent anti-RSV components.     

Antiviral flavonoids from the seeds of Aesculus chinensis

A bioassay-guided fractionation of an ethanol extract of the seeds of Aesculus chinensis led to the isolation of two new flavanoids (1 and 2), along with eight known ones (3-10). The structures of the new compounds were elucidated by spectroscopic methods including 2D NMR. All compounds were tested for antiviral activity against respiratory syncytial virus (RSV), parainfluenza virus type 3 (PIV 3), and influenza virus type A (Flu A). Compounds 1, 2, and 6 showed significant antiviral activities against RSV with IC(50) values of 4.5, 6.7, and 4.1 microg/mL and selective index (SI) values of 15.8, 32, and 63.8, respectively. Compound 8 demonstrated significant antiviral activity against Flu A with an IC(50) of 24.5 microg/mL and a SI of 16.0, respectively.     

Antiviral activities of medicinal herbs traditionally used in southern mainland China

The aqueous extracts from 21 medicinal herbs traditionally used in southern mainland China were screened for antiviral activities against human herpes simplex virus type 1 (HSV-1) and human respiratory syncytial virus (RSV) using a cytopathic effect (CPE) reduction assay. Three extracts from Agrimonia pilosa, Pithecellobium clypearia and Punica granatum, respectively, showed anti-HSV-1 activity, which was possibly contributed by the polyphenolic compounds in the herbal extracts. Six of the extracts, from Blumea laciniata, Elephantopus scaber, Laggera pterodonta, Mussaenda pubescens, Schefflera octophylla and Scutellaria indica, respectively, exhibited anti-RSV activity with 50% inhibition (IC50) concentrations ranging from 12.5 to 32 microg/mL, and selective indices (SI) ranging from 11.2 to 40. In addition to polyphenolic compounds, other constituents present in these extracts may also contribute to their anti-RSV activity.     

Antiviral flavans from the leaves of Pithecellobium clypearia

Two new antiviral flavan derivatives were isolated from a methanol extract of leaves of Pithecellobium clypearia as guided by antiviral assays. The structures were characterized, by spectroscopic analyses, as 7-O-galloyltricetifavan (1) and 7,4'-di-O-galloyltricetifavan (2). Cytopathic effect (CPE) reduction assay showed that both compounds 1 and 2 possess antiviral activity against respiratory syncytial virus (RSV), with 50% inhibition concentration (IC(50)) values of 5 and 10 microg/mL, respectively; influenza A (H1N1) virus, with IC(50) values of 15.7 and 30 microg/mL; Coxsackie B3 (Cox B3) virus, with IC(50) values of 12.5 and 25 microg/mL, respectively; and Herpes simplex virus type 1 (HSV-1) with IC(50) values of 30 and 20 microg/mL, respectively. Cytotoxicity evaluation using the MTT assay showed that both compounds 1 and 2 were also moderately toxic to several cultured cell lines.     

In vitro anti-leukemic and antiviral activities of traditionally used medicinal plants in Taiwan

Medicinal plants have been historically used as treatment for different kinds of human diseases. In this study, hot water (HW) extract of five Taiwanese traditionally used medicinal plants was evaluated for their in vitro anti-leukemic (including anti-K562, L1210, P3HR1, Raji and U937 leukemia cells) and antiviral (including HSV-1 and HSV-2) activities. Results showed that Blumea lacera exhibited broad anti-leukemic activity at magnitudes ranging from moderate to mild and Ixeris chinensis is effective at inhibiting the proliferation of K562 cells. B. lacera and Tithonia diversifolia suppressed the replication of HSV-1 and HSV-2, and had IC50 values below 100 microg/ml. The medicinal plants showed no cytotoxic effect at concentrations that inhibited HSV infection. It was, therefore, concluded that the HW extract of tested medicinal plants exhibited anti-leukemic and antiviral activities at different magnitudes of potency.