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.     

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.     

Antiviral activities of various water and methanol soluble substances isolated from Ganoderma lucidum

In order to find antiviral substances from basidiomycetes, two water soluble substances, GLhw and GLlw, and eight methanol soluble substances, GLMe-1-8, were prepared from carpophores of Ganoderma lucidum. These substances were examined for their activities against five strains of pathogenic viruses such as herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), influenza A virus (Flu A) and vesicular stomatitis virus (VSV) Indiana and New Jersey strains in vitro. Antiviral activities were evaluated by the cytopathic effect (CPE) inhibition assay and plaque reduction assay. Five substances, GLhw, GLMe-1, -2, -4 and -7 significantly inhibited the cytopathic effects of HSV and VSV. In the plaque reduction assay, GLhw inhibited plaque formation of HSV-2 with 50% effective concentrations (EC50) of 590 and 580 microg/ml in Vero and HEp-2 cells, and its selectivity indices (SI) were 13.32 and 16.26. GLMe-4 did not exhibit cytotoxicity up to 1000 microg/ml, while it exhibited potent antiviral activity on the VSV New Jersey strain with an SI of more than 5.43. These results indicate the possibility of development of antiviral agents from basidiomycetous fungi.     

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.     

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.     

Antiviral activity in Argentine medicinal plants

In an ethnopharmacological screening of selected medicinal plants used in Argentina for the treatment of infectious diseases, aqueous extracts of five species were assayed in vitro to detect antiviral activity against herpes simplex virus type 1 (HSV-1), respiratory syncytial virus (RSV) and adenovirus serotype 7 (ADV-7). Polygonum punctatum, Lithraea molleoides, Sebastiania brasiliensis and Sebastiania klotzschiana but not Myrcianthes cisplatensis showed in vitro antiherpetic activity with 50% effective dose (ED50) ranging from 39 to 169 microg/ml. P. punctatum, L. molleoides and M. cisplatensis showed antiviral activity against RSV with ED50 ranging from 78 to 120 microg/ml. None of the extracts had antiviral activity against ADV-7. The differences between their maximal non cytotoxic concentration and their antiviral activity values were high enough to justify further analysis.     

Anticancer and antiviral activities of Youngia japonica (L.) DC (Asteraceae, Compositae)

Aqueous and ethanol extracts of Youngia japonica (also known as Oriental hawksbeard) were tested in vitro for anti-tumor activity against three cell lines, human promyelocytic leukaemia (HL-60), human myelogenous leukaemia (chronic K-562) and mouse Sarcoma 180 (S-180), and for antiviral activity against respiratory syncytial virus (RSV), influenza A virus (Flu A) and herpes simplex virus type 1 (HSV-1) by cytopathic effect (CPE) reduction assay. Hot water extract of Youngia japonica inhibited cell proliferation and growth of all cancer cell lines to various extent. K-562 cells were the most sensitive to the extract whereas S-180 cells were the least. It did not show any significant cytotoxic effects on normal mammalian Vero cells up to the concentration of 450 microg/mL. The ethanol extract of whole plant of Youngia japonica exhibited antiviral activity against RSV cultured in HEp-2 cells, but did not have any activity against Flu A and HSV-1. Two partially purified fractions (Fr.10 and Fr.11) from the 95% ethanol extract exhibited significant anti-RSV with 50% inhibitory concentration (IC50) in the range of 3.0-6.0 microg/mL. The ratio of the viral titer reduction in the absence (viral control) and presence of the maximal non-cytotoxic concentration (MNCC) of the Fr.10 and Fr.11 was both estimated to be 1 x 10(4) (RF, viral titer reduction factors), indicating that their anti-RSV activity was high enough to justify for further analysis. Our preliminary analysis showed that the antiviral ingredients were likely to contain phenolic compounds including tannins by chemical tests.     

Potent antiviral potamogetonyde and potamogetonol, new furanoid labdane diterpenes from Potamogeton malaianus

New furanoid labdane diterpenes, potamogetonyde (3) and potamogetonol (4), together with two known compounds, potamogetonin (1) and 15,16-epoxy-12-oxo-8(17),13(16),14-labdatrien-20,19-olide (2), were isolated from the CH(2)Cl(2) extract of Potamogeton malaianus. The chemical structures of 1-4 were elucidated by the analyses of their spectral data, mainly by 1D and 2D NMR techniques. Potamogetonyde (3) and potamogetonol (4) exhibited potent antiviral (HSV-1) activity with respective IC(50) values of 8 and 3 microg/mL. Compounds 1-4 possessed cytotoxicity toward insect cells (fall armyworm and mosquito larvae, IC(50) of 11-72 microg/mL). Furanoid diterpenes 3 and 4 also exhibited cytotoxicity against the Vero cell line with respective IC(50)'s of 31 and 28 microg/mL, while 1 and 2 were inactive at 50 microg/mL. Compounds 1-4 were inactive (at 20 microg/mL) against KB and BC cell lines and showed only weak antimycobacterial activity against Mycobacterium tuberculosis H37Ra with minimum inhibitory concentrations of 50-100 microg/mL.     

An antiviral principle present in a purified fraction from Melia azedarach L. leaf aqueous extract restrains herpes simplex virus type 1 propagation

Meliacine (MA), an antiviral principle isolated from leaves of Melia azedarach L., exhibits potent antiviral activity against herpes simplex virus type 1 (HSV-1) by inhibiting specific infected-cell polypeptides (ICPs) produced late in infection. Some of these are involved in DNA synthesis and in the assembly of nucleocapsids. The present report provides additional evidence to elucidate the mode of action of MA against HSV-1. Time-of-addition experiments confirmed that MA affects a late event in the multiplication cycle of HSV-1. We showed that MA diminished the synthesis of viral DNA and inhibited the spread of infectious viral particles when HSV-1 that expresses beta-galactosidase activity was used. In addition, the lack of a protein with an apparent MW of 55 KD was detected in MA-treated cell extracts. Ultrastructural analysis of infected cells showed that, in the case of MA treatment, a large number of unenveloped nucleocapsids accumulated in the cytoplasm and a minor proportion of mature virus was found in cytoplasmic vesicles.These findings suggest that MA exerts an antiviral action on both the synthesis of viral DNA and the maturation and egress of HSV-1 during the infection of Vero cells.     

川楝子提取物体外抗单纯疱疹病毒1型活性的实验研究

目的在体外研究川楝子提取物抗单纯疱疹病毒1型(HSV-1)的作用。方法以Vero细胞为宿主细胞,阿昔洛韦为阳性对照药物,通过观察细胞病变效应(CPE)与空斑减数实验测定川楝子提取物抗HSV-1活性,计算其IC50与治疗指数,并从药物对病毒的直接灭活作用、对病毒吸附的影响及对病毒穿膜的影响三个方面初探川楝子提取物抗HSV-1活性的机理。结果川楝子提取物能明显抑制HSV-1的致病变作用,其IC50为18.26μg/mL,TI值为18。研究显示,川楝子提取物在体外对HSV-1直接灭活的效果明显,而对HSV-1吸附与穿入细胞的抑制作用较差。结论川楝子提取物在体外有明显的抗HSV-1感染作用,且主要是通过直接灭活HSV-1而发挥作用。     

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.     

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.     

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.     

藏药紫金标抗HSV-1的作用机理研究

目的 :探讨紫金标抗单纯疱疹Ⅰ型病毒 (HSV-1)的作用及机理 ,为进一步开发该药提供理论依据。方法 :采用不同剂量的紫金标作用于适量HSV-1感染的Vero细胞 ,以 50%组织细胞感染量 (TCID₅₀ ) ,细胞病变效应(CPE) ,MTT法和核酸分子杂交作为评价指标。结果 :MTT法测得紫金标的 50 %抑制浓度 (IC₅₀ )为 29.46mg·L^-1,50%中毒浓度 (TC₅₀ )为 1 077mg·L^-1,治疗指数 (TI)为 36.56 ,结果表明紫金标有明显抑制HSV-1的作用 ,其作用强度和有效时间与药物浓度成正比。紫金标无直接灭活HSV-1的作用 ,也不能影响病毒的释放 ;但可干扰HSV -1对宿主细胞的吸附。不同浓度的紫金标能明显抑制HSV-1gD基因复制和mRNA表达。结论 :紫金标具有显著的抗HSV-1的作用 ,能抑制HSV-1对宿主细胞的吸附以及抑制HSV-1gD基因复制与转录。     

千金藤素抗单纯疱疹病毒Ⅰ型(HSV-1)初探

目的:体外测定千金藤素抗单纯疱疹病毒Ⅰ型的作用,为筛选新型抗病毒药物提供参考依据.方法:用不同稀释度的千金藤素抑制单纯疱疹病毒Ⅰ型对Vero细胞的感染作用,48 h后观察细胞病变(CPE)效应.结果:千金藤素能明显抑制HSV-1对Vero细胞的致病变作用,使细胞存活率升高.结论:千金藤素有较显著的抗HSV-1的作用,且抗病毒作用是多方面的,是一种具有潜在开发前景的药物.     

In vitro cytotoxic,antiviral and immunomodulatory effects of Plantago major and Plantago asiatica

Plantago major linn. and P. asiatica Linn. (Plantaginaceae) are commonly used as folk medicine in Taiwan for treating infectious diseases related to the respiratory, urinary and digestive tracts. In this study, we investigated the antiviral, cytotoxic and immunomodulatory activities of hot water extracts of these two species in vitro on a series of viruses, namely herpesviruses (HSV-1 and HSV-2), adenoviruses (ADV-3, ADV-8 and ADV-11), and on various human leukemia, lymphoma and carcinoma cells with XTT, BrdU and IFN-gamma kits. Results showed that hot water extract of P. asiatica possessed significant inhibitory activity on the proliferation of lymphoma (U937) and carcinoma (bladder, bone, cervix, kidney, lung and stomach) cells and on viral infection (HSV-2 and ADV-11). P. major and P. asiatica both exhibited dual effects of immunodulatory activity, enhancing lymphocyte proliferation and secretion of interferon-gamma at low concentrations (< 50 microg/ml), but inhibiting this effect at high concentration (> 50 microg/ml). The present study concludes that hot water extracts of P. major and P. asiatica possess abroad-spectrum of antileukemia, anticarcinoma and antiviral activities, as well as activities which modulate cell-mediated immunity. Further investigations to elucidate the active component(s) of P. asiatica and P. major and to evaluate their clinical application are warranted.     

Antiviral activity of Undaria pinnatifida against herpes simplex virus

The major component of an aqueous extract of the seaweed Undaria pinnati fi da has been identified previously as a galactofucan (GFS), a sulfated polysaccharide. The galactofucan was partially purified and the material tested in this study is 75% pure galactofucan sulfate. GFS was evaluated for antiviral activity against 32 clinical strains of herpes simplex virus (HSV): 14 strains of HSV-1 and 18 strains of HSV-2. Twelve strains (four HSV-1 and eight HSV-2) were resistant to acyclovir (ACV-R) and 20 strains (10 HSV-1 and 10 HSV-2) were susceptible to ACV (ACV-S). The median IC(50) of GFS for the 14 strains of HSV-1 was 32 micro g/mL. The median IC(50) of GFS for the 18 strains of HSV-2 was 0.5 micro g/mL. GFS is significantly more active against clinical strains of HSV-2 than HSV-1, p < 0.001. The mode of action of the GFS was shown to be the inhibition of viral binding and entry into the host cell. The cytotoxicity of GFS was >4.0 mg/mL in the neutral red dye uptake assay indicating that GFS is non-toxic in this assay.     

蜈蚣藻多糖的提取分离及抗病毒活性的研究

目的:提取分离蜈蚣藻多糖,在体外检测不同浓度蜈蚣藻对单纯疱疹病毒Ⅱ型的抑制作用。方法:用五种不同的提取分离方法从蜈蚣藻中提取硫酸多糖,再从药物对细胞的保护、对HSV-2增殖的影响及对HSV-2感染细胞的综合作用三个方面研究不同浓度的蜈蚣藻抑制HSV-2对Vero细胞的感染作用,细胞病变效应法(CPE)观察和MTT法测定蜈蚣藻粗多糖抗HSV-2活性。结果:蜈蚣藻粗多糖能明显抑制HSV-2对Vero细胞的致病变作用,其中水提醇沉法所得的蜈蚣藻多糖的IC50为5.80μg/m。l结论:蜈蚣藻有较显著的抗HSV-2活性,且初步推测其抗HSV-2活性是作用在HSV-2和受体结合、吸附、侵入Vero细胞阶段,是一种具有潜在开发前景的药物。     

Effect of the Potent Antiviral 1‐Cinnamoyl‐3,11‐Dihydroxymeliacarpin on Cytokine Production by Murine Macrophages Stimulated with HSV‐2

The limonoid 1‐cinnamoyl‐3,11‐dihydroxymeliacarpin (CDM) isolated from leaf extracts of Melia azedarach L, has potent antiherpetic effect in epithelial cells. Since Meliacine, the partially purified extract source of CDM, has therapeutic effect on murine genital herpes, the potential use of CDM as microbicide against herpetic infections was studied here. To determine the cytotoxic effect of CDM, the MTT assay and acridine orange staining of living cells were performed. The antiherpetic action of CDM was measured by plaque reduction assay, and the immunomodulatory effect was determined by measuring the cytokine production using a bioassay and ELISA method. The results presented here showed that CDM inhibited Herpes Simplex Virus type 2 (HSV‐2) multiplication in Vero cells but did not affect its replication in macrophages which were not permissive to HSV infection. In macrophages, levels of TNF‐α, IFN‐γ, NO, IL‐6 and IL‐10 were increased by CDM used alone or in combination with HSV‐2. Besides, CDM not only synergized TNF‐α production combined with IFN‐γ, but also prolonged its expression in time. Results indicate that CDM inhibits HSV‐2 multiplication in epithelial cells and also increases cytokine production in macrophages, both important actions to the clearance of infecting virus in the mouse vagina. Copyright © 2013 John Wiley & Sons, Ltd.