Anti-viral activity of the extracts of a Kenyan medicinal plant Carissa edulis against herpes simplex virus

Herpes simplex virus (HSV) infection is a major opportunistic infection in immunosuppressed persons. It is therefore a serious disease in high HIV/AIDS prevalence areas as in sub-Saharan Africa where infections due to HSV have risen significantly. The development of resistant strains of HSV to the available drugs for infection management, as is evident in the first drug of choice acyclovir, has further compounded this situation. There is therefore an urgent need to identify and develop new alternative agents for management of HSV infections, more so, for those due to resistant strains. We report here on an aqueous total extract preparation from the roots of Carissa edulis (Forssk.) Vahl (Apocynaceae), a medicinal plant locally growing in Kenya that has exhibited remarkable anti-HSV activity in vitro and in vivo for both wild type and resistant strains of HSV. The extract significantly inhibited formation of plaques in Vero E6 cells infected with 100PFU of wild type strains of HSV (7401H HSV-1 and Ito-1262 HSV-2) or resistant strains of HSV (TK(-) 7401H HSV-1 and AP(r) 7401H HSV-1) by 100% at 50 microg/ml in vitro with minimal cell cytotoxicity (CC(50)=480 microg/ml). When the extract was examined for in vivo efficacy in a murine model using Balb/C mice cutaneously infected with wild type or resistant strains of HSV, the extract at an oral dose of 250 mg/kg significantly delayed the onset of HSV infections by over 50%. It also increased the mean survival time of treated infected mice by between 28 and 35% relative to the infected untreated mice (p<0.05 versus control by Student's t-test). The mortality rate for mice treated with extract was also significantly reduced by between 70 and 90% as compared with the infected untreated mice that exhibited 100% mortality. No acute toxicity was observed in mice at the oral therapeutic dose of 250 mg/kg. These results suggest that this herbal extract has potent anti-viral agents against herpes simplex viruses that can be exploited for development of an alternative remedy for HSV infections.     

Evaluation of anti-HSV-2 activities of Barleria lupulina and Clinacanthus nutans

Barleria lupulina Lindl. and Clinacanthus nutans (Burm. f.) Lindau, both belonging to the family Acantaceae, are well-known medicinal plants used in Thai folklore medicine. Virucidal effects of organic extracts of these two plants against herpes simplex virus type 2 strain G, HSV-2 (G), the standard HSV-2 strain were noted. The extracts were assessed for intracellular activities against HSV-2 (G) and five clinical HSV-2 isolates. B. lupulina extract exhibited activity against all five isolates but not the standard strain while that of C. nutans did not show any activity against these viruses as determined by plaque inhibition assay. When the activities were verified by yield reduction assay, anti-HSV-2 activities of B. lupulina extract were observed against HSV-2 (G) as well. The results suggest a therapeutic potential of B. lupulina but not C. nutans against HSV-2.     

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.     

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 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.     

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.     

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.     

In vitro and in vivo activity of eugenol on human herpesvirus

Eugenol (4-allyl-1-hydroxy-2-methoxybenzene) was tested for antiviral activity against HSV-1 and HSV-2 viruses. In vitro, it was found that the replication of these viruses was inhibited in the presence of this compound. Inhibitory concentration 50% values for the anti-HSV effects of eugenol were 25.6 microg/mL and 16.2 microg/mL for HSV-1 and HSV-2 respectively, 250 microg/mL being the maximum dose at which cytotoxicity was tested. Eugenol was virucidal and showed no cytotoxicity at the concentrations tested. Eugenol-acyclovir combinations synergistically inhibited herpesvirus replication in vitro. Topical application of eugenol delayed the development of herpesvirus induced keratitis in the mouse model.     

裸花紫珠不同提取部位体外抗单纯疱疹病毒Ⅰ型作用研究

目的:研究裸花紫珠不同提取部位体外抗单纯疱疹病毒Ⅰ型(HSV-1)的活性,为进一步分离纯化寻找抗HSV-1的活性组分做准备。方法:系统分离法分别制备裸花紫珠石油醚、乙酸乙酯、正丁醇部位以及相应的敲除部位,采用HSV-1感染的Hep-2细胞体外模型,进行体外抗HSV-1活性评价的评价,将制备的各部位抗病毒活性与全成分进行比较,确定裸花紫珠体外抗HSV-1的活性部位。结果:裸花紫珠乙酸乙酯部位的治疗指数TI与全成分组相当,正丁醇部位和敲出石油醚部位的TI略低于全成分组,其他组无效。结论:乙酸乙酯部位为裸花紫珠抗HSV-1的主要药效部位。     

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.     

Attachment and Penetration of Acyclovir‐resistant Herpes Simplex Virus are Inhibited by Melissa officinalis Extract

Medicinal plants are increasingly of interest as novel source of drugs for antiherpetic agents, because herpes simplex virus (HSV) might develop resistance to commonly used antiviral drugs. An aqueous extract of Melissa officinalis and the phenolic compounds caffeic acid, p‐coumaric acid and rosmarinic acid were examined for their antiviral activity against herpes simplex virus type 1 (HSV‐1) acyclovir‐sensitive and clinical isolates of acyclovir‐resistant strains in vitro. When drugs were added during the intracellular replication of HSV‐1 infected cells, no antiviral effect was observed by plaque reduction assay. However, Melissa extract interacted directly with free viral particles of two acyclovir‐resistant HSV strains at low IC₅₀ values of 0.13 and 0.23 µg/mL and high selectivity indices of 2692 and 1522, respectively. The Melissa extract and rosmarinic acid inhibited HSV‐1 attachment to host cells in a dose‐dependent manner for acyclovir‐sensitive and acyclovir‐resistant strains. These results indicate that mainly rosmarinic acid contributed to the antiviral activity of Melissa extract. Penetration of herpes viruses into cells was inhibited by Melissa extract at 80% and 96% for drug‐sensitive and drug‐resistant viruses, respectively. Melissa extract exhibits low toxicity and affects attachment and penetration of acyclovir‐sensitive and acyclovir‐resistant HSVs in vitro. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Herpes virus inhibitory substances from Hypericum connatum Lam., a plant used in southern Brazil to treat oral lesions

Hypericum connatum (Guttiferae) is used in southern Brazil in the treatment of lesions in the mouth, often related to acute herpetic gingivo-stomatitis. The chemical investigation of the plant revealed the presence of phloroglucinol derivatives and flavonoids. From the n-hexane extract of the aerial parts a phloroglucinol derivative, hyperbrasilol B, was isolated, while the methanolic extract afforded four flavonoids: amentoflavone, hyperoside, guaijaverine and luteoforol. The crude methanolic extract and fractions (n-hexane, dichloromethane and methanol) as well as the isolated compounds were tested for antiviral activity against herpes simplex viruses (HSV). Among the tested samples, luteoforol was the most active inhibiting the cytopathic effect (CPE) and reducing the viral titer of HSV-1 DNA viral strains KOS and VR733 (ATCC).     

夏枯草多糖及凝胶抗单纯疱疹病毒的药效学研究

目的：探究夏枯草多糖及凝胶在体外和体内条件下的抗单纯疱疹病毒作用,为抗疱疹病毒新药研究提供科学依据。方法：本研究采用空斑减数法检测夏枯草多糖的体外抗单纯疱疹病毒（Herpes Simplex Virus,HSV）活性;同时,建立HSV-1（皮肤）和HSV-2（外阴部）病毒感染豚鼠模型,以病灶病变情况、丘疱疹数、典型病变评分结果以及病灶组织中HSV-1和HSV-2病毒的DNA拷贝数等为指标,对夏枯草多糖凝胶的体内抗HSV活性进行评价。结果：夏枯草多糖在体外实验中能够抑制HSV-1和HSV-2的活性;夏枯草多糖凝胶能够削弱HSV-1和HSV-2病毒感染豚鼠的病灶病变,发挥抗单纯疱疹病毒活性。结论：夏枯草多糖及凝胶在体外和体内实验中均具有抗单纯疱疹病毒的活性,具有开发成为抗单纯疱疹病毒药物的潜在价值。     

单纯疱疹病毒实时PCR检测以及与巢式PCR和病毒培养方法的比较

目的：在Lightcycler系统上建立和评价HSV-1、HSV-2型病毒的实时定量PCR方法,并与巢式PCR和病毒分离培养的结果进行比较。方法：120例从疑似单纯疱疹病毒感染患儿口腔黏膜及唇黏膜取样,进行实时PCR,巢式PCR和病毒分离检测HSV-1、HSV-2的感染情况。结果：HSV-1阳性率：病毒分离和巢式PCR分别为34/120（28.3%）和36/120（30%）,实时PCR为38/120（31.7%）;HSV-2的检测：病毒分离阳性率为5/120（4.2%）,巢式PCR阳性率为7/120（5.8%）,实时PCR阳性率为8/120（6.7%）。结论：实时PCR增加了单纯疱疹病毒DNA检出率,尤其是和病毒分离相比。实时PCR和巢式PCR在敏感性上没有显著性差异。实时PCR技术具有快速扩增,降低污染风险的优势,它是诊断单纯疱疹病毒感染的一种合适方法。     

Anti-oxidant and inflammatory mediator's growth inhibitory effects of compounds isolated from Phyllanthus urinaria

Phyllanthus urinaria Linnea (Euphorbiaceae), is a traditional anti-hepatitis herb used in Taiwan. In continuation of our search for potent natural anti-inflammatory agents, from the ethanolic extract of this plant, nine compounds including phyllanthin (1), phyltetralin (2), trimethyl-3,4-dehydrochebulate (3), methylgallate (4), and rhamnocitrin (5), methyl brevifolincarboxylate (6), beta-sitosterol-3-O-beta-d-glucopyranoside (7), quercitrin (8), and rutin (9) were isolated. The structures of compounds 3 and 6 were established based on NMR and mass spectral studies. The isolates 1-9 were investigated for their antioxidant, and anti-inflammatory activities in vitro. In the antioxidant assay, the isolates 3, 4 and 6 exhibited significant DPPH radical scavenging activity with an IC(50) value of 9.4, 9.8 and 8.9 microM, respectively. On the other hand, in the inflammatory mediators growth inhibitory assay from LPS/interferon (IFN)-gamma-activated peritoneal macrophages, all the isolates except 7, significantly and dose-dependently inhibited the enhanced production of NO radicals, and such modulation was closely associated with the inhibition of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In addition, 30 microM of isolates 3 and 6, and 50 microM of 4, significantly arrest the mitogen-stimulated spleen cells in G0/G1 stage. This is the first report on Phyllanthus urinaria isolates for their growth inhibitory activities against inflammatory mediators, in addition to spleen cell cycle arrest in G0/G1 stage. Therefore, these isolates from Phyllanthus urinaria may be useful for the treatment of cell-mediated immune diseases.     

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.     

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.     

Antiviral activity of the red marine alga Ceramium rubrum

An extract from the red marine alga Ceramium rubrum (Huds.) Ag. from the Bulgarian Black Sea seacoast considerably inhibited the reproduction of influenza viruses type A and B in vitro and in ovo. The virus-induced cytopathogenic effect (CPE), infectious virus yields and the production of hemagglutinin were all reduced at non-toxic concentrations of the extract. The virus-inhibitory effect was selective, dose-related and strain-specific; selectivity indices ranged 9.5-68.3. The inhibition affected adsorption as well as the intracellular stages of viral replication. The extract inhibited also the reproduction of herpes simplex virus (HSV) type 1 and type 2 in cell cultures. The preparation exhibited a strong HSV-inactivating activity.     

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.