Anti-Influenza A Virus Activity of Rhamnan Sulfate from Green Algae Monostroma nitidum in Mice with Normal and Compromised Immunity.

Influenza viruses cause a significant public health burden each year despite the availability of anti-influenza drugs and vaccines. Therefore, new anti-influenza virus agents are needed. Rhamnan sulfate (RS) is a sulfated polysaccharide derived from the green alga Monostroma nitidum. Here, we aimed to demonstrate the antiviral activity of RS, especially against influenza A virus (IFV) infection, in vitro and in vivo. RS showed inhibitory effects on viral proliferation of enveloped viruses in vitro. Evaluation of the anti-IFV activity of RS in vitro showed that it inhibited both virus adsorption and entry steps. The oral administration of RS in IFV-infected immunocompetent and immunocompromised mice suppressed viral proliferation in both mouse types. The oral administration of RS also had stimulatory effects on neutralizing antibody production. Fluorescent analysis showed that RS colocalized with M cells in Peyer's patches, suggesting that RS bound to the M cells and may be incorporated into the Peyer's patches, which are essential to intestinal immunity. In summary, RS inhibits influenza virus infection and promotes antibody production, suggesting that RS is a potential candidate for the treatment of influenza virus infections.

Characterization of ent-kaurene synthase and kaurene oxidase involved in gibberellin biosynthesis from Scoparia dulcis.

Gibberellins (GAs) are ubiquitous diterpenoids in higher plants, whereas some higher plants produce unique species-specific diterpenoids. In GA biosynthesis, ent-kaurene synthase (KS) and ent-kaurene oxidase (KO) are key players which catalyze early step(s) of the cyclization and oxidation reactions. We have studied the functional characterization of gene products of a KS (SdKS) and two KOs (SdKO1 and SdKO2) involved in GA biosynthesis in Scoparia dulcis. Using an in vivo heterologous expression system of Escherichia coli, we found that SdKS catalyzed a cyclization reaction from ent-CPP to ent-kaurene and that the SdKOs oxidized ent-kaurene to ent-kaurenoic acid after modification of the N-terminal region for adaptation to the E. coli expression system. The real-time PCR results showed that the SdKS, SdKO1 and SdKO2 genes were mainly expressed in the root and lateral root systems, which are elongating tissues. Based on these results, we suggest that these three genes may be responsible for the metabolism of GAs in S. dulcis.

In vitro and in vivo antiherpetic effects of (1R,2R)-1-(5'-methylful-3'-yl)propane-1,2,3-triol.

In this study, we demonstrated the in vitro and in vivo antiherpetic activities of a stable furan derivative, (1R,2R)-1-(5'-methylful-3'-yl)propane-1,2,3-triol (MFPT), which had originally been isolated from Streptomyces sp. strain FV60. In the present study, we synthesized MFPT from (5-methylfuran-3-yl)methanol in 6 steps for use in the experiments. MFPT showed potent in vitro antiviral activities against two acyclovir (ACV)-sensitive (KOS and HF) strains and an ACV-resistant (A4-3) strain of herpes simplex virus type 1 (HSV-1) and an ACV-sensitive HSV type 2 (HSV-2) UW 268 strain, their selectivity indices ranging from 310 to 530. By intravaginal application of MFPT to mice, the virus yields decreased dose-dependently against the three strains of HSV-1 and HSV-2. When MFPT was applied at a dose of 1.0 mg/day, the lesion scores, as clinical signs manifested by viral infection, were extensively suppressed in HSV-1-infected mice, whereas the lesion scores in HSV-2-infected mice were not markedly decreased. Interestingly, MFPT exerted an inhibitory effect against ACV-resistant HSV-1 in mice to a similar degree as in ACV-sensitive HSV-1-infected mice. Therefore, the compound might have potential for developing a topical antiviral agent that could be also applied to the infections caused by ACV-resistant viruses.

Characterization of 12-Oxophytodienoic Acid Reductases from Rose-scented Geranium (Pelargonium graveolens).

Pelargonium graveolens L'Hér, also referred to as rose geranium, is a popular herbal plant with typical rosy fragrance largely based on the blend of monoterpenoid constituents. Among them, citronellol, which is biosynthesized from geraniol via double bond reduction, is the most abundant scent compound. In this study, three 12-oxophytodienoic acid reductases (PgOPRl-3) hive been cloned from P. graveolens, as -possible candidates for the double-bond reductase involved in citronellol biosynthesis. The bacterially expressed recombinant PgOPRs did not reduce geraniol to citronellol, but stereoselectively converted citral into (S)-citronellal in the presence of NADPH. Thus, the a,-unsaturated carbonyl moiety in the substrate is essential for the catalytic activity of PgOPRs; as reported for OPRs from other plants and structurally related yeast old yellow enzymes. PgOPRs promiscuously accepted linear and cyclic α,ß- uisaturated carbonyl substrates, including methacrolein, a typical reactive carbonyl compound. The possible biotechnological applications for PgOPRs in plant metabolic'engineering, based on their catalytic properties, are discussed herein.

Enhanced Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Coexpression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Escherichia coli.

Two genes involved in δ-guaiene biosynthesis in Aquilaria microcarpa, δ-guaiene synthase (GS) and famesyl diphosphate synthase (FPS), were overexpressed in Escherichia coli cells. Immunoblot analysis revealed that the concentration of GS-translated protein was rather low in the cells transformed by solely GS while appreciable accumulation of the recombinant protein was observed when GS was coexpressed with FPS. GS-transformed cells liberated only a trace amount of δ-guaiene (0.004 µg/mL culture), however, the concentration of the compound elevated to 0.08 pg/mL culture in the cells transformed by GS plus FPS. δ-Guaiene biosynthesis was markedly activated when E. coli cells coexpressing GS and FPS were incubated in enriched Terrific broth, and the content of the compound increased to approximately 0.6 µg/mL culture. These results suggest that coexpression of FPS and GS in E. coli is required for efficient δ- guaiene production in the bacterial cells, and the sesquiterpene-producing activity of the transformant is appreciably enhanced in the nutrients-enriched medium.

The biosynthetic activities of primary and secondary metabolites in suspension cultures of Aquilaria microcarpa.

Two types of suspension-cultured Aquilaria microcarpa cells, friable and aggregated, were selectively generated. The biosynthetic activities of primary and secondary metabolites in target cells were detected using laser scanning microscopy (LSM) imaging with diphenylboric acid 2-amino ethyl ester (DPBA) and 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) staining. Scanned friable cells produced weakly fluorescent images revealing low productivity of metabolites. On the other hand, scanning of aggregated cells produced clear fluorescent images depicting the accumulations of flavonoids and lipids. Furthermore, abundant deposition of an unknown resinous compound in extracellular portion of aggregated cells could be visualized. The resinous compound was white to whitish-gray in color and highly sedimented in the medium. Based on these observations, we focused our investigation of metabolite productivity on aggregated suspension cells. Some prominent extracellular compounds were detected in the used liquid medium, as well as in the resinous residue within the medium. The characteristics of these metabolites were investigated in detail via gas chromatography-mass spectrometry (GC-MS) analysis.

Characterization and biological effects of two polysaccharides isolated from Acanthopanax sciadophylloides.

Two polysaccharides abbreviated ANP and AAP were isolated from the young buds of Acanthopanax sciadophylloides. ANP consisted of L-arabinose, D-mannose, D-glucose and D-galactose in a ratio of ca 1.0:2.6:2.5:1.4 and its weight average molecular weight (Mw) was 1.07×10(4). AAP consisted of L-arabinose, D-galactose and 4-O-methyl-D-glucuronic acid in a ratio of ca 5:10:1, and its Mw was estimated to be 8.40×10(4). ANP was suggested to be an acetylated heteropolysaccharide, whereas AAP was speculated to be a type II arabinogalactan on the basis of structural analysis data. Both polysaccharides were found to stimulate NO production and induce the expression of cytokine mRNAs including IL-1ß, IL-6, IL-10 and TNF-α on RAW264.7 cells. They also induced NF-κB activation in RAW-Blue cells. NO production and NF-κB activation by both polysaccharides were decreased by pretreatment with neutralizing anti-TLR-4 and anti-CD14 antibodies but not with anti-TLR-2, anti-SR-A, anti-CD11c, and anti-Dectin-1 antibodies. Therefore, these immunostimulating effects of ANP and AAP were suggested to be promoted by the interaction through the membrane receptors, TLR-4 and CD14. In addition to immunomodulating effects, ANP showed anti-HSV-2 effects in vitro.

Topical application of polyethylenimine as a candidate for novel prophylactic therapeutics against genital herpes caused by herpes simplex virus.

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) cause genital herpes, which can enhance the acquisition of human immunodeficiency virus. The development of anti-HSV agents with novel mechanisms of action is urgently required in the topical therapy of genital herpes. In this study, the in vitro and in vivo anti-HSV effects of Epomin SP-012(®), a highly cationic polyethylenimine, were evaluated. When the in vitro antiviral effects of SP-012 were assessed, this compound showed potent activity against HSV-1 and HSV-2. It inhibited the attachment of HSV-2 to host cells and cell-to-cell spread of infection in a concentration-dependent manner and exerted a virucidal effect. No SP-012-resistant HSV-2 was found when the virus was successively passaged in the presence of SP-012. In a mouse genital herpes model, topically administered SP-012 inhibited the progression of the disease caused by HSV infection. These data illustrate that SP-012 may be a novel class of HSV inhibitor that would be acceptable for long-term topical application.

Induction, cloning and functional expression of a sesquiterpene biosynthetic enzyme, δ-guaiene synthase, of Aquilaria microcarpa cell cultures.

A homology-based cloning strategy yielded a cDNA clone presumably encoding δ-guaiene synthase, a sesquiterpene cyclase, from tissue cultures of Aquilaria microcarpa, which were treated with methyl jasmonate. Incubation of cell cultures of the plant with yeast extract also induced transcriptional activation of the sesquiterpene synthase gene. The translated protein of the gene obtained by heterologous expression in Escherichia coli catalyzed the cyclization of farnesyl diphosphate to liberate δ-guaiene with δ-guaiene and germacrene A as the minor products. The results obtained in the present study, together with the previously reported results, suggest that two classes of δ-guaiene synthase occur in Aquilaria; the enzyme proteins from A. microcarpa and A. sinensis liberate germacrene A as a minor product, while the protein from A. crassna generates α-humulene instead of germacrene A.

Daurichromenic acid-producing oxidocyclase in the young leaves of Rhododendron dauricum.

Rhododendron dauricum L., a flowering tree popular in Hokkaido, produces daurichromenic acid (DCA), a terpenophenol with a potent anti-HIV activity. The DCA-producing enzyme, named DCA synthase, could be detected in the soluble protein fraction prepared from the young leaves of R. dauricum. DCA synthase catalyzed oxidocyclization of the farnesyl group of grifolic acid to form (+)-DCA as the major reaction product. The DCA synthase reaction proceeds without the need for any cofactors and coenzymes except for molecular oxygen. Interestingly, these catalytic properties of DCA synthase are quite similar to those reported for cannabinoid synthases in the marijuana plant Cannabis sativa L.

Anti-influenza A virus effects of fructan from Welsh onion (Allium fistulosum L.).

A fructan that acts as an anti-influenza A virus substance was isolated from hot water extract of the green leafy part of a Welsh onion (Allium fistulosum L.). The structure of the fructan was characterised and elucidated by chemical and spectroscopic analyses. The fructan was composed of terminal (21.0%) and 2,1-linked ß-D-Fruf residues (65.3%) with 1,6-linked ß-D-Glcp residues (13.7%). The molecular weight of the polysaccharide and polydispersity was estimated to be 1.5×10(3) and 1.18, respectively. Although the fructan did not show anti-influenza A virus activity in vitro, it demonstrated an inhibitory effect on virus replication in vivo when it was orally administered to mice. In addition, the polysaccharide enhanced the production of neutralising antibodies against influenza A virus. Therefore, the antiviral mechanism of the polysaccharide seemed to be dependent on the host immune system, i.e., enhancement of the host immune function was achieved by the administration of the polysaccharide. From our observations, the fructan from Welsh onions is suggested to be one of the active principles which exert an anti-influenza virus effect.

Structures and anti-HSV-2 activities of neutral polysaccharides from an edible plant, Basella rubra L.

Four neutral polysaccharides (BRN-1, BRN-2, BRN-3 and BRN-4) were isolated from the hot water extract of the aerial part of Basella rubra L. They were found to consist of a large amount of D-galactose (81.0-92.4%) and small amounts of L-arabinose (5.4-7.8%), D-glucose (2.2-11.0%) and mannose (~2.9%). Linkage analysis revealed that all these neutral polysaccharides might be arabinogalactan type I polysaccharides in different molecular weight and chain length. Among them, only BRN-3 showed antiviral activity against herpes simplex virus type 2 (HSV-2) with 50% inhibitory concentration of 55 µg/mL without showing the cytotoxicity up to 2300 µg/mL. Furthermore, the main antiviral target of BRN-3 was shown to be the inhibition of virus adsorption to host cells. This is the first report on the neutral polysaccharide with anti-HSV-2 activity obtained from B. rubra L.

Antiviral and immunostimulating effects of lignin-carbohydrate-protein complexes from Pimpinella anisum.

Three antiviral and immunostimulating substances (LC1, LC2 and LC3) were isolated from a hot water extract of seeds of Pimpinella anisum by combination of anion-exchange, gel filtration and hydrophobic interaction column chromatographies. Chemical and spectroscopic analyses revealed them to be lignin-carbohydrate-protein complexes. These lignin-carbohydrate complexes (LCs) showed antiviral activities against herpes simplex virus types 1 and 2 (HSV-1 and -2), human cytomegalovirus (HCMV) and measles virus. LCs were also found to interfere with virus adsorption to the host cell surface and directly inactivate viruses. Furthermore, they enhanced nitric oxide (NO) production by inducing iNOS mRNA and protein expression in RAW 264.7 murine macrophage cells. The induced mRNA expression of cytokines including IL-1ß and IL-10 was also apparent. These results suggest that the lignin-carbohydrate-protein complexes from P. anisum possessed potency as functional food ingredients against infectious diseases.

Potent virucidal effect of pheophorbide a and pyropheophorbide a on enveloped viruses.

In this study, we evaluated the inhibitory effect of ethanol and aqueous extracts from a stem of Opuntia ficus indica on replication of three kinds of viruses: two enveloped viruses [herpes simplex virus type 2 (HSV-2), influenza A virus (IFV-A)], and one non-enveloped virus [poliovirus type 1 (PV-1)]. Only ethanol extract from the cactus stem showed significant antiviral activity in vitro. Two chlorophyll derivatives, pheophorbide a and pyropheophorbide a, were isolated as active substances exhibiting potent virucidal effects on HSV-2 and IFV-A, but no activity against PV-1 was observed. These findings suggest that these active compounds might recognize specific glycoproteins of enveloped viruses, precluding their binding to host cell receptors and inhibiting viral infections.

Inhibitory effect of methanol extract of Ganoderma lucidum on acute itch-associated responses in mice.

In this study, the antipruritic effect of the methanol extract of Ganoderma lucidum (MEGL) was studied in mice. Oral administration of MEGL (10-1000 mg/kg) produced a dose-dependent inhibition of scratching, an itch-related response, induced by intradermal 5-hydroxytryptamine (5-HT) (100 nmol/site), alpha-methyl-5-HT (100 nmol/site), and proteinase-activated receptor-2 (PAR(2))-activating peptide SLIGRL-NH(2) (50 nmol/site). However, MEGL (100-1000 mg/kg) did not inhibit the scratching induced by histamine (100 nmol/site), substance P (100 nmol/site), and compound 48/80 (10 microg/site). These results raise the possibility that MEGL is effective against pruritus mediated by proteinases and 5-HT and that primary afferents expressing PAR(2) and 5-HT(2A) receptors are the sites of its action.

Two new monoterpenes from Sibiraea angustata.

Two novel monoterpenes, sibiscolacton (1) and sibiraic acid (2), were isolated from the aerial part of Sibiraea angustata RCHD: . along with seven known compounds, namely three phenylpropanoids (3-5), two flavonoids (6,7), one glucityl ferulate (8, sibirate), and a monoterpene glucoside (9, sibiskoside). The structures of 1 and 2 were established on the basis of spectroscopic and chemical data.

Evaluation of chikusetsusaponin IVa isolated from Alternanthera philoxeroides for its potency against viral replication.

Chikusetsusaponin IVa and calenduloside E were isolated from the whole plant of Alternanthera philoxeroides (Mart.) Griseb (Amaranthaceae) and evaluated for their antiviral activities. Chikusetsusaponin IVa showed antiviral activities against HSV-1, HSV-2, human cytomegalovirus, measles virus, and mumps virus with selectivity indices (CC (50)/IC (50)) of 29, 30, 73, 25, and 25, respectively. On the other hand, calenduloside E showed no antiviral effects against any of the viruses tested. The mode of HSV-2 action of chikusetsusaponin IVa was determined under different experimental conditions. The anti-HSV-2 target of the compound might be mainly related to direct inactivation of virus particles and to the inhibition of release of progeny viruses from infected cells, but it is not related to inhibition of viral attachment, cell penetration, and viral protein synthesis. This compound also provided in vivo efficacy in a mouse model of genital herpes caused by HSV-2. These results demonstrate that chikusetsusaponin IVa might be a candidate of antiherpetic agents.

In vivo anti-influenza virus activity of an immunomodulatory acidic polysaccharide isolated from Cordyceps militaris grown on germinated soybeans.

An acidic polysaccharide (APS) was isolated from the extract of Cordyceps militaris grown on germinated soybeans. Analyses of sugar composition indicated that APS consisted of d-galactose, L-arabinose, D-xylose, L-rhamnose, and D-galacturonic acid. On the basis of the result of methylation analysis, APS was considered to be mainly composed of Araf-(1-->, -->5)-Araf-(1-->, -->4)-Galp-(1--> and -->4)-GalAp-(1--> residues. When the polysaccharide was intranasally administered, it decreased virus titers in the bronchoalveolar lavage fluid and the lung of mice infected with influenza A virus and increased survival rate. Furthermore, APS increased TNF-alpha and IFN-gamma levels in mice when compared with those of untreated mice. APS enhanced nitric oxide (NO) production and induced iNOS mRNA and protein expressions in RAW 264.7 murine macrophage cells. The induction of mRNA expression of cytokines including IL-1beta, IL-6, IL-10, and TNF-alpha was also observed. These results demonstrated that APS might have beneficial therapeutic effects on influenza A virus infection at least in part by modulation of the immune function of macrophages.

Cloning and expression of calmodulin gene in Scoparia dulcis.

A homology-based cloning strategy yielded a cDNA clone, designated Sd-cam, encoding calmodulin protein from Scoparia dulcis. The restriction digests of genomic DNA of S. dulcis showed a single hybridized signal when probed with the fragment of this gene in Southern blot analyses, suggesting that Sd-cam occurs as a sole gene encoding calmodulin in the plant. The reverse-transcription polymerase chain reaction analysis revealed that Sd-cam was appreciably expressed in leaf, root and stem tissues. It appeared that transcription of this gene increased transiently when the leaf cultures of S. dulcis were treated with methyl jasmonate and calcium ionophore A23187. These results suggest that transcriptional activation of Sd-cam is one of the early cellular events of the methyl jasmonate-induced responses of S. dulcis.

Antiherpetic activities of sulfated polysaccharides from green algae.

In order to evaluate the potency of novel antiviral drugs, 11 natural sulfated polysaccharides (SPs) from 10 green algae ( Enteromorpha compressa, Monostroma nitidum, Caulerpa brachypus, C. okamurai, C. scapelliformis, Chaetomorpha crassa, C. spiralis, Codium adhaerens, C. fragille, and C. latum) and 4 synthetic sulfated xylans (SXs) prepared from the beta-(1,3)-xylan of C. brachypus, were assayed for anti-Herpes simplex virus type 1 (HSV-1) activity. Except for one from E. compressa, all SPs showed potent anti-HSV-1 activities with 50 % inhibitory concentrations (IC (50)) of 0.38 - 8.5 microg/mL, while having low cytotoxicities with 50 % inhibitory concentrations of >2900 microg/mL. Anti-HSV-1 activities of SXs were dependent on their degrees of sulfation. To delineate the drug-sensitive phase, 4 polysaccharides, which showed potent anti-HSV-1 activities, were applied to time-of-addition experiments. Among the polysaccharides tested, 3 polysaccharides (SX4, SP4 from C. brachypus, and SP11 from C. latum) showed strong anti-HSV-1 activities with IC (50) of 6.0, 7.5, and 6.9 microg/mL, respectively, even when added to the medium 8 h post-infection. These experiments demonstrated that some sulfated polysaccharides not only inhibited the early stages of HSV-1 replication, such as virus binding to and penetration into host cells, but also interfered with late steps of virus replication. These results revealed that some sulfated polysaccharides from green algae should be promising candidates of antiviral agents which might act on different stages in the virus replication cycle.