Lignan Dimers from Forsythia viridissima Roots and Their Antiviral Effects.

Six new dimeric lignans (1-6) and one new lignan glycoside (16) were isolated from Forsythia viridissima roots along with nine known lignans (7-15). Spectroscopic analyses and chemical methods were used to determine these new structures and their absolute configurations. Among these compounds, dimatairesinol (1) and viridissimaols A-E (2-6) were assigned as dimers of dibenzylbutyrolactone analogues. Furthermore, the isolated compounds were evaluated for their antiviral activities against coxsackievirus B3 (CVB3) and human rhinovirus 1B (HRV1B). In these tests, compounds 12 and 15 showed antiviral effects against CVB3 infection with IC50 values of 15.4 and 36.4 µM, respectively, while 2, 3, 8, and 9 showed activities against HRV1B with IC50 values of 45.7, 47.5, 13.0, and 43.2 µM, respectively.

Chemical Identification of Isoflavonoids from a Termite-Associated Streptomyces sp. RB1 and Their Neuroprotective Effects in Murine Hippocampal HT22 Cell Line.

Insect-associated bacteria have been recognized as a very promising natural resource for discovering bioactive secondary metabolites with diverse pharmacological effects. One new isoflavonoid glycoside, termisoflavone D (1), together with seven known isoflavonoids (2⁻8), were identified from MeOH extracts of the fungus-growing termite-associated Streptomyces sp. RB1. The chemical structure of the new compound 1 was elucidated using comprehensive spectroscopic methods including 1D and 2D NMR, along with LC/MS analysis. The existence of two rhamnose moieties in 1 was determined with comparative NMR analysis, and the absolute configuration was elucidated using chemical reactions. The neuroprotective activities of compounds 1⁻8 were thoroughly investigated using the murine hippocampal HT22 cell line. Compound 5 prevented glutamate-induced HT22 cell death by blocking intracellular reactive oxygen species (ROS) accumulation. The present study provides the first experimental evidence for the potential use of isoflavonoids from termite-associated bacteria as lead compounds that can prevent neuronal damage induced by glutamate.

Lignan Glycosides and Flavonoid Glycosides from the Aerial Portion of Lespedeza cuneata and Their Biological Evaluations.

Lespedeza cuneata (Fabaceae), known as Chinese bushclover, has been used in traditional medicines for the treatment of diseases including diabetes, hematuria, and insomnia. As part of a continuing search for bioactive constituents from Korean medicinal plant sources, phytochemical analysis of the aerial portion of L. cuneata led to the isolation of two new lignan glycosides (1,2) along with three known lignan glycosides (3⁻7) and nine known flavonoid glycosides (8⁻14). Numerous analysis techniques, including 1D and 2D NMR spectroscopy, CD spectroscopy, HR-MS, and chemical reactions, were utilized for structural elucidation of the new compounds (1,2). The isolated compounds were evaluated for their applicability in medicinal use using cell-based assays. Compounds 1 and 4⁻6 exhibited weak cytotoxicity against four human breast cancer cell lines (Bt549, MCF7, MDA-MB-231, and HCC70) (IC50 < 30.0 µM). However, none of the isolated compounds showed significant antiviral activity against PR8, HRV1B, or CVB3. In addition, compound 10 produced fewer lipid droplets in Oil Red O staining of mouse mesenchymal stem cells compared to the untreated negative control without altering the amount of alkaline phosphatase staining.

Antiviral activity of micafungin against enterovirus 71.

BACKGROUND: Enterovirus 71 (EV71) is a major causative agent of hand-foot-mouth disease (HFMD) and also causes severe neurological complications, leading to fatality in young children. However, no effective therapy is currently available for the treatment of this infection. METHODS: We identified small-molecule inhibitors of EV71 from a screen of 968 Food and Drug Administration (FDA)-approved drugs, with which clinical application for EV71-associated diseases would be more feasible, using EV71 subgenomic replicon system. Primary hits were extensively evaluated for their antiviral activities in EV71-infected cells. RESULTS: We identified micafungin, an echinocandin antifungal drug, as a novel inhibitor of EV71. Micafungin potently inhibits the proliferation of EV71 as well as the replication of EV71 replicon in cells with a low micromolar IC50 (~5 µM). The strong antiviral effect of micafungin on EV71 replicon and the result from time-of-addition experiment demonstrated a targeting of micafungin on virion-independent intracellular process(es) during EV71 infection. Moreover, an extensive analysis excluded the involvement of 2C and 3A proteins, IRES-dependent translation, and also that of polyprotein processing in the antiviral effect of micafungin. CONCLUSIONS: Our research revealed a new indication of micafungin as an effective inhibitor of EV71, which is the first case reporting antiviral activity of micafungin, an antifungal drug.

Five new quassinoids and cytotoxic constituents from the roots of Eurycoma longifolia.

Eurycoma longifolia has been widely used for various traditional medicinal purposes in South-East Asia. In this study, five new quassinoids, eurylactone E (1), eurylactone F (2), eurylactone G (3), eurycomalide D (4), and eurycomalide E (5), along with ten known quassinoids (6-15) were isolated from the roots of E. longifolia. Their structures were determined by extensive spectroscopic methods, including 1D and 2D NMR, and MS spectra data. Among the isolated compounds, 13ß-methyl,21-dihydroeurycomanone (6) has been reported as a synthetic derivative. However, it was isolated from the natural product for the first time in this study. The cytotoxic activities of fifteen compounds were evaluated against human lung cancer cell line, A549 and human cervical cancer cell line, HeLa.

Anti-wrinkle effects of fermented and non-fermented Cyclopia intermedia in hairless mice.

BACKGROUND: The fermented leaves and stems of Cyclopia intermedia are used to brew honeybush tea, an herbal tea indigenous to South Africa with reported anti-wrinkle effects. Wrinkle formation caused by photoaging clearly involves changes in extracellular matrix components and mechanical properties of the skin. METHODS: The inhibitory effects of honeybush extract and fermented honeybush on wrinkle formation were determined by analyzing skin replicas, histologically examining epidermal thickness, and identifying damage to collagen fibers. RESULTS: Honeybush extract and fermented honeybush reduced the length and depth of skin winkles caused by UV irradiation and inhibited thickening of the epidermal layer, in addition to suppressing collagen tissue breakdown reactions, indicating its potential use as a skin wrinkle prevention agent. CONCLUSIONS: This in vivo study demonstrates that honeybush produces significant anti-wrinkle effects and is therefore of interest in anti-aging skin care products.

Protective effect of mango (Mangifera indica L.) against UVB-induced skin aging in hairless mice.

BACKGROUND: Mangifera indica L. (Anacardiaceae) is a medicinal plant whose extracts have been described as an antioxidant with anti-inflammatory and immunomodulatory activities. PURPOSE: Skin aging is a consequence of chronic sun exposure to the sun and therefore ultraviolet (UV) radiation. Naturally occurring antioxidants are known to reduce skin aging. Therefore, the aim of the present study was to evaluate the protective role of mango extract against UVB-induced skin aging in hairless mice. METHODS: HR-1 hairless male mice (6 weeks old) were divided into three groups: control (n = 5), UVB-treated vehicle (n = 5), and UVB-treated mango extract (n = 5) groups. UVB-irradiated mice from the mango extract group were orally administered 0.1 ml of water containing 100 mg of mango extract/kg body weight per day. The inhibitory activity of mango extract on wrinkle formation was determined by the analysis of the skin replica, epidermal thickness based on histological examination, and damage to collagen fiber. RESULTS: The mean length of wrinkles in UVB-treated vehicle group significantly improved after the oral administration of mango extract, which significantly inhibited the increase in epidermal thickness and epidermal hypertrophy (P < 0.05). Furthermore, a marked increase in collagen bundles was observed in the UVB-treated group after the administration of mango extract by Masson's trichrome staining. CONCLUSION: These results indicate that mango extract showed anti-photoaging activity in UVB-irradiated hairless mice.

Intranasal immunization with curdlan induce Th17 responses and enhance protection against enterovirus 71.

Mucosal surfaces are in contact with the external environment and protect the body from infection by various microbes. To prevent infectious diseases at the first line of defense, the establishment of pathogen-specific mucosal immunity by mucosal vaccine delivery is needed. Curdlan, a 1,3-ß-glucan has a strong immunostimulatory effect when delivered as a vaccine adjuvant. Here, we investigated whether intranasal administration of curdlan and antigen (Ag) could induce sufficient mucosal immune responses and protect against viral infections. Intranasal co-administration of curdlan and OVA increased OVA-specific IgG and IgA Abs in both serum and mucosal secretions. In addition, intranasal co-administration of curdlan and OVA induced the differentiation of OVA-specific Th1/Th17 cells in the draining lymph nodes. To investigate the protective immunity of curdlan against viral infection, intranasal co-administration of curdlan with recombinant VP1 of EV71 C4a was administered and showed enhanced protection against enterovirus 71 in a passive serum transfer model using neonatal hSCARB2 mice, although intranasal administration of VP1 plus curdlan increased VP1-specific helper T cells responses but not mucosal IgA. Next, Mongolian gerbils were intranasally immunized with curdlan plus VP1, and they had effective protection against EV71 C4a infection, while decreasing viral infection and tissue damage by inducing Th17 responses. These results indicated that intranasal curdlan with Ag improved Ag-specific protective immunity by enhancing mucosal IgA and Th17 against viral infection. Our results suggest that curdlan is an advantageous candidate as a mucosal adjuvant and delivery vehicle for the development of mucosal vaccines.

The chemical constituents from twigs of Hamamelis japonica and their antiviral activities.

Three new monoterpenoid glycosides (1-3) and one new flavanol (4) along with 15 known compounds were isolated from the twig of Hamamelis japonica Sieb. et Zucc. The chemical constituent study of the twig of H. japonica has performed for the first time in the present investigation. Their structures were determined based on extensive spectroscopic methods including 1 D and 2 D NMR and CD spectra data. All isolated compounds were tested for their antiviral activities against HRV1B-, EV71-, PR8- and CVB3-infected Vero cells. Among the tested compounds, (-)-epigallocatechin 3-O-gallate exhibited the most consistent and effective antiviral activities against EV71 and PR8 infections.

Vitisin B inhibits influenza A virus replication by multi-targeting neuraminidase and virus-induced oxidative stress.

The development of drug-resistant influenza and new pathogenic virus strains underscores the need for antiviral therapeutics. Currently, neuraminidase (NA) inhibitors are commonly used antiviral drugs approved by the US Food and Drug Administration (FDA) for the prevention and treatment of influenza. Here, we show that vitisin B (VB) inhibits NA activity and suppresses H1N1 viral replication in MDCK and A549 cells. Reactive oxygen species (ROS), which frequently occur during viral infection, increase virus replication by activating the NF-κB signaling pathway, downmodulating glucose-6-phosphate dehydrogenase (G6PD) expression, and decreasing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response activity. VB decreased virus-induced ROS generation by increasing G6PD expression and Nrf2 activity, and inhibiting NF-κB translocation to the nucleus through IKK dephosphorylation. In addition, VB reduced body weight loss, increased survival, decreased viral replication and the inflammatory response in the lungs of influenza A virus (IAV)-infected mice. Taken together, our results indicate that VB is a promising therapeutic candidate against IAV infection, complements existing drug limitations targeting viral NA. It modulated the intracellular ROS by G6PD, Nrf2 antioxidant response pathway, and NF-κB signaling pathway. These results demonstrate the feasibility of a multi-targeting drug strategy, providing new approaches for drug discovery against IAV infection.

Potential of a bivalent vaccine for broad protection against enterovirus 71 and coxsackie virus 16 infections causing hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a highly contagious viral infection that is mainly caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16). As there are no specific therapeutics for HFMD, the development of a bivalent vaccine is required to cover a broad range of infections. In this study, the effectiveness of novel monovalent and bivalent vaccines targeting EV71 C4a and CVA16 was investigated for their ability to prevent viral infections in neonatal human scavenger receptor class B member 2 (hSCARB2) transgenic mice. As hSCARB2 serves as a key viral receptor for EV71, these transgenic mice are susceptible to EV71 strains and facilitate viral binding, internalization, and uncoating processes. Antisera prepared by vaccine immunization were transferred to 2-day-old hSCARB2 transgenic mice, which were then infected with EV71 C4a or CVA16 virus. The antisera generated by each monovalent or bivalent vaccine effectively protected against EV71 C4a and CVA16 infections. The examination of tissue damage and viral contents in various organs indicated that both monovalent and bivalent antisera reduced EV71 C4a viral load in the brainstem, and no significant tissue damage was observed. During CVA16 infection, the monovalent and bivalent antisera significantly reduced viral contents in both the brainstem and muscles. These results suggest that passive immunity by monovalent and bivalent antisera can effectively protect against EV71 C4a and CVA16 infections. Thus, the development of a bivalent vaccine that can provide broad protection against both CV and EV infections may be a promising strategy in preventing HFMD.

Antiviral Mechanisms of Saucerneol from Saururus chinensis against Enterovirus A71, Coxsackievirus A16, and Coxsackievirus B3: Role of Mitochondrial ROS and the STING/TKB-1/IRF3 Pathway.

Enterovirus A71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus B3 (CVB3) are pathogenic members of the Picornaviridae family that cause a range of diseases, including severe central nervous system complications, myocarditis, and pancreatitis. Despite the considerable public health impact of these viruses, no approved antiviral treatments are currently available. In the present study, we confirmed the potential of saucerneol, a compound derived from Saururus chinensis, as an antiviral agent against EV71, CVA16, and CVB3. In the in vivo model, saucerneol effectively suppressed CVB3 replication in the pancreas and alleviated virus-induced pancreatitis. The antiviral activity of saucerneol is associated with increased mitochondrial ROS (mROS) production. In vitro inhibition of mROS generation diminishes the antiviral efficacy of saucerneol. Moreover, saucerneol treatment enhanced the phosphorylation of STING, TBK-1, and IRF3 in EV71- and CVA16-infected cells, indicating that its antiviral effects were mediated through the STING/TBK-1/IRF3 antiviral pathway, which was activated by increased mROS production. Saucerneol is a promising natural antiviral agent against EV71, CVA16, and CVB3 and has potential against virus-induced pancreatitis and myocarditis. Further studies are required to assess its safety and efficacy, which is essential for the development of effective antiviral strategies against these viruses.

Anti-human rhinoviral activity of polybromocatechol compounds isolated from the rhodophyta, Neorhodomela aculeata.

An extract of the red alga, Neorhodomela aculeata, exhibited antiviral activity against human rhinoviruses. Bioassay-guided purification was performed to yield six compounds, which were subsequently identified as lanosol (1) and five polybromocatechols (2-6) by spectroscopic methods, including 1D and 2D NMR and mass spectrometric analyses. Structurally, all of these compounds, except compound 5, contain one or two 2,3-dibromo-4,5-dihydroxyphenyl moieties. In a biological activity assay, compound 1 was found to possess antiviral activity with a 50% inhibitory concentration (IC50) of 2.50 µg/mL against HRV2. Compound 3 showed anti-HRV2 activity, with an IC50 of 7.11 µg/mL, and anti-HRV3 activity, with an IC50 of 4.69 µg/mL, without demonstrable cytotoxicity at a concentration of 20 µg/mL. Collectively, the results suggest that compounds 1 and 3 are candidates for novel therapeutics against two different groups of human rhinovirus.

Quercetin 3-rhamnoside exerts antiinfluenza A virus activity in mice.

Our previous report showed that quercetin 3-rhamnoside (Q3R) possessed antiviral activity against influenza A/WS/33 virus in vitro. The present study evaluated the effect of Q3R on influenza A/WS/33 virus infected mice. Mice orally treated with Q3R (6.25 mg/kg per dose) at 2 h before and once daily for 6 days after influenza virus infection showed significant decreases in weight loss, and decreased mortality. Lung virus titers of mice killed at 6 days after infection were about 2000 times lower than that of the placebo-treated control mice and about two times lower than that for the oseltamivir-treated mice. Furthermore, histological evaluation showed that administration of Q3R delayed the development and progression of pulmonary lesions. Therefore, Q3R could be an attractive lead for the development of antiviral agents against influenza virus.

Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation.

Coxsackievirus B3 (CVB3) infection causes acute pancreatitis and myocarditis. However, its pathophysiological mechanism is unclear. Here, we investigated how lipid metabolism is associated with exacerbation of CVB3 pathology using high-fat diet (HFD)-induced obese mice. Mice were intraperitoneally inoculated with 1×106 pfu/mouse of CVB3 after being fed a control or HFD to induce obesity. Mice were treated with mitoquinone (MitoQ) to reduce the level of mitochondrial ROS (mtROS). In obese mice, lipotoxicity of white adipose tissue-induced inflammation caused increased replication of CVB3 and mortality. The coxsackievirus adenovirus receptor increased under obese conditions, facilitating CVB3 replication in vitro. However, lipid-treated cells with receptor-specific inhibitors did not reduce CVB3 replication. In addition, lipid treatment increased mitochondria-derived vesicle formation and the number of multivesicular bodies. Alternatively, we found that inhibition of lipid-induced mtROS decreased viral replication. Notably, HFD-fed mice were more susceptible to CVB3-induced mortality in association with increased levels of CVB3 replication in adipose tissue, which was ameliorated by administration of the mtROS inhibitor, MitoQ. These results suggest that mtROS inhibitors can be used as potential treatments for CVB3 infection.

Quercetin 7-rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species.

BACKGROUND: On the base of our previous study we were observed relevant studies on the hypothesis that the antiviral activity of quercetin 7-rhamnoside (Q7R), a flavonoid, won't relate ability of its antioxidant. METHODS: We were investigated the effects of Q7R on the cytopathic effects (CPE) by CPE reduction assay. Production of DNA fragment and reactive oxygen species (ROS) induced by PEDV infection were studied using DNA fragmentation assay and flow cytometry. RESULTS: In the course of this study it was discovered that Q7R is an extremely potent compound against PEDV. The addition of Q7R to PEDV-infected Vero cells directly reduced the formation of a visible cytopathic effect (CPE). Also, Q7R did not induce DNA fragmentation. Furthermore, ROS increased the infection of PEDV, which was strongly decreased by N-acetyl-L-cysteins (NAC). However, the increased ROS was not decreased by Q7R. Antiviral activity of antioxidants such as NAC, pyrrolidine dithiocarbamate (PDTC), and the vitamin E derivative, trolox, were hardly noticed. CONCLUSIONS: We concluded that the inhibition of PEDV production by Q7R is not simply due to a general action as an antioxidants and is highly specific, as several other antioxidants (NAC, PDTC, trolox) are inactive against PEDV infection.

Antiviral Activity of Chrysin against Influenza Virus Replication via Inhibition of Autophagy.

Influenza viruses cause respiratory infections in humans and animals, which have high morbidity and mortality rates. Although several drugs that inhibit viral neuraminidase are used to treat influenza infections, the emergence of resistant viruses necessitates the urgent development of new antiviral drugs. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid that exhibits antiviral activity against enterovirus 71 (EV71) by inhibiting viral 3C protease activity. In this study, we evaluated the antiviral activity of chrysin against influenza A/Puerto Rico/8/34 (A/PR/8). Chrysin significantly inhibited A/PR/8-mediated cell death and the replication of A/PR/8 at concentrations up to 2 µM. Viral hemagglutinin expression was also markedly decreased by the chrysin treatment in A/PR/8-infected cells. Through the time course experiment and time-of-addition assay, we found that chrysin inhibited viral infection at the early stages of the replication cycle. Additionally, the nucleoprotein expression of A/PR/8 in A549 cells was reduced upon treatment with chrysin. Regarding the mechanism of action, we found that chrysin inhibited autophagy activation by increasing the phosphorylation of mammalian target of rapamycin (mTOR). We also confirmed a decrease in LC3B expression and LC3-positive puncta levels in A/PR/8-infected cells. These results suggest that chrysin exhibits antiviral activity by activating mTOR and inhibiting autophagy to inhibit the replication of A/PR/8 in the early stages of infection.

Anti-human rhinovirus activity of gallic acid possessing antioxidant capacity.

Human rhinoviruses (HRVs) are a major cause of the common cold and until now there is no registered clinically effective antiviral chemotherapeutic agent for treatment of diseases caused by HRVs. Our previous report showed that gallic acid from Woodfordia fruticosa flowers possessed antioxidant activity. Many studies reported that antioxidants possess antiviral activities against various viruses. Therefore, we examined antiviral activity of gallic acid against HRVs and mode of its actions by observing the effect of gallic acid on HRV-induced cytopathic effect (CPE) and the infectivity of HRV particles, and then carried out a time-addition study. As a result, gallic acid actively inhibited HRV2 and -3 replications with antiviral activity more than 55% without cytotoxicity in human epitheloid carcinoma cervix (HeLa) cells at a concentration of 100 mug/mL. Also, ribavirin showed lower anti-HRV3 activity than gallic acid and similar anti-HRV3 activity to it. The addition of gallic acid to HRV-infected HeLa cells directly reduced the formation of a visible CPE. Furthermore, gallic acid did directly interact or activate with HRV particles. Collectively, we concluded that the inhibition of HRV production by gallic acid is mainly due to a general action as an antioxidant and the mode of action derived from the inhibition of virus absorption.

Antiviral and anticancer activities of 13(E)-labd-13-ene-8alpha,15-diol isolated from Brachyglottis monroi.

The antiviral activity of 13(E)-labd-13-ene-8alpha,15-diol (1), isolated from Brachyglottis monroi, was examined against human rhinovirus 2 (HRV2) and 3 (HRV3), and the anticancer activity on human cancer cells (A549 and Hep2). Compound (1) showed strong anti-HRV2 and HRV3 activity with a 50% inhibitory concentration (IC(50)) of 2.68 and 0.87 microg/mL, respectively, and a 50% cytotoxicity concentration (CC(50)) of 59.45 microg/mL. Ribavirin only showed anti-HRV3 activity with an IC(50) of 30.48 microg/mL and a CC(50) > 100 microg/mL. The addition of compound (1) to HRV-infected HeLa cells directly reduced the formation of visible cytopathic effect (CPE) and it directly interacted with HRV particles. Furthermore, A549 and Hep2 cells incubated with 32 microg/mL of compound (1) for 48 h exhibited antilung and antilaryngeal cancer activities, with a viability of less than 50%. These results suggest that compound (1) may be used as a potential antiviral and anticancer agent.

Chemical components from the twigs of Caesalpinia latisiliqua and their antiviral activity.

Three new compounds, (3S)-dihydrobonducellin 8-O-ß-D-glucopyranoside (1), 3',5'-dimethoxy-jezonolid (2), and latisilinoid (3), along with 16 known compounds, were isolated from the twigs of Caesalpinia latisiliqua (Leguminosae). The known compounds were identified as flavonoids, stilbenes, and phenolics as determined by extensive spectroscopic methods, including 1D and 2D NMR. All the isolated compounds were evaluated for their antiviral activity in HRV1B-, CVB3-, and EV71-infected cells. Among the tested compounds, three flavonoids (4-6) and two stilbenes (12 and 14) exhibited significant antiviral activity. This is the first phytochemical investigation of C. latisiliqua twigs.