Lien Chai Chiang

Ge-Gen-Tang has anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines

ETHNOPHARMACOLOGICAL RELEVANCE Ge-Gen-Tang (GGT) has been used against adult respiratory tract infection for thousand years in ancient China. However, GGT is unable to inhibit influenza virus. The effect of GGT to manage respiratory tract viral infection has been questioned. Several ingredients of GGT and their constituents are able to inhibit various viruses. Therefore, GGT might have antiviral activity against other viruses causing respiratory tract illness. Human respiratory syncytial virus (HRSV) is one of the most important airway viruses. However, it is unknown whether GGT is effective against HRSV. AIM OF THE STUDY HRSV contributes considerably to respiratory tract illness of the elderly and immunocompromised adults. There is no effective therapeutic modality for HRSV infection. In order to find a readily available agent to manage HRSV infection, the authors tested the hypothesis that GGT can effectively minimize airway pathology by preventing HRSV-induced plaque formation in respiratory mucosal cell lines. MATERIALS AND METHODS Effect of the hot water extract of GGT on HRSV was tested by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Ability of GGT to stimulate anti-viral cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS GGT dose-dependently inhibited HRSV-induced plaque formation in both cell lines (p<0.0001), especially in A549 cells. GGT was more effective when given before viral infection (p<0.0001). GGT could dose-dependently inhibit viral attachment (p<0.0001) with or without heparin. GGT could further inhibit HRSV internalization time-dependently and dose-dependently (p<0.0001). GGT could stimulate mucosal cells to secrete IFN-β to counteract viral infection before and after viral inoculation. CONCLUSIONS GGT is effective against HRSV-induced plaque formation in airway epithelium.

Water extract of licorice had anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines.

Abstract Ethnopharmacological relevance Licorice (Glycyrrhiza uralensis Fisch., Leguminosae) has been used in herbal medicine and food supplement worldwide for centuries. Licorice is a common ingredient of several prescriptions of traditional Chinese medicine which have been proved to inhibit infection of human respiratory syncytial virus (HRSV). There are two preparations of licorice, Radix Glycyrrhizae and Radix Glycyrrhizae Preparata. However, it is unknown whether licorice or which preparation of licorice is effective against HRSV, nor is its active constituent. Aim of the study We tested the hypothesis that Radix Glycyrrhizae can effectively decrease HRSV-induced plaque formation in respiratory mucosal cell lines. We also tried to find out the active constituent. Materials and methods Anti-HRSV activities of hot water extracts of preparations of licorice, glycyrrhizin and 18β-glycyrrhetinic acid (18β-GA), the active constituents of licorice, were examined by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Abilities of crude licorice to inhibit viral replication and to stimulate IFN-β were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results Radix Glycyrrhizae and Radix Glycyrrhizae Preparata dose-dependently inhibited HRSV-induced plaque formation in both HEp-2 and A549 cell lines (p<0.0001). The effect of Radix Glycyrrhizae was better than that of Radix Glycyrrhizae Preparata on HEp-2 cells. However, there was no difference of their anti-HRSV effects on A549 cells. Besides, glycyrrhizin was ineffective at all. Nevertheless, 18β-GA showed a potent anti-HRSV activity. Radix Glycyrrhizae was more effective when given before viral inoculation (p<0.0001) which may be due to its inhibition of viral attachment on (p<0.0001) and penetration (p<0.0001) into the host cells. The anti-HRSV activity of Radix Glycyrrhizae was further confirmed by RT-PCR and qRT-PCR. 300μg/ml Radix Glycyrrhizae markedly decreased the viral amounts within the cells and in the suspension. Radix Glycyrrhizae might further stimulate mucosal cells to secrete IFN-β to counteract viral infection. Conclusions Both Radix Glycyrrhizae and Radix Glycyrrhizae Preparata are effective against HRSV infection on airway epithelial cells. Radix Glycyrrhizae inhibited HRSV mainly by preventing viral attachment, internalization, and by stimulating IFN secretion. 18β-GA may be one of its active constituents.

Excoecarianin, Isolated from Phyllanthus urinaria Linnea, Inhibits Herpes Simplex Virus Type 2 Infection through Inactivation of Viral Particles

Phyllanthus urinaria Linnea (Euphorbiaceae) is one of the traditional medicinal plants widely used by oriental people to treat various diseases. We have previously demonstrated that the acetone extract of P. urinaria inhibits herpes simplex virus type 2 (HSV-2) but not HSV-1 infection. In a continuing effort to clarify the antiviral mechanisms of P. urinaria, we isolated the pure compound excoecarianin from the whole plant of P. urinaria through acetone extraction, and investigated its anti-HSV-1 and HSV-2 activities. Our results indicated that excoecarianin protected Vero cells from HSV-2 but not HSV-1 infection, and its 50% inhibitory concentration (IC50) was 1.4 ± 0.1 μM. The antiviral effective concentration of excoecarianin did not affect the viability or the morphology of Vero cells. Although excoecarianin inhibited HSV-2 infection, the inhibitory effect, however, was most prominent when excoecarianin was concurrently added with the virus. Pretreatment of Vero cells with excoecarianin with removal of the drug prior to infection did not yield any antiviral effects, and the same observation was made for post viral entry treatment. Subsequent studies revealed that excoecarianin inactivated HSV-2 virus particles to prevent viral infection. A synergistic antiviral effect against HSV-2 was also observed when Vero cells were treated with a combination of acyclovir (ACV) and excoecarianin. These results suggested that excoecarianin merits to be further explored as an entry inhibitor against HSV-2 and could potentially be investigated for combinatorial drug treatment with nucleoside analogues such as ACV in therapeutic management of HSV-2 infection.

Antiviral effect of cimicifugin from Cimicifuga foetida against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) causes serious infection of the lower respiratory tract in children and an effective antiviral therapy against the viral pathogen remains unavailable. We previously demonstrated that the oriental medicinal plant, Cimicifuga foetida L. (C. foetida), possessed inhibitory activity against RSV. Since cimicifugin is a major constituent of C. foetida, we sought to examine in this study its anti-RSV effect on both the human upper (HEp-2) and lower (A549) respiratory tract cell lines. Results revealed that cimicifugin dose-dependently inhibited RSV-induced plaque formation in both HEp-2 and A549 cells (p < 0.0001), with a superior effect in the latter cell type (p < 0.0001). The antiviral activity of cimicifugin was time-dependent (p < 0.0001) and was most effective when cells were treated with the compound before viral inoculation. Additional experiments demonstrated that cimicifugin could inhibit viral attachment (p < 0.0001) and viral internalization (p < 0.0001). Furthermore, the drug could potentiate heparins effect against attachment of RSV, particularly in A549 cells. Enzyme-linked immunosorbent assay (ELISA) analysis of antiviral cytokines induction revealed that cimicifugin could also stimulate epithelial cells to secrete IFN-β to counteract viral infection. Taken together, these results indicate that cimicifugin is an efficient antiviral agent against RSV infection. We suggest that cimicifugin might be useful for the management of RSV pathogenesis.

Yakammaoto inhibited human coxsackievirus B4 (CVB4)-induced airway and renal tubular injuries by preventing viral attachment, internalization, and replication.

ETHNOPHARMACOLOGICAL RELEVANCE Yakammaoto is a prescription of traditional Chinese medicine (TCM) containing nine ingredients, including Ephedra sinica, Pinellia ternate, Zingiber officinale, Tussilago farfara, Aster tataricus, Ziziphus jujube, Belamcanda chinensis, Asarum sieboldii, and Schisandra chinensis. Yakammaoto has been used against flu-like symptoms for more than two thousand years in China and Japan. Coxsackievirus B4 (CVB4) causes not only flu-like symptoms but life-threatening diseases, such as pneumonia, acute kidney injury, and so forth with severe morbidity and mortality. There is no effective therapeutic modality against CVB4 infection. It is unknown whether yakammaoto is effective against CVB4 infection. We tested the hypothesis that yakammaoto can effectively inhibit CVB4-induced plaque formation in human airway and renal tubular cell lines by preventing viral attachment, internalization, and replication. MATERIALS AND METHODS The fingerprint of yakammaoto was assessed by HPLC. Effects of yakammaoto on CVB4 infection were tested by plaque reduction assay, reverse transcription polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA). RESULTS Yakammaoto dose-dependently inhibited CVB4-induced plaque formation in HK-2, A549, and HEp-2 cells (p<0.0001). Yakammaoto was both effective when supplemented prior to and after viral inoculation (p<0.0001) by preventing viral attachment (p<0.0001), internalization (p<0.0001), and replication (p<0.0001). Yakammaoto could decrease NGAL secretion before cytolysis to protect against viral injury. CONCLUSIONS Yakammaoto had antiviral activity against CVB4-induced cellular injuries in airway mucosa and renal tubular epithelia by preventing viral attachment, internalization, and replication. The current study provides a basic support of its potential use against CVB4-induced airway and concomitant renal injuries.

Xiao-Qing-Long-Tang (Sho-seiryu-to) inhibited cytopathic effect of human respiratory syncytial virus in cell lines of human respiratory tract

ETHNOPHARMACOLOGICAL RELEVANCE Xiao-Qing-Long-Tang (XQLT, TJ-19, Sho-seiryu-to, so-cheong-ryong-tang) has been used against acute airway diseases for thousands of year in ancient China. Most of the acute airway illnesses are caused by virus. However, without activity against influenza virus, XQLT has been questioned to manage respiratory tract viral infection. Nevertheless, XQLT might be active against airway viruses other than influenza. Human respiratory syncytial virus (HRSV) is one of the most common respiratory viral pathogens without effective management. However, it is unknown whether XQLT has anti-HRSV activity. AIM OF THE STUDY We tested the hypothesis that XQLT can effectively minimize HRSV-induced plaque formation in respiratory tract mucosal cell lines. MATERIALS AND METHODS Anti-HRSV activity of a hot water extract of XQLT was examined by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Its effects on syncytial formation and viral fusion (F) protein were examined directly by microscopy and by western blot, respectively. Ability of XQLT to stimulate IFN-β was evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS Hot water extract of XQLT dose-dependently inhibited HRSV-induced plaque formation in both HEp-2 and A549 cells (P<0.0001), particularly when given before viral inoculation (p<0.0001). XQLT inhibited viral attachment (p<0.0001) and internalization (p<0.0001). 300μg/ml XQLT could decrease both the number and the size of HRSV-induced syncytium without clear effect on the production of viral F protein. XQLT could stimulate epithelial cells to secrete IFN-β before and after viral inoculation to counteract viral infection (p<0.0001). CONCLUSIONS XQLT is effective against HRSV infection on airway epithelia by preventing viral attachment, internalization, syncytial formation, and by stimulating interferon secretion.

Water extract of Cinnamomum cassia Blume inhibited human respiratory syncytial virus by preventing viral attachment, internalization, and syncytium formation.

ETHNOPHARMACOLOGICAL RELEVANCE Cinnamomum cassia Blume is a popular traditional Chinese herbal medicine that has been used to manage respiratory tract disease, including common cold and chronic bronchitis for thousand years. Human respiratory syncytial virus (HRSV) is one of the leading causes of severe lower respiratory tract illness worldwide. No effective therapeutic modality against HRSV infection has been proved. It is unknown whether Cinnamomum cassia is effective against HRSV. AIM OF THE STUDY This study tested the hypothesis that Cinnamomum cassia can effectively decrease HRSV-induced plaque formation and syncytium formation in respiratory mucosal cell lines. MATERIALS AND METHODS Antiviral activity of the hot water extract of Cinnamomum cassia against HRSV was tested by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Its ability to inhibit the synthesis of viral fusion (F) protein was examined by Western blot assay. RESULTS Cinnamomum cassia dose-dependently inhibited HRSV-induced plaque formation in both HEp-2 and A549 cell lines (p<0.0001). Cinnamomum cassia was more effective when given before viral infection (p<0.0001) mainly by inhibition of viral attachment (p<0.0001) and internalization (p<0.0001). Cinnamomum cassia could inhibit F protein production and syncytium formation to interfere with HRSV spreading. CONCLUSIONS Cinnamomum cassia prevented airway epithelia from HRSV infection through inhibiting viral attachment, internalization and syncytium formation. Cinnamomum cassia could be a candidate to develop therapeutic modalities to manage HRSV infection in the future.

Cimicifuga foetida L. inhibited human respiratory syncytial virus in HEp-2 and A549 cell lines.

Human respiratory syncytial virus (HRSV) causes serious pediatric infection of the lower respiratory tract without effective therapeutic modality. Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) has been proven to be effective at inhibiting HRSV-induced plaque formation, and Cimicifuga foetida is the major constituent of SMGGT. We tested the hypothesis that C. foetida effectively inhibited the cytopathic effects of HRSV by a plaque reduction assay in both human upper (HEp2) and lower (A549) respiratory tract cell lines. Its ability to stimulate anti-viral cytokines was evaluated by an enzyme-linked immunosorbent assay (ELISA). C. foetida dose-dependently inhibited HRSV-induced plaque formation (p < 0.0001) before and after viral inoculation, especially in A549 cells (p < 0.0001). C. foetida dose-dependently inhibited viral attachment (p < 0.0001) and could increase heparins effect on viral attachment. In addition, C. foetida time-dependently and dose-dependently (p < 0.0001) inhibited HRSV internalization. C. foetida could stimulate epithelial cells to secrete IFN-β to counteract viral infection. However, C. foetida did not stimulate TNF-α secretion. Therefore, C. foetida could be useful in managing HRSV infection. This is the first evidence to support that C. foetida possesses antiviral activity.

Gan-Lu-Siao-Du-yin, a prescription of traditional Chinese medicine, inhibited enterovirus 71 replication, translation, and virus-induced cell apoptosis

ETHNOPHARMACOLOGICAL RELEVANCE Gan-Lu-Siao-Du-yin (GLSDY) is a prescription of traditional Chinese medicine. GLSDY contains 11 ingredients and is commonly used for endemic diseases. Enterovirus 71 (EV71) is an endemic disease that can cause meningoencephalitis with mortality and neurologic sequelae without any effective management. It is unknown whether GLSDY is effective against EV71 infection. AIM OF THE STUDY To test the hypothesis that GLSDY can protect cell from EV71-induced injury. MATERIALS AND METHODS Effects of a hot water extract of GLSDY on EV71 were tested in human foreskin fibroblast cells (CCFS-1/KMC) and human rhabdomyosarcoma cells (RD cells) by plaque reduction assay and flow cytometry respectively. Inhibition of viral replication was further examined by reverse quantitative RT-PCR (qRT-PCR). Its effect on viral protein translation and virus-induced apoptosis were examined by western blot. RESULTS GLSDY was dose-dependently effective against EV71 infection (p<0.0001) in both CCFS-1/KMC cells and RD cells. GLSDY was highly effective when supplemented after viral inoculation (P<0.0001) with an IC50 of 8.7μg/mL. GLSDY inhibited viral RNA replication (P<0.0001), formation of viral structural proteins (VP0, VP1, VP2 and VP3) and non-structural proteins (protease 2B and 3AB). Furthermore, 300μg/mL GLSDY is effective to inhibit virus-induced apoptosis possibly through direct inhibition of caspase-8 and indirectly by inhibition of Bax. CONCLUSIONS GLSDY is cheap and readily available to manage EV71 infection by inhibiting viral replication, viral protein formations, and EV71-induced apoptosis.

Flos Farfarae Inhibits Enterovirus 71-Induced Cell Injury by Preventing Viral Replication and Structural Protein Expression

Enterovirus 71 (EV71) infection can cause airway symptoms, brainstem encephalitis, neurogenic shock, and neurogenic pulmonary edema with high morbidity and mortality. There is no proven therapeutic modality. Flos Farfarae is the dried flower bud of Tussilago farfara L. that has been used to manage airway illnesses for thousands of years. It has neuro-protective activity and has been used to manage neuro-inflammatory diseases. However, it is unknown whether Flos Farfarae has activity against EV71-induced neuropathy. The current study used both human foreskin fibroblast (CCFS-1/KMC) and human rhabdomyosarcoma (RD) cell lines to test the hypothesis that a hot water extract of Flos Farfarae could effectively inhibit EV71 infection. The authenticity of Flos Farfarae was confirmed by HPLC-UV fingerprint. Through plaque reduction assays and flow cytometry, Flos Farfarae was found to inhibit EV71 infection ([Formula: see text]). Inhibition of viral replication and protein expression were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR), and western blot, respectively. The estimated IC[Formula: see text]s were 106.3[Formula: see text][Formula: see text]g/mL in CCFS-1/KMC, and 15.0[Formula: see text][Formula: see text]g/mL in RD cells. Therefore, Flos Farfarae could be beneficial to inhibit EV71 infection by preventing viral replication and structural protein expression.