Antiviral Amentoflavone from Selaginella sinensis

Abstract

Upper respiratory infection, especially in infants and children, is a common disease worldwide. Respiratory syncytial virus (RSV) has been recognized as a major cause of such infections.1) The nucleoside analog ribavirin has been used widely for treating RSV infections in healthcare settings such as hospitals and nursing homes. However, the treatment protocol, the potentially severe side effects and the emergence of drug-resistant mutants during long-term medication with this drug have often limited its administration to patients. 2)Therefore, the development of new antiviral agents with different mechanisms is most desirable. Traditional medicines utilizing natural products have been shown to contain antiviral compounds. [3][4][5][6][7] We screened for such compounds from natural products. In this paper, we report the isolation of active compounds from the whole herb of Selaginella sinensis (Selaginellaceae) and the content determination of amentoflavone in nine different species of Selaginella. Antiviral Assays The antiviral activity of test sample against RSV was measured by the plaque reduction assay. 8,9) Briefly, two-days old confluent monolayers of Hep 2 cells were infected with RSV at 100 plaque-forming units (PFU) per well in 0.5 ml maintenance medium . The inoculated cultures were incubated at 37°C in a CO 2 incubator with gentle agitation at 10-15 intervals to ensure uniform virus adsorption. Following 1 h virus adsorption, the inoculum was removed and 1.5 ml of agarose overlay medium containing 0.6% agarose in DMEM and 2% FBS were added to each well. When the agarose overlay became well-solidified, 1.5 ml of diluted test samples in maintenance medium were added to each well. A minimum of three different concentrations of each test sample was tested. One well per plate was added with 1.5 ml of maintenance medium without the test sample as control. The culture plates were incubated in the humidified CO 2 incubator for 4-5 d. The cultures were then fixed with 10% formalin in PBS and subsequently stained with 1% crystal violet in 20% ethanol upon removal of the agarose overlay. The number of plaques per well was enumerated macroscopically. The 50% inhibitory concentration (IC 50 ) was determined as the concentration of the test sample that reduced 50% of the plaque formation in the control wells. MATERIALS AND METHODS MaterialsExtraction and Isolation of Main Compound from the Herb of S. sinensis Air dried herb (5.0 kg) was extracted by refluxing with 90 l ethanol for three times, each time for 2 h. The ethanol solution was filtered and evaporated under reduced pressure to give a brownish ethanol extract (660 g). The ethanol extract was dissolved in water containing 0.5 N HCl and filtered. The precipitate was dissolved in water containing 0.5 N NaOH and filtered. The basic solution was adjusted to pH 1-3 with HCl, stayed overnight and filtered. The precipitate was washed with water to give a yellowish powder (195 g). Then this yellowish powder was extracted with chloroform, ethyl acetate and methanol by ...

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

“…Recently, some extracts from traditional Chinese medicinal herbs were reported to exhibit in vitro anti-RSV activity (Ma et al, 2002). A potent antiviral compound amentoflavone against RSV was isolated from the herb Selaginella sinensis (Ma et al, 2001). The result of the present study is the first report of anticancer and anti-RSV activities of Youngia japonica which is a folklore medicine used by Chinese for centuries.…”

Antiherpetic activity of a flavonoid fraction from Ocotea notata leaves

“…They are found in vegetables, fruits, seeds, fl owers as well as in wine, tea and propolis (Harborne & Williams, 2000;Grotewold, 2006 antiviral activity of flavonoids had increased in the last two decades. The most cited flavonoid classes displaying antiherpes activity are flavonols and flavones (Hayashi et al, 1997;Amaral et al, 1999;Ma et al, 2001;Chiang et al, 2003;Lyu et al, 2005;Fritz et al, 2007;Gomes et al, 2008;Schnitzler et al, 2009;Martins et al, 2011).…”

Target-guided isolation and purification of antioxidants from Selaginella sinensis by offline coupling of DPPH-HPLC and HSCCC experiments

“…Previous phytochemical investigations have indicated that seven biflavonoids (e.g. amentoflavone, 7 -O-methylamentoflavone, 4 ,7 -di-O-methylamentoflavone, 2,3-dihydroamentoflavone, robustaflavone, ginkgetin, hinokiflavone) [2][3][4][5][6], two flavonoids (e.g. genistin and quercetin) [2,5], lignans [7,8] and phenols [9] exist in S. sinensis.…”