Ethanolic Extract of Melia Fructus Has Anti-influenza A Virus Activity by Affecting Viral Entry and Viral RNA Polymerase

Abstract

Meliae Fructus (MF) is the dried ripe fruit of Melia toosendan Siebold et Zuccarini, Meliaceae family. MF is widely used in traditional medicine to treat inflammation and helminthic infection and has anti-bacterial, anti-oxidant, anti-cancer, anti-inflammatory, and analgesic activities. However, potential anti-influenza properties of MF have yet to be investigated. We determined whether an ethanolic extract of MF (EMF) has anti-viral activity via an EMF pre-, co-, and post-treatment assay, using the Influenza A/PR/8/34 and H3N2 virus on Madin-Darby canine kidney (MDCK) cells. The EMF had anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cell. EMF inhibited the activity of hemagglutinin (HA) and neuraminidase (NA) of influenza virus. EMF inhibited viral HA, nucleoprotein (NP), matrix protein 2 (M2), non-structural protein 1 (NS1), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) mRNA synthesis at 5 h post infection (hpi), however, the levels of PA, PB1, and PB2 mRNA were increased in pre- and co-EMF treated cells compared with control virus-infected and EMF post-treated cells at 18 hpi. The level of M2 protein expression was also decreased upon pre- and co-treatment with EMF. The PA protein was accumulated and localized in not only the nucleus but also the cytoplasm of virus-infected MDCK cells at 18 hpi. Pre-EMF treatment inhibited the expression of pAKT, which is induced by influenza virus infection, at the stage of virus entry. We also found that treatment of EMF up-regulated the antiviral protein Mx1, which may play a partial role in inhibiting influenza virus infection in pre- and co-EMF treated MDCK cells. In summary, these results strongly suggested that an ethanolic extract of Meliae Fructus inhibited influenza A virus infection by affecting viral entry, PA proteins of the RNA polymerase complex, and Mx1 induction and may be a potential and novel anti-influenza agent.

Protective Effect of Panax notoginseng Root Water Extract against Influenza A Virus Infection by Enhancing Antiviral Interferon-Mediated Immune Responses and Natural Killer Cell Activity

“…To evaluate the anti-influenza A virus effects of PNR, we used GFP-expressing viruses as previously described ( 18 , 45 ). Pretreatment of RAW 264.7 cells infected with A/PR8/34-GFP (MOI = 1) with PNR resulted in a marked reduction in GFP expression in a concentration-dependent manner (Figure 4 A), and virus-induced death was also concentration-dependently suppressed in pretreated cells (Figure 4 B).…”

“…KBPV-VR-32 (H3N2) was purchased from the Korea Bank for Pathogenic Viruses. All three strains were propagated in allantoic fluid from 10-day-old chicken embryos ( 18 , 45 ) and viral titers were determined as described previously ( 18 , 45 ).…”

“…The NI assay was performed using an NA-Fluor™ Influenza Neuraminidase Assay Kit (Applied Biosystems, Foster City, CA, USA), as per the manufacturer’s instructions ( 45 ).…”

“…Total RNA was extracted from cell lysates using an RNeasy mini kit (Qiagen, Hilden, Germany), according to the supplier’s instructions ( 18 , 45 ). qRT-PCR was performed using AccuPower ® 2× Greenstar qPCR Master Mix (Bioneer, Daejeon, South Korea) and a CFX96 Touch Real-Time PCR System (Bio-Rad, Hercules, CA, USA), according to the suppliers’ instructions ( 18 , 45 ). The sense and antisense primer sequences are listed in Table 1 .…”

“…Equal amounts of protein lysate in RIPA buffer were separated by 12% SDS-PAGE ( 18 , 45 ). After transfer, PVDF membranes were incubated at room temperature for 1 h with primary antibodies (1:1,000) followed by HRP-conjugated secondary antibodies (1:2,000) at room temperature for 1 h. Protein bands were detected using enhanced chemiluminescence reagent and a ChemiDoc imaging system ( 45 ). The presented data are representative of at least three independent experiments.…”