Rotaviruses are the most prevalent cause of acute viral gastroenteritis in children less than five years of age. Recent estimates indicate 2.7 million cases occur each year in the U.S. and 600,000 deaths occur annually worldwide (reviewed in [1
]). Available data on two attenuated vaccines show they both are efficacious in reducing the incidence and severity of rotavirus gastroenteritis in the U.S. and other developed countries [2
]. Reported vaccine efficacy is lower in developing nations where the majority of deaths from rotavirus infections occur, likely due to multiple factors including suboptimal immune responses associated with poor nutritional status or concurrent enteric infections [4
]. Methods to enhance natural disease resistance find value in these regions if protective immunity could be improved by complementing existing vaccines with products that have antiviral or immune adjuvant activity.
Licorice is derived from the root of the perennial herb Glycyrrhiza
and in addition to its use as a sweetening agent, has been one of the more extensively used medicinal plants [8
]. Pharmacologically active components that have been most studied include triterpene saponins, with glycyrrhizin (GA) being present in the highest concentration [9
]. 18β-glycyrrhetinic acid (GRA) is the aglycone product of GA hydrolysis mediated in the gut by bacterial glucoronidases [11
]. Both GA and GRA have been studied in several systems to evaluate their immunomodulatory properties. GA has been used in Japan for >20 years to treat chronic viral hepatitis, and patients administered a continuous regimen of an intravenous formulation of GA (Stronger Neo--Minophagen C®, SNMC) demonstrate clinical improvement and reduced incidence of hepatocellular carcinoma [13
]. GA also has been studied in an animal model of viral infection. Mice administered GA intraperitoneally survived a lethal dose of influenza virus [17
]. Antibody to IFNγ abolished this protective effect, but the mechanisms of protection in this model system remain unclear. While mechanisms of action in vivo
are not well understood, they likely are multi-factorial and could be due to direct effects on virus replication, or functions associated with modulation of inflammatory and cell protective responses.
The initial report describing antiviral activity of GA in vitro
showed reduced replication of vesicular stomatitis virus (VSV), vaccinia virus (VV), herpes simplex 1 virus (HSV-1), and Newcastle Disease virus (NDV), but not poliovirus type 1 [18
]. Subsequently, GA was shown to have antiviral activity against viruses in several families including flaviviruses, herpesviruses, influenza virus, SARS coronavirus, hepatitis C virus and others (reviewed in [19
]). Suggested mechanisms of activity in these systems include direct effects on the adsorption, penetration and particle maturation steps of the replication cycle and in some cases, direct inactivation of virus particles. For example, anti-influenza virus activity of GA in cell culture was attributed to interference with viral endocytosis, likely due to its ability to modulate membrane fluidity [20
], although alternative mechanisms recently have been proposed [22
GRA has anti-tumorigenic, anti-ulcerative, anti-inflammatory, and anti-hepatotoxic activity in vitro
and in vivo
]. The utility of GRA as an antiviral compound is not as well described as GA, but there are data to suggest GRA has immunomodulatory and cell protective activity in vitro
and in vivo.
For example, in vitro
, GRA induces NFĸB-mediated nitric oxide synthase expression in macrophages [24
], and IL-8 expression in epithelial cells [25
]. Others studies also suggest potential anti-inflammatory activity associated with reduced cytokine expression and attenuation of NFĸB activation [26
]. In vivo
, protective efficacy of GRA against protozoan and bacterial infections has been demonstrated. In a mouse model of visceral leishmaniasis [24
], analysis of pro-inflammatory cytokine transcripts in isolated spleen cells from infected animals revealed that GRA induces IFNγ, TNFα, and IL-12, indicative of a TH
1 and curative response. In a bacterial system, GRA attenuated lung pathology associated with Staphyloccus aureus
The relative lack of data regarding specific antiviral activity of GRA compared to GA led us to expand on previous data showing GRA treatment reduces rotavirus replication in cell culture [25
], and include GA in our analyses, given the wealth of data available for this compound and its current availability for clinical use. We show that GRA treatment results in a significant reduction in rotavirus titers when added to cultures post-infection. The levels of viral structural proteins VP2 and VP6, and nonstructural protein NSP2 were reduced in GRA treated cells, consistent with anti-rotavirus activity of this compound.