18β-glycyrrhetinic acid inhibits rotavirus replication in culture
© Hardy et al.; licensee BioMed Central Ltd. 2012
Received: 26 December 2011
Accepted: 4 May 2012
Published: 22 May 2012
Glycyrrhizin (GA) and primary metabolite 18β-glycyrrhetinic acid (GRA) are pharmacologically active components of the medicinal licorice root, and both have been shown to have antiviral and immunomodulatory properties. Although these properties are well established, the mechanisms of action are not completely understood. In this study, GA and GRA were tested for the ability to inhibit rotavirus replication in cell culture, toward a long term goal of discovering natural compounds that may complement existing vaccines.
Epithelial cells were treated with GA or GRA various times pre- or post-infection and virus yields were measured by immunofluorescent focus assay. Levels of viral proteins VP2, VP6, and NSP2 in GRA treated cells were measured by immunoblot to determine if there was an effect of GRA treatment on the accumulation of viral protein.
GRA treatment reduced rotavirus yields by 99% when added to infected cultures post-- virus adsorption, whereas virus yields in GA treated cultures were similar to mock treated controls. Time of addition experiments indicated that GRA-mediated replication inhibition likely occurs at a step or steps subsequent to virus entry. The amounts of VP2, VP6 and NSP2 were substantially reduced when GRA was added to cultures up to two hours post-entry.
GRA, but not GA, has significant antiviral activity against rotavirus replication in vitro, and studies to determine whether GRA attenuates rotavirus replication in vivo are underway.
KeywordsRotavirus Licorice 18beta-glycyrrhetinic acid Antiviral
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 ). 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 [2–5]. 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, 6, 7]. 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 spp., and in addition to its use as a sweetening agent, has been one of the more extensively used medicinal plants . Pharmacologically active components that have been most studied include triterpene saponins, with glycyrrhizin (GA) being present in the highest concentration [9, 10]. 18β-glycyrrhetinic acid (GRA) is the aglycone product of GA hydrolysis mediated in the gut by bacterial glucoronidases [11, 12]. 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 [13–16]. GA also has been studied in an animal model of viral infection. Mice administered GA intraperitoneally survived a lethal dose of influenza virus . 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 . 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 ). 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, 21], although alternative mechanisms recently have been proposed .
GRA has anti-tumorigenic, anti-ulcerative, anti-inflammatory, and anti-hepatotoxic activity in vitro and in vivo[19, 23]. 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 , and IL-8 expression in epithelial cells . Others studies also suggest potential anti-inflammatory activity associated with reduced cytokine expression and attenuation of NFĸB activation [26, 27]. In vivo, protective efficacy of GRA against protozoan and bacterial infections has been demonstrated. In a mouse model of visceral leishmaniasis [24, 28], 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 TH1 and curative response. In a bacterial system, GRA attenuated lung pathology associated with Staphyloccus aureus pneumonia .
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 , 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.
GRA and GA cytotoxicity in MA104 cells
GRA reduces rotavirus infectivity while GA has no effect
GRA does not reduce infectivity through a direct effect on the virus particle
GRA reduces infectivity at a post--entry step
GRA was inhibitory to RRV replication only when added to cultures post-virus adsorption. To determine whether the reduction in titer observed when cells were treated with GRA was a result of a block to virus entry, cells were inoculated with RRV at a multiplicity of infection of three pfu/cell, and virus was adsorbed for one hour at 4°C. Incubation at 4°C allows virus adsorption, but not entry into the cells [30, 31]. After the adsorption period, cultures were incubated for one hour at 37°C to allow virus entry, and then GRA or DMSO was added and infections were allowed to proceed for five hours. The results showed that infectious virus titers were reduced by 99% when GRA was added following the 37°C incubation (Figure 3B), suggesting that GRA inhibits RRV replication at a step that is subsequent to virus entry into the cell.
Viral protein levels are reduced in GRA treated cells
The antiviral properties of two principal pharmacologically active constituents of licorice root, GA and GRA, have been extensively described, with by far the most data available for GA [8, 23, 32, 33]. The mechanisms by which these compounds are antiviral, and the steps of the virus replication cycle at which they act vary depending on the virus under investigation. There are ample data to suggest antiviral activity of GA in vivo may result from induction of cytoprotective and immunomodulatory responses rather than effects on virus-specific functions per se, and such mechanisms of action could in part explain its broad-spectrum activity (reviewed in . In contrast to GA, fewer reports of in vivo activity of GRA are available. However, when administered intravenously, GA is metabolized in the liver by lysosomal β-D-glucoronidase into 3-mono--glucoronide-glycyrrhetinic acid, and then finally by glucoronidases of intestinal bacteria into GRA, which then can be reabsorbed [11, 12, 34]. Therefore, it is possible that some of the in vivo effects of GA are due to one or more of its metabolites, including GRA.
We have shown that GRA reduces rotavirus yields during a single round of replication in MA104 cells whereas GA does not. Given numerous reports of antiviral activity of GA in vitro, it was somewhat surprising that GA did not effect rotavirus replication. However, inhibitory activity of GA may have a preference for enveloped viruses, consistent with potential mechanisms of action attributed to its effects on membrane fluidity.
GRA did not directly inactivate the rotavirus particle because yields of infectious virus were not reduced when virus stocks were incubated with the compound prior to inoculation onto cells. The reduction in yields of infectious virus and viral protein levels when GRA was added to cultures up to two hours post-adsorption suggests interference with rotavirus replication occurs at a step subsequent to virus entry. These data are consistent with previous studies illustrating a similar effect of GA on in vitro replication of NDV, VSV, HSV-1, and VV when the compound was added several hours post-infection . Interestingly, the amount of VP2 present when GRA was added two-hours post-entry was similar to the levels observed when GRA was added one hour post-entry, but the amounts of VP6 and NSP2 were considerably higher. This observation could be explained by effects on viral transcription, translation or degradation of viral proteins, but how such mechanisms would explain the selective reduction are difficult to predict. Studies to conclusively define steps at which GRA exerts its inhibitory effect are in progress.
There are multiple mechanisms by which GRA could reduce virus yields, including effects on specific steps of the replication cycle or on cellular pathways that play a role in establishment of an environment conducive to maximum replication efficiency. For example, we have shown that GRA induces NFĸB activation in MA104 cells , and more recently reported that some rotavirus strains down-regulate NFĸB activity through the functions of nonstructural protein NSP1 . NFĸB activity is required for induction of a robust antiviral response, and sustained activation of NFĸB by GRA could override virus-encoded mechanisms that down-regulate its activity, resulting in reduced virus replication. Other studies also report GRA-mediated activation of NFĸB [24, 28]. Of note, however, are reports showing GRA inhibits NFĸB activation, and this has been interpreted as GRA-mediated regulation of the inflammatory response [22, 26, 36]. In these and other studies, the effects of GRA on NFĸB were measured following induction of the pathway by virus infection or by pro-inflammatory mediators, suggesting that the effects of GRA on NFĸB are context-dependent. Deciphering the interactions between GRA and the NFĸB signaling pathway at the molecular level will contribute to understanding how its activity is modulated depending on the stimulus. One possible alternative mechanism of replication inhibition that is not mutually exclusive is provided by several reports indicating GRA modulates PI3/Akt activity [27, 37, 38]. Pharmacological inhibition of the PI3/Akt pathway substantially reduces rotavirus yields in culture . The effects of GRA on these signaling pathways in the context of rotavirus infection and how GRA may modulate them currently are under investigation.
Together, the data reported here show GRA inhibits rotavirus replication at a step or steps subsequent to virus entry, and support antiviral activity of GRA reported in other systems. Rotavirus infects mature enterocytes at the villus tips in the proximal small intestine and thus in vivo, the tropism is polarized epithelial cells. Continuing studies will address whether there is a difference either in GRA toxicity or in the levels of reduction in virus replication in this cell type. We have administered GRA up to concentrations of 50 mg/kg to mice and have not observed clinical signs that would indicate toxicity (unpublished data), suggesting that inhibitory levels can be achieved in vivo. We currently are determining whether GRA-mediated replication inhibition in vitro translates to attenuation of virus replication in the mouse model of rotavirus infection.
Cells, virus, and compounds
MA104 monkey kidney epithelial cells (ATCC CRL2378) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Mediatech) supplemented with 5% fetal bovine serum (FBS, Atlanta Biologicals). Rhesus rotavirus strain RRV was propagated in MA104 cells and stock titers were determined by immunofluorescent focus assay (IFA, see below).
Glycyrrhizin (GA) and 18β-glycyrrhetinic acid (GRA) were purchased from Sigma-Aldrich. Stock solutions were prepared to a concentration of 100 mg/mL in DMSO and aliquots were stored at -80°C. Stock solutions were diluted to working concentrations in DMEM without FBS.
Cell viability under conditions of GA or GRA treatment was measured with the CellTiter-Glo Luminescent Cell Viability Assay (Promega) following the manufacturer’s protocol. Controls for 100% cell viability or 100% toxicity consisted of cells and media alone, or treatment with digitonin, respectively.
Immunofluorescent focus assay (IFA)
Rotavirus infectivity following various treatments was determined by IFA as previously described . Briefly, MA104 cells were cultured to confluence in 96-well microtiter plates. Wells were inoculated with 10-fold dilutions of RRV stock pre-treated with 10 μg/mL TPCK-trypsin (Worthington Biochemicals) for 30 minutes at 37°C. Sixteen hours post-infection, cells were fixed for 10 minutes with 2% paraformaldehyde, and then for 10 minutes with ice cold 100% methanol to permeabilize membranes. Structural protein VP6 was detected with mouse monoclonal antibody A6M  at a 1:200 dilution, followed by Alexa Fluor® 488-conjugated goat anti-mouse antibody (1:4000, Molecular Probes). VP6-positive foci indicative of infected cells were enumerated and titers expressed as fluorescent focus units (FFU) per mL.
Infections, compound treatments, and culture harvest
Infections were performed as previously described . MA104 cells were inoculated with trypsin-activated RRV at a multiplicity of infection of three pfu/cell, and virus was adsorbed to cells for one hour at 37°C. Following the adsorption step, media was removed and replaced with fresh media with or without compound according to the parameters of the experiment. Six hours post-infection, cells and supernatant were collected and subjected to three freeze-thaw cycles. Lysates were clarified by low speed centrifugation, and virus in the supernatant was titered by IFA.
GA, GRA, or DMSO vehicle control was added to cultures at the times indicated by each experiment. Pre-infection treatments were for six hours prior to addition of virus, and compound was not present in the media for the remainder of the infection. For post-infection treatments, compound was added following the virus adsorption or entry step and was present in the media for the duration of the infection.
Cell lysates for immunoblot were prepared as previously described . Proteins were separated by SDS-polyacrylamide gel electrophoresis and immunoblots were probed with anti-SA114F rotavirus polyclonal antiserum that recognizes structural proteins VP2 and VP6, anti-GAPDH (Ambion), or anti-NSP2 (kindly provided by M Estes, Baylor College of Medicine) antibodies. Proteins that reacted with antibody were visualized by chemiluminescence using the Pierce ECL kit.
This work was supported by PHS grant NCCAM AT004986 to MEH. Additional support was provided by NCRR grant RR020185-09, NIGM grant GM103500-09, equipment from the Murdock Charitable Trust, and the Montana Agriculture Experiment Station.
- Widdowson MA, Steele D, Vojdani J, Wecker J, Parashar U: Global rotavirus surveillance: determining the need and measuring the impact of rotavirus vaccines. J Infect Dis 2009, Suppl 1:S1-S8.View Article
- Vesikari T, Matson DO, Dennehy P, Van Damme P, Santosham M, Rodriguez Z, Dallas MJ, Heyse JF, Goveia MG, Black SB, et al.: Safety and efficacy of a pentavalent human--bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 2006,354(1):23–33.PubMedView Article
- Ruiz-Palacios GM, Perez-Schael I, Velazquez FR, Abate H, Breuer T, Clemens SC, Cheuvart B, Espinoza F, Gillard P, Innis BL, et al.: Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med 2006,354(1):11–22.PubMedView Article
- O'Ryan M, Lucero Y, Linhares AC: Rotarix (R): vaccine performance 6 years postlicensure. Expert Rev Vaccines 2011,10(12):1645–1659.PubMedView Article
- Ciarlet M, Schodel F: Development of a rotavirus vaccine: clinical safety, immunogenicity, and efficacy of the pentavalent rotavirus vaccine, RotaTeq. Vaccine 2009, 27 Suppl 6:G72-G81.PubMedView Article
- Zaman K, Dang DA, Victor JC, Shin S, Yunus M, Dallas MJ, Podder G, Vu DT, Le TP, Luby SP, et al.: Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, double--blind, placebo--controlled trial. Lancet 2010,376(9741):615–623.PubMedView Article
- Armah GE, Sow SO, Breiman RF, Dallas MJ, Tapia MD, Feikin DR, Binka FN, Steele AD, Laserson KF, Ansah NA, et al.: Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub--Saharan Africa: a randomised, double--blind, placebo--controlled trial. Lancet 2010, 376:606–614.PubMedView Article
- Fiore C, Eisenhut M, Ragazzi E, Zanchin G, Armanini D: A history of the therapeutic use of liquorice in Europe. J Ethnopharmacol 2005,99(3):317–324.PubMedView Article
- Robbers JSS MK, Tyler VE: Pharmacognosy and Pharmacobiotechnology. Williams and Wilkins, Baltimore; 1996.
- Barnes JA, Anderson LA, Phillipson JD: Herbal Medicine. Pharmaceutical Press, London; 2002.
- Hattori M, Sakamoto T, Kobashi K, Namba T: Metabolism of glycyrrhizin by human intestinal flora. Planta Med 1983,48(5):38–42.PubMedView Article
- Hattori M, Sakamoto T, Yamagishi T, Sakamoto K, Konishi K, Kobashi K, Namba T: Metabolism of glycyrrhizin by human intestinal flora. II. Isolation and characterization of human intestinal bacteria capable of metabolizing glycyrrhizin and related compounds. Chem Pharm Bull (Tokyo) 1985,33(1):210–217.View Article
- van Rossum TG, Vulto AG, de Man RA, Brouwer JT, Schalm SW: Review article: glycyrrhizin as a potential treatment for chronic hepatitis C. Aliment Pharmacol Ther 1998,12(3):199–205.PubMedView Article
- Orlent H, Hansen BE, Willems M, Brouwer JT, Huber R, Kullak-Ublick GA, Gerken G, Zeuzem S, Nevens F, Tielemans WC, et al.: Biochemical and histological effects of 26 weeks of glycyrrhizin treatment in chronic hepatitis C: a randomized phase II trial. J Hepatol 2006,45(4):539–546.PubMedView Article
- Ikeda K, Arase Y, Kobayashi M, Saitoh S, Someya T, Hosaka T, Sezaki H, Akuta N, Suzuki Y, Suzuki F, et al.: A long--term glycyrrhizin injection therapy reduces hepatocellular carcinogenesis rate in patients with interferon--resistant active chronic hepatitis C: a cohort study of 1249 patients. Dig Dis Sci 2006,51(3):603–609.PubMedView Article
- Arase Y, Ikeda K, Murashima N, Chayama K, Tsubota A, Koida I, Suzuki Y, Saitoh S, Kobayashi M, Kumada H: The long term efficacy of glycyrrhizin in chronic hepatitis C patients. Cancer 1997,79(8):1494–1500.PubMedView Article
- Utsunomiya T, Kobayashi M, Pollard RB, Suzuki F: Glycyrrhizin, an active component of licorice roots, reduces morbidity and mortality of mice infected with lethal doses of influenza virus. Antimicrob Agents Chemother 1997,41(3):551–556.PubMed
- Pompei R, Flore O, Marccialis MA, Pani A, Loddo B: Glycyrrhizic acid inhibits virus growth and inactivates virus particles. Nature 1979,281(5733):689–690.PubMedView Article
- Fiore C, Eisenhut M, Krausse R, Ragazzi E, Pellati D, Armanini D, Bielenberg J: Antiviral effects of Glycyrrhiza species. Phytother Res 2008,22(2):141–148.PubMedView Article
- Wolkerstorfer A, Kurz H, Bachhofner N, Szolar OH: Glycyrrhizin inhibits influenza A virus uptake into the cell. Antiviral Res 2009,83(2):171–178.PubMedView Article
- Harada S: The broad anti--viral agent glycyrrhizin directly modulates the fluidity of plasma membrane and HIV--1 envelope. Biochem J 2005,392(Pt 1):191–199.PubMed
- Michaelis M, Geiler J, Naczk P, Sithisarn P, Leutz A, Doerr HW, Cinatl J: Glycyrrhizin exerts antioxidative effects in H5N1 influenza A virus--infected cells and inhibits virus replication and pro--inflammatory gene expression. PLoS One 2011,6(5):e19705.PubMedView Article
- Armanini D, Fiore C, Mattarello MJ, Bielenberg J, Palermo M: History of the endocrine effects of licorice. Experimental and Clinical Endocrinology and Diabetes 2002, 110:257–261.PubMedView Article
- Jeong HG, Kim JY: Induction of inducible nitric oxide synthase expression by 18beta--glycyrrhetinic acid in macrophages. FEBS Lett 2002,513(2–3):208–212.PubMedView Article
- Shaneyfelt ME, Burke AD, Graff JW, Jutila MA, Hardy ME: Natural products that reduce rotavirus infectivity identified by a cell--based moderate--throughput screening assay. Virol J 2006, 3:68.PubMedView Article
- Kang OH, Kim JA, Choi YA, Park HJ, Kim DK, An YH, Choi SC, Yun KJ, Nah YH, Cai XF, et al.: Inhibition of interleukin--8 production in the human colonic epithelial cell line HT--29 by 18 beta--glycyrrhetinic acid. Int J Mol Med 2005,15(6):981–985.PubMed
- Wang CY, Kao TC, Lo WH, Yen GC: Glycyrrhizic acid and 18beta--glycyrrhetinic acid modulate lipopolysaccharide--induced inflammatory response by suppression of NF--kappaB through PI3K p110delta and p110gamma inhibitions. J Agric Food Chem 2011,59(14):7726–7733.PubMedView Article
- Ukil A, Biswas A, Das T, Das PK: 18 Beta--glycyrrhetinic acid triggers curative Th1 response and nitric oxide up--regulation in experimental visceral leishmaniasis associated with the activation of NF--kappa B. J Immunol 2005,175(2):1161–1169.PubMed
- Li HE, Qiu JZ, Yang ZQ, Dong J, Wang JF, Luo MJ, Pan J, Dai XH, Zhang Y, Song BL, et al.: Glycyrrhetinic acid protects mice from Staphylococcus aureus pneumonia. Fitoterapia 2012,83(1):241–248.PubMedView Article
- Mendez E, Lopez S, Cuadras MA, Romero P, Arias CF: Entry of rotaviruses is a multistep process. Virology 1999,263(2):450–459.PubMedView Article
- Sanchez-San Martin C, Lopez T, Arias CF, Lopez S: Characterization of rotavirus cell entry. J Virol 2004,78(5):2310–2318.PubMedView Article
- Asl MN, Hosseinzadeh H: Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytother Res 2008,22(6):709–724.PubMedView Article
- Pompei R, Laconi S, Ingianni A: Antiviral properties of glycyrrhizic acid and its semisynthetic derivatives. Mini Rev Med Chem 2009,9(8):996–1001.PubMedView Article
- Krahenbuhl S, Hasler F, Frey BM, Frey FJ, Brenneisen R, Krapf R: Kinetics and dynamics of orally administered 18 beta--glycyrrhetinic acid in humans. J Clin Endocrinol Metab 1994,78(3):581–585.PubMedView Article
- Graff JW, Ettayebi K, Hardy ME: Rotavirus NSP1 inhibits NFkappaB activation by inducing proteasome--dependent degradation of beta--TrCP: a novel mechanism of IFN antagonism. PLoS Pathog 2009,5(1):e1000280.PubMedView Article
- Chang YL, Chen CL, Kuo CL, Chen BC, You JS: Glycyrrhetinic acid inhibits ICAM--1 expression via blocking JNK and NF--kappaB pathways in TNF--alpha--activated endothelial cells. Acta Pharmacol Sin 2010,31(5):546–553.PubMedView Article
- Kao TC, Shyu MH, Yen GC: Glycyrrhizic acid and 18beta--glycyrrhetinic acid inhibit inflammation via PI3K/Akt/GSK3beta signaling and glucocorticoid receptor activation. J Agric Food Chem 2010,58(15):8623–8629.PubMedView Article
- Kao TC, Shyu MH, Yen GC: Neuroprotective effects of glycyrrhizic acid and 18beta--glycyrrhetinic acid in PC12 cells via modulation of the PI3K/Akt pathway. J Agric Food Chem 2009,57(2):754–761.PubMedView Article
- Halasz P, Holloway G, Turner SJ, Coulson BS: Rotavirus replication in intestinal cells differentially regulates integrin expression by a phosphatidylinositol 3--kinase--dependent pathway, resulting in increased cell adhesion and virus yield. J Virol 2008,82(1):148–160.PubMedView Article
- Zambrano JL, Ettayebi K, Maaty WS, Faunce NR, Bothner B, Hardy ME: Rotavirus infection activates the UPR but modulates its activity. Virol J 2011, 8:359.PubMedView Article
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