- Open Access
Antiviral activity of carnosic acid against respiratory syncytial virus
© Shin et al.; licensee BioMed Central Ltd. 2013
Received: 25 June 2013
Accepted: 2 October 2013
Published: 8 October 2013
Human respiratory syncytial virus (hRSV) is a leading cause of severe lower respiratory infection and a major public health threat worldwide. To date, no vaccine or effective therapeutic agent has been developed. In a screen for potential therapeutic agents against hRSV, we discovered that an extract of Rosmarinus officinalis exerted a strong inhibitory effect against hRSV infection. Subsequent studies identified carnosic acid as a bioactive constituent responsible for anti-hRSV activity. Carnosic acid has been shown to exhibit potent antioxidant and anti-cancer activities. Anti-RSV activity of carnosic acid was further investigated in this study.
Effects of extracts from various plants and subfractions from R. officinalis on hRSV replication were determined by microneutralization assay and plaque assay. Several constituents were isolated from ethyl acetate fraction of R. officinalis and their anti-RSV activities were assessed by plaque assay as well as reverse-transcription quantitative PCR to determine the synthesis of viral RNAs.
Among the tested bioactive constituents of R. officinalis, carnosic acid displayed the most potent anti-hRSV activity and was effective against both A- and B-type viruses. Carnosic acid efficiently suppressed the replication of hRSV in a concentration-dependent manner. Carnosic acid effectively suppressed viral gene expression without inducing type-I interferon production or affecting cell viability, suggesting that it may directly affect viral factors. A time course analysis showed that addition of carnosic acid 8 hours after infection still effectively blocked the expression of hRSV genes, further suggesting that carnosic acid directly inhibited the replication of hRSV.
The current study demonstrates that carnosic acid, a natural compound that has already been shown to be safe for human consumption, has anti-viral activity against hRSV, efficiently blocking the replication of this virus. Carnosic acid inhibited both A- and B- type hRSV, while it did not affect the replication of influenza A virus, suggesting that its antiviral activity is hRSV-specific. Collectively, this study suggests the need for further evaluation of carnosic acid as a potential treatment for hRSV.
Human respiratory syncytial virus (hRSV), a single-stranded RNA virus of the family Paramyxoviridae, is a leading cause of lower respiratory tract infection in infants and children . This is especially true for high-risk groups, including infants with congenital heart disease and immunosuppressed patients, where infection by hRSV causes severe mortality . It has been estimated that acute lower respiratory infection by hRSV caused approximately 66,000–199,000 deaths of children under the age of five worldwide in 2005 . Despite the severity of the global health threat and economic burden posed by hRSV infection, no effective hRSV-specific antiviral agents have been developed to date. Ribavirin, a nucleoside analog, is the only approved drug for severe hRSV infection, but it is not recommended owing to several factors, including its toxicity and difficulty of administration . Notably, ribavirin has shown limited efficacy in clinical trials on children with bronchiolitis caused by hRSV [5–8]. Although immunoprophylaxis with an hRSV-neutralizing antibody (palivizumab, MedImmune) confers substantial protection against severe hRSV diseases, no licensed vaccine is currently available. Thus, there is an urgent need for the development of effective and safe vaccines and therapeutics. Previous studies showed inhibition of hRSV by several natural compounds, including amentoflavone ; anagyrine, oxymatrine, sophoranol, wogonin, and oroxylin A ; 3-alpha-hydroxy-lup-20(2 9)-ene-23,28-dioic acid and 3-epi-betulinic acid 3-O-sulfate ; and cimicifugin .
Extract from Rosmarinus officinalis (Rosemary) has been known to possess strong antioxidant activity and widely used for food preservation . In traditional medicine, R. officinalis has been used to treat various conditions . In addition, R. officinalis has been used as a traditional medicine to treat various infectious diseases, and its antimicrobial effect against various bateria has been proved by several studies [15–17]. Among its constituents, carnosic acid (CAS #3650-09-7), a benzenediol abietane diterpene and its degradation product carnosol are well known antioxidative compounds . In addition to its antioxidant activity, carnosic acid exerts growth-inhibitory effects on breast cancer cells , ovarian cancer cells , and prostate cancer cells  by inducing apoptosis. Carnosic acid also possesses anti-bacterial, anti-inflammatory and neuroprotective activities [22–24]. Although carnosic acid has been shown to inhibit human immunodeficiency virus (HIV) protease activity , little is known about the antiviral actions of carnosic acid.
In this study, we found that carnosic acid from R. officinalis was capable of inhibiting hRSV infection and replication, suggesting its potential therapeutic and prophylactic use against hRSV infection.
Results and discussion
R. Officinalis extract inhibits the replication of hRSV
Isolation of compounds from R. officinalis
Identification of carnosic acid as the compound responsible for anti-hRSV activity
Effect of carnosic acid on interferon production upon hRSV infection
Analysis of cytotoxic activity of carnosic acid
Assessment of the anti-hRSV activity of carnosic acid
Inhibition of hRSV replication and infection by carnosic acid
In this study, we have shown that carnosic acid effectively suppresses hRSV replication. Carnosic acid showed similar suppressive effect against both A- and B-type hRSV, while it did not inhibit influenza A virus replication. Since carnosic acid did not induce significant cell death and interferon production at the used concentration, it is not likely that the inhibitory effect is due to the effect on host cells. Failure to suppress influenza A virus also support that notion. Moreover, carnosol, which has similar structure and possesses antioxidative activity, was unable to inhibit hRSV RNA synthesis. It suggests that the suppressive effect of carnosic acid is not due to the antioxidative activity of carnosic acid. Although the mechanism by which carnosic acid inhibit the replication of hRSV is unclear, it is likely that it affect viral factors such as viral proteins. In our study, we have shown that the levels of viral RNAs were significantly lowered by the treatment of carnosic acid on infected cells by using primers complementary to F, SH, NS2 and G proteins. Since carnosic acid is unable to inhibit the replication of influenza A virus, which also use negative sense ssRNA as their genome, it is not likely carnosic acid inhibits hRSV by directly interacting with viral RNAs. Given that carnosic acid inhibits hRSV replication by affecting viral proteins, one possible mechanism is carnosic acid act as an inhibitor of viral RNA synthesis by affecting viral proteins, which are related to viral RNA synthesis. For example, carnosic acid may directly affect RNA polymerase activity of hRSV L protein or cofactor P protein to suppress hRSV RNA synthesis. Targeting M2 protein by carnosic acid also can be a possible hypothesis, since M2 protein of hRSV has been known to be important for efficient transcription of viral mRNAs . Time-of-addition study results suggest that carnosic acid also affect the initial infection step. Thus, it can be postulated that carnosic acid may play an additional different role in inhibiting hRSV by interacting viral surface proteins such as F, G and SH proteins. Further investigations such as isolation of resistant mutant viruses or identification of viral proteins which interact with carnosic acid will reveal the more specific mechanism of inhibition.
The use of ribavirin for the treatment for hRSV infection is limited by its toxicity and limited efficacy; thus, there is an urgent need for the development of new therapeutic agents against hRSV. The current study demonstrates that carnosic acid, which has been used as a preservative and antioxidant in food, effectively inhibits the replication of hRSV. Carnosic acid not only reduced viral RNA synthesis, it also inhibited the initial infection of hRSV. Consequently, hRSV progeny virus production was greatly reduced by carnosic acid treatment. Since treatment with carnosic acid both pre- and post-exposure suppressed the replication of hRSV, this compound might be a potential prophylactic and therapeutic agent against hRSV infection.
Material and methods
Cells and viruses
The human larynx carcinoma cell line HEp-2 (CCL-23) and human adenocarcinoma alveolar basal epithelial cell line A549 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/mL). hRSV A2 and hRSV KR/B (subgroup B) were described elsewhere . Viruses were propagated by infecting HEp-2 cell monolayers with a previously prepared small-scale isolate stock at a multiplicity of infection (MOI) of 0.01. Viruses were harvested when cytopathic effects were greater than 60% and were titrated by plaque assay as described previously . Influenza A/Puerto Rico/8/1934 virus was propagated in specific pathogen-free embryonated eggs as described previously .
The rosemary plant (Rosmarinus officinalis L.) and other plants used in this study were purchased from a local market at Seoul. A voucher specimen (No. 2012-ROOF01) has been deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University.
Screening of plant extracts for anti-hRSV activity by microneutralization assay
Plant extracts were initially screened using microneutralization assays, as described previously . Briefly, methanol (MeOH) extracts of each plant were dissolved in DMSO and serially diluted with Phosphate buffered saline (PBS). MeOH extracts (20 μg/ml) of each plant were added to HEp-2 cells (5 × 104 cells/well) together with 1000 plaque-forming units (pfu) of hRSV A2 virus. Final concentration of DMSO was 2% in all samples. Three days after infection, cells were fixed with 3.7% formaldehyde. After blocking with 5% skim milk, the expression of hRSV fusion (F) protein was assessed by enzyme-linked immune sorbent assay (ELISA) using mouse anti-F monoclonal antibody (Sino Biological) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG.
Extraction and isolation of compounds
Air-dried, powdered aerial parts of R. officinalis (12.5 g) were extracted three times with 70% ethanol (EtOH) by maceration. The extracts were combined and concentrated in vacuo at 40°C to give a 70% EtOH extract (3.11 g). The extract was suspended in H2O (100 mL) and successively extracted with n- hexane (3 × 100 mL), ethyl acetate (EtOAc; 3 × 100 mL), and butanol (BuOH; 3 × 100 mL) to give n- hexane-soluble (719.7 mg), EtOAc-soluble (950.2 mg), BuOH-soluble (460.6 mg), and water-soluble extracts (966.5 mg), respectively. Based on preliminary biological test results, the EtOAc-soluble extract (940 mg) was subjected to column chromatography on silica gel to obtain pure compounds. The purities (>95%) and structures of the obtained compounds were determined by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. The structures of the isolates were identified based on comparisons of physical and spectroscopic data with published values.
RNA isolation and reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR)
List of primers used for RT-qPCR in this study
hRSV A2 F
hRSV A2 NS2
hRSV A2 SH
hRSV B G
Influenza A NP
Influenza A M2/M1
Effect of carnosic acid on interferon production upon hRSV infection
The levels of interferon mRNAs were determined by RT-qPCR. A549 cells were treated with 20 μg/ml of designated compound one hour prior to hRSV infection. Cells were then incubated for additional 6 hours, followed by RNA isolation and RT-qPCR. To analyze the effect of compounds on interferon production without viruses, cells were incubated with vehicle or designated compounds (20 μg/ml) for 6 hours without virus infection. Primers complementary to interferon-β, λ1 and λ2 (Table 1) were used to analyze mRNA levels. mRNA levels were normalized to β-actin mRNA levels.
Effect of carnosic acid on viral RNA synthesis
A549 cells were treated with different concentrations of designated compound one hour prior to hRSV infection. Cells were then infected with hRSV A2 (MOI 0.5) for an hour. After washing to remove unbound viruses, cells were further incubated for 48 hours in media containing the same concentrations of each compound. Total RNAs were isolated and used for RT-qPCR. The replication of hRSV A2 virus was analyzed using primers complementary to F protein, SH protein and non-structural protein 1 (NS1). To determine the replication of hRSV B virus, primers complementary to NS2 and G protein of hRSV B type were used for quantitative PCR. The replication of influenza A virus was analyzed using primers complementary to NP protein and M2/M1 protein. Viral RNA levels were normalized to β-actin mRNA levels. Sequences of primers used are listed in Table 1.
Cell viability was determined by a MTT (3-(4,5)-dimenthylthiahiazo(−z-y1)-di-phenytetrazoliumromide) cell viability assay. A549 cells in a 96-well plate were cultured in DMEM containing increasing concentrations of carnosic acid for 48 h. Next, the culture medium was replaced with fresh medium containing 20 μl of MTT (5 mg/ml) for 4 h. After that the MTT containing medium was aspirated and 200 μl of DMSO was added to lyse the cells and solubilize the water insoluble formazone. Absorbance of the lysates was determined on a microplate reader at 570 nm.
Carnosic acid-induced apoptosis was determined by annexin V and PI double labeling. Cells treated with or without carnosic acid for 24 or 48 hours were washed with PBS, and stained with 5 μl of FITC-conjugated annexin V and 5 μl of PI (50 μg/ml) in 100 μl binding buffer (10 mM HEPES at pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). The fluorescence of annexin V and PI were monitored by FC500 fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (Beckman Coulter, CA, USA) at an excitation wavelength of 488 nm and emission wavelengths of 525 and 625 nm, respectively. Five thousand events were collected per sample.
Virus yield-reduction assay
A549 cells were treated with designated extracts (20 μg/ml) 1 hour prior to hRSV inoculation. hRSV (MOI 0.5) was then added and cells were incubated for 1 hour to permit viral internalization. Free virus particles were removed by washing three times with phosphate-buffered saline (PBS), and infected cells were further incubated in medium containing 2% FBS and vehicle (DMSO) or 20 μg/ml of designated extracts. Viruses were harvested 3 days after infection and subsequently titrated by plaque assay. To determine the effect of carnosic acid, carnosic acid (20 μg/ml) was added to cells instead of extracts. Viruses were harvested 2 or 4 days after infection and subjected to plaque assay. Viral titer was expressed as pfu/mL.
A549 cells were seeded and incubated overnight. Cells were treated with carnosic acid (10 μg/ml) at different time points before and after viral inoculation (MOI 0.5). Total RNA was prepared from cells 48 hours after viral inoculation, followed by RT-qPCR using primers specific for F and NS2 proteins. The effect of carnosic acid on the initial infection by hRSV was examined by treating cells with carnosic acid 1 hour prior to inoculation with virus. After inoculation, cells were incubated for 1 hour to allow viral internalization. Free virus and carnosic acid were removed by extensive washing with PBS. As a control, cells were treated with carnosic acid for an hour and washed before viral inoculation to exclude the possibility that carnosic acid altered viral infection or replication by affecting cell physiology. Total RNA was prepared from cells 48 hours after viral inoculation and subjected to RT-qPCR using primers specific for F and NS2 proteins, as described above.
Microneutralization assays and RT-qPCR assays were repeated at least three times. Values were expressed as means and standard deviation from triplicate samples of a representative result. Statistical comparisons between the control and treated groups were analyzed using the Student’s t-test. The level of statistical significance was set at either P<0.05 (*), 0.01 (**) or 0.001(***).
This work was supported by a grant from the Kyung Hee University in 2012 (KHU-20120474).
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