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Extract of Scutellaria baicalensis inhibits dengue virus replication
© Zandi et al.; licensee BioMed Central Ltd. 2013
Received: 24 January 2013
Accepted: 24 April 2013
Published: 29 April 2013
Scutellaria baicalensis (S. baicalensis) is one of the traditional Chinese medicinal herbs that have been shown to possess many health benefits. In the present study, we evaluated the in vitro antiviral activity of aqueous extract of the roots of S. baicalensis against all the four dengue virus (DENV) serotypes.
Aqueous extract of S. baicalensis was prepared by microwave energy steam evaporation method (MEGHE™), and the anti-dengue virus replication activity was evaluated using the foci forming unit reduction assay (FFURA) in Vero cells. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to determine the actual dengue virus RNA copy number. The presence of baicalein, a flavonoid known to inhibit dengue virus replication was determined by mass spectrometry.
The IC50 values for the S. baicalensis extract on Vero cells following DENV adsorption ranged from 86.59 to 95.19 μg/mL for the different DENV serotypes. The IC50 values decreased to 56.02 to 77.41 μg/mL when cells were treated with the extract at the time of virus adsorption for the different DENV serotypes. The extract showed potent direct virucidal activity against extracellular infectious virus particles with IC50 that ranged from 74.33 to 95.83 μg/mL for all DENV serotypes. Weak prophylactic effects with IC50 values that ranged from 269.9 to 369.8 μg/mL were noticed when the cells were pre-treated 2 hours prior to virus inoculation. The concentration of baicalein in the S. baicalensis extract was ~1% (1.03 μg/gm dried extract).
Our study demonstrates the in vitro anti-dengue virus replication property of S. baicalensis against all the four DENV serotypes investigated. The extract reduced DENV infectivity and replication in Vero cells. The extract was rich in baicalein, and could be considered for potential development of anti-DENV therapeutics.
Many traditionally used herbs contain phytochemicals that could be beneficial for health. These plant-derived compounds and extracts have been used in many forms for the treatment of various ailments and as dietary supplements [1–3]. Traditionally used herbal preparations commonly used by different communities across the world, have been the immediate source of knowledge for further discovery of potentially beneficial phytochemicals. It is a widely believed that naturally-derived phytochemicals would have lesser side-effects as they are often derived from edible herbs [4–6]. The traditional Chinese medicinal practices advocate the use of herbs, many of which have been extensively characterized and studied [7, 8]. Among these herbs include those that have been described to possess anti-infective properties [9–11]. Traditionally, most of these herbal-based medications are dispensed in aqueous forms, and be administered orally . Given the availability of huge number of traditionally used medicinal plants, it is likely that at least a few of these would exhibit antiviral activities at concentrations suitable for direct consumption .
S. baicalensis is one of the most widely used medicinal plants, and is officially listed in the Chinese Pharmacopoeia . Extracts of its roots have been widely used in the treatment of inflammation, cancer, infectious diseases, hypercholesterolemia and hypertension [13, 14]. The roots of this plant contain a plethora of bioactives, for instance different types of flavonoids such as baicalein and wogonin . Recently, we showed that several flavonoids including quercetin, fisetin [16, 17] and baicalein  possessed significant antiviral activities against dengue viruses (DENV).
Dengue is a serious viral disease in the tropical and subtropical regions of the world, and accounts for ~500,000 hospital admissions annually and consumes massive hospital resources . Dengue is currently one of the most rapidly spreading mosquito-borne diseases that is estimated to pose health threat to ~2.5 billion people living in endemic regions [19, 20]. Dengue virus (DENV) is an enveloped virus belonging to the Flaviviridae family with four distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) . Currently, there is no specific anti-dengue medication available and treatment is only supportive . In addition, there is no approved dengue vaccine available heretofore. Hence, there is an urgent and immediate necessity to explore an effective antiviral strategy against dengue. Since, single molecule antiviral drug development is a cumbersome, time-consuming and highly expensive endeavour, an efficient traditional herbal preparation could be an immediate alternative. Herein, we investigated the potentials of aqueous extract of S. baicalensis against the four DENV serotypes in regards to inhibition of different stages of replication in vitro.
S. baicalensis dried ground roots extract (AHPE-XA-09) used in the current investigation was provided by Herbitec Sdn Bhd (Malaysia), prepared using its own propriety method. Briefly, microwave energy generated steam was used to prepare the aqueous extract. The extract prepared in deionized water as a stock solution with concentration 60 mg/ml, was stored at −20°C. Before use in the experiments, the stock solution was clarified and sterilized using a syringe filter with 0.2 micron pore size (Millipore, MA, USA). Eagle’s minimum essential medium (EMEM) (Gibco, NY, USA) was used as a diluent to prepare the different concentrations of the extract.
Standardization of extract and mass spectrometry
Identification and quantitation of the active compound, baicalein, in AHPE-XA-09 was performed by liquid chromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Briefly, the concentration of pure baicalein ranging from 0.5 μg/mL to 20 μg/mL (Indofine Chemical Company Inc., New Jersey, USA) was dissolved in methanol (BDH Laboratory Supplies, England) and injected (10 μl) into the Applied Biosystems 3200 LC/MS/MS System (California, USA). Compound separation was achieved on a Gemini C6-Phenyl 110A column (150 mm×2.00 mm, particle diameter of 5 μm) (Phenomenex, USA) with a mobile phase composed of 0.1% formic acid (BDH Laboratory Supplies, England): acetonitrile (BDH Laboratory Supplies, England) at a flow rate of 0.50 mL/min. The transition ions and retention times were used as a detection point for baicalein peak. The transition ions were monitored in the multiple reaction monitoring modes (MRM). The area under the curve based on the different intensity height in the chromatogram of the different concentrations of baicalein was used to plot a standard linear regression curve. The AHPE-XA-09 (100 μg/mL lyophilized powder) was dissolved in methanol, filtrated through 0.45 μm PVDF filter (Whatman, USA) and was subsequently injected into the LC/MS/MS System. Inverse prediction method for the best fit linear regression curve generated from the pure baicalein was used for quantitation of the concentration of baicalein in AHPE-XA-09.
Cells and virus
C6/36 mosquito cell line was used for the propagation of all DENV isolates used in the investigation . Vero (African green monkey kidney) cell line was used for the evaluation of antiviral activity as previously described . The cell lines were maintained and propagated in EMEM (Gibco, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, NY, USA). The C6/36 and Vero cells were incubated at 28°C and 37°C respectively, in the presence of 5% CO2. At the time of virus inoculation and antiviral assays, the concentration of FBS was reduced to 2%. We used four different clinical DENV isolates representing the four serotypes of DENV (DENV-1, DENV-2, DENV-3 and DENV-4) in current investigation which identified by our group using full genome sequencing method. All the four clinical isolates propagated and maintained as previously described . After titration of the virus isolates, the stocks were stored at −70°C until further use in the experiments.
In vitro cytotoxicity assay
The cytotoxic potential of AHPE-XA-09 extract against Vero cells was determined using the MTT assay as described previously . Briefly, the confluent Vero cells in 96-well microplates were treated with different concentrations of AHPE-XA-09 in triplicates. The treated cells were incubated with the extract for four days, which was similar to the time period for the antiviral assay at 37°C followed by the addition of 15 μl of MTT solution to each well. The microplate was further incubated at 37°C for four hours and subsequently a solublization/stop solution was added. The optical density (OD) of all the wells including non-treated cells was read using a plate reader (TECAN, Mannendorf, Switzerland) at 570 nm. The cytotoxicity of AHPE-XA-09 was calculated using Graph Pad Prism 5 (Graph Pad Software Inc., San Diego, CA).
Antiviral activity assays
The effect of AHPE-XA-09 on intracellular replication of DENV was examined in Vero cells plated on 24-well cell culture microplate. After attaining 80% confluency, virus inoculum consisting of 200 FFU of each DENV serotype was added to each well. Viruses were allowed to adsorb to the cells for 1 hour at 37°C. Unadsorbed viruses were removed by rinsing the cells with sterile PBS twice. Different concentrations of AHPE-XA-09 were mixed with 1.5% carboxymethylcellulose (CMC) containing a cell-growth medium supplemented with 2% FBS and the plates were incubated at 37°C for four days. DENV foci were visualized as described previously . The number of foci formed was expressed as foci-forming unit (FFU). The antiviral effects of AHPE-XA-09 was measured by calculating the percentage of foci reduction (% RF) against the controls maintained in parallel using the following formula ; RF (%) = (C-T) × 100/C, where, C is the mean of the number of foci from triplicates without AHPE-XA-09 added and T is the mean of the number of foci from triplicates of each treatment with AHPE-XA-09 extract.
Time of addition studies
Prophylactic activity assay
The effects of prophylactic activity of AHPE-XA-09 prior to DENV infection were examined by treating a confluent monolayer of Vero cell line with different concentrations of AHPE-XA-09 for 5 h before DENV inoculation. The treatment medium was aspirated after 5 h, and the cells were washed twice with sterile PBS, and then infected with 200 FFU of each DENV serotypes. The microplate was kept at 37°C for 1 h to allow virus adsorption. Following virus adsorption, the infected monolayer was rinsed twice with sterile PBS and supplemented with 2% FBS containing EMEM with 1.5% CMC and the corresponding concentrations of the extract. Later, the plates were incubated at 37°C for 4 days in the presence of 5% CO2. Viral foci were stained and counted as described above.
Anti-adsorption activity assay
The effect of AHPE-XA-09 against DENVs adsorption and attachment to the Vero cells was determined by simultaneously adding the different concentrations of AHPE-XA-09 extract and 200 FFU DENV to Vero cells. The treated cells were washed with PBS after 1 h incubation at 37°C and supplemented with 2% FBS containing EMEM with 1.5% CMC. The plates were incubated at 37°C for 4 days with 5% CO2. Viral foci were stained and counted as described above.
Extracellular virucidal activity
The potential direct virucidal effects of AHPE-XA-09 on DENV were evaluated by treating the viral suspension with 200 FFU of DENV with increasing concentrations of AHPE-XA-09 for 2 h at 37°C. The confluent Vero cells in 24-well plate were infected with the AHPE-XA-09-treated viral suspensions. After 1 h of adsorption at 37°C, the cells were washed twice with PBS and overlaid with 1.5% CMC-containing cell culture medium. The microplate was incubated for 4 days in a humidified 37°C incubator in the presence of 5% CO2. The viral foci were visualized as described above.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Primer sequences used for q-RT-PCR of DENV
Forward: 5′ CAA TAT GCT GAA ACG CGC GAG AAA 3′
Reverse: 5′ GCT CCA TTC TTC TTG AAT GA 3′
Forward: 5′ CAA TAT GCT GAA ACG CGA GAG AAA 3′
Reverse: 5′ AAG ACA TTG ATG GCT TTT GA 3′
Forward: 5′ CAA TAT GCT GAA ACG CGT GAG AAA 3′
Reverse: 5′ GAA GGT TCC CCA TCT AGC CA 3′
Forward: 5′ GAA GTG AAA ACA TGT CTG TGG CCC A 3′
Reverse: 5′ TTC ACA GCA CAA TTA CCG CCA G 3′
The half maximal cytotoxicity concentration (CC50) and half maximal inhibitory concentration (IC50) were used as the main parameters in this investigation. Selectivity index value (SI) was determined as the ratio of CC50/IC50 of the plant extract. GraphPad PRISM for Windows, version 5 (GraphPad Software Inc., San Diego, CA, 2005) was used for all statistical analyses.
Results and Discussion
Cytotoxicity of S .Baicalensis on Vero cells
Antiviral assays and mechanisms of action of AHPE-XA-09 against DENV
IC 50 values of the S. baicalensis extract, AHPE-XA-09, against the different stages of replication of DENV serotypes
Prophylactic activity IC50(μg/mL)
Anti-adsorption activity IC50(μg/mL)
After adsorption activity IC50(μg/mL)
Direct virucidal activity IC50(μg/mL)
Protective prophylactic effect
Activity against virus adsorption to cells
Ability to inhibit intracellular viral replication
Direct virucidal effects
Identification and quantitative analysis of baicalein in AHPE-XA-09
Recent lines of evidence suggest that certain compounds from the S. baicalensis extract, such as baicalein or chrysin, showed antiviral activity against hepatitis A virus (HAV), which is an RNA virus. However, the mechanism(s) underlying the mode of action of these compounds remain elusive . Indeed, antiviral activity of 5,7,4'-trihydroxy-8-methoxyflavone a flavonoid from the roots of S. baicalensis was shown against influenza viruses [24, 28].
Recently, we have shown the novel antiviral activity of pure commercial baicalein against DENV-2 (NGC strain) . Therefore, our current investigations point to the potential use of cold water extract of the roots of S. baicalensis, AHPE-XA-09 as one of the main source for baicalein, although some other bioactives present in the extract could also possess anti-DENV activities. The anti-DENV activities of S. baicalensis extract were noticeable at all stages of in vitro infection; when the cells were treated after virus adsorption, during the virus adsorption or even prior to virus infection. The tested extract exerted significant direct virucidal effects on extracellular free DENVs particles, which is a notable ability for anti-dengue candidate to neutralize free viruses at viremic stages. The mechanism of how the extract precisely inhibits DENVs replication remains ambiguous. However, our findings suggest that one possible mechanism whereby the S. baicalensis extract acts against DENV replication could be attributed to its ability to bind and/or to inactivate certain important structural and/or non-structural protein(s) of DENVs. Such inhibitory mechanism has previously been reported with some flavonoids such as pinostrobin against DENV NS2B/NS3 protease . Moreover, it has been demonstrated that baicalein, as a main flavonoid found in the tested extract, is able to bind to HIV-1 integrase . On the other hand, the activity of flavonoids such as wogonin and baicalein has been demonstrated against cellular DNA and RNA besides their ability to inactivate the cellular RNA polymerases [31–33]. Therefore, it may be another potential mechanism for the tested plant extract and its constituents to inhibit the DENV replication via interference with DENV RNA polymerase and/or viral RNA, which is open for future investigations.
In conclusion, the cold aqueous extract of the roots of S. baicalensis, one of the traditional Chinese medicinal herbs used to treat infectious diseases, exhibited a novel anti-dengue activity against all four serotypes of DENV in vitro. Our investigations have shown that this extract specifically targeted different stages of DENV infection and replication in vitro. Our study also demonstrated the potent direct virucidal activity of S. baicalensis extract, which serves as an important criteria for anti-dengue drug development as it could neutralize extracellular DENVs circulating in viremic patients. Our findings also highlight the potentials of S. baicalensis aqueous extract for use as anti-dengue agent, and the presence of baicalein, a known flavonoid with anti-dengue virus replication properties perhaps is one of the possible naturally active antiviral constituents.
The authors thank Herbitec Sdn Bhd for supplying the extract and grant for the study.
- Tang LI, Ling AP, Koh RY, Chye SM, Voon KG: Screening of anti-dengue activity in methanolic extracts of medicinal plants. BMC Complement Altern Med. 2012, 12: 3-10.1186/1472-6882-12-3.View ArticlePubMedPubMed CentralGoogle Scholar
- Zandi K, Ramedani E, Mohammadi K, Tajbakhsh S, Deilami I, Rastian Z, Fouladvand M, Yousefi F, Farshadpour F: Evaluation of antiviral activities of curcumin derivatives against HSV-1 in Vero cell line. Nat Prod Commun. 2010, 5: 1935-1938.PubMedGoogle Scholar
- Astani A, Reichling J, Schnitzler P: Screening for antiviral activities of isolated compounds from essential oils. Evid Based Complement Altern Med. 2011, 2011: 253643-View ArticleGoogle Scholar
- Han H, He W, Wang W, Gao B: Inhibitory effect of aqueous dandelion extract on HIV-1 replication and reverse transcriptase activity. BMC Complement Alternat Med. 2011, 11: 112-10.1186/1472-6882-11-112.View ArticleGoogle Scholar
- He W, Han H, Wang W, Gao B: Anti-influenza virus effect of aqueous extracts from dandelion. Virol J. 2011, 8: 538-10.1186/1743-422X-8-538.View ArticlePubMedPubMed CentralGoogle Scholar
- Li J, Huang H, Feng M, Zhou W, shi X, Zhou P: In vitro and in vivo anti-hepatitis B virus activities of a plant extract from Geranium carolinianum L. Antiviral Res. 2008, 79: 114-120. 10.1016/j.antiviral.2008.03.001.View ArticlePubMedGoogle Scholar
- Yang Y, Zhang L, Fan X, Qin C, Liu J: Antiviral effect of geraniin on human enterovirus 71 in vitro and in vivo. Bioorg Med Chem Lett. 2012, 22: 2209-2211. 10.1016/j.bmcl.2012.01.102.View ArticlePubMedGoogle Scholar
- Hudson JB: Applications of the phytomedicine Echinacea purpurea (purple coneflower) in infectious diseases. J Biomed Biotechnol. 2012, 2012: 769896-View ArticlePubMedGoogle Scholar
- Chen L, Dou J, Su Z, Zhou H, Wang H, Zhou W, Guo Q, Zhou C: Synergistic activity of baicalein with ribavirin against influenza A (H1N1) virus infections in cell culture and in mice. Antiviral Res. 2011, 91: 314-320. 10.1016/j.antiviral.2011.07.008.View ArticlePubMedGoogle Scholar
- Chingwaru W, Majinda RT, Yeboah SO, Jackson JC, Kapewangolo PT, Kandawa-Schulz M, Cencic A: Tylosema esculentum (Marama) tuber and bean extracts are strong antiviral agents against rotavirus infection. Evid Based Complement Alternat Med. 2011, 2011: 284795-View ArticlePubMedPubMed CentralGoogle Scholar
- Ge H, Wang YF, Xu J, Gu Q, Liu HB, Xiao PG, Zhou J, Liu Y, Yang Z, Su H: Anti-influenza agents from traditional Chinese medicine. Nat Prod Rep. 2010, 27: 1758-1780. 10.1039/c0np00005a.View ArticlePubMedGoogle Scholar
- An X, Zhang AL, May BH, Lin L, Xu Y, Xue CC: Oral Chinese herbal medicine for improvement of quality of life in patients with stable chronic obstructive pulmonary disease: a systematic review. J Alternat Complement Med. 2012, 18: 731-743. 10.1089/acm.2011.0389.View ArticleGoogle Scholar
- Scheck AC, Perry K, Hank NC, Clark WD: Anticancer activity of extracts derived from the mature roots of S. baicalensis on human malignant brain tumor cells. BMC Complement Alternat Med. 2006, 16: 27-View ArticleGoogle Scholar
- Zhang N, Van Crombruggen K, Holtappels G, Bachert C: A herbal composition of S. Baicalensis and eleutherococcus senticosus shows potent anti-inflammatory effects in an Ex vivo human mucosal tissue model. Evid Based Complement Alternat Med. 2012, 2012: 673145-PubMedPubMed CentralGoogle Scholar
- Li HB, Chen F, Chen F: Isolation and purification of baicalein, wogonin and oroxylin A from the medicinal plant S. baicalensis by high-speed counter-current chromatography. J Chromatogr A. 2005, 1074: 107-110. 10.1016/j.chroma.2005.03.088.View ArticlePubMedGoogle Scholar
- Zandi K, Teoh BT, Sam SS, Wong PF, Mustafa MR, Abubakar S: In vitro antiviral activity of fisetin, rutin and naringenin against dengue virus type-2. J Med Plants Res. 2011, 5: 5534-5539.Google Scholar
- Zandi K, Teoh BT, Sam SS, Wong PF, Mustafa MR, Abubakar S: Antiviral activity of four types of bioflavonoid against dengue virus type-2. Virol J. 2011, 8: 560-10.1186/1743-422X-8-560.View ArticlePubMedPubMed CentralGoogle Scholar
- Zandi K, Teoh BT, Sam SS, Wong PF, Mustafa MR, Abubakar S: Novel antiviral activity of baicalein against dengue virus. BMC Complement Alternat Med. 2012, 12: 3-10.1186/1472-6882-12-3.View ArticleGoogle Scholar
- Gubler DJ: Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol. 2002, 10: 100-103. 10.1016/S0966-842X(01)02288-0.View ArticlePubMedGoogle Scholar
- Kuno G: Research on dengue and dengue-like illness in east Asia and the western pacific during the first half of the 20th century. Rev Med Virol. 2007, 17: 327-341. 10.1002/rmv.545.View ArticlePubMedGoogle Scholar
- Che P, Wang L, Li Q: The development, optimization and validation of an assay for high throughput antiviral drug screening against Dengue virus. Int J Clin Exp Med. 2009, 2: 363-373.PubMedPubMed CentralGoogle Scholar
- Zandi K, Lani R, Wong PF, Teoh BT, Sam SS, Johari J, Mustafa MR, AbuBakar S: Flavone enhances dengue virus type-2 (NGC strain) infectivity and replication in Vero cells. Molecules. 2012, 17: 2437-2445. 10.3390/molecules17032437.View ArticlePubMedGoogle Scholar
- Wong SS, Abd-Jamil J, Abubakar S: Antibody neutralization and viral virulence in recurring dengue virus type 2 outbreaks. Viral Immunol. 2007, 20: 359-368. 10.1089/vim.2006.0100.View ArticlePubMedGoogle Scholar
- Nagai T, Suzuki Y, Tomimori T, Yamada H: Antiviral activity of plant flavonoid, 5,7,4'-trihydroxy-8-methoxyflavone, from the roots of S. baicalensis against influenza A (H3N2) and B viruses. Biol Pharm Bull. 1995, 18: 295-299. 10.1248/bpb.18.295.View ArticlePubMedGoogle Scholar
- Yu K, Gong Y, Lin Z, Cheng Y: Quantitative analysis and chromatographic fingerprinting for the quality evaluation of S. baicalensis Georgi using capillary electrophoresis. J Pharm Biomed Anal. 2007, 43: 540-548. 10.1016/j.jpba.2006.08.011.View ArticlePubMedGoogle Scholar
- Burnett BP, Jia Q, Zhao Y, Levy RM: A medicinal extract of Scutellari abaicalensis and Acacia catechu acts as a dual inhibitor of cyclooxygenase and 5-lipoxygenase to reduce inflammation. J Med Food. 2007, 10: 442-451. 10.1089/jmf.2006.255.View ArticlePubMedGoogle Scholar
- Lu Y, Joerger R, Wu C: Study of the chemical composition and antimicrobial activities of ethanolic extracts from roots of S. baicalensis Georgi. J Agric Food Chem. 2011, 59: 10934-10942. 10.1021/jf202741x.View ArticlePubMedGoogle Scholar
- Nagai T, Moriguchi R, Suzuki Y, Tomimori T, Yamada H: Mode of action of the anti-influenza virus activity of plant flavonoid, 5,7,4'-trihydroxy-8-methoxyflavone, from the roots of Scutellaria baicalensis. Antiviral Res. 1995, 26: 11-25. 10.1016/0166-3542(94)00062-D.View ArticlePubMedGoogle Scholar
- Kiat TS, Pippen R, Yusof R, Ibrahim H, Khalid N, Rahman NA: Inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, Boesenbergia rotunda (L.), towards dengue-2 virus NS3 protease. Bioorg Med Chem Lett. 2006, 16: 3337-3340. 10.1016/j.bmcl.2005.12.075.View ArticlePubMedGoogle Scholar
- Ahn HC, Lee SY, Kim JW, Son WS, Shin CG, Lee BJ: Binding aspects of baicalein to HIV-1 integrase. Mol Cells. 2001, 12: 127-130.PubMedGoogle Scholar
- Shinozuka K, Kikuchi Y, Nishino C, Mori A, Tawata S: Inhibitory effect of flavonoids on DNA-dependent DNA and RNA polymerases. Experientia. 1998, 44: 882-885.View ArticleGoogle Scholar
- Nafisi S, Shadaloi A, Feizbakhsh A, Tajmir-Riahi HA: RNA binding to antioxidant flavonoids. J Photochem Photobiol B. 2009, 94: 1-7. 10.1016/j.jphotobiol.2008.08.001.View ArticlePubMedGoogle Scholar
- Sun Y, Bi S, Song D, Qiao C, Mu D, Zhang H: Study on the interaction mechanism between DNA and the main active components in S. baicalensis Georgi. Sensor Actuat B. 2008, 129: 799-810. 10.1016/j.snb.2007.09.082.View ArticleGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/91/prepub
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