http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(3): 451–456 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.924018
ORIGINAL ARTICLE
Inhibitory effects of Pinus massoniana bark extract on hepatitis C virus in vitro Chunfeng Wang1, Lianfeng Zhang1, Peng Cheng1, and Qiao Zhang2 First Affiliated Hospital of Zhengzhou University, Zhengzhou, China and 2College of Public Health, Zhengzhou University, Zhengzhou, China
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1
Abstract
Keywords
Context: Given that Pinus massoniana Lamb (Pinaceae) bark extract (PMBE) is a safe and non-toxic flavonoid found abundantly in nature, it was considered a promising novel candidate agent in the treatment of virus infection. Object: Experiments were conducted to assay the antiviral character of PMBE against Hepatitis C virus (HCV). Materials and methods: Assay of PMBE cytotoxicity, HCV replication, infectious HCV production, and its potential influence on the pathways for IFN-ISRE and NS3 protease were conducted. Results: HCV replication was suppressed when the concentration of PMBE raised greater than 5 mg/mL and its EC50 was 9.58 mg/mL. In the 10 mg/mL group, HCV replication was suppressed for 48 h. When the concentration increased to 40 mg/mL, HCV replication was significantly suppressed (luciferase activity was only 10% at 96 h). PMBE could inhibit HCV virus production efficiently (PMBE group was 5 FFU). Cell viability was affected by 40 mg/mL of PMBE. The F/R ratio ranged from 98% to 101%. The rate of OD450 ranged from 96% to 102%. NS3 catalytic activity ranged from 5% (40 mg/mL PMBE) to 45% (5 mg/mL PMBE). Even when used in a low amount (5 mg/mL), NS3 catalytic activity was significantly inhibited (p50.01). Conclusions: The results suggest that PMBE is effective for use in the stabilization of HCV replication and active liver inflammation.
IFN-ISRE, NS3 catalytic activity, PMBE cytoxicity, replicon
Introduction The distribution areas of Pinus massoniana Lamb (Pinaceae) cover the South China and northern regions of Vietnam to India (Stephan, 1976). Its needle, bark, and turpentine have been used in Chinese folk medicine. In traditional Chinese medicine, the bark of Pinus massoniana is used to promote constringency, hemostasis, and detoxification. It is also variously prescribed for rheumatism arthralgia, hypertension, and chilblain in China and other countries in the Orient (Bi et al., 2002). In eastern Asia (mainly China and Japan), Pinus massoniana needles and bark have been used in diet therapies for thousands of years (Wu et al., 2009). Even in the last decade of the twentieth century, bioactive substances of Pinus massoniana bark have not been successfully extracted through crude sieving, centrifugation, or micro- and ultrafiltration with hollow fiber, and coiled-spiral nanofiltration membranes (Cui et al., 2005). Recently, it has been confirmed that the bioactive substances in P. massoniana bark extracts (PMBE) are flavonoids (26.0–28.3%) (mainly procyanidins),
Correspondence: Lianfeng Zhang, First Affiliated Hospital of Zhengzhou University, 1 Jianshe Street, Zhengzhou 450052, China. Tel: +86 371 69066997. Fax: +86 371 69066997. E-mail: [email protected]
History Received 3 September 2013 Revised 27 March 2014 Accepted 9 May 2014 Published online 4 December 2014
which are mainly included in the procyanidin B series (Bi et al., 2002). In recent years, the inhibitory activities of some flavonoids against human immunodeficiency virus (HIV) have stimulated the interests of researchers. The relationship between the flavonoid structure and their inhibitory activity against HIV-1 has been reported (Liu et al., 2006). Baicalin can inhibit HIV-1-infection and replication in vitro (Vlietinck et al., 1998). Flavonoids can also inhibit other viruses (Xiao et al., 2011). For example, anthocyanin, morin, rutin, dihydrofisetin, pelargonidin chloride, and catechin, whose working ingredient is flavonoid, possess inhibitory activity against seven viruses, including herpes simplex virus (HSV), respiratory syncytial virus, poliovirus, and Sindbis virus. Antiviral effects include the inhibition of viral polymerases and the binding of viral nucleic acids or capsid proteins (Xiao et al., 2011). Hepatitis C is a crucial public health problem and a major cause of chronic liver diseases. Hepatitis C virus (HCV) has been found to be the causative agent of hepatitis C (Botte et al., 1996). According to the World Health Organization (WHO), nearly 3% of the world population have been infected with HCV. More than 170 million people are chronic carriers of HCV and at the high risk of developing liver cirrhosis and/or hepatocellular carcinoma (HCC). HCV encodes one large open reading frame which is translated as a single polyprotein through an internal ribosome entry site (IRES)
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and proteolytically processed to yield structural and non-structural (NS) viral proteins (Bachmetov et al., 2012). The IRES regulates the assembly of translation initiation complexes on viral mRNA directly by a sequential pathway that is distinct from canonical eukaryotic initiation (Otto et al., 2004). The most extensively studied HCV proteins is NS3, which demonstrates multiple enzymatic activities. NS3 protease activity is also essential to generate the components of HCV RNA replication complex. Because of its especial importance for viral functionality, NS3 provides the relevant therapeutic targets against HCV (Zuo et al., 2005). Only 15–20% of patients with chronic hepatitis C have a sustained virologic response to interferon therapy. The efficacy and safety of recombinant interferon a-2b alone with those of a combination of interferon a-2 b and ribavirin for the initial treatment of patients with chronic hepatitis C were reported (McHutchison et al., 1998). In patients with chronic hepatitis C, initial therapy with interferon and ribavirin were more effective than treatment with interferon alone (McHutchison et al., 1998). In this paper, we checked the inhibitory effects of PMBEs on HCV replication in vitro. The aim of the present study was to investigate further PMBE which had shown great potential as a NS3 inhibitor. Further in vitro studies showed that it also could inhibit the replication of subgenomic HCV RNA replicons and virus production in cell culture. These results suggested that PMBE might inhibit HCV not only at the translation level indirectly but also by inhibiting HCV NS3 protease activity directly, making it a promising agent for anti-HCV therapy.
Materials and methods Preparation of PMBE Bark from P. massoniana lamb was collected from a farm in Wuhan, Hubei Province, in June 2012. All materials were identified in Huazhong Agricultural University. As P. massoniana is not in the catalogue of rare and endangered species of China, it is unnecessary for specific permissions. Bark from P. massoniana was dried, ground into powder, and put into a diffuser. Distilled water was added into the diffuser, which was then heated for the filtrate. The filters were subjected to crude sieving, centrifugation, and microfiltration to remove suspended matter and macromolecular impurities. Hollow fiber membrane ultrafiltration was used to generate permeation solution containing bioactive compounds such as flavonoids and proanthocyanidins. This solution was inspissated with a coiled-spiral nanofiltration membrane for removal of micromolecular impurities such as carbohydrates, inorganic solutes, and superabundant water. The resulting concentrate was dried to yield biologically active PMBE. To produce a stock solution, 100 mg PMBE was dissolved in 10 mL 100% ethanol or dimethylsulfoxide (DMSO), filtered through a 0.22 mm Millipore membrane and stored in 5 mL Eppendorf tubes at 4 C. To make a working solution, the stock solution was diluted with the RPMI 1640 culture medium containing 10% fetal bovine serum (FBS), 100 units/ mL penicillin, 100 mg/mL streptomycin, and 10 mM 4-(2hydroxyethyl)-1-piperazine ethanesulfonic acid. Working
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solutions of 5, 10, and 40 mg/mL PMBE were made for methylcyclopentadienyl manganese tricarbonyl proliferation assays. PMBE cytotoxicity assay A human hepatoma cell line, Huh7, was maintained in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum at 37 C under 5% CO2. Huh7 cells expressing the HCV replicon were cultured in a medium containing 200 mg/mL G418 (Beyotime, Shanghai, China). To test the cytotoxic effect of PMBE, culture cell viability was evaluated with the MST-1 assay (Beyotime, Shanghai, China). In brief, Huh-7 cells (5000 in a 96-well culture dish) were treated with varying concentrations of PMBE dissolved in DMSO. Medium was collected and replaced with freshly prepared PMBE-containing medium every 24 h. WST-1 (10 mL) was added to supernatant fluids and measured in a plate reader (450 nm) (Omega, Mu¨nchen, Germany). HCV replication assay An HCV subgenomic replicon plasmid was reconstructed by substituting the neomycin phosphotransferase gene with a fusion gene comprising the firefly luciferase and neomycin phosphotrasnferase (pRep-Feo). RNA was synthesized from pRep-Feo and transfected into Huh7 cells. After culture in the presence of G418, cell lines stably expressing the replicon were established. To study the ability of PMBE to inhibit HCV replication, 10 mg/mL of PMBE or solvent (DMSO) was added to the replicon cell line (1 105 cells in a 24-well culture dish) for 3 d. The culture medium was collected and replaced with freshly prepared PMBE-containing medium every 24 h after treatment, and the activity of the secreted alkaline phosphatase (SEAP) was measured as described (Bourne et al., 2005). Infectious HCV production assay To study the ability of PMBE to inhibit HCV production, an in vitro transcribed HCV-JFH1 RNA was transfected into Huh7.5.1 cells. Huh-7.5 cells were pretreated with PMBE or solvent for 3 h before infection with the HCV-JFH1virus at a multiplicity of infection (MOI) of 10 mg PMBE or DMSO was added every 24 h during 4 d of treatment. To prevent PMBE interfering with the focus-forming unit (FFU) reduction assay, the medium was replaced with the fresh PMBEfree medium 24 h before assaying. The titre of the virus released into the supernatant fluid was then determined by the FFU assay. HCV-IRES reporter construction A plasmid, pCIneo-Rluc-IRES-Fluc, was used to analyze HCV internal ribosome entry site (IRES)-mediated translation efficiency (Sekine-Osajima et al., 2009). The plasmid, expressing a bicistronic RNA, in which Renilla luciferase (Rluc) was translated in a cap-dependent manner and firefly luciferase (Fluc) was translated by HCV-IRES-mediated initiation, was stably transfected into Huh7 cells.
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DOI: 10.3109/13880209.2014.924018
Inhibitory effects of PMBE on HCV in vitro
After cultured in the presence of G418, Huh7/CRIF cells were established. Activities of the HCV-IRES-mediated translation were measured by culture of Huh7/CRIF cells in the presence of drugs and by dual luciferase assays after 48 h.
Statistical analyses
In vitro NS3 protease activity assay
Results and discussion
To determinate the inhibition of HCV NS3 catalytic activity, we used our throughput assay of NS3 catalysis as previously described (Berdichevsky et al., 2003). Digestion of the recombinant substrate protein (Tiangen, Beijing, China) with the sc NS3 protease was performed in 100 mL reaction volume at 37 C for 60 min. The reaction was performed in a reaction buffer (50 mM Tris(HCl) pH 7.5, 150 mM NaCl, 0.05% Tween, 20% glycerol, and 1 mM DTT) with 0.25– 1.0 mM recombinant substrate and 0.01–0.1 mM of recombinant protease. Following digestion, the reaction was transferred into an Eppendorf tube containing 20 mg cellulose preequilibrated with reaction buffer. The cellulose matrix was gently agitated at room temperature for 5 min followed by centrifugation for 2 min at maximum speed. The supernatant containing liberated green fluorescent protein was transferred to a black 96-well plate and data were collected in a fluorometer (Omega, Mu¨nchen, Germany) utilizing excitation filter 485 nm, and emission filter 538 nm. At each substrate concentration and each time point, the substrate was also incubated in the absence of the enzyme and the extent of the spontaneous cleavage reaction was determined. Specific cleavage was the difference between the fluorescence obtained in the presence and absence of the enzyme.
Suppression of HCV replication by PMBE
Real-time RT-PCR analysis The total cellular RNA was extracted from cultured cells (Tiangen, Beijing, China). Two milligrams of total cellular RNA was used to generate cDNA from each sample using the SuperScript II reverse-transcriptase (Invitrogen, Carlsbad, CA). The cDNA was amplified by PCR in a 50 mL mixture containing 10 mmol/L of Tris-HCl (pH 8.3), 50 mmol/L of potassium chloride, 1.5 mmol/L of magnesium chloride, 0.01% gelatin, 1 unit Taq DNA polymerase (TAKARA, Dalian, China), 200 mmol/L of each dNTP, 30 ng of each first stage primer, and 5 mL of the cDNA sample. The reaction was performed for 35 cycles, including denaturation at 94 C for 1 min, annealing of primers at 55 C for 1 min, and primer extension at 72 C for 1 min. Primers used were deduced from a well-conserved 50 -non-coding region that is almost identical among different HCV isolates: 50 -CTGTGAGGAACTA CTGTCTT-30 (sense; nt 28-47) and 50 -AACACTACTCGG CTAGCAGT-30 (anti-sense; nt 229-248) (Sakamoto et al., 1993). One microlitre of the first stage PCR product was transferred to the second stage 50 mL mixture. The second stage PCR was performed for 35 cycles under the same condition except that the following primers were used: 50 -TTCACGCAGAAAGCGTCTAG-Y (sense; nt 46-65), and 50 -GTTGATCCAAGAAAGGACCC-Y (anti-sense; nt 171-190) (Sakamoto et al., 1993). The replicon RNA expression levels were measured using the real-time PCR system (ABI, Glasgow, CA) and Quantifast SYBR Green PCR Kit (Qiagen, Valencia, CA).
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Statistical analyses were performed with SPSS 13 (SPSS Inc., Chicago, IL) using Student’s t-test. p values of less than 0.05 were considered statistically significant.
Results showed that HCV replicon was suppressed when the concentration of PMBE was greater than 5 mg/mL. We calculated that the EC50 was 9.58 mg/mL for PMBE. Figure 1(b) shows that there was no significant difference about MST-1 assay between the two groups, even in 40 mg/mL. It suggested that there were no adverse effects on cell growth and viability (Figure 1b), indicating that the antiviral action of PMBE is not due to cytotoxic or antiproliferative effects. Huh7/Rep-Feo cells were cultured with various concentrations of PMBE (0, 5, 10, and 40 mg/mL), and the expression was measured by luciferase assay (Figure 1c and d). There was no significant different between the control group (0 mg/ mL) with the 5 mg/mL group. In the 10 mg/mL group, HCV replicon was suppressed for 48 h (luciferase activity was 70%). When the concentration increased to 40 mg/mL, HCV replicon was significantly suppressed (luciferase activity was only 10% at 96 h). Results showed that the suppressive effect of the HCV replicon lasted for 72 h (Figure 1c and d). PMBE-mediated inhibition of infections virus production To evaluate the inhibitory potential of PMBE to cells infected HCV, supernatant fluids were collected 48 h after treatment with PMBE, and the titre of infectious virus was determined by FFU reduction assay. Comparing with the control group (88 FFU), PMBE could inhibit HCV virus production efficiently (PMBE group was 5 FFU). Compared with the titre of infectious virus released by cells treated with the solvent alone (DMSO) (Figure 2), HCV mRNA was down to 82% (the PMBE concentration was 10 mg/mL) and 30% (40 mg/mL). The results showed that 40 mg/mL of PMBE had effects on cell viability from 24 h to 72 h (Figure 3). Absence of synergistic anti-HCV effects of interferon-a with PMBE To determine whether IFN-a and PMBE had a synergistic inhibitory effect on the replicon, Huh7/Rep-Feo cells were cultured with combinations of IFN-a and PMBE at various concentrations. The relative dose-inhibition curves of IFN (0, 5, 10, 40, and 100 U/mL) were plotted under matching concentrations of PMBE of 0, 5, 10, and 40 mg/mL (Figure 4). The curves did not show synergy of PMBE and IFN against the HCV replicon. These results suggested that the action of the compounds on the intracellular replication of HCV replicon was independent to the IFN-ISRE pathway. Suppress the HCV IRES-dependent translation and HCV NS3 catalysis by PMBE The F/R ratio ranged from 98% (40 mg/mL PMBE) to 101% (10 mg/mL PMBE) (Figure 5a). The rate of OD450 (compared
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Pharm Biol, 2015; 53(3): 451–456
Figure 1. Effect of PMBE on the expression of HCV relicon. (a) Huh7/Rep-Feo cells were cultured in the presence of PMBE at concentrations of 0, 0.1, 1, 10, and 100 mg/mL. Single asterisks indicate p values of less than 0.05. Error bars indicate mean ± SD. (b) MST-1 assay of Huh7/Rep-Feo cells cultured with PMBE. Error bars indicate mean ± SD. (c) Dose-dependent suppression of HCV replication by PMBE. Error bars indicate mean ± SD of triplicate assay. (d) Time-dependent suppression of HCV replication by PMBE. The internal luciferase activities were measured at times of culture indicated. Assays were performed in triplicate. Error bars indicate mean ± SD.
Figure 2. Inhibition of infectious virus production by PMBE. Huh-7.5 cells were pretreated with 10 mg/mL PMBE or DMSO for 3 h before infection cells each day. Analysis was performed following 72 h treatment. Focus-forming units’ assay of virus released into the media by HCV-JFH1 virus-infected cells revealed the inhibition of cell production. Error bars indicate mean ± SD.
Figure 3. Real-time RT-PCR analyses of replicon RNA by PMBE. Huh7/Rep-Feo cells were cultured with indicated concentrations of PMBE, and harvested at 48 h after exposure.
with the control group) ranged from 96% (10 mg/mL PMBE) to 102% (5 mg/mL PMBE) (Figure 5b). There were no significant differences about F/R and OD450 ratio between each group. Results suggested that there were no significant changes of the internal luciferase activities at concentrations of PMBE that suppressed expression of the HCV replicon. The MST-1 assay did not show any effects on cell growth and viability at concentrations used in this assay. NS3 catalytic activity ranged from 5% (40 mg/mL PMBE) to 45% (5 mg/mL PMBE). Even when used in low amounts (5 mg/mL), NS3 catalytic activity was significantly inhibited (p50.01) (Figure 6).
Investigations into phytochemicals for antiviral activities have shown great importance in recent years. There are tremendous amount of literature available regarding is a antiviral potential of phytochemicals. In this study, we investigated the effects of PMBE on HCV NS3 catalytic activity and HCV infection, and an efficient inhibition was obtained. Flavonoid, a major ingredient of PMBE, has been reported to display antiviral activities and reduce the replication of several viruses (Andres et al., 2009; Liu et al., 2006; Rainsford, 2006). Flavonoid is a class of plant pigment, found in a wide range of green vegetables and fruits.
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DOI: 10.3109/13880209.2014.924018
Figure 4. Effect of PMBE used in combination with interferon (IFN)-a on HCV replication. Huh7/Rep-Feo cells were cultured with combination of IFN-a and PMBE at indicated concentrations. The internal luciferase activities were measured after 48 h of culture. Error bars indicate mean ± SD. Plots of 100% in each curves represent replicon expression levels that were treated with indicated amounts of PMBE and without IFN-a.
Inhibitory effects of PMBE on HCV in vitro
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Figure 6. Inhibition of HCV NS3 activities by PMBE. Inhibition of NS3 catalytic activity was tested in 96-well plates containing 30 nM scNS3 and 0.25 mM substrate that were incubated for 1 h at 37 C. For the assay, the inhibitor was mixed with the enzyme in the wells prior to the addition of the substrate. Percent catalysis was calculated from control reactions performed without inhibitors.
They also have various biochemical activities such as antioxidative and superoxide scavenging activities. Results revealed that PMBE could inhibit HCV replication in subgenomic HCV RNA replicon cell lines and block HCV viral production almost completely when using the JFH1 infectious clone. In all model systems used, PMBE efficiently inhibited NS3 protease activity and HCV replication. Thus, we provided the evidences that PMBE could affect HCV infection with a different (direct) mechanism as well. However, higher inhibition efficiency on viral production must be attributed to mutual inhibitory effection NS3 proteases. Considering the current status of limited therapy options to HCV infection and their unsatisfactory outcomes, it is urgent that anti-HCV molecules should be screened widely to develop novel antiviral therapies (Bachmetov et al., 2012). The results suggest that PMBE will be promising against HCV replication and active liver inflammation. In addition, further investigations about the action of these drugs on the expression and maturation of HCV proteins may elucidate new aspects of the viral infection and replication and may constitute novel molecular targets on anti-HCV chemotherapeutics.
Declaration of interest Figure 5. Influence of HCV IRES-mediated translations by PMBE. A biocistronic reporter gene plasmid, pCIneo-Rluc-IRES-Fluc, was stably transfected into Huh7 cells. (a) Dual luciferase assay. The cells were cultured with PMBE at the indicated concentrations, and dual luciferase activities were measured after 48 h of treatment. Values are displayed as ratios of Fluc to Rluc. (b) MST-1 assay of Huh7/neo-Rluc-IRES-Fluc cells cultured with PMBE at different concentrations. MST-1 assays at 48 h after treatment with each drug were performed in triplicate. Error bars indicate mean ± SD.
They are classified as flavone, flavonol, flavanone, flavanol, isoflavone, chalcone, anthocyanin, and catechin, according to their molecular structures. Many flavonoids have various biological functions such as antibacterial, antioxidative, and anticarcinogeninc activities (Xiao et al., 2011).
This research was supported by the National Natural Science Foundation of China (Grant no. 31372453). The authors alone are responsible for the content and writing of the paper.
References Andres A, Donovan SM, Kuhlenschmidt MS. (2009). Soy isoflavones and virus infections. J Nutr Biochem 20:563–9. Bachmetov L, Gal-Tanamy M, Shapira A, et al. (2012). Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity. J Viral Hepatitis 19:e81–8. Berdichevsky Y, Zemel R, Bachmatov L, et al. (2003). A novel high throughput screening assay for HCV NS3 serine protease inhibitors. J Virol Methods 107:245–55. Bi YF, Zheng XK, Feng WS, et al. (2002). Isolation and structure identification of ligan glycosides from pine needles of Pinus massoniana Lamb. Yao Xue Xue Bao 37:626–9.
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Botte C, Janot C. (1996). Epidemiology of HCV infection in the general population and in blood transfusion. Nephrol Dial Transplant 11: 19–21. Bourne N, Pyles RB, Yi M, et al. (2005). Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system. Antiviral Res 67: 76–82. Cui Y, Xie H, Wang J. (2005). Potential biomedical properties of Pinus massoniana bark extract. Phytother Res 19:34–8. Liu ZM, Yang YS, Wang XL, Wen RX. (2006). Recent progress on antiHIV research of traditional Chinese medicine and components. Zhongguo Zhong Yao Za Zhi 31:1753–8. McHutchison JG, Gordon SC, Schiff ER, et al. (1998). Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med 339:1485–92. Otto GA, Puglisi JD. (2004). The pathway of HCV IRES-mediated translation initiation. Cell 119:369–80. Rainsford KD. (2006). Influenza (‘‘Bird Flu’’), inflammation and anti-inflammatory/analgesic drugs. Inflammopharmacology 14: 2–9.
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Sakamoto N, Sato C, Haritani H, et al. (1993). Detection of hepatitis C viral RNA in sporadic acute non-A, non-B hepatitis by polymerase chain reaction. Its usefulness for the early diagnosis of seronegative infection. J Hepatol 17:28–33. Sekine-Osajima Y, Sakamoto N, Nakagawa M, et al. (2009). Two flavonoids extracts from Glycyrrhizae radix inhibit in vitro hepatitis C virus replication. Hepatol Res 39:60–9. Stephan, G. (1976). Studies on the physiological behavior of Pinus merkusii Jungh. and Pinus massoniana Lamb. Beitr Trop Landwirtsch Veterinarmed 14:71–8. Vlietinck AJ, De Bruyne T, Apers S, Pieters LA. (1998). Plant-derived leading compounds for chemotherapy of human immunodeficiency virus (HIV) infection. Planta Med 64:97–109. Wu C, Feng D, Ma H, et al. (2009). Effect of Pinus massoniana bark extract on IFN-gamma-induced ICAM-1 expression in HaCaT human keratinocytes. J Ethnopharmacol 122:48–53. Xiao ZP, Peng ZY, Peng MJ, et al. (2011). Flavonoids health benefits and their molecular mechanism. Mini Rev Med Chem 11:169–77. Zuo GY, Li ZQ, Chen LR, Xu XJ. (2005). In vitro anti-HCV activities of Saxifraga melanocentra and its related polyphenolic compounds. Antivir Chem Chemother 16:393–8.
ORIGINAL ARTICLE
Inhibitory effects of Pinus massoniana bark extract on hepatitis C virus in vitro Chunfeng Wang1, Lianfeng Zhang1, Peng Cheng1, and Qiao Zhang2 First Affiliated Hospital of Zhengzhou University, Zhengzhou, China and 2College of Public Health, Zhengzhou University, Zhengzhou, China
Pharmaceutical Biology Downloaded from informahealthcare.com by Mcgill University on 02/07/15 For personal use only.
1
Abstract
Keywords
Context: Given that Pinus massoniana Lamb (Pinaceae) bark extract (PMBE) is a safe and non-toxic flavonoid found abundantly in nature, it was considered a promising novel candidate agent in the treatment of virus infection. Object: Experiments were conducted to assay the antiviral character of PMBE against Hepatitis C virus (HCV). Materials and methods: Assay of PMBE cytotoxicity, HCV replication, infectious HCV production, and its potential influence on the pathways for IFN-ISRE and NS3 protease were conducted. Results: HCV replication was suppressed when the concentration of PMBE raised greater than 5 mg/mL and its EC50 was 9.58 mg/mL. In the 10 mg/mL group, HCV replication was suppressed for 48 h. When the concentration increased to 40 mg/mL, HCV replication was significantly suppressed (luciferase activity was only 10% at 96 h). PMBE could inhibit HCV virus production efficiently (PMBE group was 5 FFU). Cell viability was affected by 40 mg/mL of PMBE. The F/R ratio ranged from 98% to 101%. The rate of OD450 ranged from 96% to 102%. NS3 catalytic activity ranged from 5% (40 mg/mL PMBE) to 45% (5 mg/mL PMBE). Even when used in a low amount (5 mg/mL), NS3 catalytic activity was significantly inhibited (p50.01). Conclusions: The results suggest that PMBE is effective for use in the stabilization of HCV replication and active liver inflammation.
IFN-ISRE, NS3 catalytic activity, PMBE cytoxicity, replicon
Introduction The distribution areas of Pinus massoniana Lamb (Pinaceae) cover the South China and northern regions of Vietnam to India (Stephan, 1976). Its needle, bark, and turpentine have been used in Chinese folk medicine. In traditional Chinese medicine, the bark of Pinus massoniana is used to promote constringency, hemostasis, and detoxification. It is also variously prescribed for rheumatism arthralgia, hypertension, and chilblain in China and other countries in the Orient (Bi et al., 2002). In eastern Asia (mainly China and Japan), Pinus massoniana needles and bark have been used in diet therapies for thousands of years (Wu et al., 2009). Even in the last decade of the twentieth century, bioactive substances of Pinus massoniana bark have not been successfully extracted through crude sieving, centrifugation, or micro- and ultrafiltration with hollow fiber, and coiled-spiral nanofiltration membranes (Cui et al., 2005). Recently, it has been confirmed that the bioactive substances in P. massoniana bark extracts (PMBE) are flavonoids (26.0–28.3%) (mainly procyanidins),
Correspondence: Lianfeng Zhang, First Affiliated Hospital of Zhengzhou University, 1 Jianshe Street, Zhengzhou 450052, China. Tel: +86 371 69066997. Fax: +86 371 69066997. E-mail: [email protected]
History Received 3 September 2013 Revised 27 March 2014 Accepted 9 May 2014 Published online 4 December 2014
which are mainly included in the procyanidin B series (Bi et al., 2002). In recent years, the inhibitory activities of some flavonoids against human immunodeficiency virus (HIV) have stimulated the interests of researchers. The relationship between the flavonoid structure and their inhibitory activity against HIV-1 has been reported (Liu et al., 2006). Baicalin can inhibit HIV-1-infection and replication in vitro (Vlietinck et al., 1998). Flavonoids can also inhibit other viruses (Xiao et al., 2011). For example, anthocyanin, morin, rutin, dihydrofisetin, pelargonidin chloride, and catechin, whose working ingredient is flavonoid, possess inhibitory activity against seven viruses, including herpes simplex virus (HSV), respiratory syncytial virus, poliovirus, and Sindbis virus. Antiviral effects include the inhibition of viral polymerases and the binding of viral nucleic acids or capsid proteins (Xiao et al., 2011). Hepatitis C is a crucial public health problem and a major cause of chronic liver diseases. Hepatitis C virus (HCV) has been found to be the causative agent of hepatitis C (Botte et al., 1996). According to the World Health Organization (WHO), nearly 3% of the world population have been infected with HCV. More than 170 million people are chronic carriers of HCV and at the high risk of developing liver cirrhosis and/or hepatocellular carcinoma (HCC). HCV encodes one large open reading frame which is translated as a single polyprotein through an internal ribosome entry site (IRES)
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and proteolytically processed to yield structural and non-structural (NS) viral proteins (Bachmetov et al., 2012). The IRES regulates the assembly of translation initiation complexes on viral mRNA directly by a sequential pathway that is distinct from canonical eukaryotic initiation (Otto et al., 2004). The most extensively studied HCV proteins is NS3, which demonstrates multiple enzymatic activities. NS3 protease activity is also essential to generate the components of HCV RNA replication complex. Because of its especial importance for viral functionality, NS3 provides the relevant therapeutic targets against HCV (Zuo et al., 2005). Only 15–20% of patients with chronic hepatitis C have a sustained virologic response to interferon therapy. The efficacy and safety of recombinant interferon a-2b alone with those of a combination of interferon a-2 b and ribavirin for the initial treatment of patients with chronic hepatitis C were reported (McHutchison et al., 1998). In patients with chronic hepatitis C, initial therapy with interferon and ribavirin were more effective than treatment with interferon alone (McHutchison et al., 1998). In this paper, we checked the inhibitory effects of PMBEs on HCV replication in vitro. The aim of the present study was to investigate further PMBE which had shown great potential as a NS3 inhibitor. Further in vitro studies showed that it also could inhibit the replication of subgenomic HCV RNA replicons and virus production in cell culture. These results suggested that PMBE might inhibit HCV not only at the translation level indirectly but also by inhibiting HCV NS3 protease activity directly, making it a promising agent for anti-HCV therapy.
Materials and methods Preparation of PMBE Bark from P. massoniana lamb was collected from a farm in Wuhan, Hubei Province, in June 2012. All materials were identified in Huazhong Agricultural University. As P. massoniana is not in the catalogue of rare and endangered species of China, it is unnecessary for specific permissions. Bark from P. massoniana was dried, ground into powder, and put into a diffuser. Distilled water was added into the diffuser, which was then heated for the filtrate. The filters were subjected to crude sieving, centrifugation, and microfiltration to remove suspended matter and macromolecular impurities. Hollow fiber membrane ultrafiltration was used to generate permeation solution containing bioactive compounds such as flavonoids and proanthocyanidins. This solution was inspissated with a coiled-spiral nanofiltration membrane for removal of micromolecular impurities such as carbohydrates, inorganic solutes, and superabundant water. The resulting concentrate was dried to yield biologically active PMBE. To produce a stock solution, 100 mg PMBE was dissolved in 10 mL 100% ethanol or dimethylsulfoxide (DMSO), filtered through a 0.22 mm Millipore membrane and stored in 5 mL Eppendorf tubes at 4 C. To make a working solution, the stock solution was diluted with the RPMI 1640 culture medium containing 10% fetal bovine serum (FBS), 100 units/ mL penicillin, 100 mg/mL streptomycin, and 10 mM 4-(2hydroxyethyl)-1-piperazine ethanesulfonic acid. Working
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solutions of 5, 10, and 40 mg/mL PMBE were made for methylcyclopentadienyl manganese tricarbonyl proliferation assays. PMBE cytotoxicity assay A human hepatoma cell line, Huh7, was maintained in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum at 37 C under 5% CO2. Huh7 cells expressing the HCV replicon were cultured in a medium containing 200 mg/mL G418 (Beyotime, Shanghai, China). To test the cytotoxic effect of PMBE, culture cell viability was evaluated with the MST-1 assay (Beyotime, Shanghai, China). In brief, Huh-7 cells (5000 in a 96-well culture dish) were treated with varying concentrations of PMBE dissolved in DMSO. Medium was collected and replaced with freshly prepared PMBE-containing medium every 24 h. WST-1 (10 mL) was added to supernatant fluids and measured in a plate reader (450 nm) (Omega, Mu¨nchen, Germany). HCV replication assay An HCV subgenomic replicon plasmid was reconstructed by substituting the neomycin phosphotransferase gene with a fusion gene comprising the firefly luciferase and neomycin phosphotrasnferase (pRep-Feo). RNA was synthesized from pRep-Feo and transfected into Huh7 cells. After culture in the presence of G418, cell lines stably expressing the replicon were established. To study the ability of PMBE to inhibit HCV replication, 10 mg/mL of PMBE or solvent (DMSO) was added to the replicon cell line (1 105 cells in a 24-well culture dish) for 3 d. The culture medium was collected and replaced with freshly prepared PMBE-containing medium every 24 h after treatment, and the activity of the secreted alkaline phosphatase (SEAP) was measured as described (Bourne et al., 2005). Infectious HCV production assay To study the ability of PMBE to inhibit HCV production, an in vitro transcribed HCV-JFH1 RNA was transfected into Huh7.5.1 cells. Huh-7.5 cells were pretreated with PMBE or solvent for 3 h before infection with the HCV-JFH1virus at a multiplicity of infection (MOI) of 10 mg PMBE or DMSO was added every 24 h during 4 d of treatment. To prevent PMBE interfering with the focus-forming unit (FFU) reduction assay, the medium was replaced with the fresh PMBEfree medium 24 h before assaying. The titre of the virus released into the supernatant fluid was then determined by the FFU assay. HCV-IRES reporter construction A plasmid, pCIneo-Rluc-IRES-Fluc, was used to analyze HCV internal ribosome entry site (IRES)-mediated translation efficiency (Sekine-Osajima et al., 2009). The plasmid, expressing a bicistronic RNA, in which Renilla luciferase (Rluc) was translated in a cap-dependent manner and firefly luciferase (Fluc) was translated by HCV-IRES-mediated initiation, was stably transfected into Huh7 cells.
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DOI: 10.3109/13880209.2014.924018
Inhibitory effects of PMBE on HCV in vitro
After cultured in the presence of G418, Huh7/CRIF cells were established. Activities of the HCV-IRES-mediated translation were measured by culture of Huh7/CRIF cells in the presence of drugs and by dual luciferase assays after 48 h.
Statistical analyses
In vitro NS3 protease activity assay
Results and discussion
To determinate the inhibition of HCV NS3 catalytic activity, we used our throughput assay of NS3 catalysis as previously described (Berdichevsky et al., 2003). Digestion of the recombinant substrate protein (Tiangen, Beijing, China) with the sc NS3 protease was performed in 100 mL reaction volume at 37 C for 60 min. The reaction was performed in a reaction buffer (50 mM Tris(HCl) pH 7.5, 150 mM NaCl, 0.05% Tween, 20% glycerol, and 1 mM DTT) with 0.25– 1.0 mM recombinant substrate and 0.01–0.1 mM of recombinant protease. Following digestion, the reaction was transferred into an Eppendorf tube containing 20 mg cellulose preequilibrated with reaction buffer. The cellulose matrix was gently agitated at room temperature for 5 min followed by centrifugation for 2 min at maximum speed. The supernatant containing liberated green fluorescent protein was transferred to a black 96-well plate and data were collected in a fluorometer (Omega, Mu¨nchen, Germany) utilizing excitation filter 485 nm, and emission filter 538 nm. At each substrate concentration and each time point, the substrate was also incubated in the absence of the enzyme and the extent of the spontaneous cleavage reaction was determined. Specific cleavage was the difference between the fluorescence obtained in the presence and absence of the enzyme.
Suppression of HCV replication by PMBE
Real-time RT-PCR analysis The total cellular RNA was extracted from cultured cells (Tiangen, Beijing, China). Two milligrams of total cellular RNA was used to generate cDNA from each sample using the SuperScript II reverse-transcriptase (Invitrogen, Carlsbad, CA). The cDNA was amplified by PCR in a 50 mL mixture containing 10 mmol/L of Tris-HCl (pH 8.3), 50 mmol/L of potassium chloride, 1.5 mmol/L of magnesium chloride, 0.01% gelatin, 1 unit Taq DNA polymerase (TAKARA, Dalian, China), 200 mmol/L of each dNTP, 30 ng of each first stage primer, and 5 mL of the cDNA sample. The reaction was performed for 35 cycles, including denaturation at 94 C for 1 min, annealing of primers at 55 C for 1 min, and primer extension at 72 C for 1 min. Primers used were deduced from a well-conserved 50 -non-coding region that is almost identical among different HCV isolates: 50 -CTGTGAGGAACTA CTGTCTT-30 (sense; nt 28-47) and 50 -AACACTACTCGG CTAGCAGT-30 (anti-sense; nt 229-248) (Sakamoto et al., 1993). One microlitre of the first stage PCR product was transferred to the second stage 50 mL mixture. The second stage PCR was performed for 35 cycles under the same condition except that the following primers were used: 50 -TTCACGCAGAAAGCGTCTAG-Y (sense; nt 46-65), and 50 -GTTGATCCAAGAAAGGACCC-Y (anti-sense; nt 171-190) (Sakamoto et al., 1993). The replicon RNA expression levels were measured using the real-time PCR system (ABI, Glasgow, CA) and Quantifast SYBR Green PCR Kit (Qiagen, Valencia, CA).
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Statistical analyses were performed with SPSS 13 (SPSS Inc., Chicago, IL) using Student’s t-test. p values of less than 0.05 were considered statistically significant.
Results showed that HCV replicon was suppressed when the concentration of PMBE was greater than 5 mg/mL. We calculated that the EC50 was 9.58 mg/mL for PMBE. Figure 1(b) shows that there was no significant difference about MST-1 assay between the two groups, even in 40 mg/mL. It suggested that there were no adverse effects on cell growth and viability (Figure 1b), indicating that the antiviral action of PMBE is not due to cytotoxic or antiproliferative effects. Huh7/Rep-Feo cells were cultured with various concentrations of PMBE (0, 5, 10, and 40 mg/mL), and the expression was measured by luciferase assay (Figure 1c and d). There was no significant different between the control group (0 mg/ mL) with the 5 mg/mL group. In the 10 mg/mL group, HCV replicon was suppressed for 48 h (luciferase activity was 70%). When the concentration increased to 40 mg/mL, HCV replicon was significantly suppressed (luciferase activity was only 10% at 96 h). Results showed that the suppressive effect of the HCV replicon lasted for 72 h (Figure 1c and d). PMBE-mediated inhibition of infections virus production To evaluate the inhibitory potential of PMBE to cells infected HCV, supernatant fluids were collected 48 h after treatment with PMBE, and the titre of infectious virus was determined by FFU reduction assay. Comparing with the control group (88 FFU), PMBE could inhibit HCV virus production efficiently (PMBE group was 5 FFU). Compared with the titre of infectious virus released by cells treated with the solvent alone (DMSO) (Figure 2), HCV mRNA was down to 82% (the PMBE concentration was 10 mg/mL) and 30% (40 mg/mL). The results showed that 40 mg/mL of PMBE had effects on cell viability from 24 h to 72 h (Figure 3). Absence of synergistic anti-HCV effects of interferon-a with PMBE To determine whether IFN-a and PMBE had a synergistic inhibitory effect on the replicon, Huh7/Rep-Feo cells were cultured with combinations of IFN-a and PMBE at various concentrations. The relative dose-inhibition curves of IFN (0, 5, 10, 40, and 100 U/mL) were plotted under matching concentrations of PMBE of 0, 5, 10, and 40 mg/mL (Figure 4). The curves did not show synergy of PMBE and IFN against the HCV replicon. These results suggested that the action of the compounds on the intracellular replication of HCV replicon was independent to the IFN-ISRE pathway. Suppress the HCV IRES-dependent translation and HCV NS3 catalysis by PMBE The F/R ratio ranged from 98% (40 mg/mL PMBE) to 101% (10 mg/mL PMBE) (Figure 5a). The rate of OD450 (compared
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Figure 1. Effect of PMBE on the expression of HCV relicon. (a) Huh7/Rep-Feo cells were cultured in the presence of PMBE at concentrations of 0, 0.1, 1, 10, and 100 mg/mL. Single asterisks indicate p values of less than 0.05. Error bars indicate mean ± SD. (b) MST-1 assay of Huh7/Rep-Feo cells cultured with PMBE. Error bars indicate mean ± SD. (c) Dose-dependent suppression of HCV replication by PMBE. Error bars indicate mean ± SD of triplicate assay. (d) Time-dependent suppression of HCV replication by PMBE. The internal luciferase activities were measured at times of culture indicated. Assays were performed in triplicate. Error bars indicate mean ± SD.
Figure 2. Inhibition of infectious virus production by PMBE. Huh-7.5 cells were pretreated with 10 mg/mL PMBE or DMSO for 3 h before infection cells each day. Analysis was performed following 72 h treatment. Focus-forming units’ assay of virus released into the media by HCV-JFH1 virus-infected cells revealed the inhibition of cell production. Error bars indicate mean ± SD.
Figure 3. Real-time RT-PCR analyses of replicon RNA by PMBE. Huh7/Rep-Feo cells were cultured with indicated concentrations of PMBE, and harvested at 48 h after exposure.
with the control group) ranged from 96% (10 mg/mL PMBE) to 102% (5 mg/mL PMBE) (Figure 5b). There were no significant differences about F/R and OD450 ratio between each group. Results suggested that there were no significant changes of the internal luciferase activities at concentrations of PMBE that suppressed expression of the HCV replicon. The MST-1 assay did not show any effects on cell growth and viability at concentrations used in this assay. NS3 catalytic activity ranged from 5% (40 mg/mL PMBE) to 45% (5 mg/mL PMBE). Even when used in low amounts (5 mg/mL), NS3 catalytic activity was significantly inhibited (p50.01) (Figure 6).
Investigations into phytochemicals for antiviral activities have shown great importance in recent years. There are tremendous amount of literature available regarding is a antiviral potential of phytochemicals. In this study, we investigated the effects of PMBE on HCV NS3 catalytic activity and HCV infection, and an efficient inhibition was obtained. Flavonoid, a major ingredient of PMBE, has been reported to display antiviral activities and reduce the replication of several viruses (Andres et al., 2009; Liu et al., 2006; Rainsford, 2006). Flavonoid is a class of plant pigment, found in a wide range of green vegetables and fruits.
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DOI: 10.3109/13880209.2014.924018
Figure 4. Effect of PMBE used in combination with interferon (IFN)-a on HCV replication. Huh7/Rep-Feo cells were cultured with combination of IFN-a and PMBE at indicated concentrations. The internal luciferase activities were measured after 48 h of culture. Error bars indicate mean ± SD. Plots of 100% in each curves represent replicon expression levels that were treated with indicated amounts of PMBE and without IFN-a.
Inhibitory effects of PMBE on HCV in vitro
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Figure 6. Inhibition of HCV NS3 activities by PMBE. Inhibition of NS3 catalytic activity was tested in 96-well plates containing 30 nM scNS3 and 0.25 mM substrate that were incubated for 1 h at 37 C. For the assay, the inhibitor was mixed with the enzyme in the wells prior to the addition of the substrate. Percent catalysis was calculated from control reactions performed without inhibitors.
They also have various biochemical activities such as antioxidative and superoxide scavenging activities. Results revealed that PMBE could inhibit HCV replication in subgenomic HCV RNA replicon cell lines and block HCV viral production almost completely when using the JFH1 infectious clone. In all model systems used, PMBE efficiently inhibited NS3 protease activity and HCV replication. Thus, we provided the evidences that PMBE could affect HCV infection with a different (direct) mechanism as well. However, higher inhibition efficiency on viral production must be attributed to mutual inhibitory effection NS3 proteases. Considering the current status of limited therapy options to HCV infection and their unsatisfactory outcomes, it is urgent that anti-HCV molecules should be screened widely to develop novel antiviral therapies (Bachmetov et al., 2012). The results suggest that PMBE will be promising against HCV replication and active liver inflammation. In addition, further investigations about the action of these drugs on the expression and maturation of HCV proteins may elucidate new aspects of the viral infection and replication and may constitute novel molecular targets on anti-HCV chemotherapeutics.
Declaration of interest Figure 5. Influence of HCV IRES-mediated translations by PMBE. A biocistronic reporter gene plasmid, pCIneo-Rluc-IRES-Fluc, was stably transfected into Huh7 cells. (a) Dual luciferase assay. The cells were cultured with PMBE at the indicated concentrations, and dual luciferase activities were measured after 48 h of treatment. Values are displayed as ratios of Fluc to Rluc. (b) MST-1 assay of Huh7/neo-Rluc-IRES-Fluc cells cultured with PMBE at different concentrations. MST-1 assays at 48 h after treatment with each drug were performed in triplicate. Error bars indicate mean ± SD.
They are classified as flavone, flavonol, flavanone, flavanol, isoflavone, chalcone, anthocyanin, and catechin, according to their molecular structures. Many flavonoids have various biological functions such as antibacterial, antioxidative, and anticarcinogeninc activities (Xiao et al., 2011).
This research was supported by the National Natural Science Foundation of China (Grant no. 31372453). The authors alone are responsible for the content and writing of the paper.
References Andres A, Donovan SM, Kuhlenschmidt MS. (2009). Soy isoflavones and virus infections. J Nutr Biochem 20:563–9. Bachmetov L, Gal-Tanamy M, Shapira A, et al. (2012). Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity. J Viral Hepatitis 19:e81–8. Berdichevsky Y, Zemel R, Bachmatov L, et al. (2003). A novel high throughput screening assay for HCV NS3 serine protease inhibitors. J Virol Methods 107:245–55. Bi YF, Zheng XK, Feng WS, et al. (2002). Isolation and structure identification of ligan glycosides from pine needles of Pinus massoniana Lamb. Yao Xue Xue Bao 37:626–9.
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C. Wang et al.
Botte C, Janot C. (1996). Epidemiology of HCV infection in the general population and in blood transfusion. Nephrol Dial Transplant 11: 19–21. Bourne N, Pyles RB, Yi M, et al. (2005). Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system. Antiviral Res 67: 76–82. Cui Y, Xie H, Wang J. (2005). Potential biomedical properties of Pinus massoniana bark extract. Phytother Res 19:34–8. Liu ZM, Yang YS, Wang XL, Wen RX. (2006). Recent progress on antiHIV research of traditional Chinese medicine and components. Zhongguo Zhong Yao Za Zhi 31:1753–8. McHutchison JG, Gordon SC, Schiff ER, et al. (1998). Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med 339:1485–92. Otto GA, Puglisi JD. (2004). The pathway of HCV IRES-mediated translation initiation. Cell 119:369–80. Rainsford KD. (2006). Influenza (‘‘Bird Flu’’), inflammation and anti-inflammatory/analgesic drugs. Inflammopharmacology 14: 2–9.
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Sakamoto N, Sato C, Haritani H, et al. (1993). Detection of hepatitis C viral RNA in sporadic acute non-A, non-B hepatitis by polymerase chain reaction. Its usefulness for the early diagnosis of seronegative infection. J Hepatol 17:28–33. Sekine-Osajima Y, Sakamoto N, Nakagawa M, et al. (2009). Two flavonoids extracts from Glycyrrhizae radix inhibit in vitro hepatitis C virus replication. Hepatol Res 39:60–9. Stephan, G. (1976). Studies on the physiological behavior of Pinus merkusii Jungh. and Pinus massoniana Lamb. Beitr Trop Landwirtsch Veterinarmed 14:71–8. Vlietinck AJ, De Bruyne T, Apers S, Pieters LA. (1998). Plant-derived leading compounds for chemotherapy of human immunodeficiency virus (HIV) infection. Planta Med 64:97–109. Wu C, Feng D, Ma H, et al. (2009). Effect of Pinus massoniana bark extract on IFN-gamma-induced ICAM-1 expression in HaCaT human keratinocytes. J Ethnopharmacol 122:48–53. Xiao ZP, Peng ZY, Peng MJ, et al. (2011). Flavonoids health benefits and their molecular mechanism. Mini Rev Med Chem 11:169–77. Zuo GY, Li ZQ, Chen LR, Xu XJ. (2005). In vitro anti-HCV activities of Saxifraga melanocentra and its related polyphenolic compounds. Antivir Chem Chemother 16:393–8.
