Background
Several viruses cause malignant diseases worldwide, resulting in significant mortality and economic losses. For instance, Influenza A viruses are responsible for seasonal epidemics and have caused three pandemics in the 20th century (1918, 1957, and 1968) [
1]. In addition, viral diseases cause serious threats to livestock sector and animal welfare together with productivity losses, uncertain food security and negative impacts on human health [
2].
Although various prophylactic or therapeutic drugs and vaccines have been developed to prevent and treat viral diseases, the emergence of novel mutants or resistant virus strains reduces their efficacy and lead to the public health problems. Especially, with a view to provide effective methods for preventing viral diseases, researches have been attempted to identify novel materials with antiviral activities from natural or synthetic resources.
Natural products are one of relevant sources for antiviral substances. Therefore, many screening efforts have been made to identify antiviral substances from natural sources with high efficacy, low toxicity and minor side effects. Hence, treatments with herbal extracts having antiviral activities may assist to recover many animals through effective feed conversions and weight gains to result better economic returns. Consequently, as an alternative to conventional chemical agents, a large number of phytochemicals which containing extracts or pure substances from medical plants have been recognized as antivirals [
3].
To identify potential antiviral agents, 200 natural oriental herbal medicines were screened and among them, Cortex Phellodendri (C. Phellodendri) found to display a wide array of antiviral activities. C. Phellodendri (Huangbai in Chinese), the dried bark of
Phellodendron amurense Ruprecht (Family: Rutaceae), is one of the 50 fundamental herbs of traditional Chinese medicine and has been used against osteoarthritis, weight loss, obesity, diarrhea, diabetes, pneumonia and eye infections for a long period of time [
4]. This herb is widely found in China and the Korean peninsula, and a wide range of primary scientific articles are already available on the activities of extracts of Phellodendron bark, although the underline mechanisms in the therapeutic process remain unclear. Further, the antiviral activity of C. Phellodendri has not been scientifically described.
In this study, the antiviral activities of C. Phellodendri aqueous extracts (CP) against wide array of viruses in vitro and in vivo were evaluated. Additionally, the immune-modulatory potential of C. Phellodendri which regulates the antiviral immune response was confirmed.
Methods
Cells and viruses
RAW264.7 (ATCC® TIB-71™), HEK293T (ATCC® CRL-11268™), HeLa (ATCC® CCL-2™) and MDCK (ATCC CCL-34, NBL-2) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1 % antibiotic-antimycotic solution (Gibco, Grand Island, NY, USA) at 37 °C with 5 % CO2. Green Fluorescent Protein (GFP)-tagged Influenza A (A/PuertoRico/8/34(H1N1) (PR8-GFP), Newcastle Disease Virus (NDV-GFP) and challenge viruses of influenza A subtypes [{A/Aquaticbird/Korea/W81/2005 (H5N2)}, {A/PR/8 /34(H1N1)}, {A/Aquaticbird/Korea/W44/2005(H7N3)} and {A/Chicken /Korea/116/2004(H9N2)}] were propagated in the allantoic fluid of 10-day-old chicken embryos. Vesicular Stomatitis Virus (VSV-GFP), Herpes Simplex Virus (HSV-GFP) and Enterovirus-71 (EV-71) were propagated on confluent Vero cells (ATCC® CCL-81™) and Coxsackie virus (H3-GFP) was propagated on confluent A549 (ATCC® CCL-185™) cells.
Plant materials and total aqueous extract preparation
Crude plant material, the dried bark of Phellodendron amurense Ruprecht (specimen number: Phellodendron amurense Rupr., PSNA200005151048; Department of Pharmacology at Busan National University, Korea), was purchased from Jaecheon Oriental Herbal Market (Jaecheon, Korea) and verified by Professor Ki-Hwan Bae at the College of Pharmacy, Chungnam National University. Water-soluble herbal extract of C. Phellodendri was prepared by Vitabio Corporation (Daejeon, Korea).
Determination of Effective Concentration (EC50) of Cortex Phellodendri in vitro
The EC
50 can be defined as the observed extract concentration at which 50 % reduction in virus titer. To determine the EC
50 values of CP against divergent viruses in vitro, a modified GFP assay was developed using RAW264.7, HEK293T and HeLa cell lines [
5]. Raw264.7, HEK293T and HeLa cells were cultured in 96-well plates and after 12 h of incubation, the media was replaced with 2-fold serially diluted CP from original stock (0.1 mg/ml). At 12 h post-treatment (hpt), RAW264.7 cells were infected with PR8-GFP (MOI = 1.0), VSV-GFP (MOI = 1.0) or NDV-GFP (MOI = 3.0); HEK293T cells with VSV-GFP (MOI = 0.2) or HSV-GFP (MOI = 2.0); and HeLa cells with H3-GFP (MOI = 3.0) or EV-71 (MOI = 0.5) using DMEM containing 1 % FBS. At 2 h post-infection (hpi), the inocula were replaced with DMEM (10 % FBS). GFP expression was measured at 24 hpi using Glomax multi-detection system (Promega, WI, USA). EC
50 values were calculated as the extract concentration which yielded 50 % GFP expression or in the EV-71, 50 % reduction in viral cytopathic effects (CPE).
Cytotoxicity assay (CC50) of Cortex Phellodendri in vitro
The CC
50 assay was performed in 72-well tissue culture plates and the CC
50 was determined through trypan blue exclusion test as described previously [
6]. Increasing concentrations (0.1-16 μg/ml) of the extract were added to 75–80 % confluent RAW264.7, HEK293T and HeLa cell monolayers. After 24 h, the cell viability of each treatment group was determined by trypan blue staining. Concentrations of the extract plotted against the cell viabilities, and CC
50 was calculated as the concentration of the extract resulting 50 % cell viability.
Antiviral assays in Cortex Phellodendri-treated RAW264.7 and epithelial (HEK293T or HeLa) cells
Virus replication inhibition assay was performed using the GFP viruses as described previously with some modifications [
7]. RAW264.7 (12-well plates; 8 x 10
5 cells/well), HEK293T and HeLa cells (6-well plates; 1 x 10
6 cells/well) were incubated at 37 °C for 12 h. For the pre-treatment assay, DMEM alone (untreated and virus-only groups), positive control; DMEM with 1000 U recombinant mouse or human interferon-β (rmIFN-β or rhIFN-β, Sigma, St. Louis, USA) and DMEM with 1.0 μg/ml of CP were added to the cells. At 12 hpt, RAW264.7 cells were infected with either PR8-GFP (MOI = 1.0), NDV-GFP (MOI = 3.0) or VSV-GFP (MOI = 1.0) using DMEM supplemented with 1 % FBS. HEK293T cells were infected with VSV-GFP (MOI = 0.2) or HSV-GFP (MOI = 2.0). HeLa cells were infected with H3-GFP (MOI = 3.0) or EV-71 (MOI = 0.5). At 2 hpi, wells were gently washed with PBS and replaced the media with DMEM (10 % FBS). GFP expression or CPE, which reflects virus replication, was observed at 12 or 24 hpi under 200X magnification. The cell viability and the virus titer were determined at 12 or 24 hpi. GFP expression was measured at 24 hpi using Glomax multi-detection system (Promega, Wisconsin, USA) according to the manufacturer’s instructions.
NDV-GFP mRNA expression in RAW 264.7 cells
Total mRNA from RAW264.7 cells infected with NDV-GFP (MOI = 3.0) was extracted using the RNeasy Mini Kit (Qiagen, Seoul, Korea) and cDNA was synthesized using reverse transcriptase (Toyobo, Japan) according to manufacturer’s protocol. Real-time polymerase chain reaction (RT-PCR) was performed using gene specific primers to determine the NDV-GFP mRNA expression levels. For the NDV Matrix (M) gene, forward primer was 5′-TCGAGICTGTACAATCTTGC-3′ and the reverse primer was 5′- GTCCGAGCACATCACTGAGC-3′ [
8]. For the GAPDH (housekeeping gene), the forward primer was 5′-TGACCACAGTCCATG CCATC-3′ and the reverse primer was 5′-GACGGACACATTGGG GGTAG-3′ [
9]. Equal amount of PCR products were run on 1.5 % ethidium bromide agarose gels and visualized using GelDoc Imaging System (Bio-Rad, Seoul, Korea). The relative band intensity (RBI) of the M gene was compared with GAPDH using Band Quantification Software (Bio-Rad, Seoul, Korea).
Virus titration from Cortex Phellodendri-treated cell supernatants or cells
Viral titers were determined with cell supernatants (VSV-GFP and H3-GFP) or cells (PR8-GFP and HSV-GFP) in Vero cells using a standard plaque assay. EV-71 titer measured by the 50 % tissue culture infectious doses (TCID50) using HeLa cells with some modifications. Briefly, 75–80 % confluent HeLa cells grown in 96-well micro-titer plates were infected with 10-fold serial diluted supernatants (50 μl/well) collected from the infected cells. At 2 hpi, DMEM (10 % FBS) containing L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) trypsin (Thermo Fisher Scientific, Rockford, USA) was added to the infected wells and incubated for another 2 days. CPE were observed daily under 200X magnification, and the titers were determined by TCID50.
Oral administration of Cortex Phellodendri and virus challenge in BALB/c mice
Five-week-old 52 BALB/c mice (16 ± 1 g) were used to conduct in vivo experiments (Table
1) For the survival rate analysis, 40 mice were grouped in to 4 main groups (
n = 10), each containing 2 subgroups, control (
n = 5) and CP treated group (
n = 5). For the lung virus titration another 12 mice were grouped into 2 sub-groups, control (
n = 6) and CP treated group (
n = 6), and lung tissue was extracted at 3 (
n = 3) and 5 dpi (
n = 3) from each control and treated group. CP were orally administered to the mice (0.8 μg/g body weight) in a total volume of 200 μl at 1, 3 and 5 days before infection. PBS (200 μl) was orally administered to control groups mice. Treatment and challenge experiments were conducted in an approved BSL-2
+ laboratory facility. Mice were intranasally infected with 5 times 50 % minimum lethal dose (MLD
50) of H1N1, H5N2, H7N3 or H9N2 in 20 μl of PBS per mouse. Body weight and survival were monitored up to 13 dpi. Mice exhibiting more than 25 % body weight loss were considered as an experimental end point and were humanely killed. Animal study was conducted under appropriate conditions with the approval of the Institutional Animal Care and Use Committee of Bioleaders Corporation, Daejeon, South Korea, Protocol number: BSL-ABLS-13-008.
Table 1
Mouse groups of in vivo experiments
H1N1 | Control (n = 5) | PBS | - | Survival rate |
CP (n = 5) | CP | 0.8 | Survival rate |
H5N2 | Control (n = 5) | PBS | - | Survival rate |
CP (n = 5) | CP | 0.8 | Survival rate |
H7N3 | Control (n = 5) | PBS | - | Survival rate |
CP (n = 5) | CP | 0.8 | Survival rate |
H9N2 | Control (n = 5) | PBS | - | Survival rate |
CP (n = 5) | CP | 0.8 | Survival rate |
H1N1 | Control (n = 6) 3 dpi (n = 3), 5 dpi (n = 3) | PBS | - | Lung titration |
CP (n = 6) 3 dpi (n = 3), 5 dpi (n = 3) | CP | 0.8 | Lung titration |
Determination of lung viral titers
Lung tissues from euthanized mice were collected aseptically, and lung viral titer was calculated by the Reed and Muench method and expressed as Log
10 TCID
50/Lung. In the modified hemagglutination assay (HA), instead of turkey red blood cells (RBC), 0.5 % chicken RBC was used and wells containing HA were scored as positive [
10].
Detection of IFN-β and pro-inflammatory cytokines
Cells were treated with 1000 units/ml rmIFN-β or rhIFN-β and 1.0 μg/ml CP in DMEM supplemented with 10 % FBS or media alone, and then incubated at 37 °C with 5 % CO2. The supernatants were harvested at 0, 12 and 24 hpt. Using commercial ELISA kits, murine interleukin (IL)-6, IL-12, tumor necrosis factor-alpha (TNF-α) (BD Bioscience, USA), murine IFN-β (PBL Interferon Source, USA), human IFN-β (TFB Inc., Tokyo, Japan) and human IL-6 (Invitrogen, Carlsbad, California, USA) were measured according to manufacturer’s protocol.
Determination of the effects of Cortex Phellodendri on Type I IFN-related protein phosphorylation by Immunoblot analysis
RAW264.7 cells were treated with DMEM alone (negative control), DMEM with 100 ng/ml LPS (positive control), or DMEM with 1.0 μg/ml CP, and cells were harvested at 0, 3, 6, 12, and 24 hpt. Cell pellets were washed with PBS and whole cell lysates were subjected to SDS-PAGE followed by standard immunoblotting with indicated antibodies: anti-IRF3 (Abcam, #ab25950), anti-phospho-IRF3 (Ser396) (Cell Signaling, #4947), anti-p65 (Cell Signaling, #4764S), anti-phospho-p65 (Cell Signaling, #3031S), anti-STAT1 (Cell Signaling, #9175), anti-phospho-STAT1 (Cell Signaling, #9167), anti-TBK1 (Cell Signaling, #3504S), or anti-phospho-TBK1 (Cell Signaling, #5483S), anti-p38 (Cell Signaling, #9212), anti-phospho-p38 (Cell Signaling #4631S), anti-ERK (Cell Signaling, #9102), anti-phospho-ERK (Cell Signaling, #9102S), or anti-β-actin (Santa Cruz SC 47778).
Level of mRNA induction by Cortex Phellodendri in vitro
RAW264.7 or HEK293T cells were treated with DMEM supplemented with media alone (negative control), DMEM with 1000 units/ml rmIFN-β or rhIFN-β (positive control), or DMEM with 1.0 μg/ml CP and the cells were harvested at 0, 3, 6, 12, and 24 hpt. The total RNA isolation, cDNA synthesis, and visualization of band intensity after PCR were as previously described. Additionally, different cDNA levels from HEK293T cells were measured by quantitative RT- PCR (qRT-PCR) using a QuantiTech SYBR Green PCR kit (Qiagen, Seoul, Korea) on a Mygenie96 thermal block (Bioneer, Daejeon, Korea). The PCR primers are listed in Tables
3 and
4.
HPLC analysis for chemical characterization of aqueous plant extract of Cortex Phellodendri
The liquid chromatography-mass spectrometry (LC-MS) analysis was performed on an Agilent 1200 Series high-performance liquid chromatography (HPLC) (Agilenttechnologies CO, USA) connected to a linear ion trap mass spectrometer 4000 QTRAP system (AB Sciex CO., Canada) equipped with an ESI TurboV source. Chromatography was carried out on a column ZORBAX Eclipse XDB-C18 (4.6 x 150 mm, I.D.-5 μm) (Agilent technologies CO., USA). The mobile phase consisted of 1 % Formic acid (Solvent A) and Acetonitrile (Solvent B) in the gradient mode as follows : 0–8 min 0–25 % B; 8–12 min 25–35 % B; 12–17 min 35–50 % B; 17–25 min 50–100 % B; 25–28 min 100–0 % B at flow rate of 1.0 ml/min at 30 °C. Conditions of the MS analysis were as follows: positive ion mode, spectra range from m/z 100 to 500, nebulizer; 70.0 psi. Molecular weight identification of purified fraction (PA-6) and berberine was carried out by electrospray ionization mass spectrometry (ESI-MS).
Determination of antiviral characteristics of berberine
PA-6 antiviral activity and cytokine inducing ability was further tested in immune (RAW264.7) cells using an effective dose (10.0 μg/ml). Inhibition of virus replication (PR8-GFP) in RAW264.7 cells, virus titration and induction of cytokine secretion were monitored according to the protocols described above sections.
Statistical analysis
Statistical analysis was performed using GraphPad Prism software version 6 for Windows (GraphPad Software, USA). Data are presented as the means ± standard deviations (S.D.) and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance (ANOVA). Unpaired t-test was performed at each time points to compare the control and NDV infected extract-treated groups. Results for percent initial body weight were also compared by Student’s t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. P < 0.05 were regarded as significant.
Discussion
In the present study, the broad spectrum antiviral activity of medicinal herb C. Phellodendri both in vitro and in vivo was evaluated to suggest the rationale for its immune-modulatory and biological properties. C. Phellodendri has been reported as a well-known source for the variety of therapeutics with rare side effects after a dose [
14], and in the tested cell lines, CP also did not show any significant cytotoxic effect. Moreover, the cell CC
50 of CP was several magnitudes higher than the EC
50 of tested viruses and the SI of the herb for various viruses indicate the higher safety margin of the extract for therapeutic and/or prophylactic purposes (Table
1).
In the present study, the CP inhibited the replication of PR8-GFP (Fig.
1a), VSV-GFP (Figs.
1b and
2a), NDV-GFP (Fig.
1c), HSV-GFP (Fig.
2b), H3-GFP (Fig.
2c) and EV-71 (Fig.
2d) in immune (RAW264.7) and epithelial (HEK293T and HeLa) cells. Interestingly, oral administration of CP not only increased the survival rate of mice subjected to lethal challenges with different influenza A virus subtypes including H1N1, H5N2, H7N3 and H9N2 but also led to rapid weight recovery (Fig.
3). Although CP-inoculated mice initially displayed little weight reduction, majority did not lose more than 25 % of their body weight. However, all of the mice in the control groups displayed more than 25 % losses within 9 dpi. Collectively, it suggest that CP is strong enough to inhibit the viral replication and promoted the survival of mice against lethal infections of diverse influenza A viruses.
Upon viral infection, host cells initially recognize the virus infection and rapidly evoke the antiviral innate immune responses including secretion of type I IFN and pro-inflammatory cytokines [
15]. Secreted IFNs and cytokines induce an antiviral state which is important to protect the host cells against invading viruses, as induction of antiviral state in early time of virus infection is critical to control the spread and pathogenesis of viruses [
16]. In this study, CP induces an antiviral state via the induction of type I IFN and pro-inflammatory cytokines in vitro. To elucidate the delineate features of CP in antiviral signaling, the effect of CP on the phosphorylation of IRF-3, p65, TBK1, STAT1, ERK and p38, the important regulatory molecules which present in the type I IFN [
17‐
20] and NF-kB [
21,
22] signaling pathways were studied and it shown to be up-regulated. Additionally, CP up-regulated the transcriptional levels of IFN-β, ISGs and various antiviral genes. These findings confirm that CP can induce the innate immune responses via type I IFN and NF-kB signaling for controlling viral replication.
In this study, murine macrophages (Raw264.7) were used to evaluate the antiviral effect of CP and also used epithelial cells such as HEK293T or HeLa cells which are very susceptible for viral replication. Recently, it is acknowledged that HEK293T [
23] and HeLa [
24] cells have less prominent pattern recognition receptors (PRRs), chiefly Toll-like receptors (TLRs). Thus it could postulate that CP may contain active components which can bind or penetrate the cell membrane and those active components may stimulate the cell surface PRRs, cytoplasmic PRRs or both and ultimately, can induce the antiviral immune responses in immune cell or epithelial cells. A bacterial endotoxin, LPS (or lipid A-associated protein) is a known immunomodulator and is often a contaminant in biological preparations. Therefore, it could speculate that macrophage-stimulating properties of CP might be due to contamination from bacterial endotoxin [
25]. In this study, CP was tested with LPS using a LAL assay and was found only a trace amount of endotoxin (Fig.
4d), and antiviral function of CP was not due to LPS stimulation.
The constituents of CP are numerous and diverse, including aporphine alkaloids, protoberberine alkaloids, flavonoids, berberine, oxyberberine, palmatine, isovanillin, ferulic acid, limonin and various other molecules [
4]. HPLC analysis was conducted to identify different active compounds present in the total aqueous fraction and found six major peaks (Fig.
6a), and six fractions (PA-1 to PA-6) which were successfully purified. Among six fractions, PA-6 was identified to have significant antiviral properties similar to a CP and later confirmed as berberine (Fig.
6c). Berberine has already been studied for its anticancer properties [
26], antibacterial activity [
27], antifungal activity [
28] and antiviral activity [
29]. Especially, it was reported that berberine inhibit influenza viral replication by reducing the viral neuraminidase activity, and reducing inflammatory responses which induced by influenza virus through inhibition of nitric oxide (NO) and repression of TNF-α and monocyte chemoattractant protein 1 (MCP-1) gene transcription [
30]. Moreover, HB-13, a compound derived from berberine, also inhibits HSV-1 and HSV-2 activity [
31,
32]. However, in the present study, the antiviral effects of berberine was demonstrated with more specific mechanism via type I IFN stimulation. Consequently, antiviral or immunomodulatory effects of CP could be due to the cumulative effect of berberine or various other unknown active compounds present in the extract.
Modern pharmacological researches have further confirmed that alkaloids of C. Phellodendri could be used for treating many diseases. Among them, relationship between mechanisms of antiviral effects and active compounds including berberine should be required further study. Furthermore, the question of how to address the need for both individualizing (the basis of TCM) and standardizing (the basis of modern pharmacology) the treatments with C. Phellodendri must be settled. Once these issues are resolved, the prospects will be exists for widespread use of C. Phellodendri as a safe, effective, and affordable form of healthcare.
Conclusion
The current study propose that pre-treatment of CP has a broad spectrum of anti-viral functions against VSV-GFP, PR8-GFP, NDV-GFP, HSV-GFP, H3-GFP and EV-71 in vitro and against divergent influenza A subtypes of H1N1, H5N2, H7N3 and H9N2 in vivo mouse model. Moreover, our results describe a possible mode of CP antiviral activity via the induction of type I IFN or pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in the host cell. Additionally, active components present in CP including berberine are important for the observed antiviral and immunomodulatory properties. Thus, the use of C. Phellodendri as an orally active antiviral agent, together with present vaccines or individually, has the potential to be an effective remedy in both humans and livestock for prophylaxis applications.
Abbreviations
C. Phellodendri, Cortex Phellodendri; CC50, cytotoxicity concentration; CP, Cortex Phellodendri aqueous extract; CPE, cytopathic effects; dpi, days post-infection; EC50, effective concentration; ERK, extracellular signal-regulated kinases; ESI-MS, electrospray ionization mass spectrometry; EU, endotoxin unit; EV-71, enterovirus-71; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GBP-1, guanylate binding protein 1; GFP, green fluorescent protein; H3: coxsackie virus; HA, hemagglutination assay; hpi, hours post-infection; HPLC, high-performance liquid chromatography; HSV, herpes simplex virus; IFN, interferon; IL, interleukin; iNOS, inducible isoform nitric oxide synthases; IRF-3, interferon regulatory transcription factor 3; ISGs, interferon-stimulating genes; LAL, limulus amebocyte lysate; LC-MS, liquid chromatography-mass spectrometry: LPS, lipopolysaccharide; M-gene: matrix gene; MCP-1, monocyte chemoattractant protein 1; MLD50, 50 % minimum lethal dose; MOI, multiplicity of infection; Mx1, interferon-induced GTP-binding protein; NDV, newcastle disease virus; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; NO, nitric oxide; PA, purified fraction; PR8, influenza A virus; PRRs, pattern recognition receptors; qRT-PCR, quantitative real-time polymerase chain reaction; RBC, red blood cells; RBI: relative band intensity; rhIFN-β, recombinant human interferon-β; rmIFN-β, recombinant mouse interferon-β; RT-PCR, real-time polymerase chain reaction; SI, selection indexes; STAT1, signal transducer and activator of transcription 1; TBK1, TANK-binding kinase 1; TCID50, 50 % tissue culture infectious doses; TLRs, toll-like receptors; TNF-α, tumor necrosis factor-alpha; VSV, vesicular stomatitis virus
Acknowledgements
The authors thank Dr. J. U. Jung of the University of Southern California, USA for providing Green Fluorescence Protein (GFP)-tagged PR8, NDV, VSV, HSV and H3 and Dr. Y. K. Choi of Chungbuk National University, Cheongju, Republic of Korea for providing the challenge viruses.