In vitro anti-viral effect of fructopyrano-(1→4)-glucopyranose from Radix isatidis on infuenza virus A

To investigate the in vitro anti-viral effect of fructopyrano-(1→4)-glucopyranose (FG) from traditional Chinese medicine Radix isatidis, dog kidney cells (MDCK) were with treated with either FG and then with virus (Group A); virus and then with FG (Group B); or FG and virus simultaneous-ly (Group C). Results showed the in vitro cell protection rate and virus inhibition rate increased significantly with the increase of FG concentration when compared with cells without FG treatment (p<0.01). The anti-viral effect was the best in Group C and that in Group A was superior to that in Group B. FG could exert significant inhibited effect on neuraminidase activity with the IC 50 of 0.07 mmol/mL and on hemagglutinin of avian influenza virus with minimum inhibitory concentration of 3.13 mg/mL. Our findings indicate FG can confer obvious antiviral effects on influenza A virus in vitro .


Abstract
To investigate the in vitro anti-viral effect of fructopyrano-(1→4)glucopyranose (FG) from traditional Chinese medicine Radix isatidis, dog kidney cells (MDCK) were with treated with either FG and then with virus (Group A); virus and then with FG (Group B); or FG and virus simultaneously (Group C). Results showed the in vitro cell protection rate and virus inhibition rate increased significantly with the increase of FG concentration when compared with cells without FG treatment (p<0.01). The anti-viral effect was the best in Group C and that in Group A was superior to that in Group B. FG could exert significant inhibited effect on neuraminidase activity with the IC50 of 0.07 mmol/mL and on hemagglutinin of avian influenza virus with minimum inhibitory concentration of 3.13 mg/mL. Our findings indicate FG can confer obvious antiviral effects on influenza A virus in vitro. Eagl , s MEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, kanamycin and glutamine (pH 7.0~7.2) in an atmosphere with 5%CO2 at 37°C. Passaging was performed twice weekly.
Detection of cytotoxicity: MDCK cells were seeded into 96 -well plate (100 μL/well; 1×10 4 cells/well). When cell confluence reached about 100%, the medium was removed and then treated with FG at different concentrations (starting at 100 mg/mL; 200 μL/well) and 4 wells were included in each group. Incubation was performed in an atmosphere with 5%CO2 for 48 h at 37°C. The cell morphology was observed daily under an inverted microscope and the cytopathic effect (CPE) on MDCK cells was determined. Then MTT assay was performed. In brief, 20 μL of MTT solution (5 mg/mL) was added to each well followed by incubation in an atmosphere with 5% CO2 for 4 h at 37℃ followed by addition of 150 μL of dimethyl sulfoxide to each well and incubation under continuously shaking for 10 min. The optical density was measured at 490 nm on a microplate reader. The inhibition rate of cell growth was calculated as follow: IR = (ODcontrol -ODtreatment) / ODcontrol× 100%. The maximum non-toxic concentration (TC0) was also determined (Mosmann, 1983). Experiment was performed in triplicate and averaging was performed.
Determination of anti-viral effect: MTT assay was performed to determine the anti-viral effect of FG in three groups according to previously described (Knox et al. 2003;Oosterheert et al., 2005) . 1) Group A: MDCK cells were seeded into a 96-well plate and treated with FG at four different concentrations (the lowest concentration was TC0). In the positive control group, Ribavirin was added. Incubation was performed at 37℃ for 2h. Following washing in PBS twice, 100 TCID50 of influenza virus was added. In the blank control, none was added, and virus was added in the absence of FG in the virus control groups. Incubation was done in an atmosphere with 5%CO2 for 2 h at 37℃ and then the medium was refreshed with drug-free medium followed by incubation in an atmosphere with 5%CO2 for 24 h at 37℃ . Optical density was measured as described in MTT assay above.
2) Group B: MDCK cells seeded into 96-well plate and then treated with 100 TCID50 of influenza virus followed by incubation in an atmosphere with 5%CO2 for 2 h at 37℃ . After washing in PBS twice, FG at four concentrations above was added with the lowest concentration of TC0. In the blank control group, no treatment was performed and in virus control group, only virus was added. In the positive control group, peramivir was added. Incubation was performed in an atmosphere with 5%CO2 for 2 h at 37℃ . Then, the medium was refreshed with drug-free medium followed by incubation in an atmosphere with 5%CO2 for 24 h at 37℃ and optical density was measured as described in MTT assay above.
3) Group C: Cells were independently mixed with FG at four concentrations with the lowest concentration of TC0. Blank control group and virus control group were also incluced. In the positive control group, peramivir was added. Incubation was performed in an atmosphere with 5%CO2 for 2 h at 37℃ . Then, the medium was refreshed with drug-free medium followed by incubation in an atmosphere with 5%CO2 for 24 h at 37℃ and optical density was measured as described in MTT assay above.
Suppressive effect of FG on neuraminidase activity: The supernatant of influenza virus (bovine serum-free medium containing 1.2 mg/mL albumin, 5 μg/mL trypsin, ) was added to MDCK cells. When cell changes were present, the supernatant was collected by centrifugation at 1,000 rpm for 10 min followed by inactivation with NP-40 (final concentration: 0.1%). The supernatant was filtered through a 0.22-µm filter serving as the neuraminidase solution, which was stored at -70℃ (Peng et al., 2005;Li et al., 2009).
Inhibition for neuraminidase of FG: Neuraminidase (10 μL) was added into 96-well plate followed by addition of FG (20 μL) at different concentrations and subsequent incubation for 1 h. After addition of 20 µmol/L MUNANA (40 μL) and 33 mmol/L MES (30 μL ) (total volume: 100µL), incubation was performed at 37℃ for 30 min. Then, 25%alcohol containing 0.1 mmol/L glycine (pH=10.7; 200μL) was added to stop reaction. Blank control was also included. In the positive control group (MES), peramivir was added. In the detection of neuraminidase activity, sample solution of same volume was added. In the control group, medium of same volume was added. Neuraminidase activity was determined with a fluorescence microplate reader with EX at 355 nm, EM at 460 nm, required value of 20% and gain value of 20. The inhibition rate (IR) was calculated (Mosmann, 1983). The half inhibitory concentration (IC50) of FG on neuraminidase was calculated with the Bliss method (Nayak and Reichl, 2004).
Hemagglutination inhibition assay: PBS (pH 7.2) was added to a 96-well plate (25 μL/well), and 50 mL FG solution was added in the first tier which was dispensed by the serum(by RDE treatment, dilution of 1:10) and continuous multiproportion diluted until dumping 25 μL in the last one (No. 8). The first tier concentration was 50mg/mL. Then 25 μL influenza virus (A/PR8/34) suspensions were added to each pore of 4 hemagglutination units, and it was covered at room temperature 1 h after thoroughly misce bene. Eventually, adding 0.75%guinea pig red blood cell suspensions 50 μL which were dispensed by PBS buffer in each pore, and laying the drug control group (an action of only drugs without added liquid of influenza virus), PBS buffer control group (only the influenza virus suspensions and PBS buffer without added drugs) and serum control group (only serum and red cell suspensions of guinea pig), observing and recording after placed it in incubator for 60 min at 4℃ . Criteria for determination: (1)++++: Red blood cells arranged evenly on bottom; (2)+++: Red blood cells arranged evenly on bottom but the edge of cell layer was thin; (3) ++: Red blood cells formed a circle on the bottom, which was surrounded by a small agglutination block; (4) +: Red blood cells formed a group on the bottom without smooth edge; (5)-: Red blood cells formed a group on the bottom with smooth and regular edge.

Results
When the concentration of FG was lower than 12.50 mg/mL, there was no significant difference in cell proliferation between the FG group and control group, indicating that FG at <12.50 mg/mL has no toxic effect on MDCK cells.
In the present study, the anti-viral effect of FG was determined by MTT assay and ribavirin treatment served as a positive control. Results showed FG could exert anti-viral effect to a certain extent in three groups but the anti-viral effect in Group C was the best and the anti-viral effect in Group A was superior to that in Group B. Anti-viral effect of FG is shown in Table I. 1) Group A: When compared with virus control group, the virus inhibition rate and cell protection rate following FG treatment were markedly increased (p<0.01). However, the virus inhibition rate and cell protection rate following FG treatment were inferior to those in the positive control group (p<0.05). 2) Group B: When compared with virus control group, the virus inhibition rate and cell protection rate following FG treatment were markedly increased (p<0.01). However, the virus inhibition rate and cell protection rate following FG treatment were inferior to those in the positive control group (p<0.05). 3) Group C: When compared with virus control group, the virus inhibition rate and cell protection rate following FG treatment were markedly increased (p<0.01). However, the virus inhibition rate and cell protection rate following FG treatment were inferior to those in the positive control group (p<0.05). Data within the parenthesis is SD Suppressive effect on neuraminidase activity was observed following treatment with FG or peramivir. The dose-response curve between response rate and the Logarithmic dose was "S" shaped (Figure 2). The suppressive effect of FG was similar to that of peramivir. The IC50 of FG was 0.071 mmol/mL and that of peramivir was 0.003 nmol/mL.
FG had obvious inhibitory effect on influenza virus hemagglutinin and the minimum inhibitory concentration was 3.13 mg/mL. Non-specific agglutination was absent following serum treatment. (Table II (Colman, 1994;Cao et al., 2002). Currently, substrate fluorescence detection (FL-MU-NANA method) is one of methods for the detection of neuraminidase activity in vitro, and has become an important method to screen and evaluate anti-viral activity of anti-influenza virus drugs including herbals (Peng and Li, 1999). In the present study, FL-MU-NANA method was employed to measure the biological activity of NA. Our results showed FG had anti-viral effect and neuraminidase may be one of targets of FG.
The influenza virus mainly consists of nucleocapsid and envelope. A glycoprotein in the envelope is a key protein to bind to receptors on cells of host and can form agglutination with red blood cells of animals. If the serum hemagglutinin antibody can specifically bind to the antigens on the virus, the interaction between  hemagglutinin on virus and receptors on red blood cells is interrupted and red blood cell aggregation is then inhibited. In the present study, virus (hemagglutinin) was pre-treated with FG and then with red blood cells of guinea pig, and agglutination was observed. Results showed FG at effective concentration could significantly inhibit the hemagglutinin of virus in vitro, and the minimum inhibitory concentration was 3.13 mg/mL.

Contributions
Sun Qin and Shui Pi-xian contributed equally in this study.