Antiviral effect of baicalin phospholipid complex against duck hepatitis A virus type 1

Antiviral effect of baicalin phospholipid complex against duck hepatitis A virus type 1 ABSTRACT Duck hepatitis A virus type 1 (DHAV-1) is one of the main pathogens of ducklings and causes a high mortality rate. Baicalin (BA) has potent antiviral effect, but the solubility is very poor. In order to increase the absorption, solubility, and pharmacological activity, the phospholipid complex was used to modify BA in present study. Therefore, BA phospholipid complex (BAPC) was prepared. The anti-DHAV-1 abilities of BA and BAPC in vitro was evaluated by cell counting kit-8 and reverse transcription quantitative PCR. The curative effects of BA and BAPC on ducklings which were infected by DHAV-1 in addition to the ALT and AST levels were also detected. The results indicated the anti-DHAV-1 ability of BAPC was stronger than that of BA both in vitro and in vivo. To explore the anti-DHAV-1 mechanism, the influence of BAPC on DHAV-1 adsorption, replication, and release was studied. Furthermore, the anti-oxidative and immuno-enhancing abilities of BAPC in the treatment of infected ducklings were also determined. The results showed BAPC inhibited DHAV-1 adsorption, replication and release. Furthermore, it played anti-oxidative and immno-enhancing roles in the treatment, and the immno-enhancing role was crucial to the treatment. INTRODUCTION Duck hepatitis A virus type 1 (DHAV-1) is a member of genus Avihepatovirus in the family Picornaviridae (Sheng et al., 2014). The pathogenicity of DHAV-1 is very strong and it mainly infects ducklings (Levine and Fabricant, 1950). When ducklings less than three weeks old infected by DHAV-1, the mortality rate is even up to 100%. And the lesions of the dead ducklings’ livers are always serious with swell and bleeding (Chen et al., 2014). The peroxidation damages of the infected ducklings are also very serious (Chen et al., 2015). However, no licensed drug is able to treat ducklings infected by DHAV-1 in clinic. In our previous study, many compounds in natural sources exhibited anti-DHAV-1 abilities, such as bush sophora root polysaccharide and its sulfate (Chen et al., 2014; Chen et al., 2015), icariin and its phosphate (Xiong et al., 2014), and Codonopsis pilosula polysaccharide and its phosphate (Ming et al., 2016). Baicalin (5,6-dihydroxy-7-O-glucuronide flavone, BA), a kind of flavonoids, is a main ingredient of Chinese medicinal herb huang qin (Scutellaria baicalensis Georgi). It possesses antiviral (Moghaddam et al., 2014), anti-oxidative (Cao et al., 2011), and immuno-enhancing ability (Hu et al., 2012). However, BA has poor water solubility and poor fat solubility (Li et al., 2012). Phospholipid complex can improve the physicochemical properties of the original compound, increase the absorption, and eliminate adverse reactions (Xu et al., 2016). It is reported that BA phospholipid complex (BAPC) possesses better solubility and nasal mucosa absorption than BA (Li et al., 2012). So in present study, the BAPC was prepared and its physicochemical properties were characterized. Furthermore, the anti-DHAV-1 ability of BAPC compared with BA was detected both in vitro and in vivo. At the same time, in order to explore the anti-DHAV-1 mechanisms of BAPC, the anti-DHAV-1 links (direct antiviral mechanism) in addition to the anti-oxidative and immuno-enchancing effects (indirect antiviral mechanism) were also explored. MATERIALS AND METHODS Reagents Dulbecco's modified eagle medium (Gibco, USA) supplemented with penicillin 100 IU/mL, streptomycin 100 IU/mL, glutamine 0.75 mg/mL, and 10% fetal bovine serum, was called growth medium (GM) and used for culturing the duck embryonic hepatocytes (DEHs). Dulbecco's modified eagle medium (Gibco, USA) supplemented with penicillin 100 IU/mL, streptomycin 100 IU/mL, glutamine 0.75 mg/mL, and 1% fetal bovine serum, was called maintenance medium (MM) and used for diluting drugs and maintaining cells. D-Hank's solution was used for washing the embryo tissue and cells. The pH of GM, MM and D-Hank's was adjusted to 7.4 using 5.6% NaHCO3. The trypsin (Amresco, USA) was dissolved into 0.20% with D-Hank's. The cell counting kit-8 reagent was brought from Dojindo, Japan. RNAiso Plus Reagent, PrimeScriptTM RT Master Mix Kit, and SYBR® Premix Ex TaqTM (Tli RNaseH Plus) Kit were bought from Takara, Japan. Hinokitiol used as the pro-oxidant which is also known as 4-isopropyltropolone was brought from Tokyo Chemical Industry CO., LTD., Japan. FK506 used as the immunosuppressant which is also known as tacrolimus was brought from Aladdin Industrial Corporation, China. The solvent used to dissolve hinokitiol and FK506 as well as treat ducklings was normal saline containing 0.3% Tween-80, 0.5% glycerinum and 0.1% dimethyl sulfoxide. The superoxide dismutase (SOD), malonaldehyde (MDA), catalase (CAT) and glutathione peroxidase (GSH-Px) assay kits were brought from Nanjing Jiancheng Bioengineering Institute, China. The IL-2 and IFN-γ ELISA kits for ducks were brought from Kamiya Biomedical Company, USA. DEHs and DHAV-1 DEHs were prepared as the method described previously (Chen et al., 2014). Briefly, liver tissues from a duck embryonic at 15 days were minced and then digested with 0.20% trypsin. Then the cells were washed thrice with D-Hank's, diluted into 0.8 × 106–1.2 × 106 cells/mL with GM, and then incubated in a humid atmosphere of 5% CO2 at 37°C. After 48 hours, the hepatocytes grew into a monolayer. DHAV-1 (LQ2 strain) used for in vitro and in vivo experiments were supplied by the Shandong Institute of Poultry in China. For in vitro experiments, it was generated by inoculating DEHs using regular procedure and diluted into 50 TCID50 in the experiments. For in vivo experiments, it was generated using the following procedure: three-day-old cherry valley ducklings were intramuscular injected with 0.2 mL DHAV-1, the liver tissue homogenates were obtained after three days. After centrifugation, the DHAV-1 for in vivo experiment was harvested and diluted into 20 LD50 in the experiments. BA and BAPC BA was purchased from Sigma Chemical Company (Sigma, USA). BAPC was prepared as the method described by Li et al. (2012). Firstly, 1.25 g BA and 2.5 g soy phospholipids were mixed together. Then 500 mL of tetrahydrofuran was added. And the mixture was refluxed in a thermostatic water bath at 55°C for 1 hour. Finally, BAPC was obtained after the vacuum rotary evaporation at 50°C. For in vitro assays, BA was diluted to 31.25 μg/mL with MM, BAPC was diluted twofold serially from 1000 μg/mL to 3.906 μg/mL. For in vivo assays, both BA and BAPC were diluted into 1 mg/mL with distilled water. Structural Characterization of BAPC Infrared Spectroscopy Analysis The FT-IR spectrum of BA and BAPC in a wavenumber range from 400 cm−1 to 4000 cm−1 was recorded by KBr pellets method (Qin et al., 2013) with a Nicolet 200 Magna-IR spectrometer (Nicolet Instrument Corp., USA). The major peaks (intensity and wavenumber) were found using OMNIC software (Nicolet Instruments Corp., USA). Differential Scanning Calorimetry (DSC) The DSC was performed in a differential scanning calorimeter (Mettler-Toledo, Switzerland). BA, soy phospholipids, physical mixture, and BAPC were loaded into aluminum pans separately and heated at the rate of 10°C/min from 50 to 300°C in nitrogen atmosphere. X-Ray Diffraction (XRD) The crystalline states of BA, soy phospholipids, physical mixture, and BAPC were evaluated with XRD diffractometer (Rigaku Denki, Japan). The detection conditions were as follows: copper target, temperature 25°C, voltage 40 kV, current 40 mA, scanning rate 3 °/min, and scanning range 5–50°C. Cytotoxicity Test 100 μL BAPC diluted twofold serially from 1000 μg/mL to 3.906 μg/mL were added into a 96-well plate containing the DEHs monolayer; 100 μL MM was added into the cell control wells; 5 wells per treating. Then the plate was incubated in a humid atmosphere of 5% CO2 at 37°C. After 72 hours, the absorbance at 450 nm wavelength of each well was detected using the cell counting kit-8 reagent (Hamamoto et al., 2004; Molenaar et al., 2012). Anti-DHAV-1 Assay In vitro Cell Viability 100 μL DHAV-1 was added into a 96-well plate containing the DEHs monolayer except the cell control wells which were added with 100 μL MM. Then the plate was incubated in a humid atmosphere of 5% CO2 at 37°C for 2 hours. After that, the plate was washed thrice with D-Hank's, 100 μL BA at 31.25 μg/mL was added into the BA treating wells; 100 μL BAPC diluted twofold serially from 1000 μg/mL to 3.906 μg/mL were added into the BAPC treating wells; 100 μL MM was added into the virus control and cell control wells. Afterwards, the plate was incubated in a humid atmosphere of 5% CO2 at 37°C. After 72 hours, the absorbance at 450 nm wavelength of each well was detected using the cell counting kit-8 reagent (Hamamoto et al., 2004; Molenaar et al., 2012). DHAV-1 Titer 400 μL DHAV-1 was added into a 24-well plate containing the DEHs monolayer except the cell control wells which were added with 400 μL MM. Then the plate was incubated in a humid atmosphere of 5% CO2 at 37°C for 2 hours. After that, the plate was washed thrice with D-Hank's, 400 μL BA at 31.25 μg/mL was added into the BA treating wells; 400 μL BAPC at 31.25 μg/mL was added into the BAPC treating wells; 400 μL MM was added into the virus control and cell control wells. Afterwards, the plate was incubated in a humid atmosphere of 5% CO2 at 37°C. After 36 hours, the relative DHAV-1 titer was detected by reverse transcription quantitative PCR (RT-qPCR). In vivo 180 three-day-old cherry valley ducklings (Purchased from Tangquan Poultry Farm, Jiangsu province, China) were randomly divided into four groups: blank control group (isolation reared), virus control group, BA treating group, and BAPC treating group. Each Duckling in virus control group, BA treating group, and BAPC treating group was intramuscular injected 0.2 mL DHAV-1; which in blank control group was intramuscular injected 0.2 mL normal saline. One hour later, ducklings in BA and BAPC treating groups were respectively treated by aqueous BA and BAPC solution, both at the dosage of 2 mg per duckling and once a day for five days. While ducklings in blank control and virus control groups were treated by distilled water. Blood samples in each group were taken from five ducklings at the 4th, the 8th, and the 54th h. The DHAV-1 titer at the 8th h was detected by RT-qPCR. The alanine transaminase (ALT) and aspartate aminotransferase (AST) levels at the 4th, the 8th, and the 54th h were detected by enzymatic colorimetry used automatic biochemistry analyzer (7180 Automatic Analyzer, HITACHI, Japan) in Nanjing Shihuang Institute of Animal Science and Technology. Anti-DHAV-1 Links of BAPC Adsorption The adsorption was detected by two sample-adding modes: post-adding drug and pre-adding drug modes (Chen et al., 2014). Briefly, in the post-adding drug mode, the virus control and BPAC treating wells of a 24-well cell culture plate containing a DEHs monolayer were incubated with 400 μL DHAV-1 at 37°C in a humid atmosphere of 5% CO2 for 1 hour. Then, the plate was washed three times with D-Hank's and 400 μL MM (virus control wells) or 400 μL BAPC (BAPC-treated wells) was added, three wells per treating. The plates were then incubated at 4°C for 4 hours. After that, the DHAV-1 was detected by RT-qPCR. In the pre-adding drug mode, 400 μL MM and BAPC were respectively added into the virus control and BPAC treating wells in a 24-well cell culture plate containing a DEHs monolayer at 4°C for 4 hours, three wells per treating. Then, the plate was washed three times with D-Hank's and 400 μL DHAV-1 was added to the virus control and BPAC treating wells. The plates were then incubated at 37°C in a humid atmosphere of 5% CO2 for 1 hour. Similarly, the DHAV-1 was detected by RT-qPCR. Cell controls were used in these two assays. Replication The virus control and BPAC treating wells of a 24-well cell culture plate containing a DEHs monolayer were treated with the addition of 400 μL DHAV-1. The plate was then incubated at 37°C in a humid atmosphere of 5% CO2 for 2 hours to allow adsorption and penetration (Song et al., 2013). After that, the plate was washed three times using D-Hank's and 400 μL MM or BAPC were respectively added to the virus control wells or the BAPC treating wells, three wells per treating. No treatment was added to the cell control wells. To avoid the influence of virus release and immediate re-adsorption, the DHAV-1 was detected by RT-qPCR after the plate was incubated at 37°C in a humid atmosphere of 5% CO2 for 15 hours (Yao et al., 2016). Release The 24-well cell culture plate was treated with 400 μL DHAV-1, except for the cell control wells. Afterwards, the plate was incubated at 37°C in a humid atmosphere of 5% CO2 for 32 hours to allow adsorption, penetration, replication and release (Yao et al., 2016). The plates were then washed three times with D-Hank's and 400 μL MM was added to the virus control and cell control wells, and 400 μL BPAC was added to the BPAC-treated wells. Then, the plate was incubated at 37°C in a humid atmosphere of 5% CO2 for 2 hour. The cell supernatant was centrifuged and the sediment was removed. The RT-qPCR was used to detect virus release. Anti-Oxidative Assay The anti-oxidative effect of BAPC in treating ducklings which were infected with DHAV-1 was tested. In order to detect whether the anti-oxidative effect of BAPC was vital in the treatment, a pro-oxidant, hinokitiol, was used to block the anti-oxidative effect of BAPC. 360 one-day-old cherry valley ducklings (Purchased from Tangquan Poultry Farm, Jiangsu province, China) were randomly divided into six groups: blank control, virus control, BAPC-treated, hinokitiol control, DHAV-1 and hinokitiol control, BAPC and hinokitiol-treated. From day 1 to day 3, ducklings in the former three groups were intramuscular injected with solvent (normal saline containing 0.3% Tween-80, 0.5% glycerinum and 0.1% dimethyl sulfoxide), ducklings in the latter three groups were intramuscular injected with hinokitiol at the dosage 80 mg/kg. At the day 4, ducklings except in the blank control and hinokitiol control groups (injected with normal saline) were injected with DHAV-1 (0.2 mL per duckling). Then ducklings in the BAPC-treated and BAPC and hinokitiol -treated groups were treated by aqueous BAPC solution at the dosage of 2 mg per duckling, once a day for five days. Blood and liver samples in each group were taken from five ducklings at the 8th, and the 54th h. The GSH-Px, SOD, CAT, and MDA levels were tested by the GSH-Px, SOD, CAT, and MDA assay kits, respectively. Immuno-Enhancing Assay The immuno-enhancing effect of BAPC in treating ducklings which were infected with DHAV-1 was tested. In order to detect whether the immuno-enhancing effect of BAPC was vital in the treatment, an immunosuppressant, FK506, was used to block the immuno-enhancing effect of BAPC. 360 one-day-old cherry valley ducklings (Purchased from Tangquan Poultry Farm, Jiangsu province, China) were randomly divided into six groups: blank control, virus control, BAPC-treated, FK506 control, DHAV-1 and FK506 control, BAPC and FK506-treated. From day 1 to day 3, ducklings in the former three groups were intramuscular injected with solvent (normal saline containing 0.3% Tween-80, 0.5% glycerinum and 0.1% dimethyl sulfoxide), ducklings in the latter three groups were intramuscular injected with FK506 at the dosage 5 mg/kg. At the day 4, ducklings except in the blank control and FK506 control groups (injected with normal saline) were injected with DHAV-1 (0.2 mL per duckling). Then ducklings in the BAPC-treated and BAPC and FK506 -treated groups were treated by aqueous BAPC solution at the dosage of 2 mg per duckling, once a day for five days. Blood samples in each group were taken from five ducklings at the 4th, the 8th, and, the 54th h. The IL-2 and IFN-γ levels were tested by the IL-2 and IFN-γ ELISA kits, respectively. RT-qPCR When total RNA was extracted by RNAiso Plus Reagent, the reverse transcription was immediately performed using PrimeScriptTM RT Master Mix Kit. Then real-time PCR was operated according to SYBR® Premix Ex TaqTM (Tli RNaseH Plus) Kit. The forward primer 5΄-GCCACCCTTCCTGAGTTTGT-3΄ and the reverse primer 5΄-TACCATTCCACTTCTCCTGCTT-3΄ were used to determine the DHAV-1 gene (Chen et al., 2014). The forward primer 5΄-CTTTCTTGGGTATGGAGTCCTG-3΄ and the reverse primer 5΄-TGATTTTCATCGTGCTGGGT- 3΄ were used to determine the β-actin gene (Chen et al., 2014). Statistical Analysis The data were expressed as the mean ± S.D. 2−ΔΔCT (Kenneth and Thomas, 2001) method was used to analysis relative gene expression. Duncan's multiple range tests or t-tests were used to analyze the difference with the software SPSS 16.0. RESULTS Characterization of BAPC DSC analyses showed that melting points of both BA and soy phospholipids were observed in their physical mixture, but disappeared in BAPC (Figure 1A). What was more, the endothermic peaks in the physical mixture was the simple superposition of those in BA and soy phospholipids; however there was a new endothermic peak in BAPC. This presumably because of the reduced order between the hydrocarbon chains of phospholipids resulted from the highly dispersion of BA in soy phospholipids, after BA bounding to the polar ends of soy phospholipids. Figure 1. View largeDownload slide Characterization of BAPC. BA, soy phospholipids, the physical mixture, and BAPC were subjected to differential scanning calorimetry analyses (A), infrared spectra analyses (B), and X-ray diffraction analyses (C). Figure 1. View largeDownload slide Characterization of BAPC. BA, soy phospholipids, the physical mixture, and BAPC were subjected to differential scanning calorimetry analyses (A), infrared spectra analyses (B), and X-ray diffraction analyses (C). Additionally, infrared spectra analyses showed the absorption peaks in the physical mixture was the simple superposition of those in BA and soy phospholipids, but which of BAPC was extremely different (Figure 1B). In the physical mixture, the hydroxyl stretching vibration appeared at 3390 cm−1, however it appeared at 3350 cm−1 in BAPC. Moreover, the P-O bond stretching vibration in soy phospholipids was at 1238 cm−1 and it was observed in the physical mixture, but disappeared in BAPC. This suggested the hydroxyl in BA interacted with the P-O group in soy phospholipids forming BAPC by physical interactions such as the combination of hydrogen bonds or van der Waals force. In XRD assay, many sharp peaks were observed in both BA and the physical mixture, but soy phospholipids and BAPC existed in amorphous form (Figure 1C). It presumably because the high viscosity of soy phospholipids prevented the diffusion of BA molecules in the carrier and therefore inhibited the formation. Anti-DHAV-1 effect In vitro Figure 2 A showed that BAPC from 1000 μg/mL to 62.5 μg/mL exhibited cytotoxicity on DEHs and presented no toxicity when the concentration was lower than 31.25 μg/mL, it even promoted the cytoactive of DEHs at 31.25 μg/mL. Therefore the testing concentration of BAPC against DHAV-1 on DEHs was lower than 31.25 μg/mL. Obviously, the anti-DHAV-1 effect of BAPC was concentration dependent (Figure 2B). What was more, the A450 value of BAPC-treated at 31.25 μg/mL was significantly higher than that of BA-treated at 31.25 μg/mL. Additionally, the relative DHAV-1 titer in BAPC-treated cells was significantly lower than that in BA-treated (Figure 2C). It indicated the anti-DHAV-1 ability of BAPC was stronger than that of BA, which was also manifested by the light microscopy images of DEHs: cytopathic effect of BA-treated cells was alleviated compared with that of virus control but more serious than that of BAPC-treated (Figure 2D). Figure 2. View largeDownload slide Anti-DHAV-1 ability on DEHs of BAPC. (A) Cytotoxicity test of BAPC on DEHs was tested by CCK-8 method. (B) BA and BAPC at different concentration were respectively added to the cells which were infected by DHAV-1, and CCK-8 method was used to contrastively analyze the anti-DHAV-1 effects of BA and BAPC. (C) The influences of BA and BAPC on DHAV-1 reproduction were tested by RT-qPCR method. (D) Cytopathic effect of cell control, virus control, BA treating group, and BAPC treating group. The cytopathic alterations were marked with black arrows. *, compared with cell control, P < 0.05; **, compared with cell control, P < 0.01; ***, compared with cell control, P < 0.001; ##, compared with virus control, P < 0.01; ###, compared with virus control, P < 0.001; a, P < 0.01. Figure 2. View largeDownload slide Anti-DHAV-1 ability on DEHs of BAPC. (A) Cytotoxicity test of BAPC on DEHs was tested by CCK-8 method. (B) BA and BAPC at different concentration were respectively added to the cells which were infected by DHAV-1, and CCK-8 method was used to contrastively analyze the anti-DHAV-1 effects of BA and BAPC. (C) The influences of BA and BAPC on DHAV-1 reproduction were tested by RT-qPCR method. (D) Cytopathic effect of cell control, virus control, BA treating group, and BAPC treating group. The cytopathic alterations were marked with black arrows. *, compared with cell control, P < 0.05; **, compared with cell control, P < 0.01; ***, compared with cell control, P < 0.001; ##, compared with virus control, P < 0.01; ###, compared with virus control, P < 0.001; a, P < 0.01. In vivo Figure 3 A shows the survival curve of each group. Compared with virus control, death in BA-treated group and BAPC-treated group during this period was lower. The mortality rate of virus control was 93.3%. BA and BAPC decreased the mortality rates which of BA-treated group and BAPC-treated group were respectively 77.8% and 60.0%. Furthermore, BA and BAPC reduced the DHAV-1 titer in ducklings and apparently BAPC did better (Figure 3B). Figure 3. View largeDownload slide Anti-DHAV-1 ability on ducklings of BAPC. (A). The survival curve of blank control, virus control, BA treating group, and BAPC treating group. (B) DHAV-1 titer of each group was tested by RT-qPCR method. (C) ALT and AST levels of each group at the 4th h, the 8th h, and the 54th h. *, compared with blank control, P < 0.05; **, compared with blank control, P < 0.01; ***, compared with blank control, P < 0.001; #, compared with virus control, P < 0.05; ##, compared with virus control, P < 0.01; a, P < 0.001; b, P < 0.05. Figure 3. View largeDownload slide Anti-DHAV-1 ability on ducklings of BAPC. (A). The survival curve of blank control, virus control, BA treating group, and BAPC treating group. (B) DHAV-1 titer of each group was tested by RT-qPCR method. (C) ALT and AST levels of each group at the 4th h, the 8th h, and the 54th h. *, compared with blank control, P < 0.05; **, compared with blank control, P < 0.01; ***, compared with blank control, P < 0.001; #, compared with virus control, P < 0.05; ##, compared with virus control, P < 0.01; a, P < 0.001; b, P < 0.05. When ducklings were infected with DHAV-1, no matter in the earlier stage (4th and 8th h) or in the later stage (54th h), the ALT and AST levels were significantly increased (Figure 3C). After the treatment of BA and BAPC, the ALT and AST levels decreased to a certain extent (Figure 3C), and in general the ALT and AST levels in BAPC-treated ducklings were lower, as comparison with that of BA-treated. Anti-DHAV-1 Reproduction Links The influence of BAPC on DHAV-1 adsorption, replication, and release is showed in Figure 4. In the post-adding drug mode (Figure 4A) of the adsorption assay, there was no significant difference between the BAPC group and virus control; in the pre-adding drug mode (Figure 4B), the DHAV-1 level in BAPC group was significantly lower than that of virus control. It meant BAPC inhibited the virus adsorption on DEHs in the pre-treating way. In replication assay, the DHAV-1 level in BAPC group was lower than that of virus control with a significant difference (Figure 4C). This indicated BAPC inhibited DHAV-1 replication in DEHs. Moreover, BAPC significantly decreased DHAV-1 release (Figure 4D). Figure 4. View largeDownload slide Anti-DHAV-1 reproduction links of BAPC. (A). Influence of BAPC on DHAV-1 adsorption in the post-adding drug mode. (B) Influence of BAPC on DHAV-1 adsorption in the pre-adding drug mode. (C). Influence of BAPC on DHAV-1 replication. (D). Influence of BAPC on DHAV-1 release. *, compared with virus control, P < 0.05; **, compared with virus control, P < 0.01. Figure 4. View largeDownload slide Anti-DHAV-1 reproduction links of BAPC. (A). Influence of BAPC on DHAV-1 adsorption in the post-adding drug mode. (B) Influence of BAPC on DHAV-1 adsorption in the pre-adding drug mode. (C). Influence of BAPC on DHAV-1 replication. (D). Influence of BAPC on DHAV-1 release. *, compared with virus control, P < 0.05; **, compared with virus control, P < 0.01. Anti-Oxidative Effects The GSH-Px, SOD, CAT, and MDA levels of ducklings in each group are showed in Figure 5. Under the treatment of hinokitiol, GSH-Px, SOD, and CAT activities decreased significantly no matter in the serum or liver. And in the meantime, both in the serum or liver, the amounts of MDA increased significantly. This indicated the pro-oxidative effect of hinokitiol. Figure 5. View largeDownload slide Anti-oxidative effect of BAPC in the treatment of ducklings which were infected by DHAV-1. *, compared with blank control, P < 0.05; #, compared with virus control, P < 0.05. Figure 5. View largeDownload slide Anti-oxidative effect of BAPC in the treatment of ducklings which were infected by DHAV-1. *, compared with blank control, P < 0.05; #, compared with virus control, P < 0.05. At the 8th h, DHAV-1 did not affect the GSH-Px, SOD, CAT, and MDA levels in ducklings. But in the 54th h, DHAV-1 induced the decrease of GSH-Px, SOD, and CAT activities in addition to the increase of MDA amount. While after the treatment of BAPC, the GSH-Px, SOD, CAT, and MDA levels in ducklings returned to normalization. Obviously, such effect of BAPC was intervened by hinokitiol as the pro-oxidative effect (Figure 5). And yet for all that, the mortality rates of ducklings in BAPC-treated group and BAPC and hinokitiol-treated group were at the same level (Table 1). It indicated though hinokitiol intervened the anti-oxidative effect of BAPC, it did not affect the curative effect of BAPC on ducklings which infected by DHAV-1. Table 1. Mortality rate of each group in the anti-oxidative assay. DHAV-1  BAPC  Hinokitiol  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  95.6a  +  +  −  64.4b  +  +  +  62.2b  DHAV-1  BAPC  Hinokitiol  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  95.6a  +  +  −  64.4b  +  +  +  62.2b  a-cData within a column without the same superscripts differ significantly (P < 0.05). View Large Table 1. Mortality rate of each group in the anti-oxidative assay. DHAV-1  BAPC  Hinokitiol  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  95.6a  +  +  −  64.4b  +  +  +  62.2b  DHAV-1  BAPC  Hinokitiol  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  95.6a  +  +  −  64.4b  +  +  +  62.2b  a-cData within a column without the same superscripts differ significantly (P < 0.05). View Large Immuno-Enhancing Effects The IL-2 and IFN-γ levels of ducklings in each group are showed in Figure 6. Under the treatment of FK506, the IL-2 and IFN-γ levels decreased significantly compared with those non-FK506 treated. It manifested the immunosuppressive effect of FK506. Figure 6. View largeDownload slide Immunoregulation effect of BAPC in the treatment of ducklings which were infected by DHAV-1. *, compared with blank control, P < 0.05; #, compared with virus control, P < 0.05. Figure 6. View largeDownload slide Immunoregulation effect of BAPC in the treatment of ducklings which were infected by DHAV-1. *, compared with blank control, P < 0.05; #, compared with virus control, P < 0.05. When ducklings infected by DHAV-1, the IL-2 and IFN-γ levels increased at the 8th h, but did not last to the latter stage (54th h). After the treatment of BAPC, IL-2 and IFN-γ levels significantly increased at the 8th h and lasted to the latter stage. And the IL-2 and IFN-γ levels were very high. Obviously, the immuno-enhancing ability of BAPC was intervened by FK506 resulted from its immunosuppressive effect (Figure 6). At the same time, the mortality rate of ducklings in BAPC and FK506-treated group was significantly lower than that of BAPC-treated (Table 2). This meant FK506 intervened the immuno-enhancing effect of BAPC and affected the curative effect of BAPC. Table 2. Mortality rate of each group in the immuno-enhancing assay. DHAV-1  BAPC  FK506  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  97.8a  +  +  −  64.4b  +  +  +  93.3a  DHAV-1  BAPC  FK506  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  97.8a  +  +  −  64.4b  +  +  +  93.3a  a-cData within a column without the same superscripts differ significantly (P < 0.05). View Large Table 2. Mortality rate of each group in the immuno-enhancing assay. DHAV-1  BAPC  FK506  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  97.8a  +  +  −  64.4b  +  +  +  93.3a  DHAV-1  BAPC  FK506  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  97.8a  +  +  −  64.4b  +  +  +  93.3a  a-cData within a column without the same superscripts differ significantly (P < 0.05). View Large DISCUSSION It is well known that many compounds from natural sources have pharmacological activities. Artemisinin save millions of lives from malaria (Hien and White, 1993). Because of this, the Swedish Academy of Sciences awarded Youyou Tu, the discoverer of the artemisinin, Nobel Prize in physiology or medicine 2015. Arsenic is a kind of Chinese traditional medicine, possessing potent anti-tumor (Qian et al., 2013; Wang et al., 2017) and anti-angiogenesis (Gao et al., 2016) activities. Paclitaxel, isolated from the bark of western taxus brevifolia, possesses excellent antineoplastic ability, especially on ovarian cancer (Huang et al., 2016) and breast cancer (Zhang et al., 2016). Nowadays, it has been one antineoplastic agent in clinic. Therefore in recent years, many investigators’ attentions of drug development shifts to compounds from natural sources because of the strong biological activity (Chi et al., 2017; Xie et al., 2017; Dziewulska et al., 2018). Flavonoids are the important natural products and show various biological activity, such as antiviral (Moghaddam et al., 2014), anti-oxidative (Cao et al., 2011; Xu et al., 2016), anti-tumor (Konoshima et al., 1997; Jung et al., 2003), immuno-enhancing (Hu et al., 2012), and anti-inflammatory (Garcíalafuente et al., 2009). BA is a kind of flavonoids and the main ingredient of Chinese medicinal herb “huang qin” (Scutellaria baicalensis Georgi) which was used to treat chest distress, diarrhea, icterus, fetal irritability, and asthma. Like many other flavonoids from natural sources, it shows various pharmacological activities (Cao et al., 2011; Hu et al., 2012; Moghaddam et al., 2014; Nayak et al., 2014). Antiviral is one outstanding activity of BA. It exerts anti-influenza virus activity by disrupting NS1-p85b binding resulting in up-regulation of IFN-induced antiviral signaling and a decrease in PI3K/Akt signaling (Nayak et al., 2014). It is also reported that BA inhibited dengue virus replication with IC50 at 13.56 μg/mL, exhibited virucidal activity against dengue virus type 2 with IC50 at 8.74 μg/mL, and showed anti-adsorption effect against dengue virus type 2 with IC50 at 18.07 μg/mL (Moghaddam et al., 2014). However, BA is insoluble in water and fat, which makes it difficult to penetrate cell membrane and causes low bioavailability (Li et al., 2012). Complex with phospholipids can improve the solubility, physicochemical properties, and absorption of flavonoids (Xu et al., 2016). It is reported the water solubility of quercetin-PC is about thrice as much as that of quercetin, meanwhile the solubility in chloroform of quercetin-PC is about 734 times as much as that of quercetin (Xu et al., 2016). Similarly, the solubility of BAPC is much larger than that of BA. Complex with soy phospholipids can enhance the solubility of BA in water about 3.5 times, and about 70 times in n-octanol (Li et al., 2012). Here, we studied the structural characterization of BAPC (Figure 1). We presumed that BA is highly dispersed in soy phospholipids, and BAPC was formed by physical interactions between BA and phospholipids. Complex with phospholipids can also improve the pharmacological activity of the original compound (Guo et al., 2014; Xu et al., 2016). In present work, we found that the anti-DHAV-1 ability of BAPC was significantly higher than that of BA (Figures 2 and 3). Compared with BA, BAPC inhibited the DHAV-1 titer both in DEHs and ducklings more effectively. Additionally, BAPC decreased the ALT and AST levels more effectively in ducklings which were infected by DHAV-1. Meanwhile, the mortality rate of BAPC-treated ducklings was lower than that of BA-treated. These results indicated complex with phospholipid was a proper method to modify BA. It is well known that in the process of a virus infecting the host cell it first adsorbs on the cell surface and then enters the cell. After that, the virus starts the replication including the synthesis of protein and gene following by the virus assembly. In the end, large amount of progeny viruses release and infect new host cells. Many compounds in natural sources exhibit antiviral activity via the inhibition of virus adsorption, replication, or release. Song et al. (2013) reported sulfated Chuanminshen violaceum polysaccharide inhibited duck enteritis virus activity by preventing virus adsorption with IC50 from 82.83 μg/mL to 109.28 μg/mL, and it also prevented the cell-to-cell spread of duck enteritis virus. Moghaddam et al. (2014) reported BA inhibited dengue virus adsorption and replication. The adsorption of DHAV-1 saturates at 90 min post-infection, the replication lasted around 13 hours after the early protein synthesis for 5 hours, and the release of DHAV-1 was in steady after 32 hours (Yao et al., 2016). In our previous study, bush sophora root polysaccharide inhibited DHAV-1 replication and its sulfate inhibited DHAV-1 replication and release (Chen et al., 2014). In present work, we explored the influence of BAPC on DHAV-1 adsorption, replication, and release (Figure 4), and the results showed BAPC inhibited DHAV-1 adsorption, replication, and release. Therefore, the DHAV-1 reproduction was inhibited by BAPC (Figures 2C and 3B). The balance of free radicals is important to the health. However a lot of negative factors can stimulate cells to produce free radicals including RNA virus (Schwarz, 1996). It is reported that hepatitis virus can reduce the activities of GSH-Px, SOD, and CAT, at the same time promote MDA content (Boya et al., 1999; Demirdag et al., 2003; Sayal et al., 2013), which exacerbates the severity of hepatitis. Antioxidant therefore is also an important treatment method of hepatitis (Dikici et al., 2005; Stewart et al., 2007). DHAV-1 can decrease the activities of GSH-Px, SOD, and CAT in ducklings as well as increase the MDA amount in the later stage (Chen et al., 2015). The same result has been achieved in present work (Figure 5). BA, as a kind of flavonoids, possesses anti-oxidative ability. It is reported that BA can reduce the level of MDA and elevate the activities of SOD and GSH as well as GSH-Px in gerbils (Cao et al., 2011). Here, we found BAPC also promoted the GSH-Px, SOD, and CAT activities and dropped MDA levels in infected ducklings at the later stage. To detect whether the anti-oxidative effect of BAPC is vital to its curative effect on ducklings which were infected by DHAV-1, the hinokitiol was used at the same time. Hinokitiol with high dose is one pro-oxidant and can increase the GSH-Px, SOD, and CAT activities as well as decrease the MDA level in ducklings (Chen et al., 2015). Obviously, after the treatment of hinokitiol, the anti-oxidative effect of BAPC was neutralized. However the mortality rate of ducklings did not change. It indicated though BAPC played an anti-oxidative effect in the process of the treatment, it was not vital to the curative effect. The progress of viral disease is closely related to body's immune level (Pettersson et al., 2014). IL-2 and IFN-γ are two crucial immuno-regulative and cytokine network regulatory factors (Millán et al., 2014). Therefore, we also detected the IL-2 and IFN-γ levels in present work. The results showed BAPC promoted the IL-2 and IFN-γ secretion, which indicated the immuno-enhancing ability of BAPC. Similarly, in order to detect whether the immuno-enhancing effect of BAPC is vital to its curative effect, the FK506 was used. FK506 is an immunosuppressive drug which has been commonly used to prevent allograft rejection after transplantation discovered in the 1980s and exerts its therapeutic effects by suppression of T-cell activation (Siebelt et al., 2014). BAPC played immuno-enhancing effect in the process of the treatment (Figure 6). Apparently, FK506 blocked the immuno-enhancing effect of BAPC (Figure 6), which resulted in the increase of the mortality rate of ducklings (Table 2). It indicated when the immuno-enhancing effect was blocked, the curative effect of BAPC lost. Therefore, it manifested the immuno-enhancing effect of BAPC was vital to the curative effect. Based on these findings, it came to a conclusion that complex with soy phospholipids improved the anti-DHAV-1 ability of BA. BAPC showed direct anti-DHAV-1 ability via inhibiting DHAV-1 adsorption, replication, and release, meanwhile exhibited indirect anti-DHAV-1 ability via enhancing the immunity of ducklings. In present work, we found complex with soy phospholipids improved the antiviral effect of BA. Thus we suggested complex with phospholipid was a significative method in pharmaceutical sciences. CONCLUSION In summary, we successfully prepared BAPC by the interaction of BA and soy phospholipids in tetrahydrofuran. BAPC showed a better anti-DHAV-1 ability than BA. It showed inhibitive effect on DHAV-1 adsorption, replication, and release. In the treatment of ducklings that were infected by DHAV-1, BAPC exhibited anti-oxidative and immuno-enhancing effects. However, only the immuno-enhancing effect was crucial to the curative effect. ACKNOWLEDGMENTS The project was supported by Fundamental research funds for the central universities (Y0201700441), National Natural Science Foundation of China (Grant No. 31572557, 31772784), and the Special Fund for Agro-scientific Research in the Public Interest (201303040, 201403051), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). 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Antiviral effect of baicalin phospholipid complex against duck hepatitis A virus type 1

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

ABSTRACT Duck hepatitis A virus type 1 (DHAV-1) is one of the main pathogens of ducklings and causes a high mortality rate. Baicalin (BA) has potent antiviral effect, but the solubility is very poor. In order to increase the absorption, solubility, and pharmacological activity, the phospholipid complex was used to modify BA in present study. Therefore, BA phospholipid complex (BAPC) was prepared. The anti-DHAV-1 abilities of BA and BAPC in vitro was evaluated by cell counting kit-8 and reverse transcription quantitative PCR. The curative effects of BA and BAPC on ducklings which were infected by DHAV-1 in addition to the ALT and AST levels were also detected. The results indicated the anti-DHAV-1 ability of BAPC was stronger than that of BA both in vitro and in vivo. To explore the anti-DHAV-1 mechanism, the influence of BAPC on DHAV-1 adsorption, replication, and release was studied. Furthermore, the anti-oxidative and immuno-enhancing abilities of BAPC in the treatment of infected ducklings were also determined. The results showed BAPC inhibited DHAV-1 adsorption, replication and release. Furthermore, it played anti-oxidative and immno-enhancing roles in the treatment, and the immno-enhancing role was crucial to the treatment. INTRODUCTION Duck hepatitis A virus type 1 (DHAV-1) is a member of genus Avihepatovirus in the family Picornaviridae (Sheng et al., 2014). The pathogenicity of DHAV-1 is very strong and it mainly infects ducklings (Levine and Fabricant, 1950). When ducklings less than three weeks old infected by DHAV-1, the mortality rate is even up to 100%. And the lesions of the dead ducklings’ livers are always serious with swell and bleeding (Chen et al., 2014). The peroxidation damages of the infected ducklings are also very serious (Chen et al., 2015). However, no licensed drug is able to treat ducklings infected by DHAV-1 in clinic. In our previous study, many compounds in natural sources exhibited anti-DHAV-1 abilities, such as bush sophora root polysaccharide and its sulfate (Chen et al., 2014; Chen et al., 2015), icariin and its phosphate (Xiong et al., 2014), and Codonopsis pilosula polysaccharide and its phosphate (Ming et al., 2016). Baicalin (5,6-dihydroxy-7-O-glucuronide flavone, BA), a kind of flavonoids, is a main ingredient of Chinese medicinal herb huang qin (Scutellaria baicalensis Georgi). It possesses antiviral (Moghaddam et al., 2014), anti-oxidative (Cao et al., 2011), and immuno-enhancing ability (Hu et al., 2012). However, BA has poor water solubility and poor fat solubility (Li et al., 2012). Phospholipid complex can improve the physicochemical properties of the original compound, increase the absorption, and eliminate adverse reactions (Xu et al., 2016). It is reported that BA phospholipid complex (BAPC) possesses better solubility and nasal mucosa absorption than BA (Li et al., 2012). So in present study, the BAPC was prepared and its physicochemical properties were characterized. Furthermore, the anti-DHAV-1 ability of BAPC compared with BA was detected both in vitro and in vivo. At the same time, in order to explore the anti-DHAV-1 mechanisms of BAPC, the anti-DHAV-1 links (direct antiviral mechanism) in addition to the anti-oxidative and immuno-enchancing effects (indirect antiviral mechanism) were also explored. MATERIALS AND METHODS Reagents Dulbecco's modified eagle medium (Gibco, USA) supplemented with penicillin 100 IU/mL, streptomycin 100 IU/mL, glutamine 0.75 mg/mL, and 10% fetal bovine serum, was called growth medium (GM) and used for culturing the duck embryonic hepatocytes (DEHs). Dulbecco's modified eagle medium (Gibco, USA) supplemented with penicillin 100 IU/mL, streptomycin 100 IU/mL, glutamine 0.75 mg/mL, and 1% fetal bovine serum, was called maintenance medium (MM) and used for diluting drugs and maintaining cells. D-Hank's solution was used for washing the embryo tissue and cells. The pH of GM, MM and D-Hank's was adjusted to 7.4 using 5.6% NaHCO3. The trypsin (Amresco, USA) was dissolved into 0.20% with D-Hank's. The cell counting kit-8 reagent was brought from Dojindo, Japan. RNAiso Plus Reagent, PrimeScriptTM RT Master Mix Kit, and SYBR® Premix Ex TaqTM (Tli RNaseH Plus) Kit were bought from Takara, Japan. Hinokitiol used as the pro-oxidant which is also known as 4-isopropyltropolone was brought from Tokyo Chemical Industry CO., LTD., Japan. FK506 used as the immunosuppressant which is also known as tacrolimus was brought from Aladdin Industrial Corporation, China. The solvent used to dissolve hinokitiol and FK506 as well as treat ducklings was normal saline containing 0.3% Tween-80, 0.5% glycerinum and 0.1% dimethyl sulfoxide. The superoxide dismutase (SOD), malonaldehyde (MDA), catalase (CAT) and glutathione peroxidase (GSH-Px) assay kits were brought from Nanjing Jiancheng Bioengineering Institute, China. The IL-2 and IFN-γ ELISA kits for ducks were brought from Kamiya Biomedical Company, USA. DEHs and DHAV-1 DEHs were prepared as the method described previously (Chen et al., 2014). Briefly, liver tissues from a duck embryonic at 15 days were minced and then digested with 0.20% trypsin. Then the cells were washed thrice with D-Hank's, diluted into 0.8 × 106–1.2 × 106 cells/mL with GM, and then incubated in a humid atmosphere of 5% CO2 at 37°C. After 48 hours, the hepatocytes grew into a monolayer. DHAV-1 (LQ2 strain) used for in vitro and in vivo experiments were supplied by the Shandong Institute of Poultry in China. For in vitro experiments, it was generated by inoculating DEHs using regular procedure and diluted into 50 TCID50 in the experiments. For in vivo experiments, it was generated using the following procedure: three-day-old cherry valley ducklings were intramuscular injected with 0.2 mL DHAV-1, the liver tissue homogenates were obtained after three days. After centrifugation, the DHAV-1 for in vivo experiment was harvested and diluted into 20 LD50 in the experiments. BA and BAPC BA was purchased from Sigma Chemical Company (Sigma, USA). BAPC was prepared as the method described by Li et al. (2012). Firstly, 1.25 g BA and 2.5 g soy phospholipids were mixed together. Then 500 mL of tetrahydrofuran was added. And the mixture was refluxed in a thermostatic water bath at 55°C for 1 hour. Finally, BAPC was obtained after the vacuum rotary evaporation at 50°C. For in vitro assays, BA was diluted to 31.25 μg/mL with MM, BAPC was diluted twofold serially from 1000 μg/mL to 3.906 μg/mL. For in vivo assays, both BA and BAPC were diluted into 1 mg/mL with distilled water. Structural Characterization of BAPC Infrared Spectroscopy Analysis The FT-IR spectrum of BA and BAPC in a wavenumber range from 400 cm−1 to 4000 cm−1 was recorded by KBr pellets method (Qin et al., 2013) with a Nicolet 200 Magna-IR spectrometer (Nicolet Instrument Corp., USA). The major peaks (intensity and wavenumber) were found using OMNIC software (Nicolet Instruments Corp., USA). Differential Scanning Calorimetry (DSC) The DSC was performed in a differential scanning calorimeter (Mettler-Toledo, Switzerland). BA, soy phospholipids, physical mixture, and BAPC were loaded into aluminum pans separately and heated at the rate of 10°C/min from 50 to 300°C in nitrogen atmosphere. X-Ray Diffraction (XRD) The crystalline states of BA, soy phospholipids, physical mixture, and BAPC were evaluated with XRD diffractometer (Rigaku Denki, Japan). The detection conditions were as follows: copper target, temperature 25°C, voltage 40 kV, current 40 mA, scanning rate 3 °/min, and scanning range 5–50°C. Cytotoxicity Test 100 μL BAPC diluted twofold serially from 1000 μg/mL to 3.906 μg/mL were added into a 96-well plate containing the DEHs monolayer; 100 μL MM was added into the cell control wells; 5 wells per treating. Then the plate was incubated in a humid atmosphere of 5% CO2 at 37°C. After 72 hours, the absorbance at 450 nm wavelength of each well was detected using the cell counting kit-8 reagent (Hamamoto et al., 2004; Molenaar et al., 2012). Anti-DHAV-1 Assay In vitro Cell Viability 100 μL DHAV-1 was added into a 96-well plate containing the DEHs monolayer except the cell control wells which were added with 100 μL MM. Then the plate was incubated in a humid atmosphere of 5% CO2 at 37°C for 2 hours. After that, the plate was washed thrice with D-Hank's, 100 μL BA at 31.25 μg/mL was added into the BA treating wells; 100 μL BAPC diluted twofold serially from 1000 μg/mL to 3.906 μg/mL were added into the BAPC treating wells; 100 μL MM was added into the virus control and cell control wells. Afterwards, the plate was incubated in a humid atmosphere of 5% CO2 at 37°C. After 72 hours, the absorbance at 450 nm wavelength of each well was detected using the cell counting kit-8 reagent (Hamamoto et al., 2004; Molenaar et al., 2012). DHAV-1 Titer 400 μL DHAV-1 was added into a 24-well plate containing the DEHs monolayer except the cell control wells which were added with 400 μL MM. Then the plate was incubated in a humid atmosphere of 5% CO2 at 37°C for 2 hours. After that, the plate was washed thrice with D-Hank's, 400 μL BA at 31.25 μg/mL was added into the BA treating wells; 400 μL BAPC at 31.25 μg/mL was added into the BAPC treating wells; 400 μL MM was added into the virus control and cell control wells. Afterwards, the plate was incubated in a humid atmosphere of 5% CO2 at 37°C. After 36 hours, the relative DHAV-1 titer was detected by reverse transcription quantitative PCR (RT-qPCR). In vivo 180 three-day-old cherry valley ducklings (Purchased from Tangquan Poultry Farm, Jiangsu province, China) were randomly divided into four groups: blank control group (isolation reared), virus control group, BA treating group, and BAPC treating group. Each Duckling in virus control group, BA treating group, and BAPC treating group was intramuscular injected 0.2 mL DHAV-1; which in blank control group was intramuscular injected 0.2 mL normal saline. One hour later, ducklings in BA and BAPC treating groups were respectively treated by aqueous BA and BAPC solution, both at the dosage of 2 mg per duckling and once a day for five days. While ducklings in blank control and virus control groups were treated by distilled water. Blood samples in each group were taken from five ducklings at the 4th, the 8th, and the 54th h. The DHAV-1 titer at the 8th h was detected by RT-qPCR. The alanine transaminase (ALT) and aspartate aminotransferase (AST) levels at the 4th, the 8th, and the 54th h were detected by enzymatic colorimetry used automatic biochemistry analyzer (7180 Automatic Analyzer, HITACHI, Japan) in Nanjing Shihuang Institute of Animal Science and Technology. Anti-DHAV-1 Links of BAPC Adsorption The adsorption was detected by two sample-adding modes: post-adding drug and pre-adding drug modes (Chen et al., 2014). Briefly, in the post-adding drug mode, the virus control and BPAC treating wells of a 24-well cell culture plate containing a DEHs monolayer were incubated with 400 μL DHAV-1 at 37°C in a humid atmosphere of 5% CO2 for 1 hour. Then, the plate was washed three times with D-Hank's and 400 μL MM (virus control wells) or 400 μL BAPC (BAPC-treated wells) was added, three wells per treating. The plates were then incubated at 4°C for 4 hours. After that, the DHAV-1 was detected by RT-qPCR. In the pre-adding drug mode, 400 μL MM and BAPC were respectively added into the virus control and BPAC treating wells in a 24-well cell culture plate containing a DEHs monolayer at 4°C for 4 hours, three wells per treating. Then, the plate was washed three times with D-Hank's and 400 μL DHAV-1 was added to the virus control and BPAC treating wells. The plates were then incubated at 37°C in a humid atmosphere of 5% CO2 for 1 hour. Similarly, the DHAV-1 was detected by RT-qPCR. Cell controls were used in these two assays. Replication The virus control and BPAC treating wells of a 24-well cell culture plate containing a DEHs monolayer were treated with the addition of 400 μL DHAV-1. The plate was then incubated at 37°C in a humid atmosphere of 5% CO2 for 2 hours to allow adsorption and penetration (Song et al., 2013). After that, the plate was washed three times using D-Hank's and 400 μL MM or BAPC were respectively added to the virus control wells or the BAPC treating wells, three wells per treating. No treatment was added to the cell control wells. To avoid the influence of virus release and immediate re-adsorption, the DHAV-1 was detected by RT-qPCR after the plate was incubated at 37°C in a humid atmosphere of 5% CO2 for 15 hours (Yao et al., 2016). Release The 24-well cell culture plate was treated with 400 μL DHAV-1, except for the cell control wells. Afterwards, the plate was incubated at 37°C in a humid atmosphere of 5% CO2 for 32 hours to allow adsorption, penetration, replication and release (Yao et al., 2016). The plates were then washed three times with D-Hank's and 400 μL MM was added to the virus control and cell control wells, and 400 μL BPAC was added to the BPAC-treated wells. Then, the plate was incubated at 37°C in a humid atmosphere of 5% CO2 for 2 hour. The cell supernatant was centrifuged and the sediment was removed. The RT-qPCR was used to detect virus release. Anti-Oxidative Assay The anti-oxidative effect of BAPC in treating ducklings which were infected with DHAV-1 was tested. In order to detect whether the anti-oxidative effect of BAPC was vital in the treatment, a pro-oxidant, hinokitiol, was used to block the anti-oxidative effect of BAPC. 360 one-day-old cherry valley ducklings (Purchased from Tangquan Poultry Farm, Jiangsu province, China) were randomly divided into six groups: blank control, virus control, BAPC-treated, hinokitiol control, DHAV-1 and hinokitiol control, BAPC and hinokitiol-treated. From day 1 to day 3, ducklings in the former three groups were intramuscular injected with solvent (normal saline containing 0.3% Tween-80, 0.5% glycerinum and 0.1% dimethyl sulfoxide), ducklings in the latter three groups were intramuscular injected with hinokitiol at the dosage 80 mg/kg. At the day 4, ducklings except in the blank control and hinokitiol control groups (injected with normal saline) were injected with DHAV-1 (0.2 mL per duckling). Then ducklings in the BAPC-treated and BAPC and hinokitiol -treated groups were treated by aqueous BAPC solution at the dosage of 2 mg per duckling, once a day for five days. Blood and liver samples in each group were taken from five ducklings at the 8th, and the 54th h. The GSH-Px, SOD, CAT, and MDA levels were tested by the GSH-Px, SOD, CAT, and MDA assay kits, respectively. Immuno-Enhancing Assay The immuno-enhancing effect of BAPC in treating ducklings which were infected with DHAV-1 was tested. In order to detect whether the immuno-enhancing effect of BAPC was vital in the treatment, an immunosuppressant, FK506, was used to block the immuno-enhancing effect of BAPC. 360 one-day-old cherry valley ducklings (Purchased from Tangquan Poultry Farm, Jiangsu province, China) were randomly divided into six groups: blank control, virus control, BAPC-treated, FK506 control, DHAV-1 and FK506 control, BAPC and FK506-treated. From day 1 to day 3, ducklings in the former three groups were intramuscular injected with solvent (normal saline containing 0.3% Tween-80, 0.5% glycerinum and 0.1% dimethyl sulfoxide), ducklings in the latter three groups were intramuscular injected with FK506 at the dosage 5 mg/kg. At the day 4, ducklings except in the blank control and FK506 control groups (injected with normal saline) were injected with DHAV-1 (0.2 mL per duckling). Then ducklings in the BAPC-treated and BAPC and FK506 -treated groups were treated by aqueous BAPC solution at the dosage of 2 mg per duckling, once a day for five days. Blood samples in each group were taken from five ducklings at the 4th, the 8th, and, the 54th h. The IL-2 and IFN-γ levels were tested by the IL-2 and IFN-γ ELISA kits, respectively. RT-qPCR When total RNA was extracted by RNAiso Plus Reagent, the reverse transcription was immediately performed using PrimeScriptTM RT Master Mix Kit. Then real-time PCR was operated according to SYBR® Premix Ex TaqTM (Tli RNaseH Plus) Kit. The forward primer 5΄-GCCACCCTTCCTGAGTTTGT-3΄ and the reverse primer 5΄-TACCATTCCACTTCTCCTGCTT-3΄ were used to determine the DHAV-1 gene (Chen et al., 2014). The forward primer 5΄-CTTTCTTGGGTATGGAGTCCTG-3΄ and the reverse primer 5΄-TGATTTTCATCGTGCTGGGT- 3΄ were used to determine the β-actin gene (Chen et al., 2014). Statistical Analysis The data were expressed as the mean ± S.D. 2−ΔΔCT (Kenneth and Thomas, 2001) method was used to analysis relative gene expression. Duncan's multiple range tests or t-tests were used to analyze the difference with the software SPSS 16.0. RESULTS Characterization of BAPC DSC analyses showed that melting points of both BA and soy phospholipids were observed in their physical mixture, but disappeared in BAPC (Figure 1A). What was more, the endothermic peaks in the physical mixture was the simple superposition of those in BA and soy phospholipids; however there was a new endothermic peak in BAPC. This presumably because of the reduced order between the hydrocarbon chains of phospholipids resulted from the highly dispersion of BA in soy phospholipids, after BA bounding to the polar ends of soy phospholipids. Figure 1. View largeDownload slide Characterization of BAPC. BA, soy phospholipids, the physical mixture, and BAPC were subjected to differential scanning calorimetry analyses (A), infrared spectra analyses (B), and X-ray diffraction analyses (C). Figure 1. View largeDownload slide Characterization of BAPC. BA, soy phospholipids, the physical mixture, and BAPC were subjected to differential scanning calorimetry analyses (A), infrared spectra analyses (B), and X-ray diffraction analyses (C). Additionally, infrared spectra analyses showed the absorption peaks in the physical mixture was the simple superposition of those in BA and soy phospholipids, but which of BAPC was extremely different (Figure 1B). In the physical mixture, the hydroxyl stretching vibration appeared at 3390 cm−1, however it appeared at 3350 cm−1 in BAPC. Moreover, the P-O bond stretching vibration in soy phospholipids was at 1238 cm−1 and it was observed in the physical mixture, but disappeared in BAPC. This suggested the hydroxyl in BA interacted with the P-O group in soy phospholipids forming BAPC by physical interactions such as the combination of hydrogen bonds or van der Waals force. In XRD assay, many sharp peaks were observed in both BA and the physical mixture, but soy phospholipids and BAPC existed in amorphous form (Figure 1C). It presumably because the high viscosity of soy phospholipids prevented the diffusion of BA molecules in the carrier and therefore inhibited the formation. Anti-DHAV-1 effect In vitro Figure 2 A showed that BAPC from 1000 μg/mL to 62.5 μg/mL exhibited cytotoxicity on DEHs and presented no toxicity when the concentration was lower than 31.25 μg/mL, it even promoted the cytoactive of DEHs at 31.25 μg/mL. Therefore the testing concentration of BAPC against DHAV-1 on DEHs was lower than 31.25 μg/mL. Obviously, the anti-DHAV-1 effect of BAPC was concentration dependent (Figure 2B). What was more, the A450 value of BAPC-treated at 31.25 μg/mL was significantly higher than that of BA-treated at 31.25 μg/mL. Additionally, the relative DHAV-1 titer in BAPC-treated cells was significantly lower than that in BA-treated (Figure 2C). It indicated the anti-DHAV-1 ability of BAPC was stronger than that of BA, which was also manifested by the light microscopy images of DEHs: cytopathic effect of BA-treated cells was alleviated compared with that of virus control but more serious than that of BAPC-treated (Figure 2D). Figure 2. View largeDownload slide Anti-DHAV-1 ability on DEHs of BAPC. (A) Cytotoxicity test of BAPC on DEHs was tested by CCK-8 method. (B) BA and BAPC at different concentration were respectively added to the cells which were infected by DHAV-1, and CCK-8 method was used to contrastively analyze the anti-DHAV-1 effects of BA and BAPC. (C) The influences of BA and BAPC on DHAV-1 reproduction were tested by RT-qPCR method. (D) Cytopathic effect of cell control, virus control, BA treating group, and BAPC treating group. The cytopathic alterations were marked with black arrows. *, compared with cell control, P < 0.05; **, compared with cell control, P < 0.01; ***, compared with cell control, P < 0.001; ##, compared with virus control, P < 0.01; ###, compared with virus control, P < 0.001; a, P < 0.01. Figure 2. View largeDownload slide Anti-DHAV-1 ability on DEHs of BAPC. (A) Cytotoxicity test of BAPC on DEHs was tested by CCK-8 method. (B) BA and BAPC at different concentration were respectively added to the cells which were infected by DHAV-1, and CCK-8 method was used to contrastively analyze the anti-DHAV-1 effects of BA and BAPC. (C) The influences of BA and BAPC on DHAV-1 reproduction were tested by RT-qPCR method. (D) Cytopathic effect of cell control, virus control, BA treating group, and BAPC treating group. The cytopathic alterations were marked with black arrows. *, compared with cell control, P < 0.05; **, compared with cell control, P < 0.01; ***, compared with cell control, P < 0.001; ##, compared with virus control, P < 0.01; ###, compared with virus control, P < 0.001; a, P < 0.01. In vivo Figure 3 A shows the survival curve of each group. Compared with virus control, death in BA-treated group and BAPC-treated group during this period was lower. The mortality rate of virus control was 93.3%. BA and BAPC decreased the mortality rates which of BA-treated group and BAPC-treated group were respectively 77.8% and 60.0%. Furthermore, BA and BAPC reduced the DHAV-1 titer in ducklings and apparently BAPC did better (Figure 3B). Figure 3. View largeDownload slide Anti-DHAV-1 ability on ducklings of BAPC. (A). The survival curve of blank control, virus control, BA treating group, and BAPC treating group. (B) DHAV-1 titer of each group was tested by RT-qPCR method. (C) ALT and AST levels of each group at the 4th h, the 8th h, and the 54th h. *, compared with blank control, P < 0.05; **, compared with blank control, P < 0.01; ***, compared with blank control, P < 0.001; #, compared with virus control, P < 0.05; ##, compared with virus control, P < 0.01; a, P < 0.001; b, P < 0.05. Figure 3. View largeDownload slide Anti-DHAV-1 ability on ducklings of BAPC. (A). The survival curve of blank control, virus control, BA treating group, and BAPC treating group. (B) DHAV-1 titer of each group was tested by RT-qPCR method. (C) ALT and AST levels of each group at the 4th h, the 8th h, and the 54th h. *, compared with blank control, P < 0.05; **, compared with blank control, P < 0.01; ***, compared with blank control, P < 0.001; #, compared with virus control, P < 0.05; ##, compared with virus control, P < 0.01; a, P < 0.001; b, P < 0.05. When ducklings were infected with DHAV-1, no matter in the earlier stage (4th and 8th h) or in the later stage (54th h), the ALT and AST levels were significantly increased (Figure 3C). After the treatment of BA and BAPC, the ALT and AST levels decreased to a certain extent (Figure 3C), and in general the ALT and AST levels in BAPC-treated ducklings were lower, as comparison with that of BA-treated. Anti-DHAV-1 Reproduction Links The influence of BAPC on DHAV-1 adsorption, replication, and release is showed in Figure 4. In the post-adding drug mode (Figure 4A) of the adsorption assay, there was no significant difference between the BAPC group and virus control; in the pre-adding drug mode (Figure 4B), the DHAV-1 level in BAPC group was significantly lower than that of virus control. It meant BAPC inhibited the virus adsorption on DEHs in the pre-treating way. In replication assay, the DHAV-1 level in BAPC group was lower than that of virus control with a significant difference (Figure 4C). This indicated BAPC inhibited DHAV-1 replication in DEHs. Moreover, BAPC significantly decreased DHAV-1 release (Figure 4D). Figure 4. View largeDownload slide Anti-DHAV-1 reproduction links of BAPC. (A). Influence of BAPC on DHAV-1 adsorption in the post-adding drug mode. (B) Influence of BAPC on DHAV-1 adsorption in the pre-adding drug mode. (C). Influence of BAPC on DHAV-1 replication. (D). Influence of BAPC on DHAV-1 release. *, compared with virus control, P < 0.05; **, compared with virus control, P < 0.01. Figure 4. View largeDownload slide Anti-DHAV-1 reproduction links of BAPC. (A). Influence of BAPC on DHAV-1 adsorption in the post-adding drug mode. (B) Influence of BAPC on DHAV-1 adsorption in the pre-adding drug mode. (C). Influence of BAPC on DHAV-1 replication. (D). Influence of BAPC on DHAV-1 release. *, compared with virus control, P < 0.05; **, compared with virus control, P < 0.01. Anti-Oxidative Effects The GSH-Px, SOD, CAT, and MDA levels of ducklings in each group are showed in Figure 5. Under the treatment of hinokitiol, GSH-Px, SOD, and CAT activities decreased significantly no matter in the serum or liver. And in the meantime, both in the serum or liver, the amounts of MDA increased significantly. This indicated the pro-oxidative effect of hinokitiol. Figure 5. View largeDownload slide Anti-oxidative effect of BAPC in the treatment of ducklings which were infected by DHAV-1. *, compared with blank control, P < 0.05; #, compared with virus control, P < 0.05. Figure 5. View largeDownload slide Anti-oxidative effect of BAPC in the treatment of ducklings which were infected by DHAV-1. *, compared with blank control, P < 0.05; #, compared with virus control, P < 0.05. At the 8th h, DHAV-1 did not affect the GSH-Px, SOD, CAT, and MDA levels in ducklings. But in the 54th h, DHAV-1 induced the decrease of GSH-Px, SOD, and CAT activities in addition to the increase of MDA amount. While after the treatment of BAPC, the GSH-Px, SOD, CAT, and MDA levels in ducklings returned to normalization. Obviously, such effect of BAPC was intervened by hinokitiol as the pro-oxidative effect (Figure 5). And yet for all that, the mortality rates of ducklings in BAPC-treated group and BAPC and hinokitiol-treated group were at the same level (Table 1). It indicated though hinokitiol intervened the anti-oxidative effect of BAPC, it did not affect the curative effect of BAPC on ducklings which infected by DHAV-1. Table 1. Mortality rate of each group in the anti-oxidative assay. DHAV-1  BAPC  Hinokitiol  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  95.6a  +  +  −  64.4b  +  +  +  62.2b  DHAV-1  BAPC  Hinokitiol  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  95.6a  +  +  −  64.4b  +  +  +  62.2b  a-cData within a column without the same superscripts differ significantly (P < 0.05). View Large Table 1. Mortality rate of each group in the anti-oxidative assay. DHAV-1  BAPC  Hinokitiol  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  95.6a  +  +  −  64.4b  +  +  +  62.2b  DHAV-1  BAPC  Hinokitiol  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  95.6a  +  +  −  64.4b  +  +  +  62.2b  a-cData within a column without the same superscripts differ significantly (P < 0.05). View Large Immuno-Enhancing Effects The IL-2 and IFN-γ levels of ducklings in each group are showed in Figure 6. Under the treatment of FK506, the IL-2 and IFN-γ levels decreased significantly compared with those non-FK506 treated. It manifested the immunosuppressive effect of FK506. Figure 6. View largeDownload slide Immunoregulation effect of BAPC in the treatment of ducklings which were infected by DHAV-1. *, compared with blank control, P < 0.05; #, compared with virus control, P < 0.05. Figure 6. View largeDownload slide Immunoregulation effect of BAPC in the treatment of ducklings which were infected by DHAV-1. *, compared with blank control, P < 0.05; #, compared with virus control, P < 0.05. When ducklings infected by DHAV-1, the IL-2 and IFN-γ levels increased at the 8th h, but did not last to the latter stage (54th h). After the treatment of BAPC, IL-2 and IFN-γ levels significantly increased at the 8th h and lasted to the latter stage. And the IL-2 and IFN-γ levels were very high. Obviously, the immuno-enhancing ability of BAPC was intervened by FK506 resulted from its immunosuppressive effect (Figure 6). At the same time, the mortality rate of ducklings in BAPC and FK506-treated group was significantly lower than that of BAPC-treated (Table 2). This meant FK506 intervened the immuno-enhancing effect of BAPC and affected the curative effect of BAPC. Table 2. Mortality rate of each group in the immuno-enhancing assay. DHAV-1  BAPC  FK506  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  97.8a  +  +  −  64.4b  +  +  +  93.3a  DHAV-1  BAPC  FK506  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  97.8a  +  +  −  64.4b  +  +  +  93.3a  a-cData within a column without the same superscripts differ significantly (P < 0.05). View Large Table 2. Mortality rate of each group in the immuno-enhancing assay. DHAV-1  BAPC  FK506  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  97.8a  +  +  −  64.4b  +  +  +  93.3a  DHAV-1  BAPC  FK506  Mortality rate (%)  −  −  −  0.0c  −  −  +  0.0c  +  −  −  91.1a  +  −  +  97.8a  +  +  −  64.4b  +  +  +  93.3a  a-cData within a column without the same superscripts differ significantly (P < 0.05). View Large DISCUSSION It is well known that many compounds from natural sources have pharmacological activities. Artemisinin save millions of lives from malaria (Hien and White, 1993). Because of this, the Swedish Academy of Sciences awarded Youyou Tu, the discoverer of the artemisinin, Nobel Prize in physiology or medicine 2015. Arsenic is a kind of Chinese traditional medicine, possessing potent anti-tumor (Qian et al., 2013; Wang et al., 2017) and anti-angiogenesis (Gao et al., 2016) activities. Paclitaxel, isolated from the bark of western taxus brevifolia, possesses excellent antineoplastic ability, especially on ovarian cancer (Huang et al., 2016) and breast cancer (Zhang et al., 2016). Nowadays, it has been one antineoplastic agent in clinic. Therefore in recent years, many investigators’ attentions of drug development shifts to compounds from natural sources because of the strong biological activity (Chi et al., 2017; Xie et al., 2017; Dziewulska et al., 2018). Flavonoids are the important natural products and show various biological activity, such as antiviral (Moghaddam et al., 2014), anti-oxidative (Cao et al., 2011; Xu et al., 2016), anti-tumor (Konoshima et al., 1997; Jung et al., 2003), immuno-enhancing (Hu et al., 2012), and anti-inflammatory (Garcíalafuente et al., 2009). BA is a kind of flavonoids and the main ingredient of Chinese medicinal herb “huang qin” (Scutellaria baicalensis Georgi) which was used to treat chest distress, diarrhea, icterus, fetal irritability, and asthma. Like many other flavonoids from natural sources, it shows various pharmacological activities (Cao et al., 2011; Hu et al., 2012; Moghaddam et al., 2014; Nayak et al., 2014). Antiviral is one outstanding activity of BA. It exerts anti-influenza virus activity by disrupting NS1-p85b binding resulting in up-regulation of IFN-induced antiviral signaling and a decrease in PI3K/Akt signaling (Nayak et al., 2014). It is also reported that BA inhibited dengue virus replication with IC50 at 13.56 μg/mL, exhibited virucidal activity against dengue virus type 2 with IC50 at 8.74 μg/mL, and showed anti-adsorption effect against dengue virus type 2 with IC50 at 18.07 μg/mL (Moghaddam et al., 2014). However, BA is insoluble in water and fat, which makes it difficult to penetrate cell membrane and causes low bioavailability (Li et al., 2012). Complex with phospholipids can improve the solubility, physicochemical properties, and absorption of flavonoids (Xu et al., 2016). It is reported the water solubility of quercetin-PC is about thrice as much as that of quercetin, meanwhile the solubility in chloroform of quercetin-PC is about 734 times as much as that of quercetin (Xu et al., 2016). Similarly, the solubility of BAPC is much larger than that of BA. Complex with soy phospholipids can enhance the solubility of BA in water about 3.5 times, and about 70 times in n-octanol (Li et al., 2012). Here, we studied the structural characterization of BAPC (Figure 1). We presumed that BA is highly dispersed in soy phospholipids, and BAPC was formed by physical interactions between BA and phospholipids. Complex with phospholipids can also improve the pharmacological activity of the original compound (Guo et al., 2014; Xu et al., 2016). In present work, we found that the anti-DHAV-1 ability of BAPC was significantly higher than that of BA (Figures 2 and 3). Compared with BA, BAPC inhibited the DHAV-1 titer both in DEHs and ducklings more effectively. Additionally, BAPC decreased the ALT and AST levels more effectively in ducklings which were infected by DHAV-1. Meanwhile, the mortality rate of BAPC-treated ducklings was lower than that of BA-treated. These results indicated complex with phospholipid was a proper method to modify BA. It is well known that in the process of a virus infecting the host cell it first adsorbs on the cell surface and then enters the cell. After that, the virus starts the replication including the synthesis of protein and gene following by the virus assembly. In the end, large amount of progeny viruses release and infect new host cells. Many compounds in natural sources exhibit antiviral activity via the inhibition of virus adsorption, replication, or release. Song et al. (2013) reported sulfated Chuanminshen violaceum polysaccharide inhibited duck enteritis virus activity by preventing virus adsorption with IC50 from 82.83 μg/mL to 109.28 μg/mL, and it also prevented the cell-to-cell spread of duck enteritis virus. Moghaddam et al. (2014) reported BA inhibited dengue virus adsorption and replication. The adsorption of DHAV-1 saturates at 90 min post-infection, the replication lasted around 13 hours after the early protein synthesis for 5 hours, and the release of DHAV-1 was in steady after 32 hours (Yao et al., 2016). In our previous study, bush sophora root polysaccharide inhibited DHAV-1 replication and its sulfate inhibited DHAV-1 replication and release (Chen et al., 2014). In present work, we explored the influence of BAPC on DHAV-1 adsorption, replication, and release (Figure 4), and the results showed BAPC inhibited DHAV-1 adsorption, replication, and release. Therefore, the DHAV-1 reproduction was inhibited by BAPC (Figures 2C and 3B). The balance of free radicals is important to the health. However a lot of negative factors can stimulate cells to produce free radicals including RNA virus (Schwarz, 1996). It is reported that hepatitis virus can reduce the activities of GSH-Px, SOD, and CAT, at the same time promote MDA content (Boya et al., 1999; Demirdag et al., 2003; Sayal et al., 2013), which exacerbates the severity of hepatitis. Antioxidant therefore is also an important treatment method of hepatitis (Dikici et al., 2005; Stewart et al., 2007). DHAV-1 can decrease the activities of GSH-Px, SOD, and CAT in ducklings as well as increase the MDA amount in the later stage (Chen et al., 2015). The same result has been achieved in present work (Figure 5). BA, as a kind of flavonoids, possesses anti-oxidative ability. It is reported that BA can reduce the level of MDA and elevate the activities of SOD and GSH as well as GSH-Px in gerbils (Cao et al., 2011). Here, we found BAPC also promoted the GSH-Px, SOD, and CAT activities and dropped MDA levels in infected ducklings at the later stage. To detect whether the anti-oxidative effect of BAPC is vital to its curative effect on ducklings which were infected by DHAV-1, the hinokitiol was used at the same time. Hinokitiol with high dose is one pro-oxidant and can increase the GSH-Px, SOD, and CAT activities as well as decrease the MDA level in ducklings (Chen et al., 2015). Obviously, after the treatment of hinokitiol, the anti-oxidative effect of BAPC was neutralized. However the mortality rate of ducklings did not change. It indicated though BAPC played an anti-oxidative effect in the process of the treatment, it was not vital to the curative effect. The progress of viral disease is closely related to body's immune level (Pettersson et al., 2014). IL-2 and IFN-γ are two crucial immuno-regulative and cytokine network regulatory factors (Millán et al., 2014). Therefore, we also detected the IL-2 and IFN-γ levels in present work. The results showed BAPC promoted the IL-2 and IFN-γ secretion, which indicated the immuno-enhancing ability of BAPC. Similarly, in order to detect whether the immuno-enhancing effect of BAPC is vital to its curative effect, the FK506 was used. FK506 is an immunosuppressive drug which has been commonly used to prevent allograft rejection after transplantation discovered in the 1980s and exerts its therapeutic effects by suppression of T-cell activation (Siebelt et al., 2014). BAPC played immuno-enhancing effect in the process of the treatment (Figure 6). Apparently, FK506 blocked the immuno-enhancing effect of BAPC (Figure 6), which resulted in the increase of the mortality rate of ducklings (Table 2). It indicated when the immuno-enhancing effect was blocked, the curative effect of BAPC lost. Therefore, it manifested the immuno-enhancing effect of BAPC was vital to the curative effect. Based on these findings, it came to a conclusion that complex with soy phospholipids improved the anti-DHAV-1 ability of BA. BAPC showed direct anti-DHAV-1 ability via inhibiting DHAV-1 adsorption, replication, and release, meanwhile exhibited indirect anti-DHAV-1 ability via enhancing the immunity of ducklings. In present work, we found complex with soy phospholipids improved the antiviral effect of BA. Thus we suggested complex with phospholipid was a significative method in pharmaceutical sciences. CONCLUSION In summary, we successfully prepared BAPC by the interaction of BA and soy phospholipids in tetrahydrofuran. BAPC showed a better anti-DHAV-1 ability than BA. It showed inhibitive effect on DHAV-1 adsorption, replication, and release. In the treatment of ducklings that were infected by DHAV-1, BAPC exhibited anti-oxidative and immuno-enhancing effects. However, only the immuno-enhancing effect was crucial to the curative effect. ACKNOWLEDGMENTS The project was supported by Fundamental research funds for the central universities (Y0201700441), National Natural Science Foundation of China (Grant No. 31572557, 31772784), and the Special Fund for Agro-scientific Research in the Public Interest (201303040, 201403051), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). 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Poultry ScienceOxford University Press

Published: May 11, 2018

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