Zhuoyu WANG, Ming CHEN, Bingkun YANG, Changyou LIU, Chanjuan CHEN, Lihe FAN, Yongjun WANG*

1. College of Animal Science & Technology, Jilin Agricultural University, Changchun 130118, China; 2. Dehui Aquatic Technology Promotion Center, Dehui 130300, China; 3. Jilin Academy of Fishery Sciences, Changchun 130033, China

Abstract [Objectives] To study the toxic effect and antiviral activity of anthraquinone extract of Polygonum cuspidatum on infection of Koi herpes virus (KHV). [Methods] The MTT method and CPE microscopy were used to detect the common carp brain (CCB) cytotoxicity of the P. cuspidatum anthraquinone extract in 48 h. Eight groups of different concentrations of the P. cuspidatum anthraquinone extract 1.96, 3.91, 7.28, 15.63, 31.25, 62.5, 125, 250 μg/mL experimental groups and a control group without drug effect were set up. After determining the maximum non-toxic range of the P. cuspidatum anthraquinone extract, the viral replication inhibition test was carried out. [Results] The concentration of the P. cuspidatum anthraquinone extract 31.25 μg/mL was recognized as the maximum non-toxic concentration. The survival rate of CCB cells was higher than 80%, and the toxic dose (CC50) of the drug for 50% cell death was (72.67±2.12) μg/mL. The maximum inhibition rate of the P. cuspidatum anthraquinone extract was 78.63%±5.47% at a concentration of 31.25 μg/mL, and the 50% effective drug dose (IC50) for inhibiting the virus was (13.67±0.47) μg/mL, and the therapeutic index (TI) was 5.48±0.49. In the direct virus killing test, the highest virus inhibition rate was 32.21%. [Conclusions] Under the experimental conditions, it can be concluded that the P. cuspidatum anthraquinone extract has high anti-KHV activity, and at the same time. It is expected to lay a theoretical foundation for the research of P. cuspidatum anthraquinone extract against KHV.

Key words Polygonum cuspidatum, Koi herpes virus (KHV), Antiviral effect

1 Introduction

Cyprinuscarpiois one of the freshwater fishes with a long history of breeding, has many varieties, wide distribution[1], high yield[2], and high economic value[3]. Since the late 1990s, a highly infectious, pathogenic and lethal Koi herpes virus disease (KHVD) started to spread among carps, causing huge losses toCyprinuscarpiobreeding industry.

Polygonumcuspidatum, as a traditional Chinese herbal medicine, widely distributed in China and has huge resources and is easily available. In addition, extensive studies have shown that theP.cuspidatumanthraquinone extract has good antiviral activity[4-6]. At present, there is no effective treatment for KHV. In this study, we explored the maximum safe concentration of theP.cuspidatumanthraquinone extract and the effective and safe dose against KHV throughinvitroexperiments. This study is intended to provide certain data support for the development of new anti-KHV drugs, and provide new ideas for drug prevention and treatment of KHVD.

2 Materials and methods

2.1 Main materials and reagentsCommon carp brain (CCB) cells (donated by Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences); Koi herpes virus Jilin strain (KHV-CJ) separated and identified by our research team.Polygonumcuspidatumanthraquinone extract (donated by Jilin Provincial Pharmaceutical Research Institute, anthraquinone purity >90%). Dimethyl sulfoxide (DMSO), MTT and ribavirin (C8H12N4O5>98%) (Sigma, USA); L-15 Leibovitz culture medium, fetal bovine serum (Gibco, USA); Trypsin (Hyclone, USA); MCA-15A CO2cell incubator (SANYO, Japan); IX51 inverted microscope (Olympus, Japan); 680 microplate reader (BIO-RAD, Spain); PCR Amplifier (Eppendorf AG, Spain); Gene gel imaging analysis system (Syngene, USA).

2.2 Preparation ofP.cuspidatumanthraquinone extract

Weighed 2.0 g of theP.cuspidatumextract powder and dissolved it in 20 mL of DMSO to prepare a mother liquor of 20 mg/mL. Stirred thoroughly until the drug was completely dissolved, used a 0.22 μm microporous filter to filter and sterilize, stored at room temperature, and prepared the solution just before the experiment. At the same time, the positive control group used the broad-spectrum antiviral drug ribavirin, the preparation method was the same as above, and it was stored at 4 ℃.

2.3 CCB cell cultureResuscitated the cryopreserved CCB cells with L-15 cell culture medium containing 20% FCS. When the CCB cells grew to 80% of the bottom of the cell flask, digested the cells with 1 mL of cell digestion solution. Then, added the complete culture medium containing serum to terminate the digestion, passed the culture according to the volume ratio of the cell suspension to the culture medium 1∶2, and placed it in a 25 ℃, 5% CO2cell incubator.

2.4 Toxicity test ofP.cuspidatumanthraquinone extract on CCB cellsPrepared 10 mL of cell suspension with a concentration of 2×105cells/mL, and inoculated in a 96-well plate with 100 μL/well in a constant temperature cell incubator at 25 ℃ and 5% CO2for 24 h, with gradient concentrations of 250, 125, 62.5, 31.25 , 15.63, 7.28, 3.91 and 1.96 μg/mL theP.cuspidatumanthraquinone extract culture solution. Discarded the cell culture medium, washed the cell surface with PBS one time, and added the prepared series of gradient concentration theP.cuspidatumextract culture medium, repeated 5 wells for each dilution concentration, and set a negative control group (added 100 μL cell maintenance solution) and a blank control group (no cells, 100 μL cell maintenance solution). After incubating for 48 h at 25 ℃ and 5% CO2, took out the 96-well cell culture plate, added 10 μL of MTT (5 mg/mL) to each well, continued incubating for 4 h, discarded all the liquid in the well, and added 100 μL/well of DMSO, shook at low speed for 10 min to dissolve the crystals. Used the microplate reader to detected the absorbance at 570 nm, Cell survival rate (%)=(AverageODof experimental wells/AverageODof control group)×100%, and calculated the toxic dose of the drug for 50% cell death (CC50).

2.5 Inhibitory effect ofP.cuspidatumanthraquinone extract on KHV-CJThe experimental groups were divided into the group with the same time of addition of KHV-CJ and theP.cuspidatumanthraquinone extract (hereinafter referred to as Group A) and the group ofP.cuspidatumanthraquinone extract drug after KHV-CJ adsorption (hereinafter referred to as Group B).

In group A, when the CCB cells in the 96-well plate were filled with 75%-85% of the bottom, added 100 μL of 100 TCID50/mL KHV solution, and added the same volume ratio (1.96-31.25 μg/mL) of theP.cuspidatumextract drug solution (5 replicates per group), with a pure virus control group (100 μL+100 μL L-15 cell culture medium) and a blank control group (200 μL L-15), adsorbed in a cell incubator at 25 ℃ and 5% CO2for 2 h, then removed all the liquid in the culture plate, washed once with PBS, added new cell maintenance solution, and continued culturing in a cell incubator at 25 ℃ and 5% CO2. The CPE was observed daily with an inverted microscope and photographed and recorded. Until the typical CPE of the virus control group appeared, added 10 μL of MTT (5 mg/mL) to each well and incubated for 4 h, discarded the liquid, added 100 μL of DMSO, shook at low speed for 10 min, measured theODvalue of each well by the above-mentioned MTT method, recorded the data. Calculated the inhibition rate of KHV with different concentrations of drug solution: Inhibition rate (%)=(AverageODof experimental group drug-AverageODof virus control group)/(AverageODof normal cell control group-AverageODof virus control group)×100%.

In group B, when the CCB cells in the 96-well plate were filled with 75%-85% of the bottom, washed with PBS two times, and added 100 μL of 100 TCID50/mL KHV solution, and incubated in the CO2cell incubator for 2 h, and set the blank control group and cell control group. Discarded the virus solution, and added the prepared series of gradient concentration theP.cuspidatumextract culture medium, and incubated at a constant temperature of 25 ℃ and 5% CO2. The CPE was observed daily with an inverted microscope and photographed and recorded. Used MTT method to determine theODvalue of each well, recorded the data, calculated the 50% effective drug dose (IC50) for inhibiting the virus, and evaluate the therapeutic index (TI)=CC50/IC50.

Fig.1 Toxicity of different concentrations of Polygonum cuspidatum extract on CCB cells (A1-8) (×100)

3 Results and analysis

3.1 Toxic effect ofP.cuspidatumanthraquinone extract on CCB cellsThe higher the concentration of theP.cuspidatumanthraquinone extract, the more serious the toxic effect on CCB cells. The cell viability decreased with the increase in drug concentration, which is manifested by changes in cell morphology, condensation and rounding, falling off from the bottom, and even some cells are broken and lysed, and free in the cell culture supernatant. When the concentration of theP.cuspidatumanthraquinone extract is ≤ 31.25 μg/mL, it basically does not cause significant changes in the morphology or cell density of CCB cells, so it is recognized as the maximum non-toxic concentration in the cytotoxicity test.

The CCB cells were recovered and tested by the MTT method to detect the survival rate. It was found that theP.cuspidatumanthraquinone extract had certain cytotoxicity, and the cytotoxicity increased with the increase of the drug concentration of theP.cuspidatumanthraquinone extract. TheCC50of the drug concentration ofP.cuspidatumanthraquinone extract in CCB cells was (72.67±2.12) μg/mL. Besides, the cytotoxicity of theP.cuspidatumanthraquinone extract was basically zero in the low concentration range, and there was a certain situation that promotes the proliferation of CCB cells (Fig.2).

Fig.2 Effect of Polygonum Cuspidatum anthraquinone extract on viability of CCB cells

3.2 Inhibition of KHV virus replication by different concentrations ofP.cuspidatumanthraquinone extractAfter pre-infecting CCB cells with 100TCID50KHV for 2 h, removed the virus solution and washed twice with PBS, and then added the multiple-diluted (1.96-31.25 μg/mL) theP.cuspidatumextract drug solution for co-incubation. Used MTT method to measure the antiviral activity of each dilution. As shown in Fig.3, the drug concentration of theP.cuspidatumanthraquinone extract inhibited KHV replication in a dose-dependent manner. As the concentration ofP.cuspidatumanthraquinone extract increased, the ability to inhibit KHV replication was stronger, and the maximum inhibition rate was 78.63%±5.47% when the concentration was 31.25 μg/mL. TheP.cuspidatumanthraquinone extractIC50=(13.67±0.47) μg/mL，TI(Pc)=[(72.67±2.12) μg/mL]/[(13.67±0.47 μg/mL)]=5.48±0.49. The therapeutic index of theP.cuspidatumanthraquinone extract shows that theP.cuspidatumanthraquinone extract has certain efficacy against KHV.

Fig.3 Antiviral effect of Polygonum cuspidatum anthraquinone extract on KHV in CCB cells

3.3 Direct killing effect of different concentrations ofP.cuspidatumanthraquinone extract drugs on KHVIn order to test whether theP.cuspidatumanthraquinone extract has the ability to directly kill KHV particles, different concentrations of theP.cuspidatumanthraquinone extract were incubated with KHV before infecting CCB cells. The results of MTT method showed that various concentrations of theP.cuspidatumanthraquinone had a certain ability to directly kill KHV particles, and theP.cuspidatumconcentration was 31.25, 15.63, 7.28, 3.91, 1.96, and 0 μg/mL, respectively; the cell survival rate was 32.21%±1.23%, 25.31%±2.61%, 12.43%±1.03%, 10.11%±2.58%, 6.21%±1.54%, and 2.67%±1.79%, respectively; when the concentration was 31.25 μg/mL, it had the highest inhibition rate of KHV of 32.21%.

3.4 Pretreatment of different concentrations ofP.cuspidatumanthraquinone extract drugs before KHV infectionIn order to explore whether CCB cells incubated with pre-administration can prevent KHV adsorption and make CCB cells have the ability to inhibit KHV adsorption, CPE microscopy revealed that the drug pre-incubation group was not significantly different from the negative KHV control group, and there was no significant difference between theP.cuspidatumanthraquinone extract with different drug concentration gradients. No obvious anti-CCB virus adsorption effect was observed, and the time of CPE production in the pretreatment group of theP.cuspidatumanthraquinone extract drug was similar to the time of CPE production in the negative KHV control group, and there was no phenomenon that delayed the production of CPE by CCB. Fig.4 shows the recorded CCB cell pathological time chart and the negative control CCB cell pathological chart taking the maximum safe concentration of theP.cuspidatumanthraquinone extract drug at 31.25 μg/mL as an example.

Note: A: Negative control group was infected with KHV CCB cells (A1: 1 d, A2: 3 d, A3: 6 d, A4: 9 d, A5: 11 d) (×100); B: Test group 31.25 μg/mL the P. cuspidatum anthraquinone extract CCB cells (B1: 1 d, B2: 3 d, B3: 6 d, B4: 9 d, B5: 11 d) (×100).

4 Discussion

In the current aquaculture industry, viral diseases have become the biggest problem hindering the sustainable and healthy development of industry benefits and efficiency[7]. The breeding of Cyprinidae fishes occupies a large proportion in the aquaculture industry. Among them, carp is an extremely important species of edible fish in China, and is known as one of the four famous domestic fishes. At the same time, C. carpio has extremely high ornamental value and economic value in the global ornamental fishes. With the deterioration of the breeding environment, various infectious diseases, especially KHVD, have spread[8]. At present, Cyprinidae fishes can be infected by KHV throughout their life, and they are extremely infectious and have a high mortality rate, which has become one of the most important factors restricting the sustainable development of carp aquaculture.

By now, there is no effective therapeutic agent for KHVD, and related vaccines have certain problems, and they cannot be put into production and application[9-10]. The use of Chinese herbal medicine to treat diseases has a long history in China. Chinese herbal medicines are rich, have low toxicity, and have extremely high application value. Therefore, it is extremely beneficial to screen out antiviral drugs from Chinese herbal medicines.P.cuspidatumwas included in theChinesePharmacopoeiain 1997. It has the advantages of high safety, small side effects, and rich pharmacological effects[11-13], and it has a very high development and utilization prospect. Many studies have confirmed that theP.cuspidatumanthraquinone extract has a certain inhibitory effect on herpes simplex virus, hepatitis B virus, severe acute respiratory syndrome (SASR) coronavirus and many other viruses[4-6]. However, at present, the application of Chinese herbal medicineP.cuspidatumin the treatment of aquatic viral diseases is not common, and the related scientific research progress is relatively less than that in mammals.

In this study, we first used CCB cells to culture and amplify KHV, and determined KHV TCID50, and then used MTT method to determine the effective and safe drug concentration range of theP.cuspidatumanthraquinone extract in preparation for subsequent experiments. The experimental results indicate that the toxic effect of theP.cuspidatumanthraquinone extraction is related to the dose-dependent. The greater the concentration, the stronger the toxicity of the drug. When the concentration of theP.cuspidatumanthraquinone extract was 31.25 μg/mL, the cell survival rate was over 80%. At the concentration of 3.91 μg/mL, there was basically no cytotoxicity, and at 15.63 μg/mL, it can promote the proliferation and growth of CCB cells to a certain extent. In addition, related microscopic observations showed that when the concentration of the theP.cuspidatumanthraquinone extract was 31.25 μg/mL and below, the cells were alive and healthy, grew normally, and did not change their morphology. The broad-spectrum antiviral drug ribavirin also has certain cytotoxicity, and the theP.cuspidatumanthraquinone extract is relatively less toxic.

In this experiment, we used the MTT method to detect the protective effect of different concentration gradients of theP.cuspidatumanthraquinone extract on KHV-infected CCB cells, and to detect whetherP.cuspidatumanthraquinone extract has the ability to directly kill KHV and resist virus adsorption. The experimental results indicate that the antiviral activity of the theP.cuspidatumanthraquinone extract was enhanced after infection, withIC50of 13.67±0.47 μg/mL and TI of 5.48±0.49. In addition, the broad-spectrum antiviral drug ribavirinIC50was about 36 times that of the theP.cuspidatumanthraquinone extract, and its anti-KHV activity was lower than that of theP.cuspidatumanthraquinone extract. The anti-KHV replication experiment of theP.cuspidatumanthraquinone extract (1.96-31.25 μg/mL) showed that the optimal therapeutic concentration was 31.25 μg/mL The drug concentration of theP.cuspidatumanthraquinone extract inhibited KHV replication in a dose-dependent manner. As the concentration of theP.cuspidatumanthraquinone extract increased, the ability to inhibit KHV replication was stronger. Besides, theP.cuspidatumanthraquinone extract had a certain ability to directly kill KHV particles, but the anti-virus adsorption effect was not obvious.

In summary, in this study, we preliminarily confirmed that theP.cuspidatumanthraquinone extract has a certain effect on anti-KHV throughinvitroexperiments. However, its mechanism of action and the effects ofinvivoexperiment still need to be further studied in depth. Through this experiment, we found the maximum non-toxic concentration was ≤31.25 μg/mL) and the optimal therapeutic concentration of theP.cuspidatumanthraquinone extract was 31.25 μg/mL. This is expected to lay a theoretical foundation for the follow-up research on the anti-KHV of theP.cuspidatumanthraquinone extract.