Use of corilagin in the preparation of anti-rotavirus drugs

Abstract

The application discloses application of corilagin in preparation of an anti-rotavirus drug and belongs to the technical field of biotechnology.The application of corilagin in preparation of the anti-rotavirus drug has the effect of resisting RV biosynthesis, and can play the role of resisting RV by inhibiting expression of a VP6 gene.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the use of corilagin in the preparation of anti-rotavirus drugs. Background Technology

[0002] Rotavirus (RV), belonging to the genus Rotavirus in the family Reoviridae, is one of the leading pathogens causing acute gastroenteritis and diarrhea in infants under 5 years old, especially those in developing countries requiring hospitalization. The high infectivity and pathogenicity of RV pose a significant threat to society, with approximately 130 million people infected globally each year and about 200,000 deaths. Currently, there are no specific drugs for treating RV infection, and the variability and diversity of RV strains limit the range of strains that RV vaccines can prevent. Therefore, preventing and controlling RV infection remains a major clinical challenge.

[0003] Corilagin (Cor), 1-acyl-3,6-hexahydroxybiphenyl dicarboxylic acid glucose, is a polyphenolic tannin compound extracted from plants such as Phyllanthus urinaria, Geranium wilfordii, Citrus reticulata, and Trifolium repens. It possesses a wide range of biological activities, including antitumor, antioxidant, anti-atherosclerotic, antifibrinolytic, antihypertensive, antiviral, antibacterial, and anti-inflammatory effects. Currently, there are no reports on the anti-RV activity of Cor.

[0004] Therefore, providing information on the application of corilagin in the preparation of anti-rotavirus drugs is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the use of corilagin in the preparation of anti-rotavirus drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of corilagin in the preparation of anti-rotavirus drugs; the chemical structural formula of corilagin is shown below. Figure 1 .

[0008] Furthermore, the application of kerilagin in the preparation of anti-rotavirus biosynthetic drugs.

[0009] Furthermore, the rotavirus is RV-WA strain and RV-SA-11 strain.

[0010] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of corilagin in the preparation of anti-rotavirus drugs. Cor has the effect of inhibiting RV biosynthesis, but has no obvious anti-RV adsorption and direct inhibition effect. It can exert the anti-RV effect by inhibiting the expression of the VP6 gene. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0012] Figure 1 The attached figure shows the chemical structural formula of Cor;

[0013] Figure 2 The attached figure shows MA104 cells before and after RV virus infection according to the present invention;

[0014] Where A: normal MA104 cells; B: MA104 cells 48 hours after RV infection;

[0015] Figure 3 The attached figure shows the cell toxicity results of Cor in this invention (Control group represents the control group, compared with the Control group, ** P < 0.05 *** P < 0.001);

[0016] Figure 4 The attached figure illustrates the Cor anti-RV adsorption effect of the present invention;

[0017] Among them, A: RV-WA strain; B: RV-SA-11 strain;

[0018] Figure 5 The attached figure illustrates the Cor anti-RV synthesis effect of the present invention;

[0019] Among them, A: RV-WA strain; B: RV-SA-11 strain; compared with the Ribavirin group, * P < 0.05 ** P < 0.05 *** P < 0.001;

[0020] Figure 6 The attached figure illustrates the direct inhibition of RV by Cor according to the present invention;

[0021] Among them, A: RV-WA strain; B: RV-SA-11 strain; compared with the Ribavirin group, **** P < 0.0001;

[0022] Figure 7 The attached figure shows the relative expression level of the RV-VP6 gene in this invention;

[0023] Where A: RV-WA strain; B: RV-SA-11 strain; Control group represents the normal control group of cells, compared with the RV group, ****P<0.0001). Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Cell-grade dimethyl sulfoxide, fetal bovine serum, and high-glucose DMEM culture medium were purchased from GIBCO, USA; 0.25% trypsin digestion solution, 0.25% trypsin digestion solution containing EDTA, and anti-penicillin / streptomycin (double antibiotic) were purchased from Beijing Solarbio, Inc.; MA104 cell line was obtained from the cell bank of Sun Yat-sen University; RV-Wa and RV-SA-11 cell lines were obtained from the Institute of Immunology, Third Military Medical University; 1×PBS phosphate buffer was purchased from Beyotime Biotechnology Research Institute, Jiangsu Province.

[0026] Cor stock solution: Weigh 9.8 mg of Cor standard, purity ≥ 98%, batch number: O26IB230022 (Shanghai Yuanye Biotechnology Co., Ltd.), add 154.46 μL of cell-grade DMSO to dissolve it in a clean bench, and filter the solution through a 0.22 μM filter membrane to sterilize it using a disposable sterile syringe, so as to prepare a stock solution of 100 mM.

[0027] Ribavirin solution: 100 mg / mL Ribavirin stock solution was stored at 4°C and diluted to 1 mg / mL with high glucose DMEM culture medium as a positive control (prepared and used immediately).

[0028] DMEM culture medium containing 10% fetal bovine serum: In a clean bench, add 5 mL of fetal bovine serum and 0.5 mL of penicillin-dextrose antibody to 45 mL of high glucose DMEM culture medium, mix thoroughly, place in a labeled 50 mL centrifuge tube, seal, and store at 4 °C.

[0029] 10 μg / mL EDTA-free trypsin: 0.25% trypsin digestion solution (0.25 g trypsin / 100 mL, 2500 μg / mL) was diluted to 10 μg / mL with high glucose DMEM culture medium.

[0030] RV growth maintenance medium: 0.25% trypsin digestion solution (0.25g trypsin / 100mL, 2500μg / mL) was diluted to 1μg / mL with high glucose DMEM culture medium.

[0031] Statistical data analysis:

[0032] All experiments were repeated three times. Experimental data are expressed as mean ± standard deviation (ˉx±s). SPSS software was used for statistical analysis. The t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons of means among multiple groups. The results are expressed as mean ± standard deviation. Data analysis was performed using GraphPadprism 8.01 with Mann-Whitney statistics. P < 0.05 was considered statistically significant.

[0033] Example 1: RV infection of MA104 cells

[0034] After digesting and passaged MA104 cells, new cell culture flasks were removed, and 1 mL of cell suspension and 3 mL of DMEM medium containing 10% fetal bovine serum were added. The cells were incubated at 37°C and 5% CO2 for 48 h until they grew to a monolayer, ready for RV expansion. First, RV-Wa and RV-SA-11 strains were thawed in a 37°C water bath. 500 μL of RV virus solution was mixed thoroughly with 500 μL of 10 μg / mL EDTA-free trypsin and incubated at 37°C and 5% CO2 for 30 min. Then, the MA104 cell culture flasks that had grown to a monolayer were removed, washed once with phosphate-buffered saline (PBS, pH=7), and then rinsed twice with high-glucose DMEM medium. Next, 1 mL of the incubated virus solution was added, followed by 3 mL of RV growth maintenance medium. Virus-infected MA104 cells exhibit cytopathic effect (CPE). When the cytopathic effect reaches 75%, the cells were frozen at -20°C. After repeating the freezing and thawing process three times, centrifuge at low temperature and collect the supernatant, which is the viral fluid. Repeat the above process to amplify RV.

[0035] MA104 cells before and after infection with RV-Wa strain virus were observed. Figure 2 Normal MA104 cells are triangular or spindle-shaped with a clear cell outline. After infection with RV-Wa strain, MA104 cells show obvious pathological changes, with blurred cell boundaries, increased intercellular distance, increased intracellular black granules, and finally, the cells completely detach and float.

[0036] Example 2: Detection of Cor toxicity to MA104 cells using the CCK8 assay

[0037] Based on the commonly used concentration range of Cor, this experiment conducted a preliminary cytotoxicity test between 3.9 and 1000 μM. MA104 cells in the logarithmic growth phase were observed under a microscope. When the cells were uniform in morphology, plump, with clear edges, and reached 80% confluence, they were digested, centrifuged, and resuspended. The suspension was further diluted, and 10 μl was taken and counted on a hemocytometer to calculate the required cell volume. Subsequently, cells were seeded in 96-well plates, with 100 μl of cell suspension added to each well, resulting in a cell density of 8 × 10⁶ cells / well.4 Cells / mL. When cells grew to a monolayer, a series of drug solutions of varying concentrations were administered, while the control group received only an equal volume of high-glucose DMEM culture medium. After 48 hours of incubation, the cytotoxicity of Cor was assessed using a CCK-8 assay kit. 1 / 10 volume of CCK-8 solution was added to each well, and the cells were incubated in an incubator. After 1 hour, the absorbance was measured and recorded at 450 nm. The relative cell viability formula is:

[0038] {(A 实验组 -A 空白 ) / (A 对照组 -A 空白 )×100%}

[0039] See results Figure 3 The safe concentration of Cor is 3.9–125 μM, at which point the cell survival rate reaches over 90%.

[0040] Example 3: Detection of three effects of Cor against RV using the CCK8 method

[0041] To investigate whether Cor has an in vitro anti-RV infection effect, three effects of Cor against RV were studied in MA104 cell models infected with RV-WA and RV-SA-11 strains: adsorption, direct inhibition, and biosynthesis.

[0042] (1) Cor's anti-RV adsorption effect

[0043] Drug solution was added to 96-well plates containing monolayered MA104 cells, with 6 replicates per well, 100 μL per well. The positive control group received an equal volume of 1 mg / mL ribavirin, while the normal cell control and virus control groups received only an equal volume of high-glucose DMEM medium. Incubation was performed at 37°C and 5% CO2 for 2 h. The drug solution was then aspirated, and 100 TCID50 of virus (incubated with 10 μg / mL trypsin at 37°C for 30 min) was added to each well (100 μL per well) in the drug group, positive control group, and virus control group. The normal cell control group received only an equal volume of high-glucose DMEM medium. Incubation was performed at 37°C and 5% CO2 for 2 h. The virus solution was then aspirated, and except for the normal cell control group, 100 μL of RV growth maintenance medium was added to each well in the drug group, positive control group, and virus control group. Incubation was performed at 37°C and 5% CO2, with continuous observation. After 48 h of culture, the results were analyzed using a CCK-8 assay kit. Add 1 / 10 volume of CCK-8 solution to each well, incubate in an incubator, and detect and record the absorbance at a wavelength of 450 nm after 1 hour. Repeat the experiment 3 times.

[0044] See results Figure 4In MA104 cell models infected with RV-WA and RV-SA-11 strains, Cor at 7.8–125 μM did not significantly increase the inhibition rate of RV, with the highest inhibition rate being only about 8%. Compared with the Ribavirin group, there was no statistically significant difference, indicating that Cor had no significant anti-RV adsorption effect.

[0045] (2) Cor's anti-RV synthesis effect

[0046] 100 TCID50 viral suspension (virus reacted with 10 μg / mL trypsin for 30 minutes) was added to 96-well plates of MA104 cells grown into a monolayer, 100 μL per well. Cells were washed twice with PBS beforehand. A normal control group was established, with an equal volume of high-glucose DMEM medium added. Incubation was performed at 37°C and 5% CO2 for 2 hours. The viral suspension was then aspirated, and different concentrations of drug solutions and 1 mg / mL Ribavirin were added, 100 μL per well. A virus control group was established, with only RV growth maintenance medium added, 100 μL per well. Incubation was performed at 37°C and 5% CO2, with continuous observation. After 48 hours of continuous culture, the results were analyzed using a CCK-8 assay kit. 1 / 10 volume of CCK-8 solution was added to each well, and the cells were incubated for 1 hour. The absorbance was measured and recorded at 450 nm. The viral inhibition rate of the drug was calculated, and the experiment was repeated three times.

[0047] See results Figure 5 In MA104 cell models infected with RV-WA and RV-SA-11 strains, Cor, at concentrations ranging from 15.625 to 62.5 μM, showed viral inhibition rates of 68.52%, 53.29%, and 50.22% against RV-WA, RV-SA-11, and 57.38%, 74%, and 72.26%, respectively. Ribavirin showed inhibition rates of 67.42% and 70.64% against RV-WA and RV-SA-11, respectively, indicating that Cor possesses anti-RV biosynthetic activity.

[0048] (3) Direct inhibitory effect of Cor on RV

[0049] The drug was mixed with an equal volume of 100 TCID50 viral fluid (virus reacted with 10 μg / mL trypsin for 30 min) and incubated for 2 h. Cells were washed twice with PBS and then added to 96-well culture plates containing monolayers of MA104 cells. In the positive control group, Ribavirin and RV were treated in the same manner. The normal cell control group and the virus control group were incubated with an equal volume of high-glucose DMEM medium. The mixture was incubated at 37°C and 5% CO2 for 2 h. The mixture was then aspirated, and 100 μL of RV growth maintenance medium was added to each well. The mixture was incubated at 37°C and 5% CO2, and continuously observed. After 48 h of culture, the CCK-8 assay was performed. 1 / 10 volume of CCK-8 solution was added to each well, and the mixture was incubated in an incubator. The absorbance was measured and recorded at 450 nm after 1 h. The viral inhibition rate of the drug was calculated, and the experiment was repeated three times.

[0050] See results Figure 6 In MA104 cell models infected with RV-WA and RV-SA-11 strains, Cor did not significantly increase the inhibition rate of RV at concentrations of 7.8–125 μM. The highest inhibition rate in each concentration group was only about 10%, which was statistically significant compared with the Ribavirin group, indicating that Cor did not have a significant direct inhibitory effect on RV.

[0051] Example 4: qPCR detection of the expression level of the RV structural protein VP6 gene.

[0052] (1) Extraction and quantification of total RNA

[0053] ① To further verify whether Cor has an anti-RV biosynthetic effect, cells were seeded in 6-well plates, with 2 mL of cell suspension added to each well, and the cell density was 1 × 10⁻⁶ cells / well. 5 Cells / mL. Once cells reached a monolayer, Cor anti-RV biosynthesis was initiated. Continuous observation was performed in the incubator. After 24 hours of incubation, total RNA was extracted from the 16μM, 32μM, and 64μM drug groups, the Ribavirin group, the Control group, and the RV group using the Trizol method. The supernatant was discarded, and the cells were washed twice with PBS. 1 mL of Trizol reagent was added, and the mixture was allowed to stand for 5 minutes before being collected in 1.5 mL enzyme-free EP tubes. 200 μL of chloroform was added to the tubes, and the mixture was vortexed for 15 seconds. After standing at room temperature for 3 minutes, the tubes were centrifuged (4℃, 12000 rpm, 15 minutes). The centrifuged sample separated into three layers: a colorless upper layer, a white middle layer, and a red lower layer.

[0054] ② Carefully aspirate the supernatant into a new 1.5 mL enzyme-free EP tube (approximately 500 μL), add an equal volume of pre-chilled isopropanol, vortex vigorously to mix, incubate at 4°C for 10 min, and centrifuge (4°C, 12000 r / min, 10 min).

[0055] ③ After centrifugation, a white precipitate can be seen at the bottom of the EP tube. Remove the supernatant and keep the precipitate. Add 1 mL of the prepared 75% ethanol solution (prepared by mixing anhydrous ethanol and enzyme-free water in a 3:1 ratio), shake to mix, centrifuge (4℃, 12000r / min, 5min), discard the supernatant, let stand at room temperature for 15min-20min, and air dry.

[0056] ④ After drying, add 20 μL of DEPC water to the EP tube and gently blow on the tube wall to dissolve the RNA. After measuring the RNA concentration of the sample with a NanoDrop micro-ultraviolet spectrophotometer, it can be used directly in experiments or stored at -80℃ for later use.

[0057] (2) Reverse transcription of mRNA

[0058] ① Genomic DNA removal reaction

[0059] Prepare the reaction mixture on ice according to the following components, with a reaction volume of 20 μL. Follow the instructions for the Evo M-MLV RTKit with gDNA Clean for qPCR II. All consumables used in this experiment were Axygen enzyme-free consumables.

[0060] The genomic DNA removal reaction system is shown in Table 1.

[0061] Table 1 Genomic DNA Reaction System

[0062]

[0063] Reaction conditions: 42℃ for 2 min; 4℃.

[0064] *1: The amount of RNA can be added as needed. In a 20 μL reverse transcription system, use a maximum of 1 μg total RNA; when using the probe method, use a maximum of 2 μg total RNA.

[0065] ②Reverse transcription reaction

[0066] Prepare the reaction solution according to Table 2 and carry out the reverse transcription reaction.

[0067] Table 2 Reverse transcription reaction system

[0068]

[0069] Reaction conditions: 37℃ for 15 min; 85℃ for 5 sec; 4℃.

[0070] (3) Real-Time PCR reaction

[0071] Real-time quantitative PCR was performed using SYBR Green I fluorescent labeling to detect VP6 expression levels in the drug group, Ribavirin group, N group, and RV group. The Green Premix Pro Taq HS qPCR Kit II was used, with GAPDH as the internal control. An Axygen Real-time PCR plate was used. The real-time quantitative PCR amplification reaction system was prepared according to Table 3, and the reaction solution was prepared on ice (total reaction volume: 10 μL). qPCR reaction conditions are shown in Table 4; primer sequences are shown in Table 5.

[0072] Table 3 PCR reaction system

[0073]

[0074] Table 4 qPCR reaction conditions

[0075]

[0076] Table 5 Primer sequences

[0077]

[0078] See results Figure 7 In MA104 cell models infected with RV-WA and RV-SA-11 strains, the Control group did not express VP6. Compared with the RV group, the Ribavirin group showed significantly reduced VP6 gene expression, indicating that Ribavirin has an anti-RV effect. Compared with the RV group, the Cor group showed significantly reduced VP6 expression at concentrations of 16 μM, 32 μM, and 64 μM, with the most significant inhibition of VP6 expression at 64 μM. This indicates that Cor exerts its anti-RV effect by inhibiting the expression of the RV-VP6 gene.

[0079] Conclusion: Cor has an anti-RV biosynthesis effect, but no obvious anti-RV adsorption or direct inhibition effect. It exerts its anti-RV effect by inhibiting the expression of the VP6 gene.

[0080] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Corilagin in the preparation of anti-rotavirus drugs.

2. The application according to claim 1, characterized in that, The rotaviruses mentioned are RV-WA strain and RV-SA-11 strain.

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