Application of Ginkgolic Acid in the Preparation of Anti-Rotavirus Drugs

Abstract

This invention discloses the application of ginkgolic acid in the preparation of anti-rotavirus drugs, belonging to the field of biotechnology. The application of ginkgolic acid in the preparation of anti-rotavirus drugs disclosed in this invention demonstrates that ginkgolic acid has an anti-RV biosynthesis effect, exerting its anti-RV effect by inhibiting the expression of the VP6 gene.

Description

Technical Field

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

[0002] Rotavirus (RV) belongs to the genus Rotavirus in the family Reoviridae. It is a non-enveloped double-stranded RNA (dsRNA) virus. Its viral particles have an icosahedral structure, consisting of a genome containing 11 dsRNA segments enclosed by three concentric capsids. Rotavirus is a major cause of severe dehydrating gastroenteritis in children under 5 years of age.

[0003] In 2016, more than 258 million children under the age of five worldwide suffered from diarrhea due to rotavirus infection, resulting in an estimated 128,500 deaths and approximately 1.537 million hospitalizations. Almost all children under the age of five have been infected with rotavirus before the age of five. Rotavirus is transmitted via the fecal-oral route and is easily spread through contact with contaminated surfaces or direct contact with an infected person, making it highly contagious.

[0004] Rotavirus infection typically manifests as rotavirus gastroenteritis, primarily caused by group A rotavirus. After infection, children often experience watery or soupy stools, accompanied by fever, nausea, vomiting, and varying degrees of dehydration and electrolyte imbalance. Severe cases can lead to shock and even death. Symptoms vary depending on age; older children usually experience milder symptoms such as diarrhea, abdominal pain, and bloating.

[0005] Currently, there are no specific drugs for treating rotavirus enteritis in clinical practice. The RV vaccines available in China include the oral live RV vaccine (Lanzhou Lamp Rotavirus Vaccine LLR) launched in 2001 and the oral penta-valent human-bovine reassortant rotavirus vaccine (RV5) launched in 2018. Neither of these vaccines is currently included in the National Immunization Program and they are relatively expensive. Due to the variability and diversity of rotavirus strains, the preventive efficacy of these vaccines is also somewhat limited. Therefore, effectively preventing and treating rotavirus infection remains a significant challenge.

[0006] Ginkgolic acid (chemical structure shown in...) Figure 1Ginkgolic acid (GBA) is a derivative of 6-alkyl or 6-enyl salicylic acid, primarily found in the outer seed coat of Ginkgo biloba. Studies have shown that GBA can induce tumor cell apoptosis and inhibit tumor cell growth, exhibiting certain effects against human lung cancer cells, human breast cancer cells, human colon adenocarcinoma cells, human bladder cancer cells, and human ovarian adenocarcinoma. In terms of antiviral activity, GBA has a certain antiviral effect against Coxsackie B3 virus; it can also inhibit the RNase H activity of HIV-1 reverse transcriptase (RT), thus exerting a certain anti-HIV effect. However, there are currently no reports on the anti-RV effect of GBA.

[0007] Therefore, providing the application of ginkgolic acid 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

[0008] In view of this, the present invention provides the application of ginkgolic acid in the preparation of anti-rotavirus drugs.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] Application of ginkgolic acid in the preparation of anti-rotavirus drugs.

[0011] Furthermore, the application of ginkgolic acid in the preparation of anti-rotavirus biosynthetic drugs.

[0012] Furthermore, the rotavirus is the RV-WA strain.

[0013] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of ginkgolic acid in the preparation of anti-rotavirus drugs. Ginkgolic acid has the effect of inhibiting RV biosynthesis and can exert the anti-RV effect by inhibiting the expression of VP6 gene. Attached Figure Description

[0014] 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.

[0015] Figure 1 The attached figure shows the chemical structural formula of Ginkgolic acid of the present invention;

[0016] Figure 2 The attached figure shows MA104 cells before and after RV virus infection according to the present invention;

[0017] Where A: normal MA104 cells; B: MA104 cells 48 hours after RV infection;

[0018] Figure 3 The attached figure shows the cytotoxicity results of ginkgolic acid in this invention on cells; N represents the control group, i.e., the untreated group.

[0019] Figure 4 The attached figure illustrates the anti-RV adsorption effect of Gingolic acid in this invention;

[0020] Figure 5 The attached figure illustrates the anti-biosynthesis effect of Gingolic acid on RV according to the present invention; compared with the Ribavirin group, **p<0.01, ***p<0.001;

[0021] Figure 6 The attached figure shows the direct killing effect of Gingolic acid on RV in this invention; compared with the Ribavirin group, ****p<0.0001;

[0022] Figure 7 The attached figure shows the effect of Gingolic acid of the present invention on the expression of the viral structural protein VP6 gene in RV-infected MA104 cells; compared with the RV group, ****p<0.0001. Detailed Implementation

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] The experimental cell lines and reagents are shown in Table 1.

[0025] Table 1. Experimental cell lines and reagents

[0026]

[0027] The main instruments are listed in Table 2.

[0028] Table 2 Main Instruments

[0029]

[0030] Main reagent preparation methods:

[0031] Gingolic acid stock solution: Weigh an appropriate amount of Gingolic acid standard (purity ≥98%, catalog number: 104140-230201), add 1ml of cell-grade DMSO to dissolve it in a laminar flow hood, and filter the solution through a 0.22μM filter membrane using a disposable sterile syringe to prepare a 10mM stock solution.

[0032] DMEM medium (containing 10% fetal bovine serum and 1% penicillin antibody): In a clean bench, add 50ml of fetal bovine serum and 5ml of penicillin antibody to 445ml of high-glucose DMEM medium, mix thoroughly, divide into labeled 50ml centrifuge tubes, seal, and store at 4℃.

[0033] 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.

[0034] 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.

[0035] Statistical data analysis

[0036] 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. P < 0.05 was considered statistically significant. The results are expressed as mean ± standard deviation. Mann-Whitney statistics were performed using Graphpad Prism 9.5.0.

[0037] Example 1: RV infection of MA104 cells

[0038] After digesting and passaged MA104 cells, a new cell culture flask was taken out, and 1 mL of cell suspension and 3 mL of DMEM culture 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, at which point they could be used for RV expansion. The RV-Wa strain was first 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 flask that had grown to a monolayer was taken out, washed once with phosphate-buffered saline (PBS, pH=7), and then rinsed twice with high-glucose DMEM culture medium. 1 mL of the incubated virus solution was then added, followed by 3 mL of RV growth maintenance medium. MA104 cells infected with the virus will exhibit cytopathic effect (CPE). When the degree of cytopathic effect reaches 75%, the cells should be 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.

[0039] 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 and distinct cell outline. After infection with RV, MA104 cells show obvious pathological changes, with blurred cell boundaries, increased intercellular distance, and eventually complete cell detachment and floating.

[0040] Example 2: CCK8 assay for the toxicity of Gingolic acid to MA104 cells

[0041] Based on the commonly used concentration range of ginkgolic acid, this experiment conducted a preliminary cytotoxicity test at 1-6 μM. MA104 cells in the logarithmic growth phase were observed under a microscope. When the cells were uniformly shaped, 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. Drug administration was initiated when cells adhered to the culture vessel and formed a monolayer, while the control group received only an equal volume of high-glucose DMEM culture medium. After 48 h of incubation, the cytotoxicity of ginkgolic acid was assessed using the CCK8 reagent. 1 / 10 volume of CCK8 solution was added to each well, and the cells were incubated for 90 min. The absorbance was then measured and recorded at 450 nm after shaking for 10 s. The relative cell viability formula is:

[0042] {(A 实验组 -A 空白 ) / (A 对照组 -A空白 )×100%}

[0043] See the results Figure 3 Ginkgolic acid at concentrations between 1 and 4 μM showed no drug toxicity to cells and maintained an average cell viability of over 90%. Compared to the control group, the cell viability of the drug group at 1-4 μM was not significantly different from that of the control group. Therefore, the experimental drug concentration of gingerolic acid was selected as 1-4 μM.

[0044] Example 3: Detection of three anti-RV effects of Ginkgolic acid using the CCK8 assay

[0045] To investigate whether gingerolic acid has an in vitro anti-RV infection effect, experiments were conducted in the MA104 cell model to examine the adsorption, direct inactivation, and biosynthesis of gingerolic acid against RV virus.

[0046] (1) Anti-RV adsorption effect of Ginkgolic acid

[0047] 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 culture 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), 100 μL per well, was added to all cells except the normal cell control group. Incubation was performed at 37°C and 5% CO2 for 2 h. The normal cell control group received only an equal volume of high-glucose DMEM culture medium. The virus was then aspirated, and RV growth maintenance medium (200 μL per well) was added to all cells except the normal cell control group, the drug group, the positive control group, and the 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 90 min. Repeat the experiment 3 times.

[0048] See the results Figure 4 Compared with the Ribavirin group, the Ginkgolic acid group showed no significant increase in the inhibition rate of anti-RV at 1μM, 2μM, 3μM, and 4μM, with no statistical difference. The highest inhibition rate in the drug group was only about 5%, indicating that Ginkgolic acid has no significant anti-RV adsorption effect.

[0049] (2) Anti-RV synthesis effect of Ginkgolic acid

[0050] 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 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 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 plates were incubated for 90 minutes. The absorbance was measured and recorded at 450 nm. The viral inhibition rate was calculated, and the experiment was repeated three times.

[0051] See the results Figure 5 Compared with the ribavirin group, the inhibition rates of RV by the ginkgolic acid group at 1 μM, 2 μM, and 3 μM were 41.90%, 66.55%, and 68.95%, respectively, showing statistically significant differences. The inhibition rate of RV by ginkgolic acid at 4 μM was 53.81%, with no statistically significant difference, indicating that the anti-biosynthetic activity of the ginkgolic acid group at 4 μM was comparable to that of ribavirin against RV. The experimental results show that ginkgolic acid has a significant inhibitory effect on the biosynthesis of RV.

[0052] (3) Direct killing effect of ginkgolic acid on RV

[0053] The drug was mixed with an equal volume of 100 TCID50 viral solution (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 received only an equal volume of DMEM. 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 for 90 min. 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.

[0054] See the results Figure 6Compared with the Ribavirin group, each Ginkgolic acid group showed a lower inhibition rate of RV, and the difference was statistically significant, indicating that Ginkgolic acid has no obvious direct killing effect on RV.

[0055] Example 4: qPCR detection of the expression level of the RV structural protein VP6 gene.

[0056] (1) Extraction and quantification of total RNA

[0057] ① To further verify whether ginkgolic acid has an anti-RV synthesis effect, after 48 hours of ginkgolic acid anti-RV synthesis treatment, high, medium, and low drug groups (1 μM, 2 μM, and 4 μM), a Ribavirin group (positive control group), and an RV group (virus control group) were selected. The supernatant was discarded, the samples were washed twice with PBS, 1 ml of Trizol reagent was added, and the samples were allowed to stand for 5 min before being collected in 1.5 mL enzyme-free EP tubes. 200 μL of chloroform was added to the tubes, the mixture was vortexed for 15 s, allowed to stand at room temperature for 3 min, and then centrifuged (4℃, 12000 r / min, 15 min). The centrifuged samples separated into three layers: a colorless upper layer, a white middle layer, and a red lower layer.

[0058] 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).

[0059] 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.

[0060] 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.

[0061] (2) Reverse transcription of mRNA

[0062] ① Genomic DNA removal reaction

[0063] Prepare the reaction mixture on ice according to the components in Table 3, 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.

[0064] Table 3 Genomic DNA Reaction System

[0065]

[0066] Reaction conditions: 42℃ for 2 min; 4℃.

[0067] *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.

[0068] ②Reverse transcription reaction

[0069] Prepare the reaction solution according to Table 4 and carry out the reverse transcription reaction.

[0070] Table 4 Reverse transcription reaction system

[0071]

[0072] Reaction conditions: 37℃ for 15 min; 85℃ for 5 sec; 4℃.

[0073] (3) Real-Time PCR reaction

[0074] Real-time quantitative PCR was performed using SYBR Green I fluorescent labeling to detect VP6 expression levels in high, medium, and low drug groups (1 μM, 2 μM, 4 μM), the Ribavirin group, and the RV group. The GreenPremix Pro Taq HS qPCR Kit II was used, with GAPDH as the internal control. Axygen's dedicated Real-time 8-tube PCR system was used. The real-time quantitative PCR amplification reaction system was prepared according to Table 5, and the reaction solution was prepared on ice (total reaction volume: 10 μL). qPCR reaction conditions are shown in Table 6; primer sequences are shown in Table 7.

[0075] Table 5 PCR reaction system

[0076]

[0077] Table 6 qPCR reaction conditions

[0078]

[0079] Table 7 Primer sequences

[0080]

[0081] like Figure 7 As shown, compared with the RV group, the expression level of VP6 in the gingerolic acid group was significantly reduced at concentrations of 1 μM, 2 μM, and 4 μM, with statistically significant differences. Among them, the inhibition of VP6 expression by gingerolic acid at 4 μM was the most significant. This indicates that gingerolic acid exerts its anti-RV effect by inhibiting the gene expression of VP6.

[0082] Conclusion: Ginkgolic acid has an anti-RV biosynthesis effect, but no obvious anti-RV adsorption or direct killing effect. It exerts its anti-RV effect by inhibiting VP6 gene expression.

[0083] 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. The application of ginkgolic acid in the preparation of anti-rotavirus drugs, characterized in that, The chemical structural formula of the ginkgolic acid is shown below:

2. The application of ginkgolic acid in the preparation of anti-rotavirus biosynthetic drugs, characterized in that, The chemical structural formula of the ginkgolic acid is shown below:

3. The application according to claim 1 or 2, characterized in that, The rotavirus in question is the RV-WA strain.

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