Application of lycorine in preparation of anti-rotavirus drugs
By using marlin to inhibit the expression of the structural protein VP6 gene of rotavirus, the problem of lack of effective anti-rotavirus drugs in the prior art was solved, and effective inhibition of rotavirus was achieved.
Patent Information
- Application Number
- CN202510546633.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-07-04
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The lack of effective anti-rotavirus drugs in the prior art, especially variants against rotavirus and the risk of vaccine use, leads to difficulties in preventing and treating RV diarrhea.
Anti-rotavirus drugs were prepared using Lycorine, which exerted anti-rotavirus effects by inhibiting the expression of the structural protein VP6 gene.
Aralisine can effectively inhibit the biosynthesis of rotavirus and reduce the expression of VP6 genes, thereby exerting the role of anti-rotavirus, providing new therapeutic methods.
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Figure CN120241730A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and more particularly to the application of lycorine in the preparation of anti-rotavirus drugs. Background Art
[0002] Rotavirus (RV) is an unencapsulated RNA virus belonging to the genus Rotavirus of the family Reoviridae, and is the main pathogen causing dehydrating diarrhea in infants and young children under 5 years old. Approximately 110 million people are infected globally every year, and the number of deaths is about 200,000. Currently, there is still a lack of effective drugs clinically. The currently marketed RV vaccines mainly include Rotarix, Rotasiil, RotaTeq, and Rotavac, etc. However, due to the variation of RV strains and the potential risks such as intussusception and biliary atresia in the use of RV vaccines, the prevention scope is limited. Therefore, it is of great significance to discover new candidate compounds for the prevention and treatment of RV diarrhea.
[0003] Lycorine (C 16 H 17 NO4, the chemical structure is shown in Figure 1 ) is an alkaloid extracted from the bulbs of plants of the genus Lycoris and its congeners in the family Amaryllidaceae. Existing studies have shown that Lycorine can inhibit a variety of tumors through mechanisms such as inducing apoptosis and inhibiting proliferation of tumor cells, such as renal cancer, liver cancer, colorectal cancer, lung cancer, cervical cancer, prostate cancer, myeloma, leukemia, and breast cancer, etc. At the same time, it also has an inhibitory effect on coronavirus, hepatitis C virus, human enterovirus 71, and coxsackievirus A16. However, there is no report on the anti-RV effect of Lycorine.
[0004] Therefore, providing the application of lycorine in the preparation of anti-rotavirus drugs is an urgent problem to be solved by those skilled in the art. Summary of the Invention
[0005] In view of this, the present invention provides the application of lycorine in the preparation of anti-rotavirus drugs.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] The application of lycorine in the preparation of anti-rotavirus drugs.
[0008] Furthermore, the application of lycorine in the preparation of anti-rotavirus biosynthesis drugs.
[0009] Furthermore, the rotavirus is RV-Wa strain.
[0010] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses the application of lycorine in the preparation of anti-rotavirus drugs. Lycorine has an anti-biosynthesis effect on RV and can play an anti-RV role by inhibiting the expression of the structural protein VP6 gene. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without creative efforts.
[0012] Figure 1 is the chemical structural formula of Lycorine;
[0013] Figure 2 are MA104 cells before and after RV virus infection;
[0014] Among them, A: normal MA104 cells; B: MA104 cells 48 h after RV infection;
[0015] Figure 3 are the cytotoxicity results of Lycorine on cells; N is the control group, that is, the group without drug treatment. Compared with the N group, **p < 0.01;
[0016] Figure 4 is the anti-RV adsorption effect of Lycorine; compared with the Ribavirin group, △△△△ p < 0.0001;
[0017] Figure 5 is the anti-biosynthesis effect of Lycorine on RV; compared with the Ribavirin group, △ p < 0.1, △△ p < 0.01;
[0018] Figure 6 is the direct inhibitory effect of Lycorine on RV; compared with the Ribavirin group, △△△△ p < 0.0001;
[0019] Figure 7 is the effect of Lycorine on the expression of the RV structural protein VP6 gene; compared with the RV group, #### p < 0.0001. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0021] The experimental cell lines and reagents are shown in Table 1.
[0022] Table 1
[0023]
[0024] The main instruments are shown in Table 2.
[0025] Table 2
[0026]
[0027]
[0028] Preparation methods of main reagents:
[0029] Lycorine stock solution: Weigh an appropriate amount of Lycorine standard (purity ≥ 98%, batch number: CFS202401), add 1 ml of cell-grade DMSO to dissolve it in a laminar flow hood, filter and sterilize it with a 0.22 μM filter membrane, and prepare the stock solution to be 10 mM.
[0030] DMEM medium containing 10% fetal bovine serum: In a laminar flow hood, sequentially add 50 ml of fetal bovine serum and 5 ml of penicillin-streptomycin double antibody to 445 ml of high-glucose DMEM medium, mix well, dispense, seal, and store at 4°C.
[0031] 10 μg / mL trypsin without EDTA: Dilute 0.25% trypsin digestion solution (0.25 g trypsin / 100 mL, 2500 μg / mL) to 10 μg / mL with high-glucose DMEM medium.
[0032] Ribavirin solution: Store 100 mg / mL Ribavirin stock solution at 4°C, and dilute it to 1 mg / mL with high-glucose DMEM medium as the positive control group (prepared and used immediately).
[0033] RV growth maintenance solution: Dilute 10 μg / mL trypsin without EDTA and high-glucose DMEM medium at a ratio of 1:10 to 1 μg / mL.
[0034] Statistical data analysis
[0035] All experiments were repeated three times. The experimental data were expressed as mean ± standard deviation Presentation. SPSS 26.0 software was used to statistically analyze the data. The t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison of means among multiple groups. p < 0.05 indicates that the difference is statistically significant. Data analysis was performed using the statistical analysis software Graphpad prism 9.5.0 for Lycorinenn-Whitney statistics.
[0036] Example 1 Virus Titer Determination
[0037] When MA104 cells grew to a monolayer density, after routine digestion, the cell fluid was collected and centrifuged. The supernatant was discarded, and 4 mL - 7 mL of DMEM medium containing 10% fetal bovine serum was added to the centrifuge tube for dilution and counting. The cells were inoculated onto a 96-well plate at 8000 cells per well and cultured in a CO2 incubator for 12 h. After the cells adhered and grew, they were used for subsequent experiments. The virus strain RV-Wa and 10 μg / mL of trypsin without EDTA were mixed and sealed in a 1:1 ratio and incubated in a CO2 incubator for 30 min - 60 min. The virus solution was serially diluted 6-fold by 10-fold with high-glucose DMEM medium, and 9 replicates were set for each gradient concentration. The pre-inoculated 96-well plate was taken out, rinsed once with PBS buffer, then rinsed twice with high-glucose DMEM medium, and then the diluted virus solution was added to the 96-well plate, 100 μL per well. At the same time, a normal group was set as a control, and 100 μL of high-glucose DMEM medium was added to each well of the normal group. The 96-well plate was returned to the CO2 incubator for 2 h. After taking it out, the virus solution was aspirated and discarded, and then 100 μL of high-glucose DMEM medium was added to each well and continued to be cultured in the CO2 incubator. The cytopathic effect of the cells was observed under a microscope at regular intervals. When the cells in the lowest dilution gradient group no longer showed CPE changes, the number of wells with cytopathic changes and the cytopathic conditions of each group were observed and recorded under the microscope. At the same time, 10 μL of MTT reagent was added to each well of the 96-well plate under light-proof conditions and cultured in the incubator for 3 h - 4 h. Then the culture medium was aspirated and discarded, and 150 μL of dimethyl sulfoxide was immediately added. The absorbance of each well was detected with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 490 nm. Calculation was performed according to the method of Reed and Muench.
[0038] The results showed that TCID 50 = 10 -4.3492 / 100 μL, 100 TCID 50 = 10 -4.3492 / μL.
[0039] Example 2 RV Infection of MA104 Cells
[0040] After subculturing MA104 cells, take out a new cell culture flask, add 1 mL of cell suspension and 3 mL of DMEM medium containing 10% fetal bovine serum, and incubate at 37 °C and 5% CO2 for 48 h. When the cells grow to a monolayer, they can be used for RV amplification. First, thaw the RV-Wa strain at room temperature. Take 500 μL of RV virus solution and mix it thoroughly with 500 μL of 10 μg / mL trypsin without EDTA, and incubate at 37 °C and 5% CO2 for 30 min. Then take out the MA104 cells that have grown to a monolayer, first rinse them once with phosphate buffer (PBS, pH = 7), then rinse them twice with high-glucose DMEM medium, then add 1 mL of the incubated virus solution, and then add 3 mL of RV growth maintenance solution to the cell culture flask. The MA104 cells after virus infection will show cytopathic effect (CPE). When the degree of lesion reaches about 75%, freeze them in a -20 °C refrigerator. Repeat the freezing and thawing process 3 times, then centrifuge at low temperature and collect the supernatant, which is the virus solution. Repeat the above process to amplify RV.
[0041] MA104 cells before and after RV-Wa strain virus infection are shown in Figure 2 , the normal shape of MA104 cells is triangular or fusiform, and the cell outline is obvious and clear; after MA104 cells are infected with RV, obvious cell lesions occur, the cell boundaries become blurred, the distance between cells increases, and finally the cells completely detach and float.
[0042] Example 3 Detection of the toxicity of Lycorine to MA104 cells by CCK-8 method
[0043] According to the common drug concentration range of Lycorine, a cytotoxicity pre-experiment was carried out between 2 - 128 μM in this experiment. Take MA104 cells in the logarithmic growth phase and observe them under a microscope. The cell morphology is uniform and full, the edges are clear, and the number reaches about 80%. Digest, centrifuge and resuspend them. After further diluting the suspension, take 10 μL and count it on a hemocytometer to calculate the required cell volume. Then plate the cells in a 96-well plate, add 100 μL of cell suspension to each well, and the cell density is 8×10 4 cells / mL. Administer the drug when the cells adhere to form a monolayer. The control group only adds an equal volume of high-glucose DMEM medium. After incubating for 48 h, detect the cytotoxicity of Lycorine with CCK-8 reagent. Add 1 / 10 volume of CCK-8 solution to each well, incubate in an incubator, after 90 min, shake for 10 s, and detect and record the absorbance at a wavelength of 450 nm. The relative survival rate formula of the cells is:
[0044] {(A 实验组 -A 空白 ) / (A 对照组 -A 空白 )×100%}
[0045] The results are shown in Figure 3 , when the Lycorine drug concentration is below 64 μM, there is no obvious drug toxicity to the cells, and the average survival rate of the cells is more than 90%. Therefore, the experimental drug concentration of Lycorine is selected as 60 μM and below.
[0046] Example 4 Detection of three anti-RV effects of Lycorine by CCK-8 method
[0047] To study whether Lycorine has an anti-RV infection effect in vitro, experiments were conducted on a MA104 cell model from three aspects: the adsorption, direct inhibition, and biosynthesis effects of Lycorine against RV.
[0048] (1) Anti-RV adsorption effect of Lycorine
[0049] Add the drug solution into a 96-well culture plate of MA104 cells grown to a monolayer. Each drug solution is repeated in 6 wells, 100 μL per well. In the positive control group, an equal volume of 1 mg / mL ribavirin is added. The normal cell control group and the virus control group only add an equal volume of high-glucose DMEM medium. Incubate at 37 °C and 5% CO2 for 2 h. Aspirate the drug solution, and add 100 TCID50 of the virus (the virus is incubated with 10 μg / mL trypsin at 37 °C for 30 min) to each well except the normal cell control group, 100 μL per well. Incubate at 37 °C and 5% CO2 for 2 h. The normal cell control group only adds an equal volume of high-glucose DMEM medium. Aspirate the virus, and add the RV growth maintenance solution to the drug group, the positive control group, and the virus control group except the normal cell control group, 100 μL per well. Incubate at 37 °C and 5% CO2, and observe continuously. After culturing for 48 h, detect with a CCK-8 kit. Add 1 / 10 volume of the CCK-8 solution to each well, incubate in the incubator, and detect and record the absorbance at a wavelength of 450 nm after 90 min. Repeat the experiment 3 times.
[0050] The results are shown in Figure 4 . Compared with the Ribavirin group, the inhibition rates of the Lycorine group against RV at 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM are 6.83%, 12.48%, 8.36%, 10.78%, and 9.26% respectively, all lower than those of the Ribavirin group. There are significant differences in statistical comparison (p values are p < 0.0001, p = 0.0003, p < 0.0001, p < 0.0001, p < 0.0001 respectively). The results show that Lycorine has no anti-RV adsorption effect.
[0051] (2) Anti-RV synthesis effect of Lycorine
[0052] Add 100 TCID50 virus solution (the virus is incubated with 10 μg / mL trypsin for 30 min) to a 96-well culture plate of MA104 cells grown to confluence, 100 μL per well. The cells were rinsed twice with PBS before. Set a normal cell control and add an equal volume of high-glucose DMEM medium. Incubate at 37 °C and 5% CO2 for 2 h, then aspirate the virus solution and add different concentrations of the drug solution and 1 mg / mL Ribavirin, 100 μL per well; set a virus control group and only add the RV growth maintenance solution, 100 μL per well. Incubate at 37 °C and 5% CO2 and observe continuously. After continuous culture for 48 h, detect with a CCK-8 kit. Add 1 / 10 volume of CCK-8 solution to each well, incubate in the incubator, and detect and record the absorbance at a wavelength of 450 nm after 90 min. Calculate the virus inhibition rate of the drug and repeat the experiment 3 times.
[0053] The results are shown in Figure 5 . Compared with the Ribavirin group, the inhibition rates of the Lycorine group against RV at 20 μM and 30 μM were lower than those of the Ribavirin group, 41.26% and 39.19% respectively, and the differences were statistically significant (p values were 0.0114 and 0.0031 respectively); the inhibition rates of the Lycorine group against RV at 40 μM, 50 μM, and 60 μM reached 52.18%, 52.74%, and 54.47% respectively, all higher than those of Ribavirin, and there were no statistical differences (p values were 0.9998, 0.9937, and 0.8325 respectively), indicating that Lycorine has an anti-RV biosynthesis effect at 40 μM, 50 μM, and 60 μM.
[0054] (3) Direct inhibitory effect of Lycorine on RV
[0055] Mix the drug with 100 TCID50 virus solution (the virus is incubated with 10 μg / mL trypsin for 30 min) in equal volume and incubate for 2 h. After rinsing the cells twice with PBS, add them to a 96-well culture plate of MA104 cells grown to confluence. In the positive control group, perform the same operation as above with Ribavirin and RV, and the normal cell control group and the virus control group only add an equal volume of high-glucose DMEM medium. Incubate at 37 °C and 5% CO2 for 2 h, then aspirate the mixture and add the RV growth maintenance solution, 100 μL per well, incubate at 37 °C and 5% CO2, and observe continuously. After culturing for 48 h, detect with a CCK-8 kit. Add 1 / 10 volume of CCK-8 solution to each well, incubate in the incubator, and detect and record the absorbance at a wavelength of 450 nm after 90 min. Calculate the virus inhibition rate of the drug and repeat the experiment 3 times.
[0056] The results are shown in Figure 6。Compared with the Ribavirin group, the inhibition rates of Lycorine against RV at 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM were -0.32%, 2.32%, 1.05%, 2.53%, and 3.23% respectively, all lower than those of the Ribavirin group, and there were significant differences in statistical comparison (p values were all less than 0.0001), indicating that Lycorine has no direct inhibitory effect on RV.
[0057] Example 5 Detection of the expression level of the RV structural protein VP6 gene by qPCR
[0058] (1) Extraction and quantification of total RNA
[0059] ① To further verify whether Lycorine has an anti-biosynthesis effect on RV, after 48 hours of the anti-biosynthesis effect of Lycorine on RV, select each drug group (40 μM, 50 μM, 60 μM), Ribavirin group, N group (normal cell control group), and RV group (virus control group). Remove the supernatant, rinse twice with PBS, add 1 mL of Trizol reagent, let it stand for 5 minutes, and collect it in a 1.5 mL enzyme-free EP tube. Add 200 μL of chloroform to the tube, vortex for 15 s, then let it stand at room temperature for 3 minutes, and centrifuge (4 °C, 12,000 r / min, 15 minutes). The centrifuged sample is divided into three layers, namely the colorless upper layer, the white middle layer, and the red lower layer.
[0060] ② Carefully aspirate the colorless upper layer into a new 1.5 mL enzyme-free EP tube (volume about 500 μL), add an equal volume of pre-cooled isopropanol, mix well by vigorously shaking up and down, let it stand at 4 °C for 10 minutes, and centrifuge (4 °C, 12,000 r / min, 10 minutes).
[0061] ③ After centrifugation, a white precipitate can be seen at the bottom of the EP tube. Remove the supernatant and retain the precipitate. Add 1 mL of the prepared 75% ethanol solution (prepared by mixing anhydrous ethanol and enzyme-free sterile water in a ratio of 3:1), shake well, centrifuge (4 °C, 12,000 r / min, 5 minutes), discard the supernatant, and let it stand at room temperature for 15 - 20 minutes to dry.
[0062] ④ After drying, add 20 μL of DEPC water to the EP tube, gently pipette along the tube wall to dissolve the RNA. After measuring the RNA concentration of the sample with a NanoDrop micro ultraviolet spectrophotometer, it can be directly used for subsequent experiments, or the sample can be stored at -80 °C for later use.
[0063] (2) Reverse transcription reaction of mRNA
[0064] ① Reaction to remove genomic DNA
[0065] Prepare the reaction mixture according to the composition in Table 3 on ice, with a reaction volume of 20 μL. The operation is carried out according to the instructions of Evo M-MLV RTKit with gDNA Clean for qPCR II. All consumables used in this experiment are Axygen enzyme-free consumables.
[0066] Table 3 Reaction system for removing genomic DNA
[0067]
[0068] Reaction conditions: 42 °C for 2 min; 4 °C.
[0069] *1: The amount of RNA can be added according to needs. In a 20 μL reverse transcription system, up to 1 μg of total RNA can be used; when using the probe method, up to 2 μg of total RNA can be used.
[0070] ② Reverse transcription reaction
[0071] Prepare the reaction solution according to the content in Table 4 and carry out the reverse transcription reaction.
[0072] Table 4 Reverse transcription reaction system
[0073]
[0074] Reaction conditions: 37 °C for 15 min; 85 °C for 5 sec; 4 °C.
[0075] (3) Real Time PCR reaction
[0076] Real-time quantitative PCR uses SYBR Green I fluorescence labeling to detect the VP6 expression levels in each drug group (40 μM, 50 μM, 60 μM), Ribavirin group, and RV group. Use Green Premix Pro Taq HS qPCRKit II kit, and GAPDH is selected as the internal reference. Use Axygen's special Real-time eight-strip PCR tubes for the experiment. Prepare the real-time fluorescence quantitative PCR amplification reaction system according to Table 5, and the operation of preparing the reaction solution is carried out on ice (the total reaction system is 10 μL). The qPCR reaction conditions are shown in Table 6; the primer sequences are shown in Table 7.
[0077] Table 5 PCR reaction system
[0078]
[0079] Table 6 qPCR reaction conditions
[0080]
[0081] Table 7 Primer Sequences
[0082]
[0083] As Figure 7 shown, compared with the RV group, the expression levels of VP6 in the Lycorine group were significantly decreased at 40 μM, 50 μM, and 60 μM, with statistically significant differences (p values were all less than 0.0001). The results indicate that Lycorine can play an anti-RV role by inhibiting the gene expression of VP6 through anti-RV biosynthesis.
[0084] Conclusion: Lycorine has an anti-RV biosynthesis effect, without anti-RV adsorption and direct inhibition effects. Its anti-RV biosynthesis effect can play an anti-RV role by inhibiting the expression of the structural protein VP6 gene.
[0085] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Use of lycorine in the preparation of anti-rotavirus drugs.
2. Use of lycorine in the preparation of anti-rotavirus biosynthesis drugs.
3. The application according to claim 1 or 2, characterized in that, The rotavirus is RV-Wa strain.