WO2024001265A1 - 麦冬皂苷d在制备抗轮状病毒药物中的应用 - Google Patents

麦冬皂苷d在制备抗轮状病毒药物中的应用 Download PDF

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WO2024001265A1
WO2024001265A1 PCT/CN2023/079429 CN2023079429W WO2024001265A1 WO 2024001265 A1 WO2024001265 A1 WO 2024001265A1 CN 2023079429 W CN2023079429 W CN 2023079429W WO 2024001265 A1 WO2024001265 A1 WO 2024001265A1
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rotavirus
saponin
ophiopogon
drug
cells
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PCT/CN2023/079429
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French (fr)
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赵文昌
宋丽军
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广东医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8968—Ophiopogon (Lilyturf)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of Ophiopogon saponin D, and specifically relates to the application of Ophiopogon saponin D in the preparation of anti-rotavirus drugs.
  • Rotavirus is a member of the reovirus family (Reoviridae). Its appearance is an icosahedral structure, and its three-layer concentric protein outer capsid surrounds a genome composed of 11 dsRNAs, responsible for encoding 6 species. Structural proteins and 6 non-structural proteins. Rotavirus is the main pathogen causing diarrhea in infants and young children. The highly contagious and pathogenic nature of rotavirus causes great harm to the entire society. About 130 million people are infected and about 200,000 people die every year around the world. So far, the mechanism of rotavirus infection has not been fully studied. Clinical treatment mainly relies on symptomatic treatment, and there is a lack of specific drugs that directly target rotavirus.
  • RotaTeq the only rotavirus vaccine currently on the market in the United States, is very expensive, and due to the variability and diversity of rotavirus strains, its range of preventable strains is limited. Therefore, how to prevent and treat rotavirus infection is a major problem. In order to reduce the infection rate and mortality of rotavirus, it is urgent to propose more research and application of specific drugs against rotavirus.
  • Ophiopogonin D is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. It has anti-inflammatory and antioxidant effects. However, there is currently no anti-rotavirus Ophiopogonin saponin D. According to reports on the effects of Ophiopogon japonicus, the structural formula of Ophiopogon saponin D is shown in Figure 1.
  • the purpose of the present invention is to propose the application of Ophiopogon saponin D in the preparation of anti-rotavirus drugs in order to solve the above-mentioned deficiencies in the prior art, and provide a new strategy for drug research or clinical treatment of rotavirus infection.
  • the invention provides the application of Ophiopogon saponin D in the preparation of anti-rotavirus drugs.
  • the present invention creatively discovers that Ophiopogon saponin D can be used to prepare anti-rotavirus drugs.
  • Ophiopogon saponin D has an obvious direct killing effect on rotavirus, and the pharmaceutical drugs of Ophiopogon saponin D are There is no drug toxicity to cells at concentrations between 0.4mM and 2mM.
  • the drug concentration of Ophiopogon saponin D is 0.6mM-1.8mM.
  • Ophiopogon saponin D has a direct killing effect on rotavirus.
  • the present invention uses the MA104 cell model to explore the effect of Ophiopogon saponin D on anti-rotavirus infection in vitro.
  • the experimental results show that the drug concentration of Ophiopogon saponin D is in the range of 1.2mM-1.8mM, and Ophiopogon saponin D is effective against rotavirus. It has an obvious direct killing effect, and when the drug concentration is 1.4mM, the inhibitory rate of Ophiopogon saponin D against rotavirus is as high as 79.8%.
  • Ophiopogon saponin D inhibited the expression of structural protein VP6 gene in rotavirus-infected MA104 cells.
  • the present invention explores the effect of Ophiopogon saponin D on the expression of structural protein VP6 gene in rotavirus-infected MA104 cells.
  • the experimental results show that when the drug concentrations of Ophiopogon saponin D are 0.6mM, 1.0mM and 1.4mM, the VP6 gene expression level were significantly reduced, with statistical differences, and there was a dose-effect correlation. This shows that Ophiopogon saponin D can exert its anti-rotavirus effect by inhibiting the gene expression of VP6.
  • the Ophiopogon saponin D inhibited the expression of RV-Wa VP6 viral protein in MA104 cells.
  • the Ophiopogon saponin D can reduce the gene and protein expression of VP6 in intestinal organoids infected by RV. That is, the anti-RV effect of Ophiopogon saponin D was further confirmed in the RV-infected mouse intestinal organoid model.
  • the invention provides an anti-rotavirus drug, the active ingredient of which includes Ophiopogon japonicus saponin D.
  • anti-rotavirus drug is a single or compound preparation of Ophiopogon saponin D.
  • the preparation form of the anti-rotavirus drug is selected from tablets, capsules, granules, powders, oral liquids, injections, films, suppositories, nasal drops, semi-solid preparations, injections, emulsions or sprays. any of them.
  • Ophiopogon saponin D provided by the present invention in the preparation of anti-rotavirus drugs reveals that Ophiopogon saponin D can be used in the preparation of anti-rotavirus drugs. This application can contribute to the development of safe and effective drugs to prevent and treat rotavirus. Provide new strategies for coronavirus infection.
  • the present invention uses the MA104 cell model to explore the effect of Ophiopogon saponin D on anti-rotavirus infection in vitro.
  • the results show that the drug concentration of Ophiopogon saponin D is in the range of 1.2mM-1.8mM, and Ophiopogon saponin D is effective against rotavirus. It has obvious direct killing effect, and when the drug concentration is 1.4mM, Ophiopogon japonicus
  • the inhibitory rate of saponin D against rotavirus is as high as 79.8%.
  • the present invention also explores the effect of Ophiopogon saponin D on the expression of structural protein VP6 gene in rotavirus-infected MA104 cells.
  • Ophiopogon saponin D when the drug concentration of Ophiopogon saponin D is 0.6mM, 1.0mM and 1.4mM, the VP6 gene expression levels are equal. Significant reduction, statistical difference, and dose-effect correlation. This shows that Ophiopogon saponin D can exert its anti-rotavirus effect by inhibiting the gene expression of VP6.
  • the present invention also verified that Ophiopogon saponin D inhibited the expression of RV-Wa VP6 viral protein in MA104 cells. Moreover, the anti-RV effect of Ophiopogon saponin D was further confirmed in the RV-infected mouse intestinal organoid model. Therefore, the present invention proposes using Ophiopogon saponin D as the main active ingredient, which is expected to develop safe and effective specific anti-rotavirus drugs, and provides guiding value for the clinical application of drugs for preventing and treating rotavirus infections.
  • Figure 1 is the chemical structural formula of Ophiopogon saponin D.
  • Figure 2 is a cell morphology diagram of normal MA104 cells under a microscope in Example 4.
  • Figure 3 is a cell morphology diagram of MA104 cells 48 hours after rotavirus infection under a microscope in Example 4.
  • Figure 4 is a graph showing the experimental results of the toxic effect of Ophiopogon saponin D on MA104 cells in Example 4.
  • Figure 5 is a graph showing the experimental results of the anti-rotavirus adsorption effect of Ophiopogon saponin D in Example 5.
  • Figure 6 is a graph showing the experimental results of the anti-rotavirus biosynthesis effect of Ophiopogon saponin D in Example 6.
  • Figure 7 is a graph showing the experimental results of the direct killing effect of Ophiopogon saponin D on rotavirus in Example 7.
  • Figure 8 is a graph showing the experimental results of the effect of Ophiopogon saponin D on the expression of the structural protein VP6 gene in rotavirus-infected MA104 cells in Example 8.
  • Figure 10 shows the effect of IF detection of OPD on RV-VP6 protein expression in Example 9.
  • Figure 11 is a growth cycle diagram of mouse intestinal organoid culture in Example 10.
  • Figure 12 shows Hoechst/PI staining of intestinal organoids of different ages infected with RV in Example 10 (red: dead cells, blue: nuclei).
  • Figure 15 shows the effect of IF detection OPD on RV-VP6 protein expression in Example 10, using anti-RV VP-6 protein antibodies (green) and DAPI (blue) for immunofluorescence staining.
  • the present invention provides the experimental materials, instruments, main practical preparation methods used in the following examples, and the statistical analysis methods of the experimental data in the following examples as follows. :
  • 2DMEM medium (containing 10% fetal bovine serum, 1% double antibody): Add 50ml fetal calf serum and 5ml double antibody to 400ml of high-sugar DMEM medium in a clean workbench, mix thoroughly and mark the device. Put it in a good 50ml centrifuge tube, seal it, and store it at 4°C.
  • EDTA-free trypsin EDTA-free 0.25% trypsin digestion solution and DMEM medium without fetal bovine serum were diluted to 10 ⁇ g/mL in a ratio of 1:250.
  • Rotavirus growth maintenance medium 10 ⁇ g/mL trypsin without EDTA and DMEM medium without fetal bovine serum diluted to 1 ⁇ g/mL in a ratio of 1:10.
  • the invention provides the application of Ophiopogon saponin D in the preparation of anti-rotavirus drugs.
  • the drug concentration of Ophiopogon saponin D is 0.6-1.8mM.
  • Ophiopogon saponin D has a direct killing effect on rotavirus.
  • Ophiopogon saponin D inhibited the expression of structural protein VP6 gene in rotavirus-infected MA104 cells.
  • the invention provides an anti-rotavirus drug, the active ingredient of which includes Ophiopogon japonicus saponin D.
  • anti-rotavirus drug is a single or compound preparation of Ophiopogon saponin D.
  • the preparation form of the anti-rotavirus drug is selected from tablets, capsules, granules, powders, oral liquids, injections, films, suppositories, nasal drops, semi-solid preparations, injections, emulsions or sprays. any of them.
  • the experimental method for rotavirus infection of MA104 cells in this example is as follows:
  • the CCK8 method is used to detect the toxicity of Ophiopogon saponin D to MA104 cells.
  • the experimental method is as follows:
  • this experiment conducted a cytotoxicity pre-experiment between 0.2mM and 2mM. Take MA104 cells in the logarithmic growth phase and observe them under a microscope. If the cells are uniform and plump in shape, with clear edges and the number reaches 80%, digest, centrifuge and resuspend them. After further diluting the suspension, take 10 ⁇ l and count it on a blood cell counting plate. Calculate required cell volume. Then the cells were plated in a 96-well plate, and 100 ⁇ l of cell suspension was added to each well. The cell density was 8 ⁇ 10 4 cells/mL. The drug was administered when the cells adhered to form a monolayer.
  • the relative survival rate formula of cells is: ⁇ (A experimental group -A blank )/(A control group -A blank ) ⁇ 100% ⁇
  • the cell morphology of normal MA104 cells described in this example and MA104 cells 48 hours after rotavirus infection under the microscope is shown in Figure 2-3.
  • the shape of normal MA104 cells is triangular or spindle-shaped, and the cell outline is clearly clear; the infection is rotavirus-shaped.
  • MA104 cells showed obvious changes in the cells, the cell boundaries became blurred, the distance between cells increased, and black particles increased in the cells. Finally, the cells completely fell off and floated.
  • the specific method for detecting the anti-rotavirus adsorption effect of Ophiopogon saponin D using the CCK8 method is as follows:
  • TCID50 virus the virus reacts with 10 ⁇ g/mL trypsin at 37°C for 30 min
  • 100 TCID50 virus the virus reacts with 10 ⁇ g/mL trypsin at 37°C for 30 min
  • cell maintenance solution 200 ⁇ L per well, incubate at 37°C and 5% CO2 for continuous observation.
  • CCK-8 kit was used for detection.
  • This example explores the anti-rotavirus adsorption effect of Ophiopogon saponin D.
  • the results are shown in Figure 5.
  • ⁇ represents P ⁇ 0.01, and ⁇ represents P ⁇ 0.0001.
  • the anti-rotavirus inhibition rate of the Ophiopogon saponin D group at 1.2mM, 1.4mM, and 1.6mM was significantly increased, with statistical differences, but the highest inhibition rate was only about 30%, indicating that Ophiopogon saponin D has no obvious anti-rotavirus adsorption effect.
  • This example uses the CCK8 method to detect the anti-rotavirus biosynthetic effect of Ophiopogon saponin D.
  • the specific method is as follows:
  • TCID50 virus liquid (30 mm of virus mixed with 10 ⁇ g/mL trypsin) into the 96-well culture plate of MA104-2 cells grown to a monolayer, 100 ⁇ L per well. The cells were washed twice with PBS before. Set the cells as normal control and add an equal volume of DMEM culture medium. Incubate at 37°C and 5% CO 2 for 2 hours, then aspirate the virus liquid, add different concentrations of medicinal solutions and Ribavirin, 100 ⁇ L per well, set up virus control, add only cell maintenance solution, 100 ⁇ L per well. Incubate at 37°C, 5% CO2 and observe continuously. After continuous culture for 48 hours, CCK-8 kit was used for detection. Add 1/10 volume of CCK-8 solution to each well, incubate in an incubator, and detect and record the absorbance at 450 nm wavelength after 1 hour. Calculate the virus inhibition rate of the drug and repeat the experiment three times.
  • the CCK8 method is used to detect the direct killing effect of Ophiopogon saponin D on rotavirus.
  • the specific method is as follows:
  • the direct killing activity is equivalent; the inhibitory rate against rotavirus is obvious at 1.2mM-1.8mM, and the inhibitory rate is the strongest at 1.4mM, reaching 79.8%, which is significantly higher than the Ribavirin group, and there is a significant statistical difference (P ⁇ 0.0001), indicating that Ophiopogon saponin D has an obvious direct killing effect on rotavirus.
  • Ribavirin group, N group and RV were selected.
  • Group remove the supernatant, wash twice with PBS, add 1ml Trizol reagent, let stand for 5 minutes, and collect in a 1.5mL enzyme-free EP tube.
  • RNA concentration of the sample After measuring the RNA concentration of the sample with the NanoDrop micro-volume UV spectrophotometer, it can be used directly for experiments or the sample can be stored at -80°C for later use.
  • reaction mixture Prepare the reaction mixture on ice according to the following ingredients.
  • the reaction volume is 20 ⁇ L.
  • the operation is carried out according to the instructions of Evo M-MLV RT Kit with gDNA Clean for qPCR II. All consumables used in this experiment were Axygen enzyme-free consumables.
  • RNA can be added as needed.
  • a maximum of 1 ⁇ g total RNA can be used; when using the probe method, a maximum of 2 ⁇ g total RNA can be used.
  • Real-time quantitative PCR was performed using SYBR Green I fluorescent label to detect the expression levels of VP6 in the drug group, Ribavirin group, N group and RV group. Green Premix Pro Taq HS qPCR Kit II, GAPDH is used as the internal reference. Use Axygen's special Real-time PCR plate for the experiment. Prepare the real-time fluorescence quantitative PCR amplification reaction system according to the following table 3-5. The reaction solution is prepared and operated on ice (the total reaction system is 10 ⁇ L).
  • This example aims to explore the effect of Ophiopogon saponin D on the expression of structural protein VP6 gene in rotavirus-infected MA104 cells.
  • the results are shown in Figure 8, where, compared with the RV group, * represents P ⁇ 0.05 and *** represents P ⁇ 0.001, **** represents P ⁇ 0.0001. It can be seen from the figure that the N group does not express VP6. Compared with the RV group, the VP6 gene expression in the Ribavirin group is significantly reduced, with a statistical difference (p ⁇ 0.0001), indicating that Ribavirin has an anti-rotavirus effect.
  • the expression of VP6 in the test drug group was significantly reduced at concentrations of 0.6mM, 1.0mM and 1.4mM, with statistical differences, and there was a dose-effect correlation.
  • the test drug group inhibited the expression of VP6 most significantly at 1.4mM (p ⁇ 0.0001), but there was no statistical difference compared with the Ribavirin group. This shows that Ophiopogon saponin D can exert anti-rotation by inhibiting the gene expression of VP6.
  • the role of viruses is compared with the RV group.
  • This example uses Western blotting and immunofluorescence staining to determine the effect of Ophiopogon saponin D on the expression of structural protein VP6 in RV-infected MA104 cells.
  • the specific steps are as follows:
  • the total protein in the cell sample was dissolved in 150 ⁇ l RIPA buffer (Beyotime Biotechnology, China) containing 50 ⁇ g/ml PMSF or 1 ⁇ g/ml phosphatase inhibitor, and then used with BCA protein assay kit (Thermo Scientific, MA, USA) Perform quantification.
  • BCA protein assay kit Thermo Scientific, MA, USA
  • PVDF polyvinylidene fluoride
  • IF Immunofluorescence
  • RV-Wa and OPD were incubated at 37°C for 2 hours and then inoculated on the MA104 cell monolayer. After washing the inoculum for 2 hours, they were incubated at 37°C for 24 hours respectively.
  • the cells were fixed and stained with anti- Immunofluorescence staining was performed with antibodies to RV VP-6 protein (green) and DAPI (blue), and MA104 cells were imaged through an Olympus IX70 fluorescence microscope.
  • This example explores the inhibitory effect of Ophiopogon saponin D on RV on intestinal organoids.
  • the specific steps are as follows:
  • the small intestines of C57Bl/6J mice aged 6 to 8 weeks were sacrificed, washed and cut into pieces, and the mild dissociation reagent GCDR (Stemcell, Canada) was added to dissociate at room temperature for 15 minutes to extract the crypt structure containing intestinal stem cells. Resuspend in complete medium and Matrigel. Finally, 60 ⁇ l droplets of the mixture containing crypts were placed in the center of each well of a 24-well plate and incubated at 37 °C with 5% CO for 15 min. After the droplets have solidified, add 700 ⁇ l of culture medium to each well.
  • RV-infected mouse intestinal organoids place the intestinal organoids in good growth status into 12-well centrifuge tubes, wash twice with ice-cold PBS, centrifuge at 900 rpm for 3 minutes, take 300 ⁇ L of activated RV and resuspend the small intestinal organoids. Incubate in a 37°C incubator for 2 hours. After centrifugation at 900 rpm for 3 minutes, wash three times with ice-cold PBS to remove free viruses, and inoculate the organoids into a 24-well plate for normal culture.
  • crypts were extracted from C57BL/6J mice and embedded in Matrigel to culture into intestinal organoids.
  • the growth cycle is shown in Figure 11. The results show that on the first day, the crypts formed a globular structure in Matrigel, and on the second day Germination began. On the third day, the number of crypt buds increased. On the fourth and fifth days, a characteristic rosette structure appeared. On the sixth day, the color of the intestinal lumen of the small intestinal organoids became darker and a complex multi-leaf structure appeared, forming a mature Intestinal organoids.
  • the intestinal organoids after administration were stained with positive immunofluorescence (IF) of viral protein VP6, as shown in Figure 15.
  • IF positive immunofluorescence
  • Ophiopogon saponin D can inhibit rotavirus infection of MA104 cells, and Ophiopogon saponin D in the drug concentration range of 1.2-1.8mM has obvious direct killing effect on rotavirus. killing effect.
  • Ophiopogon saponin D can exert anti-rotavirus effects by inhibiting the gene and protein expression of structural protein VP6.
  • the anti-RV effect of OPD was further confirmed in the RV-infected mouse intestinal organoid model. It shows that OPD can be used as an anti-RV drug candidate compound, which provides experimental basis for the development of OPD anti-RV drugs.
  • Ophiopogon saponin D can be used as an active ingredient, alone or in a compound with other active ingredients, using pharmaceutically acceptable excipients and conventional preparation methods to make tablets, capsules, granules, powders, oral Drugs for the prevention and treatment of rotavirus infection in various dosage forms such as liquids, injections, films, suppositories, nasal drops, semi-solid preparations, injections, emulsions or sprays are available for clinical use.

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Abstract

麦冬皂苷D在制备抗轮状病毒药物中的应用及包括麦冬皂苷D作为有效成分的抗轮状病毒药物，实验证实，在麦冬皂苷D的药物浓度为1.2mM-1.8mM范围内，麦冬皂苷D通过抑制MA104细胞中结构蛋白VP6基因表达而对轮状病毒有直接杀灭作用，并且对细胞没有药物毒性。

Description

麦冬皂苷D在制备抗轮状病毒药物中的应用 技术领域
本发明涉及麦冬皂苷D技术领域，具体涉及麦冬皂苷D在制备抗轮状病毒药物中的应用。
背景技术
轮状病毒(rotavirus，RV)是呼肠孤病毒家族(Reoviridae)的一员，其外表为二十面体形结构，三层同心蛋白外衣壳包围着由11条dsRNA构成的基因组，负责编码6种结构蛋白和6种非结构蛋白。轮状病毒是导致婴幼儿腹泻的主要病原体，轮状病毒的高度传染性和致病性对整个社会造成极大的危害，全球每年约有1.3亿人发生感染，死亡人数约20万。迄今为止，人们对轮状病毒感染机制研究还不够充分，临床上主要是对症治疗，缺乏直接针对轮状病毒的特效药物。目前唯一的一种在美国上市的轮状病毒疫苗RotaTeq，价格非常昂贵，且由于轮状病毒毒株的变异性、多样性使其预防毒株的范围有限。因此，如何防治轮状病毒感染是重大的难题，为了降低轮状病毒感染率和死亡率，亟需提出更多对抗轮状病毒的特效药物研究及应用。
麦冬皂苷D(ophiopogonin D,OPD)是一种从麦冬(Thunb.)Ker Gawl中提取的甾体皂苷，具有抗炎和抗氧化作用等，但目前尚无麦冬皂苷D抗轮状病毒的作用报道，麦冬皂苷D的结构式如附图1所示。
发明内容
本发明的目的是为了解决上述现有技术的不足提出了麦冬皂苷D在制备抗轮状病毒药物中的应用，为防治轮状病毒感染的药物研究或临床治疗提供了一种新策略。
为了实现上述目的，本发明采用如下技术方案：
本发明提供了麦冬皂苷D在制备抗轮状病毒药物中的应用。
本发明创造性地发现可以将麦冬皂苷D用于制备抗轮状病毒药物，通过体外实验研究发现，麦冬皂苷D对轮状病毒具有明显的直接杀灭作用，并且麦冬皂苷D的药物药物浓度在0.4mM-2mM之间对细胞均没有药物毒性。
进一步的，在所述抗轮状病毒药物中，所述麦冬皂苷D的药物浓度为0.6mM-1.8mM。
进一步的，麦冬皂苷D对轮状病毒具有直接杀灭作用。
本发明通过采用MA104细胞模型探究麦冬皂苷D对体外抗轮状病毒感染的作用，实验结果表明，麦冬皂苷D的药物浓度在1.2mM-1.8mM范围内，麦冬皂苷D对轮状病毒有明显的直接杀灭作用，且在药物浓度为1.4mM时，麦冬皂苷D对轮状病毒的抑制率高达79.8％。
进一步的，所述麦冬皂苷D抑制轮状病毒感染MA104细胞中结构蛋白VP6基因表达。
本发明探究了麦冬皂苷D对轮状病毒感染MA104细胞中结构蛋白VP6基因表达的影响，实验结果发现，麦冬皂苷D的药物浓度为0.6mM、1.0mM和1.4mM时，VP6基因表达量均明显降低，具有统计学差异，且存在剂量效应相关性。由此表明，麦冬皂苷D可通过抑制VP6的基因表达来发挥其抗轮状病毒作用。
进一步的，所述麦冬皂苷D抑制了MA104细胞中RV-Wa的VP6病毒蛋白的表达。
进一步的，所述麦冬皂苷D可降低RV感染肠道类器官的VP6的基因和蛋白表达。即麦冬皂苷D的抗RV作用也在RV感染小鼠肠道类器官模型上得到进一步证实。
本发明提供了一种抗轮状病毒药物，所述抗轮状病毒药物的有效成分包括麦冬皂苷D。
进一步的，所述抗轮状病毒药物为麦冬皂苷D的单方或复方制剂。
进一步的，所述抗轮状病毒药物的制剂形式选自片剂、胶囊剂、颗粒剂、粉剂、口服液、注射剂、膜剂、栓剂、滴鼻剂、半固体制剂、注射剂、乳剂或喷雾剂中的任意一种。
与现有技术相比，本发明的有益效果在于：
(1)本发明提供的麦冬皂苷D在制备抗轮状病毒药物中的应用，揭示了麦冬皂苷D能够应用于制备抗轮状病毒药物，该应用能够为开发安全有效的药物以防治轮状病毒感染提供新策略。
(2)本发明采用MA104细胞模型探究麦冬皂苷D对体外抗轮状病毒感染的作用，结果显示麦冬皂苷D的药物浓度在1.2mM-1.8mM范围内，麦冬皂苷D对轮状病毒有明显的直接杀灭作用，且在药物浓度为1.4mM时，麦冬 皂苷D对轮状病毒的抑制率高达79.8％。本发明还探究了麦冬皂苷D对轮状病毒感染MA104细胞中结构蛋白VP6基因表达的影响，结果显示麦冬皂苷D的药物浓度为0.6mM、1.0mM和1.4mM时，VP6基因表达量均明显降低，具有统计学差异，且存在剂量效应相关性。由此表明麦冬皂苷D可通过抑制VP6的基因表达来发挥其抗轮状病毒作用。本发明还验证了麦冬皂苷D抑制了MA104细胞中RV-Wa的VP6病毒蛋白的表达。并且，麦冬皂苷D的抗RV作用也在RV感染小鼠肠道类器官模型上得到进一步证实。因此，本发明提出了以麦冬皂苷D为主要活性成分，有望研制出安全有效的特异性抗轮状病毒药物，为防治轮状病毒感染药物临床应用提供了指导价值。
附图说明
图1是麦冬皂苷D的化学结构式。
图2是实施例4中显微镜下正常MA104细胞的细胞形态图。
图3是实施例4中显微镜下轮状病毒感染48h后的MA104细胞的细胞形态图。
图4是实施例4中麦冬皂苷D对MA104细胞的毒性作用的实验结果图。
图5是实施例5中麦冬皂苷D抗轮状病毒吸附作用的实验结果图。
图6是实施例6中麦冬皂苷D抗轮状病毒生物合成作用的实验结果图。
图7是实施例7中麦冬皂苷D对轮状病毒的直接杀灭作用的实验结果图。
图8是实施例8中麦冬皂苷D对轮状病毒感染MA104细胞中结构蛋白VP6基因表达的影响的实验结果图。
图9是实施例9中western blot检测OPD对RV-VP6蛋白表达的影响(n＝3)。
图10是实施例9中IF检测OPD对RV-VP6蛋白表达的影响。
图11是实施例10中小鼠肠道类器官培养的生长周期图。
图12是实施例10中Hoechst/PI染色RV感染不同天龄的肠道类器官，(红色：死细胞，蓝色：细胞核)。
图13是实施例10中qPCR检测OPD对RV-VP6基因表达的影响(n＝6)。
图14是实施例10中WB检测OPD对RV-VP6蛋白表达的影响(n＝6)。
图15是实施例10中IF检测OPD对RV-VP6蛋白表达的影响，用抗RV VP-6蛋白的抗体(绿色)和DAPI(蓝色)进行免疫荧光染色。
具体实施方式
为了便于本领域技术人员的理解，下面结合实施例对本发明作进一步的说明，实施方式提及的内容并非对本发明的限定。
为了使本发明的目的、技术方案和技术效果更加清楚，本发明提供下述实施例所用到的实验材料、仪器、主要实际的配制方法以及下述实施例中实验数据的统计分析方法作如下说明：
(1)实验细胞株和试剂
(2)主要仪器
(3)主要试剂的配制方法
①麦冬皂苷D母液：称取麦冬皂苷D标准品8.6mg，纯度≥98％,批号：P26F9F54695，在超净台内加1ml细胞级DMSO溶解，用一次性无菌针管将药液通过0.22μM滤膜过滤除菌，配置母液为10mM。
②DMEM培养基(含10％胎牛血清、1％双抗)：在超净工作台内将50ml胎牛血清和5ml双抗依次加入400ml的高糖DMEM培养基中，充分混匀后分装置标记好的50ml离心管中，封口，4℃保存。
③10μg/mL不含EDTA的胰酶：不含EDTA的0.25％胰蛋白酶消化液和不含胎牛血清的DMEM培养基以1：250的比例稀释至10μg/mL。
④轮状病毒生长维持液：10μg/mL不含EDTA的胰酶和不含胎牛血清的DMEM培养基以1：10的比例稀释至1μg/mL。
(4)统计数据分析
本发明下述实施例的所有实验均重复三次。实验数据采用均数±标准差表示。采用SPSS软件统计数据，两组间比较采用t检验，多组间均数的比较采用单因素方差分析。得到的数据结果以平均值±标准差的形式表示。数据分析采用统计分析软件Graphpad prism8.01进行Mann–Whitney统计。(#P<0.05，##P<0.01，###P<0.001表示与N组相比；△△P<0.01，△△△△P<0.0001表示与Ribavirin组相比；*P<0.05，**P<0.01，***P<0.001，****P<0.0001表示与RV组相比)。
实施例1
本发明提供了麦冬皂苷D在制备抗轮状病毒药物中的应用。
进一步的，在所述抗轮状病毒药物中，所述麦冬皂苷D的药物浓度为0.6-1.8mM。
进一步的，麦冬皂苷D对轮状病毒具有直接杀灭作用。
进一步的，所述麦冬皂苷D抑制轮状病毒感染MA104细胞中结构蛋白VP6基因表达。
实施例2
本发明提供了一种抗轮状病毒药物，所述抗轮状病毒药物的有效成分包括麦冬皂苷D。
进一步的，所述抗轮状病毒药物为麦冬皂苷D的单方或复方制剂。
进一步的，所述抗轮状病毒药物的制剂形式选自片剂、胶囊剂、颗粒剂、粉剂、口服液、注射剂、膜剂、栓剂、滴鼻剂、半固体制剂、注射剂、乳剂或喷雾剂中的任意一种。
实施例3
本实施例中轮状病毒感染MA104细胞的实验方法如下：
将病毒置于37℃水浴锅中融化，经过胰蛋白酶预处理30min后，将病毒溶液加入到生长到80～90％的MA104细胞中，轮状病毒感染所用的细胞培养基中不添加血清。病毒感染后的MA104细胞会发生细胞病变CPE(Cytopathic  effect)，当病变程度达到75％时，-20℃冰箱中冻存。重复冻存和融化过程3次后，低温离心，收取上清，即为所得病毒液。重复上述过程，扩增轮状病毒。
实施例4
本实施例采用CCK8法检测麦冬皂苷D对MA104细胞的毒性的实验方法如下：
根据麦冬皂苷D常用药物浓度范围，本实验在0.2mM-2mM之间进行细胞毒性预实验。取对数生长期的MA104细胞于显微镜下观察，细胞形态均一饱满，边缘清晰且数量达到80％时，进行消化、离心和重悬，混悬液进一步稀释后取10μl于血细胞计数板上计数，计算所需细胞体积。随后在96孔板中细胞铺板，每孔加100μl细胞混悬液，细胞密度为8×104个/mL。待细胞贴壁成单层时给药。孵育48h后，CCK-8试剂盒检测麦冬皂苷D的细胞毒性。每孔加入1/10体积的CCK-8溶液，培养箱中孵育，1h后于450nm波长下检测并记录吸光度。细胞的相对存活率公式为：
{(A实验组-A空白)/(A对照组-A空白)×100％}
本实施例所述正常MA104细胞和轮状病毒感染48h后的MA104细胞在显微镜下的细胞形态如图2-3所示，正常MA104细胞形状为三角形或梭形，细胞轮廓明显清晰；感染轮状病毒后的MA104细胞，细胞发生明显病变，细胞边界变得模糊，细胞间距离增大，细胞内黑色颗粒增加，最后细胞完全脱落，漂浮。
本实施例探究麦冬皂苷D对MA104细胞的毒性结果如图4所示，其中，N代表未用药组，与N组比较，#代表P＜0.05。麦冬皂苷D的药物浓度在0.4mM-2mM之间，对细胞均没有药物毒性且平均存活率为100％以上，表明麦冬皂苷D对细胞具有一定的生长促进作用。与N组相比，麦冬皂苷D在药物浓度为0.4mM和2mM时，有促细胞生长作用，且具有统计学差异(P＜0.05)。而在0.6mM-1.8mM时检测药物组细胞活力与N组无明显差异。因此我们选取的麦冬皂苷D的实验药物浓度为0.6mM-1.8mM。
实施例5
本实施例采用CCK8法检测麦冬皂苷D抗轮状病毒吸附作用的具体方法如下：
在生长至单层的MA104细胞的96孔培养板内加入药液，每个药液重复6孔，每孔100μL。阳性对照组中加入等体积的利巴韦林(Ribavirin)，正常细胞对照组、病毒对照组均只加入等体积DMEM培养液(不含血清)。37℃、5％CO2孵育2h。将药液吸出，除正常细胞对照组以外加入100TCID50的病毒(病毒与10μg/mL胰酶37℃作用30min)，每孔100μL，37℃、5％CO2孵育2h，将病毒吸出，加入细胞维持液，每孔200μL，37℃、5％CO2孵育连续观察。培养48h后用CCK-8试剂盒检测。每孔加入1/10体积的CCK-8溶液，培养箱中孵育，1h后于450nm波长下检测并记录吸光度，重复实验3次。
本实施例探究麦冬皂苷D的抗轮状病毒吸附作用结果如图5所示，其中，与Ribavirin组比较，△△代表P<0.01，△△△△代表P<0.0001。与Ribavirin组相比，麦冬皂苷D组在1.2mM、1.4mM、1.6mM下抗轮状病毒的抑制率有明显的升高，有统计学差异，但最高抑制率仅为30％左右，表明麦冬皂苷D无明显抗轮状病毒的吸附的作用。
实施例6
本实施例采用CCK8法检测麦冬皂苷D抗轮状病毒生物合成作用的具体方法如下：
将100TCID50病毒液(病毒与10μg/mL胰酶作用30mm)加入到生长至单层的MA104-2细胞96孔培养板内，每孔100μL，之前用PBS将细胞冲洗2次。设细胞正常对照，加入等体积DMEM培养液。37℃、5％CO2孵育2h，然后将病毒液吸出，分别加入不同浓度药液和Ribavirin，每孔100μL，设病毒对照，只加入细胞维持液，每孔100μL。37℃、5％CO2孵育连续观察。连续培养48h后，用CCK-8试剂盒检测。每孔加入1/10体积的CCK-8溶液，培养箱中孵育，1h后于450nm波长下检测并记录吸光度。计算药物的病毒抑制率，重复实验3次。
本实施例探究麦冬皂苷D的抗轮状病毒生物合成作用如图6所示。与Ribavirin组相比，各麦冬皂苷D组对轮状病毒抑制率较低，统计学比较无显著性差异，表明麦冬皂苷D无明显抗轮状病毒生物合成的作用。
实施例7
本实施例采用CCK8法检测麦冬皂苷D对轮状病毒的直接杀灭作用的具体方法如下：
将药物与100TCID50的病毒液(病毒与10μg/mL胰酶作用30min)等体积混合作用2h。用PBS将细胞冲洗2次后将其加入到生长至单层MA104细胞的96孔培养板内。阳性对照组中将Ribavirin与轮状病毒进行与上述相同的操作，正常细胞对照组、病毒对照组均只加入等体积DMEM。37℃、5％CO2孵育2h，然后将混合液吸出，加入细胞维持液，每孔200μL，37℃、5％CO2孵育连续观察，培养48h后用CCK-8试剂盒检测。每孔加入1/10体积的CCK-8溶液，培养箱中孵育，1h后于450nm波长下检测并记录吸光度。计算药物的病毒抑制率，重复实验3次。
本实施例探究麦冬皂苷D对轮状病毒直接杀灭作用结果如图7所示，其中，与Ribavirin组比较，△△代表P<0.01，△△△代表P<0.001，△△△△代表P<0.0001。与Ribavirin组相比，麦冬皂苷D在0.8mM、1mM时对轮状病毒的抑制率分别为60.9％、65.9％，统计学比较无明显差异，表明麦冬皂苷D与Ribavirin对轮状病毒的直接杀灭活性相当；在1.2mM-1.8mM时对轮状病毒的抑制率明显，且在1.4mM时抑制率最强，达到79.8％，明显高于Ribavirin组，统计学比较有明显差异(P<0.0001)，表明麦冬皂苷D对轮状病毒有明显的直接杀灭作用。
实施例8
本实施例采用qPCR检测轮状病毒结构蛋白VP6基因的表达量的具体方法如下：
(1)总RNA的提取及定量
①为了进一步验证麦冬皂苷D是否对轮状病毒有直接杀灭作用，麦冬皂苷D对轮状病毒直接杀灭作用48h后，选择高、中、低药物组、Ribavirin组、N组和RV组，去上清液，PBS清洗两次，添加1ml Trizol试剂，静置5min，收集于1.5mL的无酶EP管中。向管中加入200μL的氯仿，涡旋震荡15s，后室温静置3min，离心(4℃，12000r/min，15min)，离心后的样品分三层，即无色的上层、白色中间层和红色的下层。
②小心吸取上清液至新的1.5mL无酶的EP管中(体积约500μL)，加入等体积的预冷的异丙醇，上下剧烈震荡混匀，4℃静置10min，离心(4℃，12000r/min，10min)
③离心后EP管底部可见白色沉淀，去上清保留沉淀，加入配制好的75％乙醇溶液(用无水乙醇和无酶水按照3：1比例配制)1mL，摇晃混匀，离心(4℃，12000r/min，5min)，弃去上清液，室温放置15min-20min，晾干。
④晾干后向EP管中加入20μL DEPC水，轻轻吹打管壁溶解RNA。NanoDrop微量紫外分光光度计测定样品的RNA浓度后可直接用于实验或-80℃保存样品备用。
(2)mRNA的逆转录反应
①去除基因组DNA反应
在冰上按照下列成分配制反应混合液，反应体积为20μL，操作按照Evo M-MLV RT Kit with gDNA Clean for qPCR II说明书进行。该实验下所用耗材皆为Axygen无酶耗材。
表1去基因组DNA反应体系
反应条件：42℃ 2min
4℃
*1：RNA量可根据需要添加。在20μL反转录体系中，最多使用1μg total RNA；使用探针法时，最多使用2μg total RNA。
②反转录反应
按照下表2内容进行反应液的配制，进行反转录反应。
表2反转录反应体系

反应条件：37℃ 15min
85℃ 5sec
4℃
(3)Real Time PCR反应
实时定量PCR的采用SYBR Green I荧光标记，检测药物组、Ribavirin组、N组和RV组的VP6表达水平，采用Green Premix Pro Taq HS qPCR Kit II试剂盒，内参选用GAPDH。实验用Axygen专用的Real-time PCR板，按照下列下表3-5配制实时荧光定量PCR扩增反应体系，反应液配制在冰上进行操作(反应总体系为10μL)。
表3 PCR反应体系
表4 qPCR反应条件
表5引物序列

本实施例旨在探究麦冬皂苷D对轮状病毒感染MA104细胞中结构蛋白VP6基因表达的影响，结果如图8所示，其中，与RV组比较，*代表P<0.05，***代表P<0.001，****代表P<0.0001。由图可知，N组不表达VP6，与RV组相比，Ribavirin组VP6基因表达明显降低，有统计学差异(p<0.0001)，表明Ribavirin具有抗轮状病毒的作用。与RV组相比，检测药物组在0.6mM、1.0mM和1.4mM浓度下的VP6表达量均明显降低，有统计学差异，且存在剂量效应相关性。其中，检测药物组在1.4mM时抑制VP6的表达最为显著(p<0.0001)，但与Ribavirin组相比无统计学差异，由此表明麦冬皂苷D可通过抑制VP6的基因表达发挥抗轮状病毒的作用。
实施例9
本实施例采用蛋白质印迹法和免疫荧光染色测定麦冬皂苷D对RV感染MA104细胞中结构蛋白VP6表达的影响，具体步骤如下：
将细胞样品中的总蛋白质溶于150μl含有50μg/ml PMSF或1μg/ml磷酸酶抑制剂的RIPA缓冲液(Beyotime Biotechnology，China)中，然后用BCA蛋白质测定试剂盒(Thermo Scientific，MA，USA)进行定量。我们用等量的蛋白质(40μg)洗涤样品，用10％SDS-PAGE分离，然后转移到聚偏二氟乙烯(PVDF)膜(Millipore，MA，USA)上。然后，我们在室温下使用2.5％脱脂奶粉(Merck)在PBST中封闭膜120分钟，然后在4℃下用指定的一级抗体(VP6抗体)孵育过夜。接下来，我们在室温下用山羊抗兔/小鼠IgG(H+L)/TRITC二级抗体孵育膜2小时。我们在奥德赛红外成像系统(LI-COR Biotechnology，Lincoln，NE，USA)上显示了蛋白质带，所有选项均设置为制造商默认值。所有实验一式三份。
免疫荧光染色(Immunofluorescence,IF)检测OPD对RV-VP6蛋白表达的影响。在37℃下将RV-Wa和OPD孵育2小时后接种于MA104细胞单层上，作用2h洗涤接种物后，然后分别在37℃下孵育24h，固定细胞，用抗 RV VP-6蛋白的抗体(绿色)和DAPI(蓝色)进行免疫荧光染色，通过Olympus IX70荧光显微镜对MA104细胞进行成像。
结果如图9、图10所示，与RV组比较，OPD不同浓度组RV-VP6蛋白的水平表达降低，统计学比较有明显差异，且以剂量依赖的方式降低了RV-VP6蛋白的水平表达。免疫荧光染色(IF)实验的结果证实，与RV组比较，OPD抑制了MA104细胞中RV-Wa的VP6病毒蛋白的表达。
实施例10
本实施例探究麦冬皂苷D在肠道类器官上对RV的抑制作用，具体步骤如下：
将6-8周的C57Bl/6J小鼠处死后取其小肠，清洗后剪碎，加入温和的解离试剂GCDR(Stemcell，Canada)室温解离15min，提取其含有肠道干细胞的隐窝结构，用完全培养基和Matrigel基质胶重悬。最后将含有隐窝的混合物液滴60μl置于24孔板的每个孔的中心，在37℃下用5％CO2孵育15分钟。液滴固化后，每个孔加入700μl培养基。
RV感染小鼠肠道类器官，取生长状态良好的肠道类器官12孔于离心管中，用冰的PBS洗涤两次，900rpm，3min离心，取300μL活化后的RV重悬小肠类器官，于37℃培养箱中孵育2h。900rpm，3min离心后，以冰的PBS洗涤三次去除游离的病毒，类器官接种至24孔板正常培养。
首先从C57BL/6J小鼠中提取隐窝包埋在基质胶中培养成肠道类器官，生长周期如图11，结果表明，第一天，隐窝在基质胶中形成球状结构，第二天开始萌芽，第三天隐窝出芽数增多，第四第五天呈现出特征性的花环结构，在第六天小肠类器官肠腔中央颜色变深，出现复杂的多叶结构，形成了成熟的肠道类器官。
将RV感染不同天龄的肠道类器官后进行Hoechst/PI染色，发现不同天数的肠道类器官都出现了凋亡现象，说明RV可感染肠道类器官，并引起肠道类器官凋亡(图12)。
本实验选择传代后的类器官感染RV，将RV-WA株和OPD在37℃下共同孵育2小时后作用于肠道类器官2小时，彻底清洗类器官以去除残留的病毒颗粒，随后将其重新嵌入Matrigel胶中24小时，分别提取肠道类器官RNA 和蛋白，通过qPCR和Western blot检测OPD对RV基因组RNA的合成和病毒蛋白表达的影响，结果如图13、图14所示。结果表明OPD可降低RV感染肠道类器官的VP6的基因和蛋白表达，有统计学差异。
为了进一步确认OPD的作用，用病毒蛋白VP6的阳性免疫荧光(IF)对给药后的肠道类器官进行染色后如图15所示，OPD处理组VP6免疫荧光明显降低。
由上述实施例1-10的结果可以判定，麦冬皂苷D能够抑制轮状病毒感染MA104细胞，并且在药物浓度范围为1.2-1.8mM内的麦冬皂苷D对轮状病毒有明显的直接杀灭作用。另外，麦冬皂苷D可通过抑制结构蛋白VP6基因和蛋白表达发挥抗轮状病毒的作用。同时OPD的抗RV作用也在RV感染小鼠肠道类器官模型上得到进一步证实。显示了OPD可作为抗RV药物候选化合物，为开发OPD抗RV药物提供了实验依据。上述结果表明，麦冬皂苷D能够作为活性成分，单独应用或与其它活性组分组成复方，采用药学上可接受的辅料和制剂常规方法，制成片剂、胶囊剂、颗粒剂、粉剂、口服液、注射剂、膜剂、栓剂、滴鼻剂、半固体制剂、注射剂、乳剂或喷雾剂等各种剂型的防治轮状病毒感染药物，供临床使用。
上述的具体实施例是对本发明技术方案和有益效果的进一步说明，并非对实施方式的限定。对本领域技术人员来说，在不脱离本发明构思的前提下任何显而易见的替换均在本发明的保护范围之内。

Claims (7)

  1. 麦冬皂苷D在制备抗轮状病毒药物中的应用。
  2. 根据权利要求1所述的麦冬皂苷D在制备抗轮状病毒药物中的应用，其特征在于：在所述抗轮状病毒药物中，所述麦冬皂苷D的药物浓度为0.6mM-1.8mM。
  3. 根据权利要求1所述的麦冬皂苷D在制备抗轮状病毒药物中的应用，其特征在于：麦冬皂苷D对轮状病毒具有直接杀灭作用。
  4. 根据权利要求1所述的麦冬皂苷D在制备抗轮状病毒药物中的应用，其特征在于：所述麦冬皂苷D抑制轮状病毒感染MA104细胞中结构蛋白VP6基因表达。
  5. 一种抗轮状病毒药物，其特征在于：所述抗轮状病毒药物的有效成分包括麦冬皂苷D。
  6. 根据权利要求5所述的抗轮状病毒药物，其特征在于：所述抗轮状病毒药物为麦冬皂苷D的单方或复方制剂。
  7. 根据权利要求5所述的抗轮状病毒药物，其特征在于：所述抗轮状病毒药物的制剂形式选自片剂、胶囊剂、颗粒剂、粉剂、口服液、注射剂、膜剂、栓剂、滴鼻剂、半固体制剂、注射剂、乳剂或喷雾剂中的任意一种。
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