GB2625845A

GB2625845A - Use of ophiopogonin D in preparing anti-rotavirus medicament

Info

Publication number: GB2625845A
Application number: GB2307811.6A
Authority: GB
Inventors: Zhao Wenchang; Song Lijun
Original assignee: Guangdong Medical University
Current assignee: Guangdong Medical University
Priority date: 2022-06-28
Filing date: 2023-03-03
Publication date: 2024-07-03
Also published as: GB202307811D0

Abstract

Provided are use of ophiopogonin D in preparing an anti-rotavirus medicament and an anti-rotavirus medicament comprising ophiopogonin D as the active ingredient. Studies demonstrate that within a concentration range of 1.2 mM to 1.8 mM, ophiopogonin D has a direct killing effect on rotavirus by inhibiting the gene expression of a structural protein VP6 in MA104 cells, and has no toxicity to the cells.

Description

APPLICATION OF OPHIOPOGONIN D IN PREPARATION OF

ANTI-ROTAVIRUS DRUGS

Technical Field

The present invention relates to the technical field of ophiopogonin D, and in particular to an application of ophiopogonin D in preparation of anti-rotavirus drugs.

Background

Rotavirus (RV) is a member of Reoviridae family, has an icosahedral structure in appearance, is a genome formed by enclosing 11 pieces of dsRNA by three layers of concentric protein outer capsid, and is responsible for encoding 6 kinds of structural proteins and 6 kinds of non-structural proteins. The rotavirus is a major pathogen that causes infantile diarrhea; high infectiousness and pathogenicity of the rotavirus cause a great harm to the whole society; and there are about 130 million people infected and about 200 thousand deaths every year all over the world. So far, researches on the mechanism of rotavirus infection are not enough; symptomatic treatment is mainly used clinically; and there is a lack of specific drugs directly against the rotavirus. As the only rotavirus vaccine currently marketed in the United States, RotaTeq is very expensive and is limited in range of virus strains prevented due to the variability and the diversity of rotavirus strains. Therefore, how to prevent and treat rotavirus infection is a great problem. In order to lower the infection rate and the death rate of the rotavirus, there is an urgent need for providing more researches and applications of specific drugs against the rotavirus.

Ophiopogonin D (OPD) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl, and has the anti-inflammatory effect, antioxidation and the like. However, there is no report on the effect of the ophiopogonin D

Description

against the rotavirus at present. A structural formula of the ophiopogonin D is shown in Fig. 1.

Summary

A purpose of the present invention is to provide an application of ophiopogonin D in preparation of anti-rotavirus drugs for solving the above deficiencies in the prior art, which provides a new strategy for drug researches or clinical treatment of rotavirus infection.

In order to achieve the above purpose, the present invention uses the following technical solution: The present invention provides an application of ophiopogonin D in preparation of anti-rotavirus drugs.

The present invention creatively finds that the ophiopogonin D may be used for preparing the anti-rotavirus drugs. It is found through in vitro experimental researches that the ophiopogonin D has an obvious direct killing effect on rotavirus; and a drug concentration of the ophiopogonin D is 0.4 mM to 2 mM, which has no drug toxicity to cells.

Further, in the anti-rotavirus drugs, the drug concentration of the ophiopogonin D is 0.6 mM to 1.8 mM.

Further, the ophiopogonin D has a direct killing effect on the rotavirus The present invention explores the effect of the ophiopogonin D on anti-rotavirus infection in vitro using an MA104 cell model. Experimental results show that when the drug concentration of the ophiopogonin D is within a range from 1.2 mM to 1.8 mM, the ophiopogonin D has an obvious direct killing effect on the rotavirus; and if the drug concentration is 1.4 mM, the inhibition rate of the ophiopogonin D to the rotavirus is up to 79.8%.

Further, the ophiopogonin D inhibits gene expression of a structural protein VP6 in MA104 cells infected with the rotavirus.

Description

The present invention explores the effect of the ophiopogonin D on gene expression of the structural protein VP6 in the MA104 cells infected with the rotavirus. Experimental results show that when the drug concentrations of the ophiopogonin D are 0.6 mM, 1.0 mM and 1.4 mM, a VP6 gene expression level is obviously lowered; and there is a statistical difference and dose-effect correlation. Thus, it shows that the ophiopogonin D may exert the anti-rotavirus effect by inhibiting gene expression of VP6.

Further, the ophiopogonin D inhibits expression of a VP6 viral protein of RV-Wa in the MA104 cells.

Further, the ophiopogonin D may reduce gene and protein expression of VP6 in intestinal organoids infected with the RV. That is, the anti-RV effect of the ophiopogonin D is further confirmed in an RV-infected mouse intestinal organoid model.

The present invention provides an anti-rotavirus drug, and the active component of the anti-rotavirus drug comprises the ophiopogonin D. Further, the anti-rotavirus drug is a monomer or compound preparation of the ophiopogonin D. Further, a form of the preparation of the anti-rotavirus drug is selected from any one of a tablet, a capsule, granules, powder, an oral liquid, an injection, a film, a suppository, nasal drops, a semi-solid preparation, an emulsion or a spray.

Compared with the prior art, the present invention has the beneficial effects: (1) The application of the ophiopogonin D in preparation of the anti-rotavirus drugs provided by the present invention discloses that the ophiopogonin D can be applied to preparation of the anti-rotavirus drugs, and the application can provide a new strategy for developing the safe and effective drugs to prevent and control rotavirus infection.

(2) The present invention explores the effect of the ophiopogonin D on anti-rotavirus infection in vitro using the MA104 cell model. Results show that when the drug concentration of the ophiopogonin D is 1.2 mM to 1.8 mM, the ophiopogonin D has an obvious direct killing effect on the rotavirus; and when the

Description

drug concentration is 1.4 mM, the inhibition rate of the ophiopogonin D to the rotavirus is up to 79.8%. The present invention further explores the effect of the ophiopogonin D on gene expression of the structural protein VP6 in the MA104 cells infected with the rotavirus. Results show that when the drug concentrations of the ophiopogonin D are 0.6 mM, 1.0 mM and 1.4 mM, a VP6 gene expression level is obviously lowered; and there is a statistical difference and dose-effect correlation. Thus, it shows that the ophiopogonin D may exert the anti-rotavirus effect by inhibiting gene expression of VP6. The present invention further verifies that the ophiopogonin D inhibits expression of the VP6 viral protein of RV-Wa in the MA104 cells. Moreover, the anti-RV effect of the ophiopogonin D is further confirmed in the RV-infected mouse intestinal organoid model. Therefore, the present invention proposes an expectation of developing safe and effective specific anti-rotavirus drugs with the ophiopogonin D as a main active component, and provides a guide value to clinical applications of the anti-rotavirus drugs.

Description of Drawings

Fig. 1 shows a chemical structural formula of ophiopogonin D Fig. 2 is a cell morphology diagram of normal MA104 cells under a microscope in embodiment 4.

Fig. 3 is a cell morphology diagram of MA104 cells after being infected with rotavirus for 48 h under a microscope in embodiment 4.

Fig. 4 is an experimental result diagram of the toxic effect of ophiopogonin D on MA104 cells in embodiment 4.

Fig. 5 is an experimental result diagram of the effect of ophiopogonin D of resisting rotavirus adsorption in embodiment 5 Fig. 6 is an experimental result diagram of the effect of ophiopogonin D of resisting rotavirus biosynthesis in embodiment 6.

Fig. 7 is an experimental result diagram of the effect of ophiopogonin D of directly killing rotavirus in embodiment 7.

Description

Fig. 8 is an experimental result diagram of the effect of ophiopogonin D on gene expression of a structural protein VP6 in MA104 cells infected with rotavirus in embodiment 8.

Fig. 9 shows the effect of Western blot detection for OPD on expression of an RV-VP6 protein in embodiment 9 (n=3).

Fig. 10 shows the effect of IF detection for OPD on expression of an RV-VP6 protein in embodiment 9.

Fig. 11 is a growth period diagram of mouse intestinal organoid culture in embodiment 10.

Fig. 12 shows intestinal organoids infected with Hoechst/PI stained RV for different days in embodiment 10 (red: dead cells; and blue: cell nuclei).

Fig. 13 shows the effect of ciPCR detection for OPD on gene expression of RV-VP6 in embodiment 10 (n=6).

Fig. 14 shows the effect of WB detection for OPD on expression of an RV-VP6 protein in embodiment 10 (n=6).

Fig. 15 shows the effect of IF detection for OPD on expression of an RV-VP6 protein in embodiment 10, wherein immunofluorescent staining is performed with an antibody (green) against an anti-RV VP-6 protein and DAPI (blue).

Detailed Description

For convenience in understanding by those skilled in the art, the present invention is further described below in combination with the embodiments, and the contents mentioned in the implementations are not the limitation to the present invention.

In order to make the purpose, the technical solutions and the technical effects of the present invention more clear, the present invention provides experimental materials, instruments and main practical preparation methods used in the following embodiments as well as statistical analysis methods of experimental data in the following embodiments, as described below:

Description

(1) Experimental cell strains and reagents Material name Manufacturer Ophiopogonin D (OPD) with a batch number Shanghai Yuanye Biotechnology Co., LTD.

of P26F9F54695 MA104 cell strain Cell bank of Zhongshan University Rotavirus-Wa strain (RV-Wa strain) Institute of Immunization of Third Military Medical University xPBS phosphate buffer Jiangshu Beyotime Biotechnology institute Cellular level dimethyl sulfoxide America GIBCO Company Fetal calf scrum America GIBCO Company High glucose DMEM culture fluid America GIBCO Company 0.25% trypsin solution Beijing Solarbio Company 0.25% trypsin solution containing EDTA Beijing Solarbio Company Penicillin-streptomycin (double antibody) Beijing Solarbio Company Co-focus culture dish Beijing Labgic Technology Co., LTD.

Pipette tip Shanghai Kirgen Biotiniotechnology Co., LTD.

Cell culture flask American Coming-Coster Company Cell culture dish American Coming-Coster Company Culture plate American Coming-Coster Company C57BL/6J mouse Animal Experimental Center of South Medical University VP6 antibody against rotavirus Abeam (Cambridge, UK) (2) Main Instruments Instrument name Manufacturer Ordinary optical microscope Japan OLYMPUS Company Inverted fluorescent microscope Japan NIKON Company Micropipette German Eppendorf Company -20°C refrigerator Midea Group -80°C ultra-low temperature refrigerator Thermo Scientific Company

Description

CO2 incubator Thermo Scientific Company High-pressure sterilizer Japan Sanyo Company Clean bench Suzhou Antai Air Technology Co., Ltd. Electric-heated thermostatic water bath Shanghai Yiheng Technology Co., Ltd Multimode reader Gene Company Linited Nucleic acid-protein determinator German Eppendoif Company Living cell imager Thermo Scientific Company Illumination incubator Shanghai Yiheng Technology Co., Ltd Thermal cycler T100 Bio-Rad Company PCR analyzer Thermo Scientific Company High-speed freezing centrifuge German Eppendorf Company Vortex oscillator Ika Instruments and Equipment Co Ltd. Liqud nitrogen container CHART Biotechnology Company (3) Preparation method for main reagents 0 Ophiopogonin D mother solution: 8.6 mg of an ophiopogonin D standard substance with a purity larger than or equal to 98% and a batch number of P26F9F54695 was weighed and dissolved with 1 ml of cellular level DMSO in a super clean bench; a medical solution was dropped on 0.22R1v1 filter membrane with a disposable sterile needle for filtration sterilization; and 10 nM of a mother solution was prepared.

0 DMEM culture medium (containing 10% fetal calf serum and 1% double antibody): 50 ml of a fetal calf serum and 5 ml of a double antibody were sequentially added to 400 ml of a high glucose DMEM culture fluid in the clean bench for full and uniform mixing; a mixture was separately charged into labelled 50 ml centrifuge tubes for sealing; and the 50 ml centrifuge tubes were preserved at 4°C 0 10 It.g/mL pancreatic enzyme without containing EDTA: 0.25% trypsin solution without containing EDTA and a DMEM culture medium without containing the fetal calf serum were diluted to 10 jig/mL in a ratio of Ito 250.

Description

0 Rotavints growth maintenance solution: 10 ug/mL pancreatic enzyme without containing the EDTA and the DMEM culture medium without containing the fetal calf serum were diluted to 1 jtgimL in a ratio of 1 to 10.

(4) Statistical data analysis All experiments in the following embodiments of the present invention are repeated for three times. Experimental data is expressed as means ± standard derivation ( X±s). Data is counted with SPSS software, comparison between two groups uses t test, and comparison between multiple groups in mean uses single factor analysis of variance. Obtained data results are expressed as the means ± standard derivation. Data analysis uses statistical analysis software Graphpad prism 8.01 for Mann-Whitney counting. C P<0.05, P<0.01, "P<0.001 represent that data is compared with that in N group; Aa P<0.01, aAAA P<0.0001 represent that the data is compared with that in Ribavirin group; and *P<0.05, **P<0.01, *** P<0.001, '*P<0.0001 represent that the data is compared with that in RV group).

Embodiment 1 The present invention provides an application of ophiopogonin D in preparation of anti-rotavirus drugs.

Further, the ophiopogonin D has a direct killing effect on the rotavirus Further, the ophiopogonin D inhibits gene expression of a structural protein VP6 in MA104 cells infected with the rotavirus.

Embodiment 2 The present invention provides an anti-rotavirus drug, and the active component of the anti-rotavints drug comprises the ophiopogonin D Further, the anti-rotavints drug is a monomer or compound preparation of the ophiopogonin D Further, a form of the preparation of the anti-rotavirus drug is selected from any one of a tablet, a capsule, granules, powder, an oral liquid, an injection, a film, a suppository, nasal drops, a semi-solid preparation, an emulsion or a spray.

Description

Embodiment 3 An experimental method of infecting 1v1A104 cells with rotavirus in this embodiment is as follows: A virus was melted in a water bath kettle at 37°C; a resultant was pretreated with trypsin for 30 min; and then, a viral solution was added to the MA104 cells growing to 80-90% without adding a serum in a cell culture medium used for rotavirus infection. The cytopathic effect (CPE) would be generated in MA104 cells infected with the rotavirus; and when the cytopathic effect reaches 75%, the MA104 cells were cryopreserved in a refrigerator at -20°C. After the cryopreserving and melting processes were repeated for 3 times, low-temperature centrifugation was performed for collection of a supernatant which is an obtained virus liquid. The above process was repeated for amplification of the rotavirus.

Embodiment 4 An experimental method of detecting the toxic effect of the ophiopogonin D on the MA104 cells with a CCK8 method in this embodiment is as follows: According to a range of a common drug concentration of the ophiopogonin D, a cytotoxicity pre-experiment was performed in the range from 0.2 mM to 2 mM in this experiment. MA104 cells in a logarithmic growth phase were observed under a microscope; and when cells were uniform and full in morphology and had clear edges, and the number of the cells reached 80%, digestion, centrifugation and resuspension were performed, a suspension was further diluted, then 10 ul of diluted suspension was placed on a blood cell counting plate for counting, and a required cell volume was calculated. Then, the cells were plated in a 96-well plate, and 100 [El of cell suspension was added to each well with a cell density being 8 xi04/mL Administration was performed after the cells were adhered to walls to form monolayer cells. After 48 h of incubation, the cytotoxicity of the ophiopogonin D was detected with a CCK-8 kit. 1/10 volume of CCK-8 solution was added to each well for incubation in an incubator; and after 1 h, a resultant was detected at a wavelength of 450 nm, and an absorbance was recorded. A formula of relative survival rate of the cells is as follows:

Description

The cell morphology of the normal MA104 cells and the MA104 cells after being infected with the rotavirus for 48 h in this embodiment under the microscope are shown in Figs. 2-3. The normal MA104 cells are triangular or fusifonn and are obvious and clear in outline. The MA104 cells infected with the rotavirus have an obvious cytopathic effect, are blurred in boundary, and are increased in distance between cells; black particles are added in the cells; and finally, the cells completely exfoliate and float.

Results of the toxicity of the ophiopogonin D to the MA104 cells, explored in this embodiment, are shown in Fig. 4, wherein N represents a non-taken group; and compared with the N group, # represents P<0.05. When a drug concentration of the ophiopogonin D is 0.4 mM to 2 mM, the ophiopogonin D has no drug toxicity to the celss, and an average survival rate of the cells is 100% or above, which shows that the ophiopogonin D has a certain growth promotion effect on the cells. Compared with the N group, when the drug concentrations of the ophiopogonin D are 0.4 mM and 2 mM, the ophiopogonin D has the effect of promoting growth of the cells, and there is a statistical difference (P< 0.05). However, when the drug concentration of the ophiopogonin D is 0.6 mM to 1.8 mM, it is detected that there is no obvious difference between a drug group and the N group in cell viability. Therefore, the selected experimental drug concentration of the ophiopogonin D is 0.6-1.8 mM.

Embodiment 5 A specific method of detecting the effect of the ophiopogonin D of resisting rotavirus adsorption with the CCK8 method in this embodiment is as follows: A medical solution was added to a 96-well culture plate with the MA104 cells growing to the monolayer cells, and 6 wells were repeated for each medical solution by 100 pt/well. An equal volume of ribavirin was added in a positive control group; and an equal volume of DMEM culture fluid (without containing the serum) was added to each of a normal cell control group and a virus control

Description

group. Incubation was performed with 5% CO2 at 37°C for 2 h. The medical solutions were sucked out, 100 TC1D50 of virus (the virus was reacted with 10 lig/mL pancreatic enzyme at 37°C for 30 min) was added to each group by 100 RL/well in addition to the normal cell control group; incubation was performed with 5% CO, at 37°C for 2 h; the viruses were sucked out; a cell maintenance solution was added by 200 gL/well; and incubation was performed at 37°C and 5% CO, for continuous observation. After 48 h of culture, detection was performed with the CCK-8 kit. 1/10 volume of CCK-8 solution was added to each well for incubation in the incubator; after 1 h, a resultant was detected at a wavelength of 450 nm, and an absorbance was recorded; and the experiment was repeated for 3 times Results of the effect of the ophiopogonin D of resisting rotavirus adsorption, explored in this embodiment, are shown in Fig. 5, wherein compared with a Ribavirin group, AA represents P<0.01, and A A AA represents P<0.0001. Compared with the Ribavirin group, the inhibition rates of the ophiopogonin D group to the rotavirus at 1.2 mM, 1.4 mM and 1.6 mM are obviously increased, and there is a statistical difference. However, the highest inhibition rate is about 30% only, which shows that the ophiopogonin D has no obvious effect of resisting rotavirus adsorption.

Embodiment 6 A specific method of detecting the effect of the ophiopogonin D of resisting rotavirus biosynthesis with the CCK8 method in this embodiment is as follows: TCID50 of viral solution (the virus was reacted with 10 ug/mL pancreatic enzyme for 30 min) was added to 96-well culture plate with the MA104-2 cells growing to the monolayer cells by 100 gL/well, wherein the cells were washed with a PBS previously. A cell normal control was set, and an equal volume of the DMEM culture fluid was added. Incubation was performed with 5% CO, at 37°C for 2 h; then, the viral solution was sucked out; different concentrations of medical solutions and Ribavirin were added by 100 pt/well respectively; and a virus control group was set, the cell maintenance solution was added by 100 uL/well

Description

only. Incubation was performed with 5% CO, at 37°C for continuous observation. Continuous culture was performed for 48 h, and detection was performed with the CCK-8 kit. 1/10 volume of CCK-8 solution was added to each well for incubation in an incubator; and after 1 h, a resultant was detected at a wavelength of 450 nm, and an absorbance was recorded. The virus inhibition of the drug was calculated, and the experiment was repeated for 3 times The effect of the ophiopogonin D of resisting rotavirus biosynthesis, explored by this embodiment, is shown in Fig. 6. Compared with the Ribavirin group, the inhibition rate of each ophiopogonin D group to the rotavirus is relatively low, and there is no significant difference in statistical comparison, which shows that the ophiopogonin D has no obvious effect of resisting rotavirus biosynthesis.

Embodiment 7 A specific method of detecting the effect of the ophiopogonin D of directly killing the rotavirus with the CCK8 method in this embodiment is as follows: The drug and 100 TC1D50 of the viral solution (the virus reacted with 10 lig/mL pancreatic enzyme for 30 min) were mixed in an equal volume and reacted for 2 h. After the cells were washed with the PBS for 2 times, the cells were added to the 96-well culture plate with the MA104 cells growing to the monolayer cells. In the positive control group, operation, the same as the above, was performed on the Ribavirin and the rotavirus; and an equal volume of the DMEM was added to each of the normal cell control group and the virus control group. Incubation was performed with 5% CO, at 37°C for 2 h; then, a mixed solution was sucked out; the cell maintenance solution was added by 200 pt/well; incubation was performed at with 5% CO2 37°C for continuous observation; and after 48 h of culture, detection was performed with the CCK-8 kit. 1/10 volume of CCK-8 solution was added to each well for incubation in the incubator; and after 1 h, a resultant was detected at a wavelength of 450 nm, and an absorbance was recorded. The virus inhibition of the drug was calculated, and the experiment was repeated for 3 times.

Description

Results of the effect of the ophiopogonin D of directly killing the rotavirus, explored in this embodiment, are shown in Fig. 7, wherein compared with the Ribavirin group, AA represents P<0.01; AAA represents P<0.001; and AAAA represents P<0.0001. Compared with the Ribavirin group, the inhibition rates of the ophiopogonin D to the rotavirus at 0.8 mM and 1 mM are 60.9% and 65.9% respectively, and there is no significant difference in statistical comparison, which shows that the effect of the ophiopogonin D of directly killing the rotavirus is equivalent to that of the Ribavirin. The inhibition rate to the rotavirus is obvious at 1.2-1.8 mM, and is highest at 1.4 mM, reaching 79.8%; the inhibition rate of the ophiopogonin D is obvious higher than that of the Ribavirin group; and there is a significant difference in statistical comparison (P<0.0001), which shows that the ophiopogonin D has obvious effect of directly killing the rotavirus.

Embodiment 8 A specific method of detecting an expression level of a structural protein VP6 gene of the rotavirus with qPCR in this embodiment is as follows: (1) Extraction and quantification of total RNA 0 In order to further verify whether the ophiopogonin D has the direct killing effect on the rotavirus, after 48 h of directly killing the rotavirus by the ophiopogonin D, a high drug group, a medium drug group, a low drug group, the Ribavirin group, the N group and the RV group were selected for removing supernatant; the cells were washed with the PBS twice; 1 ml of Trizol reagent was added; still standing was performed for 5 min; and a resultant was collected into a 1.5 mL nonenzymatic EP tube. 200 [IL of chloroform was added to the tube for vortex oscillation for 15 s; the tube was subjected to still standing at a room temperature for 3 min and then centrifugation (at 4°C and 12000 r/min for 15 min); and thee layers were formed in a sample after centrifugation, i.e. a colourless upper layer, a white middle layer and a red lower layer.

0 The supernatant was carefully sucked into a new 1 5 mL nonenzymatic EP tube (with a volume of about 500 [IL); an equal volume of pre-cooled isopropanol was added; vigorous shaking was performed up and down for uniform mixing; and a

Description

mixture was subjected to still standing at 4°C for 10 min and then centrifugation (at 4°C and 12000 r/min for 10 min) 0 White precipitates was visible at the bottom of the EP tube after centrifugation; a supernatant was removed with reserving the precipitates; 1 mL of prepared 75% ethanol solution (prepared from anhydrous ethanol and nonenzymatic water in a ratio of 3 to 1) was added; shaking was performed for uniform mixing; centrifugation was performed (at 4°C and 12000 r/min for 5 min); a supernatant was discarded; and a resultant was placed at the room temperature for 15-20 min and was aired.

0 20 p..L of DEPC water was added to the EP tube after airing, and a tube wall was gently blown and beaten for dissolving RNA. After a concentration of RNA of a sample was determined with a Nanodrop micro ultraviolet spectrophotometer, the sample could be directly used for the experiment or was preserved at -80°C for later use.

(2) Reverse transcriptional reaction of m RNA 0 Reaction for removal of genome DNA A reaction mixture was prepared from the following components on ice with a reaction volume of 20 RL; and operation was performed according to instructions of Evo M-MLV RT Kit with gDNA Clean for qPCR 11. Consumables used in this experiment are all Axygen enzyme-free consumables.

Table 1 Reaction System For Removal Of Genome DNA System Component name Quantity added gDN A Clean Reagent 1 gL xgDNA Clean Buffer 2 gL Total RNA" RNase free water Up to 10 tiL Reaction condition: 42°C 2 min 4°C

Description

*1: The quantity of RNA may be added as needed. In a 20 RL retrotranscription system, up to 1 lug of total RNA is used; and in the case of using a probe method, up to 2 lug of total RNA is used.

0 Reverse transcription reaction A reaction liquid was prepared according to the contents in the following Table 2 for reverse transcription reaction.

Table 2 Reverse Transcription Reaction System Component name Quantity added step])reaction liquid 10 ttL Evo M-MLV RTase Enzyme Mix 1 pi-RT Primer Mix 1 ttL 5xRTase Reaction Buffer Mix I 4 ttL RNase free water 4 ttL Total 20 ttL Reaction condition: 37°C 15 m in 85°C 5 sec 4°C (3) Real Time PCR reaction In real time PCR, SYBR Green I fluorescent labelling was used for detecting VP6 expression levels in the drug groups, the Ribavirin group, the N group and the RV group, wherein an SYBRO Green Premix Pro Taq HS qPCR Kit 11 kit was used, and GAPDH was selected as an internal reference. An Axygen special Real-time PCR plate was used in the experiment; a real-time fluorescent quantitative PCR amplification reaction system was prepared according to the following Tables 3-5; and a reaction liquid was prepared on ice (with the volume of a reaction total system being 10 ut).

Table 3 PCR Reaction System Component name 10 ttL of system 2X SYBRE) Green Pro Taq HS Premix II cDNA

Description

Primer F 0.2 i..tL Primer R 0,2L RNase free water Up to 10 nL Table 4 qPCR Reaction Condition Temperature Time Number of cycles Step 1 95°C 30 sec Step 2 95°C 5 sec 40 60°C 30 sec Step 3 Dissociation stage Table 5 Primer Sequence Gene Primer (5'-3') GAPDH Forward CTGGTGACCCGTGCTGCTT, SEQ ID NO.]; Reverse TTTGCCGCCTTCTGCCTTA, SEQ ID NO.2; VP6 Forward GACCAAACATGACCATCTG, SEQ ID NO.3; Reverse CATCGGCACCCGTCAGACT, SEQ ID NO.4.

This embodiment is intended to explore the effect of the ophiopogonin D on gene expression of the structural protein VP6 in the MA104 cells infected with the rotavims, and results are shown in Fig. 8, wherein compared with the RV group, * represents P<0.05, *** represents P<0.001, and **** represents P<0.0001. It can be seen from the drawings that the N group does not express VP6; and compared with the RV group, the gene expression level of VP6 in the Ribavirin group is obviously lowered, and there is a statistical difference (p<0.0001), which shows that the Ribavirin has the effect of resisting the rotavirus. Compared with the RV group, the VP6 expression levels of the detection drug group at the concentrations of 0.6 mM, 1.0 mM and 1.4 mM are all obviously lowered, and there is a statistical difference and dose-effect correlation, wherein the detection drug group is the most significant in inhibiting expression of VP6 at 1.4 mM (p<0.0001); however, compared with the Ribavirin group, there is no statistical difference. Thus, it shows that the ophiopogonin D may exert the anti-rotavirus effect by inhibiting gene expression of VP6.

Description

Embodiment 9 This embodiment uses western blot and immunofluorescence staining to determine the effect of the ophiopogonin D on expression of the structural protein VP6 in the MA104 cells infected with RV. The specific steps are as follows: A total protein in a cell sample was dissolved into 150 jil of RITA buffer solution containing 50 jig/ml PMSF or 1 jig/ml phosphatase inhibitor (Beyotime Biotechnology, China); and then, quantification was performed with a BCA protein determination kit (Thermo Scientific, MA, USA). The sample was washed with an equal quantity of protein (40 jag), separated with 10% DS-PAGE, and then transferred onto a poly(vinylidene fluoride) (PVDF) film (Millipore, MA, USA). Then, the film was occluded with 2.5% skim milk powder (Merck) in PBST at the room temperature for 120 min, and then was incubated with a specified primary antibody (a VP6 antibody) at 4°C overnight. Next, the film was incubated with a goat anti-rabbit/mouse igG (H+L)/TRITC secondary antibody at the room temperature for 2 11. A protein band was displayed on an Odyssey infrared imaging system (LT-COR Biotechnology, Lincoln, NE, USA); and all options were set as defaults provided by the manufacturer. All the experiments were conducted in triplicate.

lmmunofluorescence (IF) staining was used to detect the effect of the OPD on RV-VP6 protein expression. After the RV-Wa and the OPD were incubated at 37°C for 2 h, the RV-Wa and the OPD were inoculated to the MA104 cell monolayer for reaction for 2 h; inoculums were washed and then were incubated at 37°C for 24 h respectively; the cells were fixed, and subjected to immunofluorescence staining with an antibody (green) against the RV-VP6 protein and DAFT (blue); and the MA104 cells were imaged with an Olympus IX70 fluorescent microscope.

Results are shown in Fig. 9 and Fig. 10. Compared with the RV group, in an OPD different concentration group, the expression level of the RV-VP6 protein is lowered, and there is a significant difference in statistical comparison; and the expression level of the RV-VP6 protein is lowered depending on a dose. Results of

Description

the immunofluorescence (IF) staining experiment confirm that compared with the RV group, the OPD inhibits expression of the VP6 viral protein of RV-Wa in the MA104 cells.

Embodiment 10 This embodiment explores the inhibition effect of the ophiopogonin D on the RV in intestinal organoids. The specific steps are as follows: C57B1/6J mice aged from 6 weeks to 8 weeks were sacrificed for small intestines; the small intestines were cleaned and cut into pieces; a moderate dissociation reagent GCDR (Stemcell, Canada) was added for dissociation at the room temperature for 15 min; and a crypt structure containing intestinal stem cells was extracted and resuspended with a complete medium and matrigel. Finally, 60 jil of mixture drops containing the crypt structure were added to each well of a 24-well plate for incubation with 5% CO, at 37°C for 15 min. After the drops were solidified, 700 jtl of culture medium was added to each well.

Intestinal organoids of the mice were infected with the RV; 12 wells of the intestinal organoids growing well were added in a centrifuge tube, washed with iced PBS twice, and centrifuged at 900 rpm fir 3 min; 300 jiL of RV resuspended intestinal organoids after being activated were incubated in the incubator at 37°C for 2 h. After centrifugation at 900 rpm for 3 min, the intestinal organoids were washed with the iced PBS for three times to remove free viruses, and then inoculated to the 24-well plate for normal culture.

Crypts were extracted from the C57BL/6J mice first and embedded into the matrigel for culture of the intestinal organoids. The growth period is shown in FIG. 11. The results show that on Day 1, the crypts form a ball structure in the matrigel; on Day 2, the ball structure starts to germinate; on Day 3, the number of crypt buds are increased; on Days 4 and 5, a distinctive Kranz structure occurs; and on Day 6, a color in the center of each intestinal organoid becomes darker, with occurrence of a complex multi-leaf structure and formation of mature intestinal organoids.

The intestinal organoids after being infected with the RV for different days were subjected to Hoechst/PI staining. It is found that all the intestinal organoids

Description

with different days have the apoptosis phenomenon, which shows that the intestinal organoids may be infected with the RV, and apoptosis of the intestinal organoids may be caused (Fig. 12).

In this experiment, organoids after passage were selected and infected with the RV; after an RV-WA strain and the OPD were incubated at 37°C for 2 h together, the RV-WA strain and the OPD acted on the intestinal organoids for 2 h; the intestinal organoids were completely cleaned to remove residual virus particles, and then were re-embedded into the Matrigel for 24 h; RNA and the protein were extracted from the intestinal organoids respectively; and the effects of the OPD on synthesis of RV genome RNA and expression of the viral protein were detected with ciPCR and Western blot. Results are shown in Fig. 13 and Fig. 14. The results show that the OPD may lower gene and protein expression of VP6 in the intestinal organoids infected with the RV, and there is a statistical difference.

In order to further confirm the effect of the OPD, after the administrated intestinal organoids were stained with positive immunofluorescence (IF) of the viral protein VP6, as shown in Fig. 15, VP6 immunofluorescence in an OPD treatment group is obviously lowered.

It can be judged from the results in embodiments 1-10 that the ophiopogonin D can inhibit the MA104 cells infected with the rotavirus; and the ophiopogonin D with a drug concentration range from 1.2 mM to 1.8 mM has an obvious direct killing effect on the rotavirus. In addition, the ophiopogonin D may exert the anti-rotavirus effect by inhibiting gene and protein expression of the structural protein VP6. Meanwhile, the anti-RV effect of the OPD is further confirmed on an RV-infected mouse intestinal organoid model. It shows that the OPD may serve as a candidate compound for an anti-RV drug, which provides an experimental basis for developing OPD anti-RV drugs. The above results show that, as an active component, the ophiopogonin D can be independently applied or forms a compound preparation with other active components; and conventional methods for pharmaceutically acceptable excipients and preparations are used to prepare drugs for preventing and treating rotavirus infection in various dosages of tablets,

Description

capsules, granules, powder, oral liquids, injections, films, suppositories, nasal drops, semi-solid preparations, mulsions or sprays for clinical use.

The above specific embodiments are further description of the technical solutions and beneficial effects of the present invention, but not limitations to the implementations. For those skilled in the art, any obvious replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

Claims I. An application of ophiopogonin D in preparation of anti-rotavints drugs.
2. The application of ophiopogonin D in preparation of anti-rotavirus drugs according to claim 1, wherein in the anti-rotavirus drugs, the drug concentration of the ophiopogonin D is 0.6 mM to 1.8 mM.
3. The application of ophiopogonin D in preparation of anti-rotavirus drugs according to claim 1, wherein the ophiopogonin D has a direct killing effect on the rotavints.
4. The application of ophiopogonin D in preparation of anti-rotavints drugs according to claim 1, wherein the ophiopogonin D inhibits gene expression of a structural protein VP6 in MA104 cells infected with the rotavints.
5. An anti-rotavirus drug, wherein the active component of the anti-rotavirus drug comprises the ophiopogonin ID.
6. The anti-rotavints drug according to claim 5, wherein the anti-rotavirus drug is a monomer or compound preparation of the ophiopogonin D
7. The anti-rotavints drug according to claim 5, wherein a form of the preparation of the anti-rotavirus drug is selected from any one of a tablet, a capsule, granules, powder, an oral liquid, an injection, a film, a suppository, nasal drops, a semi-solid preparation, an emulsion or a spray.