CN114668754B - Application of 1,5-anhydro sorbitol in preparing medicine for treating and preventing diseases caused by SARS-CoV-2 virus - Google Patents
Application of 1,5-anhydro sorbitol in preparing medicine for treating and preventing diseases caused by SARS-CoV-2 virus Download PDFInfo
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Abstract
The invention provides application of compound 1, 5-dehydrated sorbitol in preparing medicines for treating diseases caused by SARS-CoV-2 virus. According to the invention, through research on the inhibition effect of 1, 5-sorbitan on SARS-CoV-2 virus in Vero cells, Caco-2 cells and ACE2-293T cells, 1, 5-sorbitan can be used as a candidate drug for preventing and treating SARS-CoV-2 virus infection. The invention has application value in the treatment of SARS-CoV-2 virus infection.
Description
Technical Field
The present invention relates to the application of compound 1,5-anhydro sorbitol in preparing medicine for preventing and treating SARS-CoV-2 virus diseases.
Background
The new coronavirus epidemic situation is pandemic in the world and becomes a serious public health problem, and no specific medicine exists at present. Although vaccines have been available for vaccination, the protective capacity of the vaccine is less than ideal due to the constant mutation of the virus.
There is an urgent need to develop drugs for treating diseases caused by SARS-CoV-2 virus. The inventor finds that 1,5-anhydro-D-glucitol (1, 5-anhydro-D-glucitol; 1,5AG) has strong inhibitory effect on SARS-CoV-2 infection, and can possibly become a candidate drug for treating SARS-CoV-2 infection.
Disclosure of Invention
The technical problem to be solved by the invention is how to prepare a medicament for preventing and/or treating diseases caused by SARS-CoV-2 virus or SARS-CoV-2 virus infection, and/or how to prepare a SARS-CoV-2 virus inhibitor.
In order to solve the above technical problems, a first object of the present invention is to provide any one of the following uses of 1, 5-sorbitan:
the application of U1 and 1, 5-dehydrated sorbitol in the preparation of medicine for preventing and/or treating SARS-CoV-2 virus caused disease or SARS-CoV-2 virus infection;
application of U2 and 1, 5-dehydrated sorbitol in preparing SARS-CoV-2 virus inhibitor.
In the above application, the SARS-CoV-2 virus-caused disease can be respiratory system infection and/or digestive system infection. The respiratory system infection is respiratory tract infection and/or lung infection, the respiratory tract infection can be nasopharyngitis, rhinitis, pharyngolaryngitis, tracheitis and/or bronchitis, and the lung infection can be pneumonia. The digestive system infection may be diarrhea. SARS-CoV-2 virus infected patients exhibit symptoms of atypical viral pneumonia characterized by high fever, dyspnea, lymphopenia, rapid progression of chest radiographs visible as lung shadows, acute lung injury due to cytokine storm induced by the virus, acute respiratory distress syndrome in critically ill patients, and even respiratory failure.
The second purpose of the invention is to provide the application of 1, 5-dehydrated sorbitol in preparing products for inhibiting the proliferation or replication of SARS-CoV-2 virus.
The third purpose of the invention is to provide the application of 1, 5-dehydrated sorbitol in preparing products for inhibiting the proliferation or replication of SARS-CoV-2 virus in animal individuals, animal organs, animal tissues or animal cells.
In the above application, the animal is a mammal.
In the above application, the cell is a human or a nonhuman green monkey cell. The cell can be a Vero cell, a Caco-2 cell or an ACE2-293T cell.
The invention also provides a SARS-CoV-2 virus inhibitor, the active component of the SARS-CoV-2 virus inhibitor contains 1, 5-dehydrated sorbitol.
The SARS-CoV-2 virus inhibitor can be 1, 5-anhydrosorbitol only, and can also contain carrier or excipient.
The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these are in particular water-soluble carrier materials. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. In order to prepare the unit dosage form into preparations for injection, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired. The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; for luminal administration, such as rectally and vaginally; administration to the respiratory tract, e.g., nasally; administration to the mucosa.
According to the invention, through research on the inhibition effect of 1, 5-sorbitan on SARS-CoV-2 virus in Vero cells, Caco-2 cells and ACE2-293T cells, 1, 5-sorbitan can be used as a candidate drug for preventing and treating SARS-CoV-2 virus infection. The invention has application value in the treatment of SARS-CoV-2 virus infection.
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FIG. 1 is a graph showing the inhibition of SARS-CoV-2 virus replication by 1, 5-sorbitan in Vero cells in example 1. Data are mean ± sd, repeat number 6, meaning that the difference is very significant compared to Mock at a 1, 5-sorbitan treatment concentration of 0 μ M (P < 0.01).
FIG. 2 is a graph showing the results of inhibiting the proliferation of SARS-CoV-2 virus by 1, 5-sorbitan in example 2.
FIG. 3 is a graph showing the results of measuring the semi-inhibitory concentration of 1, 5-sorbitan in example 3, and the data are the mean. + -. standard deviation and the number of repetitions is 3.
FIG. 4 is a graph showing the inhibition of SARS-CoV-2 virus replication by 1, 5-sorbitan in Caco-2 cells in example 4. Data are mean ± sd, repeat number 6, mean significant differences (P <0.05) and mean very significant differences (P < 0.01).
FIG. 5 is a graph showing the results of inhibiting SARS-CoV-2 virus replication in ACE2-293T cells by 1, 5-sorbitan in example 5. Data are mean ± sd, repeat number 6, meaning that the differences were very significant in the graph (P < 0.01).
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.
SARS-CoV-2 virus is from the disease prevention and control center of Shenzhen city, numbered Shenzhen03, and is described in non-patent document "Genomic epidemic of SARS-CoV-2in Guangdong Provice, China.cell.2020; 181(5) 997-1003. e9.'. The biological material is available to the applicant to the public according to the relevant national biosafety regulations and is only used for repeating the relevant experiments of the present invention and is not used for other purposes.
Vero cells are African green monkey kidney cell lines and are described in non-patent document "Flavivirus NS1 protein in induced host series renal access by mosquitoes. Nature Microbiology,2016 (9): 16087". The biological material is publicly available from the university of Qinghua, and is used only for repeating the relevant experiments of the present invention, and is not used for other purposes.
Caco-2 cells are colon cancer cell lines and are described in non-patent literature "Proteomics of SARS-CoV-2-infested host cells reveals thermal targets. Nature.2020 Jul; 583(7816):469-472". The biological material is publicly available from the university of Qinghua, and is used only for repeating the relevant experiments of the present invention, and is not used for other purposes.
ACE2-293T cells are 293T Cell lines stably integrating ACE2 receptors and are described in non-patent documents "SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a clinical Proven Protease inhibitor. cell.2020Apr 16; 181(2) 271-280.e 8'. The biological material is publicly available from the university of Qinghua, and is used only for repeating the relevant experiments of the present invention, and is not used for other purposes.
1, 5-sorbitan (cat # M1203) is a product of the company apexbio, CAS chemical No.: 154-58-5.
In the following examples, DMEM complete medium supplemented with 10% FBS, 100mg/ml streptomycin and 100U/ml penicillin was DMEM medium as a basal medium, and FBS (fetal bovine serum), streptomycin and penicillin were added thereto, wherein the content of FBS was 10% (by volume), the concentration of streptomycin was 100mg/ml and the concentration of penicillin was 100U/ml. DMEM medium (cat # 12100046) was Invitrogen.
In the following examples, a DMEM medium containing 2% serum with a final concentration of 1% low-melting agarose is a DMEM medium as a basic medium to which low-melting agarose and serum are added, wherein the content of low-melting agarose is 1% (by volume) and the content of serum is 2% (by volume).
In the following examples, the RPMI1640 complete medium supplemented with 10% FBS, 100mg/ml streptomycin and 100U/ml penicillin was prepared by using the RPMI1640 complete medium as a basal medium, and adding FBS (fetal bovine serum), streptomycin and penicillin to the basal medium, wherein the FBS content in the basal medium was 10% (by volume), the streptomycin concentration was 100mg/ml, and the penicillin concentration was 100U/ml. RPMI1640 (cat # 21875034) is a product of Invitrogen.
The quantitative tests in the following examples, unless otherwise specified, were set up in triplicate and the results averaged. All data in the following examples were analyzed for significance using GraphPad \ Prism 7 software Student's T-test.
Example 1: inhibition of SARS-CoV-2 virus replication in Vero cells by 1, 5-anhydrosorbitol
This example studies the effect of 1, 5-sorbitan on SARS-CoV-2 virus replication in Vero cells, and the specific steps of the treatment of 1, 5-sorbitan with 5 concentration gradients of 0. mu.M (mock), 100. mu.M (treatment one), 200. mu.M (treatment two), 300. mu.M (treatment three) and 400. mu.M (treatment four) were as follows:
(1) at 37 ℃ 5% CO 2 The Vero cell line was cultured in a constant temperature incubator of (1), and the medium was completely cultured in DMEM supplemented with 10% FBS and 100mg/ml streptomycin and 100U/ml penicillin. Vero cells with good growth state are planted in a 48-well plate, and the cell density is 1 multiplied by 10 4 One per ml.
(2) Mu.l of 1, 5-sorbitan solution was added to each well 1h before infection with the virus so that the final concentration of 1, 5-sorbitan in Mock was 0. mu.M, 100. mu.M in treatment one, 200. mu.M in treatment two, 300. mu.M in treatment three, and 400. mu.M in treatment four.
(3) SARS-CoV-2 virus was inoculated at 0.0001M.O.I. into Vero cells, DMSO (dimethyl sulfoxide) at the corresponding concentration as a control, 37 ℃ and 5% CO 2 And incubating for 2h in the constant temperature incubator. Treatment at each concentration and control at each concentration were set up in 6 replicates.
(4) Cell supernatants were removed and cells were washed three times with PBS buffer and the last time residual PBS was blotted. 400. mu.l of 1, 5-sorbitan solution were added at concentrations of 0. mu.M (mock), 100. mu.M (treatment one), 200. mu.M (treatment two), 300. mu.M (treatment three) and 400. mu.M (treatment four), respectively. 37 ℃ and 5% CO 2 The culture was continued for 40h in the incubator.
(5) The cell supernatant was removed, the cells were washed three times with PBS, 400. mu.l of RI solution (company: Axygen, cat. No. AP-MN-MS-RNA-250) was added to each well, and after fully lysing the cells, the cells were transferred to a 1.5ml centrifuge tube and frozen at-80 ℃.
(6) The frozen sample was sampled, RNA was extracted using an RNA extraction kit (product number AP-MN-MS-RNA-250, manufactured by Axygen) and the RNA was reverse-transcribed into cDNA using a reverse transcription kit (product number Bio-Rad).
(7) The SYBR Green qPCR technique was used to detect viral load in the samples, GAPDH values as an internal control.
The primer pair for detecting SARS-CoV-2 is as follows:
an upstream primer: 5'-AGAAGATTGGTTAGATGATGATAGT-3' (shown as sequence 1 in the sequence table);
a downstream primer: 5'-TTCCATCTCTAATTGAGGTTGAACC-3' (shown in sequence 2 of the sequence table).
The primer pairs for detecting the GAPDH gene were as follows:
an upstream primer: 5'-AGCCTCAAGATCATCAGCAATG-3' (shown as sequence 3 in the sequence table);
a downstream primer: 5'-ATGGACTGTGGTCATGAGTCCTT-3' (shown in sequence 4 of the sequence table).
The results are shown in FIG. 1: vero cell viral load with 1, 5-sorbitan added was significantly reduced and dose-dependent compared to the control group.
Example 2: 1, 5-anhydrosorbitol inhibits SARS-CoV-2 virus proliferation (immunofluorescence)
This example studies the effect of 1, 5-sorbitan on the proliferation of SARS-CoV-2 virus in Vero cells, and the specific steps of the treatment were as follows, wherein 1, 5-sorbitan was set to 5 concentration gradients of 0. mu.M (mock), 100. mu.M (treatment one), 200. mu.M (treatment two), 300. mu.M (treatment three), and 400. mu.M (treatment four):
(1) the washed and sterilized slides were placed in 24-well plates in advance, and 1mL of DMEM complete medium supplemented with 10% FBS and 100mg/mL streptomycin and 100U/mL penicillin was added per well. Inoculating Vero cell 0.5-2X 10 3 And (3) the cells are enabled to be completely attached to the wall when infected, and the cell density reaches 50-60%. At 37 ℃ 5% CO 2 Culturing the Vero cells in the constant-temperature incubator.
(2) Mu.l of 1, 5-sorbitan solution was added to each well 1h before infection with the virus so that the final concentration of 1, 5-sorbitan in Mock was 0. mu.M, 100. mu.M in treatment one, 200. mu.M in treatment two, 300. mu.M in treatment three, and 400. mu.M in treatment four.
(3) SARS-CoV-2 virus was inoculated at 0.0001M.O.I. into Vero cells, corresponding concentrations of DMSO (dimethyl sulfoxide) as a control, 37 ℃ C., 5% CO 2 And incubating for 2h in the constant temperature incubator. Treatment at each concentration and control at each concentration were set to 3 replicates.
(4) Cell supernatants were removed and cells were washed three times with PBS buffer and the last time residual PBS was blotted. 400. mu.l of 1, 5-sorbitan solution was added thereto at concentrations of 0. mu.M (mock), 100. mu.M (treatment one), 200. mu.M (treatment two), 300. mu.M (treatment three) and 400. mu.M (treatment four), respectively, and the culture was continued for 40 hours.
(5) Cell supernatants were removed, washed 3 times with PBS, and 1mL of 4% freshly prepared paraformaldehyde was added to a 24-well plate as a fixative and fixed at room temperature for 60 min. Removing the fixed solution, washing with washing solution for 3 times, each for 3-5min, and completely absorbing the liquid.
(6) Blocking with 2mL of blocking solution (BD,51-2091KZ) for 15min, can be performed by gentle shaking on a shaker. Removing blocking solution, washing with washing solution for 3 times, each for 3-5min, and completely absorbing liquid.
(7) A primary antibody (Anti-SARS-CoV-2Nucleocapsid, product of abcam, cat # ab272852) was added thereto, incubated for 2 hours, washed 3 times with PBST, the supernatant was removed, washed 3 times with PBST, and a secondary antibody labeled with cy3 (invitrogen, cat # A-11003) and DAPI (product of abcam, cat # ab228549) were added thereto, and incubated for 1 hour at 37 ℃.
(8) The supernatant was removed by aspiration, washed 3 times with PBST, 3-5min each time, and the liquid was drained. The coverslip was removed and placed on a clean paper towel, 20. mu.L of anti-quencher was added to the slide, and the slide was placed with the cell side down, avoiding the generation of bubbles. After drying around the slide, 20. mu.L of neutral gum was applied to the edge of the slide, and the slide was observed under a fluorescent microscope and photographed.
The results are shown in FIG. 2: the number of cells fluorescing in the test group with 1, 5-sorbitan added was significantly less than in the control group.
Example 3: semi-Inhibitory Concentration (IC) of 1, 5-anhydrosorbitol 50 ) Measurement of
This example measures the semi-Inhibitory Concentration (IC) of 1, 5-sorbitan 50 ) 1, 5-anhydrosorbitolsSetting a total of 9 concentration gradients of 400. mu.M, 300. mu.M, 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M and 3.125. mu.M, and the specific steps are as follows:
(1) 1, 5-sorbitan was prepared as a solution of 400. mu.M, 300. mu.M, 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, respectively.
(2) At 37 ℃ 5% CO 2 The Vero cell line was cultured in a constant temperature incubator of (1), and the medium was completely cultured in DMEM supplemented with 10% FBS and 100mg/ml streptomycin and 100U/ml penicillin. Vero cells with good growth state are planted in a 48-well plate, and the cell density is 1 multiplied by 10 4 One per ml.
(3) Mu.l of 1, 5-sorbitan solution was added to each well 1h before infection to give final concentrations of 1, 5-sorbitan of 400. mu.M, 300. mu.M, 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, respectively.
(4) SARS-CoV-2 virus was inoculated at 0.0001M.O.I. into cells, DMSO at the corresponding concentration as a control, at 37 ℃ and 5% CO 2 The incubation was carried out in an incubator for 2 h. Treatment at each concentration and control at each concentration were set to 3 replicates.
(6) Cell supernatants were removed and cells were washed three times with PBS and the last time residual PBS was blotted. Adding the 1, 5-dehydrated sorbitol solution with the corresponding concentration prepared in the step (1) into the treatment of each concentration, and continuing culturing for 40 h. The cell supernatants were transferred to 1.5ml centrifuge tubes and frozen at-80 ℃.
(7) Vero cells with good growth state are planted in a 6-well plate, and the cell density is 4.5 multiplied by 10 5 The medium was complete with DMEM supplemented with 10% FBS and 100mg/ml streptomycin and 100U/ml penicillin. 37 ℃ and 5% CO 2 The incubator was incubated overnight.
(8) Diluting the cell supernatant obtained in the step (6) by 100 times, 1000 times and 10000 times respectively, adding the diluted cell supernatant into a 6-hole plate, and incubating the cell supernatant with Vero cells for 2 hours at 37 ℃.
(9) Cell supernatants were removed and washed once with PBS, 2.5ml of 2% serum-containing DMEM medium prepared to a final concentration of 1% low melting agarose was added to each well and the plates were left for 20 minutes at room temperature to allow the gel medium to fully solidify.
(10) The solidified 6-well plate is returned to 37 ℃ with 5% CO 2 The culture is continued in the incubator for 3-4 days. When formation of plaques with significant cell lysis was observed by microscopic observation, 1ml of 4% formaldehyde solution was added to each well and fixed at room temperature for 1 hour.
(11) After the cells are fixed, the gel in the plate hole is scraped out, the residual formaldehyde solution is washed away by water, 500 mu l of 1 percent crystal violet solution is added into each hole, and the cells are dyed for 5 minutes at room temperature.
(12) Discarding the crystal violet dye, repeatedly washing off the residual crystal violet with water, drying the residual crystal violet on absorbent paper until more water is left, and standing the front side for drying.
And calculating and counting the number of the plaques in each hole. IC50 mapping and analysis were performed using GraphPad \ Prism 7 software. The results are shown in FIG. 3: IC (integrated circuit) 50 ＝27.44μM。
Example 4: 1, 5-anhydroglucitol for inhibiting SARS-CoV-2 virus replication in Caco-2 cells
This example studies the effect of 1, 5-sorbitan on SARS-CoV-2 virus replication in Caco-2 cells, and the following specific steps were performed by setting a concentration gradient of 0. mu.M (mock), 100. mu.M (treatment one), 200. mu.M (treatment two), 300. mu.M (treatment three), and 400. mu.M (treatment four) for 1, 5-sorbitan:
(1) at 37 ℃ 5% CO 2 The Caco-2 cell line was cultured in a constant temperature incubator in RPMI1640 supplemented with 10% FBS and 100mg/ml streptomycin and 100U/ml penicillin. Caco-2 cells with good growth state are planted in a 48-well plate, and the density of the Caco-2 cells is 1 multiplied by 10 4 /ml。
(2) Mu.l of 1, 5-sorbitan solution was added 1h before infection with the virus so that the final concentration of 1, 5-sorbitan in Mock was 0. mu.M, 100. mu.M in treatment one, 200. mu.M in treatment two, 300. mu.M in treatment three and 400. mu.M in treatment four.
(3) SARS-CoV-2 virus was inoculated at 0.0001M.O.I into cells, DMSO at the corresponding concentration as a control, 37 ℃ and 5% CO 2 The incubation was carried out in an incubator for 2 h. At each concentration ofTreatment and control for each concentration were set up in 6 replicates.
(4) Cell supernatants were removed and cells were washed three times with PBS and the last time residual PBS was blotted. 400. mu.l of 1, 5-sorbitan solution were added at concentrations of 0. mu.M (mock), 100. mu.M (treatment one), 200. mu.M (treatment two), 300. mu.M (treatment three) and 400. mu.M (treatment four), respectively. 37 ℃ and 5% CO 2 The culture was continued for 40h in the incubator.
(5) Cell supernatants were removed, cells were washed three times with PBS, 400. mu.l RI solution was added to each well, and after sufficient lysis of cells, they were transferred to 1.5ml centrifuge tubes and frozen at-80 ℃.
(6) The frozen sample was sampled, RNA was extracted using an RNA extraction kit (product number AP-MN-MS-RNA-250, manufactured by Axygen) and the RNA was reverse-transcribed into cDNA using a reverse transcription kit (product number Bio-Rad).
(7) The SYBR Green qPCR technique was used to detect viral load in the samples, GAPDH values as an internal control.
The primer pair for detecting SARS-CoV-2 is as follows:
an upstream primer: 5'-AGAAGATTGGTTAGATGATGATAGT-3' (shown as sequence 1 in the sequence table);
a downstream primer: 5'-TTCCATCTCTAATTGAGGTTGAACC-3' (shown in sequence 2 of the sequence table).
The primer pairs for detecting the GAPDH gene were as follows:
an upstream primer: 5'-AGCCTCAAGATCATCAGCAATG-3' (shown as sequence 3 in the sequence table);
a downstream primer: 5'-ATGGACTGTGGTCATGAGTCCTT-3' (shown in sequence 4 of the sequence table).
The results are shown in FIG. 4: compared with a control group, the Caco-2 cell virus load added with the 1, 5-dehydrated sorbitol is obviously reduced.
Example 5: 1, 5-anhydroglucitol in ACE2-293T cell for inhibiting SARS-CoV-2 virus replication
This example studies the effect of 1, 5-sorbitan on SARS-CoV-2 virus replication in ACE2-293T cells, and 1, 5-sorbitan was treated with 5 concentration gradients of 0. mu.M (mock), 100. mu.M (treatment one), 200. mu.M (treatment two), 300. mu.M (treatment three), and 400. mu.M (treatment four), as follows:
(1) at 37 ℃ 5% CO 2 The ACE2-293T cell line was cultured in an incubator with DMEM complete medium supplemented with 10% FBS and 100mg/ml streptomycin and 100U/ml penicillin. ACE2-293T cells with good growth state are planted in a 48-well plate, and the cell density is 1 x 10 4 /ml。
(2) Mu.l of 1, 5-sorbitan solution was added to each well 1h before infection with the virus so that the final concentration of 1, 5-sorbitan in Mock was 0. mu.M, 100. mu.M in treatment one, 200. mu.M in treatment two, 300. mu.M in treatment three, and 400. mu.M in treatment four.
(3) SARS-CoV-2 virus was inoculated at 0.0001M.O.I into ACE2-293T cells at a corresponding concentration in DMSO as a control at 37 ℃ with 5% CO 2 The incubation was carried out in an incubator for 2 h. Treatment at each concentration and control at each concentration were set up in 6 replicates.
(4) Cell supernatants were removed and cells were washed three times with PBS and the last time residual PBS was blotted. 400. mu.l of 1, 5-sorbitan solution were added at concentrations of 0. mu.M (mock), 100. mu.M (treatment one), 200. mu.M (treatment two), 300. mu.M (treatment three) and 400. mu.M (treatment four), respectively. 37 ℃ and 5% CO 2 The culture was continued for 40h in the incubator.
(5) Cell supernatants were removed, cells were washed three times with PBS, 400. mu.l RI solution was added to each well, and after cell lysis was complete, cells were transferred to 1.5ml centrifuge tubes and frozen at-80 ℃.
(6) The frozen sample was sampled, RNA was extracted using an RNA extraction kit (product number AP-MN-MS-RNA-250, manufactured by Axygen) and the RNA was reverse-transcribed into cDNA using a reverse transcription kit (product number Bio-Rad).
(7) The SYBR Green qPCR technique was used to detect viral load in the samples, GAPDH values as an internal control.
The primer pair for detecting SARS-CoV-2 is as follows:
an upstream primer: 5'-AGAAGATTGGTTAGATGATGATAGT-3' (shown as sequence 1 in the sequence table);
a downstream primer: 5'-TTCCATCTCTAATTGAGGTTGAACC-3' (shown in sequence 2 of the sequence table).
The primer pairs for detecting the GAPDH gene were as follows:
an upstream primer: 5'-AGCCTCAAGATCATCAGCAATG-3' (shown as sequence 3 in the sequence table);
a downstream primer: 5'-ATGGACTGTGGTCATGAGTCCTT-3' (shown in sequence 4 of the sequence table).
The results are shown in FIG. 5: the ACE2-293T cell viral load with 1, 5-sorbitan added was significantly reduced and dose dependent compared to the control group.
The research on the inhibition effect of the 1, 5-sorbitan on SARS-CoV-2 virus in Vero cells, Caco-2 cells and ACE2-293T cells respectively shows that the 1, 5-sorbitan can be used as a candidate drug for preventing and treating SARS-CoV-2 virus infection. The invention has application value in the treatment of SARS-CoV-2 virus infection.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Qinghua university
Application of <120> 1,5-anhydro sorbitol in preparing medicine for treating and preventing diseases caused by SARS-CoV-2 virus
<130> CGGNQASY216058
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agaagattgg ttagatgatg atagt 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttccatctct aattgaggtt gaacc 25
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agcctcaaga tcatcagcaa tg 22
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggactgtg gtcatgagtc ctt 23
Claims (5)
1.1, 5-use of sorbitan, characterized in that: the application is any one of the following:
the application of U1 and 1, 5-dehydrated sorbitol in preparing medicine for preventing and/or treating SARS-CoV-2 virus caused diseases or SARS-CoV-2 virus infection;
application of U2 and 1, 5-dehydrated sorbitol in preparing SARS-CoV-2 virus inhibitor is provided.
Application of 1,5-anhydro sorbitol in preparing products for inhibiting SARS-CoV-2 virus proliferation or replication.
Use of 1, 5-sorbitan in the preparation of products for inhibiting the proliferation or replication of SARS-CoV-2 virus in animal individuals, animal organs, animal tissues or animal cells.
4. Use according to claim 3, characterized in that: the animal is a mammal.
5. Use according to claim 4, characterized in that: the cells are human or non-erythematous monkey cells.
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| PCT/CN2022/091628 WO2022228581A1 (en) | 2021-04-28 | 2022-05-09 | Application of 1,5-dehydrated sorbitol in preparation of drugs for treating and preventing diseases caused by sars-cov-2 virus |
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| JP2013075895A (en) * | 2011-09-16 | 2013-04-25 | Kissei Pharmaceutical Co Ltd | Pharmaceutical composition for treatment of urinary tract infection |
| CN107835636A (en) * | 2014-12-17 | 2018-03-23 | 特雷奥斯淀粉及甜味剂比利时公司 | The bactericidal composition of isomer mixture comprising monose alkyl monoacetal or monose AlkylMonoETHer |
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| KR102330592B1 (en) * | 2015-08-26 | 2021-11-24 | 에스케이이노베이션 주식회사 | Method of Preparing Anhydrosugar Alcohols Using Steam Stripping |
| CN111329998A (en) * | 2020-03-27 | 2020-06-26 | 广东天普生化医药股份有限公司 | Application of ulinastatin in preparing medicine for preventing and treating severe coronavirus pneumonia |
| CN112255219A (en) * | 2020-10-12 | 2021-01-22 | 中拓生物有限公司 | 1, 5-sorbitan determination kit, and preparation method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013075895A (en) * | 2011-09-16 | 2013-04-25 | Kissei Pharmaceutical Co Ltd | Pharmaceutical composition for treatment of urinary tract infection |
| CN107835636A (en) * | 2014-12-17 | 2018-03-23 | 特雷奥斯淀粉及甜味剂比利时公司 | The bactericidal composition of isomer mixture comprising monose alkyl monoacetal or monose AlkylMonoETHer |
Non-Patent Citations (2)
| Title |
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| Altered Lipid Metabolism in Recovered SARS Patients Twelve Years after Infection;Wu, Qi等;《Scientific Reports》;20170822;第7卷(第1期);第1-12页 * |
| The serum metabolome of COVID-19 patients is distinctive and predictive;Ding Shi等;《Metabolism, Clinical and Experimental》;20210302(第118期);第154739（1-12）页 * |
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