CN116059229B - Pharmaceutical composition for preventing and treating new coronavirus infection, and medicine and application thereof - Google Patents
Pharmaceutical composition for preventing and treating new coronavirus infection, and medicine and application thereof Download PDFInfo
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Abstract
The invention discloses a pharmaceutical composition for preventing and treating new coronavirus infection, and a medicine and application thereof, belonging to the technical field of medicines, wherein the pharmaceutical composition for preventing and treating new coronavirus infection comprises the following active components: indirubin, syringic acid, 4-hydroxy quinazoline, dyers woad leaf flavone, isoquercitrin, chlorogenic acid, methyl gallate, rutin, mulberry leaf flavone, p-coumaric acid, ferulic acid, glycyrrhizic acid, glycyrrhizin, apioside and licoflavone. The pharmaceutical composition has definite functional components, and the pharmaceutical composition composed of the active components can effectively reduce the S protein level of the novel coronavirus, has better prevention and treatment effects on the novel coronavirus infection, and has the advantages of smaller dosage and more treatment effects on the novel coronavirus infection compared with the original Shuqing granule.
Description
Technical Field
The invention relates to the technical field of pharmaceutical compositions, in particular to a pharmaceutical composition for preventing and treating new coronavirus infection, and a medicine and application thereof.
Background
The Shuqing granule is a Chinese patent medicine which is commercially available at present and consists of five medicinal materials of gypsum, dyers woad leaf, mulberry leaf, reed rhizome and liquorice, has the functions of clearing away heat and toxic materials and freeing lung and stomach, and is used for treating the exogenous wind-heat syndrome of children.
The Chinese patent ZL202110628320.1 can obviously reduce IL-6 secretion and NF-kBp 65 protein phosphorylation level of Calu-3 cells caused by SARS-CoV-2S protein stimulation, and the clearing particles can regulate NF-kB signal channels by competing with the receptors of SARS-CoV-2 on the cells and play a role in resisting inflammatory response induced by SARS-CoV-2. On the basis of the research, the invention searches the relevant effective components of the Shuqing granule through the deep research of the Shuqing granule, and develops a pharmaceutical composition for preventing and treating the new coronavirus infection, which has definite components and better effect.
Disclosure of Invention
The invention aims to provide a pharmaceutical composition with definite components and better effect for preventing and treating new coronavirus infection, and a medicine and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a pharmaceutical composition for preventing and treating new coronavirus infection, which comprises the following active ingredients: indirubin, syringic acid, 4-hydroxy quinazoline, dyers woad leaf flavone, isoquercitrin, chlorogenic acid, methyl gallate, rutin, mulberry leaf flavone, p-coumaric acid, ferulic acid, glycyrrhizic acid, glycyrrhizin, apioside and licoflavone.
The pharmaceutical composition is based on Chinese patent medicine Shuqing granules, and is obtained by carefully selecting active ingredients of indirubin, syringic acid, 4-hydroxy quinazoline and dyers woad leaf flavone in dyers woad leaf, active ingredients of isoquercitrin, chlorogenic acid, methyl gallate, rutin and mulberry leaf flavone in dyers woad leaf, and compounding active ingredients of coumaric acid, ferulic acid and liquorice in the reed rhizome, namely glycyrrhizic acid, glycyrrhizin, apioside and licoflavone.
The pharmaceutical composition comprising the active ingredients can effectively reduce the S protein level of the novel coronavirus and has better prevention and treatment effects on the novel coronavirus infection.
In some preferred embodiments, indirubin is used in the present invention in an amount of 0.3-1 parts by weight, preferably any one of values between 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.
In some preferred embodiments, the amount of syringic acid used in the present invention is from 0.03 to 0.08 parts by weight, preferably any value between 0.03, 0.04, 0.05, 0.06, 0.07 and 0.08.
In some preferred embodiments, the 4-hydroxy quinazoline of the present invention is used in an amount of from 0.03 to 0.08 parts by weight, preferably any one of values between 0.03, 0.04, 0.05, 0.06, 0.07 and 0.08.
In some preferred embodiments, the amount of the flavonoids of dyers woad leaf in the present invention is 180 to 240 parts by weight, preferably any one of values between 180, 190, 200, 210, 220, 230 and 240.
In some preferred embodiments, isoquercitrin is used in the present invention in an amount of 20-50 parts by weight, preferably any of values between 20, 30, 40 and 50.
In some preferred embodiments, chlorogenic acid is used in the present invention in an amount of 3-10 parts by weight, preferably any of 3, 4, 5, 6, 7, 8, 9 and 10.
In some preferred embodiments, the methyl gallate is used in the present invention in an amount of 130 to 180 parts by weight, preferably any number between 130, 140, 150, 160, 170 and 180.
In some preferred embodiments, rutin is used in the present invention in an amount of 1.5 to 2.2 parts by weight, preferably any one of values between 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 and 2.2.
In some preferred embodiments, the mulberry leaf flavone is used in the present invention in an amount of 60 to 80 parts by weight, preferably any one of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and 80.
In some preferred embodiments, the p-coumaric acid is used in the present invention in an amount of 35 to 45 parts by weight, preferably any one of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45.
In some preferred embodiments, the amount of ferulic acid used in the present invention is 10-15 parts by weight, preferably any of 10, 11, 12, 13, 14 and 15.
In some preferred embodiments, the glycyrrhizic acid is used in the present invention in an amount of 75-85 parts by weight, preferably any one of values between 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 and 85.
In some preferred embodiments, the glycyrrhizin is used in the present invention in an amount of 18-22 parts by weight, preferably any of 18, 19, 20, 21 and 22.
In some preferred embodiments, the apigenin is used in an amount of 5-12 parts by weight, preferably any one of 5, 6, 7, 8, 9, 10, 11 and 12.
In some preferred embodiments, licoflavone is used in the present invention in an amount of 20-30 parts by weight, preferably any one of values between 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.
In some preferred embodiments, the pharmaceutical composition for preventing and treating a novel coronavirus infection comprises the following active ingredients in parts by weight: 0.3-1 part of indirubin, 0.03-0.08 part of syringic acid, 0.03-0.08 part of 4-hydroxy quinazoline, 180-240 parts of dyers woad leaf flavonoid, 20-50 parts of isoquercitrin, 3-10 parts of chlorogenic acid, 130-180 parts of methyl gallate, 1.5-2.2 parts of rutin, 60-80 parts of mulberry leaf flavonoid, 35-45 parts of p-coumaric acid, 10-15 parts of ferulic acid, 75-85 parts of glycyrrhizic acid, 18-22 parts of glycyrrhizin, 5-12 parts of apioside and 20-30 parts of licoflavone.
In some preferred embodiments, the pharmaceutical composition for preventing and treating a novel coronavirus infection comprises the following active ingredients in parts by weight: 0.4-0.9 part of indirubin, 0.04-0.07 part of syringic acid, 0.04-0.07 part of 4-hydroxy quinazoline, 190-230 parts of dyers woad leaf flavonoid, 30-40 parts of isoquercitrin, 4-9 parts of chlorogenic acid, 140-170 parts of methyl gallate, 1.6-2.1 parts of rutin, 61-79 parts of mulberry leaf flavonoid, 36-44 parts of p-coumaric acid, 11-14 parts of ferulic acid, 76-84 parts of glycyrrhizic acid, 19-21 parts of glycyrrhizin, 6-11 parts of apiose glycyrrhizin and 21-29 parts of licoflavone.
The pharmaceutical composition of the present invention further comprises gypsum. Specifically, the gypsum is used in an amount of 750 to 850 parts by weight, preferably any one of values between 750, 760, 770, 780, 790, 800, 810, 820, 830, 840 and 850.
In some more preferred embodiments, the pharmaceutical composition for preventing and treating a new coronavirus infection comprises the following components in parts by weight: 0.6 part of indirubin, 0.05 part of syringic acid, 0.05 part of 4-hydroxy quinazoline, 200 parts of dyers woad leaf flavone, 30 parts of isoquercitrin, 5 parts of chlorogenic acid, 150 parts of methyl gallate, 2 parts of rutin, 75 parts of mulberry leaf flavone, 40 parts of p-coumaric acid, 12 parts of ferulic acid, 80 parts of glycyrrhizic acid, 20 parts of glycyrrhizin, 8 parts of apioside, 25 parts of licoflavone and 800 parts of gypsum.
The active ingredients of indirubin, syringic acid, 4-hydroxy quinazoline and dyers woad leaf flavone are common commercial products, namely isoquercitrin, chlorogenic acid, methyl gallate, rutin and mulberry leaf flavone.
The invention also provides a preparation method of the pharmaceutical composition, which comprises the following steps: the indirubin, syringic acid, 4-hydroxy quinazoline, dyers woad leaf flavone, isoquercitrin, chlorogenic acid, methyl gallate, rutin, mulberry leaf flavone with the formula amount are mixed, crushed and sieved to obtain the pharmaceutical composition.
The invention also provides application of the pharmaceutical composition in preparing medicines for preventing and treating new coronavirus infection.
The invention also provides a medicine for preventing and treating the novel coronavirus infection, which comprises the pharmaceutical composition and pharmaceutically acceptable auxiliary materials.
Preferably, the medicament may be in the form of a tablet, capsule, granule, paste, suspension, powder, injection, spray or pill.
The beneficial effects of the invention are as follows:
(1) The invention screens the active ingredients of the formula based on the original Shuqing granules to obtain the pharmaceutical composition containing fifteen active ingredients of indirubin, syringic acid, 4-hydroxy quinazoline, dyers woad leaf flavone, isoquercitrin, chlorogenic acid, methyl gallate, rutin and mulberry leaf flavone, and the pharmaceutical composition contains coumaric acid, ferulic acid, glycyrrhizic acid, glycyrrhizin, apioside and licoflavone. Through the research of the drug effect experiments of the pharmaceutical composition, the pharmaceutical composition provided by the invention can effectively reduce the S protein level of the novel coronavirus and has a better prevention and treatment effect on the novel coronavirus infection.
(2) Compared with the original Shuqing granule, the pharmaceutical composition for preventing and treating the new coronavirus infection has the advantages of more definite ingredients, less dosage and more advantages for preventing and treating the new coronavirus infection.
Drawings
ACE2 protein expression in different cells of figure 1.
Detailed Description
The following examples are presented only to aid in understanding the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The active ingredients in this example are all common commercial products, and the purchase information is shown in table 1;
TABLE 1 information on active ingredients
Example 1
The pharmaceutical composition for preventing and treating the novel coronavirus infection is prepared from the following components in parts by weight: 0.6 part of indirubin, 0.05 part of syringic acid, 0.05 part of 4-hydroxy quinazoline, 200 parts of dyers woad leaf flavone, 30 parts of isoquercitrin, 5 parts of chlorogenic acid, 150 parts of methyl gallate, 2 parts of rutin, 75 parts of mulberry leaf flavone, 40 parts of p-coumaric acid, 12 parts of ferulic acid, 80 parts of glycyrrhizic acid, 20 parts of glycyrrhizin, 8 parts of apioside, 25 parts of licoflavone and 800 parts of gypsum.
Example 2
The pharmaceutical composition for preventing and treating the novel coronavirus infection is prepared from the following components in parts by weight: 0.3 part of indirubin, 0.03 part of syringic acid, 0.03 part of 4-hydroxy quinazoline, 240 parts of dyers woad leaf flavone, 20 parts of isoquercitrin, 3 parts of chlorogenic acid, 130 parts of methyl gallate, 1.5 parts of rutin, 80 parts of mulberry leaf flavone, 35 parts of p-coumaric acid, 10 parts of ferulic acid, 75 parts of glycyrrhizic acid, 18 parts of glycyrrhizin, 5 parts of apioside, 20 parts of licoflavone and 750 parts of gypsum.
Example 3
The pharmaceutical composition for preventing and treating the novel coronavirus infection is prepared from the following components in parts by weight: 1 part of indirubin, 0.08 part of syringic acid, 0.08 part of 4-hydroxy quinazoline, 180 parts of dyers woad leaf flavone, 50 parts of isoquercitrin, 10 parts of chlorogenic acid, 180 parts of methyl gallate, 2.2 parts of rutin, 60 parts of mulberry leaf flavone, 45 parts of p-coumaric acid, 15 parts of ferulic acid, 85 parts of glycyrrhizic acid, 22 parts of glycyrrhizin, 12 parts of apioside, 30 parts of licoflavone and 850 parts of gypsum.
Example 4
The pharmaceutical composition for preventing and treating the novel coronavirus infection is prepared from the following components in parts by weight: 0.4 part of indirubin, 0.04 part of syringic acid, 0.04 part of 4-hydroxy quinazoline, 230 parts of dyers woad leaf flavone, 30 parts of isoquercitrin, 4 parts of chlorogenic acid, 140 parts of methyl gallate, 1.6 parts of rutin, 79 parts of mulberry leaf flavone, 36 parts of p-coumaric acid, 11 parts of ferulic acid, 76 parts of glycyrrhizic acid, 19 parts of glycyrrhizin, 6 parts of apigenin, 21 parts of licoflavone and 760 parts of gypsum.
Example 5
The pharmaceutical composition for preventing and treating the novel coronavirus infection is prepared from the following components in parts by weight: 0.9 part of indirubin, 0.07 part of syringic acid, 0.07 part of 4-hydroxy quinazoline, 190 parts of dyers woad leaf flavone, 40 parts of isoquercitrin, 9 parts of chlorogenic acid, 170 parts of methyl gallate, 2.1 parts of rutin, 61 parts of mulberry leaf flavone, 44 parts of p-coumaric acid, 14 parts of ferulic acid, 76 parts of glycyrrhizic acid, 21 parts of glycyrrhizin, 11 parts of apioside, 29 parts of licoflavone and 840 parts of gypsum.
Comparative example 1
The difference between this comparative example and example 1 is that the amounts of dyers woad leaf flavone, isoquercitrin, methyl gallate, rutin, mulberry leaf flavone, p-coumaric acid, glycyrrhizic acid and apioside were different.
Specifically, the pharmaceutical composition for preventing and treating the novel coronavirus infection is prepared from the following components in parts by weight: 0.6 part of indirubin, 0.05 part of syringic acid, 0.05 part of 4-hydroxy quinazoline, 160 parts of dyers woad leaf flavone, 70 parts of isoquercitrin, 5 parts of chlorogenic acid, 200 parts of methyl gallate, 3 parts of rutin, 50 parts of mulberry leaf flavone, 30 parts of p-coumaric acid, 12 parts of ferulic acid, 70 parts of glycyrrhizic acid, 20 parts of glycyrrhizin, 20 parts of apioside, 25 parts of licoflavone and 800 parts of gypsum.
Comparative example 2
The difference between this comparative example and example 1 is that the amounts of dyers woad leaf flavone, isoquercitrin, methyl gallate, rutin, mulberry leaf flavone, p-coumaric acid, glycyrrhizic acid and apioside were different.
Specifically, the pharmaceutical composition for preventing and treating the novel coronavirus infection is prepared from the following components in parts by weight: 0.6 part of indirubin, 0.05 part of syringic acid, 0.05 part of 4-hydroxy quinazoline, 250 parts of dyers woad leaf flavone, 10 parts of isoquercitrin, 5 parts of chlorogenic acid, 100 parts of methyl gallate, 5 parts of rutin, 120 parts of mulberry leaf flavone, 50 parts of p-coumaric acid, 12 parts of ferulic acid, 90 parts of glycyrrhizic acid, 20 parts of glycyrrhizin, 3 parts of apioside, 25 parts of licoflavone and 800 parts of gypsum.
Comparative example 3
This comparative example differs from example 1 in that lupeol and ilexoside D in folium Isatidis are used to replace syringic acid and 4-hydroxy quinazoline, astragalin in folium Mori is used to replace isoquercitrin, and glycyrrhizin in Glycyrrhrizae radix is used to replace glycyrrhizic acid.
Specifically, the pharmaceutical composition for preventing and treating the novel coronavirus infection is prepared from the following components in parts by weight: 0.6 part of indirubin, 0.05 part of lupeol, 0.05 part of ilex latifolia thunb glycoside D, 200 parts of dyers woad leaf flavone, 30 parts of astragalin, 5 parts of chlorogenic acid, 150 parts of methyl gallate, 2 parts of rutin, 75 parts of mulberry leaf flavone, 40 parts of p-coumaric acid, 12 parts of ferulic acid, 80 parts of glycyrrhizin, 20 parts of glycyrrhizin, 8 parts of apigenin, 25 parts of licoflavone and 800 parts of gypsum.
Evaluation of drug efficacy
The drug effect experimental study of the pharmaceutical composition for inhibiting the novel coronavirus S protein is carried out, the influence of the pharmaceutical composition on the novel coronavirus is observed, and the prevention and treatment effect of the pharmaceutical composition on the novel coronavirus infection is evaluated.
1 Experimental materials
1.1 Experimental cell and experimental drug
(1) Calu-3 cells: calu-3 is a human lung adenocarcinoma cell cryopreserved by the basic medical college at Jilin university;
(2) A549 cells: a549 cells are adenocarcinoma human alveolar basal epithelial cells, cryopreserved by the basic medical college of the university of gilin;
(3) 16HBE cells: is a human bronchial epithelial-like cell frozen by the basic medical college of Jilin university;
(4) BEAS-2B cells: is normal lung epithelial cells of human beings, frozen by basic medical college of Jilin university;
(5) 293T cells: the high transfection efficiency derived strain produced by human embryonic kidney cell strain 293 inserted with SV 40T-antigen gene was designated 293T and frozen by Jilin university basic medical college;
(6) Shuqing granule: jilin Huakang pharmaceutical Co., ltd., approval document: the Chinese medicine standard character size Z10980132 is dispersed by equal distilled water when in use, and is uniformly shaken when in use and stored at 4 ℃;
(7) Comparative examples 1-3, example 1 high dose, example 1 medium dose, example 1 low dose, examples 2-5, all supplied by Jilin Huakang pharmaceutical Co., ltd, dispersed with equal amounts of distilled water when used, shaken well when used, and stored at 4 ℃;
(8) SARS-CoV 2S protein: the SARS-CoV-2 genome encodes one of four major structural proteins, available from Beijing Yiqiao corporation (Sino Biological Inc).
1.2 Experimental reagents
TABLE 2 Main reagents
1.3 laboratory apparatus
TABLE 3 Main instruments
2 Experimental methods
2.1 cell culture
(1) Cell resuscitation: preheating a water bath to 37 ℃ in advance, taking out frozen cells in a liquid nitrogen tank, rapidly placing the frozen cells in the water bath at 37 ℃, continuously shaking the frozen cells, centrifuging (1000 rpm,5 min) when a small part of the cells are crystallized in the frozen cells, transferring the frozen cells into an ultra-clean workbench for aseptic operation, discarding the supernatant, adding 1 mL culture solution into the precipitate, gently and repeatedly blowing and mixing the mixture until no cell mass exists, transferring the mixture into an aseptic centrifuge tube, adding 5mL culture solution into the centrifuge tube, continuously mixing the mixture, transferring the mixture into a disposable cell culture dish, and placing the disposable cell culture dish into a cell culture box (37 ℃ and 5% CO) 2 ) Cultivation methodCulturing;
(2) Cell passage: when the growth density of cells in the culture dish is observed to be about 80% -90%, the cells are passaged. The original culture medium is firstly sucked by a pipette, after the culture medium is washed 3 times by sterile PBS, 2 mL of 0.25% pancreatin solution is added to be placed in an incubator for digestion, and when most cells are observed to shrink and round under the observation of a microscope, 1 mL culture solution is added to stop the digestion. The cells attached to the petri dish were gently scraped with a pipette and all transferred to a sterile centrifuge tube for centrifugation (1000 rpm,5 min). Discarding the supernatant, re-suspending the cells with a cell culture solution, and sub-packaging the cells into 2-3 new cell culture dishes according to the requirement for continuous culture;
(3) Cell cryopreservation: when the cells are in the logarithmic growth phase and have good morphology, the original culture medium is firstly sucked by a liquid transfer device, after the cells are washed 3 times by sterile PBS, 2 mL of 0.25% pancreatin solution is added to be placed in an incubator for digestion, and when most of the cells are observed to shrink and round under a microscope, 1 mL culture solution is added to terminate the digestion. The cells attached to the petri dish were gently scraped with a pipette and all transferred to a sterile centrifuge tube for centrifugation (1000 rpm,5 min). The supernatant is discarded, a proper amount of cell freezing solution (DMSO: cell culture solution: serum=7:2:1) is added and split charging is carried out on the cell freezing solution into a freezing tube, then the cell freezing solution is filled into a cell freezing box, and the cell freezing box is placed into a refrigerator at the temperature of minus 80 ℃ for freezing 24 h and then is transferred into a liquid nitrogen tank for preservation.
2.2 Total protein extraction and protein concentration determination
(1) Cellular protein extraction
A. Preparation of cell lysate: each 1 mL lysate contains 10. Mu.L of LPMSF, 10. Mu.L of phosphatase inhibitor and 980. Mu.L of RIPA;
B. absorbing and discarding the original culture solution of the cells, and flushing the cells for 3 times by using precooled PBS buffer solution;
C. placing the six-hole plate on ice, adding 150 mu L of protein lysate into each hole, and blowing the lysate by using a pipettor to make the lysate fully contact with cells;
D. transferring the liquid into an EP tube, placing on ice, performing vortex oscillation once every 10 min, repeating for three times, centrifuging (4 ℃ C., 12000 g,5 min), and collecting the supernatant to obtain the total cell protein.
(2) Protein concentration determination
A. Preparing BCA working solution: liquid A and liquid B in the BCA kit are mixed according to the proportion of 50:1, uniformly mixing, and preparing in situ;
B. protein standard solution preparation: completely dissolving the protein standard in PBS, and diluting to the concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL respectively;
C. loading: 200 mu L of BCA working solution, 20 mu L of protein standard substances with various concentrations and samples to be tested are added into each hole of a 96-well plate, and the mixture is uniformly mixed;
D. incubation: incubating the 96-well plate at 37 ℃ for 30 min;
E. and detecting an OD value at a wavelength of 562nm by using an enzyme-labeled instrument, drawing a standard curve, and calculating the protein concentration of the sample to be detected.
2.3 Western immunoblotting (Western blot)
(1) Adding 4 mL separating glue into the fixed clean glue plate, then rapidly injecting absolute ethyl alcohol above to ensure that the glue block is horizontally bubble-free, pouring the absolute ethyl alcohol obliquely after the separating glue is solidified, adding concentrated glue, slightly inserting a glue comb, and waiting for the solidification of the concentrated glue;
(2) Pretreatment of protein samples: adding 5×loading buffer into the sample at a certain proportion, mixing, placing into boiling water for 10 min, and storing at 4deg.C until room temperature is restored;
(3) Loading: mounting an electrophoresis tank and a rubber plate to ensure no leakage, adding electrophoresis liquid into the electrophoresis tank until the liquid level overflows from the outer glass plate, slightly pulling out a rubber comb, slightly blowing a sample loading hole by using a liquid shifter, slowly adding a processed protein sample, and adding protein markers into holes on two sides;
(4) Electrophoresis: the power supply is switched on, the constant voltage electrophoresis is carried out until the sample is compressed to the same position and reaches the junction of the separation gel and the concentrated gel, the voltage is increased to 120V, the electrophoresis is continued until the bromophenol blue indicator reaches the lower edge of the separation gel, and the electrophoresis is stopped;
(5) Transferring: after electrophoresis, the sponge cushion, the filter paper and the membrane transferring liquid are soaked in advance, the target strip is cut according to the indication position of the protein Marker strip, the PVDF membrane is cut according to the size of gel, and the PVDF membrane is soaked in methanol for activation by 30 s. Then correctly assembling a film-transferring clamping plate according to the sequence of the negative electrode, the foam-rubber cushion, the filter paper, the gel-PVDF film, the filter paper, the foam-rubber cushion and the positive electrode, putting the film-transferring clamping plate into a film-transferring groove, filling film-transferring liquid, and keeping the constant flow of ice bath for 100 mA for 1 h;
(6) Closing: taking out the PVDF membrane, washing for 5min by TBST, repeating for three times, adding 5% BSA solution, and sealing for two hours at room temperature;
(7) Incubation resistance: the primary antibody was diluted to the appropriate concentration as indicated with a 5% BSA solution. Discarding the sealing liquid, putting the sealed PVDF membrane into the corresponding primary antibody, and incubating overnight at 4 ℃;
(8) Washing the film: the next day the primary antibody was recovered and stored at 4 ℃. Washing PVDF membrane with TBST buffer solution for 6 times, each time for 5min;
(9) Secondary antibody incubation: diluting the secondary antibody to a proper concentration by using TBST, putting the cleaned PVDF film into a corresponding secondary antibody solution, and incubating for 1 h at room temperature;
(10) Washing the film: absorbing and discarding the secondary antibody, and cleaning the PVDF film by using TBST buffer solution for 6 times, each time for 5min;
(11) Developing: ECL developer A, B liquid is mixed according to the proportion of 1:1, and the mixture is prepared at present. Soaking the PVDF film in a developing solution, slightly shaking to enable the film to fully contact the solution, and then exposing and developing the PVDF film by using a gel imager;
(12) Gray value analysis: the grey values of the individual bands were calculated using Image J version6.0 software for subsequent analysis.
2.4 Determination of maximum non-toxic concentration of cells by CCK8 method
5. 5g dredge particles, 0.5g of each composition of the examples and the comparative examples are weighed and dissolved in 100 mL of preheated cell maintenance liquid at 37 ℃, and the dissolved drug solution is filtered once by a sterile filter head of 0.45 mu m and then filtered once by a sterile filter head of 0.22 mu m, so that 50 mg/mL of dredge particle liquid and 5 mg/mL of drug composition are finally obtained as mother liquor. Diluting the mother solution of the dredged particles according to concentration gradient to 8 concentrations of liquid medicine which is obtained by diluting the mother solution of the dredged particles with the concentration of 40 mg/mL, 30 mg/mL, 20 mg/mL, 10mg/mL, 5 mg/mL, 1 mg/mL and 0.1 mg/mL and the mother solution respectively; diluting the mother liquor of the pharmaceutical composition to the concentration of 4 mg/mL, 3 mg/mL, 2 mg/mL and 1 mg +.The 8 concentration liquid medicine of the diluted liquid and the mother liquid of the mL, 0.5 mg/mL, 0.1 mg/mL and 0.01 mg/mL are ready for use and are prepared at present. Taking cell suspension in logarithmic growth phase, diluting to 5×10 4 -1×10 5 mu.L of the diluted cell suspension was added to each well of a 96-well plate at 37℃and 5% CO 2 Cell incubator interior 24 h. The supernatant was discarded, and 100. Mu.L of each concentration of the drug solution was added to each well in a concentration gradient, and a blank group was added thereto, i.e., 100. Mu.L of the maintenance solution was added to the wells. After 24-h drug action, the culture broth was discarded and 100. Mu.L of maintenance fluid and 10. Mu.L of CCK8 reagent were added to each well. Then put into an incubator for incubation of 1-4 h, and OD is measured 450 Calculate the maximum non-Toxic Concentration (TC) 0 ) Repeating three times.
2.5 Detection of cell growth index by real-time label-free cell function analyzer (RTCA)
Cell growth index was recorded from live cell resistance values using RTCA software. Dilution of the cell suspension to 1X 10 according to instructions 4 Cell suspension was added to the plate at 100. Mu.L per well per mL. 24 After h, the culture solution is sucked and removed, and the diluted medicinal composition liquid in the concentration of 2.4 is respectively added according to the concentration gradient, wherein each well is 100 mu L.48 After h, observing the cell growth index of each hole, calculating the relative activity of the cells, and selecting the maximum non-toxic concentration for subsequent experiments in combination with CCK8 experiment results.
2.6 ELISA (enzyme-Linked immunosorbent assay)
(1) Taking cell suspension in logarithmic phase, inoculating in 6-hole plate, culturing to cell growth density of 80-90%;
(2) Carefully washing cells with PBS for 2-3 times, adding each group of drugs, and setting a blank control group;
(3) Collecting cell supernatant, centrifuging (4deg.C, 12000 g,15 min), and collecting supernatant;
(4) The ELISA detection kit is equilibrated for 30 min at room temperature;
(5) Adding standard diluent into the standard tube, and mixing by vortex to obtain mother liquor. Diluting the mother solution by multiple ratios according to the instruction of the kit, and preparing standard substances with different concentration gradients;
(6) Respectively adding a gradient concentration standard substance and a sample to be detected into the micro-pore plate coated with the antibody, wherein each hole is 100 mu L;
(7) Adding 50 mu L of biotin-binding antibody into each hole, sealing the plates, placing the plates on a shaking table, slowly shaking and uniformly mixing, and incubating for 2-3h at room temperature;
(8) Removing the supernatant, adding 300 mu L of 1 Xwashing liquid into each hole, washing for three times, and drying on filter paper after the third washing;
(9) 100 mu L of horseradish peroxidase (HRP) -labeled detection antibody is added to each well, the plates are sealed, and 1 h is incubated on a shaker at room temperature;
(10) Discarding the supernatant, and repeating the step (8) for cleaning;
(11) Adding 100 mu L of TMB reaction substrate into each hole, sealing the plates, and incubating for 30 min at room temperature in a dark place;
(12) And adding 50 mu L of stop solution into each hole, fully and uniformly mixing, detecting an OD value at the position with the wavelength of 450 nm by using an enzyme-labeled instrument, drawing a standard curve, and calculating the content of corresponding cytokines in a sample to be detected.
2.7 data processing
All experimental data in this study were analyzed using Graphpad prism v6.0. Firstly, the variance homogeneity and normal distribution condition of the data are detected, and then the t-test is used for analysis.P<0.05 shows a statistical difference in the number of samples,P<0.01 shows a significant statistical difference. The Western blot bands were analyzed for gray value using Image J version 6.0.
3 results of experiments
3.1 cell Screen for high expression of ACE2 protein
The molecular docking results show that ACE2 is one of the receptor binding active ingredients of the dredged particles and is also SARS-CoV-2S protein binding receptor, so in order to research whether the pharmaceutical composition can inhibit the stimulation of SARS-CoV-2S protein to cells, cell strains with high expression of S protein binding receptor ACE2 are firstly screened, A549, 16HBE, BEAS-2B, calu-3 and 293T cells are respectively inoculated into a cell culture dish, when the growth density of cells in the culture dish is about 80% -90%, total proteins of various cells are respectively extracted, and the expression of ACE2 in various cells is detected by using a Western blot method, and the results are shown in figure 1 and table 4, and compared with other four cells, the expression quantity of ACE2 of the Calu-3 cells is higher. The invention thus uses Calu-3 cells for subsequent experiments.
TABLE 4 expression of ACE2 in different cell lines
Note that: "-" means that the protein is not expressed; "+" indicates that the protein is expressed.
3.2 toxicity of the groups of drugs to Calu-3 cells
To determine whether each group of drugs was toxic to Calu-3 cells, each group of drugs was tested for toxicity to Calu-3 cells using the CCK8 method, and the results are shown in Table 5. When the drug concentration is more than 30 mg/mL, the cell survival rate is obviously reducedP< 0.01); whereas at concentrations less than 20 mg/mL, there was no difference in cell viability compared to the control group. Using RTCA technology, it was further confirmed that the maximum non-toxic concentration of the lyophobic particles on Calu-3 cells decreased as the concentration of drug administered increased at 30, 40, 50 mg/mL for the lyophobic particles, and 3, 4, 5 mg/mL for each of the examples and comparative examples. Thus, we finally used examples 1.5 mg/mL, 1 mg/mL, 2 mg/mL as low, medium, and high doses, with 10mg/mL of the dredged particles, examples 2-5, and comparative examples 1-3 mg/mL for subsequent experiments.
TABLE 5 toxicity of various groups of drugs to Calu-3 cells
Note that: "+" means toxic and "-" means non-toxic.
3.3 Optimal concentration and time screening of SARS-CoV-2S protein stimulated Calu-3 cells
IL-6 is one of the important inflammatory cytokines in SARS-CoV-2-induced production of COVID-19 pneumonia and is mainly induced by activation of the NF- κB classical pathway. Therefore, in order to determine the optimal conditions for stimulating Calu-3 cells with SARS-CoV-2S protein, the levels of IL-6 in the cell supernatants were determined by ELISA using 100, 200, 400, 800, 1000ng/mL concentrations of S protein to stimulate Calu-3 cells 6h and 24 h, respectively.
The results in Table 6 show that the amount of IL-6 secretion increases with increasing S protein concentration. The stimulation 24 h group had an overall increase in IL-6 secretion compared to the S protein stimulation 6h group. The IL-6 secretion of 1000ng/mL S protein group is increased compared with that of blank group in two time pointsP＜0.05，P< 0.01). 1000 The IL-6 secretion amount of the group 24 and h stimulated by ng/mL S protein is increased compared with that of the group 6 and h stimulated by S protein with the same concentrationP< 0.05), whereby 1000ng/mL S protein stimulated 24 h is seen as the optimal stimulation condition.
TABLE 6 variation of IL-6 expression in cells (unit pg/mL) at different concentrations of S protein and time of action
Note that: * Represent significant differences compared to the blanks of each group, i.eP<0.01; # # indicates significant differences compared to 1000ng/mL for the 6h group, i.eP<0.01。
In summary, the invention selects the Calu-3 in vitro research cell model stimulated by SARS-CoV-2S protein with the S protein concentration of 1000ng/mL and the stimulation time of 24 h.
3.4 Effect of the various groups of drugs on IL-6 secretion by Calu-3 cells after stimulation with SARS-CoV-2S protein
To see if each group of drugs was able to inhibit the stimulatory effect of SARS-CoV-2S protein on Calu-3 cells, inflammatory changes were characterized by detecting IL-6 levels in the supernatants of each group of cells, as shown in Table 7.
TABLE 7 level variation of IL6 expression (unit pg/mL)
Note that: * Representing significant differences between model and blank groupsP<0.01; # indicates the difference compared with the model groupP<0.05, # is a significant difference, i.eP<0.01；&The expression shows that compared with the Shuqing granule groupThe difference of the origins is thatP<0.05，&&As a significant difference, i.eP<0.01。
3.5 Effect of each group of drugs on NF- κ B p65 protein and phosphorylation level of Calu-3 cells after stimulation with SARS-CoV-2S protein
There are a number of literature reports that IL-6 expression is closely related to activation of NF- κB signaling pathway, and that the NF- κB classical activation pathway is through NF- κ B p65 phosphorylation leading to transcription of intracellular target genes, so to investigate whether the effect of each group of drugs on IL-6 production is related to altered NF- κ B p65 phosphorylation, western Blot was used to examine the expression levels of NF- κ B p65 and its phosphorylated proteins in each of the above groups of cells. As shown in Table 8, the NF- κ B p65 protein phosphorylation level of the comparative example and the example 1-low group was not significantly changed, and the NF- κ B p65 protein phosphorylation level of the other example group was significantly reduced as compared with the S protein model groupP< 0.05). It can be seen that it can significantly inhibit the phosphorylation level of NF- κ B p65 protein after S protein stimulation, potentially affecting secretion production of IL-6.
TABLE 8 phosphorylation levels of NF- κ B p65 protein in Calu-3 cells after stimulation of S protein by groups
Note that: * The representation being different from the model set comparison, i.e.P<0.05 represents a significant difference compared to the model group, i.e.P<0.01。
Conclusion 4
The pharmaceutical composition of the invention can effectively inhibit the infection of the novel coronavirus, and the action mechanism is probably to inhibit IL-6 secretion and play a role in relieving inflammatory response caused by SARS-CoV-2 by regulating NF- κB signal path. Wherein example effect is better than comparative example and the group of dredged particles, example 1 is better than examples 2-4, example 1 is high dose optimal; and the comparative example has equivalent effect to the group of dredged particles.
The invention has been further described above in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Claims (7)
1. A pharmaceutical composition for inhibiting IL-6 secretion and NF- κ B p65 protein phosphorylation level of Calu-3 cells after SARS-CoV-2S protein stimulation, comprising the following active ingredients in parts by weight: 0.3-1 part of indirubin, 0.03-0.08 part of syringic acid, 0.03-0.08 part of 4-hydroxy quinazoline, 180-240 parts of dyers woad leaf flavonoid, 20-50 parts of isoquercitrin, 3-10 parts of chlorogenic acid, 130-180 parts of methyl gallate, 1.5-2.2 parts of rutin, 60-80 parts of mulberry leaf flavonoid, 35-45 parts of p-coumaric acid, 10-15 parts of ferulic acid, 75-85 parts of glycyrrhizic acid, 18-22 parts of glycyrrhizin, 5-12 parts of apioside glycyrrhizin, 20-30 parts of licoflavone and 750-850 parts of gypsum.
2. The pharmaceutical composition for inhibiting IL-6 secretion and NF- κ B p65 protein phosphorylation level of Calu-3 cells after SARS-CoV-2S protein stimulation according to claim 1, comprising the following active ingredients in parts by weight: 0.4-0.9 part of indirubin, 0.04-0.07 part of syringic acid, 0.04-0.07 part of 4-hydroxy quinazoline, 190-230 parts of dyers woad leaf flavonoid, 30-40 parts of isoquercitrin, 4-9 parts of chlorogenic acid, 140-170 parts of methyl gallate, 1.6-2.1 parts of rutin, 61-79 parts of mulberry leaf flavonoid, 36-44 parts of p-coumaric acid, 11-14 parts of ferulic acid, 76-84 parts of glycyrrhizic acid, 19-21 parts of glycyrrhizin, 6-11 parts of apiose glycyrrhizin, 21-29 parts of licoflavone and 750-850 parts of gypsum.
3. The pharmaceutical composition for inhibiting IL-6 secretion and NF- κ B p65 protein phosphorylation of Calu-3 cells after SARS-CoV-2S protein stimulation according to claim 2, comprising the following components in parts by weight: 0.6 part of indirubin, 0.05 part of syringic acid, 0.05 part of 4-hydroxy quinazoline, 200 parts of dyers woad leaf flavone, 30 parts of isoquercitrin, 5 parts of chlorogenic acid, 150 parts of methyl gallate, 2 parts of rutin, 75 parts of mulberry leaf flavone, 40 parts of p-coumaric acid, 12 parts of ferulic acid, 80 parts of glycyrrhizic acid, 20 parts of glycyrrhizin, 8 parts of apioside, 25 parts of licoflavone and 800 parts of gypsum.
4. A method of preparing a pharmaceutical composition for inhibiting IL-6 secretion from Calu-3 cells and the phosphorylation level of NF- κ B p65 protein after stimulation of SARS-CoV-2S protein according to any one of claims 1-3, comprising the steps of: the indirubin, syringic acid, 4-hydroxy quinazoline, dyers woad leaf flavone, isoquercitrin, chlorogenic acid, methyl gallate, rutin, mulberry leaf flavone with the formula amount are mixed, crushed and sieved to obtain the pharmaceutical composition.
5. Use of a pharmaceutical composition for inhibiting IL-6 secretion from Calu-3 cells and NF- κ B p65 protein phosphorylation level after SARS-CoV-2S protein stimulation as claimed in any one of claims 1-3 in the manufacture of a medicament for inhibiting IL-6 secretion from Calu-3 cells and NF- κ B p65 protein phosphorylation level after SARS-CoV-2S protein stimulation.
6. A medicament for inhibiting IL-6 secretion and NF- κ B p65 protein phosphorylation of Calu-3 cells after SARS-CoV-2S protein stimulation, comprising the pharmaceutical composition for inhibiting IL-6 secretion and NF- κ B p65 protein phosphorylation of Calu-3 cells after SARS-CoV-2S protein stimulation according to any one of claims 1-3 and pharmaceutically acceptable excipients.
7. The agent for inhibiting IL-6 secretion and NF- κ B p65 protein phosphorylation of Calu-3 cells after SARS-CoV-2S protein stimulation of claim 6, wherein the agent for inhibiting IL-6 secretion and NF- κ B p65 protein phosphorylation of Calu-3 cells after SARS-CoV-2S protein stimulation is in the form of a tablet, capsule, granule, paste, suspension, powder, injection, spray or pill.
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