CN111773296B - Application of kang' ai body resistance strengthening compound in preparation of novel coronavirus infection resisting medicine - Google Patents
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Abstract
The invention discloses an application of a kang ai FU ZHENG FU FANG ZHI JI in preparing a medicine for preventing/treating novel coronavirus infection. The invention discloses a compound preparation for strengthening healthy energy by constructing a drug screening model for blocking novel coronavirus spike glycoprotein and target cell receptor angiotensin converting enzyme 2(ACE2), which can inhibit the combination of the novel coronavirus spike glycoprotein and the target cell receptor ACE2 and the expression of II-type transmembrane serine protease (TMPRSS2) to prevent the activity of viruses from entering target cells, can obviously inhibit the generation of inflammatory factors (IL-2, IL-6 and the like) by virus infected cells, can be used for preventing and treating novel coronavirus infectious diseases, provides medication selection for patients with the novel coronavirus infectious diseases, and has important effect and significance for preventing and controlling the current epidemic situation.
Description
Technical Field
The invention relates to a new application of a medicament, in particular to an application of a kang' ai Fuzheng compound preparation in preparing a medicament for preventing/treating novel coronary virus infection, belonging to the technical field of Chinese medicament application.
Background
The health-care tea is a national medicine formula preparation, and comprises the following components by weight: replenishing qi, removing toxic substance, resolving hard mass, relieving swelling, regulating stomach function, and tranquilizing mind; can be used for treating leucopenia, thrombocytopenia, and asthenia, anorexia, emesis, and insomnia due to immunologic hypofunction. The main raw material components comprise ganoderma lucidum, astragalus root, roxburgh rose, prepared rehmannia root, glossy privet fruit, epimedium and ginger processed pinellia tuber.
Coronavirus is a enveloped single-stranded positive-strand RNA virus widely found in human, mammalian and avian hosts and can cause respiratory, intestinal, liver and nervous system diseases. Six of the human coronaviruses are known, four of which generally cause only mild respiratory symptoms like the common cold, and the other two, severe acute respiratory syndrome coronavirus (SARS) and middle east respiratory syndrome coronavirus (MERS), which can cause severe respiratory diseases. The novel coronavirus (COVID-19) is the seventh of the human coronaviruses, belonging to the Beta coronavirus.
The new coronavirus COVID-19 infected pneumonia patients mainly show fever, hypodynamia and dry cough, a few patients have symptoms of nasal obstruction, watery nasal discharge, diarrhea and the like, the severe cases mostly have dyspnea after 1 week, severe patients rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct and blood coagulation dysfunction, and part of the light patients have no pulmonary inflammation. The blood routine suggests that white blood cell counts are normal or reduced, lymphocytes are reduced, and viral nucleic acid is detected positively. CT mostly shows double lung multiple wear glass density shadow (GGO), mainly refers to double lung pleural distribution, and can be accompanied with air bronchus sign, lobular space thickening and pleural thickening, and few or few pleural effusion or lymph node enlargement. Relevant studies concluded that its natural host may be a bat. Human respiratory epithelial cells are infected by a molecular mechanism of binding of the S-protein to angiotensin converting enzyme (ACE2) and activation of type II transmembrane serine protease (TMPRSS2) in the human body.
The possibility of using Reidesciclovir for the treatment of novel coronaviruses is reported in Science on a near day. The Wuhan virus institute of Chinese academy of sciences and the Beijing poison drug institute are reported to perform the research on the effect of the old drug on the novel coronavirus, which provides the opportunity for the novel coronavirus patients who can not be cured by the drug. National Weissenberg et al issued a notice of "treatment protocol for pneumonia with novel coronavirus infection printed (trial third edition)" in which antiviral treatment protocol recommended lopinavir and ritonavir as drug candidates. Additional studies have shown that based on the clinical value of ambroxol for the treatment of respiratory diseases, it may be beneficial to treat new types of coronavirus infections, which have entered the diagnostic protocol of the national Weijian Commission (third and fourth edition). However, no specific medicine for treating coronavirus infection exists at present.
According to the fact that a novel coronavirus gene sequence and ACE2 reported by scientists No. 1 and No. 7 in 2020 are receptors for the virus to enter target cells, and type II transmembrane serine protease (TMPRSS2) of the target cells can promote the novel coronavirus spike protein to be combined with ACE2 and enter the cells, the invention constructs a drug screening model for blocking the combination of the coronavirus spike protein and ACE2, and carries out in-vitro activity screening on a plurality of Guizhou national drug compound preparations by utilizing the model, and the invention discovers that the Guizhou national drug vaccine compound preparation 'Kangai Fuzheng' has new application for the first time.
Disclosure of Invention
The invention aims to provide the application of a kang' ai Fuzheng compound preparation in preparing a medicine for preventing/treating novel coronavirus infection; the kang' ai FU FANG ZHI JI can effectively inhibit the novel coronavirus spike glycoprotein from being combined with a target cell receptor ACE2 and the expression of II type transmembrane serine protease (TMPRSS2) to prevent the virus from entering target cells, and can effectively reduce the production of inflammatory factors (IL-2, IL-6, TNF-a, IL-8 and CXCL-10) by virus-infected cells.
The technical scheme of the invention is as follows:
the application of the kang 'ai body resistance strengthening compound preparation in preparing a medicine for preventing/treating novel coronavirus infection is characterized in that the kang' ai body resistance strengthening compound preparation is prepared from 600 parts by weight of lucid ganoderma, 600 parts by weight of astragalus membranaceus, 500 parts by weight of rosa roxburghii tratt, 400 parts by weight of prepared rehmannia root, 400 parts by weight of privet fruit, 400 parts by weight of epimedium herb and 200 parts by weight of ginger processed pinellia tuber serving as raw material medicines.
In the application, the kang ai fuzheng compound preparation is preferably a capsule prepared by taking 40 parts by weight of starch and 10 parts by weight of talcum powder as auxiliary materials.
In the application, the preparation method of the kang' ai Fuzheng compound preparation comprises the following steps: weighing seven raw material medicines, crushing lucid ganoderma into coarse powder, soaking lucid ganoderma and glossy privet fruit in ethanol for 12 hours, adding ethanol for reflux extraction twice, extracting for 2 hours each time, filtering, recovering ethanol from filtrate under reduced pressure, and concentrating to obtain thick paste with the relative density of 1.25-1.34 at 80 ℃ for later use; decocting the residue, astragalus, rosa roxburghii tratt, prepared rehmannia root, epimedium herb and ginger processed pinellia tuber with water twice, each time for 2 hours, filtering by times, combining the filtrates, concentrating under reduced pressure to obtain thick paste with the relative density of 1.25-1.34 at 80 ℃, combining the thick paste with the standby thick paste, drying, crushing to obtain dry paste powder, and adding auxiliary materials or not to prepare various oral preparations.
The invention has the beneficial effects that: the invention constructs a drug screening model for blocking novel coronavirus spike glycoprotein and target cell receptor angiotensin converting enzyme 2(ACE2) and screens a plurality of traditional Chinese medicine compound preparations, and discovers that the kang' ai strengthening compound preparation has the activity of inhibiting the combination of the novel coronavirus spike glycoprotein and the target cell receptor ACE2 and the expression of II-type transmembrane serine protease (TMPRSS2) to prevent viruses from entering target cells, can obviously inhibit the virus-infected cells from producing inflammatory factors (IL-2, IL-6, TNF-a, IL-8 and CXCL-10), can be used for preventing and treating novel coronavirus infectious diseases, provides medication selection for patients with new coronary pneumonia, and has important function and significance for preventing and controlling current epidemic situation.
Drawings
FIG. 1 is a graph showing the effect of KANGAI FUZHENG Capsule in inhibiting the binding of RBD-Fc to ACE 2; the binding inhibition effect of RBD-Fc on ACE2 expressed by 293T/ACE2 cells is determined by flow cytometry, the concentration of the RBD-Fc is 1 mug/ml, the concentration of the kang' ai body resistance strengthening capsule is 15 mug/ml, 30 mug/ml and 60 mug/ml respectively, and DMSO is used as a control.
FIG. 2 is a graph of the effect of the kang' ai FU ZHENG Capsule on TMPRSS2 expression by infected viral cells; TMPRSS2 mRNA was measured using real-time quantitative qPCR after treating cells with various concentrations of kang' ai centralizing capsules for 24 hours. Error bars represent mean SD from three independent experiments.
FIG. 3-1 is a graph showing the effect of KANGAI FUZHENG Capsule on the inhibition of VSV-COVID-19-St19/GFP entry into alveolar type II epithelial cells (AT 2); FIG. 3-2 is a graph of the percentage of virus infected cells after treatment with various concentrations of KANGAI FUZHENG Capsule; (a) inoculating VSDVG to 293T cells expressing the glycoprotein for 24 hours, collecting culture supernatant, filtering through a filter with a pore size of 0-22 mm, inoculating to AT2 cells, and checking GFP expression under a fluorescence microscope; (b) the number of GFP positive cells after different concentrations of drug treatment was assessed by flow cytometry (n ═ 7).
FIG. 4 is a graph showing the inhibitory effect of KANGAI FUZHENG Capsule on VSV-COVID-19-St19/GFP virus infection of alveolar epithelial cells; after 24 hours of inoculating VSV-COVID-19-St19/GFP onto 293T cells expressing the indicated glycoproteins, the culture supernatant was collected, filtered through a filter having a pore size of 0 to 22mm and inoculated onto alveolar epithelial cells type II (AT2), and the VSV-COVID-19-St19/GFP virus titer, which was determined by the expression of EGFP gene (IU/mL), was obtained by the corresponding method of virus concentration.
FIG. 5 is a graph showing the effect of KANGAI FUZHENG Capsule on the production of inflammatory factors following infection of alveolar type II cells with virus; type II epithelial cells were cultured and seeded with VSV-COVID-19-St19/GFP 24 hours later, and the content of the cytokine was quantified using ELSA, and the results were obtained from 3 independent experiments.
The invention is further described with reference to the following figures and detailed description.
Detailed Description
Example 1: the application of the kang ai FU ZHENG JIAO NANG (Kangai Fuzheng Capsule) comprises the following steps:
the kang ai FU ZHENG JIAO NANG (Kangai Fuzheng Capsule for strengthening body resistance) can be purchased from the market, or prepared according to the preparation method of kang ai FU ZHENG JIAO NANG from oral tumor pediatrics book of 'national Chinese patent drug standardization compilation', and the obtained Capsule can be directly used for preventing/treating novel coronavirus infectious diseases. The usage and dosage are as follows: orally administered 2 granules/time, 3 times/day.
Example 2: the application of the kang' ai Fuzheng compound preparation:
weighing 600g of lucid ganoderma, 600g of astragalus membranaceus, 500g of rosa roxburghii tratt, 400g of prepared rehmannia root, 400g of glossy privet fruit, 400g of epimedium herb and 200g of pinellia ternata (processed by ginger), crushing the lucid ganoderma into coarse powder, soaking the coarse powder and the glossy privet fruit in ethanol for 12 hours, adding the ethanol into the mixture, carrying out reflux extraction twice for 2 hours each time, filtering the mixture, recovering the ethanol from the filtrate under reduced pressure, and concentrating the filtrate to form thick paste with the relative density of 1.25-1.34 at 80 ℃ for later use; decocting the residue, astragalus, rosa roxburghii tratt, prepared rehmannia root, epimedium and pinellia ternate (processed by ginger) in water twice, filtering for 2 hours each time, combining the filtrates, concentrating under reduced pressure to obtain thick paste with the relative density of 1.25-1.34 at 80 ℃, combining the thick paste with the above thick paste, drying, crushing to obtain dry paste powder, and preparing various oral preparations by adopting the existing preparation forming process. The obtained oral preparation can be used for preventing/treating new type coronavirus infectious diseases. The usage and dosage are as follows: orally administered at a dose of 1 g/time and 3 times/day.
Example 3: the application of the kang' ai Fuzheng compound preparation:
weighing Ganoderma 600g, radix astragali 600g, fructus Rosae Normalis 500g, radix rehmanniae Preparata 400g, fructus Ligustri Lucidi 400g, herba Epimedii 400g and rhizoma Pinelliae (processed with rhizoma Zingiberis recens) 200g, and making into various oral preparations by conventional extraction process and preparation forming process. The obtained oral preparation can be used for preventing/treating new type coronavirus infectious diseases. The usage and dosage are as follows: orally administered at a dose of 1 g/time and 3 times/day.
Experimental example:
the inventor constructs a stable COVID-19 RBD-Fc expression cell line, can secrete RBD spike protein into culture medium, and can be easily purified by protein A affinity chromatography; the constructed VSV-COVID-19-St19 pseudovirus can be combined with a receptor ACE2 to enter target cells but cannot generate progeny viruses, so that the system can be used for screening drugs for targeting VSV-COVID-19-S protein-mediated cell entry.
First, experimental material
1. Preparing a solution of a sample to be detected: a proper amount of the kang ai FU ZHENG JIAO NANG is weighed according to the requirement, and DMSO is used for preparing solutions with different concentrations (50ug/ml,25ug/ml,12.5ug/ml,6.25ug/ml and 3.125 ug/ml).
2. Cell line: human embryonic kidney 293T is used to produce Vesicular Stomatitis Virus (VSV) pseudotype virus carrying the COVID-19-S protein. Alveolar type II epithelial cells (AT2) were used for VSV pseudotype virus-infected target cells.
3. Modified Eagle Medium with high glucose concentration supplemented with 10% fetal calf serum (DMEM 10% FCS) was used to culture 293T and AT2 cells.
4. Autoclaved Phosphate Buffered Saline (PBS): 0.14. mu.M NaCl, 2. mu.M KCl, 3. mu.M Na2HPO4、1.5 μM KH2PO4，pH7.2。
5. Transfection reagent (QIAGEN, Germany).
6. A human expression plasmid encoding the COVID-19-S protein having 19 amino acids at the C-terminus. To generate a VSV pseudotype with high viral titer, we please prepare a plasmid encoding a C-terminally truncated form of the S protein, since a C-terminal 19 amino acid truncation has been shown to result in efficient incorporation of the S protein into the VSV particle, which then shows strong infection potential to the target cell.
VSVG-GFP is a VSV pseudotype with a VSV-G protein in which the VSV-G gene is replaced by the GFP gene. Pseudotypes with the VSV-G protein were used as "seed" viruses to produce S proteins with VSV prostheses. The VSVG-GFP system was purchased from America Biogen Inc.
An 8.0.22 μm pore size sterile filter.
9. Fluorescence microscopy for detection of GFP expression.
And 10, the ELISA kit is used for analyzing the content of the inflammatory cytokines in the cell culture solution.
Second, Experimental methods
1. Construction of recombinant plasmid
Amino acids 438-506 of the COVID-19 spike protein were located as the ACE2 binding domain (RBD). The cDNA fragment encoding RBD was amplified by PCR using plasmid PUC18-S containing the coding sequence of human codon-optimized COVID-19 spike protein as a template, and primers (forward: 5'-GGCGCTAGCCATCACCAACCTGTGCCCC-3', containing a NheI recognition site; reverse: 5'-CGCGGATCCGTCACGGTGGCGGGGGCGTTC-3', containing a BamHI recognition site). The PCR product was digested with NheI and BamHI and then cloned downstream of the Peak13 expression vector CD5 antigen leader and upstream of the IgG1(Fc) Fc portion, and the Peak13 expression vector was also digested with NheI and BamHI. The resulting recombinant plasmid was named Peak 13-RBD-Fc.
2. Establishment of stable expression RBD-Fc cell line
One day prior to transfection, HEK293 cells were trypsinized at 1 × 10 per well5The density of cells was seeded into 6-well plates. The following day, 0.5. mu.g of Peak13-RBD-Fc plasmid linearized with AvrII or an equal volume of H was linearized with lipofectamine (TM) 2000(Invitrogen) following the procedure described in the instructions of the reagents Inc2O was transfected into HEK293 cells. After 48 hours, aliquots of transfected cells were transferred to selection medium containing increasing concentrations of puromycin (0.3, 0.4, 0.5, 0.6 and 0.7. mu.g/ml). After 3 days, transfected HEK293 cells selected for the appropriate puromycin concentration at which the cells transfected with Peak13-RBD-Fc plasmid survived but were H-transfected2The O-transfected cells all died, and the cells transfected with the Peak13-RBD-Fc plasmid were transferred to a 96-well plate by limiting dilution. After 10 days of culture, the cells expressing the highest amount of RBD-Fc were selected for the production of the recombinant protein RBD-Fc by ELISA assay using anti-human IgG peroxidase-conjugated antibody (Sigma).
3. Enzyme-linked immunosorbent assay (ELISA)
2X10 from each RBD-Fc-293 cell clone5Cells were seeded into 6-well plates and cultured with 2ml of medium (DMEM containing 10% FBS) for 72 hours. The concentration of RBD-Fc protein in the culture supernatants was quantified by ELISA assay using the kit BD OptEIA TM (BD Biosciences), according to the procedures described in the instructions of the reagents, briefly, culture supernatants of each RBD-Fc-293 cell clone were coated overnight with a 96-well flat-bottom EIA/RIA plate (corning, N.Y.) with 100. mu.l of medium at 4 ℃ in serial dilutions of human IgG standard in culture medium (10 ng to 30. mu.g/ml). The wells were then washed once with wash solution, blocked with assay diluent for 1 hour at room temperature, and then washed 3 times with wash solution. Next, anti-human IgG peroxidase-conjugated anti-antibody diluted in Assay Diluent (1: 5000) was added to each wellBody (Sigma) and gently stirred at room temperature for 1 hour. After three washes, 100 μ l of substrate reagent was added to each well and the wells were incubated in the dark at room temperature for 30 minutes. Finally, the reaction was stopped using a stop solution and the color development was monitored at a wavelength of 450 nm.
4. Protein purification
Culture supernatants of RBD-Fc-293 cells were collected and dialyzed against solution (20mM sodium phosphate and 1mM EDTA, pH 7.0) over 8 hours. After centrifugation at 4000rpm for 30 minutes, the supernatant was filtered through a 0.45 μm dura filter (Millipore, irish). The procedure was as described in the specification, using a HiTrap Protein A HP 5ml chromatography column (GE Healthcare Amersham Biosciences AB) and EconoTMPurification of RBD-Fc protein was performed by gradient pump tube kit (Bio-Rad, USA). Briefly, the column was first washed with 10 column volumes of binding buffer (20mM sodium phosphate, pH 7.0) at a flow rate of 5 ml/min. The cell culture supernatant was then pumped onto the column. Next, the column was washed with 20 column volumes of binding buffer and then with the supernatant. Finally, the RBD-Fc protein was eluted with 2-5 column volumes of elution buffer (0.1M glycine, pH 3.1). Neutralization buffer (60-200. mu.l, 1M Tris-HCl, pH 9.0) was added to each collection tube. The RBD-Fc Protein was further purified using a HiTrap Protein A HP 1ml chromatography column (GE Healthcare Amersham Biosciences AB). Protein concentration was determined using protein assay dye reagent concentrate (Bio-Rad) using Bovine Serum Albumin (BSA) as standard according to the protocol.
Binding inhibition assay for RBD-Fc to the receptor ACE2
The inhibitory effect of the drug on the binding of RBD-Fc to the cellular receptor ACE2 was measured by flow cytometry. Collection and separation 2X106293T cells expressing ACE2 were washed with HBSS (Sigma-Aldrich). Cells were treated with 1. mu.g/ml RBD-Fc with or without 50. mu.g/ml drug, and then incubated for 30 minutes at room temperature. Cells were washed with HBSS and incubated with anti-human IgG-FITC at 1/50 dilution for 30 minutes at room temperature. After washing, cells were fixed with 1% formaldehyde in PBS and flow cytometric by FACSCalibur using CellQuest softwareAnalysis was performed in (BD Biosciences).
6. Establishment of ACE2 expressing cell line
AT2 cells were transfected with pTargeT plasmid (Promega) pcDNA carrying ACE2 gene, and then selected with G418-containing medium to obtain ACE 2-highly expressing cells (AT2-ACE 2). These cells were grown and stored in DMEM medium with 5% fetal bovine serum.
7. Construction of pseudovirus VSV-COVID-19-S protein pseudovirus
To construct VSV that produces the COVID-19-S protein pseudotype, an expression plasmid encoding the full-length COVID-19-S protein is first constructed. The cDNA of the COVID-19-S protein was amplified with forward primer S-Bam-f (5'-GGATCCAAGTGATATTCTTGTTAACAAC-3') and reverse primer S-Bam-r (5' -GGATCCAAGAGTAAAAAATTCCATCAT-3C) and cloned into the expression vector pKS 336. The cDNA of the C-terminally truncated COVID-19-S protein was amplified from pKS-SARS-S using the forward primer S-Bam-f and the reverse primer S-Bam19r (5'-GGGATCCTTAGCAGCAAGAACCACAAGAGCATG-3') and then cloned into pKS 336. The resulting plasmid pKS-COVID-19-St19, in addition to the C-terminal 19' aa, encodes the full-length COVID-19-S protein. Expression of the S protein was detected on the cell membrane after transfection of 293T cells with the expression plasmid pKS-COVID-19-St 19. To generate a VSV pseudotype with a C-terminally truncated COVID-19-St19 protein, I inoculated VSV Δ G onto 293T cells transfected with pKS-SARS-St 19. VSVG-GFP was used as a positive control, and the COVID-19-S protein VSV pseudotype, i.e., VSV-COVID-19-St19, obtained from 293T cells transfected with pKS-COVID-19-St19, was effective in infecting lung epithelial cells. The titer of VSV-COVID-19-St19 was 5.0X 105IU/ml。
Detection of entry of VSV-COVID-19-St19 into cells
To examine the ability of VSV-COVID-19-St19 to infect target cells, a time-dependent analysis of GFP expression was performed. A monolayer of alveolar cells on 24-well glass slides was infected with VSV-COVID-19-St 19. At different time points, the virus was infected and cells were photographed under a fluorescent microscope. The number of GFP expressing cells in the photographs was counted using ImageJ software (http:// rsb. info. nih. gov/ij /). Since pseudoviruses are unable to produce progeny viruses, this system can be used to evaluate the VSV-COVID-19-S protein-mediated cell entry mechanism. Time course analysis of the number of GFP positive cells indicated that VSV-COVID-19-St19 infection could be quantified 6 hours after viral infection.
mRNA determination of ACE2 and TMPRSS2
Total RNA was extracted from virus infected cells treated with different concentrations of drug using the kit according to the procedure described in the instructions of the reagents company using the quantitative qPCR apparatus LightCycler (Roche diagnostics) used. TMPRSS2 mRNA was amplified with primers 5'-CTCTACGGACCAAACTTCATC-3' and 5'-CCACTATTCCTTGGCTAGAGTAA-3'. ACE2 was amplified with primers 5'-CCGTATCAATGATGCTTTCCG-3' and 5'-CAGTGAAGATCAGGATGACAAT G-3'.
10. Determination of inflammatory cytokines
After the infected virus cells were treated with different concentrations of the drug for 24 hours, cell culture supernatants were collected. The contents of the cell inflammatory factors TNF-a, IL-6, IL-2, etc. in the cell culture solution were measured using a high sensitivity ELISA kit (QuantikineTM HS, R & D Systems) according to the procedures described in the instructions of the reagents Co. And drawing a standard curve to calculate the sensitivity.
Third, experimental results
1. The influence of the kang 'ai body resistance strengthening capsule on the combination of the spike protein RBD of the novel coronavirus and the target cell receptor ACE2 is measured by a flow cytometer, and the result shows that the kang' ai body resistance strengthening capsule can obviously inhibit the combination of the spike protein RBD of the novel coronavirus and the receptor ACE2 and has a dose-dependent effect (see figure 1). This shows that the kang' ai FU FANG ZHI JI can inhibit the novel coronavirus from entering cells.
2. The real-time quantitative qPCR is utilized to determine the influence of the kang 'ai body resistance strengthening capsules on the mRNA expression of a novel coronavirus target cell TMPRSS2, and the result shows that the kang' ai body resistance strengthening capsules have obvious inhibition effect on the expression of TMPRSS2 infected with virus cells and have dose-dependent effect (see figure 2). This indicates that the conai FUZHENG Compound preparation can inhibit the binding of the novel coronavirus spike glycoprotein to the target cell receptor ACE2 to prevent the virus from entering the target cell by inhibiting the expression of type II transmembrane serine protease (TMPRSS 2).
3. Pseudo-virus VSV-COVID-19-St 19/GFP-infected alveolar type II epithelial cells (AT2) were treated with a kang 'ai righting capsule, and the amount of virus (GFP expression amount) was examined under a fluorescence microscope, and as a result, it was found that the kang' ai righting capsule could significantly reduce the amount of virus entering the cells (see FIGS. 3-1 and 3-2). This shows that the kang' ai FU FANG ZHI JI can prevent virus infection of cells.
4. The effect of the kang 'ai body resistance strengthening capsules on the entering of the novel coronavirus into cells is measured by a virus gradient method, and the result shows that the kang' ai body resistance strengthening capsules have obvious inhibition effect on virus-infected alveolar epithelial cells and have a dose-dependent effect (see figure 4). The fact shows that the kang' ai body resistance strengthening compound preparation has obvious inhibition effect on virus entering alveolar epithelial cells and can prevent and treat novel coronavirus clinically.
5. The effect of the kang 'ai body resistance strengthening capsules on the generation of inflammatory factors after the alveolar II cell is infected with virus is quantitatively determined by enzyme linked immunosorbent assay (ELSA), and the results show that the kang' ai body resistance strengthening capsules can reduce the amount of the inflammatory factors (IL-2, IL-6, TNF-a, IL-8 and CXCL-10) generated by virus infected cells and have a dose-dependent effect (see figure 5). This shows that the kang' ai FU FANG ZHI JI can be used for clinically treating the inflammation storm caused by the novel coronavirus infection.
Claims (3)
1. The application of the kang 'ai body resistance strengthening compound preparation in preparing a medicine for preventing/treating novel coronavirus infection is characterized in that the kang' ai body resistance strengthening compound preparation is prepared from 600 parts by weight of lucid ganoderma, 600 parts by weight of astragalus membranaceus, 500 parts by weight of rosa roxburghii tratt, 400 parts by weight of prepared rehmannia root, 400 parts by weight of glossy privet fruit, 400 parts by weight of epimedium herb and 200 parts by weight of ginger processed pinellia tuber serving as raw material medicines.
2. The use of the kang ai FU FANG ZHI JI as claimed in claim 1, wherein: the kang ai FU ZHENG FU FANG ZHI JI is a capsule prepared from starch 40 weight parts and pulvis Talci 10 weight parts as adjuvants.
3. The use of the kang ai FU FANG ZHI JI as claimed in claim 1, wherein: the preparation method of the kang' ai Fuzheng compound preparation comprises the following steps: weighing seven raw material medicines, crushing lucid ganoderma into coarse powder, soaking lucid ganoderma and glossy privet fruit in ethanol for 12 hours, adding ethanol for reflux extraction twice, extracting for 2 hours each time, filtering, recovering ethanol from filtrate under reduced pressure, and concentrating to obtain thick paste with the relative density of 1.25-1.34 at 80 ℃ for later use; decocting the residue, the astragalus, the rosa roxburghii tratt, the prepared rehmannia root, the epimedium and the ginger processed pinellia tuber with water twice, each time for 2 hours, filtering in times, combining the filtrates, concentrating under reduced pressure to obtain thick paste with the relative density of 1.25-1.34 at 80 ℃, combining the thick paste with the standby thick paste, drying, crushing to obtain dry paste powder, and adding auxiliary materials or not to prepare various oral preparations.
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| CN109602859A (en) * | 2019-01-18 | 2019-04-12 | 贵州省中国科学院天然产物化学重点实验室 | Application of the seedling medicine Liao Shi Huafeng pill in preparation prevention and treatment melanoma drug |
| CN111773276A (en) * | 2020-03-24 | 2020-10-16 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Application of kalimeris indica cold-feeling compound in preparation of novel coronavirus infection resisting medicines |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109602859A (en) * | 2019-01-18 | 2019-04-12 | 贵州省中国科学院天然产物化学重点实验室 | Application of the seedling medicine Liao Shi Huafeng pill in preparation prevention and treatment melanoma drug |
| CN111773276A (en) * | 2020-03-24 | 2020-10-16 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Application of kalimeris indica cold-feeling compound in preparation of novel coronavirus infection resisting medicines |
Non-Patent Citations (2)
| Title |
|---|
| 康艾扶正片对恶性肿瘤放化疗减毒作用的疗效观察;王建民等;《中国医院药学杂志》;20100915(第17期);第1467-1469页。 * |
| 康艾扶正片的制备及临床应用;马全龙等;《中国医药指南》;20110810(第22期);第33-34页。 * |
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