Use of aurantiamacartin in preparation of a medicine for resisting novel coronavirus

Abstract

The present application relates to the technical field of medicine, and more particularly to application of aurantio-obtusin in preparation of anti-novel coronavirus drugs, and application of aurantio-obtusin and derivatives thereof in anti-virus treatment of COVID-19. Aurantio-obtusin has high inhibitory effect on wild-type SARS-CoV-2 and mutant strains in multiple cell models, and has low toxicity and side effects in multiple cell lines and animal levels, and is a new small-molecule active ingredient for treating SARS-CoV-2 infection that has not been reported at home and abroad.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of cassia seed extract in the preparation of drugs against the novel coronavirus. Background Technology

[0002] Currently available small molecule drugs for treating COVID-19, such as remdesivir, mupiravir, pasiclovir, and azvudine, have not shown the expected protective effects in clinical treatment.

[0003] Cassia seed is a common traditional Chinese medicine, often used to treat liver heat, red eyes, headaches, hypertension, and hyperlipidemia. Its main components include cassiaside, cassiain, cassia lactone, cassia anthraquinone, and cassia alkaloid. Various small molecule monomers of cassia seed have been reported to have good therapeutic effects on a variety of diseases, such as neurodegenerative diseases, allergic diseases, and breast cancer; however, there are no literature reports on the anti-SARS-CoV-2 activity of cassia seed and its main active small molecule monomers. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of cassia seed extract in the preparation of drugs against the novel coronavirus.

[0005] Application of Cassia tora extract in the preparation of drugs against the novel coronavirus.

[0006] Optionally, the use of the orange-yellow cassia extract in the preparation of anti-novel coronavirus drugs, wherein the novel coronavirus includes: wild-type SARS-CoV-2WIV-04, Delta, Omicron BA 1.1, Omicron BA 2.2, and Omicron BA 5.1.

[0007] Optionally, the application of the cassia seed extract in the preparation of anti-novel coronavirus drugs, wherein the cassia seed extract inhibits the infection and replication of the novel coronavirus by inhibiting the viral nucleic acid level in the cell supernatant and the viral NP protein level in the cell.

[0008] Beneficial effects: Compared with the prior art, the present invention has the following technical effects:

[0009] (1) The cassia seed extract in this invention is one of the effective active ingredients in the Chinese herbal medicine cassia seed. It has been widely reported for use in health care products such as weight loss and liver protection. This invention is the first to discover that cassia seed extract has a highly effective antiviral effect against SARS-CoV-2.

[0010] (2) The orange-yellow cassia extract in this invention has no obvious liver and kidney toxicity in animals, and long-term oral administration (once a day for 14 consecutive days) has no obvious damage to the various biochemical indicators of rhesus monkeys.

[0011] (3) The orange-yellow cassia extract in this invention has a significant inhibitory effect on wild-type SARS-CoV-2 and common mutant strains in multiple cell models (Vero, small airway epithelial cells and limbal epithelial cells). Attached Figure Description

[0012] Figure 1 This is the structural formula of Cassia tora extract, and the cytotoxicity results in human small airway epithelial cells (HAWEC-02014), human limbal epithelial cells (HNCWC / HL-008), and Vero cells.

[0013] Figure 2 The figure shows the antiviral efficacy of cassia seed extract against wild-type SARS-CoV-2 WIV-04 in human small airway epithelial cells (HAWEC-02014), human limbal epithelial cells (HNCWC / HL-008), and Vero cells.

[0014] Figure 3 This is a graph showing the antiviral efficacy of Cassia tora extract against Delta strain, Omicron BA1.1, BA2.2, and BA5.1 in human small airway epithelial cells (HAWEC-02014), human limbal epithelial cells (HNCWC / HL-008), and Vero cells. Detailed Implementation

[0015] This invention provides the application of cassia seed extract in the preparation of drugs against the novel coronavirus. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0016] The aurantio-obtusin used in this invention is a commercially available product, with the English name Aurantio-obtusin, CAS number 67979-25-3, and HPLC accuracy of 98.7%.

[0017] The preferred cellular dissolution conditions for the orange-yellow cassia seed extract are dissolution in DMSO at room temperature to 20 mM, followed by aliquoting and freezing at -20°C. For animal experiments, the preferred dissolution conditions are dissolution in 2% Tween 80 via ultrasonic vibration, with freshly prepared extracts for each use.

[0018] The preferred conditions for the SARS-CoV-2 immunofluorescence assay are SARS-CoV / SARS-CoV-2 Nucleocapsid Antibody (HRP), Rabbit Mab (40143-R004-H), or Goat Anti-Rabbit IgG H&L (Alexa). 488).

[0019] The preferred culture conditions for the human small airway epithelial cells (HAWEC-02014) and human corneal epithelial cells (HNCWC / HL-008) are HL culture at 37°C and 5% CO2. The HL culture medium is a mixture of DMEM and serum-free SFM at a volume ratio of 1:3, with the addition of 5% (v / v) FBS (fetal bovine serum), and 0.4 μg / mL hydrocortisone, 5 μg / mL insulin, 8.4 ng / mL choleratoxin, and 10 ng / mL epidermal growth factor. The culture medium contained 24 μg / mL adenine, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.25 μg / mL fungizone, and 30 μM fasudil. The culture medium was filtered through a 0.22 μm pore size filter membrane.

[0020] The preferred culture conditions for Vero cells are culture in DMEM with 10% FBS at 37°C and 5% CO2.

[0021] The following specific examples will further explain the application of the cassia seed extract provided by the present invention in the preparation of drugs against the novel coronavirus.

[0022] Example 1

[0023] Cytotoxicity assay of cassia seed extract at the cellular level

[0024] (1) Vero, human small airway epithelial cells or human limbal epithelial cells were seeded at 8000 / well to 96-well plates. After 24 hours, the culture medium was removed and fresh culture medium containing a concentration gradient of cassia aurantium was added to each well. The cells were then cultured for another 48 hours.

[0025] (2) After removing the culture supernatant, wash each well three times with 200 μL of room temperature equilibrated PBS;

[0026] (3) Remove the PBS, add 100 μL of CCK8 working solution (CCk8 and DMEM are mixed at a ratio of 1:9) to each well and incubate at 37°C for 2 h;

[0027] (4) OD450 and OD630 were detected using a multi-functional microplate reader (Bio-Tek). After processing the data with GraphPad Prism 6.0 software, cell viability curves were plotted. Figure 1As shown, cassia aurantium at concentrations below 200 μM showed no cytotoxicity in human small airway epithelial cells, human limbal epithelial cells, and Vero cells.

[0028] Example 2

[0029] (1) Four healthy rhesus monkeys of similar age and weight (two males and two females) were selected for the experiment. The age range was 3-5 years old. There were two monkeys in each of the experimental groups A and B (to ensure that each experimental group had both male and female monkeys).

[0030] (2) The experimental group A was administered drug at a concentration of 1 mg / kg / day / animal and the experimental group B was administered drug at a concentration of 3 mg / kg / day / animal orally by gavage at 10:00 a.m. every day for 14 consecutive days.

[0031] (3) Blood samples were drawn between 8:00 and 10:00 AM on days 1, 6, 10 and 15 of the experiment and biochemical indicators were analyzed (samples were taken before drug administration).

[0032] (4) Serum biochemical indicators and routine urine indicators of each experimental monkey were detected using a biochemical analyzer. As shown in the table below, treatment with 1 mg / kg / day / monkey and 3 mg / kg / day / monkey showed no significant liver or kidney toxicity to rhesus monkeys.

[0033]

[0034]

[0035] Example 3

[0036] (1) Vero, human small airway epithelial cells or human limbal epithelial cells were seeded at 20,000 per well into 96-well plates and transferred to BSL-3 laboratory 24 hours later.

[0037] (2) The wild-type SARS-CoV-2WIV-04 stock solution was diluted with DMEM medium containing 2% FBS, and the final viral titer was 20,000 copies / mL;

[0038] (3) Remove the supernatant from the cell culture plate, add 100 μL of infection solution with a virus titer of 20,000 copies / mL to each well, and incubate at 37°C for 1 hour;

[0039] (4) Remove the infection fluid and wash each well three times with 200 μL of room temperature equilibrated PBS.

[0040] (5) After removing PBS, add 200 μL of 2% FBS DMEM to each well of Vero cells, and add 200 μL of HL medium to each well of human small airway epithelial cells or human limbal epithelial cells and continue culturing for 2 days.

[0041] (6) After washing the cells three times with PBS at room temperature on the second day after infection, the 48-well plates were soaked in 4% paraformaldehyde fixative and completely inactivated with SARS-CoV-2 in a 4°C refrigerator for at least 48 hours before being removed from the BSL-3 laboratory. After the nucleic acid extraction of the supernatant was completed, the surface was sprayed with 2000 mg / mL chlorine disinfectant and 75% alcohol disinfectant before being removed from the BSL-3 laboratory.

[0042] (7) Remove the paraformaldehyde fixative from the 48-well plate, wash the cells at least 3 times with PBS after equilibration at room temperature, and add 0.1 mL of cell permeabilizer (Solepro Bio) to each well for 20-30 min at room temperature.

[0043] (8) After removing the cell permeabilizer, wash the cells three times with PBS equilibrated at room temperature, and add 0.1 mL of PBS buffer containing 10% (v / v) fetal bovine serum to each well and block at room temperature for 1 hour.

[0044] (9) After removing the blocking solution, add 0.1 mL of PBS buffer containing SARS-CoV-2 Nucleocapsid Antibody to each well. The antibody to PBS ratio is 1:250 (v / v). Incubate at 4°C for 12 hours.

[0045] (10) Remove the antibody incubation solution, wash each well three times with 0.2 mL of room temperature equilibrated PBS buffer, add 0.1 mL of goat anti-rabbit lgG H&L PBS buffer at a volume ratio of 1:1000 (v / v), and incubate at room temperature in the dark for 1 hour.

[0046] (11) Remove the fluorescent secondary antibody incubation solution, wash each well three times with 0.2 mL of room temperature equilibrated PBS buffer under light-protected conditions, add 0.1 mL of PBS to each well to immerse the cells, and observe the viral NP protein in the cells under a fluorescence microscope.

[0047] (12) Observe the different drug gradient wells using a fluorescence microscope under a 10x objective lens. Observe at least 3 different fields of view for each well. Representative immunofluorescence images are shown below. Figure 3 Cassia tora extract can effectively inhibit WIV-04 infection of Vero, human small airway epithelial cells, and human limbal epithelial cells. Compared with the virus control well, treatment with 100 μM and 33.3 μM of the drug showed dose-dependent antiviral effects against viral infection.

[0048] Example 4

[0049] Inhibitory effect of Cassia tora extract on Delta, Omicron BA1.1, BA2.2, and BA5.1 strains of SARS-CoV-2.

[0050] Viral infection and detection of intracellular viral NP proteins were performed in the same manner as in Example 3, except that the wild-type virus was replaced with mutant strains such as Delta, Omicron BA1.1, BA2.2, and BA5.1. Intracellular NP immunofluorescence results are as follows: Figure 3 As shown, on day 2 post-infection, 100 μM of cassia aurantium significantly inhibited the infection of Vero, human small airway epithelial cells, and human limbal epithelial cells by the SARS-CoV-2 mutant strain.

[0051] In summary, this invention provides an application of cassia seed extract in the antiviral treatment of COVID-19. The cassia seed extract exhibits highly effective inhibitory effects against wild-type SARS-CoV-2 and mutant strains in multiple cell models. It also demonstrates low toxicity in multiple cell lines and animal studies, representing a novel small-molecule active ingredient previously unreported domestically and internationally for the treatment of SARS-CoV-2 infection.

[0052] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Application of Cassia Seed Extract in the Preparation of Anti-mutant Novel Coronavirus Drugs; The novel coronaviruses mentioned are: Delta, Omicron BA 1.1, Omicron BA 2.2, and Omicron BA 5.

1.

2. The application of the cassia seed extract according to claim 1 in the preparation of anti-mutant novel coronavirus drugs, characterized in that, The orange-yellow cassia extract inhibits the infection and replication of the novel coronavirus by suppressing the viral nucleic acid level in cell supernatant and the viral NP protein level in cells.

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