Pharmaceutical composition and anti-coronavirus drug comprising the same

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a pharmaceutical composition and an anti-coronavirus drug containing the same. The pharmaceutical composition provided by the application comprises an evodia rutaecarpa extract and a radix Stephaniae tetrandrae extract with a mass ratio of (1-3):(1-3), or evodiamine and tetrandrine with a molar mass ratio of (1-5):(1-5), has a strong inhibitory effect on coronavirus infection and anti-coronavirus efficacy, and the components show a synergistic inhibitory effect. The evodia rutaecarpa extract and the radix Stephaniae tetrandrae extract are prepared into an anti-coronavirus drug, which can provide an effective means for preventing or treating coronavirus.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and specifically relates to a pharmaceutical composition and an anticoronavirus drug comprising the same. Background Technology

[0002] Coronaviruses are a large family of viruses known to cause more serious illnesses such as the common cold, Middle East Respiratory Syndrome (MERS), and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has never been found in humans before. The International Committee on Taxonomy of Viruses has named this new pathogen Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0003] According to a statement by the World Health Organization in October 2021, "long-term COVID-19" symptoms mostly appear within three months of infection and last for at least two months, and are difficult to explain medically using other diagnostic methods. Symptoms of "long-term COVID-19" include extreme drowsiness, memory and attention deficit ("brain fog"), changes in taste and smell, and a range of symptoms such as shortness of breath, chest pain, or tightness, causing long-term inconvenience and discomfort for both COVID-19 patients and those who have recovered. Therefore, finding effective candidate drugs to inhibit the COVID-19 virus remains a pressing technical problem. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide a pharmaceutical composition and an anti-coronavirus drug comprising the same.

[0005] A first aspect of this application provides a pharmaceutical composition comprising a spike protein inhibitor and a TPC2 protein inhibitor; wherein...

[0006] The spike protein inhibitor includes Evodia rutaecarpa extract, and the TPC2 protein inhibitor includes Stephania tetrandra extract, wherein the mass ratio of the Evodia rutaecarpa extract to the Stephania tetrandra extract is (1-3):(1-3); or,

[0007] The spike protein inhibitor includes evodiamine, and the TPC2 protein inhibitor includes tetrandrine. The molar mass ratio of evodiamine to tetrandrine is (1-5):(1-5).

[0008] In some embodiments of this application, the pharmaceutical composition satisfies one or more of the following conditions:

[0009] (1) The Evodia rutaecarpa extract includes Evodia rutaecarpa alcohol extract; and,

[0010] (2) The Stephania tetrandra extract includes Stephania tetrandra alcohol extract.

[0011] In some embodiments of this application, the Evodia rutaecarpa alcohol extract includes an ethanol extract of Evodia rutaecarpa; the Stephania tetrandra alcohol extract includes an ethanol extract of Stephania tetrandra.

[0012] In some embodiments of this application, the ethanol extract of Evodia rutaecarpa includes an extract of an ethanol solution with an ethanol volume percentage of 85%-95% for Evodia rutaecarpa; the ethanol extract of Stephania tetrandra includes an extract of an ethanol solution with an ethanol volume percentage of 85%-95% for Stephania tetrandra.

[0013] A second aspect of this application provides the use of the pharmaceutical composition described in the first aspect in the preparation of an anticoronavirus drug.

[0014] In some embodiments of this application, the anti-coronavirus drug includes anti-Severe Acute Respiratory Syndrome Coronavirus No. 2 drug, anti-Severe Acute Respiratory Syndrome Coronavirus drug, anti-Middle East Respiratory Syndrome Coronavirus drug, or anti-bat SARS-like coronavirus drug.

[0015] In some embodiments of this application, the anticoronavirus drug includes the pharmaceutical composition and pharmaceutically acceptable excipients.

[0016] In some embodiments of this application, the dosage forms of the anti-coronavirus drug include ointments, patches, solutions, suspensions, emulsions, creams, sprays, drops, granules, capsules, tablets, pills, teas, or tube feeding preparations.

[0017] A third aspect of this application provides an anti-coronavirus drug comprising the pharmaceutical composition described in the first aspect and pharmaceutically acceptable excipients.

[0018] In some embodiments of this application, the dosage forms of the anti-coronavirus drug include ointments, patches, solutions, suspensions, emulsions, creams, sprays, drops, granules, capsules, tablets, pills, teas, or tube feeding preparations.

[0019] Compared with traditional technical solutions, this application has the following advantages:

[0020] After extensive screening, the applicant of this application discovered that Evodia rutaecarpa extract combined with Stephania tetrandra extract or Evodia rutaecarpa alkaloid combined with Stephania tetrandra alkaloid exhibits a good synergistic effect in inhibiting the novel coronavirus. The applicant has prepared these extracts into an anti-coronavirus drug, which can provide an effective means for the prevention and / or treatment of coronavirus. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 As verified by ACE2 immunostaining in Example 2, the HEK293T pseudovirus system contains effective ACE2 receptor protein.

[0023] Figure 2 As verified by TPC2 immunostaining in Example 2, the HEK293T pseudovirus system contains effective TPC2 protein.

[0024] Figure 3 In Example 2, after screening and testing, it was found that the antiviral activity was better when the weight ratio of Evodia rutaecarpa extract (EVF) to Stephania tetrandra extract (STS) was 1:1 and the molar mass concentration ratio of Evodia rutaecarpa alkaloid (Ru) to Stephania tetrandra alkaloid (Te) was 1:1.

[0025] Figure 4 In Example 2, the pseudovirus channel test yielded Evodia rutaecarpa alcohol extract (EVF). EtOH ) and Stephania tetrandra alcohol extract (STS) EtOH The complex can effectively inhibit the invasion of host cells by both wild-type and Omega-3 SARS-CoV-2.

[0026] Figure 5 In Example 3, according to Compusyn calculations, the combination of Evodia rutaecarpa extract and Stephania tetrandra extract showed a synergistic inhibitory effect against both wild-type and Omeprone SARS-CoV-2.

[0027] Figure 6 In Example 4, the pseudovirus pathway test showed that the complex of evodiamine (Ru) and tetrandrine (Te) could effectively inhibit the invasion of wild-type and omeprazole-type SARS-CoV-2 into host cells;

[0028] Figure 7 In Example 5, according to Compusyn calculations, evodiamine combined with tetrandrine showed a synergistic inhibitory effect against both wild-type and Omeprone SARS-CoV-2.

[0029] Figure 8 In Example 6, molecular docking analysis showed that evodiamine can effectively target the spike protein of wild-type and Omeprone viruses, while tetrandrine can also effectively target the TPC2 protein. Detailed Implementation

[0030] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0031] Unless otherwise defined, 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 application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0032] the term

[0033] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0034] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0035] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0036] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0037] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0038] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0039] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0040] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0041] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0042] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0043] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0044] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0045] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0046] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0047] Numerous studies have elucidated the mechanisms by which the novel coronavirus invades cells, including ACE2, the viral spike protein S-protein, 3CL protease, and TPC2, all of which are key proteins for viral invasion of host cells. Therefore, inhibitors targeting these proteins hold promise as a treatment option for suppressing the novel coronavirus. In previous studies, this application has found that *Evodia rutaecarpa* and its alkaloid can inhibit the entry of the novel coronavirus into host cells by inhibiting the binding of the viral spike protein to ACE2, and has published research articles in internationally renowned journals (Reference 1) and applied for a patent with the State Intellectual Property Office. Furthermore, according to existing research literature, *Stephania tetrandra* and its alkaloid are both effective TPC2 protein inhibitors (References 2-3). In view of this, this application proposes the hypothesis that the combined use of spike protein inhibitors and TPC2 inhibitors can inhibit the novel coronavirus. Through rigorous screening and testing, it has been found that the combination of *Evodia rutaecarpa* ethanol extract and *Stephania tetrandra* ethanol extract, or the combination of alkaloid and alkaloid, can achieve a synergistic inhibitory effect against the novel coronavirus. *Evodia rutaecarpa* and *Stephania tetrandra* are well-known medicinal materials in the field of traditional Chinese medicine, and alkaloids and alkaloids are also used in clinical practice. In summary, the embodiments of this application can organically combine known clinical drugs and greatly reduce the process from research results to clinical antiviral application.

[0048] [1].Lin S; Wang X; Guo H; Dai N; Tang RWL.; Lee HC; Leung KW; Dong TTX; WebbSE; Miller AL, Tsim KWK. InflammatoryResponses.Int.J.Mol.Sci.2023,24,762,https: / / doi.org / 10.3390 / ijms24010762.

[0049] [2].Jin

[0050] [3].Moccia1 F; Negri1 S; Faris1 P; Perna A; De Luca A; Soda T; Berra-RomaniR; Guerra G. Targeting Endolysosomal Two-Pore Channels to Treat CardiovascularDisorders in the Novel COronaVIrus Disease 2019. Front. Physiol., 2021, 12, https: / / doi.org / 10.3389 / fphys.2021.629119.

[0051] First aspect of the embodiments of this application

[0052] This application provides a pharmaceutical composition comprising a spike protein inhibitor and a TPC2 protein inhibitor; wherein,

[0053] The spike protein inhibitor includes Evodia rutaecarpa extract, and the TPC2 protein inhibitor includes Stephania tetrandra extract, wherein the mass ratio of the Evodia rutaecarpa extract to the Stephania tetrandra extract is (1-3):(1-3) (e.g., 1:1, 1:2, 2:1, 1:3, 3:1, etc.); or,

[0054] The spike protein inhibitor includes evodiamine, and the TPC2 protein inhibitor includes tetrandrine. The molar ratio of evodiamine to tetrandrine is (1-5):(1-5) (e.g., 1:1, 1:2, 2:1, 1:3, 3:1, 1:4, 4:1, 1:5, 5:1, etc.).

[0055] "Extract" refers to a substance derived from plants through a physicochemical extraction and separation process, using plants as raw materials and according to the intended use of the final product, to selectively obtain and concentrate multiple active ingredients from the plants. The extract in this application is a mixture of multiple chemical components obtained through extraction.

[0056] The chemical structural formula of Evodia rutaecarpa alkaloid is as follows:

[0057]

[0058] The chemical structural formula of tetrandrine is as follows:

[0059]

[0060] Optionally, the pharmaceutical composition satisfies one or more of the following conditions:

[0061] (1) The Evodia rutaecarpa extract includes Evodia rutaecarpa alcohol extract; and,

[0062] (2) The Stephania tetrandra extract includes Stephania tetrandra alcohol extract.

[0063] Further, optionally, the Evodia rutaecarpa alcohol extract includes an ethanol extract of Evodia rutaecarpa; the Stephania tetrandra alcohol extract includes an ethanol extract of Stephania tetrandra.

[0064] Alternatively, the ethanol extract of Evodia rutaecarpa may comprise an extract of an ethanol solution containing 85%-95% ethanol by volume of Evodia rutaecarpa; and the ethanol extract of Stephania tetrandra may comprise an extract of an ethanol solution containing 85%-95% ethanol by volume of Stephania tetrandra.

[0065] In this embodiment of the application, the preparation steps of the extract of Evodia rutaecarpa with an ethanol solution of 85%-95% by volume include: extracting Evodia rutaecarpa with an ethanol solution of 85%-95% by volume (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%) as the extraction solvent, and collecting the extract; and removing the solvent from the extract to prepare the extract of Evodia rutaecarpa with an ethanol solution of 85%-95% by volume.

[0066] Optionally, the mass of the extraction solvent is 8 to 12 times the mass of the Evodia rutaecarpa (e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 times).

[0067] Optionally, the Evodia rutaecarpa can be soaked in the extraction solvent before extraction. More preferably, the soaking time is 0.3 hours to 0.7 hours (e.g., 0.3, 0.4, 0.5, 0.6, or 0.7 hours).

[0068] Optionally, the extraction method includes reflux extraction. More preferably, the reflux extraction time is 0.8 hours to 1.2 hours (e.g., 0.8, 0.9, 1.0, 1.1, 1.2 hours).

[0069] Optionally, the method for removing the solvent includes rotary evaporation.

[0070] In this embodiment of the application, the preparation steps of the ethanol extract of Stephania tetrandra, comprising an ethanol solution of Stephania tetrandra with an ethanol volume percentage of 85%-95%, include:

[0071] Using an ethanol solution with a volume percentage of 85%-95% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%) as the extraction solvent, Stephania tetrandra is extracted, and the extract is collected; and the solvent is removed from the extract to prepare an extract of Stephania tetrandra with an ethanol volume percentage of 85%-95%.

[0072] Optionally, the mass of the extraction solvent is 8 to 12 times the mass of the Stephania tetrandra (e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 times).

[0073] Optionally, the powdered Stephania tetrandra can be soaked in the extraction solvent before extraction. More preferably, the soaking time is 0.3 hours to 0.7 hours (e.g., 0.3, 0.4, 0.5, 0.6, or 0.7 hours).

[0074] Optionally, the extraction method includes reflux extraction. More preferably, the reflux extraction time is 0.8 hours to 1.2 hours (e.g., 0.8, 0.9, 1.0, 1.1, 1.2 hours).

[0075] Optionally, the method for removing the solvent includes rotary evaporation.

[0076] Second aspect of the embodiments of this application

[0077] This application provides the use of the pharmaceutical composition described in the first aspect in the preparation of an anticoronavirus drug.

[0078] In some examples, the anti-coronavirus drugs include anti-Severe Acute Respiratory Syndrome Coronavirus 2, anti-Severe Acute Respiratory Syndrome Coronavirus, anti-Middle East Respiratory Syndrome Coronavirus, or anti-bat SARS-like coronavirus.

[0079] Optionally, the anticoronavirus drug includes the pharmaceutical composition and pharmaceutically acceptable excipients.

[0080] The dosage form of the anti-coronavirus drug in this application embodiment is not particularly limited. The corresponding dosage form can be prepared by selecting appropriate excipients according to clinical needs, such as ointment, patch, solution, suspension, emulsion, cream, spray, drop, granule, capsule, tablet, pill, tea or tube feeding preparation, etc.

[0081] The excipients described in this application embodiment may be selected from, but are not limited to, one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusters, and isotonic or isotropic modifiers. The diluents may be selected from, but are not limited to: starches, sugars, celluloses, and inorganic salts. The wetting agents may be selected from, but are not limited to: water and ethanol. The binders may be selected from, but are not limited to: starch paste, dextrin, sugars, cellulose derivatives, gelatin, povidone, and polyethylene glycol. The disintegrants may be selected from, but are not limited to: starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, croscarmellose, surfactants, and effervescent disintegrants. The lubricants may be selected from, but are not limited to: talc, calcium stearate, magnesium stearate, magnesium dodecyl sulfate, micronized silica gel, and polyethylene glycol. The color, flavor, and aroma modifiers may be selected from, but are not limited to: pigments, fragrances, sweeteners, gelling agents, and deodorants. The solvents may be selected from, but are not limited to: water, ethanol, glycerin, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oils, and ethyl acetate. The solubilizers may be selected from, but are not limited to: Tween derivatives, methyl ethers, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates. The co-solvents may be selected from, but are not limited to: organic acids and their salts, amides and amines, inorganic salts, polyethylene glycol, povidone, and glycerin. The emulsifiers may be selected from, but are not limited to: Span derivatives, Tween derivatives, methyl ethers, benzyl ethers, glyceryl fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth gum, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxides, silica, and bentonite. The suspending agent may be selected from, but is not limited to: glycerin, syrup, gum arabic, tragacanth gum, agar, sodium alginate, cellulose derivatives, povidone, carboplatin, polyvinyl alcohol, and thixotropic gum. The antioxidant may be selected from, but is not limited to: sulfites, metabisulfites, bisulfites, ascorbic acid, gallic acid and its esters. The metal chelating agent may be selected from, but is not limited to: disodium ethylenediaminetetraacetate and polycarboxylic acid compounds. The inert gas may be selected from, but is not limited to: nitrogen and carbon dioxide. The preservative may be selected from, but is not limited to: parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils. The local analgesic may be selected from, but is not limited to: benzyl alcohol, chlorobutanol, lidocaine, and procaine. The pH adjuster may be selected from, but is not limited to: hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, acetates, and citrates. The isotonic or isotropic regulator may be selected from, but is not limited to: glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0082] In the embodiments of this application, "anti" includes prevention, treatment, and adjunctive treatment. As used herein, "prevention and treatment" refers to alleviating, delaying the progression, attenuating, preventing, or maintaining an existing disease or condition. "Prevention and treatment" also includes curing, preventing the development of, or alleviating to some extent one or more symptoms of a disease or condition.

[0083] In the embodiments of this application, "drug" includes any agent, compound, composition, or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects produced by the "drug" in vivo is not particularly limited; it may have systemic effects or only local effects. The activity of the "drug" is not particularly limited; it may be an active substance that can interact with other substances or an inert substance that does not interact with other substances.

[0084] In the embodiments of this application, the "anti-coronavirus drug" comprises an effective amount of the pharmaceutical composition described in the first aspect.

[0085] In the embodiments of this application, "effective amount" refers to the dose by which the component corresponding to the term achieves treatment, prevention, reduction and / or relief of a specific disease, condition and / or symptom in a subject. In this application, unless otherwise specified, it refers to the dose by which coronavirus infection, condition and / or symptom are treated, prevented, reduced and / or relieved.

[0086] In this application, "therapeutic effective amount" refers to the amount of a pharmaceutically active ingredient that will elicit a biological or medical response in an individual in response to a disease, condition, and / or symptom, such as the amount of the compound of this application that brings positive physiological and / or pharmacological effects to an individual, including but not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating conditions, slowing or delaying disease progression, or preventing disease.

[0087] Third aspect of the embodiments of this application

[0088] This application provides an anti-coronavirus drug comprising the pharmaceutical composition described in the first aspect and pharmaceutically acceptable excipients.

[0089] In some embodiments of this application, the dosage forms of the anti-coronavirus drug include ointments, patches, solutions, suspensions, emulsions, creams, sprays, drops, granules, capsules, tablets, pills, teas, or tube feeding preparations.

[0090] The definitions of the terms "anti", "anticoronavirus drug", "drug", "effective dose", and "therapeutic effective dose" are as per the second part. Similarly, the limitations on excipients and dosage forms are also as per the second part.

[0091] Fourth aspect of this application

[0092] This application provides a treatment for coronavirus infection, comprising administering an effective amount of the said anti-coronavirus drug to a subject.

[0093] The definitions of the terms "anti", "anticoronavirus drug", "drug", "effective dose", and "therapeutic effective dose" are as per the second part. Similarly, the limitations on excipients and dosage forms are also as per the second part.

[0094] In some embodiments, the administration of the drug includes, but is not limited to: oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection, local administration, and inhalation.

[0095] In some of these embodiments, the drug may be administered orally, via enema, or via parenteral administration.

[0096] In some embodiments, the drug administration cycle may be intermittent, periodic, continuous, or long-term.

[0097] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0098] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0099] In previous studies, the applicant team, through a series of screenings and tests, discovered that evodiamine extract and evodiamine alkaloids can effectively inhibit the invasion of host cells by inhibiting the spike protein of SARS-CoV-2, and are therefore identified as effective spike protein inhibitors. Meanwhile, according to literature, TPC2 protein is one of the important components of SARS-CoV-2 entering cells through mucosal fusion, and thus also becomes one of the target proteins for antiviral invasion. The team believes that both the spike protein-ACE2 pathway and TPC2 protein play important roles in the viral invasion pathway, and the combination of spike protein inhibitors and TPC2 inhibitors may inhibit the viral pathway through a multi-target combined mechanism to achieve a synergistic antiviral effect and significantly reduce the half-maximal efficacy (IC50) of the inhibitor complex. 50 Multiple studies have documented that Stephania tetrandra and its alkaloids exhibit good inhibitory effects on TPC2 protein, making them effective TPC2 inhibitors.

[0100] During their research on traditional Chinese medicine, the inventors established four models: a pseudovirus model of SARS-CoV-2 infection, a model of the inhibitory effect of the spike protein of the SARS-CoV-2 SARS-CoV-2 receptor binding to the angiotensin-converting enzyme 2 (ACE2) receptor, a molecular docking model, and a Compusyn analysis of synergistic effects. These models were used to explore the synergistic effects of two compositions—Evodia rutaecarpa extract combined with Stephania tetrandra extract, or Evodia rutaecarpa alkaloid combined with Stephania tetrandra alkaloid—in inhibiting SARS-CoV-2 invasion. The results showed that both compositions, namely Evodia rutaecarpa extract combined with Stephania tetrandra extract and Evodia rutaecarpa alkaloid combined with Stephania tetrandra alkaloid, effectively inhibited the invasion pathway of SARS-CoV-2, and the inhibitory effect was superior to that of a single drug (i.e., Evodia rutaecarpa extract, Evodia rutaecarpa alkaloid, Stephania tetrandra alkaloid, and Stephania tetrandra alkaloid used individually). In other words, the two compositions exhibited a good synergistic effect in inhibiting SARS-CoV-2. Based on this discovery, this application describes the preparation of an anti-coronavirus drug using either Evodia rutaecarpa extract combined with Stephania tetrandra extract or Evodia rutaecarpa alkaloid combined with Stephania tetrandra alkaloid, aiming to provide a new solution for the control of coronavirus infection.

[0101] Example 1: Preparation of a composition of Evodia rutaecarpa alcohol extract and tetrandrine alcohol extract, and a composition of Evodia rutaecarpa alkaloid and tetrandrine.

[0102] The preparation methods of 90% ethanol extract of Evodia rutaecarpa and 90% ethanol extract of Stephania tetrandra include the following steps:

[0103] (1) Take the medicinal material, place it in a 250mL round-bottom flask, add 10 times the mass of 90% (v / v) ethanol aqueous solution to soak for half an hour, reflux and heat for 1 hour, then filter out the medicinal liquid by separating the dregs;

[0104] (2) The medicinal liquid was evaporated to dryness using a rotary evaporator to prepare 90% alcohol extract of Evodia rutaecarpa and 90% alcohol extract of Stephania tetrandra.

[0105] (3) Collect the extracted Evodia rutaecarpa alcohol extract and the tetrandrine alcohol extract and mix them in a certain proportion to form a composition. The mass ratio of Evodia rutaecarpa alcohol extract and tetrandrine alcohol extract includes: 1:1, 1:2, 2:1, 1:3, 3:1, etc.

[0106] The preparation of the composition of evodiamine and tetrandrine includes the following steps:

[0107] Evodiamine and tetrandrine are both 95% or higher in purity. They are mixed in a certain ratio to form a composition. The molar mass concentration ratio of evodiamine and tetrandrine includes: 1:1, 1:2, 2:1, 1:3, 3:1, 1:4, 4:1, 1:5, 5:1, etc.

[0108] Example 2: Synergistic inhibitory effect of Evodia rutaecarpa alcohol extract combined with Stephania tetrandra alcohol extract on SARS-CoV-2 Spike pseudovirus

[0109] (1) Experimental Principle

[0110] Pseudoviruses are a type of chimeric viral particle, which is a chimeric viral particle expressing a recombinant glycoprotein of another virus on the surface of a replication-defective virus (viral vector). By replacing the envelope glycoprotein in the viral vector with the SARS-CoV-2 S protein, pseudoviruses mimicking SARS-CoV-2 infection are formed. The S protein includes both wild-type and Omicron types. If the composition can inhibit the invasion and infection of pseudoviruses, it means that the composition can inhibit the invasion of host cells by both wild-type and Omicron SARS-CoV-2.

[0111] (2) Experimental materials

[0112] High-glucose Dulbecco's Modified Eagle medium (DMEM) was purchased from Gibco (UK). Fetal bovine serum (FBS) was purchased from Gibco (Brazil). Penicillin and streptomycin (Pen / Strep) were purchased from Gibco. Trypsin-EDTA (1x) was purchased from Gibco. Luciferase assay system was purchased from Promega. Protein Assay Dye Reagent Concentration was purchased from Bio-Rad. Bovine serum albumin (BSA) was purchased from Sigma. Microplate reader was purchased from Thermo Scientific. Microplate Luminometer was purchased from GLOMAX.

[0113] (3) Experimental steps

[0114] Cell culture: HEK293T cells were cultured in high-glucose Dullbecco modified Eagle medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS) and 1% (w / w) Pen / Strep. HEK293T cell lines overexpressing ACE-2 were transfected with the pcDNA3.1-hACE2 plasmid.

[0115] As shown in Table 1, the generation and collection of SARS-CoV-2 pseudoviruses: HEK293T cells were co-transfected with plasmids NR-52514, NR-52516, NR-52517, NR-52518, NR-52519, and 179907, which produced SARS-CoV-2 wild-type and Omeprone type Spike pseudovirus particles, respectively. The pseudoviruses in the supernatant were collected 48 hours after transfection, filtered through a 0.45 μm filter, and stored at -80°C.

[0116] Table 1. Types and functions of plasmids used in establishing the SARS-CoV-2 Spike pseudovirus model.

[0117] plastid types Embedded BEI Resources Product Number Viral Entry Protein Wildtype Spike Glycoprotein NR-52514 Viral Entry Protein Omicron Spike Glycoprotein 179907 Lentiviral Backbone Luc2; ZsGreen NR-52516 Helper Plasmid Gag; pol NR-52517 Helper Plasmid Tat1b NR-52518 Helper Plasmid Rev1b NR-52519

[0118] Pseudovirus invasion assay: ACE-2-overexpressing HEK293T cells were seeded in 48-well plates. 100 μL of pseudovirus (along with the test sample) and 400 μL of culture medium were added to each well, and the cells were incubated overnight. The culture medium was then replaced with a mixture of DMEM, FBS, and Pen / Strep. After 48 hours of incubation, the cells were washed with 1×PBS, and then luciferase activity was measured.

[0119] Neutralizing antibody (A19215) was used as a positive control, while solvent blank and pseudovirus-free group were used as negative controls. The test sample was a combination of Evodia rutaecarpa alcohol extract and Stephania tetrandra alcohol extract, and was compared with Evodia rutaecarpa alcohol extract and Stephania tetrandra alcohol extract alone.

[0120] Luciferase activity was standardized using data from a solvent blank luciferase activity assay, and the percentage of virus inhibition by the sample was calculated. Three wells were added to each sample, and the average value was taken. The experiment was repeated three times.

[0121] (4) Experimental Results

[0122] First, the cells established above were verified by ACE2 immunostaining and TPC2 immunostaining methods to contain effective ACE2 receptors. Figure 1 ) and TPC2 protein ( Figure 2 This also provides evidence that spike protein inhibitors (such as Evodia rutaecarpa alcohol extract and Evodia rutaecarpa alkaloids) inhibit the binding of spike protein to ACE2 and that TPC2 inhibitors (Stephania tetrandra and Stephania tetrandra alkaloids) inhibit viral pathways. Figure 1 As shown, after HEK293T cells were transfected with ACE2 receptor (fluorescent green), the ACE2 staining analysis confirmed that the cell surface contained a certain amount of ACE2 receptor, and the spike protein on the pseudovirus could use this receptor to invade the host cell. Figure 2 As shown in the figure, in the TPC2 staining analysis, the TPC2 protein is stained fluorescent green. As can be seen from the figure, HEK293T cells contain a certain amount of TPC2 protein.

[0123] like Figure 3 To explore the inhibitory effects of different mass ratios of Evodia rutaecarpa extract and Stephania tetrandra extract, the embodiments of this application, through screening and comparison, found that when the mass ratio between Evodia rutaecarpa extract and Stephania tetrandra extract was 1:1, i.e., 15 μg / mL: 15 μg / mL, the composition exhibited superior resistance to wild-type antibodies compared to other ratios. Figure 3(Figure A) and Omeprone virus ( Figure 3 (Figure B) activity. Meanwhile, when the molar mass ratio of evodiamine to tetrandrine was 1:1, i.e., 15 μM:15 μM, the complex expressed a stronger inhibitory effect on the wild-type ( Figure 3 C) and Omecron type ( Figure 3 D) The activity of the novel coronavirus. Figure 3 The figures shown are resistance to wild-type evodia extract combined with tetrandrine extract in different proportions, as well as evodia alkaloids combined with tetrandrine. Figure 3 Figure A in the middle, Figure 3 (Figure C) and Omeckeron type ( Figure 3 China B map Figure 3 (Figure D) Viral activity. The weight ratios of the ethanol extracts 1:1, 1:2, 2:1, 1:3, and 3:1 represent the weight ratios of Evodia rutaecarpa ethanol extract to Stephania tetrandra ethanol extract at 15 μg / mL:15 μg / mL, 10 μg / mL:20 μg / mL, 20 μg / mL:10 μg / mL, 7.5 μg / mL:22.5 μg / mL, and 22.5 μg / mL:7.5 μg / mL, respectively. The molar concentration ratios of the compounds 1:1, 1:2, 2:1, 1:3, and 3:1 represent the ratios of Evodia rutaecarpa alkaloid to Stephania tetrandra alkaloid at 15 μM:15 μM, 10 μM:20 μM, 20 μM:10 μM, 7.5 μM:22.5 μM, and 22.5 μM:7.5 μM, respectively.

[0124] Evodia rutaecarpa alcohol extract (EVF) EtOH ) and stromenol (STS) EtOH Taking a weight ratio of 1:1 for the items being lifted as an example, the results are as follows: Figure 4 As shown, the neutralizing antibody A19215 solution serves as a positive control. Figure 4 As shown, in the HKE293T pseudovirus system, taking a 1:1 weight ratio of Evodia rutaecarpa ethanol extract and Stephania tetrandra ethanol extract as an example, the combination of Evodia rutaecarpa ethanol extract and Stephania tetrandra ethanol extract can effectively inhibit the invasion of wild-type (A) and Omeprón (B) SARS-CoV-2 into host cells, exhibiting a good dose-response curve. The results show that within the concentration range of 1.5625 μg-25 μg, Evodia rutaecarpa ethanol extract (EVF)... EtOH ) and Stephania tetrandra extract (STS) EtOH The composition of ) can effectively inhibit wild-type ( Figure 4 (Figure A) and Omeckeron type ( Figure 4 The novel coronavirus (COVID-19) invades host cells (Figure B in the text), and its inhibitory effect is better than that of Evodia rutaecarpa extract and Stephania tetrandra extract when used alone.

[0125] Example 3: Analysis of the synergistic effect of the combination of Evodia rutaecarpa extract and Stephania tetrandra extract using the computer analysis software Compusyn.

[0126] (1) Experimental Principle

[0127] The Compusyn computer model (https: / / www.combosyn.com / index.html) uses the "Drug Synergy Index Theorem," specifically the formula below, to calculate the synergy index. Here, D represents the dosage used, and f... a This represents the inhibition rate, and CI is the synergistic index. The synergistic index can be used to define synergistic effects (CI < 1), additive effects (CI = 1), and antagonistic effects (CI > 1) between individual compounds.

[0128]

[0129] (2) Experimental Results

[0130] like Figure 5 As shown, taking a mass ratio of 1:1 between Evodia rutaecarpa ethanol extract and Stephania tetrandra ethanol extract as an example, when the mass ratio of Evodia rutaecarpa ethanol extract (EVF) is 1:1... EtOH ) and Stephania tetrandra alcohol extract (STS) EtOH When used in combination, the CI value remained below 1, indicating that both are effective against wild-type ( Figure 5 (Left image of A in the middle) and Omeckeron type ( Figure 5 (Figure B) The novel coronavirus exhibits a synergistic inhibitory effect.

[0131] Example 4: Synergistic inhibitory effect of evodiamine combined with tetrandrine against SARS-CoV-2 Spike pseudovirus

[0132] (1) Experimental Principle

[0133] Same as Example 2.

[0134] (2) Experimental materials

[0135] Same as Example 2.

[0136] (3) Experimental steps

[0137] Same as Example 2.

[0138] Neutralizing antibody (A19215) was used as a positive control, while solvent blank and pseudovirus-free groups served as negative controls. The test samples were different molar concentrations of the rutaecarpine-tetrandrine complex, compared with rutaecarpine and tetrandrine used alone. Luciferase activity was standardized using data from the solvent blank luciferase assay, and the percentage of virus inhibition by the sample was calculated. Each sample was applied to three wells, and the average value was taken; the experiment was repeated three times.

[0139] (4) Experimental Results

[0140] Figure 6 As shown, in the HKE293T pseudovirus system, taking a molar mass ratio of 1:1 for rutaecarpine and tetrandrine as an example, the complex of rutaecarpine and tetrandrine can effectively inhibit the invasion of wild-type (A) and omeprone (B) SARS-CoV-2 into host cells and exhibits a good dose-response curve.

[0141] Depend on Figure 6 As shown, taking a molar mass ratio of 1:1 between evodiamine and tetrandrine as an example, within the concentration range of 0.625 μM to 20 μM, the complex of evodiamine and tetrandrine exhibited good inhibition of the wild-type ( Figure 6 (Figure A) and Omeckeron type ( Figure 6 (Figure B) The effect of the novel coronavirus, and this effect is better than that of evodiamine and tetrandrine when used alone.

[0142] Example 5: Analysis of the synergistic effect of the combination of evodiamine and tetrandrine using the computer analysis software Compusyn.

[0143] (1) Experimental Principle

[0144] Same as Example 3.

[0145] (2) Experimental Results

[0146] like Figure 7 As shown, taking a molar mass ratio of 1:1 for evodiamine (Ru) and tetrandrine (Te) as an example, when evodiamine and tetrandrine are used in combination, the synergistic index remains below 1, indicating that the two can target wild-type ( Figure 7 (Figure A) and Omeckeron type ( Figure 7 (Figure B) The novel coronavirus exhibits a synergistic inhibitory effect.

[0147] Example 6: Analysis of the affinity between evodiamine and spike protein using molecular docking technology.

[0148] (1) Experimental Principle

[0149] The RBD is where the viral spike protein binds to human ACE2. If a chemical component can bind to the RBD, it can reduce the chance of the virus binding to human ACE2, thereby preventing human infection with the novel coronavirus. The binding energy of the chemical component to the RBD is calculated. The more negative the value of the binding energy, the stronger the binding and the higher the efficacy in inhibiting the novel coronavirus.

[0150] (2) Experimental Operation and Results

[0151] Using SEESAR software (Version 13.0, https: / / www.biosolveit.de / products / seesar / ), the RBD (residues 438-506) of the spike protein (PDB code: wild type 6LZG; omeprion type 7T9L) was selected as the target site to detect the affinity of tebufenozide for this target site. Furthermore, the TPC2 ligand-binding domain (PDB code: 6NQ0) was selected as the target site to further detect the binding affinity of tetrandrine to TPC2.

[0152] The results are as follows Figure 8 The results showed that evodiamine can effectively target wild-type ( Figure 8 (Figure A) and Omeckeron type ( Figure 8 (Figure B) The spike protein's RBD region releases energy, proving that evodiamine can effectively bind to the spike protein, thereby inhibiting the binding of the novel coronavirus to the ACE2 receptor and preventing it from attacking host cells.

[0153] At the same time, tetrandrine can also effectively bind to the TPC2 ligand binding domain and express an effective inhibitory effect. Figure 8 (See Figure C). The two compounds bind to spike protein and TPC2 protein, respectively, providing strong evidence that the corresponding Chinese medicinal materials inhibit the binding of spike protein to the ACE2 receptor and TPC2 protein, respectively.

[0154] Example 7

[0155] This embodiment relates to the application of two compositions in the preparation of anti-novel coronavirus drugs: one is a combination of Evodia rutaecarpa alcohol extract and the other is a combination of Evodia rutaecarpa alkaloids and the other is a combination of Evodia rutaecarpa alkaloids. The dosage form of the drug is a granule.

[0156] Example 8

[0157] This embodiment relates to the application of two compositions in the preparation of anti-novel coronavirus drugs: one is a combination of Evodia rutaecarpa alcohol extract and the other is a combination of Evodia rutaecarpa alkaloids and the other is a combination of Evodia rutaecarpa alkaloids. The dosage form of the drug is granules.

[0158] Example 9

[0159] This embodiment relates to the application of two compositions in the preparation of anti-novel coronavirus drugs: one is a combination of Evodia rutaecarpa alcohol extract and the other is a combination of Evodia rutaecarpa alkaloids and the other is a combination of Evodia rutaecarpa alkaloids. The dosage form of the drug is capsules.

[0160] Example 10

[0161] This embodiment relates to the application of two compositions in the preparation of an anti-novel coronavirus drug: a combination of Evodia rutaecarpa alcohol extract and tetrandrine alcohol extract, and a combination of Evodia rutaecarpa alkaloid and tetrandrine. The dosage form of the drug is tablets.

[0162] Example 11

[0163] This embodiment relates to the application of two compositions in the preparation of anti-novel coronavirus drugs: one is a combination of Evodia rutaecarpa alcohol extract and the other is a combination of Evodia rutaecarpa alkaloid and tetrandrine. The dosage form of the drug is a tea.

[0164] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0165] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A drug composition for treating COVID-19, comprising a spike protein inhibitor and a TPC2 protein inhibitor; wherein, The spike protein inhibitor is Evodia rutaecarpa extract, and the TPC2 protein inhibitor is Stephania tetrandra extract, wherein the mass ratio of the Evodia rutaecarpa extract to the Stephania tetrandra extract is 1:1; or... The spike protein inhibitor is evodiamine, the TPC2 protein inhibitor is tetrandrine, and the molar mass ratio of evodiamine and tetrandrine is 1:

1. The Evodia extract is an extract of an ethanol solution containing 85%-95% ethanol by volume of Evodia rutaecarpa. The Stephania tetrandra extract is an extract of Stephania tetrandra ethanol solution with a volume percentage of 85%-95%.

2. The use of the pharmaceutical composition of claim 1 in the preparation of a medicament against the wild-type strain of SARS-CoV-2 and its Omeprone variant.

3. The application according to claim 2, wherein, The drug includes the pharmaceutical composition and pharmaceutically acceptable excipients.

4. The application according to claim 3, wherein, The dosage form of the drug is ointment, patch, solution, suspension, emulsion, cream, spray, drops, granules, capsule, tablet, pill, or tea.

5. A drug for combating the wild-type strain of SARS-CoV-2 and its Omeprone variant, comprising the pharmaceutical composition of claim 1 and pharmaceutically acceptable excipients.

6. The drug against the wild-type strain of SARS-CoV-2 and its Omeprone variant as described in claim 5, wherein the dosage form is an ointment, patch, solution, suspension, emulsion, cream, spray, drops, granules, capsule, tablet, pill or tea.

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