TWI876111B - Methods for treating sars-cov-2 infection - Google Patents

Abstract

Disclosed herein are methods for the treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. The method includes the step of administering to a subject suffering from a SARS-CoV-2 infection an effective amount of an agent selected from the group consisting of an anti-viral agent, an anti-neoplastic agent, an anti-parasitic agent, an ion channel modulator, an antibiotic, an herbal extract, Soho tea, Reishi polysaccharide fraction 3 (RF3) of Ganoderma lucidum, and a combination thereof, so as to alleviate or ameliorate symptoms associated with the SARS-CoV-2 infection.

Description

Methods for treating severe acute respiratory syndrome coronavirus 2 infection

Cross-reference to related applications

This application claims priority to and the benefits of U.S. Patent Provisional Application No. 63/119,573, filed on November 30, 2020, the entire contents of which are incorporated by reference as a part of this disclosure.

This disclosure is about drugs used to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans.

SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA coronavirus in the family Betacoronavirus and is the cause of the 2019 coronavirus disease (COVID-19) pandemic. Compared with the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East respiratory syndrome–related coronavirus (MERS-CoV) outbreaks in 2002 and 2012, respectively, SARS-CoV-2 exhibits a low mortality rate but a high infection rate, posing a greater threat to public health and a huge social and economic burden.

SARS-CoV-2 infection begins with the interaction between the viral spike (S) protein triad and the angiotensin-converting enzyme 2 (ACE2) receptor of human respiratory epithelial cells, followed by interaction with transmembrane protease serine 2 (TMPRSS2) after the virus enters the cell, which cleaves the S protein and initiates viral fusion. The viral genomic RNA is translated into polyprotein 1a (PP1a) and polyprotein 1ab (PP1ab), which are then cleaved by papain-like (PL) and 3C-like proteases to form 16 non-structural proteins (Nsp1-16) as a replication-transcription complex (RTC). Four structural proteins (spike protein, coat protein, membrane protein, and nucleocapsid protein) are encoded at the 3' end and play an important role in viral maturation and infection. From the N to C terminus of PP1ab, viral RNA replication is completed by RTC proteins, such as RNA-dependent RNA polymerase (Rdrp, Nsp12). The viral proteins further undergo post-translational modifications (such as glycosylation) in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), after which the viral proteins are transported to the cell membrane for exocytosis.

Although several agents targeting viral replication and inflammation have been reported, clinical management of COVID-19 to date has been primarily supportive. Remdesivir, a Rdrp inhibitor prodrug, is the only antiviral agent to have received FDA emergency use authorization for the treatment of COVID-19. Favipiravir, an influenza Rdrp inhibitor, has been approved for the treatment of COVID-19 in Russia, China, and India, but patients taking the drug must be closely monitored to avoid adverse events; recently, results from a Phase III clinical trial in Japan showed some positive results. Hydroxychloroquine has been reported to exhibit antiviral activity against RNA viruses, especially when used in combination with zinc supplements, but due to a lack of significant benefit, hydroxychloroquine is no longer used alone in the clinical treatment of COVID-19.

RNA viruses are known to mutate more easily than DNA viruses. Recently, a protein interaction map revealed that 332 human proteins interact with 27 SARS-CoV-2 proteins; and phosphoproteomic approaches have been further used to expand the study of virus-host interactions. However, the recently reported proteomic analyses focused only on the S protein, and the detailed functions of its glycosylation are still unknown. Nevertheless, the S protein has become the most promising target for the development of neutralizing antibodies and vaccines because it is expressed on the virus surface and participates in the process of viral entry into host cells. The S protein itself is highly glycosylated and contains a large number of mutations, with nearly 85% of the sequence being altered, posing a challenge to the development of effective vaccines, antibodies with broad protective activity, and alternative therapies. However, the development of new treatments often takes years; therefore, repurposing or repositioning existing drugs and herbal remedies to treat COVID-19 has been considered an attractive approach.

In this disclosure, a drug library consisting of 2,855 drugs approved for the treatment of human and animal diseases, as well as supplements and traditional Chinese herbal medicines, was screened in the hope of finding inhibitors that can effectively inhibit SARS-CoV-2 infection in Vero E6 cells. After further study of the screened effective compounds, the dose-response relationship of these compounds was established, as well as the in vivo anti-SARS-CoV-2 effects of these effective compounds in hamsters infected with SARS-CoV-2. Accordingly, these compounds and/or extracts are candidates with the potential to be developed into drugs for the treatment of SARS-CoV-2 infection.

The present disclosure is based on the unexpected discovery that some known compounds and herbal extracts can effectively inhibit the activity of SARS-CoV-2 in vitro or in vivo. Therefore, these compounds and herbal extracts are potential candidates for development into drugs for the treatment of SARS-CoV-2 infection.

Accordingly, the first aspect of the present disclosure is to provide a method for treating a patient infected with SARS-CoV-2. The method comprises administering to the subject an effective amount of a drug selected from the group consisting of an antiviral agent, an antitumor agent, an antiparasitic agent, an ion channel modulator, an antibiotic, a herbal extract, Soho tea, Lingzhi polysaccharide fraction 3 (RF3) of Ganoderma lucidum, and combinations thereof, to alleviate or reduce symptoms associated with SARS-CoV-2 infection.

Examples of antiviral agents suitable for use in the present method include, but are not limited to, nelfinavir (N-Bn), nelfinavir mesylate, and boceprevir.

Examples of anti-tumor agents suitable for use in the present method include, but are not limited to, cepharanthine.

Examples of antiparasitic agents suitable for use in the present method include, but are not limited to, emetine, ivermectin, moxidectin, and mefloquine.

Examples of ion channel modulators suitable for use in the present method include, but are not limited to, azelnidipine, dronedarone, ivacaftor, and penfluridol.

Examples of antibiotics suitable for use in the present method include, but are not limited to, monensin and maduramicin.

According to a preferred embodiment of the present disclosure, the drug is a combination of nelfinavir and ivermectin at a molar ratio of 1:1, a combination of cephalanthrin and nelfinavir at a molar ratio of 2:1, or a combination of cephalanthrin and ivermectin at a molar ratio of 2:1.

According to a preferred embodiment of the present disclosure, the herbal extract suitable for use in the present method is an aqueous extract of a plant selected from the group consisting of Lamiaceae family, Mentheae family, Asteraceae family, Theaceae family, Fabaceae family, Compositae family, Saururaceae family, Sapindaceae family and Caprifoliaceae family.

Examples of plants of the Lamiaceae family include, but are not limited to, wild mint ( Mentha arvensis ), peppermint ( Mentha haplocalyx ), Nepeta tenuifolia , basil ( Ocimum basilicum ), perilla leaves ( Perillae folium ), perilla ( Perilla frutescens ) , Prunellae spica, Prunella vulgaris , Salvia hispanica , and rosemary ( Salvia rosmarinus ).

Examples of plants in the mint family include, but are not limited to, peppermint or wild mint.

Examples of plants in the Asteraceae family include, but are not limited to, dandelion ( Taraxacum mongolicum ), coltsfoot ( Tussilago farfara ), and chrysanthemum ( Chrysanthemum morifolium ).

Examples of plants of the Theaceae family include, but are not limited to, tea tree ( Camellia sinensis ).

Examples of plants in the Leguminosae family include, but are not limited to, peanut ( Arachis hypogaea ), licorice ( Glycyrrhiza uralensis ), licorice ( Glycyrrhiza glabra L. ), licorice ( Glycyrrhiza inflate Batalin ), and bean ( Spatholobus suberectus ).

Examples of plants of the Saururaceae family include, but are not limited to, Houttuynia cordata and dried whole herb of Houttuynia cordata in bloom ( Herba Houttuyniae ).

Examples of plants of the Compositae family include, but are not limited to, Artemisia annua L. and Artemisia scoparia Waldst .

Examples of plants in the Sapindaceae family include, but are not limited to, longan ( Dimocarpus longan ) and litchi ( Litchi chinensis ).

Examples of plants of the Caprifoliaceae family include, but are not limited to, honeysuckle ( Lonicera japonica ).

According to an embodiment of the present disclosure, the herbal extract comprises at least one compound selected from the group consisting of flavonoids, flavan-3-ols, caffeic acid derivatives, monoterpenes, diterpenes and triterpenes.

According to an embodiment of the present disclosure, the sohuo tea is a water extract extracted from a combination of perilla leaves, mint, dried fruit ears of Prunella vulgaris, Houttuynia cordata, Artemisia annua and licorice, wherein the perilla leaves, the mint, the dried fruit ears of Prunella vulgaris, the Houttuynia cordata, the Artemisia annua and the licorice have a weight percentage or volume percentage of 40-75%, 10-30%, 0.1-2%, 1-4%, 5-15% and 5-15% respectively in the combination.

According to an embodiment of the present disclosure, the Suhuo tea is a water extract extracted from a combination of perilla leaves, mint, dried fruit ears of Prunella vulgaris, Houttuynia cordata, honeysuckle, coltsfoot and licorice, wherein the perilla leaves, mint, dried fruit ears of Prunella vulgaris, Houttuynia cordata, honeysuckle, coltsfoot and licorice have a weight percentage or volume percentage of 40-75%, 10-30%, 0.1-2%, 1-4%, 5-15% and 5-15% respectively in the combination.

According to an embodiment of the present disclosure, the agent is selected from the group consisting of antiviral agents, antitumor agents, antiparasitic agents, ion channel modulators, antibiotics and combinations thereof, and is administered to the subject in an amount of 0.01-500 mg/Kg.

According to an embodiment of the present disclosure, the medicament is selected from the group consisting of herbal extracts, soothing tea, RF3 of Ganoderma lucidum, and combinations thereof, and is administered to the subject in an amount of 0.01-5 g/kg.

Examples of individuals suitable for treatment according to the present disclosure include, but are not limited to, mammals. Preferably, the individual is a human.

The use of any agent described herein for the production of a medicament for treating the SARS-CoV-2 infection described herein is also within the scope of this disclosure.

After referring to the following embodiments in conjunction with the attached drawings, a person having ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other invention purposes of the present invention, as well as the technical means and implementation modes adopted by the present invention.

In order to make the description of the disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation method covers the features of multiple specific embodiments and the method steps and their sequences for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.

1. Definition

For convenience, certain terms used in the specification, embodiments and the attached patent application are integrated here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those with ordinary knowledge in the field to which the present invention belongs.

The term "administered, administering or administration" herein refers to a form of delivery, including but not limited to, intravenous, intramuscular, intraperitoneally, intraarterially, intracranially or subcutaneously administration of a medicament (e.g., a compound or a composition) of the present invention. In certain embodiments, the compound or its salt or solvent complex of the present disclosure is formulated into a tablet for oral administration. In other embodiments, the compound or its salt or solvent complex of the present disclosure is formulated into a powder and mixed with a suitable carrier (e.g., a buffer solution) before use, such as intravenous injection, and then administered.

As used herein, the term "an effective amount" refers to an amount that is effective in dosage and for a necessary period of time to achieve the desired disease treatment result. For example, in the treatment of SARS-CoV-2 infection, an agent (i.e., a compound or herbal extract of the present disclosure) that can reduce, prevent, delay, inhibit or arrest any symptoms of SARS-CoV-2 infection should be effective. An effective amount of an agent does not need to cure the disease or condition, but will provide treatment for the disease or condition to delay, stop or prevent the onset of the disease or condition, or to improve the symptoms of the disease or condition. The effective amount can be divided into one, two or more doses in an appropriate form and administered one, two or more times over a specified period of time.

Although the numerical ranges and parameters describing the broad scope of the present invention are approximate, the numerical values set forth in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains certain errors, which originate from the standard deviation found in each detection means. Moreover, the term "about" used herein generally refers to within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, when considered by a person of ordinary skill in the art, the term "about" means within an acceptable standard error of the mean. Except for operational/operational examples, or unless otherwise expressly stated, all numerical ranges, quantities, values and percentages disclosed herein, such as for describing material quantities, durations of time, temperatures, operating conditions, quantitative ratios and the like, should be understood as being modified by the word "about" in all cases. Therefore, unless otherwise stated to the contrary, the numerical parameters set forth in this disclosure and the attached patent claims are approximate values and may be changed as required. In any case, each numerical parameter should at least be interpreted with the number of significant digits reflected and by applying ordinary rounding and addition calculation techniques.

Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents.

2. Treatment methods

The present disclosure relates to agents suitable for treating SARS-CoV-2 infection in an individual. Therefore, the primary object of the present disclosure is to provide a method for treating an individual infected with SARS-CoV-2. The method comprises administering to the individual an effective amount of an agent selected from the group consisting of antiviral agents, antitumor agents, antiparasitic agents, ion channel modulators, antibiotics, herbal extracts, Soho tea, Lingzhi polysaccharide fraction 3 (RF3) of Ganoderma lucidum, and combinations thereof, to alleviate or reduce symptoms associated with SARS-CoV-2 infection.

Examples of antiviral agents suitable for use in the present method include, but are not limited to, nelfinavir (N-Bn), nelfinavir mesylate, and boceprevir.

Examples of anti-tumor agents suitable for use in the present method include, but are not limited to, cepharanthine.

Examples of antiparasitic agents suitable for use in the present method include, but are not limited to, emetine, ivermectin, moxidectin, and mefloquine.

Examples of ion channel modulators suitable for use in the present method include, but are not limited to, azelnidipine, dronedarone, ivacaftor, and penfluridol.

Examples of antibiotics suitable for use in the present method include, but are not limited to, monensin and maduramicin.

According to a preferred embodiment of the present disclosure, the drug is a combination of nelfinavir and ivermectin at a molar ratio of 1:1, a combination of cephalanthrin and nelfinavir at a molar ratio of 2:1, or a combination of cephalanthrin and ivermectin at a molar ratio of 2:1.

According to a preferred embodiment of the present disclosure, the herbal extract suitable for use in the present method is an aqueous extract of a plant selected from the group consisting of Lamiaceae, Menthaceae, Asteraceae family, Theaceae, Leguminosae, Compositae family, Saururaceae, Sapindaceae and Caprifoliaceae.

Examples of plants of the Lamiaceae family include, but are not limited to, wild mint, peppermint, nephrolepis, basil, perilla leaf, perilla, dried fruit spikes of selfheal, selfheal, salvia officinalis, and rosemary.

Examples of plants in the mint family include, but are not limited to, peppermint or wild mint.

Examples of plants in the Asteraceae family include, but are not limited to, dandelions, coltsfoot and chrysanthemum.

Examples of plants of the Theaceae family include, but are not limited to, tea trees.

Examples of plants from the Leguminosae family include, but are not limited to, peanut, licorice, Glycyrrhiza glabra, Glycyrrhiza glabra, and Glycyrrhiza uralensis.

Examples of plants of the Saururaceae family include, but are not limited to, Houttuynia cordata and dried whole herb of Houttuynia cordata in bloom.

Examples of plants of the Compositae family include, but are not limited to, Artemisia annua and Artemisia littoralis.

Examples of plants in the Sapindaceae family include, but are not limited to, longan and lychee.

Examples of plants of the Caprifoliaceae family include, but are not limited to, honeysuckle.

According to an embodiment of the present disclosure, the herbal extract comprises at least one compound selected from the following group, including flavonoids (e.g., myricetin), flavan-3-ols (e.g.,

The group consisting of catechin or epigallocatechin gallate), caffeic acid derivatives (e.g., caftaric acid, rosmarinic acid methyl ester or chlorogenic acid), monoterpenes (e.g., 1,8-cineole or camphor), diterpenes (e.g., carnosic acid or patchouli alcohol) and triterpenes (e.g., ursolic acid).

According to certain embodiments of the present disclosure, the agent can be an antiviral agent, an antitumor agent, an antiparasitic agent, an ion channel modulator, an antibiotic, and combinations thereof, which is administered to the subject in an amount of 0.01-500 mg/kg, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 8 2, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 14 0, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 mg/kg. Preferably, the medicament is administered at 10-300 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 and 300 mg/kg. More preferably, the medicament is administered at 20-250 mg/kg is administered to the subject, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 and 250 mg/Kg.

According to certain embodiments of the present disclosure, the medicament is selected from the group consisting of herbal extracts, soda tea, RF3 of Ganoderma lucidum and combinations thereof, and is administered to the subject in an amount of 0.01-5 g/kg, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 .8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3. 4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 g/Kg. Preferably, the agent is administered to the subject in an amount of 0.05-3.5 g/Kg, for example 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g/Kg. More preferably, the agent is administered to the subject in an amount of 1.0-3.0 g/Kg, for example 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 g/Kg.

In certain embodiments, the whole plant (herbs) and its rhizome (rhizome) are extracted with hot water at a weight or volume ratio of between 1:1 and 1:1,000 to prepare a drink or "soda tea", which is then administered to an individual to protect the individual from infection by the SARS-CoV-2 virus or to reduce the viral load in the individual after the individual is exposed to the SARS-CoV-2 virus.

In a specific embodiment, one part (weight or volume) of a combination of Perilla Leaf (or Perilla), Wild Mint Herb, Prunella Spike, Heartleaf Houttuynia, Capillary Wormwood Herb (which is the seedling of Artemisia capillaris of the Asteraceae family) and Licorice (or Glycyrrhiza glabra root and rhizome) is thoroughly mixed with 1 to 1,000 parts (weight or volume) of hot water for a period of time, for example, 20 minutes, and then the whole plant and its rhizome are removed (for example, by filtering) to produce the beverage in the form of a solution. Preferably, the volume percentage or weight percentage of the perilla leaf, the mint, the dried fruit ear of Prunella vulgaris, the Houttuynia cordata, the Artemisia annua and the Licorice in the combination is 40-75%, 10-30%, 0.1-2%, 1-4%, 5-15% and 5-15%, respectively.

In other embodiments, one part (weight or volume) of a plant combination comprising Perilla leaf (or Perilla), Peppermint (Wild Mint Herb), Prunella Spike, Heartleaf Houttuynia, Honeysuckle (or Lonicerae Japonicae Flos), Butterbur and Licorice (or Glycyrrhiza uralensis root and rhizome) is thoroughly mixed with 1 to 1,000 parts (weight or volume) of hot water for about 20 minutes, and then the combination consisting of the whole plant and its rhizome is removed (for example, by filtering) to produce a beverage in the form of a solution. Preferably, in the combination, the volume percentage or weight percentage of the perilla leaf, the mint, the dried fruit ear of Prunella vulgaris, the Houttuynia cordata, the honeysuckle, the coltsfoot and the liquorice are 40-75%, 10-30%, 0.1-2%, 1-4%, 1-10%, 1-10% and 5-15%, respectively.

According to an embodiment of the present disclosure, the soda tea is administered to the individual as a beverage, which can be consumed without restriction for at least 12 days, thereby producing a protective effect (i.e., against SARS-CoV-2) on the individual, and when exposed to the SARS-CoV-2 virus, the viral load in the individual is lower than the viral load in the control group of individuals (individuals who received water instead of soda tea for 12 days).

3. Preparation

A further aspect of the present disclosure is to provide a formulation for use in the present method. In certain embodiments, one or more of the aforementioned agents are formulated into a dosage form that can be used for administration to the individual.

The preparation comprises one or more of the above-mentioned drugs and a pharmaceutically acceptable excipient. In certain embodiments, the drug (e.g., mequinine, nelfinavir, nelfinavir/ivermectin (Nel/Iver) (1:1), cephalanthus capitis/ivermectin (Cepha/Iver) (2:1), cephalanthus capitis/nelfinavir (Cepha/Nel) (2:1)) or extracts of Ganoderma lucidum, Perilla frutescens and Mentha chinensis) is mixed with a pharmaceutically acceptable excipient to form a preparation that can be administered to an individual. In other embodiments, the drug (e.g., mequinine, nelfinavir, Nel/Iver (1:1), Cepha/Iver (2:1), Cepha/Nel (2:1)) or extracts of Ganoderma lucidum, Perilla frutescens and Mentha chinensis) is mixed with a pharmaceutically acceptable excipient to form a preparation for administration to an individual.

The agent (e.g., mequinine, nelfinavir, Nel/Iver (1:1), Cepha/Iver (2:1), Cepha/Nel (2:1)), extracts of Ganoderma lucidum, Perilla frutescens and mint, or extracts of Ganoderma lucidum, Perilla frutescens and wild mint) accounts for about 0.1% to 99% of the total weight of the preparation. In certain embodiments, the agent accounts for at least 1% of the total weight of the preparation. In a specific embodiment, the agent accounts for at least 5% of the total weight of the preparation. In other embodiments, the agent accounts for at least 10% of the total weight of the preparation. In still other embodiments, the agent accounts for at least 25% of the total weight of the preparation.

The formulation is prepared according to acceptable pharmaceutical procedures, such as those described in Remington's Pharmaceutical Sciences, ^17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985). Pharmaceutically acceptable excipients are those that are compatible with the other ingredients in the formulation and are biologically acceptable.

The formulation is manufactured according to its intended route of administration. For example, if the formulation is intended for oral administration, an enteric coating may be applied to the formulation to prevent the agent of the present invention from being degraded in an acidic environment or being degraded before reaching the intestinal tract of the individual. The formulation may further include additional ingredients to assist in transporting the agent of the present invention to its desired target site. In some instances, the agents are encapsulated in a liposome to prevent degradation and to assist in transporting the drugs through the individual's circulatory system and/or to cross cell membranes to reach their desired cellular target sites.

Furthermore, the least soluble drug can be formulated into a liquid preparation together with additional agents, such as a solvating agent, an emulsifying agent and/or a surfactant. Examples of the additional agent include, but are not limited to, cyclodextrin (e.g., α-cyclodextrin or β-cyclodextrin) and non-aqueous solvents, including, but not limited to, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, dimethyl sulfoxide, biocompatible oils (e.g., cottonseed oil, peanut oil, corn oil, wheat germ oil, castor oil, olive oil, sesame oil, glycerol, tetrahydrofuran, polyethylene glycol (PEG), fatty acid esters of sorbitan, and mixtures thereof).

The amount of the drug or herbal extract in the formulation varies with the route of administration. For example, a formulation for acute care will contain a larger amount of the drug than a formulation for clinical treatment. Similarly, a parenteral formulation will contain a smaller amount of the drug than a formulation for oral ingestion. Formulations suitable for other routes of administration are also within the scope of the present disclosure.

The dosage form may be a liquid, solution, suspension, emulsion, elixir, syrup, tablet, lozenge, granules, powder, capsule, cachet, pill, ampoule, suppository, vaginal suppository pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

3.1 Preparations for oral ingestion

The medicaments of the present disclosure may be formulated as compositions suitable for oral ingestion. Formulations suitable for oral administration (e.g., by ingestion) may be present as discrete units, such as capsules, cachets, or tablets, each of which contains a predetermined amount of active compound; such as powders or granules; such as solutions or suspensions in aqueous or non-aqueous liquids; or such as oil-in-water liquid emulsions or water-in-oil liquid emulsions; such as boluses; such as licks; or such as pastes.

Tablets may be manufactured by conventional means, such as pressing or molding, with one or more auxiliary ingredients as desired. Compressed tablets may be prepared by extruding in a suitable machine the active compound in a free-flowing form such as powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, gum arabic, sorbitol, tragacanth, hydroxypropyl methylcellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silicon dioxide); disintegrants (e.g., sodium starch glycolate, cross-linked polyvinyl pyrrolidone, povidone), cross-linked sodium carboxymethyl cellulose); a surfactant or dispersant or wetting agent (e.g. sodium lauryl sulfate); and a preservative (e.g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid). Moulded tablets can be prepared by moulding in a suitable machine with the powdered compound moistened with an inert liquid diluent. Tablets may be film-coated or scored as desired and may be formulated to release the active compound therein in a slow or controlled manner, for example with varying proportions of hydroxypropylmethylcellulose to provide the desired release profile. Tablets may optionally have an enteric film coating for release in parts of the intestine other than the stomach.

3.2 Preparations for parenteral administration

Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous, and intradermal) include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain antioxidants, buffers, preservatives, stabilizers, bacteriostats, and solutes which give the formulation the same osmotic pressure as the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which may contain suspending and thickening agents, and liposomes or other particulate systems designed to direct the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for such formulations include sodium chloride injection, Ringer's Solution or Lactated Ringer's Injection. The formulation may be in sealed containers, such as ampoules and vials, in unit doses or multi-dose quantities and may be stored in lyophilized conditions requiring only the addition of a sterile liquid carrier, such as water for injection, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. The formulation may be in the form of liposomes or other nanoparticle or microparticle systems designed to direct the active compound to blood components or one or more organs.

3.3 Film-through Agent

Transmucosal preparations are suitable for topical and transmucosal use and include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, suspensions, skin patches and the like. The patches include esservoir type and matrix type skin patches and can be attached to the skin for a period of time to allow the active ingredient to be absorbed into the body by the individual.

For topical administration, a variety of dermatologically acceptable inert excipients known in the art may be used. Typical inert excipients may be, for example, water, ethanol, polyvinylpyrrolidone, propylene glycol, mineral oils, stearyl alcohol, and gel-forming substances. All of the above dosage forms and excipients are known in the pharmaceutical art. The choice of dosage form is not critical to the efficacy of the compositions disclosed herein.

For transmucosal administration, the compounds of the present disclosure may also be formulated into a variety of dosage forms for mucosal administration, such as buccal and/or sublingual drug dosage units for delivery of the drug through the oral mucosa. A variety of biodegradable polymeric excipients may be used that are pharmaceutically acceptable, provide suitable adhesion and the desired drug release profile, and are compatible with the active agent being administered and with any other ingredients that may be present in the buccal and/or sublingual drug dosage unit. In general, polymer excipients include a hydrophilic polymer that adheres to the moist surface of the oral mucosa. Examples of polymeric excipients include, but are not limited to, acrylic acid polymers and copolymers; hydrolyzed polyvinyl alcohol; polyethylene oxides; polyacrylates; vinyl polymers and copolymers; polyvinyl pyrrolidone; dextran; guar gum; pectins; starch; and cellulosic polymers. In certain embodiments, the medicaments of the present disclosure may also be formulated for nasal or oral inhalation, comprising a solution or suspension of the medicaments of the present disclosure and a pharmaceutical excipient generally acceptable for such purpose, inhaled via an inhalant aerosol spray. Alternatively, the medicament of the present disclosure in powder form can be administered through an inhaler that allows the powder to directly contact the lungs. For such preparations, if necessary, a pharmaceutically acceptable carrier such as an isotonic agent, a preservative, a dispersant or a stabilizer can be added. Further, if necessary, such preparations can be sterilized by filtration, or by applying heat or light.

The present invention is illustrated by the following examples.

Embodiment

Materials and methods

Cell culture

African green monkey kidney cells Vero _E6 were cultured in minimal essential medium (Eagle) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM pyruvate, 0.1 mM non-essential amino acids, and 1.5 g/L sodium bicarbonate in Earle's balanced salt solution (Earle's BSS) at 37°C and 5% CO2.

Compound Library

1. Known compounds

In this study, 2,855 unique compounds that have been approved and clinically evaluated were screened for antiviral activity. These compounds were dissolved in dimethyl sulfoxide (DMSO) (concentration 1 mM) and transferred to 96-well microtiter plates for antiviral activity assays against SARS-CoV-2-mediated cytopathic effects.

2. Nelfinavir derivatives (Bn) Preparation

Nelfinavir derivatives (Bn) were synthesized according to the steps described in Scheme 1.

Scheme 1. Synthesis of Nelfinavir Derivatives (Bn)

Nefinavir derivatives (Bn) 1H NMR (600 MHZ, CD3OD): δ 7.42 (d, J =7.7 Hz, 2H, Ar-H), 7.22-7.17 (m, 4H, Ar-H), 7.15-7.08 (m, 4H, Ar-H), 6.92 (t, J = 7.7 Hz, 1H, Ar-H), 6.81 (d, J = 7.5 Hz, 1H, Ar-H), 6.72 (d, J = 7.4 Hz, 1H, Ar-H), 4.32 (m, 1H), 4.09 (dd, J = 22.0, 14.9 Hz, 2H), 3.96 (m, 1H), 3.45 (dd, J = 13.7, 4.2 Hz, 1H), 3.18 (dd, J = 13.7, 10.3 Hz, 1H), 2.98 (dd, J = 11.7, 2.4 Hz, 1H), 2.64 (dd, J = 10.9, 3.0 Hz, 1H), 2.56 (dd, J = 12.7, 8.6 Hz, 1H), 2.19 (s, 3H, Ar-CH3), 2.13-2.05 (m, 2H), 1.96 (q, J = 12.6 Hz, 1H), 1.72-1.62 (m, 2H), 1.58-1.51 (m, 2H), 1.49-1.39 (m,2H), 1.33-1.20 (m, 4H), 1.17-1.08 (m, 1H); 13C NMR (150 MHz, CD3OD): δ = 177.0, 173.8, 157.3, 140.2, 137.9, 131.2, 130.3, 129.8, 128.9, 128.5, 127.6, 127.4, 123.7, 119.6, 117.1, 71.3, 70.9, 60.3, 60.2, 54.2, 43.9, 37.6, 35.4, 32.3, 31.8, 27.6, 27.3, 22.2, 13.5; high resolution mass spectrometry (high resolution mass spectrometry, HRMS) (electrospray ionization-time-of-flight (ESI-TOF)) calculated value for C35H44N3O4S [M+H]+: 602.3053, experimental value 602.3047.

3. Herbal Extracts

About 200 herbs and supplements were collected by the following procedure. 1 gram of ground herbs was mixed with water (20 mL) for about 4 hours, and the mixture was centrifuged to collect the supernatant. Then, 100 μL of the supernatant was mixed with 100 μL of Dulbecco’s minimum essential medium (DMEM) and used for subsequent assays (e.g., cytotoxicity assay).

4. Ganoderma lucidum Differentiation of Lingzhi 3

The raw material of sesame protein was obtained from Wyntek and fractionated according to the method described by previous researchers (S. F. Kiao et al., Proc. Natl. Acad. Sci. U. S. A. 110, 13809-13814 (2013)).

5. Su Huo Tea Preparation

According to the ratio disclosed in Table 1, 11 grams of herbs were extracted with 350 mL of hot water for 20 minutes to prepare two different formulations of Su Huo tea.

surface 1 Soda Tea Agent   Soda Tea #1 Soda Tea #2 Weight (g) Weight percentage (%) Weight (g) Weight percentage (%) Perilla leaves 6.3 57.3 6.6 60.0 Mint 2.2 20.0 1.8 16.4 Prunella vulgaris dried fruit spike 0.2 1.8 0.4 3.6 Houttuynia cordata 0.3 2.7 0.4 3.6 Licorice 1.0 9.1 1.0 9.1 Artemisia annua 1.0 9.1 - - Honeysuckle - - 0.4 3.6 Coltsfoot - - 0.4 3.6

Screening Antibody SARS-CoV-2 Compound

Any selected compound from the above compound library was prepared into 1 mM solution in DMSO and transferred to a 96-well plate. Each compound was diluted to a final concentration of 10, 3.3 or 1 μM (or lower concentration for potent compounds) in DMEM (2% FBS) for screening. Herbal extract (1.0 g/20 mL H ₂ O) and Ganoderma lucidum extract (0.25 mg/mL) were serially diluted 2-fold in DMEM (2% FBS) and used for screening. Vero E6 cells (1 × 10 ⁴ per well) were cultured in DMEM supplemented with 10% FBS. After 1 day of culture, when the cells reached 80-90% confluence, the medium was removed. 100 μL of DMEM solution containing the test compound and 2% FBS was added to each well of triplicate. The cells were cultured with SARS-CoV-2 (hCoV-19/Taiwan/NTU04/2020, isolated from the throat swab of a 39-year-old male confirmed patient in Taiwan) at a dose of 100 TCID50 (50% tissue infectious dose) per well in a 37°C, 5% CO ₂ incubator; the cytopathic morphology of the cells was examined at 72 hours and 120 hours, respectively, using an inverted microscope.

Cytotoxicity studies

The cytotoxicity of the test compounds to Vero E6 was determined using the CCK-8 cell counting kit (Dojindo Laboratory) according to the manufacturer's experimental procedures. Briefly, after 24 hours of incubation with different concentrations of the designated compounds, 10 μL of CCK-8 reagent was added to each well of the 96-well plate and placed in a CO ₂ incubator for 1 to 4 hours. The absorbance was measured at 450 nm using a spectrophotometer (SpectraMax M2, Meigu Molecular Instruments). The data are expressed as a percentage of the control tissue cells incubated without the compound (taken as 100%).

3CL Protease assay

Nsp5 was reacted with luciferin-AVLQSGFRK(QXL520)-NH2 in 20 mM Tris(hydroxymethyl)aminomethane (Tris)-HCl (pH 7.0) at 30°C for 30 min in the presence or absence of designated inhibitors. The reaction was monitored by disk analyzer with fluorescence (incident 480/exit 530) and the initial velocity was calculated. The curve was fitted using Prism software (Graphpad).

Animal research

Female Syrian hamsters aged 5-7 years were used in this study. On day 0, hamsters were tested for simulated infection by intranasal instillation of 100 μL phosphate buffered saline (PBS) or 100 μL PBS containing 1x10 ⁵ TCID50 of SARS-CoV-2 under intraperitoneal anesthesia with Zoletil 50 (5 mg/kg). Body weight and clinical signs of hamsters were monitored daily during the study to measure disease progression. The treatment groups were orally administered with either mequinine, nelfinavir, salinomycin or thioguanine (30 mg/kg/day), perilla leaf (200 mg/kg/day), mint (200 mg/kg/day), or extract of Ganoderma lucidum (30 mg/kg/day) twice a day, while the control group was given the same volume of drinking water. On the third day after infection, all hamsters were sacrificed and lung tissues were collected from each hamster to measure the live viral load by TCID50 assay in Vero E6 cells.

Embodiment 1 Identify the antibody SARS-CoV-2 Compounds and their properties

In this embodiment, a compound library (including herbal extracts) was constructed according to the steps described in the "Materials and Methods" section, and a cell-based assay was used to screen compounds with the ability to inhibit SARS-CoV-2 from the established compound library.

1.1 Drug Screening

The antiviral activity of each candidate compound at 10 μM, 3.3 μM and 1 μM (or 1 nM to 100 nM for potent compounds) was evaluated by the degree of cytopathic effect (CPE) when Vero E6 cells were infected with one of the strains of SARS-CoV-2. Of the 2,855 compounds tested, 15 were found to be protective against Vero E6 cells (the activity of these compounds was assessed on days 3 and 5, respectively). The lowest concentrations at which these compounds showed protective effects are summarized in Table 2 , which presents the drug concentration required to inhibit 50% of viral replication (IC ₅₀ ) and the cytotoxic concentration that can kill 50% of cells (CC ₅₀ ).

Table 2 In vitro anti-SARS-CoV-2 activity results Name Drug Class Drug Indications CPE ( M) ^a IC ₅₀ ( M) ^b CC ₅₀ ( M) ^c Day 3 Day 5 Nefinavir ( 1) Antiviral agents Anti-HIV infection 5.0 10.0 3.3 12.3 Paksepiwe ( 2) Antiviral agents Anti-HCV infection 10.0 10.0 50.1 >10 Thioguanine (3) Antineoplastic Agents Anti-cancer 1.25 2.5 1.7 25.4 Celastrin (4) Antineoplastic Agents Anti-cancer 3.75 7.5 2.8 12.9 Ipecacine( 5) Antiprotozoan Anti-amoeba 5.0 10.0 4.0e-4 >10 Ivermectin (6) Insecticide Antiparasitic 2.5 ^-d 4.1 13.2 Moxidectin (7) Insecticide Against Onchocerciasis Infection 5.0 10.0 3.1 6.9 Melquinin ( 8) Anti-malaria agent Prevention and treatment of malaria 5.0 10.0 3.2 >10 Ivacaftor( 9) CFTR ^e enhancers Cystic fibrosis 5.0 ^NI 3.7 12.9 Azelnidipine (10) Calcium channel blockers Anti-hypertension 5.0 ^NI 5.3 12.9 Penfluridol ( 11) First generation antipsychotic drugs Schizophrenia 5.0 10.0 2.4 12.9 Dronedarone (12) Ion channel blockers Irregular heart rhythm 7.5 ^NI 4.5 12.1 Thalinomycin (13) Polyether antibiotics Prevention of coccidiosis in animals 0.039 0.156 4.8e-4 13.1 Monensin (14) Polyether antibiotics Prevention of Coccidiosis in Animals 0.117 2.5 6.4 6.6 Maduramicin ( 15) Polyether antibiotics Prevention of Coccidiosis in Animals 0.039 0.117 1.3 3.4 a Minimum dose that exhibits a protective effect at 72 hours (day 3) and 120 hours (day 5) after virus inoculation ^{; b} 100 plaque forming units (PFU) of SARS-CoV-2 (multiplicity of infection (MOI) of 0.025) in Vero E6 cells; ^c Vero E6 cells; ^d Cytotoxicity was observed; ^e Cystic fibrosis transmembrane conductance regulator; ^f No inhibitory effect.

The results showed that the concentrations of nelfinavir, nelfinavir mesylate and nelfinavir derivative (Bn) that inhibited 50% 3CL main protease activity (IC ₅₀ ) were 56±15.8 μM, 33.4±6.2 μM and 35±11 μM, respectively.

The compound library used for this screening also included several other clinically approved HIV protease inhibitors, including indinavir sulfate, saquinavir mesylate, atazanavir, ritonavir, darunavir, amprenavir and lopinavir, as well as the HCV protease inhibitors daclatasvir, danoprevir and telaprevir, however, none of these compounds had an inhibitory effect on SARS-CoV-2 at the screening concentrations selected.

In addition, several known viral protease inhibitors, including acyclovir, famciclovir, penciclovir, ribavirin, cidofovir, and entecavir, and reverse transcriptase inhibitors, including zalcitabine, nevirapine, efavirenz, abacavir sulfate, tenofovir disoproxil fumarate, adefovir disoproxil fumarate, and sirolimus, are also available. dipivoxyl, delavirdine, and telbivudine were also screened by cell-based assays, but no active compounds were identified (data not shown).

1.2 Combined use

This embodiment also determines the effects of nelfinavir (Nel), cephalaenopsis (Cepha) and ivermectin (Iver) used alone or in combination at different doses against SARS-CoV-2. The synergistic or antagonistic interactions of the combination are analyzed by the median-effect principle. The combination index (CI) is calculated according to the formula (I) described below. The values of <1, 1 and >1 represent synergism, additive effect and antagonism, respectively. D1 and D2 represent the half-maximal effective concentration (EC50) of the antiviral effect of the individual drugs after combination, and (Dx)1 and (Dx)2 represent the EC50 of the individual drugs administered alone. (I)

The three drugs including Nel, Cepha and Iver were tested in combination at molar ratios of Nel/Iver = 1:1, Cepha/Iver = 2:1 and Cepha/Nel = 2:1, respectively. The results are summarized in Table 3 .

The data in Table 3 show that the three combinations above all exhibited synergistic inhibitory effects against the delta strain, and the Cepha/Iver group exhibited synergistic inhibitory effects against the alpha strain. In summary, the results in Table 3 show that the combination of these compounds is suitable for further evaluation in animal models.

Table 3 In vitro compatibility index of three compound combinations against SARS-CoV-2 Concentration ratio Drugs EC ₅₀ (uM) Combination Index (CI) NTU49 (B.1.1.7) NTU92 (B.1.617) NTU49 (B.1.1.7) NTU92 (B.1.617) Nel+Iver 1:1 Nel 1.15 1.25 1.20 0.85 Iver 1.15 1.25 Cepha+Iver 2:1 Cepha 1.94 2.72 0.75 0.95 Iver 0.97 1.36 Cepha+Nel 2:1 Cepha 2.53 2.64 1.14 0.88 Nel 1.26 1.32

1.3 Screening Traditional Chinese Herbal Medicines

In this embodiment, the antiviral activity of some well-known traditional Chinese herbal medicines was tested using a cell experiment. The herbal medicine extracts were prepared by extracting 1 gram of the herbal medicine with 20 ml of water at room temperature and then diluting it with a buffer solution. The test results are shown in Figure 1 .

The results showed that water extracts of herbs from the Lamiaceae family (perilla), the Menthaceae family (mint), the Asteraceae family (dandelion, coltsfoot and chrysanthemum), the Theaceae family (tea tree), the Lamiaceae family (Prunella vulgaris, basil, salvia officinalis, Nephrolepis chinensis, rosemary), the Leguminosae family (peanut, bean), and the Sapindaceae family (longan, litchi) could reduce the CPE of SARS-CoV-2 in Vero E6 cells when diluted 16-960 times. These herbs contain flavonoids (such as cinnamomea), flavan-3-ols (such as catechins and epigallocatechin gallate), and caffeic acid derivatives (such as kaftanic acid, methyl rosmarinic acid or chlorogenic acid), however, whether these compounds are associated with antiviral activity remains to be studied. Monoterpenes (1,8-cineole and camphor from basil leaves), diterpenes (carnosic acid and pelargonol), and triterpenes (such as ursolic acid) have been reported to block viral entry and replication. However, the exact mechanism by which herbal medicines inhibit SARS-CoV-2 infection remains unknown.

In addition, several fractions of L-fucose-containing polysaccharides from Ganoderma lucidum were tested in a cell-based anti-SARS-CoV-2 activity assay, and it was found that L-fucose-containing fraction 3 (RF3) of Ganoderma lucidum polysaccharide exhibited excellent cell protection efficiency at a concentration of 2 μg/mL, and it still retained antiviral activity and was non-cytotoxic after being diluted 1,280 times (Figure 1 ) .

Embodiment 2. Embodiment 1 Compounds identified in the herbal extracts or herbal extracts are effective against SARS-CoV-2 Effect

In this example, the drugs or herbal extracts identified by cell tests in Example 1 were tested in animals according to the steps described in the "Materials and Methods" section. In particular, the viral eradication efficiency of four drugs including mequinine, nelfinavir, thalidomycin and thioguanine, and three active herbal extracts including RF3 , perilla and mint were evaluated in female Syrian hamsters. The results are shown in Figure 2 .

Compared with the control group (administered water), salinomycin caused a higher degree of weight loss, while the other drugs and extracts did not significantly reduce the weight of the animals. Therefore, except for salinomycin, the selected drugs and extracts had no acute toxicity issues. In the animal experiment, hamsters were infected with SARS-CoV-2 by intranasal infection on day 0, followed by three days of oral administration of drugs (at a dose of 30 mg/kg/day) and extracts (200 mg/kg/day). The hamsters were sacrificed and their lungs were collected to test the viral load. Unexpectedly, the two compounds that showed the best activity in the cell-based assay (i.e., thioguanine and thalidomycin) did not show significant viral eradication in the animal experiment; this unexpected result may be related to the high hydrophilicity and low oral bioavailability of these compounds. However, compared with the control group, the extracts of mequinine and mint significantly reduced the viral load (P = 0.005, Figure 2 ), while the extracts of nelfinavir , RF3, and perilla also showed good antiviral effects (P = 0.03 for the control group, Figure 2 ). In in vivo tests, mequinine and nelfinavir were confirmed as potential drug-repurposing agents, while extracts of mint, perilla and RF3 were potential herbal candidates against SARS-CoV-2.

Embodiment 3 Su Huo Tea In vivo SARS-CoV-2 Effect

In this example, hamsters that had been exposed to the SARS-CoV-2 virus were given the Su Huo tea prepared according to the "Materials and Methods" section as their drinking water, and the changes in body weight and lung virus titer were measured. The results are shown in Figure 3 and Table 4 .

After giving hamsters sohuo tea as drinking water, the virus titer in the lungs decreased to below the detection limit in both the virus clearance ( Figure 3B ) and protection experiments (Figure 3C ) , and no significant weight loss was caused ( Figure 3A ).

The efficacy of two Suhuo Tea preparations (Preparation #1 and Preparation #2) against SARS-CoV-2 was compared, where mice infected with SARS-CoV-2 were treated with Preparation #1 and #2 for 6 days, and then the viral titer in the lungs was measured. The results are summarized in Table 4. The results showed that Suhuo Tea Preparation #2 conferred a better protective effect than Preparation #1.

Table 4 The protective effect of Suhuo tea against SARS-CoV-2 Treatment No. SARS-CoV-2 titer in lungs (TCID50/ml) water 1 5.62x10 ⁶ 5.55x10 ⁶ 2 3.98x10 ⁶ 3 ^2.51.x106 4 5.62x10 ⁶ 5 1.00x10 ⁷ Soda Tea Preparation #1 1 2.51x10 ⁵ 5.38x10 ⁵ 2 1.00x10 ⁶ 3 5.62x10 ⁵ 4 5.62x10 ⁵ 5 3.16x10 ⁵ Soda Tea Preparation #2 1 3.16x10 ⁵ 3.82x10 ⁵ 2 5.62x10 ⁴ 3 3.16x10 ⁵ 4 3.98x10 ⁵ 5 3.16x10 ⁵

The above embodiments are presented only as examples, and a person skilled in the art to which the present invention belongs may make various modifications thereto. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various embodiments of the present disclosure have been described above with a certain degree of specificity, or with reference to one or more individual embodiments, a person skilled in the art to which the present invention belongs may make many modifications to the disclosed embodiments without departing from the spirit and scope of the present disclosure.

To make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying figures are described as follows: FIG. 1 Evaluation of the cytoprotective effect of Chinese herbal medicines under continuous dilution. The anti-SARS-CoV-2 infection effect of the selected Chinese herbal medicines in water extracts (1.0 g/20 mL H ₂ O) and RF3 (0.25 mg/mL) dissolved in water is presented as Log ₂ (dilution multiple); and FIG . 2 In vivo anti- SARS -CoV-2 assay in female Syrian hamsters . FIG . 2A Body weight change after 3 days of treatment, n=5 for the test group and n =6 for the control group. FIG. 2B Virus clearance effect of drugs and extracts. Hamsters were infected with SARS-CoV-2 by intranasal instillation on day 0 and treated with drug and extract orally twice a day (30 mg/Kg/d for drug and 200 mg/Kg/d for extract) for 3 consecutive days. After 3 days, lungs were collected to measure viral load (n = 5), *P <0.05; **P < 0.005. Fig . 3 Evaluation of the anti- SARS-CoV-2 effect of Suhuo Tea ( Formulation #1) in mice . Fig. 3A Body weight change after 6 days of treatment. Fig. 3B Virus elimination effect of Suhuo Tea during 3 days of treatment. Fig . 3C Protection against viruses after 12 hours of exposure to Suhuo Tea.

According to conventional operation methods, various features and components in the figures are not drawn to scale, and the drawing method is to present the specific features and components related to the present invention in the best way. In addition, the same or similar component symbols are used to refer to similar components/parts between different figures.

Claims (4)

A use of a medicament for manufacturing a drug for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in an individual, wherein the medicament is a mint extract, a perilla extract, a soda tea, or a ganoderma lucidum polysaccharide fraction 3 (RF3) of Ganoderma lucidum, wherein the soda tea is a water extract of a combination of perilla leaves, mint, dried fruit ears of Prunella vulgaris, Houttuynia cordata, Artemisia annua, and licorice, or a water extract of a combination of perilla leaves, mint, dried fruit ears of Prunella vulgaris, Houttuynia cordata, honeysuckle, coltsfoot, and licorice. The use as described in claim 1, wherein the perilla leaf, the mint, the dried fruit spike of Prunella vulgaris, the Houttuynia cordata, the Artemisia annua and the licorice are present in the combination in an amount of 40-75%, 10-30%, 0.1-2%, 1-4%, 5-15% and 5-15% by weight or volume respectively. The use as described in claim 1, wherein the perilla leaf, the mint, the dried fruit spike of Prunella vulgaris, the Houttuynia cordata, the honeysuckle, the coltsfoot and the licorice are present in the combination in an amount of 40-75%, 10-30%, 0.1-2%, 1-4%, 1-10%, 1-10% and 5-15% by weight or volume respectively. The use as described in claim 1, wherein the individual is a human.

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