Herbal compositions, methods of making same and methods of administering same for the prevention or treatment of viral infections

Abstract

The present disclosure provides an herbal composition comprising extracts derived from herbal materials, the herbal materials comprising at least one of mugwort, wormwood, fish needle grass, dwarf lilyturf, gotu kola, platycodon, licorice, perilla, and chrysanthemum. The present disclosure also provides a method for preparing the herbal composition and a method for administering an effective amount of the herbal composition to a subject in need thereof to prevent or treat viral infection.

Description

Technical Field

[0001] This disclosure relates to an herbal composition, and more particularly to an herbal composition for the prevention or treatment of viral infections. Background Technology

[0002] Viral infections have been identified as, and remain a serious problem for both animals and humans. Coronaviruses (CoVs) are a large family of viruses that can cause illnesses ranging from the common cold to more severe diseases. For example, infections with human coronavirus strains, CoV-229E, CoV-0C43, CoV-NL63, and CoV-HKU1, often result in mild, self-limiting upper respiratory tract infections, such as the common cold, with symptoms like runny nose, sneezing, headache, cough, sore throat, and fever. Other infections can lead to more severe illnesses, such as Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which cause severe acute respiratory syndrome, kidney failure, and death, and have triggered global public health emergencies. In particular, COVID-19, caused by SARS-CoV-2, has infected more than 194 million people globally and caused more than 4,160,000 deaths.

[0003] To date, the fastest way to save lives is to reuse existing FDA-approved drugs originally intended for other conditions related to COVID-19. However, most antiviral drugs cause serious side effects such as nausea, diarrhea, dizziness, and fever, and there are still no available or proven specific antiviral treatments effective in treating or preventing coronavirus infection in trial participants.

[0004] Therefore, there is an unmet need to provide effective and safe therapies to prevent or treat coronavirus infection. Summary of the Invention

[0005] In view of this, the present disclosure provides a herbal composition that can interfere with the interaction between the coronavirus spike (S) protein and ACE2 on the surface of host cells and suppress the expression of proteins necessary for coronavirus entry and replication in the host, thereby protecting subjects from viral infection.

[0006] In at least one embodiment of this disclosure, the herbal composition comprises an extract derived from a herbal raw material and a pharmaceutically acceptable carrier, wherein the herbal raw material comprises at least one of Ohwia caudata and Anisomelesindica (L.) O. Ktze. In some embodiments, the Ohwia caudata is Ohwia root, Ohwia leaves, or a combination thereof.

[0007] In at least one embodiment of this disclosure, the herbal composition comprises an extract derived from herbal raw materials, the herbal raw materials comprising at least one of jasmine and needlegrass, and at least one of Artemisia argyi, Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorus, Glycyrrhiza uralensis, Perilla fructose, and Chrysanthemum. In some embodiments, the herbal composition comprises an extract derived from herbal raw materials, the herbal raw materials comprising at least one of jasmine and needlegrass, and Artemisia argyi, Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorus, Glycyrrhiza uralensis, Perilla fructose, and Chrysanthemum. In some embodiments, the extract derived from the herbal raw materials is an aqueous extract or an alcoholic extract.

[0008] In at least one embodiment of this disclosure, the herbal composition comprises an extract derived from herbal raw materials, which, based on their total weight, comprise at least one of jasmine and sedge by weight of 18% to 25%, and at least one of wormwood, 10% to 17% ophiopogon japonicus, 10% to 17% houttuynia cordata, 10% to 17% platycodon grandiflorus, 4% to 11% licorice, 4% to 11% perilla frutescens, and 0.4% to 11% chrysanthemum by weight.

[0009] In at least one embodiment of this disclosure, the herbal composition is prepared by a method comprising: providing the herbal raw material as described above; extracting the herbal raw material with an extractant to obtain a crude extract, wherein the extractant comprises water, alcohol, or a combination thereof; and removing solids from the crude extract to obtain a liquid portion.

[0010] In at least one embodiment of this disclosure, the method for preparing the herbal composition further comprises grinding the herbal raw material into powder or fragments.

[0011] In at least one embodiment of this disclosure, extracting the herbal raw material comprises boiling the herbal raw material in the extract for at least 5 minutes, for example, 5 minutes to 2 hours, and / or immersing the herbal raw material in the extract for at least 10 minutes, for example, 10 minutes to 1 hour, wherein the temperature of the extract is below its boiling point. In some embodiments, the weight ratio of the herbal raw material to the extract is 2:1 to 30:1, for example, 2:1, 3:1, 5:1, 8:1, 10:1, 15:1, 20:1, 25:1, and 30:1.

[0012] In at least one embodiment of this disclosure, the method for preparing the herbal composition further comprises concentrating the liquid portion to obtain a concentrated extract.

[0013] In at least one embodiment of this disclosure, a method is provided for preventing or treating viral infection in a subject in need. The method comprises administering an effective amount of at least one of the above-described herbal compositions to the subject.

[0014] In at least one embodiment of this disclosure, the viral infection is caused by a coronavirus. In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mouse hepatitis virus (MHV), or porcine epidemic diarrhea virus (PEDV). In some embodiments, the coronavirus is a variant of SARS-CoV-2, such as the D614G mutant, the B.1.1.7(α) mutant, the B.1.351(β) mutant, and the P1 mutant.

[0015] In at least one embodiment of this disclosure, the extract derived from the herbal raw material in the herbal composition is administered to the subject at an effective amount of about 25 mg / kg / day to about 2,500 mg / kg / day, for example, about 30 mg / kg / day to about 1,000 mg / kg / day and about 50 mg / kg / day to about 500 mg / kg / day.

[0016] In this disclosure, the herbal composition provided acts as an antiviral agent, inhibiting viral replication and reducing the number of viruses in host cells. Furthermore, the herbal composition provided in this disclosure is safe and addresses the side effect problems of prior art. Therefore, this disclosure provides an effective strategy for combating viral infection, which can be used to control coronavirus outbreaks. Attached Figure Description

[0017] This disclosure can be more fully understood by reading the following description of the embodiments and referring to the accompanying drawings.

[0018] Figures 1A to 1G Bar graphs showing the binding activity of viral spike proteins to the ACE2 receptor inhibited by the herbal compositions disclosed herein. Ctrl.: No treatment group; Ctrl.1: Arbidol 20 mg / mL; Ctrl.2: Compound; Ctrl.3: Lianhua Qingwen capsule; Ctrl.4: Olive leaf extract; Ctrl.5: Elderberry extract; Ctrl.6: Traditional Chinese medicine formula derived from Hualien Tzu Chi Hospital; Exp.1 to Exp.6: Herbal compositions disclosed herein.

[0019] Figures 2A to 2C Bar graphs showing the cell viability of CTX, H9c2, and HFL-1 cells treated with the herbal composition disclosed herein are displayed separately. Ctrl.: No treatment group. *: p-value < 0.05, **: p-value < 0.01.

[0020] Figures 3A to 3D A bar graph showing blood parameters of mice treated with the herbal composition disclosed herein. Figure 3A The levels of creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate aminotransferase (GOT), and alanine aminotransferase (GPT) in the blood are shown. Figures 3B to 3D The levels of creatinine (CRE), total bilirubin (T-Bil), and glucose (Glu) in the blood are shown separately. Ctrl.: No treatment group; Exp. 1: The herbal composition disclosed herein.

[0021] Figure 4A and Figure 4B The performance of transmembrane serine protease 2 (TRPMSS2) as analyzed by Western blotting is shown. Ctrl.: No treatment group; Ctrl.1: Arbidol 20 mg / mL; Ctrl.2: Compound preparation; Ctrl.3: Lianhua Qingwen capsules; Exp.1: Herbal composition disclosed herein; L: Low dose, 50 μg / mL; H: High dose, 150 μg / mL.

[0022] Figure 5A and Figure 5B The image shows the performance of FK506-binding protein 51 (FKBP51) as analyzed by Western blotting. Ctrl.: No treatment group; Ctrl.2: Compound preparation; Ctrl.3: Lianhua Qingwen capsules; Exp.1: Herbal composition disclosed herein; L: Low dose, 50 μg / mL; H: High dose, 150 μg / mL.

[0023] Figure 6 and Figure 7 Bar graphs showing the activities of 3CL protease and RNA-dependent RNA polymerase (RdRp) inhibited by the herbal composition (Exp.1) disclosed herein are shown. *: p value < 0.05, **: p value < 0.01, **: p value < 0.001.

[0024] Figure 8A and Figure 8B The inhibitory efficacy of the herbal compositions disclosed herein against wild-type, D614G mutant, B.1.1.7 mutant, and B.1.13513 mutant infections of SARS-CoV-2 in Caco-2 and Calu-3 cells is shown, respectively. Ctrl: No treatment group; Exp.7 to Exp.9: The herbal compositions disclosed herein.

[0025] Figures 9A to 9D This demonstrates the effect of the herbal composition disclosed herein on wild-type SARS-CoV-2 in mice. Figure 9A ), D614G mutant ( Figure 9B B.1.1.7 mutant ( Figure 9C ) and B.1.1351 mutant ( Figure 9D The inhibitory effect of the herbal composition on infection. Ctrl.: No treatment group; Ctrl.7: Viral treatment only; Exp.1-L: Low dose of the herbal composition disclosed herein; Exp.1-H: High dose of the herbal composition disclosed herein.

[0026] Figure 10A The inhibitory efficacy of herbal compositions according to different embodiments of this disclosure against SARS-CoV-2 B.1.1.7 mutant infection in Caco-2 cells is shown. Ctrl.: No treatment group; Exp.1, Exp.10, and Exp.11: Herbal compositions of this disclosure.

[0027] Figure 10B The inhibitory efficacy of herbal compositions according to different embodiments of this disclosure against SARS-CoV P1 mutant infection in mice is shown. Ctrl: No treatment group; Ctrl.7: Virus treatment only; Exp.1 and Exp.10: Herbal compositions of this disclosure. Detailed Implementation

[0028] The technical solutions described in the embodiments of this disclosure will be described more clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of this disclosure and are not intended to be limiting. This disclosure can also be implemented or applied as described in different embodiments. Other embodiments obtained by those skilled in the art without creation are all within the scope of this disclosure.

[0029] It should be further noted that, as used in this disclosure, the singular forms “a,” “an,” and “the” include plural referents unless explicitly and unambiguously limited to a single referent. Unless the context clearly indicates otherwise, the term “or” is used interchangeably with the term “and / or.”

[0030] As used herein, the terms "comprising" or "comprises" are used to mean including the compositions, methods and their respective components contained in this disclosure, but are open to including unspecified elements or steps, whether or not necessary.

[0031] This disclosure relates to herbal compositions, methods for preparing said herbal compositions, and methods for using said herbal compositions in subjects in need of preventing or treating viral infections.

[0032] In at least one embodiment, the viral infection treated by the method disclosed herein may be caused by a coronavirus (CoV).

[0033] The structural proteins of CoV include the nucleocapsid (N), small envelope (E), matrix (M), and trimeric protrusion (S) glycoprotein, which are essential for virion assembly and the completion of the viral life cycle during infection. In some embodiments, the methods disclosed herein for preventing or treating viral infection comprise administering an herbal composition to a subject in need. This herbal composition blocks the interaction between the coronavirus S protein and the angiotensin-converting enzyme 2 (ACE2) receptor, and suppresses the expression of proteins necessary for coronavirus entry and / or replication in the host, thereby affecting the risk of viral infection or exacerbating disease progression. Therefore, the herbal compositions disclosed herein may possess antiviral capabilities and can be used for the effective prevention or treatment of viral infection.

[0034] As used herein, the terms "preventing" or "prevention" refer to measures to prevent or avoid a disease or its symptoms or conditions, including but not limited to applying or administering one or more active agents to a subject who has not been diagnosed with the disease or its symptoms or conditions but may be susceptible to or prone to the disease. The preventive measures disclosed herein are provided to avoid, prevent, or delay the occurrence of a disease or its symptoms or conditions.

[0035] As used herein, the term "treating" or "treatment" means achieving a desired pharmacological and / or physiological effect, such as inhibiting viral entry and / or replication in a host. Such effects may be preventative in the complete or partial prevention of the disease or its symptoms or condition, or therapeutic in the complete or partial cure, relief, mitigation, remedy, or improvement of the disease or its adverse effects attributable to the disease or its symptoms or condition.

[0036] As used herein, the terms "patient" and "subject" are used interchangeably. The term "subject" refers to a human or animal. Examples of subjects include, but are not limited to, humans, monkeys, mice, rats, marmots, ferrets, rabbits, hamsters, cattle, horses, pigs, deer, dogs, cats, foxes, wolves, chickens, emus, ostriches, and fish. In some embodiments disclosed herein, the subject is a mammal, such as a primate like a human.

[0037] As used herein, the phrase "effective dose" refers to the amount of active agent required to impart the desired preventive or therapeutic effect (e.g., reduction of viral load in the host) to a subject in need. As is recognized by those skilled in the art, the effective dose can vary depending on the route of administration, the use of excipients, the possibility of combination with other therapeutic treatments, and the condition to be treated.

[0038] As used herein, the terms "administering" or "administration" refer to the placement of an active agent on a subject by a method or route, resulting in the active agent being at least partially located at a desired site to produce a desired effect. The active agents described herein can be administered by any suitable route known in the art. For example, the herbal compositions disclosed herein can be administered to a subject orally.

[0039] In at least one embodiment, the herbal composition disclosed herein comprises an extract derived from a herbal raw material and a pharmaceutically acceptable carrier thereof, wherein the herbal raw material comprises at least one of jasmine and needlewort. In some embodiments, the herbal raw material further comprises artemisia, ophiopogon japonicus, houttuynia cordata, platycodon grandiflorus, licorice, perilla, chrysanthemum, and any combination thereof.

[0040] In at least one embodiment, the herbal raw material comprises, based on the total weight of the herbal raw material, at least one of Jasminum nudiflorum and Clematis armandii, by weight 18% to 25% (e.g., 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%) of Artemisia argyi, by weight 10% to 17% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, and 17%) of Ophiopogon japonicus, and by weight 10% to 17% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, and 17%) of houttuynia cordata, 10% to 17% of platycodon grandiflorus by weight, 4% to 11% (e.g., 4%, 5%, 6%, 7%, 8%, 9%, 10%, and 11%) of licorice root by weight, 4% to 11% (e.g., 4%, 5%, 6%, 7%, 8%, 9%, 10%, and 11%) of perilla leaf by weight, and 0.4% to 11% (e.g., 0.4%, 0.6%, 0.8%, 1%, 3%, 5%, 7%, 8%, 9%, 10%, and 11%) of chrysanthemum by weight.

[0041] In at least one embodiment, the herbal raw material, ranging from about 20 grams to about 80 grams, comprises at least 5 grams to 7 grams of one of *Ipomoea purpurea* and *Houttuynia cordata*, 5 grams to 7 grams of *Artemisia argyi*, 3 grams to 5 grams of *Ophiopogon japonicus*, 3 grams to 5 grams of *Houttuynia cordata*, 3 grams to 5 grams of *Platycodon grandiflorus*, 1 gram to 3 grams of licorice, 1 gram to 3 grams of *Perilla frutescens*, and 0.1 grams to 3 grams of chrysanthemum. In some embodiments, the herbal raw material may range from about 20 grams to about 70 grams, about 20 grams to about 60 grams, about 20 grams to about 50 grams, or about 20 grams to about 40 grams.

[0042] In at least one embodiment, the pharmaceutically acceptable carrier in the herbal composition may be a diluent, disintegrant, binder, lubricant, slip agent, surfactant, or any combination thereof. The carrier in the composition is "acceptable" because it is compatible with the active agent in the composition (e.g., capable of stabilizing the active agent) and is harmless to the subject to which it is administered. One or more solubilizers may be used as pharmaceutical excipients to deliver the active ingredient. Examples of other carriers include colloidal silica, magnesium stearate, cellulose, and sodium lauryl sulfate. In some embodiments, the herbal composition comprises a pharmaceutically acceptable carrier selected from water, alcohol, maltodextrin, crystalline cellulose, and any combination thereof.

[0043] Many embodiments have been used to illustrate this disclosure. The following embodiments should not be considered as limiting the scope of this disclosure.

[0044] Example

[0045] To describe this disclosure in more detail, the herbal compositions, preparation methods, and uses of the compositions provided herein will be described in detail with reference to the following examples. Materials used in this disclosure, but not noted herein, are commercially available.

[0046] Preparation Example 1-1

[0047] Take 6g of Artemisia argyi, 6g of Jasminum sambac, 4g of Ophiopogon japonicus, 4g of Houttuynia cordata, 4g of Platycodon grandiflorus, 2g of Glycyrrhiza uralensis, 2g of Perilla frutescens, and 0.2g of Chrysanthemum morifolium, and grind them into powder or small pieces. Mix all the herbal materials with 600ml of water and boil the mixture for 5 to 10 minutes. Collect 550ml of the filtrate (qualitative filter paper No. 1, TOYO Advantec) and filter it through a 0.22-micron sterile syringe filter to obtain the herbal tea. After drying, measure the content of the herbal extract in the obtained herbal tea to be approximately 20 to 40 mg / ml.

[0048] Preparation Examples 1-2

[0049] Take 6g of Artemisia argyi, 6g of Jasminum sambac, 4g of Ophiopogon japonicus, 4g of Houttuynia cordata, 4g of Platycodon grandiflorus, 2g of Glycyrrhiza uralensis, 2g of Perilla frutescens, and 0.2g of Chrysanthemum morifolium, and grind them into powder or small pieces. Mix all the herbal materials with 600ml of water and boil the mixture down to about 60ml. Collect the filtrate (qualitative filter paper No. 1, TOYO Advantec) and filter it through a 0.22-micron sterile syringe filter to obtain a herbal tea concentrate. After drying, measure the content of the herbal extract in the obtained herbal tea concentrate to be about 150 to 200 mg / ml.

[0050] Preparation Example 2

[0051] Take 20% by weight of Artemisia argyi, 20% by weight of Jasminum sambac, 13.33% by weight of Ophiopogon japonicus, 13.33% by weight of Houttuynia cordata, 13.33% by weight of Platycodon grandiflorus, 6.67% by weight of Glycyrrhiza uralensis, 6.67% by weight of Perilla frutescens, and 6.67% by weight of Chrysanthemum morifolium, with a total weight of approximately 14 grams of herbs. Crush these herbs into small pieces and place them in a tea bag. Steep the tea bag in 300 ml of hot water (around 100°C) for 15 to 20 minutes to obtain herbal tea.

[0052] Preparation Example 3

[0053] Take 20% by weight of Artemisia argyi, 20% by weight of Jasminum sambac, 13.33% by weight of Ophiopogon japonicus, 13.33% by weight of Houttuynia cordata, 13.33% by weight of Platycodon grandiflorus, 6.67% by weight of Glycyrrhiza uralensis, 6.67% by weight of Perilla frutescens, and 6.67% by weight of Chrysanthemum morifolium, with a total weight of approximately 28 grams of herbal materials. Crush these herbal materials into small pieces and place them in a tea bag. Steep the tea bag in 600 ml of 37% alcohol for 30 minutes, then boil for 5 to 10 minutes to obtain an alcoholic extract.

[0054] Preparation Example 4

[0055] Take 6g of Artemisia argyi, 6g of Jasminum sambac, 4g of Ophiopogon japonicus, 4g of Houttuynia cordata, 4g of Platycodon grandiflorus, 2g of Glycyrrhiza uralensis, 2g of Perilla frutescens, and 0.2g of Chrysanthemum morifolium, and grind them into powder or small pieces. Mix all the herbal materials with 600ml of water and boil the mixture for 60 minutes. Collect the filtrate (qualitative filter paper No. 1, TOYO Advantec) and filter it through a 0.22-micron sterile syringe filter. Then boil it to concentrate the aqueous extract (30ml, about 5g).

[0056] Preparation Example 5

[0057] Take 6g of Artemisia argyi, 6g of Jasminum sambac, 4g of Ophiopogon japonicus, 4g of Houttuynia cordata, 4g of Platycodon grandiflorus, 2g of Glycyrrhiza uralensis, 2g of Perilla frutescens, and 0.2g of Chrysanthemum morifolium, and grind them into powder or small pieces. Mix all the herbal materials with 225.6 ml of water (8 times the total weight of the herbal materials), boil the mixture for 60 minutes, and then filter to obtain the first filtrate. Add 141.0 ml of water (5 times the total weight of the herbal materials) to the residue, boil for another 60 minutes, and then filter to obtain the second filtrate. Combine the first and second filtrates and filter through a 0.22-micron sterile syringe filter, then boil to concentrate and obtain an aqueous extract (36.66 ml, approximately 5g).

[0058] Preparation Example 6

[0059] In this embodiment, the water extract is prepared by the method described in Preparation Example 4 or 5, except that the leaves of jasmine used in Preparation Example 4 or 5 are replaced with the roots of jasmine.

[0060] Preparation Example 7

[0061] In this embodiment, the water extract is prepared by the method described in Preparation Example 4 or 5, except that Jasminum isomorphum is replaced with Nelumbo nucifera.

[0062] Preparation Example 8

[0063] In this embodiment, the Artemisia argyi aqueous extract is prepared by the method described in Preparation Example 4 or 5, except that the herbal material used in this embodiment is 6 grams of Artemisia argyi alone.

[0064] Preparation Example 9

[0065] In this embodiment, an aqueous extract of jasmine is prepared by the method described in Preparation Example 4 or 5, except that the herbal material used in this embodiment is 6 grams of jasmine alone.

[0066] Pharmacological Example 1: Materials and Methods

[0067] The therapeutic efficacy of the herbal compositions disclosed herein for the prevention or treatment of coronavirus infection was determined by the following pharmacological examples 2 to 7. Test samples were taken from preparation examples 1 to 9, and control samples serving as the control group are listed below:

[0068] Control group (Ctrl.) - No treatment group;

[0069] Control group 1 (Ctrl.1) - Arbidol, also known as Umifenovir, is a broad-spectrum antiviral drug produced in Russia, which is used against some enveloped and non-enveloped viruses;

[0070] Control group 2 (Ctrl.2) - compound, containing lactoferrin, colloidal silver, olive leaf extract and elderberry extract;

[0071] Control group 3 (Ctrl.3) - Lianhua Qingwen Capsules, a traditional Chinese medicine dosage form used to treat influenza, containing Forsythia suspensa, Lonicera japonica, Ephedra sinica, bitter almond, gypsum, Isatis indigotica root, Dryopteris crassirhizoma, Houttuynia cordata, Pogos emoncablin, rhubarb, Rhodiola rosea, menthol, and Glycyrrhiza uralensis;

[0072] Control group 4 (Ctrl.4) - Olive leaf extract;

[0073] Control group 5 (Ctrl.5) - elderberry extract; and

[0074] Control group 6 (Ctrl.6) - a Chinese medicine formulation derived from Hualien Tzu Chi Hospital (Taiwan, China), containing Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorus, Chrysanthemum morifolium, Glycyrrhiza uralensis, and Perilla frutescens.

[0075] Furthermore, the experimental methods used in these embodiments are described below:

[0076] (1) Detection of inhibition of viral spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptor

[0077] According to the manufacturer's instructions, the COVID-19 spike protein-ACE2 binding assay kit II (RayBiotech) is used to measure the effect of test samples on inhibiting the binding of SARS-CoV-2 spike protein to human ACE2 receptor.

[0078] In summary, all reagents should be kept at room temperature (approximately 18°C ​​to 25°C) before use. Next, add 100 μL of each test sample to the wells of a removable 8-well plate, seal the plate with the sealing film, and incubate at room temperature for 2.5 hours or overnight at 4°C with gentle shaking. Then discard the solution in the wells and wash each well four times with 1× washing buffer. Additionally, wash each well using a multichannel pipette filled with 300 μL of 1× washing buffer or an automated washer. After the final wash, remove the remaining 1× washing buffer by aspirating or decanting, add 100 μL of 1× mouse secondary horseradish peroxidase (HRP)-conjugated IgG to each well, and incubate at room temperature with gentle shaking for 1 hour. Afterward, discard the solution in each well and wash each well as described above. Subsequently, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) one-step substrate reagent was added to each well, and the mixture was incubated at room temperature in the dark for 30 minutes with gentle shaking. Finally, 50 μL of stop solution was added to each well, and the optical absorbance at 450 nm was immediately read.

[0079] (2) Detection of viral 3C-like (3CL) protease

[0080] According to the manufacturer's instructions, the SensoLyte SARS-CoV-2 3CL protease activity assay kit (fluorescence) is used to determine the effect of test samples on inhibiting the 3CL protease of SARS-CoV-2 to resist viral replication in human cells.

[0081] In short, the working solutions are prepared first. Specifically, a 1× test buffer is prepared by adding 10 mL of 2× test buffer to 10 mL of deionized water. The 3CL protease receptor solution is prepared by diluting the 3CL protease receptor 100-fold with the test buffer. The 3CL protease diluent is prepared by diluting the 3CL protease 80-fold with the test buffer. The inhibitor (GC376) diluent is prepared by diluting the 10 μm inhibitor solution 100-fold with the test buffer.

[0082] Next, for the enzymatic reaction, the test sample and 3CL protease diluent were added to the wells of the microplate at volumes of 10 μL / well and 40 μL / well, respectively. Simultaneously, the following control wells were set up: a positive control group containing 3CL protease but no test sample; an inhibitor control group containing 3CL protease and the inhibitor GC376; a mediator control group containing 3CL protease and a mediator for delivering the test sample (e.g., dimethyl sulfoxide (DMSO) at a concentration not exceeding 1%); a test sample control group containing assay buffer and the test sample; and a substrate control group containing assay buffer. The total volume of all control groups was brought to 50 μL using assay buffer.

[0083] Subsequently, 50 μL of 3CL protease acceptor solution was added to each well, and the fluorescence signal was measured using kinetic or endpoint readings. For kinetic readings, fluorescence intensity was immediately and continuously measured at excitation / emission (Ex / Em) = 490 nm / 520 nm, with data recorded every 5 minutes for 30 to 60 minutes. For endpoint readings, the reaction solution was incubated at 37°C for 30 to 60 minutes without direct light, followed by fluorescence intensity measurement at Ex / Em = 490 nm / 520 nm.

[0084] (3) Detection of virus-dependent RNA polymerase (RdRp)

[0085] According to the manufacturer's instructions, the SARS-CoV-2 RNA polymerase (RdRp) assay kit (ProFoldin) is used to measure the effect of test samples on inhibiting the RNA polymerase of SARS-CoV-2 to resist viral replication in human cells.

[0086] In summary, SARS-CoV2 RdRp assays were performed in a 96-well disc format. First, 1 μL of the test sample in DMSO was added to each well of the 96-well assay disc. Then, a 48 μL premix consisting of 41 μL H2O, 5 μL 10× buffer, 1 μL 50× template, and 1 μL 50× RdRp was added, followed by incubation for 5 minutes. Next, 1 μL 50× NTP was added and incubated at 34°C for 60 to 120 minutes. After the reaction was complete, 150 μL of 1× fluorescent dye solution was added to the incubated reaction mixture, and the fluorescence intensity was measured at 450 nm within 5 minutes.

[0087] (4) Detection of inhibition of transmembrane serine protease 2 (TRPMSS2) and FK506 binding protein 51 (FKBP51)

[0088] Culture 4×10⁴ cells / year in a 10 cm petri dish. 5 Caco-2 cells were treated with test samples at concentrations of 50 μg / mL (low dose) or 150 μg / mL (high dose) for 24 hours. After 12 hours, cells were collected, and the levels of TRPMSS2 and FKBP51 were analyzed by Western blotting. This determined the effectiveness of the test samples in inhibiting TRPMSS2 to resist SARS-CoV-2 attachment and invasion of host cells, and in reducing FKBP51 to resist stress responses.

[0089] (5) Detection of SARS-CoV-2 pseudotyped lentivirus

[0090] SARS-CoV-2 pseudotyped lentivirus is a type of lentivirus that possesses a green fluorescent protein gene or a fluorescence enzyme gene in its genome and expresses the SARS-CoV-2 spike protein on its surface envelope. Human intestinal epithelial cell line Caco-2 and human lung cell line Calu-3 were cultured for 12 hours, followed by co-culturing with 10 μg / mL or 30 μg / mL of test samples for 24 hours to determine the efficacy of the test samples in preventing SARS-CoV-2 infection in vitro. Subsequently, the cultured cells were infected with wild-type, D614G mutant, α mutant (B.1.1.7), or β mutant (B.1.351) SARS-CoV-2 pseudotyped lentivirus. After 24 hours, the infected cells were observed using a fluorescence microscope.

[0091] On the other hand, SKH2 / J mice were treated with test samples for 7 consecutive days via gastric tube feeding (16.22 mg or 48.66 mg / 0.3 mL / mouse / day) for in vivo assays. From day 4 to day 6, the mice were fed with the test sample via an Aerogen Solo nebulizer (10 μL 1.2 × 10⁻⁶). 6The mice were infected with a pseudotyped lentivirus (particle / mouse / day) via intranasal delivery, consisting of wild-type SARS-CoV-2, the D614G mutant, the α mutant (B.1.1.7), the β mutant (B.1.351), or the P1 mutant (Brazilian variant). Viral infection was assessed in mice on day 8 using an in vivo imaging system (IVIS).

[0092] The parameters of the Aerogen Solo sprayer are as follows: Flow rate: greater than 0.2 mL / min (average: about 0.38 mL / min); Particle size: (1) Specification range: 1 μm to 5 μm, average: 3.1 μm, measured by Anderson cascade impact sampler; (2) Specification range: 1.5 μm to 6.2 μm, average: 3.9 μm, measured by Marple 298 cascade impact sampler. According to EN 13544-1, the starting dose is 2 mL, and the aerosol output rate is 0.30 mL / min. In addition, the aerosol output is 1.02 mL / dose, and the residual gas volume is less than 0.1 mL per 3 mL dose.

[0093] (6) Cytotoxicity and cell viability assays

[0094] To determine the in vitro toxicity of the test samples, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to assess cell viability and proliferation. Specifically, astrocytes (CTX), cardiomyocytes (H9c2), and lung fibroblasts (HFL-1) were co-cultured with the test samples at concentrations ranging from 31.25 μg / mL to 4,000 μg / mL for 24 hours. MTT was then added to the cultures and incubated for 2 hours. The absorbance at 590 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader by dissolving MTT crystals in dimethyl sulfoxide (DMSO).

[0095] On the other hand, C57BL / 6 mice were treated with the test sample at a dose of 5,200 mg / kg / day via gastric tube feeding to determine the in vivo toxicity of the test sample. Twenty-four hours later, the levels of creatine phosphokinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, creatinine, total bilirubin, and glucose in the mouse blood were measured.

[0096] Pharmacological Example 2: Inhibition of viral spike protein binding to ACE2 receptor

[0097] The efficacy of the herbal compositions prepared in Preparation Examples 4 or 5 (Exp. 1, 6 to 30 mg / mL) in inhibiting the binding of SARS-CoV-2 spike protein to human ACE2 receptor was determined and compared with Arbidol (Ctrl. 1, 20 mg / mL), Compound (Ctrl. 2), Lianhua Qingwen Capsules (Ctrl. 3), Olive Leaf Extract (Ctrl. 4), Elderberry Extract (Ctrl. 5), and a Chinese medicine dosage form derived from Hualien Tzu Chi Hospital (Ctrl. 6).

[0098] These results show Figures 1A to 1F This indicates that Exp.1 exhibits significantly enhanced blocking activity against the binding of viral spike proteins to the ACE2 receptor compared to Ctrl.2 through Ctrl.6. Furthermore, reference... Figure 1B and Figure 1C The binding activity of the spike protein treated with Exp.1 at 6 mg / mL and 12 mg / mL was comparable to that treated with Ctrl.3 at 24 mg / mL and 30 mg / mL; that is, at the same dosage, the blocking activity of the herbal composition of this application is about 2.5 to 4 times higher than that of Lianhua Qingwen capsules. Furthermore, Figure 1C The results show that Exp.1 at 18 mg / mL achieves a higher blocking effect than Arbidol at 20 mg / mL. This means that low doses of the herbal composition disclosed herein can effectively block the interaction between viral spike proteins and ACE2 receptors, thereby avoiding serious side effects such as nausea, diarrhea and dizziness caused by the use of common antiviral drugs (such as Arbidol).

[0099] also, Figure 1G To demonstrate the effectiveness of inhibiting the binding of SARS-CoV-2 spike protein to the human ACE2 receptor, herbal compositions prepared by different extraction methods were compared. The aqueous extract prepared in Preparation Example 4 or 5 was labeled Exp. 1; the herbal tea prepared in Preparation Example 1 was labeled Exp. 2; the herbal tea prepared in Preparation Example 2 was labeled Exp. 3; the alcoholic extract prepared in Preparation Example 3 was labeled Exp. 4; the Artemisia argyi extract prepared in Preparation Example 8 was labeled Exp. 5; and the Jasminum nudiflorum extract prepared in Preparation Example 9 was labeled Exp. 6. These results indicate that the herbal compositions disclosed herein, prepared by different extraction methods, can also block the interaction between the viral spike protein and the ACE2 receptor.

[0100] Pharmacological Example 3: Safety of the Herbal Compositions Disclosed herein

[0101] The toxicity of the herbal compositions prepared by preparation examples 4 or 5 (Exp. 1) was determined by MTT assay and animal model assay.

[0102] The MTT assay results for cell lines CTX, H9c2, and HFL-1 are as follows: Figures 2A to 2C As shown, these results demonstrate that amounts of the herbal composition disclosed herein below 1,000 μg / mL do not affect the physiological activity of the brain, heart, and lungs. Since the herbal composition disclosed herein, as a preparation of the drinkable herbal tea provided in Examples 1-1, contains 20 to 40 mg / mL of herbal raw material extract, this implies that even if a 70 kg adult human were to consume a large quantity of 5.6 liters of the herbal composition disclosed herein, it would not cause toxicity.

[0103] In an animal model, C57BL / 6 mice were fed the herbal composition disclosed herein at a dose of 5,200 mg / kg / mouse / day, which is 20 times the recommended daily intake for a human. The results showed that the levels of creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate aminotransferase (GOT), alanine aminotransferase (GPT), creatinine (CRE), total bilirubin (T-Bil), and glucose (Glu) in mouse blood were as shown in Table 1 below. Figure 3A and Figure 3B CPK and LDH are indicators of cardiac and cerebral function; GOT and GPT are indicators of liver function; CRE is an indicator of kidney function; T-Bil contains direct and indirect bilirubin, which are indicators of biliary function. These results indicate that there were no significant differences in blood parameters between mice treated with and without the herbal composition, meaning that the herbal composition disclosed herein does not cause adverse effects even at extremely high doses.

[0104] Table 1. Blood parameters of mice after administration of the herbal composition disclosed herein.

[0105] logo Ctr1. Exp.1 unit CPK 126.73 122.77 International Units (IU / L) LDH 316.37 311.87 International Units (IU / L) GOT 137.03 130.70 International Units (IU / L) GPT 53.27 55.83 International Units per Liter CRE 134.17 137.03 millimoles per liter T-Bil 1.35 1.46 g / L Glu 2.06 2.01 millimoles per liter

[0106] Pharmacological Example 4: Inhibition of TRPMSS2 and FKBP51 expression levels

[0107] The efficacy of the herbal composition prepared by Preparation Example 4 or 5 (Exp. 1) in inhibiting the expression of TRPMSS2 and FKBP51 was determined and compared with Arbidol (Ctrl. 1, 20 mg / mL), Compound (Ctrl. 2), and Lianhua Qingwen Capsules (Ctrl. 3).

[0108] The results of suppressing TRPMSS2 expression levels are as follows Figure 4A and Figure 4BAs shown, these results indicate that Exp.1 significantly inhibited TRPMSS2 expression compared to Ctrl.2 and Ctrl.3. Furthermore, when the dosage of Exp.1 was increased from 50 μg / mL (low dose, L) to 150 μg / mL (high dose, H), TRPMSS2 expression decreased by 70%, while when the dosage of Ctrl.3 was increased from 50 μg / mL (low dose, L) to 150 μg / mL (high dose, H), TRPMSS2 expression decreased by only 10%. In other words, compared to Lianhua Qingwen capsules, the herbal composition disclosed herein is 7 times more effective in inhibiting TRPMSS2 expression, implying an effective role in blocking viral entry into host cells.

[0109] The inhibition results of FKBP51 showed that... Figure 5A and Figure 5B As shown, when the dosage of Exp.1 was increased from 50 μg / mL (low dose, L) to 150 μg / mL (high dose, H), the performance of FKBP51 decreased by 40%. FKBP51 is a stress-related molecular linker, so these results suggest that the herbal composition disclosed herein has an effect against emotional stress, anxiety, and depression that may be caused by COVID-19.

[0110] Pharmacological Example 5: Inhibition of 3CL protease and RdRp activity

[0111] The efficacy of the herbal composition prepared by Preparation Example 4 or 5 (Exp. 1) in inhibiting the activity of SARS-CoV-2 3CL protease and RdRp was determined.

[0112] The results are as follows: Figure 6 (3CL) and Figure 7 As shown in (RdRp), these results indicate that the herbal composition disclosed herein reduces the activity of RdRp and 3CL proteases by 35% to 40% in a dose-dependent manner. For SARS-CoV-2, 3CL is involved in the cleavage of polyproteins, producing viral proteins crucial to the viral life cycle, and RdRp is used for viral genome replication and viral gene transcription. Therefore, these results suggest that the herbal composition disclosed herein is effective in treating COVID-19.

[0113] Pharmacological Example 6: Prevention of SARS-CoV-2 Infection

[0114] The efficacy of the herbal composition disclosed herein for preventing SARS-CoV-2 infection was determined by using wild-type and variants of SARS-CoV-2 pseudotyped lentivirus.

[0115] For in vitro experiments, the herbal compositions to be tested were as follows: Exp.7, containing 1.5 g of the aqueous extract prepared in Preparation Example 4 or 5 and 1.5 g of excipient (a mixture of maltodextrin and crystalline cellulose) (concentration of aqueous extract: 10 μg / mL); Exp.8, containing 3 g of the aqueous extract prepared in Preparation Example 4 or 5 and 3 g of excipient (a mixture of maltodextrin and crystalline cellulose) (concentration of aqueous extract: 30 μg / mL); and Exp.9, containing 3 g of the aqueous extract prepared in Preparation Example 4 or 5 (concentration of aqueous extract: 30 μg / mL). Each composition was added to Caco-2 or Calu-3 cells. After 24 hours, wild-type pseudotype lentivirus and its three variants (i.e., D614G mutant, B.1.1.7 mutant, and B.1.351 mutant) were added.

[0116] The results are as follows Figure 8A (Caco-2) and Figure 8B As shown in (Calu-3), these results indicate that the herbal composition disclosed herein can reduce viral infection in wild-type and variant Caco-2 by 51% to 74%, and in Calu-3 by 64% to 86%.

[0117] For in vivo testing, the herbal composition prepared in Preparation Example 4 or 5 was administered to SKH2 / J mice via gastric tube for 7 consecutive days at a dose of 16.22 mg / 0.3 mL / mouse / day (Exp. 1-L) or 48.66 mg / 0.3 mL / mouse / day (Exp. 1-H). From day 4 to day 6, wild-type pseudotyped lentivirus and its three variants (i.e., D614G mutant, B.1.1.7 mutant, and B.1.351 mutant) were administered to mice via intranasal administration.

[0118] The results are as follows Figure 9A (wild type), Figure 9B (D614G mutant) Figure 9C (B.1.1.7 mutant) and Figure 9D As shown in (B.1.351 mutant), Ctrl. represents the mouse group that received no drug or viral treatment, and Ctrl.7 represents the mouse group that received only pseudotyped lentivirus. Figure 9A As shown, mice treated with the herbal composition disclosed herein exhibited lower brightness levels in their tissues, which means that the herbal composition disclosed herein can effectively prevent infection with wild-type SARS-CoV-2.

[0119] In addition, such as Figure 9BAs shown, the infection rate of the D614G mutant was reduced by 5-fold and 25-fold, respectively, in Exp.1-L and Exp.1-H mice, which means that the herbal composition disclosed herein can effectively prevent the infection of the D614G mutant.

[0120] like Figure 9C As shown, Ctrl.7 mice suffered from severe lung infection caused by the B.1.1.7 mutant (as indicated by the arrows), while Exp.1-L and Exp.1-H effectively prevented the B.1.1.7 mutant from causing severe lung infection (as indicated by the circled areas).

[0121] like Figure 9D As shown, B.1.351 mutant infections were reduced in Exp.1-L and Exp.1-H; in particular, as indicated by the circled areas, nasal and intestinal infections caused by B.1.351 mutants were significantly reduced in Exp.1-L and Exp.1-H.

[0122] These results indicate that the herbal composition disclosed herein can significantly prevent viral infections in both wild-type and mutant variants.

[0123] Pharmacological Example 7: Effects of the herbal composition disclosed herein containing alternative ingredients

[0124] In this embodiment, the efficacy of the herbal compositions prepared by Preparation Example 4 or 5 (i.e., those containing *Jasminum nudiflorum* leaf extract) for preventing SARS coronavirus-2 infection was compared with that of the compositions prepared by Preparation Example 6 (i.e., those containing *Jasminum nudiflorum* root extract). Additionally, since *Jasminum nudiflorum* and *Needleia serratifolia* can often be identified as the same traditional Chinese medicine material, the efficacy of the herbal compositions prepared by Preparation Example 4 or 5 was also compared with that of the compositions prepared by Preparation Example 7 (i.e., those containing *Needleia serratifolia* extract). This comparison was determined by assessing the efficacy of these herbal compositions for preventing SARS-CoV-2 pseudotype lentivirus infection.

[0125] The results are as follows Figure 10A and Figure 10B As shown, the herbal composition containing jasmine leaf extract is labeled Exp.1; the herbal composition containing jasmine root extract is labeled Exp.10; the herbal composition containing needlegrass extract is labeled Exp.11; and Ctrl.7 is a group of mice treated only with the P1 mutant of SARS-CoV-2 pseudotype lentivirus.

[0126] exist Figure 10AIn this study, Caco-2 cells treated with 30 μL / mL test sample were infected with the B.1.1.7 mutant of SARS-CoV-2 pseudotype lentivirus. The results showed that, similar to Exp.1, Exp.10 and Exp.11 also reduced viral infection by approximately 48% and 64%, respectively.

[0127] exist Figure 10B In this study, SKH2 / J mice were fed with test samples at a dose of 48.66 mg / 0.3 mL / mouse / day before P1 mutant infection. The results showed that in Exp.1 and Exp.10, the infection rate of P1 mutant in mice was reduced by 25-fold and 40-fold, respectively.

[0128] These results indicate that herbal compositions containing jasmine root extract or needlegrass extract can also significantly prevent SARS-CoV-2 infection.

[0129] While some embodiments of this disclosure have been described in detail above, those skilled in the art can make various modifications and changes to the illustrated embodiments without substantially departing from the teachings and advantages of this disclosure. Therefore, such modifications and changes are included within the scope of this disclosure as set forth in the appended claims.

Claims

1. An herbal composition for preventing or treating a viral infection, consisting of an extract derived from an herbal material and a pharmaceutically acceptable carrier, characterized in that the herbal material consisting of, by weight based on the total weight thereof, 20% of Artemisia indica, 20% of Artemisia vulgaris, 13% of Ophiopogon japonicus, 13% of Houttuynia cordata, 13% of Platycodon grandiflorum, 7% of Glycyrrhiza uralensis, 7% of Perilla frutescens, and 7% of Chrysanthemum morifolium; and wherein the viral infection is caused by severe acute respiratory syndrome coronavirus 2, its D614G mutant, its B.1.1.7 mutant, its B.1.351 mutant, and / or its P1 mutant.

2. The herbal composition according to claim 1, wherein, the Artemisia indica is Artemisia indica root, Artemisia indica leaf, or a combination thereof.

3. The herbal composition according to claim 1, wherein, the extract derived from the herbal material is a water extract or an alcohol extract.

4. A method of preparing the herbal composition of claim 1, comprising: providing an herbal material consisting of Artemisia indica, Artemisia vulgaris, Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorum, Glycyrrhiza uralensis, Perilla frutescens, and Chrysanthemum morifolium; extracting the herbal material with the extraction liquid to obtain a crude extract, wherein the extraction liquid contains at least one of water and alcohol; and removing solids in the crude extract to obtain a liquid fraction.

5. The method of claim 4, wherein, the extracting comprises boiling the herbal material in the extraction liquid for at least 5 minutes.

6. The method of claim 4, wherein, the extracting comprises immersing the herbal material in the extraction liquid having a temperature lower than its boiling point for at least 10 minutes.

7. The method of claim 4, wherein, the weight ratio of the herbal material to the extraction liquid is 2:1 to 30:

1.

8. The method of claim 4, further comprising concentrating the liquid fraction to obtain a concentrated extract of the herbal material.

9. Use of the herbal composition of claim 1 for the manufacture of a medicament for preventing or treating a viral infection in a subject in need thereof, wherein the viral infection is caused by severe acute respiratory syndrome coronavirus 2, its D614G mutant, its B.1.1.7 mutant, its B.1.351 mutant, and / or its P1 mutant.

10. Use according to claim 9, wherein, the extract derived from the herbal material in the herbal composition is administered to the subject in an effective amount of 25 mg / kg / day to 2,500 mg / kg / day.

11. Use according to claim 10, wherein, the extract derived from the herbal material in the herbal composition is administered to the subject in an effective amount of 50 mg / kg / day to 500 mg / kg / day.

12. The use according to claim 9, wherein, the herbal composition interferes with the binding of spike protein of the virus to angiotensin-converting enzyme 2 (ACE2) receptor.

13. The use according to claim 9, wherein, the herbal composition inhibits the expression of at least one of 3CL protease, RNA-dependent RNA polymerase (RdRp), transmembrane serine protease 2 (TRPMSS2), and FK506 binding protein 5 (FKBP5).

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