Patent Description:
Viral infection has been established and remains as a serious animal and human affliction. Coronaviruses (CoVs) are a large family of viruses that can cause illness ranging from the common cold to more severe diseases. For example, infections with the human coronavirus strains, CoV-229E, CoV-OC43, CoV-NL63, and CoV-HKU1, usually result in mild, self-limiting upper respiratory tract infections, such as a common cold, e.g., runny nose, sneezing, headache, cough, sore throat, and fever. Other infections may result in more severe diseases, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and severe acute respiratory syndrome coronavirus <NUM> (SARS-CoV-<NUM>), which cause severe acute respiratory syndrome, kidney failure, and death, and have triggered a global public health emergency. Particularly, coronavirus disease <NUM> (COVID-<NUM>) caused by SARS-CoV-<NUM> has infected more than <NUM> million people and caused over <NUM>,<NUM>,<NUM> deaths worldwide.

The fastest remedy to save lives thus far is the repurposing of existing FDA-approved drugs originally targeting other diseases for COVID-<NUM>. However, most antiviral drugs cause serious side effects such as nausea, diarrhea, dizziness, and fever, and there is still no specific antiviral treatment available or proven to be effective to treat or prevent coronavirus infection in a subject.

Therefore, there exists an unmet need to provide an effective and safe therapeutics to prevent or treat coronavirus infections.

In view of the foregoing, the present disclosure provides an herbal composition that is capable of interfering with the interaction between coronavirus spike (S) protein and ACE2 on the host cell surface, and suppressing the expression of proteins that are necessary for entry and replication of coronavirus in the host, thereby protecting a subject from a viral infection.

In at least one embodiment of the present disclosure, the herbal composition comprises an extract from an herbal raw material and a pharmaceutically acceptable carrier thereof, wherein the herbal raw material comprises Ohwia caudata. The Ohwia caudata is an Ohwia caudata root, an Ohwia caudata leaf, or a combination thereof.

In at least one embodiment of the present disclosure, the herbal composition comprises an extract from an herbal raw material comprising Ohwia caudata, and at least one of Artemisia argyi, Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorus, Glycyrrhiza uralensis, Perilla frutescens, and chrysanthemum. In some embodiments, the herbal composition comprises an extract from an herbal raw material comprising Ohwia caudata, and Artemisia argyi, Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorus, Glycyrrhiza uralensis, Perilla frutescens, and chrysanthemum. The extract from the herbal raw material is a water extract.

In at least one embodiment of the present disclosure, the herbal composition comprises an extract from an herbal raw material comprising, based on a total weight thereof, <NUM>% to <NUM>% by weight of Ohwia caudata, and at least one of <NUM>% to <NUM>% by weight of Artemisia argyi, <NUM>% to <NUM>% by weight of Ophiopogon japonicus, <NUM>% to <NUM>% by weight of Houttuynia cordata, <NUM>% to <NUM>% by weight of Platycodon grandiflorus, <NUM>% to <NUM>% by weight of Glycyrrhiza uralensis, <NUM>% to <NUM>% by weight of Perilla frutescens, and <NUM>% to <NUM>% by weight of chrysanthemum.

The herbal composition may be prepared by a method comprising: providing the herbal raw material as mentioned above; extracting the herbal raw material with an extracting solution to obtain a crude extract, wherein the extracting solution comprises water; and removing solid from the crude extract to obtain a liquid portion.

The method for preparing the herbal composition may further comprise crushing the herbal raw material into powder or pieces.

Extracting the herbal raw material may comprise boiling the herbal raw material in the extracting solution for at least <NUM> minutes, such as <NUM> minutes to <NUM> hours, and/or immersing the herbal raw material in the extracting solution with a temperature lower than a boiling point thereof for at least <NUM> minutes, such as <NUM> minutes to <NUM> hour. The weight ratio of the herbal raw material to the extracting solution may be from <NUM>:<NUM> to <NUM>:<NUM>, such as <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM> and <NUM>:<NUM>.

The method for preparing the herbal composition may further comprise concentrating the liquid portion to obtain a concentrated extract.

The herbal composition of the invention is for use in for preventing or treating a viral infection caused by a coronavirus.

In some embodiments, the coronavirus is SARS-CoV, MERS-CoV, SARS-CoV-<NUM>, mouse hepatitis virus (MHV), or porcine epidemic diarrhea virus (PEDV). In some embodiments, the coronavirus is a variant of SARS-CoV-<NUM>, such as a D614G mutant strain, a B. <NUM> (Alpha) mutant strain, a B. <NUM> (Beta) mutant strain, and a P1 mutant strain.

In at least one embodiment of the present disclosure, the extract from the herbal raw material in the herbal composition is administered to the subject in an effective amount of from about <NUM>/kg/day to about <NUM>,<NUM>/kg/day, such as from about <NUM>/kg/day to about <NUM>,<NUM>/kg/day, and from about <NUM>/kg/day to about <NUM>/kg/day.

In the present disclosure, the herbal composition provided in the present disclosure as an antivirus agent may inhibit virus replication and reduce the amount of viruses in a host cell. In addition, the herbal composition provided in the present disclosure is safe and may solve the prior-art problems of side effects. Hence, the present disclosure provides an effective strategy against viral infections, which is useful in controlling the outbreak of coronaviruses.

The present disclosure can be more fully understood by reading the following descriptions of the embodiments, with reference made to the accompanying drawings.

The technical solutions illustrated in the examples of the present disclosure will now be described more clearly and completely, and it will be apparent that the described examples are merely part of the examples of the present disclosure and are not intended to be exhaustive. The present disclosure can also be implemented or applied as described in different examples. All other examples obtained without creative work by those skilled in the art are within the scope of the present disclosure.

It is further noted that, as used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless expressly and unequivocally limited to one referent. The term "or" is used interchangeably with the term "and/or" unless the context clearly indicates otherwise.

As used herein, the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, which are included in the present disclosure, yet open to the inclusion of unspecified elements or steps, whether essential or not.

The present disclosure is directed to an herbal composition, a method for preparing the herbal composition, and a method for preventing or treating a viral infection in a subject in need thereof by using the herbal composition.

The viral infection treated by the herbal composition of the present disclosure may be caused by coronavirus (CoV).

The structural proteins of CoVs including nucleocapsid (N), small envelope (E), matrix (M) and trimeric spike (S) glycoproteins, which are essential for virion assembly and function to complete the viral life cycle during infections. In some embodiments, the herbal composition may block the interaction of coronavirus S-protein and angiotensin-converting enzyme <NUM> (ACE2) receptor, as well as suppress the expression of proteins that are necessary for entry and/or replication of the coronavirus in a host, thereby influencing the risk of contracting the viral infection or aggravating the disease progression. Accordingly, the herbal composition of the present disclosure may have antiviral activity and be useful for effectively preventing or treating viral infections caused by a coronavirus.

As used herein, the term "preventing" or "prevention" refers to preventive or avoidance measures for a disease or symptoms or conditions of a disease, which include, but are not limited to, applying or administering one or more active agents to a subj ect who has not yet been diagnosed as a patient suffering from the disease or the symptoms or conditions of the disease but may be susceptible or prone to the disease. The preventive measures of the present disclosure are provided to avoid, prevent, or postpone the occurrence of the disease or the symptoms or conditions of the disease.

As used herein, the term "treating" or "treatment" refers to obtaining a desired pharmacologic and/or physiologic effect, e.g., inhibition of viral entry and/or replication in a host. The effect may be prophylactic in terms of completely or partially preventing a disease or symptoms or conditions thereof, or may be therapeutic in terms of completely or partially curing, alleviating, relieving, remedying, or ameliorating a disease or an adverse effect attributable to the disease or symptoms or conditions thereof.

As used herein, the terms "patient" and "subject" are used interchangeably. The term "subject" means a human or animal. Examples of the subject include, but are not limited to, human, monkey, mice, rat, woodchuck, ferret, rabbit, hamster, cow, horse, pig, deer, dog, cat, fox, wolf, chicken, emu, ostrich, and fish. In some embodiments of the present disclosure, the subject is a mammal, e.g., a primate such as a human.

As used herein, the phrase "an effective amount" refers to the amount of an active agent that is required to confer a desired preventive or therapeutic effect on a subject in need thereof (e.g., reducing the amount of viruses in a host). Effective doses may vary, as recognized by those skilled in the art, depending on routes of administration, excipient usage, the possibility of co-usage with other therapeutic treatment, and the condition to be treated.

As used herein, the term "administering" or "administration" refers to the placement of an active agent into a subject by a method or route which results in at least partial localization of the active agent at a desired site to produce the desired effect. The active agent described herein may be administered by any appropriate route known in the art. For example, the herbal composition of the present disclosure is administered to the subject by oral administration.

The herbal composition of the present disclosure comprises an extract from an herbal raw material and a pharmaceutically acceptable carrier thereof, wherein the herbal raw material comprises Ohwia caudata. In some embodiments, the herbal raw material further comprises Artemisia argyi, Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorus, Glycyrrhiza uralensis, Perilla frutescens, chrysanthemum, or any combination thereof.

In at least one embodiment, the herbal raw material comprises <NUM>% to <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%) by weight of Ohwia caudata, <NUM>% to <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%) by weight of Artemisia argyi, <NUM>% to <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%) by weight of Ophiopogon japonicus, <NUM>% to <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%) by weight of Houttuynia cordata, <NUM>% to <NUM>% by weight of Platycodon grandiflorus, <NUM>% to <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%) by weight of Glycyrrhiza uralensis, <NUM>% to <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%) by weight of Perilla frutescens, and <NUM>% to <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%) by weight of chrysanthemum, based on a total weight of the herbal raw material.

In at least one embodiment, the herbal raw material in a range of from around <NUM> to around <NUM> comprises <NUM> to <NUM> of Ohwia caudata, <NUM> to <NUM> of Artemisia argyi, <NUM> to <NUM> of Ophiopogon japonicus, <NUM> to <NUM> of Houttuynia cordata, <NUM> to <NUM> of Platycodon grandiflorus, <NUM> to <NUM> of Glycyrrhiza uralensis, <NUM> to <NUM> of Perilla frutescens, and <NUM> to <NUM> of chrysanthemum. In some embodiments, the herbal raw material may be in a range of from around <NUM> to around <NUM>, from around <NUM> to around <NUM>, from around <NUM> to around <NUM>, or from around <NUM> to around <NUM>.

In at least one embodiment, the pharmaceutically acceptable carrier in the herbal composition may be diluents, disintegrants, binders, lubricants, glidants, surfactants, or any combination thereof. The carrier in the composition is "acceptable" in the sense that it is compatible with the active agent of the composition (e.g., capable of stabilizing the active agent) and not deleterious to the subject to be administered. One or more solubilizing agents may be utilized as pharmaceutical excipients for delivery of an active ingredient. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. In some embodiments, the herbal composition comprises a pharmaceutically acceptable carrier selected from water, ethanol, maltodextrin, crystalline cellulose, and any combination thereof.

Many examples have been used to illustrate the present disclosure. The examples below should not be taken as a limit to the scope of the present disclosure.

For a more detailed description of the present disclosure, the herbal composition, the preparing method and the use of the composition will be provided and described in detail with reference to the following examples. The materials used in the present disclosure but unannotated herein are commercially available.

<NUM> of Artemisia argyi, <NUM> of Ohwia caudata, <NUM> of Ophiopogon japonicus, <NUM> of Houttuynia cordata, <NUM> of Platycodon grandiflorus, <NUM> of Glycyrrhiza uralensis, <NUM> of Perilla frutescens, and <NUM> of chrysanthemum were taken and crushed into powder or small pieces. After mixing all the herbal materials with addition of <NUM> water, the mixture was boiled for <NUM> to <NUM> minutes. <NUM> of the filtrate was collected by filtration (qualitative filter paper No. <NUM>, TOYO Advantec), and filtered through a <NUM> sterile syringe filter, so as to obtain herbal tea. After drying, the amount of the extract of the herbal materials in the obtained herbal tea was measured as about <NUM> to <NUM>/mL.

<NUM> of Artemisia argyi, <NUM> of Ohwia caudata, <NUM> of Ophiopogon japonicus, <NUM> of Houttuynia cordata, <NUM> of Platycodon grandiflorus, <NUM> of Glycyrrhiza uralensis, <NUM> of Perilla frutescens, and <NUM> of chrysanthemum were taken and crushed into powder or small pieces. After mixing all the herbal materials with addition of <NUM> water, the mixture was boiled down to approximately <NUM>. The filtrate was collected by filtration (qualitative filter paper No. <NUM>, TOYO Advantec), and filtered through a <NUM> sterile syringe filter, so as to obtain herbal tea concentrate. After drying, the amount of the extract of the herbal materials in the obtained herbal tea concentrate was measured as about <NUM> to <NUM>/mL.

<NUM> wt% of Artemisia argyi, <NUM> wt% of Ohwia caudata, <NUM> wt% of Ophiopogon japonicus, <NUM> wt% of Houttuynia cordata, <NUM> wt% of Platycodon grandiflorus, <NUM> wt% of Glycyrrhiza uralensis, <NUM> wt% of Perilla frutescens, and <NUM> wt% of chrysanthemum were taken, and the total weight of the herbal materials was about <NUM>. Such herbal materials were crushed into small pieces, and packed into a tea bag. The tea bag was immersed in <NUM> of hot water (around <NUM>) for <NUM> to <NUM> minutes, so as to obtain herbal tea.

<NUM> wt% of Artemisia argyi, <NUM> wt% of Ohwia caudata, <NUM> wt% of Ophiopogon japonicus, <NUM> wt% of Houttuynia cordata, <NUM> wt% of Platycodon grandiflorus, <NUM> wt% of Glycyrrhiza uralensis, <NUM> wt% of Perilla frutescens, and <NUM> wt% of chrysanthemum were taken, and the total weight of the herbal materials was about <NUM>. Such herbal materials were crushed into small pieces, and packed into a tea bag. The tea bag was immersed in <NUM> of <NUM>% alcohol for <NUM> minutes, followed by boiling for <NUM> to <NUM> minutes, so as to obtain an ethanol extract.

<NUM> of Artemisia argyi, <NUM> of Ohwia caudata, <NUM> of Ophiopogon japonicus, <NUM> of Houttuynia cordata, <NUM> of Platycodon grandiflorus, <NUM> of Glycyrrhiza uralensis, <NUM> of Perilla frutescens, and <NUM> of chrysanthemum were taken and crushed into powder or small pieces. After mixing all the herbal materials with addition of <NUM> water, the mixture was boiled for <NUM> minutes. The filtrate was collected by filtration (qualitative filter paper No. <NUM>, TOYO Advantec), filtered through a <NUM> sterile syringe filter, and then boiled for concentration to give a water extract (<NUM>, about <NUM>).

<NUM> of Artemisia argyi, <NUM> of Ohwia caudata, <NUM> of Ophiopogon japonicus, <NUM> of Houttuynia cordata, <NUM> of Platycodon grandiflorus, <NUM> of Glycyrrhiza uralensis, <NUM> of Perilla frutescens, and <NUM> of chrysanthemum were taken and crushed into powder or small pieces. After mixing all the herbal materials with addition of <NUM> water (<NUM> times of the total weight of the herbal materials), the mixture was boiled for <NUM> minutes, and then filtered to obtain the first filtrate. After adding <NUM> of water (<NUM> times of the total weight of the herbal materials) to the residue, the mixture was boiled for further <NUM> minutes, and then filtered to obtain the second filtrate. The first and second filtrates were combined and filtered through a <NUM> sterile syringe filter, and then boiled for concentration to give a water extract (<NUM>, about <NUM>).

In this example, a water extract was prepared by the process described in Preparation Example <NUM> or <NUM>, except that the leaves of Ohwia caudate used in Preparation Example <NUM> or <NUM> were replaced with the roots of Ohwia caudate in this example.

In this example, a water extract was prepared by the process described in Preparation Example <NUM> or <NUM>, except that Ohwia caudate was replaced with Anisomeles indica (L.

In this example, a water extract of Artemisia argyi was prepared by the process described in Preparation Example <NUM> or <NUM>, except that the herbal material used in this example was <NUM> of Artemisia argyi alone.

In this example, a water extract of Ohwia caudata was prepared by the process described in Preparation Example <NUM> or <NUM>, except that the herbal material used in this example was <NUM> of Ohwia caudata alone.

The therapeutic effect of the herbal composition provided in the present disclosure for the prevention or treatment of coronavirus infection was determined in the following Pharmacological Examples <NUM> to <NUM>. The samples to be tested were obtained from Preparation Examples <NUM> to <NUM>, and the comparative samples as control groups were listed as follows:.

Further, the experimental methods used in these examples were described as follows:.

The effect of the test samples for inhibition of SARS-CoV-<NUM> spike protein binding to human ACE2 receptor was determined by using the COVID-<NUM> Spike-ACE2 Binding Assay Kit II (RayBiotech) according to the manufacturer's instructions.

Briefly, all reagents were brought to room temperature (about <NUM> to <NUM>) before use. Next, <NUM>µL of each test sample was added into the well of removable <NUM>-well strips, and covered with the plate sealing film and incubated for <NUM> hours at room temperature or overnight at <NUM> with gentle shaking. Then, the solution in wells was discarded, and each well was washed <NUM> times with <NUM> × Wash Solution. Further, each well was washed by filling with <NUM> × Wash Buffer (<NUM>µL) using a multi-channel pipette or autowasher. After the last wash, the remaining <NUM>× Wash Buffer was removed by aspirating or decanting, and <NUM>µL of <NUM>× mouse secondary horseradish peroxidase (HRP)-conjugated IgG was added to each well and incubated for <NUM> hour at room temperature with gentle shaking. After that, the solution in wells was discarded, and each well was washed as described above. Subsequently, <NUM>µL of <NUM>,<NUM>',<NUM>,<NUM>'-tetramethylbenzidine (TMB) One-Step Substrate Reagent was added to each well, and incubated for <NUM> minutes at room temperature in the dark with gentle shaking. Finally, <NUM>µL of Stop Solution was added to each well, and the optical absorbance at <NUM> was read immediately.

The effect of the test samples for inhibition of 3CL protease of SARS-CoV-<NUM> to resist viral replication in human cells was determined by using SensoLyte SARS-CoV-<NUM>3CL Protease Activity Assay Kit (Fluorimetric) according to the manufacturer's instructions.

Briefly, the working solutions were prepared firstly. Specifically, <NUM> × assay buffer was prepared by adding <NUM> of <NUM>× assay buffer to <NUM> of deionized water. The 3CL protease substrate solution was prepared by diluting the 3CL protease substrate <NUM>-fold with the assay buffer. The 3CL protease diluent was prepared by diluting the 3CL protease <NUM>-fold with the assay buffer. The inhibitor (GC <NUM>) diluent was prepared by diluting the <NUM> inhibitor solution <NUM>-fold with the assay buffer.

Next, for the enzymatic reaction, the test samples and the 3CL protease diluent were added to the microplate wells in an amount of <NUM>µL/well and <NUM>µL/well, respectively. Simultaneously, the following control wells were set up: Positive control, containing 3CL protease without the test sample; Inhibitor control, containing 3CL protease and inhibitor GC <NUM>; Vehicle control, containing 3CL protease and vehicle used in delivering the test sample (e.g., dimethyl sulfoxide (DMSO) with concentration not exceeding <NUM>%); Test sample control, containing assay buffer and test sample; and Substrate control, containing assay buffer. The total volume of all controls was brought to <NUM>µL by using the assay buffer.

Subsequently, <NUM>µL of the 3CL protease substrate solution was added into each well, and then the fluorescence signal was measured by kinetic reading or end-point reading. For kinetic reading, the fluorescence intensity was measured immediately at excitation/emission (Ex/Em) = <NUM>/<NUM> continuously, and the data were recorded every <NUM> minutes for <NUM> to <NUM> minutes. For end-point reading, the reaction solution was incubated at <NUM> for <NUM> to <NUM> minutes and kept from direct light, and then the fluorescence intensity was measured at Ex/Em = <NUM>/<NUM>.

The effect of the test samples for inhibition of RNA polymerase of SARS-CoV-<NUM> to resist viral replication in human cells was determined by using SARS-CoV-<NUM> RNA Polymerase (RdRp) Assay Kit (ProFoldin) according to the manufacturer's instructions.

Briefly, the SARS-CoV2 RdRp assay was performed in <NUM>-well plate format. Firstly, <NUM>µL of the test sample in DMSO was added into each well of the <NUM>-well assay plate. Further, <NUM>µL of a premix composed of <NUM>µL of H<NUM>O, <NUM>µL of <NUM>× buffer, <NUM>µL of <NUM>× template, and <NUM>µL of <NUM>× RdRp was added, followed by incubating for <NUM>. Subsequently, <NUM>µL of <NUM>× NTP was added and further incubated at <NUM>°Cfor <NUM> to <NUM> minutes. After reaction, <NUM>µL of <NUM>× fluorescence dye solution was added to the incubated reaction mixture, and the fluorescence intensity was measured at <NUM> in <NUM> minutes.

The effects of the test samples for inhibition of TRPMSS2 to resist the attachment and invasion of SARS-CoV-<NUM> into host cells and for reduction of FKBP51 to resist the stress responses were determined by culturing <NUM>×<NUM><NUM> Caco-<NUM> cells in a <NUM> petri dish for <NUM> hours, adding the test samples in a concentration of <NUM>µg/mL (low dose) or <NUM>µg/mL (high dose), and after <NUM> hours, collecting the cells and analyzing the levels of TRPMSS2 and FKBP51 by western blotting.

The SARS-CoV-<NUM> pseudotyped lentivirus was provided by the RNAi Core of Academia Sinica (Taiwan), which was a lentivirus having a green fluorescent protein gene or luciferase gene in its genome and expressing SARS-CoV-<NUM> spike protein on its surface envelope. For determining the effect of the test samples for prevention of SARS-CoV-<NUM> infection in vitro, the human intestinal epithelial cell line Caco-<NUM> and human lung cell line Calu-<NUM> were cultured for <NUM> hours, and then co-cultured with <NUM>µg/mL or <NUM>µg/mL of the test samples for <NUM> hours. Afterward, the cultured cells were infected with wild type, D614G mutant, Alpha mutant (B. <NUM>), or Beta mutant (B. <NUM>) of the SARS-CoV-<NUM> pseudotyped lentivirus. After <NUM> hours, the infected cells were observed by fluorescence microscope.

On the other hand, for the in vivo assay, the SKH2/J mice were treated with the test samples by gavage (<NUM> or <NUM>/<NUM>/mouse/day) for seven consecutive days. On day <NUM> to day <NUM>, the mice were infected with wild type, D614G mutant, Alpha mutant (B. <NUM>), Beta mutant (B. <NUM>), or P1 mutant (Brazil variant) of the SARS-CoV-<NUM> pseudotyped lentivirus by intranasal delivery with Aerogen Solo nebulizer (<NUM>µL of <NUM>×<NUM><NUM> particles/mouse/day). On day <NUM>, the viral infection in mice was determined by in vivo imaging system (IVIS).

The parameters of Aerogen Solo nebulizer were as follows. Flow rate: greater than <NUM>/min (average: about <NUM>/min); Particle size: (<NUM>) specification range: <NUM> to <NUM>, average value: <NUM>, measured by Andersen cascade impact sampler; (<NUM>) specification range: <NUM> to <NUM>, average value: <NUM>, measured by Marple <NUM> cascade impact sampler. According to Standard EN <NUM>-<NUM>, the starting dose was <NUM>, and the aerosol output speed was <NUM>/min. Further, the aerosol output was <NUM>/dose, and the residual volume was less than <NUM> per <NUM> dose.

For determining the toxicity of the test samples in vitro, MTT (<NUM>-(<NUM>,<NUM>-dimethylthiazol-<NUM>-yl)-<NUM>,<NUM>-diphenyltetrazolium bromide) assay was performed to evaluate the cell activity and proliferation. Specifically, astrocytes CTX, myocardial cells H9c2, and lung fibroblast HFL-<NUM> were co-cultured with the test samples in a concentration of from <NUM>µg/mL to <NUM>,<NUM>µg/mL for <NUM> hours. Afterward, MTT was added to the culture and incubated for <NUM> hours. By dissolving the MTT crystals in dimethyl sulfoxide (DMSO), the optical absorbance at <NUM> was measured by an enzyme-linked immunosorbent assay (ELISA) reader.

On the other hand, for determining the toxicity of the test samples in vivo, the C57BL/<NUM> mice were treated with the test samples by gavage at a dose of <NUM>,<NUM>/kg/day. After <NUM> hours, the levels of creatine phosphokinase, lactate dehydrogenase, aspartate transaminase, alanine transaminase, creatinine, total bilirubin, and glucose in blood of mice were measured.

The effect of the herbal composition prepared from Preparation Example <NUM> or <NUM> (Exp. <NUM>, <NUM> to <NUM>/mL) for inhibition of SARS-CoV-<NUM> spike protein binding to human ACE2 receptor was determined and compared with that of Arbidol (Ctrl. <NUM>, <NUM>/mL), Compound prescription (Ctrl. <NUM>), Lianhua Qingwen capsule (Ctrl. <NUM>), olive leaf extract (Ctrl. <NUM>), elderberry extract (Ctrl. <NUM>), and Chinese medicine formulation from Hualien Tzu Chi Hospital (Ctrl.

The results were shown in <FIG>, indicating that as compared with Ctrl. <NUM> to Ctrl. <NUM>, Exp. <NUM> exhibited greatly improved blocking activity for the viral spike protein binding to ACE2 receptor. In addition, referring to <FIG>, the binding activities of the spike protein treated with <NUM>/mL and <NUM>/mL of Exp. <NUM> were comparable to that treated with <NUM>/mL and <NUM>/mL of Ctrl. <NUM>, respectively; that is to say, under the same administered dose, the blocking activity of the herbal composition of the present application was about <NUM> to <NUM> times higher than Lianhua Qingwen capsule. Furthermore, <FIG> showed that <NUM>/mL of Exp. <NUM> may achieve a higher blocking effect than <NUM>/mL of Arbidol, implying that a low dose of the herbal composition of the present disclosure may effectively block the interaction between the viral spike protein and ACE2 receptor, such that the severe side effects such as nausea, diarrhea, and dizziness caused by the administration of conventional antiviral drugs (e.g., Arbidol) can be avoided.

In addition, <FIG> showed the comparison of the herbal compositions prepared by different extraction methods in view of the effect for inhibition of SARS-CoV-<NUM> spike protein binding to human ACE2 receptor, in which the water extract prepared from Preparation Example <NUM> or <NUM> was notated as Exp. <NUM>; the herbal tea prepared from Preparation Example <NUM> was notated as Exp. <NUM>; the herbal tea prepared from Preparation Example <NUM> was notated as Exp. <NUM>; the ethanol extract prepared from Preparation Example <NUM> was notated as Exp. <NUM>; the extract of Artemisia argyi prepared from Preparation Example <NUM> was notated as Exp. <NUM>; and the extract of Ohwia caudate prepared from Preparation Example <NUM> was notated as Exp. These results demonstrated that the herbal compositions of the present disclosure prepared by different extraction methods may also block the interaction between the viral spike protein and ACE2 receptor.

The toxicity of the herbal composition prepared from Preparation Example <NUM> or <NUM> (Exp. <NUM>) was determined by MTT assay and animal model.

The results of MTT assay of cell lines CTX, H9c2, and HFL-<NUM> were shown in <FIG>, respectively, illustrating that the herbal composition of the present disclosure in an amount of less than <NUM>,<NUM>µg/mL would not have impact on the physiological activities of brain, heart and lung. Since the herbal composition of the present disclosure as the drinkable herbal tea provided in Preparation Example <NUM>-<NUM> comprises the extract of the herbal raw materials in an amount of <NUM> to <NUM>/mL, it is implied that the herbal composition of the present disclosure would not cause toxicity even though a <NUM> adult human swigged <NUM> of the herbal composition.

In the animal model, C57BL/<NUM> mice were fed with the herbal composition of the present disclosure at a dose of <NUM>,<NUM>/kg/mouse/day, which is <NUM> times of the recommended daily intake for a person. As a result, the levels of creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate transaminase (GOT), alanine transaminase (GPT), creatinine (CRE), total bilirubin (T-Bil), and glucose (Glu) in blood of mice were shown in Table <NUM> below and <FIG>. CPK and LDH are indicators of heart and brain functions; GOT and GPT are indicators of liver function; CRE is an indicator of kidney function; and T-Bil includes direct bilirubin and indirect bilirubin, which are indicators of gallbladder function. These results indicated that there is no significant difference in the blood parameters between the mice treated with and without the herbal composition, implying the herbal composition of the present disclosure would not result in an adverse effect even in an extremely high dose.

The effect of the herbal composition prepared from Preparation Example <NUM> or <NUM> (Exp. <NUM>) for inhibition of expressions of TRPMSS2 and FKBP51 was determined and compared with that of Arbidol (Ctrl. <NUM>, <NUM>/mL), Compound prescription (Ctrl. <NUM>), and Lianhua Qingwen capsule (Ctrl.

The results of inhibition of TRPMSS2 expression were shown in <FIG> and <FIG>, indicating that Exp. <NUM> significantly inhibited TRPMSS2 expression in comparison with Ctrl. <NUM> and Ctrl. Moreover, when the administrated dose of Exp. <NUM> was increased from <NUM>µg/mL (low dose, L) to <NUM>µg/mL (high dose, H), the TRPMSS2 expression was decreased by <NUM>%, whereas when the administrated dose of Ctrl. <NUM> was increased from <NUM>µg/mL (low dose, L) to <NUM>µg/mL (high dose, H), the TRPMSS2 expression was only decreased by <NUM>%. That is to say, the herbal composition of the present disclosure may inhibit TRPMSS2 expression with seven times better efficacy in comparison with Lianhua Qingwen capsule, implying the potent effect of the herbal composition of the present disclosure for blocking viral entry into the host cell.

The results of inhibition of FKBP51 expression were shown in <FIG> and <FIG>, indicating that when the administrated dose of Exp. <NUM> was increased from <NUM>µg/mL (low dose, L) to <NUM>µg/mL (high dose, H), the FKBP51 expression was decreased by <NUM>%. FKBP51 is a molecular link to stress, such that these results implied that the herbal composition of the present disclosure has an effect against emotional stress, anxiety and depression which may be caused by COVID-<NUM>.

The effect of the herbal composition prepared from Preparation Example <NUM> or <NUM> (Exp. <NUM>) for inhibition of activities of 3CL protease and RdRp of SARS-CoV-<NUM> was determined.

The results were shown in <FIG> (3CL) and <FIG> (RdRp), respectively, indicating that the herbal composition of the present disclosure reduced the activities of RdRp and 3CL protease by <NUM>% to <NUM>% in a dose-dependent manner. For SARS-CoV-<NUM>, the 3CL involves in the cleavage of polyproteins, giving rise to viral proteins essential for the life cycle of the virus, and RdRp is used for the replication of viral genome and the transcription of viral genes. Accordingly, these results implied that the herbal composition of the present disclosure has an effect on the treatment of COVID-<NUM>.

The effect of the herbal composition of the present disclosure for prevention of SARS-CoV-<NUM> infection was determined by using wild type and variants of SARS-CoV-<NUM> pseudotyped lentivirus.

For the in vitro assay, the herbal compositions to be tested were as follows: Exp. <NUM>, containing <NUM> of the water extract prepared from Preparation Example <NUM> or <NUM> and <NUM> of excipient (a mixture of maltodextrin and crystalline cellulose) (the concentration of the water extract: <NUM>µg/mL); Exp. <NUM>, containing <NUM> of the water extract prepared from Preparation Example <NUM> or <NUM> and <NUM> of excipient (a mixture of maltodextrin and crystalline cellulose) (the concentration of the water extract: <NUM>µg/mL); and Exp. <NUM>, containing <NUM> of the water extract prepared from Preparation Example <NUM> or <NUM> (the concentration of the water extract: <NUM>µg/mL). Each composition was added to Caco-<NUM> cells or Calu-<NUM> cells. After <NUM> hours, the wild type pseudotyped lentivirus and three variants thereof (i.e., D614G mutant, B. <NUM> mutant, and B. <NUM> mutant) were added.

The results were shown in <FIG> (Caco-<NUM>) and <FIG> (Calu-<NUM>), indicating that the herbal composition of the present disclosure may reduce viral infection of both wild type and variants by <NUM>% to <NUM> % for Caco-<NUM> and by <NUM>% to <NUM> % for Calu-<NUM>.

For the in vivo assay, the herbal composition prepared from Preparation Example <NUM> or <NUM> was administrated to SKH2/J mice by gavage at a dose of <NUM>/<NUM>/mouse/day (Exp. <NUM> - L) or <NUM>/<NUM>/mouse/day (Exp. <NUM> - H) for seven consecutive days, and the wild type pseudotyped lentivirus and three variants thereof (i.e., D614G mutant, B. <NUM> mutant, and B. <NUM> mutant) were administrated to the mice by intranasal delivery on day <NUM> to day <NUM>.

The results were shown in <FIG> (wild type), <FIG> (D614G mutant), <FIG> (B. <NUM> mutant) and <FIG> (B. <NUM> mutant), in which Ctrl. was the group of mice without any drug or virus treatment, and Ctrl. <NUM> was the group of mice only treated with the pseudotyped lentivirus. As shown in <FIG>, the mice treated with the herbal composition of the present disclosure exhibited lower luminance levels in their tissues, implying that the herbal composition of the present disclosure may effectively prevent infection of wild type SARS-CoV-<NUM>.

Further, as shown in <FIG>, the infection of the D614G mutant in the mice was reduced by <NUM> times and <NUM> times in Exp. <NUM> - L and Exp. <NUM> - H, respectively, implying that the herbal composition of the present disclosure may effectively prevent infection of the D614G mutant.

As shown in <FIG>, the mice of Ctrl. <NUM> had severe lung infection caused by the B. <NUM> mutant (as indicated by the arrow), while Exp. <NUM> - L and Exp. <NUM> - H may effectively prevent the B. <NUM> mutant from strongly infecting the lungs (as indicated by circle areas).

As shown in <FIG>, the infection of the B. <NUM> mutant was reduced in Exp. <NUM> - L and Exp. <NUM> - H; in particular, as indicated by the circle areas, the nasal and intestinal infection caused by the B. <NUM> mutant was significantly reduced in Exp. <NUM> - L and Exp.

These above results indicated that the herbal composition of the present disclosure may significantly prevent viral infection of both wild type and mutant variants.

In this example, the effect of the herbal composition prepared from Preparation Example <NUM> or <NUM> (i.e., those containing the extract of Ohwia caudate leaves) for prevention of SARS-CoV-<NUM> infection was compared to that prepared from Preparation Example <NUM> (i.e., those containing the extract of Ohwia caudate roots). Also, since Ohwia caudate and Anisomeles indica (L. Ktze may generally be identified as the same Chinese medicinal material, the effect of the herbal composition prepared from Preparation Example <NUM> or <NUM> was also compared to that prepared from Preparation Example <NUM> (i.e., those containing the extract of Anisomeles indica (L. The comparison was performed by determining the effect of these herbal compositions for preventing the infection of SARS-CoV-<NUM> pseudotyped lentivirus.

The results were shown in <FIG> and <FIG>, in which the herbal composition containing the extract of Ohwia caudate leaves was notated as Exp. <NUM>; the herbal composition containing the extract of Ohwia caudate roots was notated as Exp. <NUM>; the herbal composition containing the extract of Anisomeles indica (L. Ktze was notated as Exp. <NUM>; and Ctrl. <NUM> was the group of mice only treated with the P1 mutant of SARS-CoV-<NUM> pseudotyped lentivirus.

In <FIG>, the B. <NUM> mutant of SARS-CoV-<NUM> pseudotyped lentivirus was used to infect Caco-<NUM> cells treated with <NUM>µL/mL of the test sample, and the results showed that like Exp. <NUM>, Exp. <NUM> and Exp. <NUM> may also reduce viral infection by about <NUM>% and about <NUM>%, respectively.

In <FIG>, the SKH2/J mice were fed with the test sample at a dose of <NUM>/<NUM>/mouse/day before the infection of the P1 mutant, and the results showed that the infection of the P1 mutant in the mice was reduced by <NUM> times and <NUM> times in Exp. <NUM> and Exp. <NUM>, respectively.

Claim 1:
An herbal composition for use in preventing or treating a viral infection caused by a coronavirus, comprising an extract from an herbal raw material and a pharmaceutically acceptable carrier, wherein the herbal raw material comprises Ohwia caudata, wherein the Ohwia caudata is an Ohwia caudata root, or a combination of the Ohwia caudata root and an Ohwia caudata leaf, and wherein the extract from the herbal raw material is a water extract.