Patent Application
20240016873
The present disclosure relates to herbal compositions, and in particular relates to herbal compositions for preventing or treating viral infections.
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 2 (SARS-CoV-2), which cause severe acute respiratory syndrome, kidney failure, and death, and have triggered a global public health emergency. Particularly, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has infected more than 194 million people and caused over 4,160,000 deaths worldwide.
The fastest remedy to save lives thus far is the repurposing of existing FDA-approved drugs originally targeting other diseases for COVID-19. 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 at least one of Ohwia caudata and Anisomeles indica (L.) O. Ktze. In some embodiments, 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 at least one of Ohwia caudata and Anisomeles indica (L.) O. Ktze, 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 at least one of Ohwia caudata and Anisomeles indica (L.) O. Ktze, and Artemisia argyi, Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorus, Glycyrrhiza uralensis, Perilla frutescens, and chrysanthemum. In some embodiments, the extract from the herbal raw material is a water extract or an ethanol 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, 18% to 25% by weight of at least one of Ohwia caudata and Anisomeles indica (L.) O. Ktze, and at least one of 18% to 25% by weight of Artemisia argyi, 10% to 17% by weight of Ophiopogon japonicus, 10% to 17% by weight of Houttuynia cordata, 10% to 17% by weight of Platycodon grandiflorus, 4% to 11% by weight of Glycyrrhiza uralensis, 4% to 11% by weight of Perilla frutescens, and 0.4% to 11% by weight of chrysanthemum.
In at least one embodiment of the present disclosure, the herbal composition is 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, ethanol or a combination thereof; and removing solid from the crude extract to obtain a liquid portion.
In at least one embodiment of the present disclosure, the method for preparing the herbal composition further comprises crushing the herbal raw material into powder or pieces.
In at least one embodiment of the present disclosure, extracting the herbal raw material comprises boiling the herbal raw material in the extracting solution for at least 5 minutes, such as 5 minutes to 2 hours, and/or immersing the herbal raw material in the extracting solution with a temperature lower than a boiling point thereof for at least 10 minutes, such as 10 minutes to 1 hour. In some embodiments, the weight ratio of the herbal raw material to the extracting solution is from 2:1 to 30:1, such as 2:1, 3:1, 5:1, 8:1, 10:1, 15:1, 20:1, 25:1 and 30:1.
In at least one embodiment of the present disclosure, the method for preparing the herbal composition further comprises concentrating the liquid portion to obtain a concentrated extract.
In at least one embodiment of the present disclosure, a method for preventing or treating a viral infection in a subject in need thereof is provided. The method comprises administering to the subject an effective amount of at least one of the above-mentioned herbal compositions.
In at least one embodiment of the present disclosure, the viral infection is caused by a coronavirus. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV, 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 a D614G mutant strain, a B.1.1.7 (Alpha) mutant strain, a B.1.351 (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 25 mg/kg/day to about 2,500 mg/kg/day, such as from about 30 mg/kg/day to about 1,000 mg/kg/day, and from about 50 mg/kg/day to about 500 mg/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.
In at least one embodiment, the viral infection treated by the method 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 method for preventing or treating a viral infection of the present disclosure comprises administering to a subject in need thereof the herbal composition that may block the interaction of coronavirus S-protein and angiotensin-converting enzyme 2 (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.
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 subject 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.
In at least one embodiment, 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 at least one of Ohwia caudata and Anisomeles indica (L.) O. Ktze. In some embodiments, the herbal raw material further comprises Artemisia argyi, Ophiopogon japonicus, Houttuynia cordata, Platycodon grandiflorus, Glycyrrhiza uralensis, Perilla frutescens, chrysanthemum, and any combination thereof.
In at least one embodiment, the herbal raw material comprises 18% to 25% (e.g., 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%) by weight of at least one of Ohwia caudata and Anisomeles indica (L.) O. Ktze, 18% to 25% (e.g., 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%) by weight of Artemisia argyi, 10% to 17% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, and 17%) by weight of Ophiopogon japonicus, 10% to 17% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, and 17%) by weight of Houttuynia cordata, 10% to 17% by weight of Platycodon grandiflorus, 4% to 11% (e.g., 4%, 5%, 6%, 7%, 8%, 9%, 10%, and 11%) by weight of Glycyrrhiza uralensis, 4% to 11% (e.g., 4%, 5%, 6%, 7%, 8%, 9%, 10%, and 11%) by weight of Perilla frutescens, and 0.4% to 11% (e.g., 0.4%, 0.6%, 0.8%, 1%, 3%, 5%, 7%, 8%, 9%, 10%, and 11%) 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 20 g to around 80 g comprises 5 g to 7 g of at least one of Ohwia caudata and Anisomeles indica (L.) 0. Ktze, 5 g to 7 g of Artemisia argyi, 3 g to 5 g of Ophiopogon japonicus, 3 g to 5 g of Houttuynia cordata, 3 g to 5 g of Platycodon grandiflorus, 1 g to 3 g of Glycyrrhiza uralensis, 1 g to 3 g of Perilla frutescens, and 0.1 g to 3 g of chrysanthemum. In some embodiments, the herbal raw material may be in a range of from around 20 g to around 70 g, from around 20 g to around 60 g, from around 20 g to around 50 g, or from around 20 g to around 40 g.
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.
6 g of Artemisia argyi, 6 g of Ohwia caudata, 4 g of Ophiopogon japonicus, 4 g of Houttuynia cordata, 4 g of Platycodon grandiflorus, 2 g of Glycyrrhiza uralensis, 2 g of Perilla frutescens, and 0.2 g of chrysanthemum were taken and crushed into powder or small pieces. After mixing all the herbal materials with addition of 600 mL water, the mixture was boiled for 5 to 10 minutes. 550 mL of the filtrate was collected by filtration (qualitative filter paper No. 1, TOYO Advantec), and filtered through a 0.22 μm 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 20 to 40 mg/mL.
6 g of Artemisia argyi, 6 g of Ohwia caudata, 4 g of Ophiopogon japonicus, 4 g of Houttuynia cordata, 4 g of Platycodon grandiflorus, 2 g of Glycyrrhiza uralensis, 2 g of Perilla frutescens, and 0.2 g of chrysanthemum were taken and crushed into powder or small pieces. After mixing all the herbal materials with addition of 600 mL water, the mixture was boiled down to approximately 60 mL. The filtrate was collected by filtration (qualitative filter paper No. 1, TOYO Advantec), and filtered through a 0.22 μm 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 150 to 200 mg/mL.
20 wt % of Artemisia argyi, 20 wt % of Ohwia caudata, 13.33 wt % of Ophiopogon japonicus, 13.33 wt % of Houttuynia cordata, 13.33 wt % of Platycodon grandiflorus, 6.67 wt % of Glycyrrhiza uralensis, 6.67 wt % of Perilla frutescens, and 6.67 wt % of chrysanthemum were taken, and the total weight of the herbal materials was about 14 g. Such herbal materials were crushed into small pieces, and packed into a tea bag. The tea bag was immersed in 300 mL of hot water (around 100° C.) for 15 to 20 minutes, so as to obtain herbal tea.
20 wt % of Artemisia argyi, 20 wt % of Ohwia caudata, 13.33 wt % of Ophiopogon japonicus, 13.33 wt % of Houttuynia cordata, 13.33 wt % of Platycodon grandiflorus, 6.67 wt % of Glycyrrhiza uralensis, 6.67 wt % of Perilla frutescens, and 6.67 wt % of chrysanthemum were taken, and the total weight of the herbal materials was about 28 g. Such herbal materials were crushed into small pieces, and packed into a tea bag. The tea bag was immersed in 600 mL of 37% alcohol for 30 minutes, followed by boiling for 5 to 10 minutes, so as to obtain an ethanol extract.
6 g of Artemisia argyi, 6 g of Ohwia caudata, 4 g of Ophiopogon japonicus, 4 g of Houttuynia cordata, 4 g of Platycodon grandiflorus, 2 g of Glycyrrhiza uralensis, 2 g of Perilla frutescens, and 0.2 g of chrysanthemum were taken and crushed into powder or small pieces. After mixing all the herbal materials with addition of 600 mL water, the mixture was boiled for 60 minutes. The filtrate was collected by filtration (qualitative filter paper No. 1, TOYO Advantec), filtered through a 0.22 μm sterile syringe filter, and then boiled for concentration to give a water extract (30 mL, about 5 g).
6 g of Artemisia argyi, 6 g of Ohwia caudata, 4 g of Ophiopogon japonicus, 4 g of Houttuynia cordata, 4 g of Platycodon grandiflorus, 2 g of Glycyrrhiza uralensis, 2 g of Perilla frutescens, and 0.2 g of chrysanthemum were taken and crushed into powder or small pieces. After mixing all the herbal materials with addition of 225.6 mL water (8 times of the total weight of the herbal materials), the mixture was boiled for 60 minutes, and then filtered to obtain the first filtrate. After adding 141.0 mL of water (5 times of the total weight of the herbal materials) to the residue, the mixture was boiled for further 60 minutes, and then filtered to obtain the second filtrate. The first and second filtrates were combined and filtered through a 0.22 μm sterile syringe filter, and then boiled for concentration to give a water extract (36.66 mL, about 5 g).
In this example, a water extract was prepared by the process described in Preparation Example 4 or 5, except that the leaves of Ohwia caudate used in Preparation Example 4 or 5 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 4 or 5, except that Ohwia caudate was replaced with Anisomeles indica (L.) O. Ktze.
In this example, a water extract of Artemisia argyi was prepared by the process described in Preparation Example 4 or 5, except that the herbal material used in this example was 6 g of Artemisia argyi alone.
In this example, a water extract of Ohwia caudata was prepared by the process described in Preparation Example 4 or 5, except that the herbal material used in this example was 6 g 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 2 to 7. The samples to be tested were obtained from Preparation Examples 1 to 9, 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-2 spike protein binding to human ACE2 receptor was determined by using the COVID-19 Spike-ACE2 Binding Assay Kit II (RayBiotech) according to the manufacturer's instructions.
Briefly, all reagents were brought to room temperature (about 18° C. to 25° C.) before use. Next, 100 μL of each test sample was added into the well of removable 8-well strips, and covered with the plate sealing film and incubated for 2.5 hours at room temperature or overnight at 4° C. with gentle shaking. Then, the solution in wells was discarded, and each well was washed 4 times with 1× Wash Solution. Further, each well was washed by filling with 1× Wash Buffer (300 μL) using a multi-channel pipette or autowasher. After the last wash, the remaining 1× Wash Buffer was removed by aspirating or decanting, and 100 μL of 1× mouse secondary horseradish peroxidase (HRP)-conjugated IgG was added to each well and incubated for 1 hour at room temperature with gentle shaking. After that, the solution in wells was discarded, and each well was washed as described above. Subsequently, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) One-Step Substrate Reagent was added to each well, and incubated for 30 minutes at room temperature in the dark with gentle shaking. Finally, 50 μL of Stop Solution was added to each well, and the optical absorbance at 450 nm was read immediately.
The effect of the test samples for inhibition of 3CL protease of SARS-CoV-2 to resist viral replication in human cells was determined by using SensoLyte SARS-CoV-2 3CL Protease Activity Assay Kit (Fluorimetric) according to the manufacturer's instructions.
Briefly, the working solutions were prepared firstly. Specifically, 1× assay buffer was prepared by adding 10 mL of 2× assay buffer to 10 mL of deionized water. The 3CL protease substrate solution was prepared by diluting the 3CL protease substrate 100-fold with the assay buffer. The 3CL protease diluent was prepared by diluting the 3CL protease 80-fold with the assay buffer. The inhibitor (GC 376) diluent was prepared by diluting the 10 μM inhibitor solution 100-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 10 μL/well and 40 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 376; Vehicle control, containing 3CL protease and vehicle used in delivering the test sample (e.g., dimethyl sulfoxide (DMSO) with concentration not exceeding 1%); Test sample control, containing assay buffer and test sample; and Substrate control, containing assay buffer. The total volume of all controls was brought to 50 μL by using the assay buffer.
Subsequently, 50 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)=490 nm/520 nm continuously, and the data were recorded every 5 minutes for 30 to 60 minutes. For end-point reading, the reaction solution was incubated at 37° C. for 30 to 60 minutes and kept from direct light, and then the fluorescence intensity was measured at Ex/Em=490 nm/520 nm.
The effect of the test samples for inhibition of RNA polymerase of SARS-CoV-2 to resist viral replication in human cells was determined by using SARS-CoV-2 RNAPolymerase (RdRp) Assay Kit (ProFoldin) according to the manufacturer's instructions.
Briefly, the SARS-CoV2 RdRp assay was performed in 96-well plate format. Firstly, 1 μL of the test sample in DMSO was added into each well of the 96-well assay plate. Further, 48 μL of a premix composed of 41 μL of H2O, 5 μL of 10× buffer, 1 μL of 50× template, and 1 μL of 50× RdRp was added, followed by incubating for 5 min. Subsequently, 1 μL of 50× NTP was added and further incubated at 34° C. for 60 to 120 minutes. After reaction, 150 μL of 1× fluorescence dye solution was added to the incubated reaction mixture, and the fluorescence intensity was measured at 450 nm in 5 minutes.
The effects of the test samples for inhibition of TRPMSS2 to resist the attachment and invasion of SARS-CoV-2 into host cells and for reduction of FKBP51 to resist the stress responses were determined by culturing 4×105 Caco-2 cells in a 10 cm petri dish for 24 hours, adding the test samples in a concentration of 50 μg/mL (low dose) or 150 μg/mL (high dose), and after 12 hours, collecting the cells and analyzing the levels of TRPMSS2 and FKBP51 by western blotting.
The SARS-CoV-2 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-2 spike protein on its surface envelope. For determining the effect of the test samples for prevention of SARS-CoV-2 infection in vitro, the human intestinal epithelial cell line Caco-2 and human lung cell line Calu-3 were cultured for 12 hours, and then co-cultured with 10 μg/mL or 30 μg/mL of the test samples for 24 hours. Afterward, the cultured cells were infected with wild type, D614G mutant, Alpha mutant (B.1.1.7), or Beta mutant (B.1.351) of the SARS-CoV-2 pseudotyped lentivirus. After 24 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 (16.22 mg or 48.66 mg/0.3 mL/mouse/day) for seven consecutive days. On day 4 to day 6, the mice were infected with wild type, D614G mutant, Alpha mutant (B.1.1.7), Beta mutant (B.1.351), or P1 mutant (Brazil variant) of the SARS-CoV-2 pseudotyped lentivirus by intranasal delivery with Aerogen Solo nebulizer (10 μL of 1.2×106 particles/mouse/day). On day 8, 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 0.2 mL/min (average: about 0.38 mL/min); Particle size: (1) specification range: 1 μm to 5 μm, average value: 3.1 μm, measured by Andersen cascade impact sampler; (2) specification range: 1.5 μm to 6.2 μm, average value: 3.9 μm, measured by Marple 298 cascade impact sampler. According to Standard EN 13544-1, the starting dose was 2 mL, and the aerosol output speed was 0.30 mL/min. Further, the aerosol output was 1.02 mL/dose, and the residual volume was less than 0.1 mL per 3 mL dose.
For determining the toxicity of the test samples in vitro, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the cell activity and proliferation. Specifically, astrocytes CTX, myocardial cells H9c2, and lung fibroblast HFL-1 were co-cultured with the test samples in a concentration of from 31.25 μg/mL to 4,000 μg/mL for 24 hours. Afterward, MTT was added to the culture and incubated for 2 hours. By dissolving the MTT crystals in dimethyl sulfoxide (DMSO), the optical absorbance at 590 nm 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/6 mice were treated with the test samples by gavage at a dose of 5,200 mg/kg/day. After 24 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 4 or 5 (Exp. 1, 6 to 30 mg/mL) for inhibition of SARS-CoV-2 spike protein binding to human ACE2 receptor was determined and compared with that of Arbidol (Ctrl. 1, 20 mg/mL), Compound prescription (Ctrl. 2), Lianhua Qingwen capsule (Ctrl. 3), olive leaf extract (Ctrl. 4), elderberry extract (Ctrl. 5), and Chinese medicine formulation from Hualien Tzu Chi Hospital (Ctrl. 6).
The results were shown in
In addition,
The toxicity of the herbal composition prepared from Preparation Example 4 or 5 (Exp. 1) was determined by MTT assay and animal model.
The results of MTT assay of cell lines CTX, H9c2, and HFL-1 were shown in
In the animal model, C57BL/6 mice were fed with the herbal composition of the present disclosure at a dose of 5,200 mg/kg/mouse/day, which is 20 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 1 below and
The effect of the herbal composition prepared from Preparation Example 4 or 5 (Exp. 1) for inhibition of expressions of TRPMSS2 and FKBP51 was determined and compared with that of Arbidol (Ctrl. 1, 20 mg/mL), Compound prescription (Ctrl. 2), and Lianhua Qingwen capsule (Ctrl. 3).
The results of inhibition of TRPMSS2 expression were shown in
The results of inhibition of FKBP51 expression were shown in
The effect of the herbal composition prepared from Preparation Example 4 or 5 (Exp. 1) for inhibition of activities of 3CL protease and RdRp of SARS-CoV-2 was determined.
The results were shown in
The effect of the herbal composition of the present disclosure for prevention of SARS-CoV-2 infection was determined by using wild type and variants of SARS-CoV-2 pseudotyped lentivirus.
For the in vitro assay, the herbal compositions to be tested were as follows: Exp. 7, containing 1.5 g of the water extract prepared from Preparation Example 4 or 5 and 1.5 g of excipient (a mixture of maltodextrin and crystalline cellulose) (the concentration of the water extract: 10 μg/mL); Exp. 8, containing 3 g of the water extract prepared from Preparation Example 4 or 5 and 3 g of excipient (a mixture of maltodextrin and crystalline cellulose) (the concentration of the water extract: 30 μg/mL); and Exp. 9, containing 3 g of the water extract prepared from Preparation Example 4 or 5 (the concentration of the water extract: 30 μg/mL). Each composition was added to Caco-2 cells or Calu-3 cells. After 24 hours, the wild type pseudotyped lentivirus and three variants thereof (i.e., D614G mutant, B.1.1.7 mutant, and B.1.351 mutant) were added.
The results were shown in
For the in vivo assay, the herbal composition prepared from Preparation Example 4 or 5 was administrated to SKH2/J mice by gavage 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) for seven consecutive days, and the wild type pseudotyped lentivirus and three variants thereof (i.e., D614G mutant, B.1.1.7 mutant, and B.1.351 mutant) were administrated to the mice by intranasal delivery on day 4 to day 6.
The results were shown in
Further, as shown in
As shown in
As shown in
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 4 or 5 (i.e., those containing the extract of Ohwia caudate leaves) for prevention of SARS-CoV-2 infection was compared to that prepared from Preparation Example 6 (i.e., those containing the extract of Ohwia caudate roots). Also, since Ohwia caudate and Anisomeles indica (L.) O. Ktze may generally be identified as the same Chinese medicinal material, the effect of the herbal composition prepared from Preparation Example 4 or 5 was also compared to that prepared from Preparation Example 7 (i.e., those containing the extract of Anisomeles indica (L.) O. Ktze). The comparison was performed by determining the effect of these herbal compositions for preventing the infection of SARS-CoV-2 pseudotyped lentivirus.
The results were shown in
In
In
These above results indicated that the herbal composition containing the extract of Ohwia caudate roots or the extract of Anisomeles indica (L.) O. Ktze may also significantly prevent SARS-CoV-2 infection.
While some of the embodiments of the present disclosure have been described in detail above, it is, however, possible for those of ordinary skill in the art to make various modifications and changes to the embodiments shown without substantially departing from the teaching and advantages of the present disclosure. Such modifications and changes are thus encompassed in the scope of the present disclosure as set forth in the appended claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/MY2021/050118 | 12/10/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63124939 | Dec 2020 | US |
