Ginsenoside M1 as a modulator of angiotensin-regulating enzymes and its use for the treatment of diseases or illnesses, including symptoms caused by coronaviruses

Abstract

The present invention relates to a novel use of ginsenoside M1 as a modulator of angiotensin-regulating enzymes and the treatment of infections caused by coronaviruses with ginsenoside M1.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 978,995, filed February 20, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a novel use of ginsenoside M1 as a modulator of angiotensin-regulating enzymes and related therapeutic methods using same. In particular, the present invention provides a method for treating diseases or conditions associated with an imbalance of angiotensin-regulating enzymes, including those caused by viral infections and the resulting organ or tissue damage, using ginsenoside M1. [Background technology]

[0003] The renin-angiotensin system (RAS) or renin-angiotensin-aldosterone system (RAAS) is a hormonal system. When massive blood loss or a drop in blood pressure occurs, this system is activated, which is an important hormone that regulates blood pressure and fluid levels in the body. Both angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) play important roles in the RAS. ACE2 converts angiotensin I (Ang I) to angiotensin (Ang)-(1-9) or Ang II to angiotensin (Ang)-(1-7) to maintain fluid balance. The RAS plays an important role in many cardiovascular diseases. Current research has shown that ACE2 deficiency can lead to acute or chronic lung injury or even pulmonary fibrosis. Meanwhile, excessive activation of Ang II can lead to vascular and kidney damage, progression to necrosis and fibrosis. Therefore, ACE2 deficiency can lead to excessive accumulation of Ang II, which can cause damage to the lungs and kidneys. However, recent research has shown that the S protein of the novel coronavirus binds to ACE2 and enters the lungs. Excessive accumulation of Ang II in the body can lead to pulmonary fibrosis and kidney damage.[1]

[0004] Coronaviruses pose serious health threats to humans and other animals. From 2002 to 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) infected 8,000 people with a case fatality rate of ~10%. Since 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has infected over 1,700 people with a case fatality rate of ~36%. Since 2013, porcine epidemic diarrhea coronavirus (PEDV) has swept across the United States, causing a nearly 100% case fatality rate among piglets and wiping out over 10% of the U.S. pig population in less than a year. Furthermore, a novel coronavirus (also known as SARS-CoV-2) has caused the COVID-19 pandemic. According to the Wikipedia website, as of February 6, 2021, more than 105 million confirmed cases and over 2.29 million deaths have been attributed to COVID-19.

[0005] In general, coronaviruses cause a wide range of respiratory, gastrointestinal, and central nervous system diseases in humans and other animals, threatening human health and resulting in economic losses. Coronaviruses can adapt to new environments through relatively easy mutation and recombination, and are therefore programmed to efficiently change their host range and tissue tropism. Therefore, the health threat posed by coronaviruses is persistent and long-term. Understanding coronavirus virology and controlling their spread has important implications for global health and economic stability.

[0006] Coronaviruses belong to the Coronaviridae family in the Nidovirales order. They can be classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. Among them, alphacoronaviruses and betacoronaviruses infect mammals, gammacoronaviruses infect birds, and deltacoronaviruses infect both mammals and birds. Representative alphacoronaviruses include human coronavirus NL63 (HCoV-NL63), transmissible porcine gastroenteritis coronavirus (TGEV), PEDV, and porcine respiratory coronavirus (PRCV). Representative betacoronaviruses include SARS-CoV, MERS-CoV, bat coronavirus HKU4, murine hepatitis coronavirus (MHV), bovine coronavirus (BCoV), and human coronavirus OC43. Representative gammacoronaviruses and deltacoronaviruses include avian infectious bronchitis coronavirus (IBV) and porcine deltacoronavirus (PdCV), respectively. Coronaviruses are large, enveloped, positive-strand RNA viruses. They have the largest genomes of all RNA viruses, typically ranging from 27 to 32 kb. The genome is packaged within a helical capsid formed by the nucleocapsid protein (N protein), which is further surrounded by an envelope. At least three structural proteins are associated with the viral envelope. The membrane protein (M protein) and envelope protein (E protein) are involved in virus assembly, while the S protein mediates virus entry into host cells. Some coronaviruses also encode an envelope-associated hemagglutinin esterase protein (HE protein). Among these structural proteins, spikes form large protrusions from the surface of the virus, giving coronaviruses a crowned appearance (hence their name; the Latin word corona means crown). In addition to mediating virus entry, spikes are important determinants of viral host range and tissue tropism and are major inducers of the host immune response. [2]

[0007] Ginsenosides, the major active components of ginseng (Panax ginseng), are known to possess various pharmacological activities, such as antitumor, antifatigue, antiallergic, and antioxidant effects. Ginsenosides share a basic structure consisting of a gonane steroid nucleus with 17 carbon atoms arranged in four rings. Ginsenosides are metallated in the body, and many recent studies have suggested that ginsenoside metabolites are more readily absorbed and act as active ingredients than naturally occurring ginsenosides. Among these, ginsenoside M1, also known as compound K (CK), is known as one of the metabolites of protopanaxadiol-type ginsenosides metabolized by human intestinal bacteria via the gypenoside pathway. To date, there have been no prior art studies reporting the effect of ginsenoside M1 on regulating angiotensin-regulating enzymes and its use for the treatment of related diseases or conditions. Summary of the Invention

[0008] In the present invention, it has been unexpectedly found that ginsenoside M1 is effective in regulating angiotensin-regulating enzymes, including upregulating ACE2 and downregulating ACE, promoting the degradation of angiotensin II, and preventing the accumulation of angiotensin II, and is therefore useful in treating diseases or illnesses associated with an abnormal balance between ACE and ACE2, or the accumulation of angiotensin II, particularly organ or tissue damage caused by viral infections (e.g., coronaviruses).

[0009] Thus, the present invention provides the use of ginsenoside M1 for the manufacture of a medicament as a modulator of angiotensin-regulating enzymes in a subject in need thereof.

[0010] In some embodiments of the invention, the angiotensin-regulating enzyme is selected from the group consisting of angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2).

[0011] In some embodiments, the modulator is an ACE inhibitor, or an ACE2 activator, or both.

[0012] In some embodiments, the modulator is effective in preventing the breakdown of angiotensin II and the accumulation of angiotensin II in a subject.

[0013] In some embodiments, the medicament is effective in treating a disease or condition associated with an imbalance between ACE and ACE2, or an accumulation of angiotensin II, in a subject.

[0014] The present invention also provides a method for treating a disease or condition associated with an abnormal balance between ACE and ACE2 or accumulation of angiotensin II in a subject in need thereof, comprising administering to the subject an amount of ginsenoside M1 effective to treat the subject.

[0015] In some embodiments, ginsenoside M1 is administered in an amount effective to inhibit ACE or activate ACE2, or both, in a subject.

[0016] In some embodiments, ginsenoside M1 is administered in an amount effective to degrade angiotensin II and prevent the accumulation of angiotensin II in a subject.

[0017] In some embodiments, the disease or condition being treated is one associated with an abnormal balance of ACE and ACE2 or accumulation of angiotensin II, including organ or tissue damage of the subject.

[0018] In some embodiments, the damage includes damage in the lungs, gastrointestinal tract, spleen, lymph nodes, heart, kidneys, bladder, liver, gallbladder, adrenal glands and / or testes.

[0019] In some embodiments, the damage is caused by a coronavirus infection.

[0020] In some embodiments, the coronavirus is selected from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and novel coronavirus (2019-nCoV).

[0021] In some embodiments, ginsenoside M1 is administered in combination with one or more additional therapeutic methods or agents useful for degrading angiotensin II and preventing the accumulation of angiotensin II. In one example, such one or more additional therapeutic agents include ACE2.

[0022] In some embodiments, ginsenoside M1 is administered in combination with one or more additional therapeutic agents selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), cytotoxic drugs, immunosuppressants, and vasodilators.

[0023] In some embodiments, ginsenoside M1 is administered in combination with one or more additional therapeutic agents selected from the group consisting of antibiotics, interferons, and antiviral agents.

[0024] Specifically, the present invention provides the use of ginsenoside M1 for manufacturing a medicine for treating lung injury.The present invention also provides a method for treating lung injury by administering an effective amount of ginsenoside M1 to a subject in need thereof.In particular, lung injury is associated with the abnormal balance of ACE and ACE2 or the accumulation of angiotensin II in the subject.

[0025] In some embodiments, the lung injury includes alveolitis, lymphocytic infiltration and / or fibrosis in the lung.

[0026] In some embodiments, ginsenoside M1 is administered by parenteral or enteral routes.

[0027] The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will become apparent from the following detailed description of several embodiments, and from the appended claims. [Brief explanation of the drawings]

[0028] For the purpose of illustrating the invention, there are shown in the drawings embodiments. It is to be understood, however, that the invention is not limited to the preferred embodiments shown. The drawings are as follows:

[0029] [Figure 1] Angiotensin levels in rat kidney tissue are shown. Renal tissue Ang II levels (top panel). Renal tissue Ang-(1-7) levels (bottom panel). The same superscript indicates no significant difference between groups (P>0.05). Groups without the same superscript were significantly different (P<0.05).

[0030] [Figure 2] The levels of angiotensin-converting enzyme (ACE) in rat kidney tissues are shown. The quantitative results of IHC staining, expressed as IOD / area, were proportional to ACE (upper panel) and ACE2 (lower panel) levels. The same superscript letter indicates no significant difference between groups (P>0.05). Groups without the same superscript letter were significantly different (P<0.05).

[0031] [Figure 3]Figure 1 shows angiotensin levels in kidney tissues of mice (infusion model). Renal tissue Ang II levels (upper panel) and Ang-(1-7) levels (lower panel). Identical superscripts indicate no significant difference between groups (P>0.05). Groups without identical superscripts were significantly different (P<0.05).

[0032] [Figure 4] The levels of ACE in mouse kidney tissues are shown. The quantitative results of IHC staining, expressed as IOD / area, were proportional to the ACE level (upper panel) and ACE2 level (lower panel). The same superscript letter indicates no significant difference between groups (P>0.05). Groups without the same superscript letter were significantly different (P<0.05).

[0033] [Figure 5] Figure 1 shows the local pulmonary renin-angiotensin system. ACE2 and M1 ameliorated the histological changes of bleomycin-induced pulmonary fibrosis. In different groups, recombinant mice were administered ACE2 or ACE2 together with M1 and bleomycin on day 0. Szapiel scores were obtained to quantify the degree of alveolitis (upper panel) and fibrosis (lower panel) and the inhibitory efficacy of ACE2 and ACE2 together with M1. Data are expressed as mean ± SEM. *p<0.05 compared to saline group, #p<0.05 compared to BLM. DETAILED DESCRIPTION OF THE INVENTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0035] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0036] The terms "comprise" or "comprising" are generally used in the sense of including / comprising and mean allowing for the presence of one or more features, components, or ingredients. The terms "comprise" or "comprising" encompass the terms "consist of" or "consisting of."

[0037] Ginsenoside M1, also known as compound K(CK) or 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, is one of the saponin metabolites known in the art. The chemical structure of ginsenoside M1 is as follows: [ka]

[0038] Ginsenoside M1 is known as one of the metabolites of protopanaxadiol-type ginsenosides via the gypenosidic pathway by human intestinal bacteria. After ingestion, ginsenoside M1 can be detected in blood or urine. Ginsenoside M1 can be produced from ginseng plants by fungal fermentation using methods known in the art, such as Taiwan Patent Application No. 094116005 (I280982) and U.S. Patent No. 7,932,057 (the contents of which are incorporated herein by reference in their entirety). In certain embodiments, ginseng plants for producing ginsenoside M1 include the Araliaceae family, the genus Panax, such as Korean ginseng (P. ginseng; Panax ginseng) and Himalayan ginseng (P. pseudoginseng) (also known as Sanqi). Generally, the method for producing ginsenoside M1 includes the steps of: (a) providing a powder of ginseng plant material (e.g., leaves or stems); (b) providing a fungus for fermenting the ginseng plant material, wherein the fermentation temperature is in the range of 20-50°C, the fermentation humidity is in the range of 70-100%, the pH value is in the range of 4.0-6.0, and the fermentation period is in the range of 5-15 days; (c) extracting and collecting the fermentation product; and (d) isolating 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol from the fermentation product.

[0039] When ginsenoside M1 is described as "isolated" or "purified" in the present invention, it should be understood that it is not completely isolated or purified, but is isolated or purified to a certain extent. For example, purified ginsenoside M1 represents a more purified form compared to its naturally occurring form. In one embodiment, a preparation containing purified ginsenoside M1 may contain ginsenoside M1 in an amount of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% (w / w) of the total amount of the preparation. When a specific number is used herein to indicate a ratio or dosage, the number should generally be understood to include a range of 10% more or less than that number, more specifically, a range of 5% more or less than that number.

[0040] As used herein, the term "individual" or "subject" includes humans and non-human animals, such as pet animals (dogs, cats, etc.), livestock (cows, sheep, pigs, horses, etc.), or laboratory animals (rats, mice, guinea pigs, etc.).

[0041] As used herein, the term "treating" means applying or administering a composition comprising one or more active agents to a disorder, a symptom or condition of a disorder, the progression of a disorder, or a subject at risk of a disorder, for the purpose of treating, curing, alleviating, mitigating, altering, curing, ameliorating, improving, or affecting the disorder, a symptom or condition of a disorder, the harm caused by a disorder, or the onset or progression of a disorder.

[0042] As used herein, the term "effective amount" refers to the amount of active ingredient that can provide the desired therapeutic effect to the treatment subject.In some embodiments, the effective amount herein can be the amount that is effective for preventing the downregulation of ACE, the upregulation of ACE2, the degradation of angiotensin II, and / or the accumulation of angiotensin II.In some embodiments, the effective amount herein can be the amount that is effective for alleviating or reducing organ or tissue damage caused by the accumulation of angiotensin II, for example, lung damage (such as alveolitis, pulmonary lymphocytic infiltration, and / or pulmonary fibrosis).

[0043] The therapeutically effective amount may vary depending on various factors, such as the route and frequency of administration, the body weight and species of the individual receiving the drug, and the purpose of administration. Those skilled in the art can determine the dosage for each case based on the disclosures herein, established methods, and their own experience. For example, in certain embodiments, the oral dosage of ginsenoside M1 used in the present invention is 10-1,000 mg / kg per day. In some examples, the oral dosage of ginsenoside M1 used in the present invention is 100-300 mg / kg per day, 50-150 mg / kg per day, 25-100 mg / kg per day, 10-50 mg / kg per day, or 5-30 mg / kg per day. Furthermore, in some embodiments of the present invention, ginsenoside M1 is administered regularly, for example, by daily administration, for a specific period of time, for example, at least 15 days, one month, two months, or more.

[0044] In one embodiment, a therapeutically effective amount of the active ingredient can be formulated with a pharmaceutically acceptable carrier into a pharmaceutical composition in a form suitable for delivery and absorption purposes. Depending on the method of administration, the pharmaceutical composition of the present invention preferably contains about 0.1% to about 100% by weight of the active ingredient, where the weight percentage is calculated based on the weight of the total composition.

[0045] As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition, preferably capable of stabilizing the active ingredient, and safe for the individual being treated. The carrier can be a diluent, vehicle, excipient, or matrix for the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, gum arabic, calcium phosphate, alginate, gum tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The composition may further contain lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; and flavoring agents. The compositions of the present invention can provide rapid, sustained, or delayed release of the active ingredient after administration to a patient.

[0046] According to the present invention, the compositions may be in the form of tablets, pills, powders, lozenges, packets, troches, elixirs, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injection solutions, and packaged powders.

[0047] The compositions of the present invention can be delivered via physiologically acceptable routes, including oral, parenteral (intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal administration. For parenteral administration, they are preferably used in the form of a sterile aqueous solution, which may contain sufficient salts or other substances, such as glucose, to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of 3 to 9), if necessary. Preparation of suitable parenteral compositions under sterile conditions can be accomplished using standard pharmacological techniques well known to those skilled in the art and requires no additional creative effort.

[0048] According to the present invention, ginsenoside M1 can be used as an active ingredient for treating diseases or conditions associated with an abnormal balance between ACE and ACE2 or the accumulation of angiotensin II in a subject. In some embodiments, such diseases or conditions include damage to a subject's organs or tissues, such as damage to the lungs, gastrointestinal tract, spleen, lymph nodes, heart, kidneys, bladder, liver, gallbladder, adrenal glands, and / or testes. In certain examples, the damage is caused by infection with a coronavirus, including, but not limited to, SARS-CoV, MERS-CoV, and 2019-nCoV.

[0049] In some embodiments, ginsenoside M1 or a composition comprising ginsenoside M1 as an active ingredient can be used in combination with existing treatment methods or therapeutic agents, such as those for treating diseases or conditions associated with the abnormal balance between ACE and ACE2 or the accumulation of angiotensin II, as described herein.In one example, ginsenoside M1 is administered together with ACE2.Therefore, a specific combination of ginsenoside M1 and ACE2 can be administered to a subject in need thereof, as described herein.

[0050] In some embodiments, ginsenoside M1 or compositions containing ginsenoside M1 as an active ingredient can be used in combination with corticosteroids (such as prednisolone), nonsteroidal anti-inflammatory drugs (NSAIDs), cytotoxic drugs (such as cyclophosphamide, chlorambucil, and azathioprine), immunosuppressants (such as cyclosporine and mycophenolate mofetil), and vasodilators (such as angiotensin-converting enzyme inhibitors (ACE inhibitors)).

[0051] In one particular embodiment, ginsenoside M1 is administered in combination with ACE2 for use in treating lung injury, such as alveolitis, lymphocytic infiltration and / or fibrosis in the lung.

[0052] In some embodiments, ginsenoside M1 is administered in combination with an antibiotic, an interferon, and an antiviral agent.

[0053] In one embodiment, the combined agents or treatment methods can be used simultaneously (concurrently) or sequentially. When combined, the agents can be mixed in the same formulation or can be in separate formulations, such as separate capsules, pills, tablets, and injections.

[0054] The present invention is further described by the following examples, which are offered for purposes of illustration and not limitation. Those of skill in the art will, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present invention. [Example]

[0055] Ginsenoside M1, 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (hereinafter referred to as LCHK168), also known as compound K (CK), was prepared by methods known in the art, such as those described in Taiwan Patent Application No. 094116005 (I280982) and U.S. Patent No. 7,932,057.

[0056] Previous studies have shown that the spike protein (S protein) on coronaviruses binds to ACE2, and the amount of ACE2 affects lung and kidney health (ACE2 Expression in Kidney and Testis May Cause Kidney and Testis Damage After 2019-nCoV Infection https: / / doi.org / 10.1101 / 2020.02.12.20022418, Posted February 13, 2020; and Angiotensin-Converting Enzyme 2 Attenuates Bleomycin-Induced Lung Fibrosis in Mice, Cellular Physiology and Biochemistry, 2015;36:697-711). In this study, ginsenoside M1 was used to regulate ACE2 content and maintain a healthy body.

[0057] Angiotensin II (Ang II)-mediated kidney injury represents a major pathogenic mechanism in most chronic kidney diseases. We found that ginsenoside M1 attenuates Ang II expression by upregulating angiotensin-converting enzyme 2 (ACE2) in renal tissue. We confirmed this finding in a mouse model of kidney injury infused with exogenous Ang II, and showed that the results were consistent across the two models. Local renin-angiotensin system (RAS) activation plays an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). ACE2 may inhibit RAS-mediated epithelial injury and fibrosis, and ACE2 deficiency may exacerbate acute and chronic lung injury.

[0058] 1. Materials and Methods 1.1 Animals and treatments 1.1.1 Rats (renal injury) A total of 20 Wistar-Kyoto rats (WKY) and 20 SHR (male, 16-17 weeks old) were divided into four groups: WKY group (8 WKY, orally administered 0.5% CMC-Na); SHR group (8 SHR, orally administered 0.5% CMC-Na); WKY + ginsenoside M1 group (8 WKY, orally administered 20 mg / kg ginsenoside M1); and SHR + ginsenoside M1 group (8 SHR, orally administered 20 mg / kg ginsenoside M1). Ginsenoside M1 or placebo was administered once daily for 42 days, after which the animals were sacrificed and blood and kidney tissue samples were collected. Renal tissue specimens were either fixed in 4% formalin (histopathology) or snap-frozen in liquid nitrogen and stored at −80°C for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA).

[0059] 1.1.2 Mouse (injection model) (renal injury) A total of 20 C57BL / 6 mice (male, 10 weeks old) were fed rodent chow and water ad libitum. Ang II (1.5 mg / kg) was either subcutaneously implanted in a 1002 osmotic minipump on the back of the neck or infused with normal saline. Animals were then assigned to one of four groups: saline group (5 mice, infused with saline and orally administered 0.5% CMC-Na); Ang II group (5 mice, infused with Ang II and orally administered 0.5% CMC-Na); saline + ginsenoside M1 group (5 mice, infused with saline and orally administered ginsenoside M1 at 20 mg / kg-1 d-1); and Ang II + ginsenoside M1 group (5 mice, infused with Ang II and orally administered ginsenoside M1 at 20 mg / kg-1 d-1). Ginsenoside M1 or placebo was administered daily for 14 days, after which the animals were sacrificed and blood and kidney tissue samples were collected. Kidney tissue specimens were either fixed in 4% formalin (histopathology) or snap-frozen in liquid nitrogen and stored at -80°C (RT-qPCR and ELISA).

[0060] 1.1.3 Mice (lung injury) Animals were randomly divided into five groups: (1) saline group: 0.9% saline (200 μL) was injected via the tail vein; (2) ACE2 group: ACE2 (0.2 mg / kg) was injected intraperitoneally once; (3) BLM group: 150 mg / kg bleomycin was injected via the tail vein once; (4) BLM + ACE2 group: 150 mg / kg bleomycin was injected via the tail vein followed by 0.2 mg / kg ACE2 intraperitoneally; (5) BLM + ACE2 + M1 group: 150 mg / kg bleomycin was injected via the tail vein followed by 0.2 mg / kg ACE2 and 60 mg / kg M1 intraperitoneally. Ten randomly selected animals from each group were euthanized by cervical dislocation on days 7, 14, and 28.

[0061] 1.2 Histopathological evaluation Renal tissue specimens were fixed in 4% formalin, embedded in paraffin, sectioned at 4 μm, stained with hematoxylin and eosin (H&E) and Masson's trichrome stain, respectively, and analyzed under a Nikon E100 light microscope.

[0062] 1.3 Immunohistochemistry (IHC) ACE and ACE2 primary antibodies were produced by Bioss Antibodies. Peroxidase-conjugated goat anti-rabbit secondary antibody, DAB, and the 2-step rabbit IHC kit were provided by ZSGB-BIO. IHC was performed according to their recommendations.

[0063] 1.4 Assessment of Ang II and Ang-(1-7) levels in renal tissue The supernatants from kidney specimens were prepared as follows: a total of 100 mg of kidney tissue was homogenized in 900 μL of ice-cold saline and centrifuged (1000 g, 4°C for 15 minutes). The resulting supernatants were stored at -80°C until analyzed with Ang II and Ang-(1-7) ELISA kits, respectively, according to the manufacturer's protocol.

[0064] 2.Results 2.1 Ginsenoside M1 reduces Ang II levels in kidney tissue of SHR: There are two main ACEs that regulate Ang II levels in vivo: ACE and ACE2. ACE converts angiotensin I to Ang II, and ACE2 converts Ang II to Ang-(1-7). Therefore, upregulation of ACE induces an increase in Ang II, whereas upregulation of ACE2 attenuates this increase. Spontaneously hypertensive rats (SHRs) are an animal model of genetic hypertension. ELISA data (Figure 1) showed that Ang II levels in the renal tissue of SHRs were significantly higher than those of the WKY and WKY + ginsenoside M1 groups. Treatment with ginsenoside M1 significantly reduced Ang II levels in the renal tissue of SHRs, whereas it had limited effect in the WKY group. Ang-(1-7) levels in the SHR group were significantly higher than those of the WKY and WKY + ginsenoside M1 groups, and the SHR + ginsenoside M1 group showed even significantly higher levels compared to the SHR group. The IHC results (Figure 2) showed that ACE levels were significantly elevated in the two SHR groups compared with the two WKY groups. The amount of ACE2 in renal tissue was compensatory upregulated in the SHR group compared with the WKY and WKY + ginsenoside M1 groups. Meanwhile, ginsenoside M1 treatment further upregulated ACE2 in renal tissue of SHR. This was the primary mechanism by which ginsenoside M1 treatment downregulated Ang II and upregulated Ang-(1-7) in the kidneys of SHR. Meanwhile, ginsenoside M1 did not significantly affect ACE expression in either WKY or SHR.

[0065] 2.2 Ginsenoside M1 reduces Ang II levels in kidney tissue of Ang II-injected mice: ELISA data (Figure 3) showed that Ang II infusion significantly increased Ang II levels in mouse kidney tissues. Ginsenoside M1 treatment significantly attenuated this increase; however, Ang II levels in the Ang II + ginsenoside M1 group were still significantly higher than those in the two saline-injected groups. Meanwhile, Ang-(1-7) levels in the Ang II group were significantly higher than those in the saline and saline + ginsenoside M1 groups. The Ang II + ginsenoside M1 group showed a significant increase compared with the Ang II group. IHC data (Figure 4) showed no significant differences in ACE expression among the various group pairs. ACE2 expression levels in the Ang II + ginsenoside M1 group were significantly elevated compared with the other three groups, which may be the primary mechanism of exogenous angiotensin degradation.

[0066] 2.3 Treatment of alveolitis and fibrosis by M1 and ACE2: As shown in Figure 5, a single injection of bleomycin at a dose of 150 mg / kg via the tail vein of mice induced mild to moderate alveolitis on day 7. Bleomycin administration is a well-characterized model of alveolitis or fibrosis in mice. Lung fibrosis was determined using the Szapiel scoring method, with higher scores indicating greater disease severity. The lungs showed focal alveolitis, primarily subpleural and perivascular, with mildly thickened alveolar septa with edema and inflammatory cell infiltration, but no obvious fibrosis was observed. At the same time, ACE2 administration did not significantly alter the pathological changes on day 7. On day 14, the BLM group showed disease progression. The lungs showed moderate to severe alveolitis and moderate fibrosis with patchy inflammatory consolidation and collagen deposition. ACE2 administration significantly reduced both alveolitis and fibrosis. On day 28, alveolitis spontaneously resolved in the BLM group; however, abnormalities in alveolar structure persisted, and fibrosis persisted, characterized by diffuse, dense, thick collagen bundles and fibroblastic foci. ACE2 significantly reflected the long-term therapeutic effect on day 28; sections from the BLM + ACE2 group showed only mild to moderate alveolitis, while sections from the BLM + ACE2 + M1 group showed only mild alveolitis and a significant reduction in fibrosis. No signs of alveolitis or fibrosis were observed in the saline or ACE2 groups at various time points.

[0067] References 1. Journal of Molecular Medicine / October 2006, Volume 84, Issue 10, pp 814-820| Cite as / Lessons from SARS: control of acute lung failure by the SARS receptor ACE2. Pharmacology & Therapeutics / Volume 128, Issue 1, October 2010, Pages 119-128 / Trilogy of ACE2: A peptidase in the renin-angiotensin system, a SARS receptor, and a partner for amino acid transporters. 2. Published in final edited form as:Annu Rev Virol. 2016 September 29; 3(1): 237-261. doi:10.1146 / annurev-virology-110615-042301. / Structure, Function, and Evolution of Coronavirus Spike Proteins / Fang Li Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 3. Antioxidant Treatment and Alcoholism / Camila S. Silva PhD, ... Helio Vannucchi MD, PhD, in Molecular Aspects of Alcohol and Nutrition, 2016 4. Beta-Glucosidase From Penicillium / Gustavo Molina, ... Glaucia M. Pastore, in New and Future Developments in Microbial Biotechnology and Bioengineering, 2018 5. Ginseng and Gastrointestinal Protection* / Min-Hyun Kim, Hyeyoung Kim, in Gastrointestinal Tissue, 2017

Claims

1. A pharmaceutical composition for treating alveolitis and associated fibrosis in a subject in need thereof, comprising effective amounts of ginsenoside M1 and angiotensin-converting enzyme 2 (ACE2), and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the effective amount of ginsenoside M1 is an amount effective to activate ACE2 in a subject.

3. The pharmaceutical composition of claim 1, wherein ginsenoside M1 is combined with one or more additional therapeutic agents selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), cytotoxic drugs, immunosuppressants, and vasodilators.

4. 10. The pharmaceutical composition of claim 1, wherein ginsenoside M1 is combined with one or more additional therapeutic agents selected from the group consisting of antibiotics, interferons, and antiviral agents.

5. Use of ginsenoside M1 and angiotensin-converting enzyme 2 (ACE2) for the manufacture of a medicine for treating alveolitis and associated fibrosis.

6. An agent for treating alveolitis and associated fibrosis, comprising a combination of ginsenoside M1 and angiotensin-converting enzyme 2 (ACE2).

7. The agent described in claim 6, wherein the combination of ginsenoside M1 and angiotensin-converting enzyme 2 (ACE2) is further combined with one or more additional therapeutic agents or treatment methods, and the one or more additional therapeutic agents or treatment methods are selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), cytotoxic drugs, immunosuppressants, vasodilators, antibiotics, interferons, and antiviral agents.

8. The agent according to claim 7, wherein ginsenoside M1 is administered before, after, or simultaneously with angiotensin-converting enzyme 2 (ACE2) and an additional therapeutic agent or method.

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