A traditional Chinese medicine compound and use thereof

Abstract

The present application relates to the field of traditional Chinese medicine, and specifically relates to the use of a Chinese herbal medicine combination in the preparation of a medicament for the treatment of inflammatory bowel disease, in particular ulcerative colitis, a method of preparing a medicament comprising the Chinese herbal medicine combination, and a method of treating an individual having inflammatory bowel disease, in particular ulcerative colitis.

Description

[0001] Cross-reference to related applications

[0002] This invention claims the benefit of U.S. Provisional Application 63 / 346,332, filed May 27, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of traditional Chinese medicine, specifically to the use of a combination of traditional Chinese herbs in the preparation of a medicament for treating inflammatory bowel disease, a method for preparing the medicament comprising the traditional Chinese herbal compound, and a method for treating an individual suffering from inflammatory bowel disease. Background Technology

[0004] Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract that affects the entire gastrointestinal tract and colon. IBD is inflammation or destruction of the intestinal wall, which can lead to intestinal ulcers and strictures. The two most common types of IBD are ulcerative colitis and Crohn's disease. For many patients, IBD is hereditary, but no single gene has been proven to cause it. Typically, the severity of the disease depends on a combination of genetic abnormalities and the effects of any number of microbes on the immune system. The most common symptom of IBD is diarrhea. Other symptoms include abdominal cramps, bloody stools, bowel obstruction, fever, fluid loss, loss of appetite, extreme weight loss, and anemia.

[0005] In today's industrialized world, approximately 5 million people suffer from inflammatory bowel disease (IBD). In Europe, the incidence rates of ulcerative colitis and Crohn's disease are 0.5% and 1%, respectively. In recent years, the number of IBD patients in Asia, particularly China, has increased dramatically. The main characteristics of IBD are diarrhea, abscesses, fistulas, abdominal pain, and strictures, all of which significantly impact patients' quality of life.

[0006] Current treatments for inflammatory bowel disease include immunosuppressants and steroids. Humanized antibodies that neutralize inflammatory cytokines or anti-integrins to block the interaction of circulating leukocytes and intestinal blood vessels. The first FDA-approved neutralizing antibody for the treatment of inflammatory bowel disease is anti-tumor necrosis factor (TNF) (a monoclonal antibody), infliximab (IFLX). Infliximab specifically binds to cytosolic and membrane-bound TNF-alpha and subsequently induces apoptotic cell death in cells in a proinflammatory state. However, long-term use of infliximab can increase the remission of clinical symptoms in patients with inflammatory bowel disease. Infliximab and many other biologics as the nature of exogenous proteins also lead to the production of anti-drug antibodies and an increased risk of malignancy. A five-year study showed that long-term use of ustekinumab, a humanized monoclonal antibody against interleukin (IL)-12 / IL-23, can relieve the condition of patients with moderate to severe Crohn's disease. Anti-integrin drugs, such as vedolizumab, also show a high maintenance rate of remission. Long-term use of immunosuppressants such as mesalazine increases the susceptibility of patients to infection. In addition, corticosteroids are not recommended for use in maintaining remission due to serious side effects. There is an urgent need for more effective therapies with different modes of action and lack of immunogenicity for long-term use in patients with inflammatory bowel disease, especially for patients in remission. SUMMARY

[0007] To solve the above technical problems, in one aspect, the present application provides a Chinese herbal compound JCM-16021, which comprises Fructus Chebulae, Radix Paeoniae Alba, Magnolia officinalis, Corydalis, Radix Polygoni Multiflori, Rhizoma Atractylodis Macrocephalae, and Coicis Semen.

[0008] In another aspect, the present application also relates to the use of the Chinese herbal compound JCM-16021 in the preparation of a medicament for treating inflammatory bowel disease. In some embodiments, the disease is ulcerative colitis and Crohn's disease.

[0009] In another aspect, the present application also relates to a method for preparing a medicament comprising the Chinese herbal compound JCM-16021.

[0010] The advantage of the present application is that the in vivo experimental results show that JCM-16021 has significant therapeutic effect in CD4 + JCM-16021 showed significant therapeutic effect in two mouse models of ulcerative colitis induced by naive T cell transfer and dextran sulfate sodium. At a dose of 2.0 g / kg body weight, JCM-16021 significantly reduced the pathological indicators of ulcerative colitis, promoted the proliferation of stem cells in the colonic crypt area, and reduced systemic inflammation in experimental colitis model mice. In addition, JCM-16021 treatment can increase the proportion of CD4 + Foxp3 + Regulatory T cells (T regproportion of the total bacteria, restore the composition of intestinal microbiome, and increase the expression of intestinal aryl hydrocarbon receptor. And the overall therapeutic effect of JCM-16021 is superior to the clinically used IFLX, especially in promoting intestinal epithelial cells and CD4 + Foxp3 + T reg cell proliferation.

[0011] Unless otherwise indicated herein or otherwise evident in context, all of the methods described herein can be performed in any suitable order according to an understanding of a person of ordinary skill in the art.

[0012] All patents, patent applications, and references cited herein are incorporated by reference in their entirety as if each had been individually incorporated. In the event of a conflict between the present disclosure and the references incorporated herein by reference, the present disclosure controls. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Therapeutic effects of JCM-16021 in CD4 + Therapeutic effects of JCM-16021 in CD4 - / - Immune-deficient mice were injected intraperitoneally with 2 x 10 5 CD4 + naive T cells. JCM-16021 treatment (oral, twice daily) was initiated three weeks after injection, with IFLX as the positive control drug (intravenous, once a week), and the sham control group was given normal saline orally. (B) Body weight changes in mice of each group during treatment, with arrows indicating the start of JCM-16021 or IFLX treatment. (C) Fecal occult blood index. (D) Ulcerative colitis pathological index. (E) Hematoxylin-eosin staining (H&E staining), periodic acid-schiff staining (PAS staining), Claudin and NF-κB p65 immunohistochemical results of mouse colon tissue sections. Values in (B)-(D) are expressed as mean ± standard error (SEM). Scale bar = 100 μm. Results from three groups of experiments (*, p < 0.05; more than 5 mice per group).

[0014] Figure 2 Therapeutic effects of JCM-16021 in CD4 + naive T cells. JCM-16021 treatment (oral, twice daily) was initiated three weeks after injection, with IFLX as the positive control drug (intravenous, once a week), and the sham control group was given normal saline orally. (B) Body weight changes in mice of each group during treatment, with arrows indicating the start of JCM-16021 or IFLX treatment. (C) Fecal occult blood index. (D) Ulcerative colitis pathological index. (E) Hematoxylin-eosin staining (H&E staining), periodic acid-schiff staining (PAS staining), Claudin and NF-κB p65 immunohistochemical results of mouse colon tissue sections. Values in (B)-(D) are expressed as mean ± standard error (SEM). Scale bar = 100 μm. Results from three groups of experiments (*, p < 0.05; more than 5 mice per group). - / -Inflammatory detection in immunodeficient mice. (A) Immunofluorescence detection of the colocalization of intestinal macrophages (Ibal) with p65 and c-Rel in mouse colon tissue. Relative expression of (B) Tnf-a and (C) Mcp-1 mRNA in mouse colon tissue. Values from qRT-PCR experiments in triplicate (n > 5 per group), data expressed as mean ± standard error (SEM) (**, p < 0.01).

[0015] Figure 3 CD4 + Ulcerative colitis induced by naive T cell transfer in Rag1 - / - Immunodeficient mice (A) Immunofluorescence staining results of PCNA and CD44 in colon tissue. (B) Immunofluorescence results of aryl hydrocarbon receptor in small intestine tissue. Scale bar = 100 pm (more than 5 mice per group).

[0016] Figure 4 CD4 + Ulcerative colitis induced by naive T cell transfer in Rag1 - / - Regulatory T cell proportion in the intestinal lamina propria immune cells of immunodeficient mice. (A) Flow cytometry analysis schematic. (B) CD4 + Foxp3 + T cells in the intestinal lamina propria of immunodeficient mice. (A) Flow cytometry analysis schematic. (B) CD4 + T cell proportion. (B) Values expressed as mean ± standard error (SEM) (*, p < 0.05; more than 5 mice per group).

[0017] Figure 5 CD4 + Ulcerative colitis induced by naive T cell transfer in Rag1 - / - Relative abundance of intestinal microbiota at the species level in immunodeficient mice. (A) Bacteroidales S24-7, (B) Prevotellaceae, (C) Rikenellaceae, (D) Helicobacteraceae, (E) Deferribacteraceae, (F) Rhodospirillaceae, (G) Clostridiaceae, (H) Christensenellaceae species. Values expressed as mean ± standard error (SEM) (more than 5 mice per group; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant).

[0018] Figure 6Therapeutic effects of JCM-16021 in a long-term dextran sulfate sodium-induced ulcerative colitis mouse model. (A) Body weight changes of mice in each group during the treatment. (B) Colon length of mice in each group. (C) Fecal occult blood index. (D) Colitis pathological index. Values are expressed as mean ± standard error (SEM) (*, p < 0.05; **, p < 0.01; ***, p < 0.005; n.s., no significant difference).

[0019] Figure 7 Changes in colonic histopathological indices of 2% dextran sulfate sodium-induced ulcerative colitis mice treated with JCM-16021. (A) Hematoxylin-eosin staining, periodic acid-Schiff staining results of mouse colon tissue sections, scale bar = 100 μm. (B) PCNA and CD44 immunofluorescence staining results in colon tissue, results from three sets of repeated immunofluorescence experiments (n ≥ 5 per group).

[0020] Figure 8 Serum levels of proinflammatory cytokines (A) Ifn-γ, (B) Tnf-α and chemokines (C) Mcp-1, (D) Gm-csf in 2% dextran sulfate sodium-induced ulcerative colitis mice treated with JCM-16021. Values are expressed as mean ± standard error (SEM) (*, p < 0.05; **, p < 0.01; ***, p < 0.005; n.s., no significant difference).

[0021] Figure 9 Relative abundance of intestinal flora at the species level in long-term dextran sulfate sodium-induced ulcerative colitis mice treated with JCM-16021. (A) Dubosiella, (B) Fissicatena, (C) Clostridium, (D) Dubosiella, (E) Lachnospiraceae bacterium. Values are expressed as mean ± standard error (SEM) (more than 5 mice per group; *, p < 0.05; **, p < 0.01; ***, p < 0.005). DETAILED DESCRIPTION

[0022] For the purpose of promoting an understanding of the principles of the application, reference will now be made to the embodiments illustrated in the drawings. It is expressly understood that the application can be performed by other embodiments and modifications, and is not limited to the embodiments described and shown here. The purpose of providing these embodiments is to make the disclosure of the application more thorough and comprehensive.

[0023] The gut microbiota and aryl hydrocarbon receptor (AhR) axis are important modulators in the pathogenesis of inflammatory bowel disease cases such as ulcerative colitis. One major reason for the limited treatment strategies is that the causative factors that initiate inflammatory bowel disease are very complex and largely unresolved. Gut commensal bacteria are fundamental to intestinal homeostasis and metabolism. Loss of balance within the gut microbiota, or dysbiosis, directly activates the immune response within the gut by initiating an innate immune response. Dysbiosis also leads to impaired metabolism, including the biosynthesis of 5-hydroxytryptamine (5-HT). Microbiota-mediated tryptophan metabolites, e.g., the tryptophan metabolite indole-3-aldehyde, directly activate AhR-dependent IL-22 transcription as ligands. IL-22-dependent mucosal immune responsiveness thus alters the composition of the microbiota and the intestinal immune response. Thus, the gut microbiota-AHR axis is an important therapeutic target for the treatment of inflammatory bowel disease.

[0024] T reg Cell therapy is promising in the treatment of inflammatory bowel disease. In inflammatory bowel disease, chronic intestinal inflammation leads to CD4 + Foxp3 + T reg cell dysfunction in vivo. In turn, impaired T reg cells impair robust tissue repair and regeneration, thus further amplifying the inflammatory response. Therefore, restoring the suppressive function of T reg cells in the context of intestinal inflammation is very promising in the treatment of inflammation and autoimmune diseases, including inflammatory bowel disease. However, manipulating T reg cell numbers or abundance to achieve homeostasis of the immune response is very challenging. Only a few compounds show activity to alter T reg / T helper (T h )17 balance or to activate T reg cell immunosuppressive function. Alternatively, approaches to engineer chimeric antigen receptor co-receptors on the cell surface are used to direct T reg cells to target antigens. Research to manipulate T reg cells in inflammation and autoimmune diseases is still in its initial stages to assess the efficacy of autologous ex vivo expanded T reg cells back to the donor patient. There is still a critical gap between bioengineering T reg cells to a clinically approved therapy for inflammatory bowel disease.

[0025] Herbal composition JCM-16021 showed significant efficacy for ulcerative colitis treatment with a novel mechanism of action. Herbal composition JCM-16021 was modified from Tong Xie Yao Fang, which has been used in China since the 12th century for the treatment of abdominal pain and diarrhea. JCM-16021 reduced visceral allodynia in rats of the post-inflammatory (PI)-IBS model induced by trinitrobenzene sulfonic acid. Treatment with JCM-16021 significantly improved the number of intestinal chromaffin cells, intestinal 5-HT content, tryptophan hydroxylase expression, and 5-HT release induced by mechanical stimulation. In addition, JCM-16021 reduced T h 1Cell-associated levels of the pro-inflammatory cytokine TNF-a.

[0026] The inventors' preliminary data showed that JCM-16021 in Rag 1 - / - naive CD4 + T cell transfer-induced ulcerative colitis and dextran sulfate sodium (DSS)-induced experimental colitis in C57BL / 6J mice. In the experimental colitis model, JCM 16021 at a dose of 2.0 g / kg significantly induced recovery of ulcerative colitis pathology, promoted proliferation of stem cells in the colonic crypt area, and reduced systemic inflammation. In addition, JCM-16021 treatment promoted the proportion of CD4 + Foxp3 + T reg cells in the lamina propria (LP), restored the composition of the intestinal microbiome, and the intestinal expression of AHR. The inventors' findings provide key evidence that the overall efficacy of JCM 16021 is superior to IFLX, which is used clinically, especially in promoting intestinal regeneration and CD4 + Foxp3 + T reg cell populations. The effective dose of JCM-16021 on the experimental ulcerative colitis model is 1-4 g / kg body weight, preferably 2 g / kg body weight.

[0027] Definitions

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those of a person of ordinary skill in the art to which the application belongs, but in case of conflict, the meaning in the present specification prevails.

[0029] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0030] All numerical values or expressions involving amounts of components, process conditions, and so forth as used in the specification and claims are understood to be preceded by the word "about", unless otherwise expressly stated. The term "about" when used in reference to a quantity or a numerical range means that the quantity or the numerical range is an approximation within experimental variability (or within statistical experimental error), in the present application, the term "about" shall have the meaning of within 10%, preferably within 5%, of the indicated value or range.

[0031] All ranges involving same components or properties include the endpoints, which can be combined independently. Because the ranges are continuous, they include every value between the minimum and the maximum. It is also understood that any numerical range recited in this application is intended to include all sub-ranges of the same entire range.

[0032] When the application is defined as a range in reference to a physical property such as molecular weight or to a chemical property, all combinations and subcombinations of the range as well as specific embodiments therein are intended to be included. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes such embodiments as those "consisting essentially of" or "consisting of."

[0033] "and / or", when used in the specification and in the claims, is to be interpreted that the associated components are "one or the other or both". Multiple components listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the associated components. Other components can be added to those components explicitly identified, whether related or unrelated, and additional components can be eliminated from those components explicitly identified, without departing from the spirit and scope of the application. Thus, as a non-limiting example, a reference to "A and / or B", when used in the conjunctive form, refers to A alone (optionally including components other than B); to B alone (optionally including components other than A); or to both A and B (optionally including additional components). As a non-limiting example, a reference to "A, B, and / or C", when used in the disjunctive form, refers to A alone; to B alone; to C alone; to A and B together; to A and C together; to B and C together; or to A and B and C together.

[0034] The terms "administering", "administered", or "administration" refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a Chinese herbal compound, an extract thereof, or a pharmaceutical composition described herein into a subject.

[0035] The terms "treat" and "treatment" refer to reversing, alleviating, delaying the onset of or inhibiting the progress of a disease described herein. In some embodiments, treatment can be administered after one or more signs or symptoms of the disease have developed. In other embodiments, treatment can be administered in the absence of signs or symptoms of the disease. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen) in order to delay or prevent development of the disease. Treatment can also be continued after symptoms have resolved, for example, to delay and / or prevent recurrence.

[0036] The terms "condition," "disease," and "disorder" are used interchangeably.

[0037] An "effective amount" of a compound described herein refers to an amount that is sufficient to elicit the desired biological response. The effective amount of a compound described herein can vary depending on the intended biological endpoint, the pharmacodynamics of the compound, the disorder being treated, the mode of administration, and the age and health of the subject. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactically effective amount. In some embodiments, the effective amount is the amount of a Chinese herbal compound or drug described herein in a single dose. In some embodiments, the effective amount is the combined amount of a Chinese herbal compound or drug described herein in multiple doses.

[0038] A "therapeutically effective amount" of a compound described herein is an amount that is sufficient to provide a therapeutic benefit in the treatment of a disorder or to delay or minimize one or more symptoms associated with the disorder. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disorder. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or eliminates symptoms, signs, or causes of a disorder, and / or enhances the therapeutic efficacy of another therapeutic agent.

[0039] The term "pharmaceutically acceptable" indicates that the substance or composition is chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the subject being treated with it.

[0040] A "subject" or "individual in need thereof for administration includes, but is not limited to, a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)) and / or a non-human animal, e.g., a mammal, e.g., a primate (e.g., a cynomolgus monkey, a rhesus monkey), a cow, a pig, a horse, a sheep, a goat, a rodent, a cat, and / or a dog. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," "individual in need thereof, and "subject" are used interchangeably herein.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0042] Chinese herbal compound and pharmaceutical composition

[0043] In one aspect of the application, the present application relates to a Chinese herbal compound JCM-16021 (herein also referred to as JCM-16021) comprising the following components: Fructus Terminaliae Chebulae, Radix Paeoniae Lactiflorae, Cortex Magnoliae Officinalis, Rhizoma Corydalis Yanhusuo, Herba Polygoni Chinensis, Rhizoma Atractylodis Macrocephalae, Semen coicis Lachryma-jobi. In one embodiment, the present application relates to a Chinese herbal compound consisting of the following components: Fructus Terminaliae Chebulae, Radix Paeoniae Lactiflorae, Cortex Magnoliae Officinalis, Rhizoma Corydalis Yanhusuo, Herba Polygoni Chinensis, Rhizoma Atractylodis Macrocephalae, Semen coicis Lachryma-jobi.

[0044] In some embodiments, the components in the above Chinese herbal compound have the following weight ratios: Fructus Terminaliae Chebulae, 6-12% w / w; Radix Paeoniae Lactiflorae, 11-17% w / w; Cortex Magnoliae Officinalis, 6-12% w / w; Rhizoma Corydalis Yanhusuo, 11-17% w / w; Herba Polygoni Chinensis, 15-21% w / w; Rhizoma Atractylodis Macrocephalae, 15-21% w / w; Semen coicis Lachryma-jobi, 15-21% w / w.

[0045] In some embodiments, the weight ratio of Fructus Terminaliae Chebulae in the above Chinese herbal compound is: 6-11% w / w, 6-10% w / w, 6-9% w / w, 6-8% w / w, 6-7% w / w, preferably 9% w / w.

[0046] In some embodiments, the weight ratio of Radix Paeoniae Lactiflorae in the above Chinese herbal compound is: 11-16% w / w, 11-15% w / w, 11-14% w / w, 11-13% w / w, 11-12% w / w, preferably 14% w / w.

[0047] In some embodiments, the weight ratio of Cortex Magnoliae Officinalis in the above Chinese herbal compound is: 6-11% w / w, 6-10% w / w, 6-9% w / w, 6-8% w / w, 6-7% w / w, preferably 9% w / w.

[0048] In some embodiments, the weight ratio of Corydalis in the above-mentioned Chinese herbal compound is: 11-16% w / w, 11-15% w / w, 11-14% w / w, 11-13% w / w, 11-12% w / w, preferably 14% w / w.

[0049] In some embodiments, the weight ratio of Polygonum in the above-mentioned Chinese herbal compound is: 15-20% w / w, 15-19% w / w, 15-18% w / w, 15-17% w / w, 15-16% w / w, preferably 18% w / w.

[0050] In some embodiments, the weight ratio of Atractylodes in the above-mentioned Chinese herbal compound is: 15-20% w / w, 15-19% w / w, 15-18% w / w, 15-17% w / w, 15-16% w / w, preferably 18% w / w.

[0051] In some embodiments, the weight ratio of Coix in the above-mentioned Chinese herbal compound is: 15-20% w / w, 15-19% w / w, 15-18% w / w, 15-17% w / w, 15-16% w / w, preferably 18% w / w.

[0052] In some preferred embodiments, the present application relates to a Chinese herbal compound comprising components with the following weight ratios: Chebula (9%), White Peony Root (14%), Magnolia Bark (9%), Corydalis (14%), Polygonum (18%), Atractylodes (18%), and Coix (18%).

[0053] In some embodiments, the Chinese herbal compound is used for promoting the increase of regulatory T cells in an individual in need thereof. In particular, in some embodiments, the Chinese herbal compound is used for treating inflammatory bowel disease in an individual in need thereof. In other embodiments, the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease. In other embodiments, the inflammatory bowel disease is ulcerative colitis.

[0054] In another aspect, the present application also relates to an extract of the above-mentioned Chinese herbal compound. In some embodiments, the extract is an aqueous solvent extract. In some embodiments, the extract is a polyethylene glycol (15)-hydroxystearate extract. In some embodiments, the extract is a distilled water extract. In some embodiments, the extract is a physiological saline extract.

[0055] In another aspect, the present application also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a material or chemical substance from the plant species of Fructus Terminaliae Chebulae, Radix Paeoniae Lactiflorae, Cortex Magnoliae Officinalis, Rhizoma Corydalis Yanhusuo, Herba Polygoni Chinensis, Rhizoma Atractylodis Macrocephalae, Semen coicis Lachryma-jobi. In some embodiments, the pharmaceutical composition consists essentially of Fructus Terminaliae Chebulae, Radix Paeoniae Lactiflorae, Cortex Magnoliae Officinalis, Rhizoma Corydalis Yanhusuo, Herba Polygoni Chinensis, Rhizoma Atractylodis Macrocephalae, Semen coicis Lachryma-jobi. In some embodiments, the pharmaceutical composition is used for treating inflammatory bowel disease in a mammal. In some embodiments, the pharmaceutical composition is used for treating ulcerative colitis in a mammal. In some embodiments, the pharmaceutical composition is used for treating Crohn's disease in a mammal. In some embodiments, the pharmaceutical composition is used as an immunotherapy for promoting the increase of regulatory T cells to treat inflammatory bowel disease, particularly ulcerative colitis.

[0056] In another aspect, the present application also relates to a pharmaceutical composition comprising the above-mentioned Chinese herbal compound and a pharmaceutically acceptable carrier. Preferably, in some embodiments, the pharmaceutically acceptable carrier comprises physiological saline, saccharides, gelatin, starch, Ringer's solution, cellulose.

[0057] In some embodiments, the pH of the pharmaceutically acceptable carrier is 3-11. In some embodiments, the pH of the pharmaceutically acceptable carrier is 4-11. In some embodiments, the pH of the pharmaceutically acceptable carrier is 4-10. In some embodiments, the pH of the pharmaceutically acceptable carrier is 5-10. In some embodiments, the pH of the pharmaceutically acceptable carrier is 5-9. In some embodiments, the pH of the pharmaceutically acceptable carrier is 6-9. In some embodiments, the pH of the pharmaceutically acceptable carrier is 6-8. In some embodiments, the pH of the pharmaceutically acceptable carrier is 7-8. Preferably, in some embodiments, the pH of the pharmaceutically acceptable carrier is 5-9, and more preferably, in some embodiments, the pH of the pharmaceutically acceptable carrier is 7-8.

[0058] In some embodiments, the herbal compound is provided in the medicament in an effective amount. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is an amount effective to treat inflammatory bowel disease in a subject in need thereof. In some embodiments, the effective amount is an amount effective to treat ulcerative colitis in a subject in need thereof. In some embodiments, the effective amount is an amount effective to treat Crohn's disease in a subject in need thereof. In some embodiments, the effective amount is an amount effective to promote an increase in regulatory T cells in a subject.

[0059] In some embodiments, the pharmaceutical composition is administered at a dosage of about 1-4 g / kg (subject body weight). In some preferred embodiments, the pharmaceutical composition is administered at a dosage of about 2 g / kg (subject body weight).

[0060] In some embodiments, the pharmaceutical composition can be administered in multiple intervals of a particular dosage, for example, 2 times per day, 1 time per day, 6 times per week, 5 times per week or less.

[0061] In some embodiments, the pharmaceutical composition can be administered by inhalation, intranasally, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, orally.

[0062] In some embodiments, the pharmaceutical composition can be used in combination with other drugs, preferably other drugs in a therapeutically effective amount, to treat inflammatory bowel disease.

[0063] In some embodiments, the pharmaceutical composition can be used in combination with other drugs, preferably other drugs in a therapeutically effective amount, to treat ulcerative colitis.

[0064] In some embodiments, the pharmaceutical composition can be used in combination with other drugs, preferably other drugs in a therapeutically effective amount, to treat Crohn's disease.

[0065] In some embodiments, the pharmaceutical composition is provided in the form of tablets, pills, dripping pills, capsules, granules, powders, suppositories, powders, pastes, patches, injection solutions, solutions, suspensions, sprays, lotions, drops, rubs, emulsions. The preparation can be conveniently presented in unit dosage form and can be prepared by methods well known in the pharmaceutical art. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences 18th Ed. (1995) Mack Publishing Co., Easton, PA. These methods include the step of bringing into association active ingredient with the carrier which constitutes one or more accessory ingredients. In general the pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product.

[0066] In some embodiments, when the pharmaceutical composition is prepared as a liquid dosage form, the pharmaceutical composition is provided at a concentration of about 0.09-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.10-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.11-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.12-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.13-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.14-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.15-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.16-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.17-0.18 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.09 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.10 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.11 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.12 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.13 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.14 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.15 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.16 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.17 mg / mL. In some embodiments, the pharmaceutical composition is provided at a concentration of about 0.18 mg / mL. In some preferred embodiments, the pharmaceutical composition is provided at a concentration of 0.18 mg / mL.

[0067] Pharmaceutically acceptable carriers (or therapeutically acceptable carriers) are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and / or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent or excipient used will depend upon the means and purpose for which the compound of the application is being applied. Solvents are generally chosen for their medical applicability, i.e., their safety in the subject, as recognized by those skilled in the art (GRAS). Generally, safe solvents are non-toxic water soluble solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG 400, PEG 300), and the like, and mixtures thereof. The pharmaceutically acceptable carriers can further include one or more buffers, excipients, diluents, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, and other known additives to provide extra quality or ease of manufacturing of the drug.

[0068] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, sugars such as sucrose, mannitol, trehalose or sorbitol, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry

[0069] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch acetate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, croscarmellose sodium (Ac-di-sol), sodium starch glycolate (Explotab), carboxymethylcellulose calcium, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0070] Exemplary binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethyl cellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, ethanol, and / or mixtures thereof.

[0071] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, glycerylphosphocholine, calcium lactate, propionic acid, calcium levulinate, valeric acid, monobasic calcium phosphate, phosphoric acid, calcium phosphate, basic calcium phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and mixtures thereof.

[0072] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0073] Liquid dosage forms for oral and parenteral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active agent, the liquid dosage forms can contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butanediol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. alcohols, oils, modified oils, glycols, polysorbate esters, cyclodextrins, polymers, and combinations thereof.

[0074] Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Injectable sterile preparations can be formulated as injectable sterile solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, for example solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, saline, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable

[0075] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is admixed with at least one inert, pharmaceutically-acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents.

[0076] Use of Chinese herbal compound

[0077] In another aspect of the present application, the present application relates to the use of the above-mentioned Chinese herbal compound or pharmaceutical composition in the preparation of a medicament for treating inflammatory bowel disease in an individual in need thereof. In further embodiments, the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease. In further embodiments, the inflammatory bowel disease is ulcerative colitis. In some embodiments, the Chinese herbal compound or pharmaceutical composition is administered in a therapeutically effective amount. In some embodiments, the Chinese herbal compound or pharmaceutical composition is administered with a pharmaceutically acceptable carrier.

[0078] In some embodiments, the present application also relates to the use of the above-mentioned Chinese herbal compound or pharmaceutical composition in the preparation of a medicament for promoting the increase of regulatory T cells in an individual in need thereof.

[0079] In some embodiments, the present application also relates to the use of the above-mentioned Chinese herbal compound or pharmaceutical composition in the preparation of a medicament for increasing CD4 + Foxp3 + the proportion of regulatory T cells in an individual in need thereof.

[0080] In some embodiments, the present application also relates to the use of the above-mentioned Chinese herbal compound or pharmaceutical composition in the preparation of a medicament for restoring the composition of intestinal microbiota and restoring the expression of intestinal aryl hydrocarbon receptor in an individual in need thereof.

[0081] In another aspect of the present application, the present application relates to the use of the above-mentioned Chinese herbal compound or pharmaceutical composition in the preparation of a medicament for treating a disease in a mammal. Preferably, the mammal is a human.

[0082] A method for preparing a medicament

[0083] Another aspect of the present application also provides a method for preparing a medicament comprising the Chinese herbal compound, the method comprising: grinding the Chinese herbal compound into dry powder; adding the dry powder into polyethylene glycol (15)-hydroxystearate, incubating at 60°C for 30 minutes; adding distilled water to constant volume; centrifuging at room temperature at 7500-12000 rpm (preferably 12000 rpm) for 10 minutes, separating the supernatant from the debris; storing the supernatant at 4°C.

[0084] In another aspect, the present application also relates to the following embodiments:

[0085] Embodiment 1. A Chinese herbal compound, characterized in that the Chinese herbal compound comprises components with the following weight ratio: Cheqizi, 6-12% w / w; Baishao, 11-17% w / w; Houpu, 6-12% w / w; Yansu, 11-17% w / w; Huocanmucao, 15-21% w / w; Baizhu, 15-21% w / w; Yiyiren, 15-21% w / w.

[0086] Embodiment 2. A Chinese herbal formula, characterized in that the Chinese herbal formula consists of components with the following weight ratio: Che Qizi, 6-12% w / w; Bai Shao, 11-17% w / w; Hou Po, 6-12% w / w; Yan Hu Suo, 11-17% w / w; Huo Tan Mu Cao, 15-21% w / w; Bai Zhu, 15-21% w / w; Yi Yi Ren, 15-21% w / w.

[0087] Embodiment 3. The Chinese herbal formula according to Embodiment 1 or 2, characterized in that each component has the following weight ratio: Che Qizi, 9% w / w; Bai Shao, 14% w / w; Hou Po, 9% w / w; Yan Hu Suo, 14% w / w; Huo Tan Mu Cao, 18% w / w; Bai Zhu, 18% w / w; Yi Yi Ren, 18% w / w.

[0088] Embodiment 4. A pharmaceutical composition, characterized in comprising the Chinese herbal formula according to any one of Embodiments 1-3 and a pharmaceutically acceptable carrier.

[0089] Embodiment 5. The pharmaceutical composition according to Embodiment 4, prepared by the following method: grinding the Chinese herbal formula and optionally a pharmaceutically acceptable carrier into dry powder; adding the dry powder into a water-soluble solvent, incubating at 60°C for 30 minutes; constant volume with distilled water; centrifuging at room temperature at 7500-12000 rpm (preferably 12000 rpm) for 10 minutes, separating the supernatant from the debris; storing the supernatant at 4°C.

[0090] Embodiment 6. The pharmaceutical composition according to Embodiment 5, wherein the water-soluble solvent is selected from polyethylene glycol (15)-hydroxystearate, distilled water and normal saline.

[0091] Embodiment 7. The pharmaceutical composition according to any one of Embodiments 4-6, wherein the pharmaceutical composition is provided in the form of a tablet, a pill, a dripping pill, a capsule, a granule, a powder, a suppository, a powder, a paste, a patch, an injection, a solution, a suspension, a spray, a lotion, a drop, a rub, an emulsion.

[0092] Embodiment 8. The pharmaceutical composition according to Embodiment 7, wherein the pharmaceutical composition is provided in the form of an injection, a solution, a suspension, a spray, a lotion, a drop, a rub, an emulsion.

[0093] Embodiment 9. The pharmaceutical composition according to Embodiment 8, wherein the pharmaceutical composition is provided at a concentration of about 0.09-0.18 mg / ml.

[0094] Embodiment 10. The pharmaceutical composition according to Embodiment 9, wherein the pharmaceutical composition is provided at a concentration of about 0.18 mg / ml.

[0095] Embodiment 11. The pharmaceutical composition of any one of embodiments 4-10, wherein the pharmaceutically acceptable carrier comprises: physiological saline, saccharides, gelatin, starch, Ringer's solution, cellulose.

[0096] Embodiment 12. The pharmaceutical composition of any one of embodiments 4-11, wherein the pH of the carrier is 3-11, preferably 5-9, more preferably 7-8.

[0097] Embodiment 13. Use of the Chinese herbal formula of any one of embodiments 1-3 or the pharmaceutical composition of any one of embodiments 4-12 in the manufacture of a medicament for treating inflammatory bowel disease in a subject in need thereof.

[0098] Embodiment 14. The use of embodiment 13, wherein the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease, preferably the inflammatory bowel disease is ulcerative colitis.

[0099] Embodiment 15. Use of the Chinese herbal formula of any one of embodiments 1-3 or the pharmaceutical composition of any one of embodiments 4-12 in the manufacture of a medicament for promoting the increase of regulatory T cells in a subject in need thereof.

[0100] Embodiment 16. Use of the Chinese herbal formula of any one of embodiments 1-3 or the pharmaceutical composition of any one of embodiments 4-12 in the manufacture of a medicament for increasing the proportion of CD4 + Foxp3 + regulatory T cells in a subject in need thereof.

[0101] Embodiment 17. Use of the Chinese herbal formula of any one of embodiments 1-3 or the pharmaceutical composition of any one of embodiments 4-12 in the manufacture of a medicament for restoring the composition of intestinal microbiota and restoring the expression of intestinal aryl hydrocarbon receptor in a subject in need thereof.

[0102] Embodiment 18. The use of embodiments 13-17, wherein the subject in need thereof is a mammal, preferably the mammal is a human.

[0103] Embodiment 19. A method of preparing a pharmaceutical composition containing a Chinese herbal formula, comprising: grinding a Chinese herbal formula and optionally a pharmaceutically acceptable carrier into a dry powder; adding the dry powder into a water-soluble solvent (preferably selected from polyethylene glycol (15)-hydroxystearate, distilled water and physiological saline), incubating at 60°C for 30 minutes; constant volume with distilled water; centrifuging at 7500-12000 rpm (preferably 12000 rpm) at room temperature for 10 minutes, separating the supernatant from the debris; storing the supernatant at 4°C;

[0104] wherein the Chinese herbal formula comprises components in the following weight ratios:

[0105] Cheqizi, 6-12% w / w;

[0106] Baishao, 11-17% w / w;

[0107] Hupo, 6-12% w / w;

[0108] Yanshu, 11-17% w / w;

[0109] Huatangmucao, 15-21% w / w;

[0110] Baizhu, 15-21% w / w;

[0111] Yiyiren, 15-21% w / w,

[0112] Preferably, wherein the Chinese herbal formula comprises components in the following weight ratios:

[0113] Cheqizi, 9% w / w; Baishao, 14% w / w; Hupo, 9% w / w; Yanshu, 14% w / w; Huatangmucao, 18% w / w; Baizhu, 18% w / w; Yiyiren, 18% w / w.

[0114] In another aspect, the present application also relates to the following embodiments:

[0115] Embodiment 1. Use of a Chinese herbal formula in the preparation of a medicament for treating a disease by promoting an increase in regulatory T cells, the Chinese herbal formula comprising the following components: Cheqizi; Baishao; Hupo; Yanshu; Huatangmucao; Baizhu; Yiyiren; the medicament further comprising an optional pharmaceutically acceptable carrier.

[0116] Embodiment 2. Use according to embodiment 1, wherein the Chinese herbal formula comprises components in the following weight ratios: Cheqizi, 6-12% w / w; Baishao, 11-17% w / w; Hupo, 6-12% w / w; Yanshu, 11-17% w / w; Huatangmucao, 15-21% w / w; Baizhu, 15-21% w / w; Yiyiren, 15-21% w / w.

[0117] Embodiment 3. Use according to embodiment 2, wherein the Chinese herbal formula comprises components in the following weight ratios: Cheqizi, 9% w / w; Baishao, 14% w / w; Hupo, 9% w / w; Yanshu, 14% w / w; Huatangmucao, 18% w / w; Baizhu, 18% w / w; Yiyiren, 18% w / w.

[0118] Embodiment 4. Use according to embodiment 1, wherein the Chinese herbal compound consists of components with the following weight ratio: Che Qizi, 6-12% w / w; Bai Shao, 11-17% w / w; Hou Po, 6-12% w / w; Yan Hu Suo, 11-17% w / w; Huo Tan Mu Cao, 15-21% w / w; Bai Zhu, 15-21% w / w; Yi Yi Ren, 15-21% w / w.

[0119] Embodiment 5. Use according to embodiment 4, wherein the Chinese herbal compound consists of components with the following weight ratio: Che Qizi, 9% w / w; Bai Shao, 14% w / w; Hou Po, 9% w / w; Yan Hu Suo, 14% w / w; Huo Tan Mu Cao, 18% w / w; Bai Zhu, 18% w / w; Yi Yi Ren, 18% w / w.

[0120] Embodiment 6. Use according to any one of embodiments 1-5, wherein the disease is inflammatory bowel disease.

[0121] Embodiment 7. Use according to embodiment 6, wherein the inflammatory bowel disease comprises ulcerative colitis and Crohn’s disease, preferably the inflammatory bowel disease is ulcerative colitis.

[0122] Embodiment 8. Use according to embodiments 1-7, wherein the medicament is provided in the form of a tablet, a pill, a dripping pill, a capsule, a granule, a powder, a suppository, a powder, a paste, a patch, an injection, a solution, a suspension, a spray, a lotion, a drop, a rub, an emulsion.

[0123] Embodiment 9. Use according to embodiment 8, wherein the medicament is provided in the form of an injection, a solution, a suspension, a spray, a lotion, a drop, a rub, an emulsion.

[0124] Embodiment 10. Use according to embodiment 9, wherein the medicament is provided at a concentration of about 0.09-0.18 mg / ml.

[0125] Embodiment 11. Use according to any one of embodiments 1-10, wherein the pharmaceutically acceptable carrier comprises: physiological saline, saccharides, gelatin, starch, Ringer’s solution, cellulose.

[0126] Embodiment 12. Use according to any one of embodiments 1-11, wherein the pH of the carrier is 3-11, preferably 5-9, more preferably 7-8.

[0127] Embodiment 13. Use according to any one of embodiments 1-12, wherein the medicament is administered at a dose of 1-4 g / kg body weight, preferably at a dose of 2 g / kg body weight.

[0128] Embodiment 14. Use of a Chinese herbal formula in the manufacture of a medicament for increasing CD4 + Foxp3 + modulation of the proportion of regulatory T cells, wherein the Chinese herbal formula comprises the following components: Cheqianzi; Baishao; Hupo; Yanshuo; Huotanmucao; Baizhu; Yiyiren; and optionally a pharmaceutically acceptable carrier.

[0129] Embodiment 15. The use according to embodiment 14, wherein the Chinese herbal formula comprises components in the following weight ratios: Cheqianzi, 6-12% w / w; Baishao, 11-17% w / w; Hupo, 6-12% w / w; Yanshuo, 11-17% w / w; Huotanmucao, 15-21% w / w; Baizhu, 15-21% w / w; Yiyiren, 15-21% w / w.

[0130] Embodiment 16. The use according to embodiment 15, wherein the Chinese herbal formula comprises components in the following weight ratios: Cheqianzi, 9% w / w; Baishao, 14% w / w; Hupo, 9% w / w; Yanshuo, 14% w / w; Huotanmucao, 18% w / w; Baizhu, 18% w / w; Yiyiren, 18% w / w.

[0131] Embodiment 17. The use according to embodiment 14, wherein the Chinese herbal formula consists of components in the following weight ratios: Cheqianzi, 6-12% w / w; Baishao, 11-17% w / w; Hupo, 6-12% w / w; Yanshuo, 11-17% w / w; Huotanmucao, 15-21% w / w; Baizhu, 15-21% w / w; Yiyiren, 15-21% w / w.

[0132] Embodiment 18. The use according to embodiment 17, wherein the Chinese herbal formula consists of components in the following weight ratios: Cheqianzi, 9% w / w; Baishao, 14% w / w; Hupo, 9% w / w; Yanshuo, 14% w / w; Huotanmucao, 18% w / w; Baizhu, 18% w / w; Yiyiren, 18% w / w.

[0133] Embodiment 19. Use of a Chinese herbal formula in the manufacture of a medicament for restoring the composition of the gut microbiota and restoring the expression of the aryl hydrocarbon receptor in the gut in an individual in need thereof, wherein the Chinese herbal formula comprises the following components: Cheqianzi; Baishao; Hupo; Yanshuo; Huotanmucao; Baizhu; Yiyiren; and optionally a pharmaceutically acceptable carrier.

[0134] Embodiment 20. Use according to Embodiment 19, wherein the Chinese herbal compound comprises components in the following weight ratio: Che Qizi, 6-12% w / w; Bai Shao, 11-17% w / w; Hou Po, 6-12% w / w; Yan Hu Suo, 11-17% w / w; Huo Tan Mu Cao, 15-21% w / w; Bai Zhu, 15-21% w / w; Yi Yi Ren, 15-21% w / w.

[0135] Embodiment 21. Use according to Embodiment 20, wherein the Chinese herbal compound comprises components in the following weight ratio: Che Qizi, 9% w / w; Bai Shao, 14% w / w; Hou Po, 9% w / w; Yan Hu Suo, 14% w / w; Huo Tan Mu Cao, 18% w / w; Bai Zhu, 18% w / w; Yi Yi Ren, 18% w / w.

[0136] Embodiment 22. Use according to Embodiment 19, wherein the Chinese herbal compound consists of components in the following weight ratio: Che Qizi, 6-12% w / w; Bai Shao, 11-17% w / w; Hou Po, 6-12% w / w; Yan Hu Suo, 11-17% w / w; Huo Tan Mu Cao, 15-21% w / w; Bai Zhu, 15-21% w / w; Yi Yi Ren, 15-21% w / w.

[0137] Embodiment 23. Use according to Embodiment 22, wherein the Chinese herbal compound consists of components in the following weight ratio: Che Qizi, 9% w / w; Bai Shao, 14% w / w; Hou Po, 9% w / w; Yan Hu Suo, 14% w / w; Huo Tan Mu Cao, 18% w / w; Bai Zhu, 18% w / w; Yi Yi Ren, 18% w / w.

[0138] Example

[0139] The preferred embodiments described in the present application can be modified by those skilled in the art with reference to the present application. Therefore, the present application claims include all equivalent modifications and changes to the subject matter of the claims of the present application within the scope permitted by law.

[0140] Animals and experimental materials

[0141] All animal experiments were reviewed and approved by the Ethics Committee of the University of Macau. C57BL / 6J mice and Rag 1 - / -Immune-deficient mice were purchased from the Jackson Laboratory, USA, and bred in-house at the specific pathogen free (SPF) animal center of the University of Macau. One week prior to experiments, mice were acclimatized to a 12-hour light / dark cycle with free access to food and water, and temperature was controlled at 22.5 ± 0.5 °C. Infliximab (IFLX) was purchased from Schering-Plough (Kenilworth). DSS (M.W 36,000-50,000, Colitis grade) was obtained from MP Biomedicals (Santa Ana, CA USA). The Chinese herbal medicine material of JCM-16021 was provided by Peili (Nanning Pharmaceutical Co. Ltd (Nanninig, China)). All other materials and reagents were purchased from Thermo Fisher Scientific and Sigma-Aldrich or as otherwise indicated.

[0142] Experimental methods

[0143] Studies were performed at the animal center of the University of Macau, and animals were housed by the center.

[0144] The first day of the experiment was defined as i) the day of adoptive transfer of neonatal CD4 + T cells to Rag 1 - / - mice, or ii) the first day of DSS administration. JCM-16021 was administered orally to mice (body weight approximately 22-24 grams) at doses of 1.0 and 2.0 grams per kilogram. Body weight and fecal blood were monitored every three days. Mice were sacrificed, and serum, colon tissue, spleen, mesenteric lymph nodes, and feces were collected for further analysis.

[0145] 1. Fecal occult blood assay

[0146] Fecal occult blood assay kits were purchased from Nanjing Jiancheng Biological Engineering Institute, and assays were performed according to the kit instructions.

[0147] 2. Histological staining, Periodic acid-Schiff staining, immunohistochemical staining and immunofluorescent staining:

[0148] At the end of the experiment, mice colon tissues were reserved, fixed with 4% paraformaldehyde at 4°C for 2 hours, and embedded with paraffin. After deparaffination, mouse colon tissue sections were washed with PBS and blocked with 3% H2O2. After blocking, the tissue sections were incubated with blocking solution (10% horse serum, 0.1% bovine serum albumin, 0.2% gelatin) for 1 hour at room temperature to block non-specific sites. After blocking, the colon tissues were incubated with different primary antibodies, including PCNA, CD44, Ibal, Claudin, p65, c-Rel, and AhR, at 4°C overnight, washed with PBS-T buffer, and incubated with corresponding secondary antibodies. The sections incubated without primary antibodies were used as negative controls at the time of analysis.

[0149] For immunohistochemical staining, the signal of HRP-conjugated secondary antibody was detected by DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA), counterstained with hematoxylin, and fixed for immunohistochemical analysis.

[0150] For immunofluorescence staining, the nuclei were observed with Hoechst 33342, and the signal was measured with a Leica TCS SP8 confocal microscope.

[0151] For H&E staining and PAS staining, the slides were deparaffinated and stained with a commercial kit from Nanjing Jiancheng Biological Engineering Institute.

[0152] 3. Ulcerative colitis pathological index score

[0153] The histological grading of colitis will be determined based on the inflammatory features, extent, regeneration, crypt damage, and percentage in colon sections stained with hematoxylin-eosin and periodic acid-schiff. The colitis score will be performed by two independent researchers who are blinded to the animal experiment.

[0154] 4. Real-time quantitative PCR (qRT-PCR)

[0155] Total RNA was extracted from tissues using Trizol reagent (Invitrogen) and reverse transcribed into cDNA using a cDNA synthesis kit (Bio-Rad Laboratories). The diluted 50-fold cDNA was used as a template for qRT-PCR. The expression level of mRNA was detected using SYBR Green fluorescent probe according to the kit operation instruction. The primers used for qRT-PCR were as follows: Tnf-a, forward 5'-GACGTGGAACTGGCAGAAGAG-3', reverse 5'-TTGGTGGTTTGTGAGTGTGAG-3'; Mcp-1, forward 5'-3', reverse 5'-3'; Il-6, forward 5'-3', reverse 5'-3'; 18S rRNA, forward 5'-GTAACCCGTTGAACCCCATT-3', reverse 5'-CCATCCAATCGGTAGTAGCG-3'

[0156] 5. Microbead-based serum cytokine level assay

[0157] The levels of pro-inflammatory cytokines (#740150) and chemokines (#740007) in mouse serum were determined using LEGENDplex TM microbeads (BioLegend, San Diego, CA, USA) according to the manufacturer's instructions.

[0158] 6. Colonic lamina propria lymphocyte isolation

[0159] After dissection of the colon from mice, the colon was washed four times in ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and then cut into approximately 1 cm long pieces. The intestinal epithelial cells and lamina propria will be separated by washing in HBSS buffer containing 2 mM EDTA for 30 min at 37 °C with vigorous shaking. The intestinal epithelial cells will be collected by centrifugation at 1,000 rpm at 4 °C. The remaining colon tissue will be incubated in digestion buffer (DMEM medium, 5% fetal bovine serum, 1 mg / ml collagenase IV and DNase I) at 37 °C with shaking for 40 min to separate the colon lamina propria lymphocytes. The digested colon tissue cell suspension will be filtered through a 70-μm cell strainer and centrifuged. The cells will then be resuspended in 10 ml of a 40:80 Percoll gradient, layered on top of 5 ml of 80% Percoll gradient in a 15-ml Falcon tube. The colon lamina propria lymphocytes will be separated by Percoll gradient by centrifugation at 1,800 rpm for 20 min at room temperature. The colon lamina propria lymphocytes collected between the Percoll density gradients will be washed with DMEM medium and used for the following flow cytometric analysis.

[0160] 7. Flow cytometric analysis of regulatory T cells

[0161] The colon lamina propria lymphocytes were diluted to 1 x 10 5 cells / ml and subjected to intracellular staining after fixation and permeabilization. The cells were stained with 1:100 diluted primary antibodies for 30 min at 4 °C in the dark, and the antibodies used for flow cytometry were CD3 (APC, BD Biosciences), CD4 (FITC, BD Biosciences), and Foxp3 (PE, BD Biosciences). The analysis was performed using a BD LSRFortessa flow cytometer (BD Biosciences), and the data were analyzed by FlowJo software. TM

[0162] 8. Intestinal microbiota 16S rRNA sequencing

[0163] Bacterial DNA was extracted from mouse feces using phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.9), and the 16S rRNA conserved region was amplified using a PCR method. V4 region 16S rRNA bacterial genome sequencing was performed using a PE250 / PE300 dual-end sequencing platform according to the recommendations of Illumina MiSeq / Novaseq (Illumina, San Diego, CA, USA). Data analysis was performed using the QIIME software package by GENEWIZ (Suzhou, China).​

[0164] 9. Statistical analysis

[0165] Data from none of the animals were excluded from analysis. All data obtained will be presented as mean ± standard error of the mean (SEM). All experiments were repeated at least three times or more, and differences in measured variables between groups will be analyzed by GraphPad Prism 5.0 software using one-way or two-way ANOVA method. Results were considered significantly different, statistically significant, when p < 0.05.

[0166] Example 1: Preparation of a medicament comprising Chinese herbal compound JCM-16021

[0167] Two grams of JCM-16021 dry powder containing 10% starch excipient (JCM-16021 composition: Terminalia chebula, 9% w / w; Radix Paeoniae Alba, 14% w / w; Magnolia officinalis, 9% w / w; Corydalis, 14% w / w; Polygonum, 18% w / w; Rhizoma Atractylodis Macrocephalae, 18% w / w; Coix, 18% w / w) was added to two grams of polyethylene glycol (15)-hydroxystearate, incubated at 60°C for 30 minutes, and then made up to a final volume of 10 ml with distilled water. The supernatant was separated from the debris by centrifugation (12,000 rpm, 10 minutes) at room temperature (25°C). The concentration of JCM-16021 was calculated to be 0.18 mg / ml JCM-16021 (2 g / 10 ml x 0.9) based on the dry weight of the dry powder. The herbal extract was stored at 4°C and used within 24 hours. Any residual precipitate that was produced upon standing was vortexed into a suspension and used to treat animals.

[0168] Example 2: Therapeutic effect of Chinese herbal compound JCM-16021 on CD4 + Therapeutic effect of naive T cell transfer in inducing ulcerative colitis

[0169] 1. CD4 + Naive T cell transfer induces ulcerative colitis model

[0170] Naive CD4 + T cells were isolated and purified from the spleens of wild-type C57BL / 6J mice using Naive CD4 + T cells. Isolated CD4 + T cells were incubated with CD4 (#100406, BioLegend), CD25 (#17025182, eBiosciences), and CD45RB (#103314, BioLegend) antibodies and analyzed for purity by a BD Fortessa flow cytometer (BD Biosciences). 4 x 10​5 CD4 + naive T cells were injected intraperitoneally into Rag 1 - / - immunodeficient mice. Mice were then monitored every three days for clinical signs of colitis, including fecal occult blood, loose stool / diarrhea, rough / hunched posture, and weight loss. Any mice with greater than 20% weight loss from starting weight or showing signs of severe illness were euthanized. CD4 + naive T cell transfer, JCM-16021 treatment was initiated and administered twice daily by gavage (doses of 1.0, 2.0 g / kg body weight) for three weeks. Infliximab was administered as a positive control drug by intraperitoneal injection at 5 mg / kg at the same time as the first JCM-16021 dose and once weekly for three weeks.

[0171] 2. Experimental results

[0172] 2.1. JCM-16021 in naive CD4 + T cell transfer-induced ulcerative colitis mice showed significant treatment Therapeutic effect

[0173] To determine the efficacy of JCM-16021 in naive T cell transfer-induced colitis in Rag 1 - / - immunodeficient mouse model, purified CD4 + CD25 - CD45RB 高 naive T cells were injected intraperitoneally into Rag 1 - / - immunodeficient mice. JCM-16021 was administered by gavage at doses of 1.0 g / kg and 2.0 g / kg body weight three weeks after naive T cell transfer ( + T cells were injected intraperitoneally into Rag 1 - / - immunodeficient mice ( Figure 1 A). JCM-16021 was administered by gavage at doses of 1.0 g / kg and 2.0 g / kg body weight three weeks after naive T cell transfer ( Figure 1 B). Infliximab (IFLX) was used as a positive control drug ( Figure 1 ) compared to Rag 1 - / - CD4 + naive T cell transfer mice showed significant weight loss ( Figure 1 B). JCM-16021 treatment dose-dependently reduced fecal occult blood ( Figure 1 C) and ulcerative colitis pathology index ( Figure 1 D); while infliximab treatment did not show similar activity. Hematoxylin-eosin (H&E) and periodic acid-schiff (PAS) staining results showed that mice receiving CD4 + naive T cell transfer had abnormal colon tissue morphology with obvious epithelial cell damage and abnormal mucin ( Figure 1E). In contrast, mice treated with 2.0 g / kg JCM-16021 exhibited similar normal epithelial barrier and mucin expression levels to Rag 1 - / - Mice similar normal epithelial barrier and mucin expression Figure 1 E). Mice receiving CD4 + Claudin expression levels in colonic epithelium of mice with suppressed colitis were restored by JCM-16021 treatment, but not in mice treated with Infliximab Figure 1 E). JCM-16021 also significantly reduced high levels of p65 expression in colitic mice Figure 1 E). JCM-16021 exhibited better therapeutic effect at 2.0 g / kg than at 1.0 g / kg dose; while Infliximab was less effective than JCM-16021 under the same experimental conditions.

[0174] 2.2. JCM-16021 treatment suppresses intestinal inflammation in ulcerative colitis mice

[0175] Immunofluorescence staining results showed that naive CD4 + NF-κΒ p65 and c-Rel expression in Iba 1 positive macrophages in colonic epithelium of mice with T cell transfer-induced ulcerative colitis exhibited apparent nuclear localization Figure 2 A). In particular, p65 and c-Rel activation were co-localized with ionized calcium binding adaptor molecule 1 (Iba1), a macrophage-specific calcium binding protein. Inhibitory effects of JCM-16021 on NF-κΒ p65 nuclear translocation and c-Rel activation were dose-dependent, indicating that colonic infiltrating macrophages were effectively suppressed in CD4 + T cell transfer-induced colitis. Both JCM-16021 and IFLX treatment significantly reduced mRNA expression levels of proinflammatory cytokine Tnf-α and chemokine Mcp-1 in colonic tissue of colitic mice Figure 2 B-2C).

[0176] 2.3. JCM-16021 treatment induces crypt area stem cell proliferation, aryl hydrocarbon receptor (AhR) expression and increased proportion of regulatory T cells Figure 3

[0177] Proliferation of colonic crypt stem cells is essential for recovery of intestinal epithelial barrier. In CD4 + CD44 + PCNA expression levels in CD44 + PCNA expression levels in CD44 Figure 3 A).

[0178] High-dose (2.0 g / kg) JCM-16021 treatment also restored aryl hydrocarbon receptor expression in the small intestine (ileum) of mice to the level of the sham-operated group. Figure 3 B). Under the same conditions, the expression level of aryl hydrocarbon receptors in the ileum of mice was increased to some extent after treatment with infliximab. Figure 3 B). In IFLX-treated mice, Ahr expression in the ileum was partially increased, but not as strongly as in JCM-16021-treated mice. Figure 4 B).

[0179] The balance of regulatory T cell proportions and numbers is crucial for treating inflammatory diseases, particularly restoring intestinal homeostasis in ulcerative colitis. Flow cytometry analysis revealed that after treatment with JCM-16021 at 2.0 g / kg, CD4+ levels in mouse intestinal lamina propria lymphocytes were significantly reduced. + FoxP3 + T reg The proportion of cells was significantly increased compared to mice with ulcerative colitis. Figure 4 ). And infliximab treatment for CD4 + FoxP3 + T reg The cell ratio was not significantly different from that of colitis mice. 2.4. JCM-16021 treatment reshapes intestinal microbiota in mice ).

[0180] Figure 5

[0181] Gut microbiota plays a crucial role in the pathogenesis and treatment mechanisms of inflammatory bowel diseases such as ulcerative colitis. We analyzed the composition of fecal microbiota in mice treated with JCM-16021 using 16S rRNA gene sequencing. Fecal samples from all mice were immediately frozen in liquid nitrogen after collection. Sequencing results revealed that at a dose of 2.0 g / kg, JCM-16021 significantly improved the gut microbiota and CD4+ of mice after treatment. + The gut microbiota of mice with ulcerative colitis induced by naive T cell transfer showed significant differences at the species level, but was very similar to that of the sham-operated group mice. Figure 5 Of the eight bacterial species that showed the most significant changes, three species recovered to the same abundance levels as the sham-operated mice after JCM-16021 treatment, including Bacteroidales_S24-7, Prevotellaceae, and Christensenellaceae. Figure 5 The abundance of five other bacterial species that were significantly elevated in colitis mice decreased dramatically to sham-operated levels after JCM-16021 treatment, including Rikenellaceae, Helicobacteraceae, Deferribacteraceae, Rhodospirillaceae, Clostridiaceae_1.1. Dextran sulfate salt (DSS)-induced ulcerative colitis mouse model )。

[0182] Example 3: Therapeutic effects of JCM-16021 in 2% dextran sulfate sodium (DSS)-induced colitis mice

[0183] 2. Experimental results

[0184] Male and female 6-8 week-old C57BL / 6J mice were treated with 2.5% dextran sulfate sodium (DSS) for 5 days followed by 2 days of recovery. 2% DSS was used for long-term administration of JCM-16021 for 4 weeks.

[0185] 2.1. JCM-16021 exhibits superior therapeutic effect in 2% dextran sulfate salt (DSS)-induced ulcerative colitis mice

[0186] Figure 6 Figure 6

[0187] We further examined the therapeutic effects of JCM-16021 in 2% DSS-induced colitis mice Figure 6 ). Although the body weight of each group of DSS-induced colitis mice did not change throughout the treatment period Figure 6 A), JCM-16021 treatment dose-dependently reversed DSS-induced colon shortening Figure 6 B), reduced fecal occult blood index Figure 6 C), and colitis pathological index Figure 7 D). These results are consistent with the therapeutic effects in CD4 + naive T cell transfer-induced colitis mice. In addition, the therapeutic effects of JCM-16021 2.0 g / kg-treated mice were significantly reduced when co-housed with non-treated colitis mice Figure 7 B-6D).

[0188] Hematoxylin-eosin and periodic acid-Schiff staining results showed that the colon tissues of 2% DSS-induced colitis mice exhibited abnormal pathological features including loss of intestinal epithelial integrity, thickening of the lamina propria, and infiltration of immune cells Figure 7 A). After JCM-16021 treatment, the above colitis pathological features were dose-dependently restored to the level of sham-operated mice Figure 7 A). The expression of PCNA in the colon crypt stem cells of JCM-16021-treated mice also gradually increased with increasing doses to the same level as that of sham-operated mice 2.2. JCM-16021 treatment reduces systemic inflammation and reshapes intestinal microbiota in dextran sulfate salt-induced ulcerative colitis mice B). However, when JCM-16021-treated mice were co-housed with non-treated colitis mice, the above therapeutic effects were similar to those of non-treated colitis mice Figure 8 .

[0189] Figure 8 Figure 9

[0190] After JCM-16021 treatment, the levels of markers of systemic inflammation in DSS-induced ulcerative colitis mice, including proinflammatory cytokines Ifn-γ, Tnf-α and chemotactic factors Mcp-1, Gm-csf in serum were significantly reduced with the increase of JCM-16021 dose Figure 9 ). Among them, the inhibitory effect of JCM-16021 on Tnf-α and Mcp-1 in the serum of colitis mice disappeared after caging with colitis mice; while the serum levels of Ifn-γ and Gm-csf were not related to whether caging with colitis mice ​

[0191] To analyze whether JCM-16021 treatment can change the composition of intestinal flora, we collected the intestinal contents of the sham operation group, DSS-induced colitis and JCM-16021 treatment group mice. Changes in specific intestinal flora were determined using 16S rRNA gene sequencing. The sequencing results showed that the composition of intestinal flora in DSS-induced colitis mice after JCM-16021 treatment returned to the level comparable to that of sham operation group mice ​ ), and the changed bacterial species mainly included Dubosiella, Fissicatena, Clostridium_sensu_stricto, Dubosiella_newyorknesis and Lachnospiraceae bacterium ​

[0192] Conclusion:

[0193] The present application provides a Chinese herbal medicine composition for treating inflammatory bowel disease, particularly ulcerative colitis. The present application also provides a method for improving colon-intestinal barrier repair by promoting the proliferation of colon crypt area stem cells. The present application improves the treatment of the disease by increasing the proportion of CD4 + Foxp3 + regulatory T (T reg ) cells in the lamina propria. The present application also provides a treatment method for restoring the composition of the intestinal microbiome and the intestinal expression of the aryl hydrocarbon receptor. In addition, the present application provides a pharmaceutical composition with a treatment effect superior to that of the clinically used biological agent infliximab.​​

Claims

1. A Chinese herbal formula for use in the preparation of a medicament for promoting CD4+CD25+Foxp3+ T cells by increasing the expression of aryl hydrocarbon receptor in colonic epithelial cells. + Foxp3 + regulatory T cells in the treatment of inflammatory bowel disease, said Chinese herbal formula consisting of components in the following weight ratio: Terminalia chebula, 6-12% w / w; Radix Paeoniae Alba, 11-17% w / w; Magnolia officinalis, 6-12% w / w; Corydalis, 11-17% w / w; Polygonum chinense, 15-21% w / w; Rhizoma Atractylodis Macrocephalae, 15-21% w / w; Coix lacryma-jobi, 15-21% w / w; The medicine also comprises an optional pharmaceutically acceptable carrier.

2. The use according to claim 1, wherein the Chinese herbal compound consists of components with the following weight ratio: Terminalia chebula, 9% w / w; Radix Paeoniae Alba, 14% w / w; Magnolia officinalis, 9% w / w; Corydalis, 14% w / w; Polygonum chinense, 18% w / w; Rhizoma Atractylodis Macrocephalae, 18% w / w; Coix lacryma-jobi, 18% w / w.

3. The use according to any one of claim 1, wherein the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease.

4. The use according to claim 1, wherein the medicine is provided in the form of tablets, pills, capsules, granules, powders, suppositories, dispersions, pastes, patches, solutions, emulsions.

5. The use according to claim 1, wherein the medicine is provided in the form of drop pills, injection solutions, suspensions, sprays, lotions, drops.

6. The use according to claim 4, wherein the medicine is provided in the form of a solution or an emulsion, wherein the solution is selected from injection solutions, suspensions, sprays, lotions, drops, liniments.

7. The use according to claim 4, wherein the medicine is provided at a concentration of 0.09-0.18 mg / mL.

8. The use of claim 1, wherein the pharmaceutically acceptable carrier comprises: Normal saline, sugar, Ringer's solution.

9. The use of claim 1, wherein the pharmaceutically acceptable carrier comprises: Gelatin, starch, cellulose.

10. The use according to claim 1, wherein the pH of the carrier is 3-11.

11. The use according to claim 1, wherein the pH of the carrier is 5-9.

12. The use according to claim 1, wherein the pH of the carrier is 7-8.

13. The use according to claim 1, wherein the medicine is administered at a dose of 1-4 g / kg body weight.

14. The use according to claim 1, wherein the medicine is administered at a dose of 2 g / kg body weight.

15. A method of preparing a pharmaceutical composition containing a Chinese herbal compound, characterized by comprise: grinding the Chinese herbal compound and the optional pharmaceutically acceptable carrier into dry powder; adding the dry powder into a water-soluble solvent, incubating at 60°C for 30 minutes; constant volume with distilled water; centrifuging at room temperature at 7500-12000 rpm for 10 minutes, separating the supernatant from the precipitate; storing the supernatant at 4°C; wherein the Chinese herbal compound consists of components with the following weight ratio: Terminalia chebula, 6-12% w / w; Radix Paeoniae Alba, 11-17% w / w; Magnolia officinalis, 6-12% w / w; Corydalis, 11-17% w / w; Polygonum chinense, 15-21% w / w; Rhizoma Atractylodis Macrocephalae, 15-21% w / w; Coix lacryma-jobi, 15-21% w / w.

16. The method according to claim 15, wherein the water-soluble solvent is selected from polyethylene glycol (15)-hydroxystearate, distilled water and normal saline.

17. The method according to claim 15, wherein the centrifuging is at room temperature at 7500-12000 rpm for 10 minutes, separating the supernatant from the precipitate.

18. The method according to claim 15, wherein the Chinese herbal compound consists of components with the following weight ratio: Terminalia chebula, 9% w / w; Radix Paeoniae Alba, 14% w / w; Magnolia officinalis, 9% w / w; Corydalis, 14% w / w; Rumex, 18% w / w; Atractylodes, 18% w / w; Coix, 18% w / w.

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