Small molecule composition and application thereof in treatment of immunosuppression agent induced immunosuppression

Abstract

The invention discloses a small molecule composition and application thereof in treating immunosuppression induced by an immunosuppressor, and relates to a compound active composition. Specifically, the invention provides a composition composed of 15 kinds of small molecules. The composition is prepared from the following raw materials: cimicifugin, 5-O-emamectin aspartame, formononetin, cimicifugin, calycosin, formononin, astragaloside, calycosin glucoside, astragaloside III, astragaloside, sec-o-glucosylhamaudol, isoastragaloside II, astragaloside I, cycloastragaloside II and isoastragaloside I. The invention further discloses a preparation method of the composition. The composition disclosed by the invention can be used for remarkably improving immunosuppression induced by the immunosuppressant.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals and relates to a small molecule composition and its use in the treatment of immunosuppression induced by immunosuppressants. Background Art

[0002] Immunosuppression is a common side effect of chemotherapeutic drugs. For example, in the metabolic process of the chemotherapeutic drug cyclophosphamide of the nitrogen mustard type, intermediate active metabolites acrolein and phosphoramide mustard will be produced. Both are cytotoxic alkylating agents that can cause serious damage to the immune system, including the depletion of susceptible immune cells, the functional attenuation of more resilient immune cells, and the systemic effects affecting immune function. Currently, there is no clear therapeutic drug for chemotherapy-induced immunosuppression.

[0003] In previous studies, we identified 58 components from Yupingfeng granules using UPLC-Q-TOF-MS. Through serum pharmacochemistry research, it was found that flavonoid components, astragaloside components in astragalus, and chromone components in ledebouriella seseloides enter the blood in the form of prototypes or metabolites. Network pharmacology research on the blood components showed that they play an immune regulation role through multiple inflammation-related targets, and the contents of these components were determined using LC-MS.

[0004] Based on the above research background, it is necessary to try to fully study the active ingredients of Yupingfeng granules in order to develop drugs that can effectively inhibit immunosuppression. Summary of the Invention

[0005] The purpose of the present invention is to develop a pharmaceutical composition for the treatment of immunosuppression induced by immunosuppressants.

[0006] In the first aspect of the present invention, a compound active combination is provided, and the compound active combination is composed of the following components:

[0007] 30 - 50 parts by weight of prim-O-glucosylcimifugin;

[0008] 55 - 70 parts by weight of 5-O-methylvisammioside;

[0009] 1 - 5 parts by weight of formononetin;

[0010] 5 - 20 parts by weight of cimifugin;

[0011] 2 - 15 parts by weight of calycosin;

[0012] 5 - 10 parts by weight of formononetin glucoside;

[0013] 20 - 40 parts by weight of astragaloside IV;

[0014] 20 - 40 parts by weight of calycosin-7-O-β-D-glucoside;

[0015] Astragaloside III: 1 - 5 parts by weight;

[0016] Astragaloside II: 10 - 30 parts by weight;

[0017] Sec-O-glucosylhamaudol: 1 - 10 parts by weight;

[0018] Isoastragaloside II: 5 - 20 parts by weight;

[0019] Astragaloside I: 3 - 30 parts by weight;

[0020] Cycloastragenol II: 10 - 30 parts by weight;

[0021] Isoastragaloside I: 20 - 60 parts by weight.

[0022] In another preferred example, at least one or more components in the compound active combination are synthetic.

[0023] In another preferred example, at least one or more components in the compound active combination are traditional Chinese medicine extracts.

[0024] In another preferred example, all components in the compound active combination are synthetic.

[0025] In another preferred example, the compound active combination does not include Yupingfeng granules.

[0026] In another preferred example, the compound active combination consists of the following components:

[0027] Prim-O-glucosylcimifugin: 35 - 45 parts by weight;

[0028] 5-O-methylvisammioside: 60 - 75 parts by weight;

[0029] Formononetin: 2 - 4 parts by weight;

[0030] Cimifugin: 10 - 15 parts by weight;

[0031] Calycosin: 4 - 10 parts by weight;

[0032] Ononin: 7 - 10 parts by weight;

[0033] Astragaloside IV: 25 - 35 parts by weight;

[0034] Calycosin-7-O-β-D-glucoside: 25 - 35 parts by weight;

[0035] Astragaloside III: 2 - 4 parts by weight;

[0036] Astragaloside II: 15 - 25 parts by weight;

[0037] Sec-O-glucosylhamaudol: 2 - 8 parts by weight;

[0038] 8 - 15 parts by weight of Isoastragaloside II;

[0039] 5 - 20 parts by weight of Astragaloside I;

[0040] 12 - 25 parts by weight of Cycloastragenol II; and

[0041] 30 - 50 parts by weight of Isoastragaloside I.

[0042] In another preferred embodiment, the compound active combination comprises the following components:

[0043] 35 - 45 parts by weight of Prim-O-glucosylcimifugin;

[0044] 60 - 75 parts by weight of 5 - O-methylvisammioside;

[0045] 2 - 4 parts by weight of Formononetin;

[0046] 10 - 15 parts by weight of Cimifugin;

[0047] 4 - 8 parts by weight of Calycosin;

[0048] 7 - 10 parts by weight of Ononin;

[0049] 25 - 35 parts by weight of Astragaloside IV;

[0050] 25 - 35 parts by weight of Calycosin - 7 - O - β - D - glucoside;

[0051] 2 - 4 parts by weight of Astragaloside III;

[0052] 15 - 20 parts by weight of Isoastragaloside II;

[0053] 2 - 8 parts by weight of Sec-O-glucosylhamaudol;

[0054] 8 - 15 parts by weight of Isoastragaloside II;

[0055] 10 - 15 parts by weight of Astragaloside I;

[0056] 15 - 20 parts by weight of Cycloastragenol II; and

[0057] 30 - 40 parts by weight of Isoastragaloside I.

[0058] The second aspect of the present invention provides a pharmaceutical composition, which comprises:

[0059] (i) The compound active combination as described in the first aspect of the present invention; and

[0060] (ii) A pharmaceutically acceptable carrier.

[0061] In another preferred embodiment, the pharmaceutical composition is an oral preparation.

[0062] In another preferred embodiment, the pharmaceutical composition is a liquid preparation or a freeze-dried preparation.

[0063] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: oral liquid, tablet, granule, capsule, or a combination thereof.

[0064] The third aspect of the present invention provides a medicine box, which comprises:

[0065] (a) a first container, and the compound active combination described in the first aspect of the present invention located in the first container or a drug containing the compound active combination described in the first aspect of the present invention; and

[0066] (b) a pharmaceutically acceptable carrier.

[0067] In another preferred embodiment, the dosage form of the drug is selected from the group consisting of: capsule, tablet, granule preparation, liquid preparation, or a combination thereof.

[0068] In another preferred embodiment, the dosage form of the drug is an oral dosage form.

[0069] In another preferred embodiment, the medicine box further contains an instruction manual.

[0070] The fourth aspect of the present invention provides the use of the compound active combination described in the first aspect of the present invention for preparing a drug or preparation for preventing and / or treating and / or alleviating immunosuppression induced by immunosuppressants.

[0071] In another preferred embodiment, the immunosuppressants are selected from: cyclophosphamide, acrolein, phosphamide mustard, dexamethasone, cyclosporine.

[0072] [[ID=,33]]In another preferred embodiment, the drug or preparation further has one or more of the following uses:

[0073] (1) inhibiting the weight loss of a subject induced by an immunosuppressant;

[0074] (2) inhibiting the decrease in the organ indices of the spleen and thymus of a subject induced by an immunosuppressant;

[0075] (3) inhibiting the decrease in the number of white blood cells in the blood of a subject induced by an immunosuppressant;

[0076] (4) inhibiting the increase in the expression of cytokine IL-10 in the serum of a subject induced by an immunosuppressant.

[0077] In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient selected from the group consisting of thymosin, gamma globulin, and interferon.

[0078] The fifth aspect of the present invention provides a method for preventing and / or treating and / or alleviating immunosuppression induced by an immunosuppressant in a subject, by administering to the subject a therapeutically effective amount of the compound active combination as described in the first aspect of the present invention or the pharmaceutical composition as described in the second aspect of the present invention.

[0079] In another preferred embodiment, the immunosuppression induced by the immunosuppressant has one or more characteristics selected from the group consisting of:

[0080] (z1) Weight loss;

[0081] (z2) Decrease in spleen and thymus organ indices;

[0082] (z3) Decrease in the number of white blood cells in the blood;

[0083] (z4) Increase in the expression of cytokine IL-10 in the serum.

[0084] In another preferred embodiment, the subjects include rodents (such as mice, rats), and primate mammals (humans).

[0085] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Shows the body weights of mice in different groups on the 16th day. Normal is the normal group, Model is the model group, YPFG is the Yupingfeng Granule group, EC is the small molecule group, and LH is the positive drug group. indicates a highly significant difference, p < 0.005 (compared between the normal group and the model group), * indicates a significant difference, p < 0.05 (compared between the drug administration group and the model group).

[0087] Figure 2 Shows the spleen and thymus organ indices of mice in different groups. Normal is the normal group, Model is the model group, YPFG is the Yupingfeng Granule group, EC is the small molecule group, and LH is the positive drug group. indicates a highly significant difference, p < 0.005 (compared between the normal group and the model group), * indicates a significant difference, p < 0.05, *** indicates a highly significant difference, p < 0.005 (compared between the drug administration group and the model group).

[0088] Figure 3Show the white blood cell counts of mice in different groups after modeling. Normal is the normal group, Model is the model group, YPFG is Yupingfeng Granule, EC is the small molecule group, and LH is the positive drug group. indicates extremely significant difference, p < 0.005 (compared between the normal group and the model group), * indicates significant difference, p < 0.05 (compared between the drug administration group and the model group).

[0089] Figure 4 Show the expression of IL-10 in the sera of mice in each group. Normal is the normal group, Model is the model group, YPFG is Yupingfeng Granule, EC is the small molecule group, and LH is the positive drug group. ## indicates very significant difference, p < 0.01 (compared between the normal group and the model group), ** indicates very significant difference, p < 0.01, * indicates significant difference, p < 0.05 (compared between the drug administration group and the model group). Detailed implementation manners

[0090] Through extensive and in-depth research, during the process of analyzing and detecting the components of the Chinese patent medicine Yupingfeng Granule, the present inventors identified 58 compounds from Yupingfeng Granule. Based on this, the present inventors further explored and also achieved the simultaneous quantitative analysis of 15 small molecule compounds among them. The present invention was completed on this basis.

[0091] The present inventors unexpectedly found that compared with directly using Yupingfeng Granule, a composite formula containing only 15 small molecule compounds showed significantly better therapeutic effects in mice when used to treat immune deficiency caused by the chemotherapy drug cyclophosphamide.

[0092] Terms

[0093] Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0094] As used herein, the terms "comprising", "including", and "containing" can be used interchangeably, and include not only closed definitions but also semi-closed and open definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0095] In the present invention, the term "prevention" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. "Prevention" as used herein also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of the subject getting the disease.

[0096] "Treatment" as described in the present invention includes delaying and terminating the progression of a disease, or eliminating the disease, and does not require 100% inhibition, eradication, and reversal. In some embodiments, compared to the levels observed in the absence of the compositions, kits, food kits, or health product kits, or combinations of active ingredients described in the present invention, the compositions or pharmaceutical compositions of the present invention reduce, inhibit, and / or reverse immunosuppression induced by immunosuppressants by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0097] As used herein, the term "effective amount" refers to an amount that produces a function or activity in a human and / or animal and is acceptable to the human and / or animal. Those of ordinary skill in the art should understand that the "effective amount" may vary depending on the form of the pharmaceutical composition, the severity of the disease, and the co-administration of other drugs, etc.

[0098] As used herein, the terms "parts by weight" and "number of parts by weight" are used interchangeably, and the parts by weight can be any fixed weight expressed in milligrams, grams, or kilograms (such as 1 mg, 1 g, 2 g, or 1 kg, etc.). For example, a composition composed of 1 part by weight of component a and 9 parts by weight of component b can be a composition composed of 1 g of component a + 9 g of component b, or a composition composed of 10 g of component a + 90 g of component b, etc. In the said composition, the percentage content of a certain component = (the number of parts by weight of this component / the sum of the number of parts by weight of all components) × 100%. Therefore, in a composition composed of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and the content of component b is 90%.

[0099] Cyclophosphamide

[0100] Cyclophosphamide is a nitrogen mustard derivative and is commonly used as an immunosuppressant and a drug for chemotherapy. During the metabolic process of cyclophosphamide in the body, intermediate active metabolites acrolein and phosphoramide mustard are produced, both of which are cytotoxic alkylating agents that can cause serious damage to the immune system, including the depletion of susceptible immune cells, the functional attenuation of more resilient immune cells, and systemic effects that affect immune function. Currently, there is no clear therapeutic drug for cyclophosphamide-induced immunosuppression. Existing research has found that flavonoids, saponins, and polysaccharides in traditional Chinese medicine have a certain ameliorating effect on cyclophosphamide-induced immunosuppression.

[0101] Yupingfeng Granules

[0102] Yupingfeng Granules is a traditional Chinese medicine preparation composed of three herbs, Astragalus membranaceus, Atractylodes macrocephala, and Saposhnikovia divaricata, in a ratio of 3:1:1. The chemical components in Yupingfeng Granules are complex, mainly including flavonoids, chromones, saponins, monosaccharides, and polysaccharides, and also include sesquiterpenoids, phenylpropanoids, volatile oils, and other components.

[0103] Drug composition

[0104] The present invention also provides a drug composition for preparing a medicament for preventing or treating immunosuppression induced by immunosuppressants, and the active ingredients thereof are selected from prim-O-glucosylcimifugin, 5-O-methylvisammioside, formononetin, cimifugin, calycosin, ononin, astragaloside IV, calycosin-7-O-β-D-glucoside, astragaloside III, astragaloside II, sec-O-glucosylhamaudol, isoastragaloside II, astragaloside I, cycloastragenol II and isoastragaloside I.

[0105] In a preferred embodiment of the present invention, the drug composition comprises: (i) a compound active combination selected from the group consisting of 30-50 parts by weight of prim-O-glucosylcimifugin; 55-70 parts by weight of 5-O-methylvisammioside; 1-5 parts by weight of formononetin; 10-20 parts by weight of cimifugin; 2-15 parts by weight of calycosin; 7-10 parts by weight of ononin; 20-40 parts by weight of astragaloside IV; 25-40 parts by weight of calycosin-7-O-β-D-glucoside; 1-5 parts by weight of astragaloside III; 10-30 parts by weight of astragaloside II; 2-10 parts by weight of sec-O-glucosylhamaudol; 5-20 parts by weight of isoastragaloside II; 3-30 parts by weight of astragaloside I; 10-30 parts by weight of cycloastragenol II; and 20-60 parts by weight of isoastragaloside I; and (ii) a pharmaceutically acceptable carrier.

[0106] There is no particular limitation on the dosage form of the drug composition of the present invention, and it may be any dosage form suitable for mammals. Preferably, the dosage form may include tablets, capsules, granules, pills, powders, oral liquids, buccal tablets, or aerosols; most preferably, tablets, capsules or granules. From the standpoint of easy preparation, administration or taking, the preferred composition is a solid composition. Oral administration is preferred.

[0107] Various conventional carriers and / or excipients required for preparing different dosage forms can be added to the composition of the present invention, such as fillers (such as starch, cellulose, dextrin), disintegrants (sodium carboxymethyl starch), lubricants (magnesium stearate), solvents (purified water), cosolvents (ethanol), coating materials. It can be prepared into any common dosage form by conventional traditional Chinese medicine preparation methods, such as tablets, capsules, granules, capsules, pills, powders.

[0108] As used herein, the components of the term "pharmaceutically acceptable carrier" refer to substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffering agents, chelating agents, thickening agents, pH regulators, transdermal promoters, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, bacteriostatic agents, pyrogen-free water, etc.

[0109] Routes of administration and treatment methods

[0110] The pharmaceutical compositions and their extracts of the present invention can be used to prepare pharmaceutical compositions or preparations for preventing and / or treating immunosuppression induced by immunosuppressants, and / or preventing and / or treating symptoms caused by immunosuppression induced by immunosuppressants. The pharmaceutical compositions or preparations of the present invention may also contain other optional medicinal materials or their extracts or contain other drugs effective for treating and / or preventing symptoms caused by immunosuppression induced by immunosuppressants.

[0111] The pharmaceutical composition or preparation is matched with the route of administration. When using the pharmaceutical composition or preparation, a safe and effective amount of the drug is administered to the desired subject (such as a human or non-human mammal). Generally, the pharmaceutical composition or preparation of the present invention is administered at a dose of 0.001 - 20 g / kg of the animal body weight per day, preferably in 1 - 4 divided doses per day, or in a sustained-release form. For most mammals, the total daily dose is 0.05 - 600 g. Of course, the specific dose also varies with the mode of administration, dosage form, and severity of the disease to be treated, and the route of administration, all of which are within the skills of a skilled physician. Administration can be carried out by conventional routes, including (but not limited to): oral, intramuscular, cutaneous, or topical administration. Oral administration is preferred.

[0112] The present invention also provides a method for preventing and / or treating symptoms caused by immunosuppression induced by immunosuppressants, by administering the pharmaceutical composition and / or the pharmaceutical extract of the present invention to the desired subject.

[0113] In another preferred example, the subject is a human or non-human mammal (such as a dog, pig, cat, monkey, sheep, horse, cow, etc.).

[0114] The main advantages of the present invention are:

[0115] (1) The pharmaceutical composition of the present invention can significantly relieve the immunosuppression induced by cyclophosphamide.

[0116] (2) Compared with the traditional Chinese medicine Yupingfeng Granules and the positive drug, the pharmaceutical composition of the present invention has a better effect in improving immunosuppression.

[0117] (3) Compared with Yupingfeng Granules, the pharmaceutical composition of the present invention has simple components and can avoid potential side effects of other components.

[0118] (4) The pharmaceutical composition of the present invention has better pharmacokinetic effects and bioavailability in vivo.

[0119] (5) Compared with Yupingfeng Granules, the pharmaceutical composition of the present invention has simple components and is more convenient for quality control of the drug during the production process.

[0120] Instruments and Test Drugs

[0121] Microplate reader (USA, Thermo Fisher Scientific), fully automatic serum biochemical analyzer 7080 (Japan, Hitachi), mouse weighing balance (China, Nanjing Emmanuel Medical Instrument Co., Ltd.), high-speed centrifuge 5810 (Germany, Eppendorf), electronic analytical balance Sartorius ALC-210.3 (Germany, Sartorius).

[0122] The reference substance of cimifugin (batch number 111522 - 201712) was purchased from the National Institutes for Food and Drug Control, with a purity of 96.2%; the reference substance of calycosin - 7 - O - β - D - glucopyranoside (batch number 111920 - 201505) was purchased from the National Institutes for Food and Drug Control, with a purity of 97.1%; the reference substance of cimifugin (batch number 0130) was purchased from Shanghai Standard Technology Co., Ltd., with a purity of 99.8%; the reference substance of 5 - O - methylvisamminol glucoside (batch number 111523 - 201509) was purchased from the National Institutes for Food and Drug Control, with a purity of 95.8%; the reference substance of ononin (batch number 3818), the reference substance of calycosin (batch number 3815), the reference substance of astragaloside III (batch number 4053), the reference substance of formononetin (batch number 3518) were purchased from Shanghai Standard Technology Co., Ltd., with a purity of 98%; the reference substance of astragaloside IV (batch number 3510) was purchased from Shanghai Standard Technology Co., Ltd., with a purity of 100%; the reference substance of sec - O - glucosylhamaudol (batch number 20161121), the reference substance of astragaloside II (batch number HR16529S1), the reference substance of isoastragaloside II (batch number HS1691C1), the reference substance of cycloastragenol II (batch number HS16816B1), the reference substance of astragaloside I (batch number HR16327S1), the reference substance of isoastragaloside I (HS21908B1) were purchased from Baoji Chenguang Biotechnology Co., Ltd., with a purity of 98%. Levamisole hydrochloride (batch number P1785704) was purchased from Adamas Beta Chemical Reagent Co., Ltd.

[0123] SPF - grade normal male bablc mice (18 - 22 g) were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. and raised in the animal house of Shanghai Institute of Pharmaceutical Industry. Approved by the Animal Experiment Ethics Committee of Shanghai Institute of Pharmaceutical Industry, the use license number is SYXK (Shanghai) 2019 - 0027. The room temperature was 25 ± 1 °C, the relative humidity was 50 - 70%, and there was a 12 - hour day - night cycle. The mice could freely access food and water.

[0124] Example 1 Preparation of the solution of small - molecule compound component group

[0125] Precisely weigh 4.0 mg of cimifugin, 6.2 mg of 5 - O - methylvisamminol glucoside, 0.3 mg of formononetin, 1.1 mg of cimifugin, 0.6 mg of calycosin, 0.8 mg of ononin, 2.8 mg of astragaloside IV, 3.2 mg of calycosin - 7 - O - β - D - glucopyranoside, 0.3 mg of astragaloside III, 1.6 mg of astragaloside II, 0.4 mg of sec - O - glucosylhamaudol, 1.1 mg of isoastragaloside II, 1.2 mg of astragaloside I, 1.7 mg of cycloastragenol II, and 3.8 mg of isoastragaloside I. Add the above reference substances into 50 mL of 0.5% CMC - Na (carboxymethyl cellulose sodium) solution, and ultrasonicate for 30 min to fully dissolve them to obtain the solution of small - molecule compound component group.

[0126] Improvement Effect of Small Molecule Compound Component Group on Cyclophosphamide-Induced Immunosuppressed Mice

[0127] Thirty-two mice were randomly divided into 5 groups and adaptively fed for 1 week. They were divided into a normal group, a model group, a Yupingfeng Granule group (YPFG), a small molecule group (EC), and a positive drug group, with 8 mice in each group. The normal group and the model group were respectively intragastrically administered 10 mL / kg of 0.5% CMC-Na solution every day; the Yupingfeng Granule group (YPFG) was intragastrically administered Yupingfeng Granule solution (dissolved with 0.5% CMC-Na, concentration 0.23 g / mL) at a dose of 10 mL / kg every day; the small molecule group was intragastrically administered small molecule chemical component group solution at a dose of 10 mL / kg every day; the positive drug group was intragastrically administered the positive drug levamisole at a dose of 40 mg / kg every day (the positive drug was dissolved with 0.5% CMC-Na); continuous intragastric administration was carried out for 15 days. On the 8th, 9th, and 10th days after administration, except for the normal group, all mice were intraperitoneally injected with 80 mg / kg of cyclophosphamide 2 hours after administration for modeling. On the 11th day (the end of modeling), 100 μL of blood was collected from the mice in each group, and the white blood cell count was detected using a fully automatic blood biochemical analyzer. On the 16th day, the mice were weighed, sacrificed after blood collection, and the spleen and thymus were taken and various indicators were detected.

[0128] 1.1 Body Weight and Organ Index

[0129] In terms of body weight ( Figure 1 ), on the 16th day, it was found that the body weight of the mice in the model group was significantly lower than that of the normal group, and the body weights of the small molecule group and the positive drug group were significantly higher than that of the model group. The two had a comparable effect of inhibiting body weight loss, while the Yupingfeng Granule group had basically no change compared with the model group, indicating that Yupingfeng Granule had little effect on improving the body weight loss of mice caused by cyclophosphamide.

[0130] In terms of organ index ( Figure 2 ), the spleen and thymus organ indices of the model group were significantly lower than those of the normal group, and the other groups were significantly higher than those of the model group. Compared with the Yupingfeng Granule group, the small molecule group had a more significant increase, and the increase in the thymus organ index was better than that of the positive drug group.

[0131] 1.2 White Blood Cell Count

[0132] The results were as Figure 3 shown. At the end of modeling, it was found that the white blood cell count of the model group was significantly lower than that of the normal group, and the white blood cell counts in the administration groups increased to varying degrees. Among them, the increase in the white blood cell count in the small molecule group was the largest, better than that of the Yupingfeng Granule group and the model group.

[0133] 1.3 Detection of the Expression of Cytokine IL-10 in Serum by Elisa

[0134] The collected blood was allowed to stand and then centrifuged at 8000 rpm / min for 10 min. The supernatant was taken, and the cytokine IL-10 in the serum was measured using an Elisa kit. The specific operation was referred to the instruction manual. IL-10 is an inflammatory and immunosuppressive factor. The results showed ( Figure 4 ) that the IL-10 in the model group was significantly increased compared with the normal group, indicating obvious immunosuppression in the model group, suggesting successful modeling. The expression of IL-10 in the drug administration groups was significantly decreased compared with the model group, indicating that immunosuppression was significantly weakened after drug administration. Among them, the decrease in the expression of IL-10 in the small molecule group was the largest, and its IL-10 expression level was basically equivalent to that of the normal group, suggesting that the small molecule group successfully resisted the immunosuppression caused by cyclophosphamide and had a better effect than the Yupingfeng granule group.

[0135] Discussion

[0136] In summary, this small molecule compound component group can not only relieve the weight loss, spleen and thymus index decline caused by cyclophosphamide-induced immunosuppression, but also improve the decrease in the number of white blood cells and reduce the abnormal increase in IL-10 caused by immunosuppression. And compared with the Yupingfeng granule group under the same drug administration dose, the small molecule compound component group has better pharmacodynamic effects. In terms of quality control, the small molecule compound component group is simpler in composition than the components involved in Yupingfeng granules, excluding the interference of excipients and other components on the quality control of its main components. And in the experiment, it was found that the small molecule component group has better solubility than Yupingfeng granules, and basically does not show precipitation or suspension phenomena, which is conducive to the absorption and action of the components in the body. Based on the above results, we developed the small molecule compound component group composed of these 15 components as a drug to improve cyclophosphamide-induced immune deficiency.

[0137] All the documents mentioned in this invention are cited in this application as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A compound active combination, characterized in that, The compound active combination consists of the following components: 30 - 50 parts by weight of prim-O-glucosylcimifugin; 55 - 70 parts by weight of 5-O-methylvisammioside; 1 - 5 parts by weight of formononetin; 5 - 20 parts by weight of cimifugin; 2 - 15 parts by weight of calycosin; 5 - 10 parts by weight of ononin; 20 - 40 parts by weight of astragaloside IV; 20 - 40 parts by weight of calycosin-7-O-β-D-glucoside; 1 - 5 parts by weight of astragaloside III; 10 - 30 parts by weight of astragaloside II; 1 - 10 parts by weight of sec-O-glucosylhamaudol; 5 - 20 parts by weight of isoastragaloside II; 3 - 30 parts by weight of astragaloside I; 10 - 30 parts by weight of cycloastragenol II; 20 - 60 parts by weight of isoastragaloside I.

2. The compound active combination according to claim 1, characterized in that, At least one or more components in the compound active combination are synthetic.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the compound active combination as claimed in claim 1; and (ii) a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition is a liquid preparation or a freeze-dried preparation.

5. The pharmaceutical composition according to claim 3, characterized in that, The dosage form of the composition is selected from the group consisting of oral liquid, tablets, granules, capsules, or a combination thereof.

6. A medicine box, characterized in that, The kit comprises: (a) a first container, and the compound active combination as claimed in claim 1 located in the first container or a drug containing the compound active combination as claimed in claim 1; and (b) a pharmaceutically acceptable carrier.

7. Use of the compound active combination according to claim 1, characterized in that, For preparing a drug or a preparation for preventing and / or treating and / or alleviating immunosuppression induced by immunosuppressants.

8. The use according to claim 7, wherein, The immunosuppressants are selected from: cyclophosphamide, acrolein, phosphoramide mustard, dexamethasone, cyclosporine.

9. The use according to claim 7, characterized in that, The drug or the preparation also has one or more of the following uses: (1) In a subject induced by an immunosuppressant, inhibiting the weight loss of the subject; (2) In a subject induced by an immunosuppressant, inhibiting the decrease in the organ indices of the spleen and thymus of the subject; (3) In a subject induced by an immunosuppressant, inhibiting the decrease in the number of white blood cells in the blood of the subject; (4) In a subject induced by an immunosuppressant, inhibiting the increase in the expression of the cytokine IL-10 in the serum of the subject.

10. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition further comprises a second active ingredient, and the second active ingredient is selected from the group consisting of thymosin, gamma globulin, interferon.

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