Application of punicalagin in preparation of medicine for preventing and treating multiple sclerosis

Abstract

The invention discloses application of punicalagin in preparation of drugs for preventing and treating multiple sclerosis, and belongs to the technical field of drugs for resisting autoimmune diseases. Experiments show that punicalagin can significantly relieve occurrence and development of experimental encephalomyelitis of mice and reduce infiltration of inflammatory cells in spinal cords of mice, and has no obvious side effects; the punicalagin is mainly used for relieving mouse experimental encephalomyelitis by inhibiting CD4 + T cells from differentiating into Th17 cells. The punicalagin can be used for developing a novel medicine for treating multiple sclerosis, and has wide medical prospect and economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-autoimmune disease drugs, and specifically relates to the application of punicalagin in the preparation of drugs for preventing and treating multiple sclerosis. Background Art

[0002] Multiple sclerosis (MS) is an immune-mediated disease characterized by inflammatory demyelinating lesions of the central nervous system (CNS). It is more common in young and middle-aged people, more common in women, and usually twice as common in women as in men. The main clinical manifestations of the disease include sensory disorders, such as numbness, tingling, burning sensation, etc., which usually occur in the fingers, feet, face and other parts; muscle movement disorders, such as limb fatigue, muscle weakness, muscle spasms, and imbalance; vision problems, such as different binocular vision, nystagmus, and instantaneous eye movement; and other symptoms, such as language disorders, thinking and memory difficulties, etc. At present, the cause of multiple sclerosis is still unclear, and it may be related to viral infection, autoimmune reaction and genetics. The autoreactive T cells in patients with multiple sclerosis enter the central nervous system through the blood-brain barrier, secrete a large number of inflammatory factors (IFN-γ, IL-17 or TNF-α), attack the myelin sheath formed by oligodendrocytes in the central nervous system, and cause demyelination. This damage will destroy the function of part of the nervous system and cause various problems. There is great heterogeneity among multiple sclerosis patients, and effective treatment options are still lacking.

[0003] Experimental autoimmune encephalomyelitis (EAE) is a disease characterized by specific sensitization of CD4 + The EAE model is a T cell-mediated autoimmune disease model characterized by mononuclear cell infiltration and demyelination around small blood vessels in the central nervous system. This pathological change is very similar to human multiple sclerosis (MS), so it is the most common animal model used to study MS. Currently, EAE models are often used for preclinical testing of drugs for the treatment of MS.

[0004] Punicalagin (C 48 H 28 O 30 ; The structure is shown in the formula below), also known as punicalagin, is the main active ingredient in pomegranate peel extract and has broad application prospects in the cosmetics industry, health care products industry, and pharmaceutical industry. In the pharmaceutical field, punicalagin has many important pharmacological effects, such as anti-oxidation, anti-tumor, antibacterial, anti-viral, and hypoglycemic and hypolipidemic effects. After being absorbed by the human body, punicalagin can be decomposed into ellagic acid under the action of human enzymes. It has excellent antioxidant properties and has been used as a food antioxidant. However, there is no report on its efficacy in treating multiple sclerosis.

[0005] Summary of the invention

[0006] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the use of punicalagin in the preparation of drugs for preventing and treating multiple sclerosis.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention discloses the use of punicalagin in the preparation of drugs for preventing and treating multiple sclerosis.

[0008] Preferably, the dosage of punicalagin is 4 mg / kg.

[0009] Preferably, the drug for preventing and treating multiple sclerosis is a drug that reduces the severity of multiple sclerosis.

[0010] Preferably, the drug for preventing and treating multiple sclerosis is a drug that inhibits Th17 differentiation.

[0011] More preferably, the drug for preventing and treating multiple sclerosis is a drug that inhibits CD4 + Drugs that induce T cell differentiation into Th17 to alleviate autoimmune encephalomyelitis.

[0012] Preferably, the drug for preventing and treating multiple sclerosis is a drug for reducing lymphocyte infiltration in the spinal cord.

[0013] Preferably, the drug for preventing and treating multiple sclerosis is a drug that reduces the number of TH17 cells and TH1 cells in spinal cord tissue.

[0014] Preferably, the drug for preventing and treating multiple sclerosis is in the form of capsules, tablets, pills, powders, granules or injections.

[0015] The second aspect of the present invention discloses a pharmaceutical composition for preventing and treating multiple sclerosis, comprising punicalagin, other drugs for treating multiple sclerosis and pharmaceutically acceptable excipients.

[0016] Preferably, the pharmaceutically acceptable excipients include one or more of starch, lactose, microcrystalline cellulose, dextrin, calcium phosphate, polyethylene glycol-4000, polyethylene glycol-6000, sodium carboxymethyl cellulose, hydroxypropyl cellulose or cross-linked polyvinylpyrrolidone.

[0017] The present invention has the following beneficial effects: The present invention provides an application of punicalagin in the preparation of a drug for the prevention and treatment of multiple sclerosis. Through in vitro differentiation experiments, it is found that punicalagin can inhibit the initial CD4 +The differentiation of T cells into Th17 cells indicates that punicalagin has the potential to treat multiple sclerosis. The effect of punicalagin on multiple sclerosis was further explored by constructing a mouse EAE model. The clinical evaluation results showed that punicalagin treatment could significantly inhibit the onset of EAE; HE staining results showed that punicalagin could significantly alleviate the occurrence and development of experimental encephalomyelitis in mice and reduce the infiltration of inflammatory cells in the spinal cord of mice; the flow cytometry staining results of spinal cord tissue mononuclear cell suspension further showed that punicalagin alleviated experimental encephalomyelitis in mice mainly by inhibiting CD4 + T cells differentiated into Th17 cells, and no obvious side effects were observed. Therefore, punicalagin can be used as a new drug for the treatment of multiple sclerosis, with broad medical prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 2 shows the effect of punicalagin on the in vitro differentiation of Th17 cells; A is the wild-type initial CD4 + Flow cytometry of Th17 differentiation induced by T cells after adding DMSO or punicalagin, B is the statistical result of A; Figure 2 The figure shows the effect of punicalagin on the clinical manifestations of the EAE model in mice; A is a statistical diagram of the clinical scores of wild-type mice treated with DMSO or punicalagin to construct the EAE model, and B is a hematoxylin-eosin staining (HE staining) of the spinal cord tissue of the EAE model in wild-type mice treated with DMSO or punicalagin; Figure 3 Figure 3 is the effect of punicalagin on Th1 and Th17 cells in the spinal cord tissue of the EAE model of mice; wherein, A is the flow cytometric graph of the proportion of Th1 and Th17 cells in the spinal cord tissue of the EAE model of wild-type mice after treatment with DMSO or punicalagin, B is the statistical result of Figure A, and C is the statistical graph of the number of Th1 and Th17 cells in the spinal cord tissue of the EAE model of wild-type mice after treatment with DMSO or punicalagin. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0020] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0021] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0022] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0023] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0024] Herein, the administration routes of punicalagin include but are not limited to oral administration, injection, implantation or external application.

[0025] In the specification of the present invention and the following examples, unless otherwise specified, the term "dosage form" is also referred to as "preparation form", "medicament", "pharmaceutical preparation", etc. According to the present invention, the term "preparation form of unit dose" is also referred to as "unit dosage form", for example, refers to a single tablet, a single capsule, etc., and the unit dosage form can provide, for example, 1 / 3, 1 / 2, 1 or 2 daily doses. For example, for tablets, one tablet can contain a daily dose for one day, which is preferred; in addition, one tablet can also contain a daily dose for 2 days, which can be scored in the middle of the tablet to facilitate the user to divide the tablet into two halves. For those with a heavier body weight who are tolerant to the drug, one tablet can be used a day, and for those who are effective for half a tablet, half a tablet can be used every day.

[0026] The present invention provides a pharmaceutical composition comprising punicalagin formulated with one or more non-toxic pharmaceutically acceptable carriers. The pharmaceutical composition can be specifically formulated for oral administration or rectal administration in solid or liquid form, or formulated for injection. Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or the following: a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; c) humectants such as glycerol; d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarder such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glyceryl monostearate; h) adsorbents such as kaolin and bentonite; i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain a buffer. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms may contain, in addition to the active compound, inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0028] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. In the following examples, the punicalagin used was purchased from MCE, CAS No.: HY-N0063. The MOG35-55 antigen peptide used was purchased from Sangon Biotech (Shanghai) Co., Ltd.; complete Freund's adjuvant was purchased from Sigma, CAS No.: F5881; pertussis toxin was purchased from ApexBio, CAS No.: B7273. Unless otherwise specified, all other raw materials used are conventional commercial products, and their specifications are conventional specifications in the art.

[0029] Example 1 Effect of Punicalagin on Differentiation of Th17 Cells in Vitro DMSO was used as the control group, and punicalagin was used as the experimental drug group. Peripheral lymph nodes of 8-week-old male wild-type C57BL / 6 mice were used to prepare mononuclear cell suspensions, which were stained with anti-mouse CD4 antibody, anti-mouse CD25 antibody, anti-mouse CD44 antibody, and anti-mouse CD62L antibody. Then, the initial CD4 + T cells (CD4+CD25-CD44-CD62L+) were cultured under Th17 polarization conditions. The Th17 cell culture conditions were: 100 ng / mL IL-6, 5 ng / mL TGFβ, 10 ng / mL IL-1β, 50 ng / mL IL-23, 2.5 μg / mL anti-mouse IFNγ and 2.5 μg / mL anti-mouse IL-4. At the same time, punicalagin was added at a drug concentration of 2.5 μM. The control group was added with an equal amount of DMSO. After 72 hours, the differentiation of Th17 cells in each group was detected by flow cytometry.

[0030] Test results such as Figure 1 As shown, Figure 1 The flow cytometry results in Figure A showed that the differentiation ratio of Th17 cells in the experimental group was significantly lower than that in the control group; Figure 1 The statistical results of B showed that the differentiation ratio of Th17 cells in the experimental group was about 4 times lower than that in the control group. The above results showed that compared with the control group, punicalagin inhibited the initial CD4 + The differentiation of T cells into Th17 cells indicates that punicalagin has the potential to treat multiple sclerosis.

[0031] Example 2 Effect of Punicalagin on Autoimmune Encephalomyelitis (EAE) 1. Effect of punicalagin on clinical manifestations of autoimmune encephalomyelitis (EAE) 1) Establishment of mouse EAE model Wild-type adult mice were randomly divided into a control group (dimethyl sulfoxide (DMSO)) and an experimental group (punicalagin), with 6 mice in each group.

[0032] Complete Freund's adjuvant (with a final concentration of 4 mg / mL of Mycobacterium tuberculosis H37Ra) and 2 mg / mL of MOG35-55 antigen peptide were mixed in equal volumes at a ratio of 1:1 and completely emulsified to obtain an emulsion. Then, 100 μL of the emulsion was subcutaneously injected at two points on both sides of the spine of each mouse in the control group and the experimental group.

[0033] The punicalagin powder was dissolved in DMSO solvent to obtain a punicalagin solution with a concentration of 25 mg / mL.

[0034] Punicalagin solution and an equal amount of DMSO were dissolved in normal saline to obtain experimental group drugs and control group drugs.

[0035] On the 0th and 2nd day of immunization, mice in each group were intraperitoneally injected with 0.2 mL pertussis toxin (200 ng / mouse), and on the 4th, 7th and 10th day after modeling, punicalagin was intraperitoneally injected at a dose of 4 mg / kg. The control group was injected with an equal amount of DMSO.

[0036] 2) Clinical evaluation The mice in each group were continuously observed and scored comprehensively. The scoring criteria were as follows: 0 points: no symptoms; 1 point: loss of tension in the tail; 2 points: weakness in the hind limbs; 3 points: paralysis of the hind limbs; 4 points: paralysis of the hind limbs and forelimbs; 5 points: dying or dead. Symptoms between the two (weak hind limbs and paralysis of the hind limbs) were scored as ±0.5.

[0037] The rating results are as follows Figure 2 As shown in Figure A, compared with the control group, punicalagin treatment could significantly inhibit the onset of EAE.

[0038] 3) HE staining After the scoring was completed, the mice in each group were killed, and the spinal cord tissues of the mice were taken for HE staining (hematoxylin-eosin staining).

[0039] The staining results are as follows Figure 2 As shown in B, the lymphocyte infiltration in the spinal cord of mice in the punicalagin-treated group was significantly less than that in the control group.

[0040] 2. Effect of punicalagin on Th1 and Th17 cells in the spinal cord tissue of autoimmune encephalomyelitis (EAE) model.

[0041] 1) Establishment of mouse EAE model Wild-type adult mice were randomly divided into a control group (DMSO) and an experimental group (punicalagin), with 6 mice in each group.

[0042] Complete Freund's adjuvant (with a final concentration of 4 mg / mL of Mycobacterium tuberculosis H37Ra) and 2 mg / mL of MOG35-55 antigen peptide were mixed in equal volumes at a ratio of 1:1 and completely emulsified to obtain an emulsion. Then, 100 μL of the emulsion was subcutaneously injected at two points on both sides of the spine of each mouse in the control group and the experimental group.

[0043] The punicalagin powder was dissolved in DMSO solvent to obtain a punicalagin solution with a concentration of 25 mg / mL.

[0044] Punicalagin solution and an equal amount of DMSO were dissolved in physiological saline to obtain experimental group drugs and control group drugs.

[0045] On the 0th and 2nd day of immunization, mice in each group were intraperitoneally injected with 0.2 mL pertussis toxin (200 ng / mouse), and on the 4th, 7th and 10th day after modeling, punicalagin was intraperitoneally injected at a dose of 4 mg / kg. The control group was injected with an equal amount of DMSO.

[0046] 2) Preparation of mouse spinal cord tissue mononuclear cell suspension At the peak of the onset of the disease in mice, spinal cord tissues of two groups of mice were taken to prepare single cell suspensions. First, the Percoll stock solution was prepared into a 90% Percoll solution, i.e., the Percoll working solution, with 10×PBS, and then the Percoll working solution was prepared into 40% and 80% Percoll working solutions with 1640 medium for standby use. The mice were dissected, and the spinal cord tissues of the mice were taken and ground in a grinder containing 2 mL of 1× PBS solution; the grinding liquid was collected in a 15mL centrifuge tube, centrifuged at 1500 rpm and 4°C for 5 min, the supernatant was discarded and resuspended with 5 mL of 40% Percoll working solution, and then 5 mL of 80% Percoll working solution was slowly added to the lower layer of 5 mL of 40% Percoll working solution with an electric pipette, and the upper and lower layers were maintained during the pipetting process. After completion, the centrifuge tube was placed at 23°C and 500 g for centrifugation for 30 min, and the centrifuge speed was adjusted to 0; after the centrifugation, the centrifuge tube was slowly removed to maintain the stratified state, and the required mononuclear cells were the middle white film layer. The middle white film layer was sucked by a pipette and collected into a new 15 mL centrifuge tube. The tube was centrifuged at 1500 rpm and 4°C for 5 min. After centrifugation, the supernatant was discarded and the cells were resuspended in 300 μL FACS solution (1× PBS solution containing 2% serum) to obtain a mouse spinal cord tissue mononuclear cell suspension.

[0047] 3) Streaming analysis The mononuclear cell suspension of mouse spinal cord tissue was taken for flow cytometry staining to analyze the proportion and number of Th1 and Th17 cells in the mouse spinal cord tissue.

[0048] Test results such as Figure 3 As shown in the figure, compared with the control group, the proportion and number of TH17 cells in the spinal cord tissue of mice in the punicalagin-treated group were significantly lower than those in the control group; the proportion of TH1 cells in the spinal cord tissue of mice in the punicalagin-treated group was equivalent to that in the control group, but the number of TH1 cells was significantly lower than that in the control group. Since TH17 cells can secrete IL-17 and TNF-α, and TH1 cells can secrete IFN-γ and TNF-α, TNF-α has a synergistic effect with the cytokines secreted by TH17 and TH1 cells, and jointly promotes inflammatory and autoimmune reactions. Therefore, with the decrease in the number of TH17 cells and TH1 cells, the levels of multiple sclerosis-related inflammatory factors IL-17, IFN-γ and TNF-α will also decrease, indicating that punicalagin can reduce the severity of EAE, has a certain therapeutic effect on multiple sclerosis, and can be used as a potential drug for the treatment of multiple sclerosis.

[0049] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Application of punicalagin in the preparation of drugs for the prevention and treatment of multiple sclerosis.

2. The use according to claim 1, characterized in that: The dosage of punicalagin was 4 mg / kg.

3. The use according to claim 1, characterized in that: The drug for preventing and treating multiple sclerosis is a drug for alleviating the severity of multiple sclerosis.

4. The use according to claim 1, characterized in that: The drug for preventing and treating multiple sclerosis is a drug for inhibiting Th17 differentiation.

5. The use according to claim 4, characterized in that: The drug for preventing and treating multiple sclerosis is to inhibit CD4 + Drugs that induce T cell differentiation into Th17 to alleviate autoimmune encephalomyelitis.

6. The use according to claim 1, characterized in that: The drug for preventing and treating multiple sclerosis is a drug for reducing lymphocyte infiltration in the spinal cord.

7. The use according to claim 1, characterized in that: The drug for preventing and treating multiple sclerosis is a drug for reducing the number of TH17 cells and TH1 cells in spinal cord tissue.

8. The use according to claim 1, characterized in that: The dosage form of the drug for preventing and treating multiple sclerosis is capsule, tablet, pill, powder, granule or injection.

9. A pharmaceutical composition for preventing and treating multiple sclerosis, characterized in that: It comprises punicalagin, other drugs for treating multiple sclerosis and pharmaceutically acceptable excipients.

10. The pharmaceutical composition for preventing and treating multiple sclerosis according to claim 9, characterized in that: The pharmaceutically acceptable excipients include one or more of starch, lactose, microcrystalline cellulose, dextrin, calcium phosphate, polyethylene glycol-4000, polyethylene glycol-6000, sodium carboxymethyl cellulose, hydroxypropyl cellulose or cross-linked polyvinylpyrrolidone.

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