Application of punicalagin in the preparation of drugs for preventing and treating multiple sclerosis

Abstract

The present invention discloses the use of punicalagin in the preparation of drugs for the prevention and treatment of multiple sclerosis, belonging to the technical field of anti-autoimmune disease drugs. Experiments have found that punicalagin can significantly alleviate the occurrence and development of experimental encephalomyelitis in mice, reduce the infiltration of inflammatory cells in the spinal cord of mice, and has no obvious side effects; punicalagin alleviates experimental encephalomyelitis in mice mainly by inhibiting CD4 + T cells differentiate into Th17 cells. Punicalagin can be used as a new drug for the treatment of multiple sclerosis, with broad medical prospects and economic value.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-autoimmune disease drugs, and particularly 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 changes in the central nervous system (CNS). It typically strikes young adults and is more common in women, who typically have a prevalence twice as high as men. Key clinical manifestations include sensory disturbances such as numbness, tingling, and burning sensations, typically in the fingers, feet, and face; motor impairments such as limb fatigue, muscle weakness, cramps, and impaired balance; vision problems such as binocular vision discrepancies, nystagmus, and sudden eye movements; and other symptoms such as speech impairment and difficulty thinking and remembering. The cause of MS remains unclear, but it may be related to viral infection, autoimmune reactions, and genetics. In MS patients, autoreactive T cells cross the blood-brain barrier and enter the CNS. They secrete large amounts of inflammatory cytokines (IFN-γ, IL-17, or TNF-α), attacking the myelin sheath formed by oligodendrocytes in the CNS, leading to demyelination. This damage disrupts the function of parts of the nervous system and causes 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 + EAE is a T-cell-mediated autoimmune disease model characterized by mononuclear cell infiltration and demyelination around small blood vessels in the central nervous system. These pathological changes closely resemble those of human multiple sclerosis (MS), making it the most commonly used animal model for MS research. Currently, EAE is a common preclinical testing method for drugs treating MS.

[0004] Punicalagin (C 48 H 28 O 30 Punicalagin (also known as punicalagin, whose structure is shown below) is the main active ingredient in pomegranate peel extract and has broad application prospects in the cosmetics, health care, and pharmaceutical industries. In the pharmaceutical field, punicalagin has many important pharmacological effects, including antioxidant, anti-tumor, antibacterial, antiviral, and hypoglycemic and lipid-lowering properties. After absorption by the human body, punicalagin is broken down by enzymes into ellagic acid, which has excellent antioxidant properties and has been used as a food antioxidant. However, there are currently no reports of 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:

[0008] The first aspect of the present invention discloses the use of punicalagin in the preparation of a drug for preventing and treating multiple sclerosis.

[0009] Preferably, the dosage of punicalagin is 4 mg / kg.

[0010] Preferably, the drug for preventing and treating multiple sclerosis is a drug that alleviates the severity of multiple sclerosis.

[0011] Preferably, the drug for preventing and treating multiple sclerosis is a drug that inhibits Th17 differentiation.

[0012] 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.

[0013] Preferably, the drug for preventing and treating multiple sclerosis is a drug that reduces lymphocyte infiltration in the spinal cord.

[0014] 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.

[0015] Preferably, the dosage form of the drug for preventing and treating multiple sclerosis is capsule, tablet, pill, powder, granule or injection.

[0016] 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.

[0017] 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.

[0018] The present invention has the following beneficial effects:

[0019] The present invention provides an application of punicalagin in the preparation of drugs for the prevention and treatment of multiple sclerosis. In vitro differentiation experiments have shown 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 can significantly inhibit the onset of EAE; HE staining results showed that punicalagin can 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 alleviates experimental encephalomyelitis in mice mainly by inhibiting CD4 + T cells differentiated into Th17 cells without significant side effects. Therefore, punicalagin can be developed as a new drug for the treatment of multiple sclerosis, with broad medical prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 2 is 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 Figure A.

[0021] Figure 2 Figure 2 shows the effect of punicalagin on the clinical manifestations of EAE model in mice. Figure A shows the clinical score statistics of EAE model established in wild-type mice after treatment with DMSO or punicalagin. Figure B shows the hematoxylin-eosin staining (HE staining) of spinal cord tissue in EAE model in wild-type mice after treatment with DMSO or punicalagin.

[0022] 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; A is a flow cytometric graph of the ratio 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 a 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

[0023] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0024] 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.

[0025] All features, such as values, amounts, contents, and concentrations, described herein as numerical ranges or percentage ranges are provided for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0026] In this document, unless otherwise specified, “include,” “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.”

[0027] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0028] Herein, the administration routes of punicalagin include but are not limited to oral administration, injection, implantation or external application.

[0029] In the present specification 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," and refers to, for example, a single tablet, a single capsule, etc., which can provide, for example, 1 / 3, 1 / 2, 1, or 2 daily doses. For example, a tablet can contain a daily dose for one day, which is preferred; in addition, a tablet can also contain a daily dose for two days. For example, a score can be made in the middle of the tablet to facilitate the user to divide the tablet in half. For those who are heavier and can tolerate the drug, one tablet can be used once a day, while for those who can effectively take half a tablet, half a tablet can be used daily.

[0030] The present invention provides pharmaceutical compositions comprising punicalagin formulated with one or more non-toxic pharmaceutically acceptable carriers. The pharmaceutical compositions can be specifically formulated for oral or rectal administration in solid or liquid form, or 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 retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) adsorbents such as kaolin and bentonite; 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 form may also contain a buffer. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers such as ethanol, isopropyl alcohol, 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, tetrahydrofuranol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0031] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, 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 equally within the scope limited by the appended claims of the application.

[0032] The following examples use conventional instruments and equipment in the art. Experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or according to the 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; and pertussis toxin was purchased from ApexBio, CAS No.: B7273. Unless otherwise specified, all other raw materials used were conventional commercial products, and their specifications were conventional specifications in the art.

[0033] Example 1 Effect of Punicalagin on Differentiation of Th17 Cells in Vitro

[0034] 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 collected 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: 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. Punicalagin was added at a drug concentration of 2.5 μM. An equal amount of DMSO was added to the control group. After 72 hours, the differentiation of Th17 cells in each group was detected by flow cytometry.

[0035] 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 differentiation of initial CD4 + The differentiation of T cells into Th17 cells indicates that punicalagin has the potential to treat multiple sclerosis.

[0036] Example 2 Effect of Punicalagin on Autoimmune Encephalomyelitis (EAE)

[0037] 1. Effect of Punicalagin on the Clinical Manifestations of Autoimmune Encephalomyelitis (EAE)

[0038] 1) Establishment of mouse EAE model

[0039] 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.

[0040] Complete Freund's adjuvant (with a final concentration of 4 mg / mL Mycobacterium tuberculosis H37Ra) and 2 mg / mL MOG35-55 antigen peptide were mixed in a 1:1 ratio of equal volumes and emulsified to form an emulsion. 100 μL of this emulsion was then subcutaneously injected into each mouse at two points on either side of the spine in both the control and experimental groups.

[0041] Punicalagin powder was dissolved in DMSO solvent to obtain a punicalagin solution with a concentration of 25 mg / mL.

[0042] Punicalagin solution and an equal amount of DMSO were dissolved in normal saline to obtain experimental group drugs and control group drugs.

[0043] On the 0th and 2nd day after immunization, mice in each group were intraperitoneally injected with 0.2 mL of pertussis toxin (200 ng / mouse), and on the 4th, 7th, and 10th day after modeling, they were intraperitoneally injected with punicalagin at a dose of 4 mg / kg. The control group was injected with the same amount of DMSO.

[0044] 2) Clinical evaluation

[0045] Mice in each group were continuously observed and scored comprehensively. The scoring criteria were as follows: 0: no symptoms; 1: loss of tail tone; 2: hindlimb weakness; 3: hindlimb paralysis; 4: hindlimb and forelimb paralysis; 5: impending death or death. Symptoms between hindlimb weakness and hindlimb paralysis were scored as ±0.5.

[0046] The scoring results are as follows Figure 2 As shown in Figure A, compared with the control group, punicalagin treatment can significantly inhibit the onset of EAE.

[0047] 3) HE staining

[0048] 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).

[0049] The staining results are as follows Figure 2 As shown in Figure B, the lymphocyte infiltration in the spinal cord of mice in the punicalagin-treated group was significantly less than that in the control group.

[0050] 2. Effect of punicalagin on Th1 and Th17 cells in the spinal cord tissue of autoimmune encephalomyelitis (EAE) model.

[0051] 1) Establishment of mouse EAE model

[0052] Wild-type adult mice were randomly divided into a control group (DMSO) and an experimental group (punicalagin), with 6 mice in each group.

[0053] Complete Freund's adjuvant (with a final concentration of 4 mg / mL Mycobacterium tuberculosis H37Ra) and 2 mg / mL MOG35-55 antigen peptide were mixed in a 1:1 ratio of equal volumes and emulsified to form an emulsion. 100 μL of this emulsion was then subcutaneously injected into each mouse at two points on either side of the spine in both the control and experimental groups.

[0054] Punicalagin powder was dissolved in DMSO solvent to obtain a punicalagin solution with a concentration of 25 mg / mL.

[0055] Punicalagin solution and an equal amount of DMSO were dissolved in normal saline to obtain experimental group drugs and control group drugs.

[0056] On the 0th and 2nd day after immunization, mice in each group were intraperitoneally injected with 0.2 mL of pertussis toxin (200 ng / mouse), and on the 4th, 7th, and 10th day after modeling, they were intraperitoneally injected with punicalagin at a dose of 4 mg / kg. The control group was injected with the same amount of DMSO.

[0057] 2) Preparation of mouse spinal cord mononuclear cell suspension

[0058] At the peak of disease onset, spinal cord tissue was obtained from two groups of mice for single-cell suspension preparation. First, a 90% Percoll solution (the Percoll working solution) was prepared using 10× PBS. The Percoll working solution was then prepared using 1640 medium to prepare 40% and 80% Percoll working solutions, respectively. Mice were dissected, and spinal cord tissue was removed and ground in a grinder containing 2 mL of 1× PBS. The ground solution was collected in a 15 mL centrifuge tube and centrifuged at 1500 rpm at 4°C for 5 minutes. The supernatant was discarded and the tissue was resuspended in 5 mL of 40% Percoll working solution. Using an electric pipette, 5 mL of 80% Percoll working solution was slowly added to the bottom layer of the 5 mL 40% Percoll working solution, maintaining the upper and lower layers. Following this, the tube was centrifuged at 23°C and 500 g for 30 minutes, with the centrifuge speed set to zero. After centrifugation, the tube was slowly removed to maintain the layers. The desired mononuclear cells were the intermediate buffy coat layer. The middle buffy coat layer was aspirated with a pipette and collected into a new 15 mL centrifuge tube. The cells were centrifuged at 1500 rpm and 4°C for 5 min. 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.

[0059] 3) Flow Cytometry

[0060] Mononuclear cell suspensions from mouse spinal cord tissue were collected for flow cytometry staining to analyze the proportions and numbers of Th1 and Th17 cells in the mouse spinal cord tissue.

[0061] Test results such as Figure 3 As shown in the results, compared with the control group, the proportion and number of TH17 cells in the spinal cord tissue of mice treated with punicalagin were significantly lower. The proportion of TH1 cells in the spinal cord tissue of mice treated with punicalagin was comparable to that of the control group, but the number of TH1 cells was significantly lower. Because TH17 cells can secrete IL-17 and TNF-α, and TH1 cells can secrete IFN-γ and TNF-α, TNF-α interacts synergistically with cytokines secreted by TH17 and TH1 cells, promoting inflammatory and autoimmune responses. Therefore, as the number of TH17 and TH1 cells decreases, the levels of multiple sclerosis-related inflammatory factors IL-17, IFN-γ, and TNF-α also decrease. This suggests that punicalagin can alleviate the severity of EAE and has a certain therapeutic effect on multiple sclerosis, making it a potential drug for the treatment of multiple sclerosis.

[0062] The above content is 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 as the sole active ingredient 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 that reduces 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 that inhibits 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 that reduces 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.

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