WO2023035130A1 - Use of aloesome in preparation of drug for treating ulcerative colitis - Google Patents

Use of aloesome in preparation of drug for treating ulcerative colitis Download PDF

Info

Publication number
WO2023035130A1
WO2023035130A1 PCT/CN2021/117079 CN2021117079W WO2023035130A1 WO 2023035130 A1 WO2023035130 A1 WO 2023035130A1 CN 2021117079 W CN2021117079 W CN 2021117079W WO 2023035130 A1 WO2023035130 A1 WO 2023035130A1
Authority
WO
WIPO (PCT)
Prior art keywords
pine
ulcerative colitis
aloe vera
aloe
mpo
Prior art date
Application number
PCT/CN2021/117079
Other languages
French (fr)
Chinese (zh)
Inventor
李友宾
王雪松
龚晶雯
曾婷婷
谭银丰
王燕
徐俊裕
Original Assignee
海南医学院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 海南医学院 filed Critical 海南医学院
Priority to PCT/CN2021/117079 priority Critical patent/WO2023035130A1/en
Publication of WO2023035130A1 publication Critical patent/WO2023035130A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/886—Aloeaceae (Aloe family), e.g. aloe vera
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants

Definitions

  • the invention relates to the field of medicines for ulcerative colitis, in particular to the use of aloe vera pine in the preparation of medicines for treating ulcerative colitis.
  • IBD Inflammatory bowel disease
  • IBD inflammatory bowel disease
  • UC ulcerative colitis
  • CD Crohn's disease
  • the commonly used drugs for the treatment of ulcerative colitis in internal medicine mainly include aminosalicylic acid drugs, glucocorticoid drugs, immunosuppressant drugs and biotherapeutic agents.
  • Aminosalicylates include drugs such as sulfasalazine, mesalamine, and olsalazine. Sulfasalazine is currently the drug of choice for the clinical treatment of ulcerative colitis. After oral administration, it is split into 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) under the action of intestinal flora after oral administration.
  • Salicylic acid is its main active ingredient.
  • Sulfasalazine is cheap, but in the process of treating ulcerative colitis, the effective rate is low, the recurrence rate is high, and there are certain toxic and side effects.
  • Common adverse reactions include gastrointestinal symptoms, allergic dermatitis, hemolysis anemia, agranulocytosis, hepatotoxicity, aplastic anemia.
  • Mesalazine and olsalazine have clear effects in the treatment of ulcerative colitis and have a low recurrence rate.
  • the adverse reactions are similar to those of sulfasalazine, and the incidence and severity are significantly reduced.
  • patients have to bear high drug costs, which is a disadvantage in the clinical application of such drugs.
  • Glucocorticoids and immunosuppressant drugs also have clear effects in treatment, but the adverse reactions and side effects that accompany the treatment also limit the application of the above drugs.
  • biotherapeutic drugs due to the rapid development of cell and molecular biology and immunology, the research and application of biotherapeutic drugs have been paid more and more attention. Ulcerative colitis can be treated by anti-inflammation and immune response regulation, but its expensive drug costs also limit The application of the above drugs. Screening drugs with clear therapeutic effects, fewer adverse reactions and moderate treatment costs, and studying the main mechanism of action of related drugs and their active ingredients have become an important part of the current scientific research work and drug development process.
  • cytokines are directly involved in the pathogenesis of inflammatory bowel disease. Cytokines play an important role in the regulation of intestinal inflammation and its related clinical symptoms. Animal experiments on inflammatory bowel disease have shown that regulating the function of cytokines can be used to treat inflammatory bowel disease. Inhibition of tumor necrosis cytokine (TNF- ⁇ ) is usually used as the gold standard for clinical treatment of inflammatory bowel disease. Regulation of cytokines can inhibit the production of tumor necrosis factor and its corresponding effect, thus playing a vital role in the treatment of inflammatory bowel disease.
  • TNF- ⁇ tumor necrosis cytokine
  • MPO Myeloperoxidase
  • the inflammatory mediator leukotriene B4 (LTB4) produced by arachidonic acid under the action of 5-lipoxygenase is an important chemokine and aggregation factor, which is important for the activation and recruitment of inflammatory cells.
  • Cells include neutrophils, eosinophils, monocytes/macrophages, mast cells, dendritic cells and effector T cells.
  • LTB4 is also known to be directly involved in the pathophysiology of inflammatory bowel disease, and inhibiting the release of LTB4 helps to inhibit the development of UC.
  • NLRP3 inflammasome can activate downstream pro-Caspase-1 and cut it into biologically active Caspase-1.
  • Activated Caspase-1 acts on pro-IL -1 ⁇ and pro-IL-18, and finally form mature IL-1 ⁇ , IL-18 and other inflammatory factors, cut the N-terminal sequence of GSDMD, make it bind to the cell membrane to produce membrane holes, cause cell pyroptosis, and aggravate the intestinal tract Inflammation, inhibition of IL-1 ⁇ , IL-18 production suppresses excessive inflammation in UC.
  • Aloe is a perennial evergreen succulent herb of the Liliaceae family (liliaceae) and the genus Aloe.
  • human beings have known it for more than three or four thousand years, and it has also been recognized in my country. It has been used for more than a thousand years.
  • aloe vera in the national pharmacopoeia as a herbal medicine.
  • the main components of aloe are anthraquinones, chromones, and polysaccharides.
  • Anthraquinone has strong anti-inflammatory activity, and anthraquinone components are cytotoxic, mutagenic and carcinogenic.
  • Aloe pine (aloesone) is the aglycon of aloin, and it is also a unique chemical component in the epidermis of aloe. Aloe pine belongs to chromone compounds, and its safety is better than anthraquinone. However, the content of aloe vera pine in aloe is low and there are few studies on related pharmacological activities.
  • the present invention proposes the use of aloe vera pine in the preparation of medicines for treating ulcerative colitis, and provides new active components for medicines for treating ulcerative colitis, and the safety is higher than anthraquinone, an active ingredient of aloe.
  • the invention finds that the aloe vera pine can be applied to the preparation of medicine for treating ulcerative colitis. It has been found through experiments that aloe vera pine can better inhibit the production of IL-1 ⁇ and IL-18, inhibit the activity of MPO, inhibit the production of LTB4 and TNF ⁇ , and can inhibit the pyroptosis of cells.
  • the concentration of the aloe pine is 2-5 ⁇ M.
  • the use of the aloe vera pine in the preparation of a medicine for treating ulcerative colitis by inhibiting MPO activity Preferably, the concentration of the aloe pine is 50-1000 ⁇ M.
  • the use of the aloe vera pine in the preparation of medicines for treating ulcerative colitis by inhibiting the production of LTB4 and TNF ⁇ Preferably, the dosage of the aloe vera pine is 20-40 mg/kg/d.
  • aloe vera pine is used in the preparation of a medicine for treating ulcerative colitis by simultaneously inhibiting the production of IL-1 ⁇ and IL-18, inhibiting the activity of MPO, and inhibiting the production of LTB4 and TNF ⁇ .
  • the present invention studies the therapeutic effect of aloe vera pine on ulcerative colitis (UC) in vitro and in vivo, and finds that aloe vera pine can inhibit the activity of MPO in vitro, inhibit macrophages from producing inflammatory factors such as IL-1 ⁇ and IL-18, thereby inhibiting the pyroptosis; in vivo can reduce DSS model mice plasma LTB4, TNF ⁇ . Therefore, aloe vera pine can improve the inflammatory response of colitis mice, thereby effectively inhibiting the process of UC, and can be used to prepare medicines for treating ulcerative colitis.
  • UC ulcerative colitis
  • Fig. 3A, Fig. 3B the effect of aloe pine on LPS and ATP on macrophage IL-1 ⁇ , IL-18 inflammatory factor generation; Among them, Fig. 3A is the inhibitory activity of aloe pine to IL-18, Fig. 3B is aloe pine on Inhibitory activity of IL-1 ⁇ ; *p ⁇ 0.5 vs LPS+ATP, **p ⁇ 0.01, ***p ⁇ 0.001, ##p ⁇ 0.01 vs KB.
  • Fig. 4A, Fig. 4B Effect of aloe vera pine on plasma leukotriene B4 and tumor necrosis factor- ⁇ concentrations in DSS ulcerative colitis mice, wherein, Fig. 4A: leukotriene B4, Fig. 4B: tumor necrosis factor- ⁇ .
  • Aloe pine can be obtained by extracting plants of the genus Aloe or rhubarb, or by chemical synthesis.
  • Substrate solution preparation Add 1 ⁇ l hydrogen peroxide to 175 ⁇ l MPO detection buffer to prepare 5 mM hydrogen peroxide solution.
  • RFU(EC) is the fluorescence readout of the enzyme control
  • RFU(S) is the fluorescence readout of the candidate inhibitor
  • mice of the Balb/c strain were kept at 23°C, humidity: 55 ⁇ 5%, alternated between light and dark for 12 hours, fed with conventional feed, and all mice had free access to water and food.
  • mice Two weeks after the start of the mouse experiment, DSS containing 2.5% concentration was dissolved in the drinking water of the mice, and it was prepared and used every day. After continuously giving the experimental animals drinking water containing 2.5% DSS for 7 days, they were given normal drinking water for 3 days. The normal control group was given normal drinking water for 10 consecutive days. Mice were then sacrificed by cardiac exsanguination under light ether anesthesia, and blood was collected in vials of 5% EDTA by cardiac puncture. The separated plasma was quickly stored at -70°C for testing. The contents of LTB4 and TNF- ⁇ in mouse plasma were determined according to the ELISA detection kit (R&D Company, USA).
  • the inhibitory activity of different concentrations of aloe vera pine on MPO is shown in Table 1. The results show that aloe vera pine has better inhibitory activity on MPO, and the inhibition rate of 50 ⁇ M concentration reaches 66.7%.
  • Ulcerative colitis is a chronic and relapsing inflammatory disease of the colon and rectum with unknown etiology. It is widely accepted that environmental interactions, genetic inheritance, and abnormal immune responses are factors in the development of ulcerative colitis. Ulcerative colitis is closely related to inflammatory mediators secreted by inflammatory cells, the most important of which are IL-18, IL-1 ⁇ , MPO, TNF ⁇ , and LTB4. The increase of IL-18 and IL-1 ⁇ will cause cell pyroptosis, which will aggravate the deterioration of IBD.
  • aloe vera pine can effectively inhibit the production of IL-18 and IL-1 ⁇ , indicating that aloe vera pine is beneficial to colitis; MPO and The infiltration of macrophages is related. Compared with non-colitis rats, the MPO activity in the colon of DSS control rats was significantly increased, indicating that neutrophils play an important role in DSS colitis. In this study, aloe vera pine can effectively Inhibits the activity of MPO, which also has anti-inflammatory activity associated with neutrophils by inhibiting MPO activity in target tissues, thereby effectively inhibiting the progression of UC.
  • LTB4 is important for the activation and recruitment of inflammatory cells, and LTB4 is also directly involved in the pathophysiology of inflammatory bowel disease.
  • the results of this study showed that the levels of plasma LTB4 in mice taking aloe vera pine were significantly reduced, and the levels of LTB4 in the high-dose group were all reduced to the level of non-colitis.
  • Aloe pine was proved to be a strong inhibitor of LTB4 release, indicating that aloe pine could reduce the accumulation of neutrophils and leukocytes in the mucosa.
  • TNF- ⁇ tumor necrosis cytokine
  • Aloe pine can inhibit pyroptosis, inhibit MPO activity, and inhibit the production of LTB4 and TNF- ⁇ in vivo, thereby improving the inflammatory response of ulcerative colitis, weakening the recruitment of immune cells, and achieving the effect of alleviating and treating UC. Therefore, aloe vera pine can be used to prepare medicine for treating ulcerative colitis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Medical Informatics (AREA)
  • Botany (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The present invention provides a use of aloesome in the preparation of a drug for treating ulcerative colitis. In the present invention, the therapeutic effect of aloesome on ulcerative colitis is studied in vivo and in vitro. It has been found that aloesome can inhibit the activity of MPO in vitro and inhibit macrophages from producing inflammatory factors, such as IL-1β and IL-18, thereby inhibiting pyroptosis; moreover, mice plasma LTB4 and TNFα in a DSS model can be reduced in vivo. Therefore, aloesome can improve the inflammatory response of mice suffering from colitis, thereby effectively inhibiting the progression of ulcerative colitis, and can be used for preparing a drug for treating ulcerative colitis.

Description

芦荟松在制备治疗溃疡性结肠炎药物中的用途Application of aloe vera pine in preparation of medicine for treating ulcerative colitis 技术领域technical field
本发明涉及溃疡性结肠炎药物领域，特别涉及芦荟松在制备治疗溃疡性结肠炎药物中的用途。The invention relates to the field of medicines for ulcerative colitis, in particular to the use of aloe vera pine in the preparation of medicines for treating ulcerative colitis.
背景技术Background technique
炎症性肠病(inflammatory bowel disease,IBD)是一种以消化道溃疡为显著特点的慢性自身免疫疾病，近些年世界各地其发病率成倍增加，患者长期遭受消化道出血、穿孔、肠梗阻等病痛的折磨。IBD主要包括溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(Crohn's disease,CD)，两者临床特点是活动期和缓解期交替出现，多呈反复发作的慢性病程。IBD发病原因有很多，主要包括环境、遗传、感染和免疫等多种因素。我国UC比CD的发病率高得多。Inflammatory bowel disease (inflammatory bowel disease, IBD) is a chronic autoimmune disease characterized by peptic ulcer. In recent years, its incidence has doubled all over the world. Wait for the torment of illness. IBD mainly includes ulcerative colitis (UC) and Crohn's disease (CD), both of which are clinically characterized by alternating active phases and remission phases, and most of them are chronic and recurrent. There are many causes of IBD, mainly including environmental, genetic, infection and immune factors. The incidence of UC in my country is much higher than that of CD.
目前内科治疗溃疡性结肠炎常用的药物主要包括氨基水杨酸类药物、糖皮质激素类药物、免疫抑制剂类药物以及生物治疗制剂类药物。氨基水杨酸类药物包括柳氮磺胺吡啶、美沙拉嗪和奥沙拉嗪等药物。柳氮磺胺吡啶是目前临床治疗溃疡性结肠炎的首选药物，经口服后在肠道内菌群的作用下裂解为5-氨基水杨酸(5-ASA)和磺胺吡啶(SP)，5-氨基水杨酸是其主要的有效成分。柳氮磺胺吡啶药物价格便宜，但在治疗溃疡性结肠炎的过程中有效率偏低、复发率偏高，同时还存在一定的毒副作用，常见的不良反应有消化道症状、过敏性皮炎、溶血性贫血、粒细胞缺乏、肝毒性、再生障碍性贫血。美沙拉嗪和奥沙拉嗪在治疗溃疡性结肠炎的过程中效果明确且复发率较低，不良反应与柳氮磺吡啶类似，发生率和严重程度明显降低。但是在长期的治疗过程中，患者要负担高额的药物费用，是此类药物在临床应用中的不利方面。糖皮质激素类及免疫抑制剂类 药物在治疗中也有明确的效果，但是伴随治疗过程中存在的不良反应及毒副作用也限制了上述药物的应用。近年来由于细胞分子生物学和免疫学的迅速发展，生物治疗制剂类药物的研究与应用日益受到重视，可通过抗炎和调节免疫反应治疗溃疡性结肠炎，但其昂贵的药物费用也限制了上述药物的应用。筛选治疗效果明确、不良反应较少且治疗费用适中的药物，研究相关药物及其有效成分的主要作用机制成为目前科研工作及药物研发过程中的一项重要内容。At present, the commonly used drugs for the treatment of ulcerative colitis in internal medicine mainly include aminosalicylic acid drugs, glucocorticoid drugs, immunosuppressant drugs and biotherapeutic agents. Aminosalicylates include drugs such as sulfasalazine, mesalamine, and olsalazine. Sulfasalazine is currently the drug of choice for the clinical treatment of ulcerative colitis. After oral administration, it is split into 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) under the action of intestinal flora after oral administration. Salicylic acid is its main active ingredient. Sulfasalazine is cheap, but in the process of treating ulcerative colitis, the effective rate is low, the recurrence rate is high, and there are certain toxic and side effects. Common adverse reactions include gastrointestinal symptoms, allergic dermatitis, hemolysis anemia, agranulocytosis, hepatotoxicity, aplastic anemia. Mesalazine and olsalazine have clear effects in the treatment of ulcerative colitis and have a low recurrence rate. The adverse reactions are similar to those of sulfasalazine, and the incidence and severity are significantly reduced. However, in the long-term treatment process, patients have to bear high drug costs, which is a disadvantage in the clinical application of such drugs. Glucocorticoids and immunosuppressant drugs also have clear effects in treatment, but the adverse reactions and side effects that accompany the treatment also limit the application of the above drugs. In recent years, due to the rapid development of cell and molecular biology and immunology, the research and application of biotherapeutic drugs have been paid more and more attention. Ulcerative colitis can be treated by anti-inflammation and immune response regulation, but its expensive drug costs also limit The application of the above drugs. Screening drugs with clear therapeutic effects, fewer adverse reactions and moderate treatment costs, and studying the main mechanism of action of related drugs and their active ingredients have become an important part of the current scientific research work and drug development process.
经过大量遗传学和免疫学研究显示，细胞因子直接地参与炎症性肠病的发病机制。细胞因子对于肠内炎症及其相关临床症状的调节发挥着重要的作用。炎症性肠病的动物实验表明，调节细胞因子的功能可以用于治疗炎症性肠病。抑制肿瘤坏死细胞因子(TNF-α)通常作为临床上治疗炎症性肠病的金标准。调节细胞因子可以抑制肿瘤坏死因子产生及其相应作用效果，从而对于治疗炎症性肠病发挥着至关重要的作用。A large number of genetic and immunological studies have shown that cytokines are directly involved in the pathogenesis of inflammatory bowel disease. Cytokines play an important role in the regulation of intestinal inflammation and its related clinical symptoms. Animal experiments on inflammatory bowel disease have shown that regulating the function of cytokines can be used to treat inflammatory bowel disease. Inhibition of tumor necrosis cytokine (TNF-α) is usually used as the gold standard for clinical treatment of inflammatory bowel disease. Regulation of cytokines can inhibit the production of tumor necrosis factor and its corresponding effect, thus playing a vital role in the treatment of inflammatory bowel disease.
髓过氧化物酶(MPO)是在嗜中性粒细胞中发现的一种酶，MPO可以通过催化氧化氯离子产生次氯酸在吞噬细胞内杀灭微生物，破坏多种靶物质，对机体产生和调节炎症反应等多方面发挥作用。它在结肠中的活性被认为是组织炎症的标志，并且与中性粒细胞浸润线性相关。如能通过抑制靶组织MPO活性而与嗜中性粒细胞相关的抗炎活性，从而有效抑制UC的进程。Myeloperoxidase (MPO) is an enzyme found in neutrophils. MPO can catalyze the oxidation of chlorine ions to produce hypochlorous acid, kill microorganisms in phagocytes, destroy a variety of target substances, and cause damage to the body. and regulation of inflammatory responses. Its activity in the colon is considered a marker of tissue inflammation and is linearly correlated with neutrophil infiltration. For example, it can effectively inhibit the process of UC by inhibiting the anti-inflammatory activity related to neutrophils by inhibiting the activity of MPO in target tissues.
由花生四烯酸在5-脂氧合酶作用下产生的炎症介质白三烯B4(LTB4)是一种重要的趋化因子和聚集因子，对于炎性细胞的激活和募集很重要，炎性细胞包括嗜中性粒细胞，嗜酸性粒细胞，单核细胞/巨噬细胞，肥大细胞，树突状细胞和效应T细胞。还已知LTB4直接参与炎症性肠病的病理生理学，抑制LTB4的释放有助于抑制UC的发展。The inflammatory mediator leukotriene B4 (LTB4) produced by arachidonic acid under the action of 5-lipoxygenase is an important chemokine and aggregation factor, which is important for the activation and recruitment of inflammatory cells. Cells include neutrophils, eosinophils, monocytes/macrophages, mast cells, dendritic cells and effector T cells. LTB4 is also known to be directly involved in the pathophysiology of inflammatory bowel disease, and inhibiting the release of LTB4 helps to inhibit the development of UC.
大量研究表明UC的发生发展与细胞焦亡存在着一定的相关性。NLRP3炎 性小体在UC中作用研究最为广泛，NLRP3炎症小体可通过激活下游pro-Caspase-1并使其剪切成具有生物活性的Caspase-1，活化的Caspase-1作用于pro-IL-1β及pro-IL-18，最终形成成熟的IL-1β，IL-18等炎症因子，切割GSDMD的N端序列，使其结合到细胞膜上产生膜孔，导致细胞焦亡，从而加重肠道炎症，抑制IL-1β，IL-18的产生可抑制UC的过度炎症。A large number of studies have shown that there is a certain correlation between the occurrence and development of UC and pyroptosis. The role of NLRP3 inflammasome in UC is the most widely studied. NLRP3 inflammasome can activate downstream pro-Caspase-1 and cut it into biologically active Caspase-1. Activated Caspase-1 acts on pro-IL -1β and pro-IL-18, and finally form mature IL-1β, IL-18 and other inflammatory factors, cut the N-terminal sequence of GSDMD, make it bind to the cell membrane to produce membrane holes, cause cell pyroptosis, and aggravate the intestinal tract Inflammation, inhibition of IL-1β, IL-18 production suppresses excessive inflammation in UC.
芦荟为百合科(liliaceae)芦荟属(aloe)多年生常绿肉质草本植物，作为一种世界性古老而神奇的植物，人类对它的认知已超过三、四千年的历史，在我国也已应用一千余年。当前，世界上有20多个国家将芦荟作为植物草药载入国家药典。芦荟主要成分为蒽醌类、色酮类、多糖。蒽醌具有较强的抗炎活性，而蒽醌类成分具有细胞毒性，致突变性和致癌性。芦荟松(aloesone)是芦荟苦素的苷元，也是芦荟表皮中特有的化学成分。芦荟松属于色酮类化合物，安全性比蒽醌好。但芦荟松在芦荟中的含量较低且相关药理活性的研究较少。Aloe is a perennial evergreen succulent herb of the Liliaceae family (liliaceae) and the genus Aloe. As an ancient and magical plant in the world, human beings have known it for more than three or four thousand years, and it has also been recognized in my country. It has been used for more than a thousand years. At present, more than 20 countries in the world have included aloe vera in the national pharmacopoeia as a herbal medicine. The main components of aloe are anthraquinones, chromones, and polysaccharides. Anthraquinone has strong anti-inflammatory activity, and anthraquinone components are cytotoxic, mutagenic and carcinogenic. Aloe pine (aloesone) is the aglycon of aloin, and it is also a unique chemical component in the epidermis of aloe. Aloe pine belongs to chromone compounds, and its safety is better than anthraquinone. However, the content of aloe vera pine in aloe is low and there are few studies on related pharmacological activities.
发明内容Contents of the invention
鉴于此，本发明提出芦荟松在制备治疗溃疡性结肠炎药物中的用途，为治疗溃疡性结肠药物提供新的活性组分，而且安全性高于芦荟活性成分蒽醌。In view of this, the present invention proposes the use of aloe vera pine in the preparation of medicines for treating ulcerative colitis, and provides new active components for medicines for treating ulcerative colitis, and the safety is higher than anthraquinone, an active ingredient of aloe.
本发明的技术方案是这样实现的：Technical scheme of the present invention is realized like this:
本发明发现可将芦荟松应用于制备治疗溃疡性结肠炎药物。经试验发现芦荟松能够较好地抑制IL-1β和IL-18产生、抑制MPO活性、抑制LTB4和TNFα产生，而能够抑制细胞焦亡。The invention finds that the aloe vera pine can be applied to the preparation of medicine for treating ulcerative colitis. It has been found through experiments that aloe vera pine can better inhibit the production of IL-1β and IL-18, inhibit the activity of MPO, inhibit the production of LTB4 and TNFα, and can inhibit the pyroptosis of cells.
进一步的，所述芦荟松在制备通过抑制IL-1β、IL-18产生来治疗溃疡性结肠炎药物中的用途。优选地，所述芦荟松的浓度为2-5μM。Further, the use of the aloe vera pine in the preparation of medicines for treating ulcerative colitis by inhibiting the production of IL-1β and IL-18. Preferably, the concentration of the aloe pine is 2-5 μM.
进一步的，所述芦荟松在制备通过抑制MPO活性来治疗溃疡性结肠炎药物中的用途。优选地，所述芦荟松的浓度为50-1000μM。Further, the use of the aloe vera pine in the preparation of a medicine for treating ulcerative colitis by inhibiting MPO activity. Preferably, the concentration of the aloe pine is 50-1000 μM.
进一步的，所述芦荟松在制备通过抑制LTB4、TNFα产生来治疗溃疡性结肠炎药物中的用途。优选地，所述芦荟松的剂量为20-40mg/kg/d。Further, the use of the aloe vera pine in the preparation of medicines for treating ulcerative colitis by inhibiting the production of LTB4 and TNFα. Preferably, the dosage of the aloe vera pine is 20-40 mg/kg/d.
进一步的，所述芦荟松在制备通过同时抑制IL-1β和IL-18产生、抑制MPO活性、抑制LTB4和TNFα产生来治疗溃疡性结肠炎药物中的用途。Further, the aloe vera pine is used in the preparation of a medicine for treating ulcerative colitis by simultaneously inhibiting the production of IL-1β and IL-18, inhibiting the activity of MPO, and inhibiting the production of LTB4 and TNFα.
与现有技术相比，本发明的有益效果是：Compared with prior art, the beneficial effect of the present invention is:
本发明从体内外研究了芦荟松对溃疡性结肠炎(UC)的治疗作用，发现芦荟松体外能抑制MPO的活性，抑制巨噬细胞产生IL-1β，IL-18等炎症因子，从而抑制细胞焦亡；体内能降低DSS模型小鼠血浆LTB4、TNFα。因此，芦荟松可以改善结肠炎小鼠的炎症反应，从而有效抑制UC进程，可用于制备治疗溃疡性结肠炎药物。The present invention studies the therapeutic effect of aloe vera pine on ulcerative colitis (UC) in vitro and in vivo, and finds that aloe vera pine can inhibit the activity of MPO in vitro, inhibit macrophages from producing inflammatory factors such as IL-1β and IL-18, thereby inhibiting the pyroptosis; in vivo can reduce DSS model mice plasma LTB4, TNFα. Therefore, aloe vera pine can improve the inflammatory response of colitis mice, thereby effectively inhibiting the process of UC, and can be used to prepare medicines for treating ulcerative colitis.
附图说明Description of drawings
图1芦荟松的氢谱图；The hydrogen spectrogram of Fig. 1 aloe pine;
图2芦荟松的碳谱图；The carbon spectrogram of Fig. 2 aloe vera pine;
图3A、图3B：芦荟松对LPS和ATP对巨噬细胞IL-1β，IL-18炎症因子生成的影响；其中，图3A为芦荟松对IL-18的抑制活性，图3B为芦荟松对IL-1β的抑制活性；*p＜0.5vs LPS+ATP，**p＜0.01,***p＜0.001，##p＜0.01vs KB。Fig. 3A, Fig. 3B: the effect of aloe pine on LPS and ATP on macrophage IL-1β, IL-18 inflammatory factor generation; Among them, Fig. 3A is the inhibitory activity of aloe pine to IL-18, Fig. 3B is aloe pine on Inhibitory activity of IL-1β; *p<0.5 vs LPS+ATP, **p<0.01, ***p<0.001, ##p<0.01 vs KB.
图4A、图4B：芦荟松对DSS溃疡性肠炎小鼠血浆白三烯B4和肿瘤坏死因子α浓度的影响，其中，图4A:白三烯B4，图4B：肿瘤坏死因子α。Fig. 4A, Fig. 4B: Effect of aloe vera pine on plasma leukotriene B4 and tumor necrosis factor-α concentrations in DSS ulcerative colitis mice, wherein, Fig. 4A: leukotriene B4, Fig. 4B: tumor necrosis factor-α.
具体实施方式Detailed ways
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.
本发明实施例所用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.
本发明实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.
芦荟松可以由芦荟属或大黄属植物提取制得，或者由化学合成制得。Aloe pine can be obtained by extracting plants of the genus Aloe or rhubarb, or by chemical synthesis.
中文名：芦荟松，分子式C 13H 12O 4；分子量：232.23198；英文名：aloesone； 其它命名：2-acetonyl-7-hydroxy-5-methylchromone(2-丙酮基-7-羟基-5-甲基-色酮)、2-acetonyl-7-hydroxy-5-methyl-chromen-4-one(2-丙酮基-7-羟基-5-甲基-色烯-4-酮)、2-(2-oxopropyl)-7-hydroxy-5-methyl-4H-1-benzopyran-4-one(2-(2-氧丙基)-7-羟基-5-甲基-4H-1-苯并吡喃-4-酮)、7-hydroxy-5-methyl-2-(2-oxopropyl)chromen-4-one(7-羟基-5-甲基-2-(2-氧丙基)色烯-4-酮)。 Chinese name: Aloe pine, molecular formula C 13 H 12 O 4 ; molecular weight: 232.23198; English name: aloesone; other names: 2-acetonyl-7-hydroxy-5-methylchromone base-chromone), 2-acetonyl-7-hydroxy-5-methyl-chromen-4-one (2-acetonyl-7-hydroxyl-5-methyl-chromen-4-one), 2-(2 -oxopropyl)-7-hydroxy-5-methyl-4H-1-benzopyran-4-one(2-(2-oxopropyl)-7-hydroxy-5-methyl-4H-1-benzopyran- 4-keto), 7-hydroxy-5-methyl-2-(2-oxopropyl)chromen-4-one(7-hydroxy-5-methyl-2-(2-oxopropyl)chromen-4-one ).
一、芦荟松合成1. Synthesis of aloe vera pine
1.1芦荟松合成路线1.1 Aloe pine synthetic route
Figure PCTCN2021117079-appb-000001
Figure PCTCN2021117079-appb-000001
1.2芦荟松的合成方法：1.2 The synthetic method of aloe vera pine:
(1)3，5-二羟基甲苯和乙酸和三氟化硼***按摩尔比10：8：20混合，在80℃油浴下磁力搅拌反应12h。反应完毕后冷却至室温，用乙酸乙酯和水萃取，萃取三次，取乙酸乙酯层，加入到饱和NaHCO 3溶液中，每摩尔3，5-二羟基甲苯加入饱和NaHCO 3溶液体积为1L，将混合物搅拌10min，使溶液中的乙酸反应完全。然后用乙酸乙酯和水萃取，萃取三次，取乙酸乙酯层，减压浓缩除去溶剂，得到浓缩物，加入乙酸乙酯使浓缩物溶解，再加入适量石油醚后静置重结晶，抽滤后得到化合物1-1。 (1) Mix 3,5-dihydroxytoluene with acetic acid and boron trifluoride ether at a molar ratio of 10:8:20, and react with magnetic stirring in an oil bath at 80°C for 12 hours. After the reaction was completed, cool to room temperature, extract with ethyl acetate and water, and extract three times, take the ethyl acetate layer, add it to a saturated NaHCO solution, add 3,5 - dihydroxytoluene to the saturated NaHCO solution to make the volume 1L , The mixture was stirred for 10 min to completely react the acetic acid in the solution. Then extract with ethyl acetate and water, extract three times, take the ethyl acetate layer, concentrate under reduced pressure to remove the solvent, obtain a concentrate, add ethyl acetate to dissolve the concentrate, add an appropriate amount of petroleum ether, stand for recrystallization, and filter with suction Compound 1-1 was obtained.
(2)化合物1-1、二氯甲烷和DIPEA(N,N-二异丙基乙胺)混合，每摩尔化合物1-1加入二氯甲烷体积为3.5L，化合物1-1和DIPEA的摩尔比为1:6.5；冰浴磁力搅拌反应至0℃后，加入MEM-Cl(2-甲氧基乙氧基甲基氯)，化合物1-1和MEM-Cl的摩尔比为1:1；室温下磁力搅拌反应12h。反应完毕后用乙酸乙酯和水萃取，萃取三次，取乙酸乙酯层，减压浓缩除 去溶剂，得到浓缩物用柱层析(石油醚︰乙酸乙酯＝6︰1，v/v)纯化得到化合物1-2，即化合物C。(2) Compound 1-1, dichloromethane and DIPEA (N,N-diisopropylethylamine) are mixed, and the volume of dichloromethane added per mole of compound 1-1 is 3.5L, and the moles of compound 1-1 and DIPEA The ratio is 1:6.5; after the reaction is heated to 0°C with magnetic stirring in an ice bath, MEM-Cl (2-methoxyethoxymethyl chloride) is added, and the molar ratio of compound 1-1 and MEM-Cl is 1:1; Magnetic stirring was carried out at room temperature for 12 h. After the reaction is completed, extract with ethyl acetate and water, extract three times, take the ethyl acetate layer, concentrate under reduced pressure to remove the solvent, and obtain a concentrate that is purified by column chromatography (petroleum ether: ethyl acetate = 6: 1, v/v) Compound 1-2, namely compound C, was obtained.
(3)取化合物C、苹果酸、4-二甲氨基吡啶和二氯甲烷混合，化合物C、苹果酸和4-二甲氨基吡啶的摩尔比为1：1.0：1.0，化合物C和二氯甲烷的摩尔体积比mol/L为1:4.0；冰浴搅拌反应至0℃后，加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐，25℃下搅拌反应6.0h；反应后用二氯甲烷和水萃取，取二氯甲烷层，减压浓缩除去溶剂，得到浓缩物，将浓缩物经柱层析纯化，所述柱层析的溶剂由体积比为6.0：1的石油醚和乙酸乙酯混合制得，得到化合物1-3，即化合物B；(3) Mix compound C, malic acid, 4-dimethylaminopyridine and dichloromethane, the molar ratio of compound C, malic acid and 4-dimethylaminopyridine is 1:1.0:1.0, compound C and dichloromethane The molar volume ratio mol/L is 1:4.0; after stirring the reaction in an ice bath to 0°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir the reaction at 25°C 6.0h; After the reaction, extract with dichloromethane and water, take the dichloromethane layer, concentrate under reduced pressure to remove the solvent, and obtain a concentrate, which is purified by column chromatography, and the solvent of the column chromatography is 6.0 by volume. : Prepared by mixing petroleum ether and ethyl acetate in 1 to obtain compound 1-3, namely compound B;
(4)取化合物B、氢氧化钠和二甲基亚砜混合，化合物B和氢氧化钠的摩尔比为1:2.4，化合物B和二甲基亚砜的摩尔体积比mol/L为1:4.0，25℃下搅拌反应8.0h，反应后用乙酸乙酯和水萃取，取乙酸乙酯层，减压浓缩除去溶剂，得到浓缩物，将浓缩物经柱层析纯化，所述柱层析的溶剂由体积比为5.0：1的石油醚和乙酸乙酯混合制得，得到化合物1-4，即化合物A；(4) Mix compound B, sodium hydroxide and dimethyl sulfoxide, the mol ratio of compound B and sodium hydroxide is 1:2.4, and the molar volume ratio mol/L of compound B and dimethyl sulfoxide is 1: 4.0, stirred and reacted at 25°C for 8.0h, extracted with ethyl acetate and water after the reaction, took the ethyl acetate layer, concentrated under reduced pressure to remove the solvent, and obtained a concentrate, which was purified by column chromatography, the column chromatography The solvent is prepared by mixing petroleum ether and ethyl acetate with a volume ratio of 5.0:1 to obtain compound 1-4, namely compound A;
(5)将化合物A、氯化氢溶液和异丙醇混合，化合物A和氯化氢的摩尔比为1：4.0，异丙醇和化合物A的摩尔比为32.7：1，加热至45℃搅拌反应1.5h；反应后减压浓缩除去溶剂，得到浓缩物，将浓缩物用柱层析纯化，柱层析的溶剂由体积比为5：1的石油醚和乙酸乙酯混合制得；纯化、干燥后得到化合物1-5，即目标产物芦荟松。(5) Mix compound A, hydrogen chloride solution and isopropanol, the molar ratio of compound A to hydrogen chloride is 1:4.0, the molar ratio of isopropanol to compound A is 32.7:1, heat to 45°C and stir for 1.5h; react Afterwards, the solvent was concentrated under reduced pressure to obtain a concentrate, which was purified by column chromatography. The solvent for column chromatography was prepared by mixing petroleum ether and ethyl acetate with a volume ratio of 5:1; after purification and drying, compound 1 was obtained. -5, the target product aloe vera pine.
芦荟松核磁数据：Aloe pine NMR data:
白色固体粉末， 1H NMR(400MHz,D 6)δ10.61(s,1H),6.62(s,1H),6.60(s,1H),6.03(s,1H),3.85(s,2H),2.65(s,3H),2.21(s,3H)； 13C NMR(100MHz,D 6)δ202.87,178.22,161.09,160.62,159.27,141.65,116.70,114.39,112.92,100.58,47.58,29.89,22.48. White solid powder, 1 H NMR(400MHz,D 6 )δ10.61(s,1H),6.62(s,1H),6.60(s,1H),6.03(s,1H),3.85(s,2H), 2.65(s,3H),2.21(s,3H); 13 C NMR(100MHz,D 6 )δ202.87,178.22,161.09,160.62,159.27,141.65,116.70,114.39,112.92,100.58,47.58,29.89.22.
芦荟松的氢谱图如图1所示；芦荟松的碳谱图如图2所示。The hydrogen spectrum of aloe pine is shown in Figure 1; the carbon spectrum of aloe pine is shown in Figure 2.
二、应用试验2. Application test
(一)实验方法(1) Experimental method
1.芦荟松对LPS和ATP对巨噬细胞IL-1β，IL-18炎症因子生成的影响1. The effect of aloe vera pine on the production of macrophage IL-1β and IL-18 inflammatory factors by LPS and ATP
制备巨噬细胞悬液：细胞计数，接种到12孔板中：每孔4 x 10 5个/ml细胞，同样的样本做3个重复。37℃培养箱中培养：细胞接种后贴壁培养24h。除空白孔外，每孔加入500μL含200ng/mL LPS的无血清DMEM高糖培养基。空白孔加入等体积不含药的无血清DMEM高糖培养基。37℃培养箱中培养4h。加入不同浓度的芦荟松50μL，使终浓度达到0.1、0.5、 1、5、10μg/mL。37℃培养箱中作用2h。其余孔加入等体积不含药的无血清DMEM高糖培养基。芦荟松也由无血清培养基配置。作用时间结束后，每孔加入50μL ATP，使其终浓度为2.5mM.作用30min，取上清液。使用Abcam Elisa试剂盒进行IL-1β，IL-18检测。 Prepare macrophage suspension: count cells, inoculate into 12-well plate: 4 x 10 5 cells/ml per well, and do 3 replicates for the same sample. Cultivate in a 37°C incubator: After cell inoculation, adhere to the wall and culture for 24 hours. Except for blank wells, 500 μL of serum-free DMEM high-glucose medium containing 200 ng/mL LPS was added to each well. An equal volume of drug-free serum-free DMEM high-glucose medium was added to the blank well. Incubate for 4 hours in a 37°C incubator. Add 50 μL of different concentrations of aloe vera pine to make the final concentration reach 0.1, 0.5, 1, 5, 10 μg/mL. Incubate at 37°C for 2 hours. Add an equal volume of drug-free serum-free DMEM high-glucose medium to the remaining wells. Aloe pine is also formulated with serum-free medium. After the action time is over, add 50 μL of ATP to each well to make the final concentration 2.5 mM. After 30 min of action, take the supernatant. IL-1β, IL-18 detection was performed using Abcam Elisa kit.
2.MPO过氧化抑制剂筛选方法2. MPO peroxidation inhibitor screening method
(1)筛选化合物、抑制剂对照和酶对照液的制备：将候选抑制剂以待测试的最高最终浓度的100倍溶解到适当的溶剂中。用MPO检测缓冲液稀释至所需测试浓度的2倍。将45μl稀释的候选抑制剂或MPO检测缓冲液添加到所需的孔中，作为样品筛选[S]或酶控制[EC](无抑制剂)。对于抑制剂对照(IC)，将5μl抑制剂对照加入245μl MPO检测缓冲液中，将抑制剂对照稀释50倍。将45μl稀释的抑制剂对照添加到所需孔中。(1) Preparation of screening compound, inhibitor control and enzyme control solutions: Dissolve candidate inhibitors in an appropriate solvent at 100 times the highest final concentration to be tested. Dilute to 2x the desired assay concentration with MPO Assay Buffer. Add 45 μl of diluted candidate inhibitor or MPO assay buffer to desired wells as sample screen [S] or enzyme control [EC] (no inhibitor). For the inhibitor control (IC), dilute the inhibitor control 50-fold by adding 5 μl of the inhibitor control to 245 μl of MPO assay buffer. Add 45 μl of the diluted inhibitor control to the desired wells.
(2)酶溶液的制备：对于每个孔，准备5μl MPO酶溶液，其中MPO分析缓冲液2.5μl、PMO酶液2.5μl。(2) Preparation of enzyme solution: For each well, prepare 5 μl of MPO enzyme solution, including 2.5 μl of MPO analysis buffer and 2.5 μl of PMO enzyme solution.
将5μl MPO酶溶液混合并加入样品、酶对照和抑制剂对照孔中。 Mix 5 μl of MPO enzyme solution and add to sample, enzyme control and inhibitor control wells.
(3)底物溶液制备：将1μl过氧化氢加入175μl MPO检测缓冲液中，制备5mM过氧化氢溶液。底物溶液包括混合。为要进行的检测数量准备足够的试剂。对于每个孔，准备50μl底物溶液，其中含有：175μl MPO分析缓冲液46μl、MPO氯化底物2μl、稀释的过氧化氢2μl，混合并在每个孔中加入50μl底物溶液。搅拌均匀。孵育5分钟。在室温下轻轻摇动，避光。然后测量荧光(Ex/Em＝535/587nm)。(3) Substrate solution preparation: Add 1 μl hydrogen peroxide to 175 μl MPO detection buffer to prepare 5 mM hydrogen peroxide solution. Substrate solutions include mixing. Prepare enough reagents for the number of assays to be performed. For each well, prepare 50 µl of substrate solution containing: 175 µl of MPO assay buffer 46 µl, MPO chloride substrate 2 µl, diluted hydrogen peroxide 2 µl, mix and add 50 µl of substrate solution to each well. Stir well. Incubate for 5 minutes. Shake gently at room temperature, protected from light. Fluorescence was then measured (Ex/Em = 535/587 nm).
计算：将酶对照(EC)的RFU设为100％，计算候选抑制剂的相对抑制(％)为：Calculation: Set the RFU of the enzyme control (EC) as 100%, and calculate the relative inhibition (%) of the candidate inhibitor as:
Figure PCTCN2021117079-appb-000002
Figure PCTCN2021117079-appb-000002
其中：RFU(EC)是酶对照的荧光读数；RFU(S)是候选抑制剂的荧光读数Where: RFU(EC) is the fluorescence readout of the enzyme control; RFU(S) is the fluorescence readout of the candidate inhibitor
3.葡聚糖硫酸钠(dextran sodium sulfate,DSS)诱导的溃疡性结肠炎小鼠血浆LTB4、TNFα的测定3. Determination of plasma LTB4 and TNFα in mice with ulcerative colitis induced by dextran sodium sulfate (DSS)
6周龄的Balb/c品系雄性小鼠，23℃，湿度：55±5％，12h明暗交替，常规饲料喂养，所有小鼠均自由饮水和进食。小鼠(n＝60)适应1周，随机分为6组(n＝10)；KS组(正常对照组)、DSS模型组(结肠炎对照组)，给药组(口服给予芦荟松60mg/kg/d组、30mg/kg/d组、15mg/kg/d组)，药物对照组(口服给予5-氨基水杨酸50mg/kg/d)。小鼠实验开始两周后，用含有2.5％浓度DSS溶解于小鼠饮用水中，每天现配现用。连续给予实验动物含2.5％浓度DSS的饮用水7天后，给予正常饮用水3天。正常对照组连续10天给予正常饮水。之后在轻度***麻醉下通过心脏放血处死小鼠，并通过心脏穿刺将血液收集在5％EDTA小瓶中。分离血浆迅速储存于-70℃待检。按照ELISA检测试剂盒(美国R&D公司) 测定小鼠血浆LTB4、TNF-α的含量。6-week-old male mice of the Balb/c strain were kept at 23°C, humidity: 55±5%, alternated between light and dark for 12 hours, fed with conventional feed, and all mice had free access to water and food. Mice (n=60) were adapted for 1 week, and were divided into 6 groups (n=10) at random; KS group (normal control group), DSS model group (colitis control group), administration group (administered aloe vera pine 60mg/ kg/d group, 30mg/kg/d group, 15mg/kg/d group), drug control group (5-aminosalicylic acid 50mg/kg/d orally administered). Two weeks after the start of the mouse experiment, DSS containing 2.5% concentration was dissolved in the drinking water of the mice, and it was prepared and used every day. After continuously giving the experimental animals drinking water containing 2.5% DSS for 7 days, they were given normal drinking water for 3 days. The normal control group was given normal drinking water for 10 consecutive days. Mice were then sacrificed by cardiac exsanguination under light ether anesthesia, and blood was collected in vials of 5% EDTA by cardiac puncture. The separated plasma was quickly stored at -70°C for testing. The contents of LTB4 and TNF-α in mouse plasma were determined according to the ELISA detection kit (R&D Company, USA).
(二)实验结果(2) Experimental results
1.芦荟松对LPS和ATP对巨噬细胞IL-1β，IL-18炎症因子生成的影响结果1. The effect of aloe vera pine on the production of macrophage IL-1β and IL-18 inflammatory factors by LPS and ATP
芦荟松对LPS和ATP对巨噬细胞IL-1β，IL-18炎症因子生成的影响结果如图3A、图3B所示。结果表明，与空白对照(KB)比巨噬细胞加入LPS刺激IL-18和IL-1β浓度明显升高，再加入ATP刺激，IL-18和IL-1β浓度显著的升高，而0.5μg/mL(2.15μM)、1μg/mL(4.3μM)芦荟松能显著抑制IL-18、IL-1β产生，浓度再增加抑制效果下降。通过抑制巨噬细胞产生IL-18、IL-1β，从而抑制细胞焦亡的产生，避免炎症的进一步加剧。The effect of aloe vera pine on LPS and ATP on the production of IL-1β and IL-18 inflammatory factors in macrophages is shown in Figure 3A and Figure 3B. The results showed that compared with the blank control (KB), the concentrations of IL-18 and IL-1β were significantly increased by adding LPS to macrophages, and then added ATP stimulation, the concentrations of IL-18 and IL-1β were significantly increased, while 0.5 μg/ mL (2.15μM), 1μg/mL (4.3μM) aloe vera pine can significantly inhibit the production of IL-18 and IL-1β, and the inhibitory effect decreases when the concentration increases. By inhibiting the production of IL-18 and IL-1β by macrophages, the production of pyroptosis can be inhibited, and further inflammation can be avoided.
2.芦荟松对MPO的抑制活性2. Inhibitory activity of aloe vera pine on MPO
芦荟松不同浓度对MPO的抑制活性见表1，结果表明，芦荟松对MPO有较好的抑制活性，50μM浓度抑制率达到66.7％。The inhibitory activity of different concentrations of aloe vera pine on MPO is shown in Table 1. The results show that aloe vera pine has better inhibitory activity on MPO, and the inhibition rate of 50 μM concentration reaches 66.7%.
表1.芦荟松不同浓度对MPO的抑制活性结果Table 1. The results of the inhibitory activity of different concentrations of aloe vera pine on MPO
Figure PCTCN2021117079-appb-000003
Figure PCTCN2021117079-appb-000003
注：4-氨基苯甲酰肼为阳性对照Note: 4-aminobenzoic hydrazide is a positive control
3.芦荟松对DSS模型小鼠血浆LTB4和TNFα浓度的影响3. Effect of aloe vera pine on plasma LTB4 and TNFα concentrations in DSS model mice
经葡聚糖硫酸钠(DSS)诱导溃疡性结肠炎病变小鼠，应用酶联免疫吸附法检测小鼠大肠组织中LTB4和TNFα的水平。Ulcerative colitis mice were induced by dextran sodium sulfate (DSS), and the levels of LTB4 and TNFα in the large intestine of the mice were detected by enzyme-linked immunosorbent assay.
芦荟松对DSS溃疡性肠炎小鼠血浆白三烯B4和肿瘤坏死因子α浓度的影响如图4A、图4B所示。结果表明，与DSS对照水平相比，DSS引起模型小鼠血浆LTB4和TNFα浓度升高，芦荟松能剂量依赖性地降低结肠炎小鼠血浆TNF-α的浓度；芦荟松也能显著降低了LTB4的血浆水平(p<0.05)，口服20mg/kg/d和40mg/kg/d剂量的芦荟松将LTB4水平降低到了基础水平(p<0.05)。The effect of aloe vera pine on the concentration of plasma leukotriene B4 and tumor necrosis factor α in DSS ulcerative colitis mice is shown in Figure 4A and Figure 4B. The results showed that compared with the DSS control level, DSS caused the increase of plasma LTB4 and TNFα concentrations in model mice, and aloe vera pine could dose-dependently reduce the plasma TNF-α concentration of colitis mice; aloe vera pine could also significantly reduce LTB4 Oral administration of 20mg/kg/d and 40mg/kg/d doses of aloe vera pine reduced LTB4 levels to basal levels (p<0.05).
(三)讨论(3) Discussion
溃疡性结肠炎是一种病因尚不十分清楚的结肠和直肠慢性及复发性炎症性疾病，其发病原因有很多。已经被广泛接受的是，环境的相互作用、基因遗传以及免疫反应异常等都是溃 疡性结肠炎发生发展的因素。溃疡性结肠炎与炎症细胞分泌的炎症介质密切相关，其中最主要的就是IL-18、IL-1β、MPO、TNFα、LTB4。IL-18、IL-1β的升高会引发细胞焦亡，从而加剧IBD的恶化，细胞实验证明芦荟松可有效抑制IL-18、IL-1β的产生，预示芦荟松对结肠炎有益；MPO与巨噬细胞的浸润有关，与非结肠炎大鼠相比，DSS对照大鼠结肠MPO活性显著增加，表明中性粒细胞在DSS结肠炎中起重要作用，在这项研究中，芦荟松能有效抑制MPO的活性，它也具有通过抑制靶组织MPO活性而与嗜中性粒细胞相关的抗炎活性，从而有效抑制UC的进程。Ulcerative colitis is a chronic and relapsing inflammatory disease of the colon and rectum with unknown etiology. It is widely accepted that environmental interactions, genetic inheritance, and abnormal immune responses are factors in the development of ulcerative colitis. Ulcerative colitis is closely related to inflammatory mediators secreted by inflammatory cells, the most important of which are IL-18, IL-1β, MPO, TNFα, and LTB4. The increase of IL-18 and IL-1β will cause cell pyroptosis, which will aggravate the deterioration of IBD. Cell experiments have proved that aloe vera pine can effectively inhibit the production of IL-18 and IL-1β, indicating that aloe vera pine is beneficial to colitis; MPO and The infiltration of macrophages is related. Compared with non-colitis rats, the MPO activity in the colon of DSS control rats was significantly increased, indicating that neutrophils play an important role in DSS colitis. In this study, aloe vera pine can effectively Inhibits the activity of MPO, which also has anti-inflammatory activity associated with neutrophils by inhibiting MPO activity in target tissues, thereby effectively inhibiting the progression of UC.
LTB4对于炎性细胞的激活和募集非常重要，LTB4还直接参与炎症性肠病的病理生理学。本研究结果表明，服用芦荟松小鼠血浆LTB4水平均显著降低，高剂量组LTB4水平都降低到了非结肠炎的水平。证明芦荟松是LTB4释放的强抑制剂，表明芦荟松可以减少黏膜中嗜中性粒细胞和白细胞的积累。LTB4 is important for the activation and recruitment of inflammatory cells, and LTB4 is also directly involved in the pathophysiology of inflammatory bowel disease. The results of this study showed that the levels of plasma LTB4 in mice taking aloe vera pine were significantly reduced, and the levels of LTB4 in the high-dose group were all reduced to the level of non-colitis. Aloe pine was proved to be a strong inhibitor of LTB4 release, indicating that aloe pine could reduce the accumulation of neutrophils and leukocytes in the mucosa.
抑制肿瘤坏死细胞因子(TNF-α)通常作为临床上治疗炎症性肠病的金标准。芦荟松能剂量依赖性地降低结肠炎小鼠血浆TNF-α的浓度，从而对于治疗炎症性肠病具有作用。Inhibition of tumor necrosis cytokine (TNF-α) is usually used as the gold standard for clinical treatment of inflammatory bowel disease. Aloe pine can dose-dependently reduce the concentration of plasma TNF-α in mice with colitis, and thus has an effect on the treatment of inflammatory bowel disease.
(四)结论(4) Conclusion
芦荟松具有抑制细胞焦亡、抑制MPO活性、体内抑制LTB4和TNF-α的产生，从而改善溃疡性结肠炎的炎症反应，减弱免疫细胞的募集，达到缓解和治疗UC的作用。因此，芦荟松可用于制备治疗溃疡性结肠炎药物。Aloe pine can inhibit pyroptosis, inhibit MPO activity, and inhibit the production of LTB4 and TNF-α in vivo, thereby improving the inflammatory response of ulcerative colitis, weakening the recruitment of immune cells, and achieving the effect of alleviating and treating UC. Therefore, aloe vera pine can be used to prepare medicine for treating ulcerative colitis.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection.

Claims (8)

  1. 芦荟松在制备治疗溃疡性结肠炎药物中的用途。Application of aloe vera pine in the preparation of medicine for treating ulcerative colitis.
  2. 根据权利要求1所述的用途，其特征在于，所述芦荟松在制备通过抑制IL-1β、IL-18产生来治疗溃疡性结肠炎药物中的用途。The use according to claim 1, characterized in that the aloe vera pine is used in the preparation of a medicine for treating ulcerative colitis by inhibiting the production of IL-1β and IL-18.
  3. 根据权利要求2所述的用途，其特征在于，所述芦荟松的浓度为2-5μM。The use according to claim 2, characterized in that the concentration of the aloe vera pine is 2-5 μM.
  4. 根据权利要求1所述的用途，其特征在于，所述芦荟松在制备通过抑制MPO活性来治疗溃疡性结肠炎药物中的用途。The use according to claim 1, characterized in that the aloe vera pine is used in the preparation of a medicament for treating ulcerative colitis by inhibiting MPO activity.
  5. 根据权利要求4所述的用途，其特征在于，所述芦荟松的浓度为50-1000μM。The use according to claim 4, characterized in that the concentration of the aloe vera pine is 50-1000 μM.
  6. 根据权利要求1所述的用途，其特征在于，所述芦荟松在制备通过抑制LTB4、TNFα产生来治疗溃疡性结肠炎药物中的用途。The use according to claim 1, characterized in that the aloe vera pine is used in the preparation of a medicine for treating ulcerative colitis by inhibiting the production of LTB4 and TNFα.
  7. 根据权利要求1所述的用途，其特征在于，所述芦荟松的剂量为20-40mg/kg/d。purposes according to claim 1, is characterized in that, the dosage of described aloe vera pine is 20-40mg/kg/d.
  8. 根据权利要求1所述的用途，其特征在于，所述芦荟松在制备通过抑制IL-1β和IL-18产生、抑制MPO活性以及抑制LTB4和TNFα产生来治疗溃疡性结肠炎药物中的用途。The use according to claim 1, characterized in that the aloe vera pine is used in the preparation of a medicine for treating ulcerative colitis by inhibiting the production of IL-1β and IL-18, inhibiting the activity of MPO and inhibiting the production of LTB4 and TNFα.
PCT/CN2021/117079 2021-09-08 2021-09-08 Use of aloesome in preparation of drug for treating ulcerative colitis WO2023035130A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/117079 WO2023035130A1 (en) 2021-09-08 2021-09-08 Use of aloesome in preparation of drug for treating ulcerative colitis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/117079 WO2023035130A1 (en) 2021-09-08 2021-09-08 Use of aloesome in preparation of drug for treating ulcerative colitis

Publications (1)

Publication Number Publication Date
WO2023035130A1 true WO2023035130A1 (en) 2023-03-16

Family

ID=85506107

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/117079 WO2023035130A1 (en) 2021-09-08 2021-09-08 Use of aloesome in preparation of drug for treating ulcerative colitis

Country Status (1)

Country Link
WO (1) WO2023035130A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110092959A (en) * 2010-02-11 2011-08-18 숙명여자대학교산학협력단 Composition for improving inflammatory bowel disease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110092959A (en) * 2010-02-11 2011-08-18 숙명여자대학교산학협력단 Composition for improving inflammatory bowel disease

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHE, QINGMING ET AL.: "Metabolism of Aloesin and Related Compounds by Human Intestinal Bacteria: A Bacterial Cleavage of the C-Glucosyl Bond and the Subsequent Reduction of the Acetonyl Side Chain", CHEMICAL AND PHARMACEUTICAL BULLETIN, vol. 39, no. 3, 31 March 1991 (1991-03-31), XP001246851 *
MA JIAN, CAO BIXUAN, CHEN XIU, XU MINJUAN, BI XIAOXU, GUAN PEIPEI, JIANG YI, XU JUN, HAN LI, HUANG XUESHI: "Violacin A, a new chromanone produced by Streptomyces violaceoruber and its anti-inflammatory activity", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM NL, vol. 28, no. 5, 1 March 2018 (2018-03-01), Amsterdam NL , pages 947 - 951, XP093046255, ISSN: 0960-894X, DOI: 10.1016/j.bmcl.2018.01.051 *
MI-YOUNG PARK; HOON-JEONG KWON; MI-KYUNG SUNG;: "Dietary aloin, aloesin, or aloe-gel exerts anti-inflammatory activity in a rat colitis model", LIFE SCIENCE, PERGAMON PRESS, OXFORD, GB, vol. 88, no. 11, 27 December 2010 (2010-12-27), GB , pages 486 - 492, XP028185847, ISSN: 0024-3205, DOI: 10.1016/j.lfs.2011.01.010 *

Similar Documents

Publication Publication Date Title
Liang et al. Therapeutic roles of polysaccharides from Dendrobium Officinaleon colitis and its underlying mechanisms
RU2746707C1 (en) Medical application of artemisinin derivatives for treatment of inflammatory intestinal disease
WO2005003145A1 (en) Shanzhuyu extract and uses thereof
JP2016523898A (en) An unripe fruit extract of Satsuma mandarin, or a composition for treating or preventing inflammatory skin diseases containing synephrine or a salt thereof as an active ingredient
Guo et al. Hydroxysafflor yellow A (HSYA) targets the platelet-activating factor (PAF) receptor and inhibits human bronchial smooth muscle activation induced by PAF
Fei et al. Acanthopanax senticosus attenuates inflammation in lipopolysaccharide-induced acute lung injury by inhibiting the NF-κB pathway
Long et al. Prophylactic effect of Kudingcha polyphenols on oxazolone induced colitis through its antioxidant capacities
WO2011047529A1 (en) Use of flavonoid glycoside compounds in manufacturing medicaments for treating malaria
US7399825B2 (en) Synthetic peptide, inhibitor to DNA viruses
WO2023035130A1 (en) Use of aloesome in preparation of drug for treating ulcerative colitis
Peng et al. Dehydrocostus lactone inhibits the proliferation of esophageal cancer cells in vivo and in vitro through ROS-mediated apoptosis and autophagy
Wu et al. Cantharidin analogue alleviates dextran sulfate sodium-induced colitis in mice by inhibiting the activation of NF-κB signaling
CN108888623A (en) Application of tyrosine protein kinase JAK2 inhibitor BX795
CN113521060B (en) Application of NEEDOLIDE in resisting novel coronavirus
CN113577054A (en) Application of aloe vera pine in preparing medicine for treating ulcerative colitis
Long et al. Inhibition of colon cancer cell growth by imidazole through activation of apoptotic pathway
Yun-Ru et al. Apoptosis induced by caudatin in human hepatoma cell line SMMC7721
CN109908151A (en) A kind of pentacyclic triterpene compound treats the application of the drug of inflammatory bowel disease in preparation
CA3097722C (en) Methods and compositions for the treatment of multiple sclerosis
JP2018526457A (en) Medicinal Ambrosia plant extract
CN112028963B (en) 23-norursane triterpenoid, preparation method thereof and application thereof in preparing glycosidase inhibitor medicine
KR102287982B1 (en) Composition comprising Novel Ferulic Acid Derivative for Preventing, Treating or Improving Autoimmune Disease and Preparation Method Thereof
TWI376384B (en)
AU2015101724A4 (en) Use of steroid compounds in the treatment of inflammatory diseases
CN106188179B (en) Sharp leaf vacation Radix Gentianae extract, compound and pharmaceutical composition with anti-diarrhea effect

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21956319

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE