WO2020252937A1 - 茄科植物提取的杂环化合物在制备治疗多发性硬化症的药物中的应用及药物 - Google Patents

茄科植物提取的杂环化合物在制备治疗多发性硬化症的药物中的应用及药物 Download PDF

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WO2020252937A1
WO2020252937A1 PCT/CN2019/104002 CN2019104002W WO2020252937A1 WO 2020252937 A1 WO2020252937 A1 WO 2020252937A1 CN 2019104002 W CN2019104002 W CN 2019104002W WO 2020252937 A1 WO2020252937 A1 WO 2020252937A1
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multiple sclerosis
medicine
solanaceae
heterocyclic compound
cells
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PCT/CN2019/104002
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English (en)
French (fr)
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董云
钱政江
朱恩妮
任真
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深圳先进技术研究院
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    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/465—Nicotine; Derivatives thereof
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • A61K36/815—Lycium (desert-thorn)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to the field of biomedical technology, in particular to the application of a heterocyclic compound extracted from a Solanaceae plant in the preparation of a medicine for treating multiple sclerosis and the medicine.
  • Multiple sclerosis is a demyelinating disease of the central nervous system in young people, which is mainly manifested in white matter inflammatory demyelinating spots. Because the disease is more extensive and diffuse, the symptoms caused are also extremely complex, such as neuritis, limb paralysis, and mental symptoms.
  • the pathological mechanism of multiple sclerosis is mainly considered to be myelin damage, neuroinflammation and neurodegenerative diseases induced by T cells and B cells of the immune system. Studies have shown that oligodendrocyte damage is considered to be the main predisposing factor of multiple sclerosis, but the cause of the disease is still unclear.
  • tumor necrosis factor has been developed from the direction of neuroinflammation to play an important role in multiple sclerosis.
  • the first object of the present invention is to provide the application of heterocyclic compounds extracted from Solanaceae plants in the preparation of medicines for treating multiple sclerosis, so as to at least alleviate one of the technical problems in the prior art.
  • the second object of the present invention is to provide a medicine that can effectively treat multiple sclerosis.
  • the invention provides the application of heterocyclic compounds extracted from Solanaceae plants in the preparation of medicines for preventing and/or treating multiple sclerosis.
  • Solanaceae plant includes one or more of wolfberry, eggplant, pepper or tomato.
  • heterocyclic compound extracted from the Solanaceae plant includes nicotine.
  • the present invention also provides a medicine for treating multiple sclerosis, the active ingredient of which is a heterocyclic compound extracted from a Solanaceae plant.
  • Solanaceae plant includes one or more of wolfberry, eggplant, pepper or tomato.
  • heterocyclic compound extracted from the Solanaceae plant includes nicotine.
  • the medicine also includes pharmaceutically acceptable excipients.
  • the pharmaceutical dosage form includes an oral preparation or an injection preparation.
  • oral preparations include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions;
  • the injection preparation includes injection or powder injection.
  • the effective dosage of the drug is 2-5 ⁇ M/kg/day.
  • the present invention has confirmed through a large number of experiments such as modeling that the heterocyclic compounds extracted from Solanaceae plants can significantly inhibit the oxidative stress induced by reactive oxygen species, thereby protecting neurons from damage, indicating that the heterocyclic compounds extracted from Solanaceae plants can be
  • the inhibitory effect of oxidative stress has a potential preventive and/or therapeutic effect on clinical neurological disease multiple sclerosis.
  • the present invention can effectively prevent and/or treat multiple sclerosis by applying heterocyclic compounds extracted from Solanaceae plants in the preparation of drugs for preventing and/or treating multiple sclerosis, thereby reducing multiple sclerosis
  • the incidence of disease and/or greatly improve the therapeutic effect of multiple sclerosis provide a basis for clinical trials, and provide a new way for the development of new drugs.
  • the drug for treating multiple sclerosis provided by the present invention has an active ingredient of a heterocyclic compound extracted from a Solanaceae plant. Therefore, it can effectively inhibit multiple sclerosis by inhibiting oxidative stress, and has a significant effect on multiple sclerosis.
  • the preventive and/or therapeutic effects of serotonin while also having the advantage of less side effects.
  • Figure 1 is a diagram showing the cell morphology of HT-22 cells provided in Example 1 of the present invention after being treated with different concentrations of hydrogen peroxide for 24 hours;
  • Example 2 is a diagram showing the cell viability detection results of HT-22 cells provided in Example 1 of the present invention after being treated with different concentrations of hydrogen peroxide for 24 hours;
  • Example 3 is a diagram showing the cell morphology of T-22 cells provided in Example 2 of the present invention after being treated with different concentrations of nicotine for 24 hours, and then treated with 600 ⁇ M hydrogen oxide for 24 hours;
  • Example 4 is a diagram showing the cell viability detection results of T-22 cells provided in Example 2 of the present invention after being treated with different concentrations of nicotine for 24 hours and then treated with 600 ⁇ M hydrogen oxide for 24 hours;
  • Figure 5 is a graph showing the results of nicotine inhibiting the detection level of reactive oxygen species ROS in HT-22 cells by nicotine provided in Example 3 of the present invention.
  • heterocyclic compounds extracted from Solanaceae plants in the preparation of drugs for the prevention and/or treatment of multiple sclerosis.
  • the heterocyclic compounds extracted from Solanaceae plants are heterocyclic compounds such as pyrrolidine.
  • the heterocyclic compound extracted from the Solanaceae plant in the present invention can be prepared in a laboratory or directly purchased commercially.
  • Multiple sclerosis is a neurological disease. Brain tissue requires high oxygen and is sensitive to hypoxia. Because it is rich in polyunsaturated fatty acids but low in antioxidant enzymes, when free radicals are excessively produced and/or cannot be eliminated in time , The brain's oxidation and antioxidant effects are out of balance, and nerve tissues and cells are susceptible to oxidative damage. Therefore, oxidative stress plays a key role in multiple sclerosis.
  • the present invention has confirmed through a large number of experiments such as modeling that the heterocyclic compounds extracted from Solanaceae plants can significantly inhibit the oxidative stress induced by reactive oxygen species, thereby protecting neurons from damage, indicating that the heterocyclic compounds extracted from Solanaceae plants can be
  • the inhibitory effect of oxidative stress has a potential preventive and/or therapeutic effect on clinical neurological disease multiple sclerosis.
  • the present invention can effectively prevent and/or treat multiple sclerosis by applying heterocyclic compounds extracted from Solanaceae plants in the preparation of drugs for preventing and/or treating multiple sclerosis, thereby reducing multiple sclerosis
  • the incidence of disease and/or greatly improve the therapeutic effect of multiple sclerosis provide a basis for clinical trials, and provide a new way for the development of new drugs.
  • the multiple sclerosis described in the present invention includes relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis and progressive relapsing multiple sclerosis
  • Various types of multiple sclerosis such as multiple sclerosis, and all diseases that are clinically determined to be multiple sclerosis fall within the protection scope of the present invention.
  • prevention and/or treatment includes prevention and treatment, and prevention or treatment.
  • the Solanaceae plant includes one or more of wolfberry, eggplant, pepper or tomato.
  • the Solanaceae plant can be wolfberry, eggplant, pepper, tomato, wolfberry and tomato, eggplant and pepper, or a combination of wolfberry, eggplant, pepper and tomato.
  • the heterocyclic compound extracted from the Solanaceae plant includes nicotine (1-methyl-2-(3-pyridyl)pyrrolidine).
  • Nicotine commonly known as nicotine, is an alkaloid found in Solanaceae plants and is a nicotinic acetylcholine receptor agonist.
  • nicotine nicotinic acetylcholine receptors nAChRs
  • the present invention has found through experiments that nicotine can also Improve multiple sclerosis by reducing oxygen stress.
  • a medicine for treating multiple sclerosis the active ingredient of which is a heterocyclic compound extracted from a Solanaceae plant.
  • the drug for treating multiple sclerosis provided by the present invention has an active ingredient of a heterocyclic compound extracted from a Solanaceae plant. Therefore, it can effectively inhibit multiple sclerosis by inhibiting oxidative stress, and has a significant effect on multiple sclerosis.
  • the preventive and/or therapeutic effects of serotonin at the same time, it also has the advantages of safety, non-toxicity and small side effects.
  • the Solanaceae plant includes one or more of wolfberry, eggplant, pepper or tomato.
  • the Solanaceae plant can be wolfberry, eggplant, pepper, tomato, wolfberry and tomato, eggplant and pepper, or a combination of wolfberry, eggplant, pepper and tomato.
  • the heterocyclic compound extracted from the Solanaceae plant includes nicotine.
  • the drug further includes pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients refer to the excipients and additives used in the production of drugs and formulating prescriptions. They refer to those that have been reasonably evaluated in terms of safety in addition to the active ingredients and are included in the pharmaceutical preparations. substance.
  • the same pharmaceutical excipients can be used in pharmaceutical preparations of different administration routes, and have different functions and uses.
  • the pharmaceutically acceptable excipients added to the medicament provided by the present invention can serve as an excipient, act as a carrier or improve stability. In addition, they also have important functions such as solubilization, solubilization, or sustained and controlled release.
  • Typical but non-limiting pharmaceutically acceptable excipients include: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators Agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, enhancers Plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, or release retardants Or multiple.
  • the dosage form of the drug includes an oral preparation or an injection preparation.
  • the above-mentioned drugs can be made into any orally acceptable preparation form, such as, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions. .
  • the carriers used in tablets generally include lactose and corn starch, and lubricants such as magnesium stearate can also be added.
  • Diluents used in capsules generally include lactose and dried corn starch.
  • Oral suspensions are usually used by mixing the active ingredients with suitable emulsifiers and suspending agents.
  • sweeteners, fragrances or coloring agents can be added to the above oral preparation forms.
  • the above-mentioned drugs can be made into any injection-acceptable preparation form, such as, but not limited to, injection or powder injection.
  • the usable carriers and solvents include water, Ringer's solution and isotonic sodium chloride solution.
  • sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.
  • the effective dose of the drug is 2-5 ⁇ M/kg/day, for example, it can be, but not limited to, 2 ⁇ M/kg/day, 3 ⁇ M/kg/day, 4 ⁇ M/kg/day or 5 ⁇ M/kg/day.
  • the frequency of administration can be, for example, but is not limited to twice a day, once a day, once every two days, once a week, or once a month.
  • the drug provided by the present invention may be administered in the form of a sustained-release preparation, in which case, less frequency of administration is required.
  • the dosage and frequency of administration vary according to the half-life of the preparation in the body of the user, and in addition, it can also vary according to whether the treatment is prophylactic or therapeutic.
  • relatively low doses such as 2 ⁇ M/kg/day
  • relatively high doses such as 5 ⁇ M/kg/day
  • relatively short intervals such as 5 ⁇ M/kg/day
  • Nicotine 1-methyl-2-(3-pyridyl)pyrrolidine (Sigma), Cell Counting Kit-8 (CCK8, JinDo, Japan), hydrogen peroxide (Sigma), DMEM medium (Gibco), fetal bovine serum FBS (Gibco), PBS (HyClone), streptomycin double antibody (Gibco), DCFH-DA detection kit (Biyuntian).
  • the HT-22 cells in the examples of the present invention are cultured in a culture flask of DMEM medium (containing 1% penicillin, streptomycin double antibody and 10% fetal bovine serum), and placed at 37°C, 5% Cultivate in a CO 2 incubator.
  • DMEM medium containing 1% penicillin, streptomycin double antibody and 10% fetal bovine serum
  • CCK8 detecting cell activity includes the following steps:
  • HT-22 cells hippocampal neuron cells
  • H 2 O 2 solutions 200, 400, 600, 800 ⁇ M
  • the cell morphology was photographed with an inverted microscope.
  • Figure 1. As the concentration of H 2 O 2 increases, the density of viable cells decreases and the number decreases; Cell Counting Kit 8 (CCK8) was used to detect the activity of HT-22 cells.
  • Figure 2. The cell activity varies with H 2 The O 2 concentration increases and decreases.
  • the data of this example shows that H 2 O 2 induces a dose-dependent cytotoxicity in HT-22 cells, and the cell viability of the 600 ⁇ M H 2 O 2 group is about 50% (the cell survival rate of the control group with PBS is 100%).
  • Example 2 The protective effect of nicotine on HT-22 cell damage induced by hydrogen peroxide
  • the concentration of 600 ⁇ M H 2 O 2 that caused nearly half of the cell death was selected to perform the following experiment. Treat HT-22 cells with different concentrations of nicotine (1, 2, 5, 10 ⁇ M) and incubate them for 24 hours, then incubate them in a 600 ⁇ M H 2 O 2 solution for 24 hours, and then use an inverted microscope to photograph the cell morphology, as shown As shown in 3, compared with the PBS control group, the H 2 O 2 group has a low cell density and a small number; compared with the H 2 O 2 group, the number of cells in each group with different concentrations of nicotine increased significantly. CCK8 was used to detect cell activity.
  • HT-22 cells were incubated with different concentrations of nicotine (1, 2, 5 and 10 ⁇ M) for 24 hours, and then incubated in 600 ⁇ M H 2 O 2 for 24 hours to verify the connection between nicotine protection and oxidative stress.
  • the present embodiment detects the level of intracellular ROS reactive oxygen species, the results were shown as compared to the PBS control group, group 2 ROS levels increased significantly 2 O H 5; Comparative H 2 O 2 groups, each with different concentrations of nicotine
  • the reactive oxygen species (ROS) level of the group decreased, and 5 ⁇ M nicotine can minimize the ROS level.
  • ROS reactive oxygen species
  • the cell reactive oxygen detection test includes the following steps:
  • HT-22 cells are cultured in a 12-well plate at a concentration of 1 ⁇ 10 5 /ml, each well contains 1ml of cell suspension. After 24 hours of culture, the cells are pretreated with different concentrations of nicotine or PBS and incubated for 24 hours. Add appropriate concentration of H 2 O 2 or gradient concentration of H 2 O 2 to the cultured cells, and incubate for 24 hours;
  • PBS 1 ⁇ phosphate buffer solution
  • DCFH-DA (10mM) and serum-free cell culture medium are diluted at a ratio of 1:1000 to 10 ⁇ M/L working solution. Add 500 ⁇ l working solution to each EP tube and incubate in a 37°C cell culture box for 20min , Mix upside down every 3-5min (make probe DCFH-DA and cell fully contact);
  • the image generated by the imaging device may be a two-dimensional ultrasound image or a three-dimensional ultrasound image.

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Abstract

本发明公开了一种茄科植物提取的杂环化合物在制备治疗多发性硬化症的药物中的应用，以及含有所述杂环化合物药物，其中茄科植物提取的杂环化合物包括尼古丁。本发明通过抑制氧化应激有效抑制多发性硬化症，对多发性硬化症起到有效的预防和/或治疗作用。

Description

茄科植物提取的杂环化合物在制备治疗多发性硬化症的药物中的应用及药物 技术领域
本发明涉及生物医学技术领域，尤其是涉及一种茄科植物提取的杂环化合物在制备治疗多发性硬化症的药物中的应用及药物。
背景技术
多发性硬化症，是一种青年的中枢神经脱髓鞘的疾病，其主要表现在白质炎性脱髓鞘斑。由于此病病变较为广泛和弥散，所引发的症状也极为复杂，例如可表现为神经炎、肢体瘫痪以及精神症状等。多发性硬化症的病理机制主要被认为是由免疫***T细胞和B细胞诱导的髓鞘损伤、神经炎症和神经退行性疾病。研究显示少突胶质细胞损伤被认为是多发性硬化症的主要诱发因素，但是该疾病的病因尚不清楚。
目前从神经炎症方向已经研发出肿瘤坏死因子在多发性硬化症上起着很重要的作用。研究发现在多发性硬化症的小鼠模型上神经胶质细胞释放肿瘤坏死因子要显著地高于正常小鼠的神经胶质细胞产生的肿瘤坏死因子，由此可推出肿瘤坏死因子的高水平的表达可是多发性硬化症疾病的一个重要病理机制。然后科学家们针对肿瘤坏死因子研究出其抑制剂，发现肿瘤坏死因子抑制剂可显著的改善多发性硬化症小鼠的病症。自此，经过多方面研究，将肿瘤坏死因子抑制剂应用于临床治疗多发性硬化症，只是病人的病症减轻。但是，服用该药的多发性硬化症的病人多半会头晕，头疼，甚至还有报道肿瘤坏死因子抑制剂导致了多发性硬化症患者死亡案例。由此可见，科学家们对多发性硬化症的病理机制以及有效治疗药物的研究还任重道远。
发明内容
本发明的第一个目的在于提供茄科植物提取的杂环化合物在制备治疗多发性硬化症的药物中的应用，以至少缓解现有技术中存在的技术问题之一。
本发明的第二个目的在于提供一种药物，该药物能够有效治疗多发性硬化 症。
本发明提供了茄科植物提取的杂环化合物在制备预防和/或治疗多发性硬化症的药物中的应用。
进一步地，所述茄科植物包括枸杞、茄子、辣椒或西红柿中的一种或多种。
进一步地，所述茄科植物提取的杂环化合物包括尼古丁。
另外，本发明还提供了一种治疗多发性硬化症的药物，所述药物的活性成分为茄科植物提取的杂环化合物。
进一步地，所述茄科植物包括枸杞、茄子、辣椒或西红柿中的一种或多种。
进一步地，所述茄科植物提取的杂环化合物包括尼古丁。
进一步地，所述药物还包括药学上可接受的辅料。
进一步地，所述药物剂型包括口服制剂或注射制剂。
进一步地，所述口服制剂包括片剂、胶囊剂、颗粒剂、丸剂、糖浆剂、口服溶液剂、口服混悬剂或口服乳剂；
优选地，所述注射制剂包括注射液或粉针剂。
进一步地，所述药物的有效给药剂量为2-5μM/kg/天。
本发明通过建模等大量的实验证实，茄科植物提取的杂环化合物能够显著抑制活性氧诱导的氧化应激，从而保护神经元不受损伤，说明茄科植物提取的杂环化合物能够通过对氧化应激的抑制作用，对临床神经疾病多发性硬化症具有潜在的预防和/或治疗作用。本发明通过将茄科植物提取的杂环化合物应用在制备预防和/或治疗多发性硬化症的药物中，能够对多发性硬化症起到有效的预防和/或治疗作用，从而减少多发性硬化症的发病率和/或大大提高多发性硬化症的治疗效果，为临床实验提供依据，也为新药的研发提供新途径。
本发明提供的治疗多发性硬化症的药物，该药物的活性成分为茄科植物提取的杂环化合物，因此，能够通过抑制氧化应激有效抑制多发性硬化症，对多发性硬化症起到明显的预防和/或治疗作用，同时还具有副作用小的优点。
附图说明
图1为本发明实施例1提供的HT-22细胞经不同浓度的过氧化氢处理24小时后的细胞形态结果图；
图2为本发明实施例1提供的HT-22细胞经不同浓度的过氧化氢处理24小时后的细胞活性检测结果图；
图3为本发明实施例2提供的T-22细胞经不同浓度的尼古丁处理24小时，再经过600μM氧化氢处理24小时后的细胞形态结果图；
图4为本发明实施例2提供的T-22细胞经不同浓度的尼古丁处理24小时，再经过600μM氧化氢处理24小时后的细胞活性检测结果图；
图5为本发明实施例3提供的尼古丁抑制H2O2在HT-22细胞内活性氧ROS检测水平的结果图。
具体实施方式
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本申请，并不用于限定本申请。
需要说明的是：
除非另有说明，本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法或材料也可应用于本发明中。
根据本发明的一个方面，提供了茄科植物提取的杂环化合物在制备预防和/或治疗多发性硬化症的药物中的应用。
茄科植物提取的杂环化合物是吡咯烷等杂环化合物。本发明中的茄科植物提取的杂环化合物，既可以为实验室制备得到的，也可以是市售直接购得的。
多发性硬化症属于神经性疾病，脑组织需氧量高，对缺氧敏感，因其含丰富的多不饱和脂肪酸但抗氧化物酶含量低，当自由基过度产生和/或无法及时清除时，脑内氧化与抗氧化作用失衡，神经组织和细胞就易受氧化损伤。因此，氧化应激在多发性硬化症中起着关键作用。
本发明通过建模等大量的实验证实，茄科植物提取的杂环化合物能够显著抑制活性氧诱导的氧化应激，从而保护神经元不受损伤，说明茄科植物提取的杂环化合物能够通过对氧化应激的抑制作用，对临床神经疾病多发性硬化症具有潜在的预防和/或治疗作用。本发明通过将茄科植物提取的杂环化合物应用在制备预防和/或治疗多发性硬化症的药物中，能够对多发性硬化症起到有效的预防和/或治疗作用，从而减少多发性硬化症的发病率和/或大大提高多发性硬化症的治疗效果，为临床实验提供依据，也为新药的研发提供新途径。
需要说明的是，在本发明中所述的多发性硬化症，包括复发缓解型多发性硬化症、继发进展型多发性硬化症、原发进展型多发性硬化症和进展复发型多发性硬化症等多种类型的多发性硬化症，凡是临床上判定属于多发性硬化症的疾病，均在本发明的保护范围内。
可以理解的是，“和/或”用于表示所说明的情况的一者或两者均可能发生，例如预防和/或治疗包括预防和治疗，以及预防或治疗。
在一些优选的实施方式中，所述茄科植物包括枸杞、茄子、辣椒或西红柿中的一种或多种。例如，茄科植物可以为枸杞、可以为茄子、可以为辣椒、可以为西红柿、可以为枸杞和西红柿、可以为茄子和辣椒、也可以为枸杞、茄子、辣椒和西红柿的组合。
在一些优选的实施方式中，所述茄科植物提取的杂环化合物包括尼古丁(1-甲基-2-(3-吡啶基)吡咯烷)。
尼古丁，俗称烟碱，是一种存在于茄科植物中的生物碱，是烟碱型乙酰胆碱受体激动剂。目前已知尼古丁的烟碱型乙酰胆碱受体(nicotinic acetylcholine receptors，nAChRs)广泛分布于体内，负责调节中枢及外周神经***、心血管 ***、免疫***等的功能，本发明通过实验发现，尼古丁还可通过降低氧应激来改善多发性硬化症。
根据本发明的另一方面，提供了一种治疗多发性硬化症的药物，所述药物的活性成分为茄科植物提取的杂环化合物。
本发明提供的治疗多发性硬化症的药物，该药物的活性成分为茄科植物提取的杂环化合物，因此，能够通过抑制氧化应激有效抑制多发性硬化症，对多发性硬化症起到明显的预防和/或治疗作用，同时还具有安全无毒、副作用小的优点。
在一些优选的实施方式中，所述茄科植物包括枸杞、茄子、辣椒或西红柿中的一种或多种。例如，茄科植物可以为枸杞、可以为茄子、可以为辣椒、可以为西红柿、可以为枸杞和西红柿、可以为茄子和辣椒、也可以为枸杞、茄子、辣椒和西红柿的组合。
在一些优选的实施方式中，所述茄科植物提取的杂环化合物包括包括尼古丁。
在一些优选的实施方式中，所述药物还包括药学上可接受的辅料。
药学上可接受的辅料是指生产药品和调配处方时，使用的的赋形剂和附加剂，是指除活性成分外，在安全性方面已进行了合理的评估，并且包含在药物制剂中的物质。同一药用辅料可用于不同给药途径的药物制剂，且有不同的作用和用途。在本发明提供的药物中添加的药学上可接受的辅料，能够起到赋型、充当载体或提高稳定性的作用，此外，还具有增溶、助溶或缓控释等重要功能。
典型但非限制性的药学上可接受的辅料包括：溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、湿润剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂或释放阻滞剂中的一种或多种。
在一个优选的实施方式中，药物的剂型包括口服制剂或注射制剂。
当口服用药时，上述药物可制成任意口服可接受的制剂形式，例如可以为，但不限于片剂、胶囊剂、颗粒剂、丸剂、糖浆剂、口服溶液剂、口服混悬剂或口服乳剂。
其中，片剂使用的载体一般包括乳糖和玉米淀粉，另外也可加入润滑剂如硬脂酸镁。胶囊剂使用的稀释剂一般包括乳糖和干燥玉米淀粉。口服混悬剂则通常是将活性成分与适宜的乳化剂和悬浮剂混合使用。
任选地，以上口服制剂形式中还可加入一些甜味剂、芳香剂或着色剂。
当以注射的形式给药时，上述药物可制成任意注射可接受的制剂形式，例如可以为，但不限于注射液或粉针剂。
其中，可使用的载体和溶剂包括水、林格氏溶液和等渗氯化钠溶液。另外，灭菌的非挥发油也可用作溶剂或悬浮介质，如单甘油酯或二甘油酯。
在一些优选的实施方式中，所述药物的有效给药剂量为2-5μM/kg/天，例如可以为，但不限于2μM/kg/天、3μM/kg/天、4μM/kg/天或5μM/kg/天。
在一个优选的实施方式中，给药频率例如可以为，但不限于每天两次、每天一次、每两天一次、每周一次或每月一次给药。或者，可以以缓释制剂的形式给予本发明提供的药物，在这种情况下，需要较少的给药频率。
给药剂量和频率根据制剂在用药者体内的半衰期而不同，此外，也可以根据是预防性处理还是治疗性处理而不同。在预防性应用中，以相对低频率的间隔长期给予相对低的剂量，例如2μM/kg/天。在治疗性应用中，有时需要以相对短的间隔给予相对高的剂量，例如5μM/kg/天，直至疾病的进展被延缓或停止，并优选地直至个体表现出疾病症状的部分或完全改善，在此之后，可以给予患者预防方案。
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本发明。
本发明实施例中用到的主要试剂信息如下：
尼古丁1-甲基-2-(3-吡啶基)吡咯烷(Sigma)，Cell Counting Kit-8(CCK8，JinDo，Japan)，过氧化氢(Sigma)，DMEM培养基(Gibco)，胎牛血清FBS(Gibco)，PBS(HyClone)，链霉素双抗(Gibco)，DCFH-DA检测试剂盒(碧云天)。
如无特殊说明，本发明实施例中的HT-22细胞培养于DMEM培养基(含1％青霉素、链霉素双抗和10％胎牛血清)的培养瓶中，放置于37℃、5％CO 2的培养箱中进行培养。
本发明实施例中，CCK8检测细胞活性包括如下步骤：
1、取适量CCK8试剂原液，用等量的PBS以1∶1等体积混合得CCK8稀释液；
2、取适量CCK8稀释液，与培养基以1∶9的比例混合，配得工作液；
3、从CO 2培养箱中取出96孔板，吸走培养基，每个孔中各加入约100μl的工作液；
4、将96孔板放于CO 2培养箱中，孵育40min-1.5h；
5、使用酶标仪，测定450nm下的吸光度，获得数据。
实施例1 过氧化氢诱导HT-22细胞损伤
将HT-22细胞(海马神经元细胞)分别在一系列浓度(200、400、600、800μM)的H 2O 2溶液中孵育24小时后，用倒置显微镜拍摄细胞形态，结果如图1所示：随着H 2O 2浓度的增加，活细胞密度下降，数量减少；用Cell Counting Kit 8(CCK8)试剂盒进行HT-22细胞的活性检测，结果如图2所示，细胞活性随H 2O 2浓度升高而下降。本实施例的数据显示H 2O 2在HT-22细胞中诱导剂量依赖的细胞毒性，其中600μM H 2O 2组细胞活性约为50％(加入PBS的对照组细胞存活率100％)。
实施例2 尼古丁对过氧化氢诱导的HT-22细胞损伤的保护作用
根据本发明实施例1中得到的H 2O 2梯度引起细胞损伤的结果，选择引起近半数细胞死亡的600μM H 2O 2浓度进行以下实验。用尼古丁的不同浓度(1、2、5、10μM)处理HT-22细胞孵育24小时后，再在浓度600μM的H 2O 2溶液中共同孵育24小时，然后用倒置显微镜拍摄细胞形态，如图3所示，对比PBS对照组，H 2O 2组的细胞密度低，数量少；对比H 2O 2组，不同浓度尼古丁各组细胞数量有较为明显地增加。用CCK8检测细胞活性，如图4所示，1、2、5、10μM的尼古丁显著地抑制了H 2O 2对HT-22细胞的损伤。当尼古丁达到10μM时，尼古丁对HT-22细胞神经保护作用明显减少。从以上结论可以得出，低浓度的尼古丁可以显著地抑制H 2O 2诱导海马神经元细胞死亡。
实施例3 尼古丁通过抑制ROS形成而保护HT-22细胞
将HT-22细胞加入不同浓度尼古丁中(1、2、5和10μM)孵育24小时后，再在浓度600μM的H 2O 2中孵育24小时，为验证尼古丁保护作用与氧化应激的联系，本实施例检测了细胞内活性氧ROS的水平，结果如图5所示，相比PBS对照组，H 2O 2组的活性氧水平明显升高；对比H 2O 2组，不同浓度尼古丁各组的活性氧(ROS)水平均降低，5μM尼古丁可使ROS水平降至最低。从上述实验可以说明，尼古丁能够通过抑制ROS形成，有效地保护HT-22细胞。
其中，细胞活性氧检测试包括如下步骤：
1、HT-22细胞以1×10 5/ml的浓度培养在12孔板中，每孔含1ml细胞悬液，培养24h后，用不同浓度的尼古丁或PBS预处理细胞，并孵育24h，之后加适当浓度的H 2O 2或梯度浓度H 2O 2到培养的细胞中，继续孵育24h；
2、从CO 2培养箱中取出12孔板，吸走培养基，每个孔中各加入约500μl的1×磷酸盐缓冲溶液(PBS)清洗细胞层，可重复1-2次，完成后吸走PBS，尽量吸净。
3、每孔加入约500μl的0.25％的无EDTA-胰酶消化液消化贴壁细胞2-4min。然后加约500μl的1×PBS或培养基终止细胞消化，将消化下的细胞收集在EP管里，室温5000RPM离心30s-1min，离心后小心去除上层的液体；
4、DCFH-DA(10mM)和无血清的细胞培养液以1∶1000的比例稀释成10μM/L浓度的工作液，每个EP管加入500μl工作液，放于37℃细胞培养箱内孵育20min，每隔3-5min颠倒混匀一下(使探针DCFH-DA和细胞充分接触)；
5、孵育结束后，使用离心机室温5000rpm离心30s-1min，使细胞沉淀并去除上液；
6、EP管中加入1ml无血清的细胞培养液，轻轻吹打重悬细胞(洗涤细胞)，之后室温5000RPM离心30s-1min，使细胞沉淀并去除上液，重复该步骤，一共用无血清的细胞培养液洗涤细胞三次；
7、最后，用适量无血清的细胞培养液重悬细胞并用流式细胞仪检测DCF的荧光，获得数据并进行后续分析。
需要说明的是，此处通过成像装置产生的图像可以是二维超声图像，也可以是三维超声图像。虽然本发明参照当前的较佳实施方式进行了描述，但本领域的技术人员应能理解，上述较佳实施方式仅用来说明本发明，并非用来限定本发明的保护范围，任何在本发明的精神和原则范围之内，所做的任何修饰、等效替换、改进等，均应包含在本发明的权利保护范围之内。

Claims (10)

  1. 茄科植物提取的杂环化合物在制备预防和/或治疗多发性硬化症的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述茄科植物包括枸杞、茄子、辣椒或西红柿中的一种或多种。
  3. 根据权利要求1或2所述的应用，其特征在于，所述茄科植物提取的杂环化合物包括尼古丁。
  4. 一种治疗多发性硬化症的药物，其特征在于，所述药物的活性成分为茄科植物提取的杂环化合物。
  5. 根据权利要求4所述的药物，其特征在于，所述茄科植物包括枸杞、茄子、辣椒或西红柿中的一种或多种。
  6. 根据权利要求4所述的药物，其特征在于，所述茄科植物提取的杂环化合物包括尼古丁。
  7. 根据权利要求4-6任一项所述的药物，其特征在于，所述药物还包括药学上可接受的辅料。
  8. 根据权利要求4-6任一项所述的药物，其特征在于，所述药物剂型包括口服制剂或注射制剂。
  9. 根据权利要求8所述的药物，其特征在于，所述口服制剂包括片剂、胶囊剂、颗粒剂、丸剂、糖浆剂、口服溶液剂、口服混悬剂或口服乳剂；
    优选地，所述注射制剂包括注射液或粉针剂。
  10. 根据权利要求4-8任一项所述的药物，其特征在于，所述药物的有效给药剂量为2-5μM/kg/天。
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