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WO2009062374A1 - Utilisation pharmaceutique de liquiritigénine pour préparer un médicament destiné au traitement de maladies neurodégénératives - Google Patents

Utilisation pharmaceutique de liquiritigénine pour préparer un médicament destiné au traitement de maladies neurodégénératives Download PDF

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Publication number
WO2009062374A1
WO2009062374A1 PCT/CN2008/001705 CN2008001705W WO2009062374A1 WO 2009062374 A1 WO2009062374 A1 WO 2009062374A1 CN 2008001705 W CN2008001705 W CN 2008001705W WO 2009062374 A1 WO2009062374 A1 WO 2009062374A1
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glycyrrhizin
disease
group
mice
neurodegenerative disease
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Fugui Cui
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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

Definitions

  • Glycyrrhizin is prepared for treatment
  • the present invention relates to the use of glycyrrhizin for the preparation of a medicament for the treatment of neurodegenerative diseases, and in particular to the use of glycyrrhizin for the preparation of a medicament for the treatment of senile dementia and Parkinson's disease.
  • Background technique
  • Glycyrrhiza is the root and rhizome of various plants of the genus Leguminosae (Glycyrrhiza Linn). It is listed in the "Shen Nong's Herbal Classic" and is listed as the top grade. It is beneficial to qi, clearing away heat and detoxifying, stopping bleeding, relieving pain, and relieving pain. Reconcile the effects of various drugs. In recent years, it has been found that licorice not only has strong detoxification effect, but also anti-ulcer, anti-inflammatory, antitussive and antitussive, lowering blood pressure, lowering blood fat and anti-cancer effects.
  • the chemical constituents of licorice are mainly flavonoids and triterpenoids, as well as a small amount of alkaloids, lignin and coumarin.
  • the dihydroflavonoid Liquir it in has a molecular formula of C 2i H 22 0 9 and a molecular weight of 418. 4.
  • the chemical name is 7-hydroxyxanthone-4, -0-glucoside (7- Hydroxyf lavanone 4' -0-glucos ide ).
  • the glycyrrhizin, or Liquir it igenin has a molecular formula of C 15 H 12 0 4 and a molecular weight of 256.3.
  • Alzheimer's disease is a chronic central nervous system degenerative disease with progressive cognitive decline as the main clinical symptom.
  • the incidence rate increases sharply with age.
  • the prevalence rate of the elderly over 65 years old in China is 1% ⁇ 5 %, and the age above 85 years old reaches 20% ⁇ 40%.
  • Pathological changes in Alzheimer's disease include extensive neuronal loss (mainly through apoptosis), numerous neuronal fiber tangles (neurof ibr i l lary tangles), and senile plaques (seni le plaque).
  • the currently marketed treatments for Alzheimer's disease include acetylcholinesterase inhibitors Arishin, Tacrine, etc., glutamate receptor blockers, memantine, etc. These drugs can improve or alleviate the symptoms of dementia in mild patients to some extent. , but can not prevent or reverse the further development of the lesion.
  • Parkinsons disease is also a common neurodegenerative disease.
  • the main pathological changes are degeneration and necrosis of dopaminergic neurons in the substantia nigra pars compacta, resulting in insufficient neurotransmitter dopamine, which ultimately leads to PD-specific Motor dysfunction.
  • levodopa remains the most effective drug for the treatment of Parkinson's disease.
  • such preparations have large side effects, and after several years of application, their efficacy is weakened, and switching phenomena and end-of-agent phenomena occur.
  • Recent experimental results suggest that levodopa may have a certain neurotoxic effect and accelerate the death of residual dopaminergic neurons.
  • the object of the present invention is to provide a novel use of glycyrrhizin, in particular for the use of glycyrrhizin for the preparation of novel medicaments for the treatment of neurodegenerative diseases, mainly Alzheimer's disease and Parkinson's disease.
  • glycyrrhizin has neuroprotective and nutritional effects in drugs selected for anti-neurodegenerative diseases, and no significant female activity (female activity refers to promotion of breast or endometrial epithelial cells). Proliferation, hyperplasia, etc., further studies have found that it promotes learning and memory in dementia model mice and enhances the ability of random movement in Parkinson's disease model mice. Therefore, glycyrrhizin can be used for the preparation of a medicament for preventing and treating senile dementia and Parkinson's disease.
  • glycyrrhizin pharmacological tests and results of glycyrrhizin will be used to illustrate their use in the preparation of novel drugs for the treatment of neurodegenerative diseases, mainly Alzheimer's disease and Parkinson's disease.
  • glycyrrhizin (1) Preparation of glycyrrhizin. After the commercially available glycyrrhizin was dissolved in methanol, an acetic acid buffer containing ⁇ -glucosidase (Sigma) was added. The ether was extracted three times and the ether layer was taken. The ether solution was then spun dry using a rotary evaporator, and the solid was scraped off and evaporated. The solid was dissolved in 10 to 20% acetonitrile, and the resolution was determined by high performance liquid chromatography (HPLC). Hirose determines the isolated compound.
  • HPLC high performance liquid chromatography
  • Hirose determines the isolated compound.
  • the glycyrrhizin of the present invention can be prepared into a dosage form such as a capsule, a tablet, a granule, a dropping pill and an injection after being added to an auxiliary material.
  • Glycyrrhizin has anti-neuronal apoptosis effect.
  • Flow cytometry was used to detect the apoptosis of rat pheochromocytoma cells (PC12) against glycyrrhizin against
  • the results showed that glycyrrhizin showed a significant anti-apoptotic effect at 0.2 g/ml.
  • Glycyrrhizin promotes the promotion of axonal growth in primary hippocampal neurons. Separation Primary rat hippocampal neurons were observed in vitro to promote the growth of neuronal axons by glycyrrhizin. The results showed that glycyrrhizin showed significant promotion of axonal growth at 0. 02 g/ml.
  • glycyrrhizin enhances memory.
  • the mouse jumping test and the Morri s water maze test were used to observe the promotion of learning and memory in scopolamine-induced Alzheimer's model mice.
  • the results showed that glycyrrhizin can significantly reduce the number of errors in the platform test, prolong the incubation period, shorten the latency of the Morr is water maze test, and increase the expression of cholinergic receptors in the brain of model mice, indicating that glycyrrhizin has enhanced learning and memory. effect.
  • glycyrrhizin has protective and nutritive effects on neurons, and can improve the learning and memory ability of the dementia model mice and the random exercise ability of the Parkinson's disease model mice without obvious female reproduction. Potential side effects of the system, etc.
  • the new drug prepared by the method provided by the invention has significant neuroprotective and neurotrophic effects without obvious female activity of flavonoids, and may have significant clinical effect without obvious toxicity, thereby generating great social benefits and economy. benefit.
  • detailed description Example 1 Preparation of glycyrrhizin
  • Example 5 Preparation of glycyrrhizin injection Take 30 g of glycyrrhizin raw material, 40 ml of propylene glycol, polyethylene glycol-400 100 ml, mix, heat in water bath for 30 min, add water for injection to 600 ml, treat in ultrasonic for 20 min, then heat in water bath to adjust pH 7.0-7.2. Filtration, potting, sterilization is available. Each 2ml, intramuscular injection, 1 time 2ml, 3 times a day, or mixed with normal saline, 5% glucose solution 500ml, intravenous drip, once a day, 3 times each time.
  • Example 6 Anti-apoptotic effect of glycyrrhizin
  • Flow cytometry was used to detect the apoptosis of rat pheochromocytoma cells (PC12) induced by glycyrrhizin against ⁇ 25 - 35 .
  • the experimental groups were as follows: control group; ⁇ 25 ⁇ 35 treatment group; ⁇ 25 - 35 different doses of drug group. Take 100 ⁇ of the cell suspension in a flow tube, add 5 ⁇ l Annexin V-FITC and ⁇ PI solution (2 (mg/ml), mix at room temperature for 15 minutes in the dark, add 400 ⁇ l PBS, and analyze by flow cytometry.
  • Rats were sterilized by volume fraction of 75% ethanol, and then decapitated in Pingya containing anatomical fluid. The whole brain was isolated and the hippocampus was separated under a dissecting microscope. It was placed in 0.25% trypsin and cut with scissors. Trypsin was pipetted into the serum containing tube every 4 min and fresh trypsin was added until no visible tissue pieces were visible. After filtering through a 200 mesh metal mesh, it was centrifuged at 1000 rpm ⁇ mirT 1 for 10 min.
  • the images were taken under a fluorescence microscope, and the length of the axons was counted by: 10 sheets per group, each measuring the length of axons of each neuron, and dividing by the total number of neurons after summation.
  • Glycyrrhizin itself has no promoting effect on the axon growth of primary hippocampal neurons, but
  • glycyrrhizin promoted axonal growth of primary hippocampal neurons in the concentration range of 0, 0 2 ⁇ 2 ⁇ ⁇ ⁇ mL- 1 (Table 2).
  • Table 2 Promoting effects of glycyrrhizin on axonal growth of primary hippocampal neurons Group concentration ( ⁇ g ⁇ mL - 1 ) Axon length ( ⁇ )
  • mice The promoting effect of glycyrrhizin on learning and memory in mice with Alzheimer's disease
  • the mouse jumping test and Morri s water maze test were used to observe the promoting effect of glycyrrhizin on learning and memory in scopolamine model mice induced by scopolamine.
  • Kunming mice weighing 14 ⁇ 16g, female. The mice were administered in groups on a random basis according to the principle of weight balance (see the table below for the dose). After 7 weeks of administration, the mold was molded and tested.
  • the mouse platform test face is as follows: The animal is placed on the platform, adapted to the environment for 3 min, and then subjected to 36 V voltage for 5 min. After 24 hours, the test recorded the latency of the first trip to the platform, the number of errors within 5 minutes, and the total shock time as a memory score.
  • the Morr i s water maze test was as follows: The mice were placed in a water maze one day before the test and free to swim for 2 min to suit the environment. Intraperitoneal injection of scopolamine 5 mg ⁇ kg 15 min before morning training. On the first day, the mice were trained 3 times. Each time, the mice were placed in the water from the 3 different water inlet points. If the mice could not find the underwater platform within 90s, they were placed directly on the platform. Stay for 30s. The next day, the training was conducted every day and afternoon. The time and path of the mice climbing the platform after entering the water were recorded as indicators for detecting the space and clues of the mice. After 24 hours, the test was repeated step by step. 3 days.
  • the results of the platform test showed that compared with the normal control group, the number of errors in the model group increased significantly within 5 minutes, and the latency was significantly shortened. Compared with the model group, the number of errors in the glycyrrhizin group was significantly reduced, and the incubation period was also significantly prolonged (see Table 3).
  • mice Male or female, weigh 18-22 grams.
  • CS-2 type mouse locomotor activity program instrument Development Institute of Materia Medica, Chinese Academy of Medical Sciences, 1-methyl-4-phenyl - 1, 2, 3, 6-tetrahydropyridine ⁇ set (1- methyl-4-phenyl -l, 2, 3, 6-tetrahydropyr idine, MPTP) is a product of Sigma.
  • the mice were randomly divided into four groups, 12 in each group, which were the normal control group, the model group, and the glycyrrhizin group (dose 30, 90 rag ⁇ kg -1 ).
  • mice After 0.54 hours of intraperitoneal injection of MPTP, except for the normal control group, the other groups of mice showed different degrees of dyskinesia, which showed slowness of movement, tremor, vertical hair, vertical tail and reduced activity. Compared with the model group, the number of autonomous activities in the glycyrrhizin group increased significantly during the test period, suggesting that glycyrrhizin can increase the central nervous system excitability and voluntary exercise ability of the model mice, and has a therapeutic effect on PD. The results are shown in Table 6. Effects of glycyrrhizin on the spontaneous activity of MPTP-induced Parkinson's disease model mice (5/5 min) Normal control 12 114. 8 ⁇ 26. 9 *** Model control 12 46. 1 ⁇ 27. 7
  • MCF-7 cells were passaged twice with phenol red-free 1640 medium containing 10% serum-free serum, digested by conventional methods, and seeded in 96-well plates at a concentration of 1.2 x 107180 ⁇ M/well. After 24 hours of cell adherence, add appropriate concentration of glycyrrhizin, glycyrrhizin or estradiol 20 ⁇ l/well, continue to culture for 72 hours, add MTT (5mg/ml) 20 ⁇ l / well before the end of the experiment, culture 4 Discard the supernatant after an hour, add DMS0 180 ⁇ ⁇ / hole, 0D value was measured at 540 nm after shaking and dissolved, and the cell proliferation rate was calculated. The results are shown in Table 7.
  • glycyrrhizin had no obvious cell proliferation at a concentration of 10 g/ml or less, and its glycoside glycoside significantly stimulated the proliferation of MCF-7 cells, indicating that glycyrrhizin had no obvious female activity, while glycyrrhizin had obvious effects.
  • the female activity suggests that the use of glycyrrhizin does not stimulate the potential side effects of proliferation or proliferation of the breast or endometrial epithelial cells. Effect of glycyrrhizin on proliferation of MCF-7 cells

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Description

甘草素在制备用于治疗
神经退行性疾病的药物中的用途 技术领域
本发明涉及甘草素在制备用于治疗神经退行性疾病药物中的 用途， 尤其涉及甘草素在制备治疗老年性痴呆和帕金森病药物中 的用途。 背景技术
甘草为豆科 (Leguminosae)植物甘草属 ( Glycyrrhiza Linn ) 多种植物的根及根茎， 始载于 《神农本草经》 ， 列为上 品， 有益气补中、 清热解毒、 祛痰止血、 緩急止痛、 调和诸药的 功效。 近年来发现甘草不但有较强的解毒作用， 还有抗溃疡、 抗 炎症、 镇痉镇咳、 降血压、 降血脂、 抗癌作用等。 甘草的化学成 分以黄酮类和三萜化合物为主， 还有少量的生物碱、 木质素和香 豆素等。 其中的二氢黄酮类化合物甘草苷 （Liquir i t in ) 的分子 式为 C2iH2209， 分子量为 418. 4， 化学名为 7-羟基黄垸酮 -4， -0- 葡萄糖苷 （ 7-Hydroxyf lavanone 4' -0-glucos ide ) 。 甘草苷 元， 或 甘草素（Liquir i t igenin)的分子式为 C15H1204， 分子量为 256. 3 , 化学名为 (2S) -7-Hydroxy-2- (4-hydroxyphenyl) -2， 3- dihydro-4H-l- benzopyran-4-one , 或 4' , 7-Dihydroxyf lavanone ( 4， ，7-二羟基黄烷酮） ， 结构式见式 （ I ) 。 文献报道， 甘草 素具有抗急性肝损伤 （Chem Biol Interact 2006, 161 (2)： 125- 138 ) 、 抑制黄嘌呤氧化酶 （Cel l mol Life Sci, 2000， 57 (3) : 500-505)等活性。
-I-
Figure imgf000003_0001
老年性痴呆病是一种以进行性认知功能减退为主要临床症状 的慢性中枢神经系统退行性疾病。 其发病率随着年龄增长而急剧 上升， 我国 65岁以上老年人的患病率为 1% ~ 5 % , 85岁以上达到 20% ~ 40 %。 老年痴呆的病理改变包括广泛的神经元丢失 （主要通 过凋亡） 、 脑内出现无数神经元纤维缠结 （neurof ibr i l lary tangles ) 和老年斑（seni le plaque)等。 目前已上市的老年痴呆 治疗药物有乙酰胆碱酯酶抑制剂安理申、 他克林等、 谷氨酸受体 阻滞剂美金刚等， 这些药物可以在一定程度上改善或緩解轻度患 者的痴呆症状， 但不能阻止或逆转病变的进一步发展。
帕金森病（ Parkinsons disease, PD)也是一种常见的神经 退行性疾病，其主要病理改变为黑质致密部多巴胺能神经元的变性 和坏死，导致神经递质多巴胺的不足，最终造成 PD特有的运动功能 障碍。 由于 PD 的病因复杂，社会危害大，随着高龄化社会的到来， 寻找具有抗 PD功效的天然药物尤为重要。 至今， 左旋多巴仍是治 疗帕金森病的最有效药物。 但这类制剂具有较大的副作用， 应用 数年后， 其疗效減弱， 出现开关现象、 剂末现象等。 近年的实验 结果提示， 左旋多巴可能具有一定的神经毒作用， 加速残存多巴 胺能神经元的死亡。
因此， 迫切需要一种疗效确切、 具有神经营养作用而毒性轻 微的新型药物。 发明内容
本发明的目的是在于提供甘草素的新用途， 具体涉及甘草素 在制备用于治疗神经退行性疾病 （主要为老年性痴呆和帕金森 病） 的新型药物中的用途。
本发明经过广泛深入的研究， 在歸选抗神经退行性疾病的药 物中出人意料地发现甘草素具有神经元保护及营养作用， 而无明 显的雌活性（雌活性指促进乳腺或子宫内膜上皮细胞增殖、 增生 等） ， 进一步研究发现其对痴呆模型小鼠学习记忆有促进作用和 增强帕金森病模型小鼠的随意运动能力。 因此， 可将甘草素用于 制备防治老年性痴呆病和帕金森病的药物。
为了更好的理解本发明的实质， 下面将用甘草素的药理试验 及结果来说明其在制备治疗神经退行性疾病 （主要为老年性痴呆 和帕金森病） 的新型药物中的应用。
( 1 )甘草素的制备。 将市售甘草苷溶解于甲醇后， 加入含 β -葡糖苷酶（S igma 公司） 的醋酸緩冲液。 乙醚萃取三次， 留取 乙醚层。 然后用旋转蒸发器旋干乙醚液， 刮取蒸干后固体备用。 以 10 ~ 20 % 乙腈溶解分离出固体后， 采用高效液相色谱法 ( HPLC )检测其分离度。 廣谮确定分离的化合物。 本发明所述的 甘草素加入辅料后可制成胶嚢、 片剂、 颗粒剂、 滴丸和针剂等剂 型。
( 2 )甘草素抗神经细胞凋亡作用。 采用流式细胞术检测甘草 素对抗 |3淀粉样蛋白 Α β 25 - 35导致大鼠嗜铬细胞瘤细胞（PC12 ) 凋 亡作用。 结果显示， 甘草素在 0. 2 g/ml 即显示明显的抗细胞凋 亡作用。
( 3 )甘草素促进原代海马神经元轴突生长促进作用。 分离大 鼠原代海马神经元， 在体外观察甘草素对神经元轴突生长的促进 作用。 结果显示， 甘草素在 0. 02 g/ml即显示明显的促进轴突生 长。
( 4 )甘草素增强记忆作用。 采用小鼠跳台试验和 Morri s 水 迷宫试验观测其对东莨菪碱所致老年痴呆模型小鼠学习记忆的促 进作用。 试验结果显示， 甘草素能明显减少跳台试验的错误次 数， 延长潜伏期， 缩短 Morr is水迷宫试验的潜伏期， 同时增加模 型小鼠脑组织胆碱能 受体表达的作用， 表明甘草素具有增强学 习记忆作用。
( 5 )甘草素抗帕金森病的实验研究。 采用 MPTP 致帕金森病 模型模型， 观察给药后模型小鼠自发活动数的变化， 结果显示甘 草素組小鼠在测试时间内的自主活动次数显著增多，提示甘草素能 提高模型小鼠的中枢神经系统兴奋輊度和随意运动能力。
( 6 )甘草素对 MCF- 7细胞增殖的影响。 结果显示甘草素无明 显的细胞增殖作用， 而其糖苷甘草苷则具有明显的刺激 MCF-7 细 胞增殖作用， 表明甘草素无明显的雌活性， 提示使用甘草素无刺 激雌性生殖系统细胞增殖、 增生等的潜在副作用。
通过上述的药理试验和结果可以得出甘草素对神经元具有保 护及营养作用， 能够改善痴呆模型小鼠的学习记忆能力和帕金森 病模型小鼠的随意运动能力， 而无明显的对雌性生殖系统等的潜 在副作用。
由本发明提供的方法制备的新药具有显著的神经保护和神经 营养作用而无明显的黄酮类化合物的雌活性， 在临床上可能会有 显著疗效而无明显的毒性， 因而产生巨大的社会效益和经济效 益。 具体实施方式 实施例 1 甘草素的制备
将市售甘草苷溶解于甲醇后， 加入含 β -葡糖苷酶（Sigma 公 司） 的醋酸緩冲液。 乙醚萃取三次， 留取乙醚层。 然后用旋转蒸 发器旋干乙醚液， 刮取蒸干后固体备用。 以 10 ~ 20 %乙腈溶解分 离出固体后， 采用高效液相色谱法 （HPLC )检测其分离度。 经分 析可知该反应 48h 后仅得一种产物， 说明甘草苷已完全水解， "归一法" 计算其纯度为 98. 74 %。 经质谱分析其分子量为 256 , 可以确定该物质为甘草素。 实施例 2 甘草素胶囊剂
取甘草素原料 500克， 药用淀粉 1000克， 搅拌均匀， 装入 0 号胶囊， 每粒 0. 3克， 每次口服 2粒， 每日三次。 实施例 3 甘草素片剂制备
取甘草素原料 500克， 药用淀粉 500克， 搅拌均匀， 用适量 乙醇制粒， 经整粒， 压片， 每片 0. 2克， 口服， 每次 2 片， 每曰 三次。 实施例 4 甘草素滴丸的制备
称取 300克聚乙二醇 4000为基质， 在水浴上溶化， 加入甘草 素原料 100 克， 搅拌均匀， 倾入保温管中， 用恒温装置保温， 使 含甘草素的聚乙二醇液体在 条件下滴入预冷的液体石蜡 中（4-l (TC)，滴完后， 将药丸倾入滤纸上吸干石蜡油， 再加入少量 滑石粉， 混匀， 得甘草素滴丸 1000粒。 口服， 1次 2粒， 1 日 3 次。 实施例 5 甘草素注射剂的制备 取甘草素原料 30 克， 丙二醇 40ml, 聚乙二醇- 400 100ml, 混合， 水浴加热 30min， 加注射用水至 600ml， 超声波中处理 20min， 再在水浴中加热， 调节 pH值 7.0-7.2。 过滤， 灌封， 灭 菌即得。 每支 2ml，肌肉注射， 1 次 2ml， 每日 3次， 或与生理盐 水， 5%葡萄糖液 500ml，混匀后， 静脉滴注，每日 1 次， 每次 3 支。 实施例 6 甘草素抗凋亡作用
采用流式细胞术检测甘草素对抗 Αβ 25 - 35导致大鼠嗜铬细胞瘤 细胞 （PC12) 凋亡作用。 实验分组如下： 对照组； Αβ25~35 处理 组； Αβ 25 - 35 不同剂量药物组。 取 100 μΐ 的细胞悬液于流式管 中 ，加入 5μ1 Annexin V-FITC和 ΙΟμΙ PI 溶液（2(mg/ml)混匀， 室温避光孵育 15分钟，加 400μ1 PBS ，流式细胞仪分析。
与正常組比， A β处理模型組凋亡率明显增加， 且有统计学意 义 （ Ρ<0.01 ) ； 各加药组凋亡率与模型组比显著降低 (Ρ<0.01 ) , 并随培养体系中药物浓度增加， 凋亡率降低更明 显， 结果表明甘草素有抗 A Ρ致 PC12细胞凋亡的作用。 表 1 甘草素抗 A β 25 ~ 35致 PC 12细胞的凋亡作用 組 别 浓度（ μ g · mL- 1 ) 凋亡率（％) 对照組 - 2.10±1.80
模型组 10 34.85士 2.16'' 甘草素 0.2 20.35 ±1.21*
2.0 17.18±1.67*
20 12.33±1.43.
注： 与对照組比 P<0.01; '与模型組比 P<0.01 实施例 7 甘草素促进原代培养海马神经元轴突生长作用
SD大鼠乳鼠， 出生 12h 内， ？ δ兼用。 大鼠乳鼠经体积分数 为 75%乙醇消毒后， 断头置于含解剖液的平亚中， 分离出全脑， 解剖显微镜下分离海马组织。 将其置于 0.25%胰蛋白酶中并用剪 刀剪碎。 每隔 4min吸取胰酶至含血清离心管中， 并加入新鲜胰酶 直至无肉眼可见的組织块。 200 目 金属筛 网过滤后以 lOOOrpm · mirT1 离心 10min。 将其重悬于 10 %胎牛血清的 DMEM/F-12种植液， 接种于预先包被多聚赖氨酸的 24孔板， 培养 过夜后换为 3%胎牛血清、 1%N2、 1%B27和 3ng ' πθ 1阿糖胞苷 的 DMEM/F-12 培养液。 加入终浓度质量分数为 0.02、 0.2、 2 μ g · mL— 1甘草素， 同时加入 2ng · mL_1NGF; 以 2ng · mL_1NGF为对照 组。 2d 后于荧光显微镜下拍照， 并统计轴突长度， 方法为： 每组 10张， 每张测量各神经元轴突长度， 求和后除以其神经元总数。
甘草素本身对原代海马神经元轴突生长无促进作用， 但在
2ng · mL^NGF 存在下可明显促进其轴突生长， 其轴突长度明显长 于对照組（P<0.01) 。 在 2ng · mL— ^GF存在下， 甘草素在 0， 02 ~ 2μδ · mL— 1的浓度范围内对原代海马神经元轴突生长有促进作用 (表 2) 。 表 2 甘草素对原代海马神经元轴突生长的促进作用 組 别 浓度（ μ g · mL-1 ) 轴突长度 ( μιη)
对照组 - 18.1 ± 2.4 甘草素 0.02 38.7±4· 6'
0.2 54.3±9.7'
2 86.2 ± 12.2.
注： η = 10 *与对照组比 Ρ<0.01 实施例 8 甘草素对老年痴呆模型小鼠学习记忆的促进作用 采用小鼠跳台试验和 Morri s水迷宫试验观测甘草素对东莨菪 碱所致老年痴呆模型小鼠学习记忆的促进作用。 昆明小鼠， 体重 14 ~ 16g, 雌性。 小鼠按体重均衡的原则随机原则分組给药 （剂量 见下表） 。 给药 7ά后, 造模并进行试验。 小鼠跳台试脸如下： 将 动物放置在平台上， 适应环境 3min,然后通以 36V 电压， 训练 5min。 24h 后测验， 记录第一次跳下平台的潜伏期、 5min 内的错 误次数和总电击时间， 以此作为记忆成绩。
Morr i s 水迷宫试验如下： 试验前一天先将小鼠放入水迷宫中 自由游泳 2min以适应环境。 每日上午训练前 15min腹腔注射东莨 菪碱 5mg · kg— 每曰给予甘草素一次。 第一天小鼠训练 3 次， 每 次分别从 3 个不同入水点将小鼠面向容器壁放入水中游泳， 若小 鼠在 90s 内不能找到水下平台， 则将其直接置于平台上并停留 30s。 第二天开始每天上下午各训练一次， 记录小鼠入水后爬上平 台所用的时间、 路经轨迹等作为检测小鼠对空间和线索学习的指 标， 24小时后按步进行复测， 共训练 3天。
每組取 4只鼠脑組织做免疫组化测定胆碱能 受体水平， 每 只鼠做两张切片， 免疫组化采用 SABC法， DAB显色， 最后用苏木 素进行核复染。 每组取一张切片作为同型对照。 数据分析时， 每 张切片按海马和皮层相同部位选取 5个视野， 结果用 MIAS医学图 像分析管理系统 4. 0 进行处理， 分析平均光密度和积分光密度。
跳台试猃结果显示， 与正常对照组比， 模型组 5min内错误次 数明显增加， 潜伏期显著缩短。 与模型組比， 甘草素组错误次数 明显減少， 潜伏期也有明显延长（见表 3 ) 。 表 3 甘草素对东茛菪碱小鼠学习记忆障碍模型跳台成缋的影响 組别 剂量 (mg · kg"1) 错误次数（n) 潜伏期 （ s ) 正常对照组 一 0.3 ± 0.2 250.9 ± 85.1 模型組 5 6.0 ± 2.7*** 10.1 ± 3.1*** 甘草素 30 2.8±1.2# 91.3 ±26.5#
90 1.3±1.1## 130.8±45.3## 注： 与正常对照组相比： *** P<0.001; 与模型組相比： #<0.05 ## P<0.01.
Morris 水迷宫结果显示， 随着训练次数的增加， 各組小鼠找 到平台的潜伏期逐渐缩短。 与正常对照组相比， 模型组（东莨菪 碱 5mg . kg"1)各次训练的潜伏期明显延长。 甘草素组从第 5次训 练开始与模型組显示出显著差异， 潜伏期明显缩短。 结果如表 4 所示： 表 4 甘草素对东茛菪碱小鼠学习记忆障碍模型水迷宫上台潜伏
期的影响
潜伏期 （ s )
甘草素 甘草素 正常对照組
Figure imgf000010_0001
(90 mg · kg"1) (30 mg · kg'1) 第 1次 50.10±19.53 87.54 ±9.67* 83.45 ±4.32 83.81士17.28 第 2次 44.31 ±21.12 87.32 ± 1.26*** 71.53±11.35 80.19 ± 14.39 第 3次 39.53 ± 17.30 79.93 ± 26.37** 54.08 ±29.33 63.04 ±23.80 第 4次 25.77 ±10.21 79.92士 25.31*** 43.35 ±28.43 52.70 ± 34.51 第 5次 18.80± 11.24 77.53 ±11.54*** 32.99 ± 19.82### 48.33士 26.33## 第 6次 12, 32士 6.33 86.50 ± 21.05*** 30.41 ± 14.2### 35.57 ±11.13### 第 7次 18.39±11.16 63.22 ± 26.84** 34.27 ± 12.30## 35.62 ±13.46#
(1) 与正常組相比: *P<0.05, **p<0.01, *** P<0.001;
与模型组相比： # P<0.05, ## P<0.01, ### P<0.001. ( 2 ) 第 1 - 3次为笫一天训练结果， 笫 4、 5次为第二天训练 结果， 笫 6、 7次为第三天训练结果 平均光密度结果显示， 与正常对照组相比， 模型对照组平均 光密度值显著下降， 说明细胞染色程度下降， ！^受体水平降低。 而甘草素组平均光密度值均比模型组有所提高， 说明药物可能使 M,受体水平提高。 积分光密度结果亦显示模型组比正常对照组显 著降低， 而甘草素组比模型組显著升高， 提示甘草素能促进！^受 体表达。 结果见表 5。 表 5 甘草素对东茛菪碱造成小鼠获得性学习记忆障碍模型脑组织
M,受体水平的影响
平均光密度 积分光密度 正常对照 0. 20 ± 0. 03 1156. 70 ± 542. 46 模型組 0， 16 ± 0. 02*** 271. 34 ± 93. 56*** 甘草素（90 mg · kg'1) 0. 25土 0. 03 ### 930. 12 ± 432. 82### 甘草素（30 mg · kg"1) 0. 20 ± 0. 02## 530. 26 ± 136. 52## 注： 与正常组相比： *** P<0. 001; 与模型组相比： # P<0. 05, ## Ρ<0· 01, ### Ρ<0. 001. 实施例 9 甘草素对 ΜΡΤΡ致帕金森病模型小鼠自发活动的影响
成年 057BL小鼠， 雌雄不拘， 体重 18-22克。 CS-2型小鼠自 主活动程序仪，中国医学科学院药物研究所研制， 1-甲基 -4-苯基- 1， 2, 3， 6— 四 氢 吡 ρ定 ( 1- methyl-4-phenyl-l, 2， 3， 6- tetrahydropyr idine, MPTP ) 为 Sigma公司产品。 将小鼠随机分 成四組，每组 12 只，分别为正常对照组、 模型組以及甘草素组（剂 量为 30， 90 rag■ kg-1) 。 连续灌胃给药 14 d，于第 11 天开始灌 胃给药前 1 h腹腔注射 MPTP 30 mg · kg— 1 (对照组给予等体积的生 理盐水） ，连续 4d，最后 1 次给药后 1 d,进行行为学指标测试， 将小鼠放入自主活动箱中，每次同时测定 4 只小鼠，每个活动箱中 1 只， 由记录仪自动记录小鼠活动，测定每只小鼠 5 min内的活动 次数。
腹腔注射 MPTP后 0. 5h ,除正常对照組外，其余各組小鼠均出 现不同程度的运动障碍，表现为动作迟緩、 震颤、 竖毛、 竖尾和活 动减少等。 与模型組比较， 甘草素组小鼠在测试时间内的自主活 动次数显著增多，提示甘草素能提高模型小鼠的中枢神经系统兴奋 程度和随意运动能力，对 PD有治疗作用。 结果见表 6。 甘草素对对 MPTP致帕金森病模型小鼠自发活动的影响 组 别 动物数 活动数（次 /5min ) 正常对照 12 114. 8 ± 26. 9 *** 模型对照 12 46. 1 ± 27. 7
甘草素（90 mg · kg") 12 102. 1 ± 24. 2### 甘草素（30 mg ' kg"1) 12 90. 3 ± 27. 9## 注： 与正常组相比： *** P<0. 001; 与模型組相比： # P<0. 05， ## P<0. 01, ### P<0. 001. 实施例 10 甘草素对 MCF-7细胞增殖的影响
将 MCF-7细胞用含 10%无雌血清的无酚红 1640培养液传代 2 次， 按常规方法消化离心， 以 1. 2 x 107180 μ ΐ/孔的浓度接种于 96孔板， 待 18-24小时细胞贴壁完全后加入适当浓度的甘草苷、 甘草素或雌二醇 20 μ 1/孔， 继续培养 72 小时， 于试验结束前加 MTT ( 5mg/ml ) 20 μ 1 /孔， 培养 4 小时后弃去上清， 加 DMS0 180 μ ΐ /孔， 震荡溶解后 540nm处测定 0D值， 计算细胞增殖率。 结果 见表 7。
结果发现， 甘草素在浓度 10 g/ml 以下无明显的细胞增殖作 用， 而其糖苷甘草苷则具有明显的刺激 MCF-7 细胞增殖作用， 表 明甘草素无明显的雌活性， 而甘草苷有明显的雌活性， 提示使用 甘草素无刺激乳腺或子宫内膜上皮细胞增殖或增生等的潜在副作 用。 甘草素对 MCF-7细胞增殖的影响
药 物 浓度 ( μ g/ml ) 增殖率（½ )
甘草苷 10 44. 1
1 36. 7
0. 1 20. 1
0. 01 9. 6
0. 001 8. 5
甘草素 10 10. 8
1 8. 7
0. 1 2. 0
0. 01 3. 6
0. 001 0. 4
17 β -雌二醇 0. 2 35. 3

Claims

权 利 要 求
1、 下式（ I ) 的甘草素在制备用于治疗神经退行性疾病的药 物中的用途
Figure imgf000014_0001
( I )
2、 根据权利要求 1的用途， 其中所述的神经退行性疾病是老 年性痴呆。
3、 根据权利要求 1的用途， 其中所述的神经退行性疾病是帕 金森病。
4、 用于治疗神经退行性疾病的药物组合物， 其包含作为活性 成分的式（ I )甘草素及药用赋形剂或载体。
5、 根据权利要求 4的药物组合物， 其中所述的神经退行性疾病是 老年性痴呆。
6、 根据权利要求 4的药物組合物， 其中所述的神经退行性疾 病是帕金森病。
7、 治疗患者神经退行性疾病的方法， 它包括将治疗上有效量 的式 （ I ) 的甘草素给予该患者。
8、 根据权利要求 7的方法， 其中所述的神经退行性疾病是老 年性痴呆。
9、 根据权利要求 7 的方法， 其中所述的神经退行性疾病是帕 金森病。
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