WO2023066216A1 - 一种包含c21甾体皂苷的药物组合物及其应用 - Google Patents

一种包含c21甾体皂苷的药物组合物及其应用 Download PDF

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WO2023066216A1
WO2023066216A1 PCT/CN2022/125749 CN2022125749W WO2023066216A1 WO 2023066216 A1 WO2023066216 A1 WO 2023066216A1 CN 2022125749 W CN2022125749 W CN 2022125749W WO 2023066216 A1 WO2023066216 A1 WO 2023066216A1
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邱佐成
陈家旭
唐紫灵
庞倩倩
李小叁
鄢黎
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暨南大学
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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
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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  • the invention belongs to the field of medicine, and relates to a pharmaceutical composition containing C21 steroidal saponin and its application, in particular to the use of a pharmaceutical composition containing C21 steroidal saponin in the preparation of A ⁇ amyloid formation inhibitors and neuroprotective agents application.
  • Nerve cell damage is one of the main causes of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.
  • the death of nerve cells can lead to cognitive, learning and memory dysfunction in patients with neurodegenerative diseases.
  • Oxidative stress caused by neurotoxic substances such as ⁇ -amyloid and neurotransmitters is considered to be the main cause of neuronal cell death.
  • glutamate is the main endogenous neurotransmitter of the central nervous system, but high concentrations of glutamate can cause neurofibrillary tangles and necrosis of neurons in the brain, leading to cognitive dysfunction.
  • extracellular glutamate toxicity reduces cellular uptake of cysteine by damaging the cystine/glutamate antagonist, leading to depletion of the intracellular antioxidant glutathione.
  • the imbalance of antioxidant content levels such as calcium ion influx, intracellular reactive oxygen species (ROS) generation, lipoxygenase (LOX)-dependent lipid peroxidation, etc., will also accelerate a series of downstream processes leading to neuronal cell death.
  • ROS reactive oxygen species
  • LOX lipoxygenase
  • Amyloid plaques formed by the deposition of neurotoxic ⁇ -amyloid (A ⁇ ) atheromatous amyloid are one of the main causes of Alzheimer's disease. It is mainly the proteolysis of ⁇ -amyloid precursor protein (amyloid precursor protein, APP) by ⁇ -secretase to produce CTF- ⁇ , which is further hydrolyzed by ⁇ -secretase to produce 40 or 42 abnormally folded A ⁇ peptides.
  • the generation and abnormal aggregation mechanism of A ⁇ are crucial to the occurrence of AD. Therefore, inhibiting the generation of A ⁇ and increasing the clearance rate of A ⁇ may be a potential therapeutic strategy to delay the development of AD.
  • Cynanchum otophyllum Schneid. is a common ethnic medicine in the Ascariaceae velvet genus widely distributed in Southwest China, Hunan, Guangxi, Cambodia and other places. Also known as white stone ginseng and poisonous dog medicine, the folks in Lijiang, Yunnan Republic call its root “white head root”. It is slightly warm in nature, sweet and slightly bitter. According to "Yunnan Chinese Herbal Medicine”, “National Chinese Herbal Medicine Compilation”, “Yi Medicine Chronicle” and other records, the dried rhizome of Qingyang ginseng can be used for rheumatic arthralgia, kidney deficiency, low back pain and lumbar muscle strain, tumbling, flashback, food accumulation, etc.
  • Qingyang ginseng has anticonvulsant, antiepileptic, antidepressant, sedative, analgesic, immune regulation, anti-hepatitis, and anti-Mégro syndrome effects.
  • C21 steroidal saponins rich in Qingyang ginseng are its representative components and main medicinal components.
  • most of the C21 saponins are embedded in glycosides, which is difficult to separate, so only a few types of free C21-steroidal aglycones have been found.
  • Studies in recent years have shown that C21 steroidal saponins have anti-hepatic fibrosis and anti-epileptic activities; they have strong anti-proliferation activities against various human tumor cell lines.
  • neuroprotective activity and anti- ⁇ -amyloid activity of C21 steroidal saponins there is no report about the neuroprotective activity and anti- ⁇ -amyloid activity of C21 steroidal saponins.
  • the purpose of the present invention is to solve the problems existing in the treatment of neurodegenerative diseases in the prior art, thereby providing a pharmaceutical composition for the treatment of neurodegenerative diseases, which can significantly reduce ⁇ -amyloid
  • the expression of the precursor protein APP can obviously promote the proliferation of neuron cells, and can inhibit the cytotoxicity induced by glutamate at the same time, and has neuroprotective activity on neuron cells.
  • the present invention is achieved by the following means:
  • the first aspect of the present invention provides a pharmaceutical composition for preventing and/or treating neurodegenerative diseases, comprising one or more of C21 steroidal saponins, pharmaceutically acceptable salts, and solvates thereof, and pharmaceutical An acceptable carrier, the C21 steroidal saponin has the structure shown in the following formula I:
  • R 1 is selected from H, O, hydroxyl, optionally substituted C 1 -C 4 alkyl, optionally substituted C 1 -C 4 alkoxy, one or more of
  • R 2 is selected from one or more of H, O, hydroxyl, optionally substituted C 1 -C 4 alkyl, optionally substituted C 1 -C 4 alkoxy;
  • R 3 is selected from one or more of H, O, hydroxyl, optionally substituted C 1 -C 4 alkyl, optionally substituted C 1 -C 4 alkoxy;
  • R 4 is selected from H, O, hydroxyl, optionally substituted C 1 -C 4 alkyl, optionally substituted C 1 -C 4 alkoxy, one or more of
  • Each adjacent carbon atom in the polycyclic ring of the compound of formula I is connected by a single bond, a double bond or a triple bond.
  • the neurodegenerative disease includes one or more of Alzheimer's disease, Parkinson's disease, and Huntington's disease.
  • the Alzheimer's disease includes one or more of Alzheimer's disease, vascular dementia, Lewy body dementia and frontotemporal dementia.
  • the pharmaceutically acceptable carrier includes one or more of fillers, binders, disintegrants, solvents, preservatives, lubricants, and flavoring agents.
  • the C21 steroidal saponin is selected from one or more of the compounds 1-5 shown in the following structures:
  • the second aspect of the present invention provides the application of one or more of C21 steroidal saponins, pharmaceutically acceptable salts or solvates thereof in the preparation of products for the prevention and/or treatment of neurodegenerative diseases, said C21 steroidal saponins have the structure shown in the following formula I:
  • R 1 is selected from H, O, hydroxyl, optionally substituted C 1 -C 4 alkyl, optionally substituted C 1 -C 4 alkoxy, one or more of
  • R 2 is selected from one or more of H, O, hydroxyl, optionally substituted C 1 -C 4 alkyl, optionally substituted C 1 -C 4 alkoxy;
  • R 3 is selected from one or more of H, O, hydroxyl, optionally substituted C 1 -C 4 alkyl, optionally substituted C 1 -C 4 alkoxy;
  • R 4 is selected from H, O, hydroxyl, optionally substituted C 1 -C 4 alkyl, optionally substituted C 1 -C 4 alkoxy, one or more of
  • Each adjacent carbon atom in the polycyclic ring of the compound of formula I is connected by a single bond, a double bond or a triple bond.
  • the neurodegenerative disease includes one or more of Alzheimer's disease, Parkinson's disease, and Huntington's disease.
  • the Alzheimer's disease includes one or more of Alzheimer's disease, vascular dementia, Lewy body dementia and frontotemporal dementia.
  • the C21 steroidal saponin is selected from one or more of the compounds 1-5 shown in the following structures:
  • the product includes one or more of medicines, health products and foods.
  • the degradation of extracellular A ⁇ is mainly completed by insulin-degrading enzyme (IDE) and neprilysin (NEP), while the degradation of intracellular A ⁇ is mainly carried out in lysosomes.
  • IDE insulin-degrading enzyme
  • NEP neprilysin
  • intracellular A ⁇ is mainly carried out in lysosomes.
  • IDE insulin-degrading enzyme
  • NEP neprilysin
  • intracellular A ⁇ is mainly carried out in lysosomes.
  • the process of AD is accompanied by the disorder of lysosomal system, and the accumulation of A ⁇ in lysosome is one of the pathological characteristics of AD.
  • AD autophagy-lysosome system
  • a ⁇ A ⁇ or neurofibrillary tangles
  • NFTs neurofibrillary tangles
  • Autophagy a degradation pathway mediated by both vesicles and lysosomes, is important for protein homeostasis and the cellular environment.
  • autophagy plays an important role in the production and metabolism of A ⁇ .
  • extracellular aggregates of A ⁇ (amyloid plaques) and abnormal phosphorylation of Tau protein in neurons are obvious pathological signs, autophagy - Defects in the lysosomal pathway may precede the development of these pathological hallmarks.
  • Autophagy disorder is an important mechanism for the excessive accumulation of A ⁇ : under normal physiological conditions, a small amount of A ⁇ produced in cells can activate autophagy by inhibiting mTOR, since autophagosomes are formed around axons, while lysosomes are mainly located around the nucleus, Autophagosomes are reversely transported to the cell body through the microtubule system of the axon, and degrade A ⁇ after combining with lysosomes. The production and degradation of A ⁇ are in balance; Increased aggregation, and the aggregated autophagosomes are rich in APP, A ⁇ , and ⁇ - and ⁇ -secretase complexes, so the abnormal increase of autophagosomes is considered to be the source of A ⁇ production.
  • LC3B is the first autophagosome marker protein discovered, and it has two forms, LC3B-I and LC3B-II.
  • LC3B synthesized in cells is processed into cytoplasmic soluble type I LC3B, which is regularly expressed.
  • type I LC3B is transformed into type II LC3B and localized on the intracellular autophagosome membrane, and its content is proportional to the number of autophagosomes. Therefore, LC3B-II is often used as a marker of intracellular autophagy to detect the conversion of LC3B, that is, the ratio of LC3B-II/LC3B-I. An increase in the ratio indicates active autophagy, and a decrease in the ratio indicates a decrease in autophagy.
  • Beclin 1 is a specific gene of autophagy, which plays a key regulatory role in the formation of autophagy, and its upregulation can stimulate the occurrence of autophagy. Beclin 1 is an important molecule in the connection between autophagy and apoptosis, and its regulation of apoptosis mainly inhibits excessive autophagy by combining with Bcl-2.
  • P62 is a common autophagy lysosome substrate, its content is negatively correlated with autophagy level, and it is an important bridge connecting LC3 and ubiquitinated substrates to be degraded.
  • the protein polymer formed by P62 can be degraded by the autophagosome, and P62 binds to the autophagosome membrane protein LC3/ATG8, thereby transporting the protein polymer containing P62 to the autophagosome.
  • the present invention has the following beneficial effects:
  • the active ingredient C21 steroidal saponin on the one hand, has a significant down-regulation effect on the APP protein of N2a cells overexpressing human APP695, and can increase the clearance rate of excess A ⁇ amyloid protein; On the other hand, it promotes the proliferation of neuronal cells, and has a protective effect on the neuronal cytotoxicity induced by neurotransmitters such as glutamate caused by the excessive accumulation of A ⁇ amyloid, so as to comprehensively play the role of treating neurodegenerative diseases. Therefore, it has a good application prospect in the preparation of A ⁇ amyloid formation inhibitors and neuroprotective agents, drugs for preventing or treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and the like.
  • the active ingredient of the present invention can inhibit glutamate-induced cytotoxicity and has neuroprotective activity on neuron cells, thereby significantly reducing severe toxic and side effects caused by inhibiting secretase activity, and has higher safety.
  • Figure 1 is a schematic diagram of the experimental results of 20-O-VK on promoting the proliferation of HT22 cells.
  • Fig. 2 is a schematic diagram of the neuroprotective effect of 20-O-VK on glutamate-induced injury of HT22 hippocampal neuron cells.
  • Fig. 3 is a cell morphology diagram of 20-O-VK on glutamate-induced injury to HT22 hippocampal neuron cells.
  • Fig. 4 is a schematic diagram of flow cytometry results of 20-O-VK inhibition of glutamate-induced apoptosis in HT22 cells.
  • Fig. 5 is a schematic diagram showing the results of 20-O-VK's inhibition of glutamate-induced apoptosis in HT22 cell percentage.
  • Fig. 6 is a schematic diagram of the percentage of apoptotic cells in the early stage of the glutamate-induced HT22 cell apoptosis inhibition experiment by 20-O-VK.
  • Fig. 7 is a schematic diagram of the effect of 20-O-VK on the survival rate of N2a-APP695 cells.
  • Figure 8 is a schematic diagram of the WB results of the effect of 20-O-VK on the expression of full-APP and CTF proteins in N2a-APP695 cells.
  • Figure 9 is a schematic diagram of the results of quantitative analysis of full-APP expression in N2a-APP695 cells by 20-O-VK.
  • Figure 10 is a schematic diagram of the results of quantitative analysis of CTF expression in N2a-APP695 cells by 20-O-VK.
  • Figure 11 is a schematic diagram of the WB results of the effect of 20-O-VK on the expression of BACE1 protein in N2a-APP695 cells.
  • Figure 12 is a schematic diagram of the results of quantitative analysis of BACE1 expression in N2a-APP695 cells by 20-O-VK.
  • Figure 13 is a schematic diagram of the WB results of the effect of 20-O-VK on the expression of LC3B-I and LC3B-II proteins in N2a-APP695 cells.
  • Figure 14 is a schematic diagram of the results of quantitative analysis of LC3B expression in N2a-APP695 cells by 20-O-VK.
  • Figure 15 is a schematic diagram of the WB results of the effect of 20-O-VK on the expression of APP-full and CTF proteins in N2a-APP695 cells.
  • Figure 16 is a schematic diagram of the results of quantitative analysis of APP-full expression in N2a-APP695 cells by 20-O-VK.
  • Figure 17 is a schematic diagram of the results of quantitative analysis of CTF expression in N2a-APP695 cells by 20-O-VK.
  • Figure 18 is a schematic diagram of the WB results of the effect of 20-O-VK on the expression of P62 protein in N2a-APP695 cells.
  • Figure 19 is a schematic diagram of the results of quantitative analysis of P62 expression in N2a-APP695 cells by 20-O-VK.
  • Figure 20 is a schematic diagram of the WB results of the effect of 20-O-VK on the expression of Beclin 1 protein in N2a-APP695 cells.
  • Figure 21 is a schematic diagram of the results of quantitative analysis of Beclin 1 expression in N2a-APP695 cells by 20-O-VK.
  • the compounds 1-5 listed in the context of the present invention are all extracted from Qingyang ginseng by conventional methods in the prior art, and their structures are determined by 1 H NMR and/or 13 C NMR. Identification. Cell lines used in the context of the present invention, including HT22, N2a, etc., were cultured according to ATCC guidelines. All cell lines were identified by short tandem repeat analysis at China Type Culture Collection (Wuhan) and verified for mycoplasma contamination using a PCR detection kit (Shanghai Biothrive Sci), while being cryopreserved in liquid nitrogen and used for subsequent experiments. The reagents and consumables used in the present invention are all commercially available or prepared according to conventional methods.
  • the experimental methods used in the present invention such as cell culture, cell proliferation experiment, cell apoptosis experiment, flow cytometry, Western Blot experiment, etc. are all conventional methods and techniques in the art. All the instruments and equipment used in the present invention are commercially available, wherein the microplate reader is the American BioTEK Synergy H1 Hybrid Multi-Mode Reader; the flow cytometer is the American BECKMAN CONLTER, CytoFLEX S.
  • step (3) The EtOAc-soluble fraction obtained in step (2) was subjected to silica gel column chromatography (CC), and CH 2 Cl 2 -MeOH (gradient 95:1 ⁇ 80:1 ⁇ 60:1 ⁇ 30:1 ⁇ 15:1 ⁇ 10:1 ⁇ 5:1 ⁇ 2:1, v/v, 5 column volumes per gradient) to obtain compound 1-5.
  • CC silica gel column chromatography
  • CH 2 Cl 2 -MeOH gradient 95:1 ⁇ 80:1 ⁇ 60:1 ⁇ 30:1 ⁇ 15:1 ⁇ 10:1 ⁇ 5:1 ⁇ 2:1, v/v, 5 column volumes per gradient
  • Example 2 C21 steroidal saponins promote the proliferation of hippocampal neuron cells
  • Groups 1-6 were added with compound 3 (20-O-VK) at concentrations of 0.01 ⁇ M, 0.1 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M, and 10 ⁇ M, respectively;
  • Group C is the control group, and an equal volume of culture medium is added;
  • the test results are shown in Figure 2-3.
  • the results showed that the hippocampal neuron injury model was constructed by adding 5 mM glutamic acid, and after administration of C21 steroidal saponins (0.5, 1, 5, 10 ⁇ M), the rate of cell damage decreased significantly, indicating that C21 steroidal saponins had an effect on glutamic acid.
  • the induced hippocampal neuron injury had significant neuroprotective activity, and the difference from group C was statistically significant (*P ⁇ 0.05, ***P ⁇ 0.001).
  • Example 4 C21 steroidal saponins inhibit glutamate-induced apoptosis of hippocampal neurons
  • the test results are shown in Figure 4-6.
  • the results showed that the hippocampal neuron injury model was constructed by adding glutamic acid, and after administration of C21 steroidal saponin (5, 10 ⁇ M), the cell survival rate was significantly increased and the cell apoptosis rate was significantly decreased by flow cytometry detection. It shows that C21 steroidal saponins have significant protective activity on hippocampal neuron apoptosis induced by glutamate, and can inhibit the apoptosis of hippocampal neuron cells, and the difference from group C is statistically significant (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001).
  • mouse neuroblastoma cell N2a (mouse Neuroblastoma-2a) stably expressing the human amyloid precursor protein APP695 gene can stably generate A ⁇ amyloid protein, which is an in vitro AD screening model provided by ATCC, USA.
  • its active ingredient C21 steroidal saponin can significantly reduce the generation of ⁇ -amyloid protein in N2a-APP695 cells and increase the clearance rate of excess A ⁇ amyloid protein;
  • it promotes the proliferation of neuronal cells, and has a protective effect on the neuronal cytotoxicity induced by neurotransmitters such as glutamate caused by the excessive accumulation of A ⁇ amyloid, so as to comprehensively play the role of treating neurodegenerative diseases. Therefore, it has a good application prospect in the preparation of A ⁇ amyloid formation inhibitors and neuroprotective agents, drugs for preventing or treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and the like.

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Abstract

本发明涉及一种治疗预防和/或治疗神经退行性疾病的药物组合物，其活性成分C21甾体皂苷一方面能够显著降低N2a-APP695细胞中β-淀粉样蛋白的生成，增加过量Aβ淀粉样蛋白的清除率；另一方面促进神经元细胞的增殖，对Aβ淀粉样蛋白过量累积导致的神经递质如谷氨酸诱导的神经元细胞毒性具有保护作用，从而综合发挥治疗神经退行性疾病的作用。因而在制备Aβ淀粉样蛋白形成抑制剂及神经保护剂，预防或治疗阿尔茨海默病、帕金森症、亨廷顿舞蹈病等神经退行性疾病的药物中具有良好的应用前景。

Description

一种包含C21甾体皂苷的药物组合物及其应用 技术领域
本发明属于医药领域，涉及一种包含C21甾体皂苷的药物组合物及其应用，具体涉及一种包含C21甾体皂苷的药物组合物在制备Aβ淀粉样蛋白形成抑制剂及神经保护剂中的应用。
背景技术
神经细胞损伤是阿尔茨海默病、帕金森症、亨廷顿舞蹈病等神经退行性疾病的主要发病原因之一。神经细胞的死亡会导致神经退行性疾病患者的认知，学习及记忆功能障碍。而神经毒性物质如β淀粉样蛋白、神经递质等造成的氧化应激反应被认为是神经细胞死亡的主要原因。其中，谷氨酸是中枢神经系统的主要内源性神经递质，但高浓度的谷氨酸可引起脑内神经纤维缠结和神经元细胞坏死，导致认知功能障碍。而且，细胞外谷氨酸毒性会通过损伤胱氨酸/谷氨酸抗器，从而减少细胞对半胱氨酸的摄入，导致细胞内抗氧化剂谷胱甘肽的耗尽。抗氧化剂含量水平的失衡，如钙离子流入，细胞内活性氧(ROS)产生，脂氧酶(LOX)依赖的脂质过氧化等，亦会加速导致神经细胞死亡的一系列下游过程。
具有神经毒性的β-amyloid(Aβ)粥样淀粉沉积而形成的淀粉样斑块是阿尔茨海默病的主要病因之一。它主要是β分泌酶蛋白水解β淀粉样前体蛋白(amyloid precursor protein，APP)，产生CTF-β，CTF-β进一步经γ-分泌酶水解产生40或42个异常折叠的Aβ肽段。Aβ的产生和异常聚集机制对AD的发生至关重要。因此，抑制Aβ的生成、增加Aβ的清除率，可能是延缓AD发展的潜在治疗策略。
青阳参(Cynanchum otophyllum Schneid.)是广泛分布于中国西南及湖南、广西、西藏等地的萝藦科鹅绒藤属植物中的一种常见民族药物，始载于《植物名实图考》，又名白石参、毒狗药，云南丽江地区民间称其根部为“白首乌”。其性微温，味甘微苦，根据《云南中草药》《全国中草药汇编》《彝药志》等记载青阳参的干燥根茎可用于风湿痹痛、肾虚腰痛和腰肌劳损、跌扑闪挫、食积、脘腹胀痛、小儿疳积、蛇犬咬伤等。现代药理研究发现，青阳参具有抗惊厥，抗癫痫，抗抑郁，镇静、镇痛，及免疫调节、抗肝炎、抗美尼尔综合征等作用。
化学成分研究表明，青阳参中富含的C21甾体皂苷类化合物，是其代表性成分及主要药效成分。但大部分的C21皂苷都被嵌入到糖苷中，分离难度大，因此只有少数类型的游离C21-甾体苷元被发现。近年来研究表明，C21甾体皂苷类化合物具有抗肝纤维化活性、抗癫痫活性；对多种人类肿瘤细胞系有较强的抗增殖活性。但关于C21甾体皂苷类化合物的神经保护活性及抗β淀粉样蛋白活性尚未见报道。
因此，研究具有对海马神经元毒性损伤有神经保护活性及抗淀粉样蛋白活性的化合物，在预防及治疗阿尔茨海默病、帕金森症、亨廷顿舞蹈病等神经退行性疾病有重要的临床意义。
发明内容
本发明的目的在于解决现有技术中针对神经退行性疾病治疗所存在的问题，从而提供了一种用于治疗神经退行性疾病的药物组合物，所述药物组合物能够显著降低β-淀粉样前体蛋白APP的表达，对神经元细胞的增殖具有明显的促进作用，同时能够抑制谷氨酸诱导的细胞毒性，对神经元细胞具有神经保护活性。
为达到上述目的，本发明是通过如下手段得以实现的：
本发明第一方面提供了一种预防和/或治疗神经退行性疾病的药物组合物，包含C21甾体皂苷、其药学上可接受的盐、其溶剂化物中的一种或多种，以及药学上可接受的载体，所述C21甾体皂苷具有如下式I所示结构：
Figure PCTCN2022125749-appb-000001
其中，R 1选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基、
Figure PCTCN2022125749-appb-000002
中的一种或多种；
R 2选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基中的一种或多种；
R 3选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基中的一种或多种；
R 4选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基、
Figure PCTCN2022125749-appb-000003
Figure PCTCN2022125749-appb-000004
中的一种或多种；
式I结构化合物多元环内各相邻碳原子之间通过单键、双键或三键进行连接。
作为优选地，所述神经退行性疾病包括老年痴呆症、帕金森病、亨廷顿舞蹈症中的一种或多种。
作为优选地，所述老年痴呆症包括阿尔茨海默病、血管性痴呆病、路易体痴呆症以及额颞痴呆症中的一种或多种。
作为优选地，所述药学上可接受的载体包括填充剂、粘合剂、崩解剂、溶剂、防腐剂、润滑剂、矫味剂中的一种或多种。
作为优选地，所述C21甾体皂苷选自如下结构所示化合物1-5中的一种或多种：
Figure PCTCN2022125749-appb-000005
本发明第二方面提供了C21甾体皂苷、其药学上可接受的盐或其溶剂化物中的一种或多种用于制备预防和/或治疗神经退行性疾病的产品中的应用，所述C21甾体皂苷具有如下式I所示结构：
Figure PCTCN2022125749-appb-000006
其中，R 1选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基、
Figure PCTCN2022125749-appb-000007
中的一种或多种；
R 2选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基中的一种或多种；
R 3选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基中的一种或多种；
R 4选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基、
Figure PCTCN2022125749-appb-000008
Figure PCTCN2022125749-appb-000009
中的一种或多种；
式I结构化合物多元环内各相邻碳原子之间通过单键、双键或三键进行连接。
作为优选地，所述神经退行性疾病包括老年痴呆症、帕金森病、亨廷顿舞蹈症中的一种或多种。
作为优选地，所述老年痴呆症包括阿尔茨海默病、血管性痴呆病、路易体痴呆症以及额颞痴呆症中的一种或多种。
作为优选地，所述C21甾体皂苷选自如下结构所示化合物1-5中的一种或多种：
Figure PCTCN2022125749-appb-000010
Figure PCTCN2022125749-appb-000011
作为优选地，所述产品包括药物、保健品、食品中的一种或多种。
关于Aβ的产生，现有研究表明：抑制β-分泌酶(BACE1)和γ-分泌酶活性可使Aβ产生量降低。但是γ-分泌酶除了参与APP的降解外还参与机体许多其他必须蛋白质的代谢，如：Notch、CD44、E-/N-/P-钙粘蛋白和低密度脂蛋白受体相关蛋白(LRP)，故阻断γ-分泌酶活性可能会引起其他意想不到的严重副作用。如敲除早老素1(PS1)基因(γ-分泌酶复合体的核心成分)导致胚胎期小鼠的死亡。因而在本发明中考察了C21甾体皂苷对BACE1蛋白的作用。
关于Aβ的清除，细胞外Aβ的降解主要由胰岛素降解酶(IDE)和脑啡肽酶(NEP)完成，而细胞内Aβ的降解主要在溶酶体进行。生理条件下，神经元溶酶体内Aβ含量极少，而在病理条件下，神经元溶酶体内Aβ的含量显著增加。AD进程伴随溶酶体系统失调，Aβ在溶酶体内的聚集是AD的病理特征之一。
最新的研究表明：AD中自噬-溶酶体系统的缺陷可能早于Aβ或神经纤维缠结(NFTs)形成，导致清除废弃蛋白质或细胞器的功能受损进而加重AD的病理进程。自噬是在囊泡和溶酶体共同介导作用下的一种降解途径，对蛋白质稳态和细胞环境非常重要。在AD的早期阶段，自噬在Aβ的产生和代谢中起重要作用，虽然Aβ的细胞外聚集体(淀粉样斑块)和神经元内Tau蛋白异常磷酸化是明显的病理标志，但自噬-溶酶体途径中的缺陷可能早于这些病理标志的形成。
自噬障碍是Aβ过度蓄积的重要机制：在正常生理状态下，细胞内产生的微量Aβ可通过抑制mTOR激活自噬，由于自噬体形成于轴突周围，而溶酶体主要位于细胞核周围，自噬体通过轴突的微管系统逆向运输至胞体，与溶酶体结合后降解Aβ，Aβ产生与降解保持平衡；在病理状态下，一方面，AD患者脑内自噬激活，自噬体增多聚集，而集聚的自噬体中富含APP、Aβ以及β-和γ-分泌酶复合体，因而自噬体的异常增多被认为是Aβ产生的源头。
另一方面，由于自噬流不通畅或溶酶体的降解功能缺陷等因素，导致自噬降解障碍，Aβ清除减少。因此，调控自噬加速Aβ的清除可能成为AD治疗的重要靶点，因而在本发明中进一步考察了C21甾体皂苷对自噬标志性蛋白LC3B、P62、Beclin 1的作用。
LC3B是第一个被发现的自噬体标记蛋白，有LC3B-Ⅰ和LC3B-Ⅱ两种形式。未发生自噬时，细胞内合成的LC3B经过加工，成为胞质可溶性Ⅰ型LC3B，常规表达。当自噬体吞噬发生时，Ⅰ型LC3B转化为Ⅱ型LC3B并定位于胞内自噬体膜上，其含量与自噬泡数量成正比。因此，LC3B-Ⅱ常被作为细胞内自噬的标记物，检测LC3B的转换，即LC3B-Ⅱ/LC3B-Ⅰ比值，比值上升代表自噬水平活跃，比值下降代表自噬水平降低。
Beclin 1是自噬的特异性基因，在自噬形成中起着关键调控作用，其上调可刺激自噬的发生。Beclin 1是自噬和凋亡发生联系的重要分子，其对凋亡的调控主要通过与Bcl-2相互结合发挥抑制过度的细胞自噬。
P62是一种常见的自噬溶酶体底物，其含量与自噬水平呈负相关，是连接LC3与待降解泛素化底物的重要桥梁。自噬流活化时，由P62形成的蛋白聚合物可被自噬体降解，P62结合自噬体膜蛋白LC3/ATG8，从而将包含P62的蛋白聚合物转运到自噬体。
本发明相对于现有技术具有如下有益效果：
(1)本发明所提供的药物组合物中，其活性成分C21甾体皂苷一方面对过表达人源APP695的N2a细胞的APP蛋白有显著下调作用，能增加过量Aβ淀粉样蛋白的清除率；另一方面促进神经元细胞的增殖，对Aβ淀粉样蛋白过量累积导致的神经递质如谷氨酸诱导的神经元细胞毒性具有保护作用，从而综合发挥治疗神经退行性疾病的作用。因而在制备Aβ淀粉样蛋白形成抑制剂及神经保护剂，预防或治疗阿尔茨海默病、帕金森症、亨廷顿舞蹈病等神经退行性疾病的药物中具有良好的应用前景。
(2)本发明的活性成分能够抑制谷氨酸诱导的细胞毒性，对神经元细胞具有神经保护活性，从而可以显著降低因抑制分泌酶活性而导致的严重毒副作用，安全性更高。
附图说明
图1为20-O-VK对HT22细胞增殖促进实验结果示意图。
图2为20-O-VK对谷氨酸诱导的HT22海马神经元细胞损伤的神经保护作用结果示意图。
图3为20-O-VK对谷氨酸诱导的HT22海马神经元细胞损伤实验细胞形态图。
图4为20-O-VK对谷氨酸诱导的HT22细胞凋亡抑制实验流式结果示意图。
图5为20-O-VK对谷氨酸诱导的HT22细胞凋亡抑制实验活细胞百分比结果示意图。
图6为20-O-VK对谷氨酸诱导的HT22细胞凋亡抑制实验早期凋亡细胞百分比结果示意图。
图7为20-O-VK对N2a-APP695细胞的生存率影响结果示意图。
图8为20-O-VK对N2a-APP695细胞中full-APP和CTF蛋白的表达影响WB结果示意图。
图9为20-O-VK对N2a-APP695细胞中full-APP表达定量分析结果示意图。
图10为20-O-VK对N2a-APP695细胞中CTF表达定量分析结果示意图。
图11为20-O-VK对N2a-APP695细胞中BACE1蛋白的表达影响WB结果示意图。
图12为20-O-VK对N2a-APP695细胞中BACE1表达定量分析结果示意图。
图13为20-O-VK对N2a-APP695细胞中LC3B-I和LC3B-II蛋白的表达影响WB结果示意图。
图14为20-O-VK对N2a-APP695细胞中LC3B表达定量分析结果示意图。
图15为20-O-VK对N2a-APP695细胞中APP-full和CTF蛋白的表达影响WB结果示意图。
图16为20-O-VK对N2a-APP695细胞中APP-full表达定量分析结果示意图。
图17为20-O-VK对N2a-APP695细胞中CTF表达定量分析结果示意图。
图18为20-O-VK对N2a-APP695细胞中P62蛋白的表达影响WB结果示意图。
图19为20-O-VK对N2a-APP695细胞中P62表达定量分析结果示意图。
图20为20-O-VK对N2a-APP695细胞中Beclin 1蛋白的表达影响WB结果示意图。
图21为20-O-VK对N2a-APP695细胞中Beclin 1表达定量分析结果示意图。
具体实施方式
为使本发明的目的、技术方案及效果更加清楚、明确，以下参照实施例对本发明作进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
在无特别说明的情况下，本发明上下文中所列出的化合物1-5等均采用现有技术中常规方法从青阳参中进行提取，并利用 1H NMR和/或 13C NMR进行结构鉴定。本发明上下文中所使用的包括HT22、N2a等细胞系均根据ATCC指南进行培养。所有细胞系均通过中国典型培养物保藏中心(武汉)的短串联重复分析鉴定，并使用PCR检测试剂盒(上海Biothrive Sci)验证是否存在支原体污染，同时在液氮中冷冻保存并用于后续实验。本发明所使用的试剂、耗材等，均通过市售获得或按常规方法配置而得。本发明所使用的实验方法，例如细胞培养、细胞增殖实验、细胞凋亡实验、流式细胞术、Western Blot实验等均为本领域的常规方法和技术。本发明所实用的仪器设备均为市售获得，其中酶标仪为美国BioTEK Synergy H1Hybrid Multi-Mode Reader；流式细胞仪为美国BECKMAN CONLTER，CytoFLEX S。
生物学实验重复中选择具有代表性的结果呈现在上下文附图中，数据按照图示中规定的以mean±SD展示。所有实验至少重复三次。数据采用GraphPad Prism 5.0进行分析。采用t检验或方差分析比较两组或两组以上的平均值差异。p＜0.05被认为是一个显著的差异。
实施例1 C21甾体皂苷的提取和分离
(1)取15kg干燥青阳参根粉末在90℃回流条件下用95％EtOH-H 2O(4×30L)萃取2h，获得粗提物2.25kg；
(2)将粗提物溶于含5％HCl的MeOH/H 2O(2:1，30L)溶液中回流3h，采用10％NaOH溶液缓慢调节反应溶液pH至7.0；随后真空除去有机溶剂后，将残余物溶于H 2O中并用EtOAc(3×30L)得到可溶于EtOAc的组分(1.48kg)；
(3)将步骤(2)获得的可溶于EtOAc的部分进行硅胶柱层析(CC)，用CH 2Cl 2-MeOH(梯度为95:1→80:1→60:1→30:1→15:1→10:1→5:1→2:1，v/v，每个梯度5个柱体积)进行洗脱，获得化合物1-5。
采用 1H NMR和/或 13C NMR对化合物1-5进行鉴定，结果如下所示。
化合物1： 1H NMR(300MHz,CD 3OD):δ H 2.12(3H,d(1.2),H-7'),1.08(3H,d(6.8),H-6'),1.08(3H,d(6.8),H-5'),2.38(1H,m,H-4'),5.55(1H,s,H-2'),2.17(3H,s,H-21),1.16(3H,s,H-19),1.57(3H,s,H-18),3.20(1H,t(8.5),H-17),1.66(1H,m,H-16),2.17(1H,m,H-16),1.65(1H,m,H-15),1.92(1H,m,H-15),4.61(1H,dd(11.5,4.2),H-12),1.69(1H,m,H-11),1.86(1H,m,H-11),1.52(1H,m,H-9),2.20(2H,m,H-7),5.33(1H,br s,H-6),2.28(2H,d(7.3),H-4),3.45(1H,m,H-3),1.56(1H,m,H-2),1.78(1H,m,H-2),1.14(1H,m,H-1),1.85(1H,m,H-1)。 13C NMR(75MHz,CD 3OD):δ c 167.4(C-1'),114.4(C-2'),167.4(C-3'),39.8(C-4'),21.3(C-5'),21.3(C-6'),16.7(C-7'),39.3(C-1),31.7(C-2),72.6(C-3),42.8(C-4),140.9(C-5),119.2(C-6),35.5(C-7),75.2(C-8),45.5(C-9),38.1(C-10),25.5(C-11),73.0(C-12),56.6(C-13),88.4(C-14),34.5(C-15),22.2(C-16),61.3(C-17),15.8(C-18),18.7(C-19),212.6(C-20),32.3(C-21)。
化合物2： 1H NMR(600MHz,CD 3OD):δ H 7.34(1H,m e,H-9'),7.53(1H,m e, H-8'),7.35(1H,m,H-7'),7.53(1H,m e,H-6'),7.34(1H,m e,H-5'),7.06(1H,d(12.1),H-3'),5.84(1H,d(12.1),H-2'),2.15(3H,s,H-21),1.18(3H,s,H-18),1.67(1H,m,H-16),2.85(1H,m,H-16),1.88(1H,m,H-15),1.98(1H,m,H-15),4.53(1H,dd(11.5,4.2),H-12),1.73(1H,m,H-11),1.84(1H,m,H-11),1.50(1H,m,H-9),2.14(2H,m,H-7),5.32(1H,br s,H-6),2.29(2H,d(7.3),H-4),3.45(1H,m,H-3),1.57(1H,m,H-2),1.79(1H,m,H-2),1.12(1H,m,H-1),1.85(1H,m,H-1)。 13C NMR(150MHz,CD 3OD):δ c 166.8(C-1'),120.8(C-2'),144.5(C-3'),136.6(C-4'),129.0(C-5'),130.6(C-6'),129.9(C-7'),130.6(C-8'),129.0(C-9'),39.8(C-1),31.7(C-2),72.6(C-3),42.8(C-4),140.7(C-5),119.2(C-6),35.1(C-7),74.9(C-8),45.2(C-9),38.0(C-10),25.0(C-11),74.4(C-12),58.5(C-13),92.9(C-14),34.1(C-15),33.2(C-16),89.9(C-17),10.1(C-18),18.6(C-19),212.0(C-20),27.5(C-21)。
化合物3： 1H NMR(300MHz,CD 3OD):δ H 7.63(1H,dd(8.0,2.0),H-7”),6.92(1H,d(8.0),H-6”),3.89(3H,s,H-4”-OMe),7.63(1H,d(2.0),H-3”),7.16(1H,m e,H-9'),6.85(1H,m e,H-8'),7.19(1H,m,H-7'),6.85(1H,m e,H-6'),7.16(1H,m e,H-5'),6.71(1H,d(12.1),H-3'),5.69(1H,d(12.1),H-2'),1.29(3H,d(6.0),H-21),4.64(1H,dd(12.6,6.0),H-20),1.17(3H,s,H-19),1.13(3H,s,H-18),1.89(2H,m,H-16),1.91(2H,m,H-15),4.71(1H,dd(11.5,4.2),H-12),1.62(1H,m,H-11),1.85(1H,m,H-11),1.47(1H,m,H-9),2.12(2H,m,H-7),5.32(1H,br s,H-6),2.28(2H,d(7.5),H-4),3.44(1H,m,H-3),1.58(1H,m,H-2),1.80(1H,m,H-2),1.10(1H,m,H-1),1.86(1H,m,H-1)。 13C NMR(150MHz,CD 3OD):δ c 167(C-1”),123.4(C-2”),114.5(C-3”),148.7(C-4”),56.5(C-4”-OMe),152.8(C-5”),115.9(C-6”),125.8(C-7”),168.2(C-1'),122.8(C-2'),142.5(C-3'),136.8(C-4'),128.6(C-5'),130.0(C-6'),129.3(C-7'),130.0(C-8'),128.6(C-9'),39.8(C-1),31.7(C-2),72.6(C-3),42.8(C-4),140.5(C-5),119.4(C-6),35.1(C-7),74.9(C-8),44.7(C-9),37.9(C-10),25.6(C-11),75.6(C-12),57.6(C-13),89.5(C-14),34.3(C-15),33.9(C-16),88.5(C-17),10.8(C-18),18.7(C-19),75.8(C-20),15.2(C-21)。
化合物4： 1H NMR(400MHz,CD 3OD):δ H 7.51(1H,d,J＝7.6Hz,H-7”),6.74(1H,d,J＝8.3Hz,H-6”),6.08(1H,d,J＝15.9Hz,H-2'),4.70(1H,q,J＝5.8Hz,H-20),3.67(1H,br s,H-3),3.57(3H,s,OCH 3-4”),1,61(3H,s,CH 3-18),1,31(3H,d,J＝6.1Hz CH 3-21),1.07(3H,s,CH 3-19)。 13C NMR(100MHz,CD 3OD):δ c 168.0(C-1'),167.1(C-1”),152.7(C-5”),148.6(C-4”),145.3(C-3'),135.5(C-4'),131.3(C-7'),129.8(C-6'),129.8(C-6'),129.2(C-5'),129.1(C-9'),125.3(C-7”),123.0(C-2”),119.9(C-2'),115.8(C-6”),113.9(C-3”),89.0(C-14),88.2(C-17),76.7(C-20),75.9(C-8),74.8(C-12),69.6(C-3),66.6(C-6),65.4(C-5),57.7(C-13),56.1(OMe-4”),45.1(C-9),42.3(C-4),39.0(C-1),36.8(C-10),35.0(C-7),32.7(C-15),31.5(C-2),31.0(C-16),26.6(C-11),17.8(C-19),15.3(C-21),11.2(C-18).ESI-MS m/z 679.5[M+H] +。
化合物5： 1H NMR(400MHz,CD 3OD):δ H 7.53(1H,d,J＝7.6Hz,H-7”),6.73(1H,d,J＝8.3Hz,H-6”),6.08(1H,d,J＝15.9Hz,H-2'),4.73(1H,q,J＝5.8Hz,H-20),3.63(1H,br s,H-3),3.55(3H,s,OCH 3-4”),1,64(3H,s,CH 3-18),1,31(3H,d,J＝6.1Hz CH 3-21),1.27(3H,s,CH 3-19). 13C NMR(100MHz,CD 3OD):δ c 168.0(C-1'),167.0(C-1”),152.7(C-5”),148.6(C-4”),145.2(C-3'),135.6(C-4'),131.3(C-7'),129.8(C-6'),129.8(C-6'),129.1(C-5'),129.1(C-9'),125.3(C-7”),123.0(C-2”),119.9(C-2'),115.8(C-6”),113.9(C-3”),89.5(C-14),88.5(C-17),78.8(C-8),75.7(C-5),76.0(C-20),75.9(C-12),77.9(C-6),68.1(C-3),58.2(C-13),56.1(OMe-4”),41.0(C-9),40.0(C-4),39.3(C-1),34.7(C-10),34.4(C-16),34.3(C-15),32.9(C-7),30.9(C-2),25.0(C-11),18.2(C-19),15.4(C-21),11.6(C-18).ESI-MS m/z 697.4[M+H] +。
实施例2 C21甾体皂苷对海马神经元细胞增殖促进实验
(1)取对数生长期的HT22细胞，将细胞以5000个/孔的密度种于96孔板，每组分别设置3个复孔；
(2)培养24h后，加入不同浓度化合物继续培养48h；其中组1-组6分别加入浓度为0.01μM、0.1μM、0.5μM、1μM、5μM、10μM的化合物3(20-O-VK)；C组为对照组，加入等体积的培养基；
(3)48h后去除含有化合物的培养基后，每孔加入含10μL的CCK-8的DMEM培养基；
(4)于5％CO 2，37℃培养箱，孵育2h；
(5)在室温下振荡15s，用酶标仪450nm处测定各孔的吸光度(OD)。
检测结果如图1所示。结果显示，与未经C21甾体皂苷处理的C组相比，C21甾体皂苷处理组随着浓度的增加(1，5，10μM)，细胞增殖活力明显提升，与C组的差异具有统计学意义(**P<0.01，***P<0.001)，说明C21甾体皂苷具有显著的促HT22细胞增殖活性。
实施例3 C21甾体皂苷对谷氨酸诱导的海马神经元损伤的神经保护作用
(1)取对数生长期的HT22细胞，将细胞以5000个/孔的密度种于96孔板，每组分别设置3个复孔；
(2)培养24h后，加入不同浓度化合物继续培养24h；其中组1-组5中分别加入浓度为0.1μM、0.5μM、1μM、5μM、10μM的化合物3；NC组为空白组，加入等体积的培养基；C组为对照组，加入等体积的培养基；
(3)24h后于组1-组5、C组中分别加入10mM谷氨酸，NC组不作任何处理，继续培养24h；
(4)24h后去除含有化合物的培养基后，每孔加入含10μL的CCK-8的DMEM培养基；
(5)于5％CO 2，37℃培养箱，孵育2h；
(6)在室温下振荡15s，用酶标仪(450nm)测定各孔的吸光度(OD)。
检测结果如图2-3所示。结果显示，通过加入5mM谷氨酸构建海马神经元损伤模型，经过C21甾体皂苷(0.5，1，5，10μM)给药处理后，细胞损伤率明显下降，说明C21甾体皂苷对谷氨酸诱导的海马神经元损伤有显著的神经保护活性，与C组的差异具有统计学意义(*P<0.05，***P<0.001)。
实施例4 C21甾体皂苷对谷氨酸诱导的海马神经元凋亡抑制实验
(1)取对数生长期的HT22细胞，将细胞以10 5个/孔的密度种于6孔板；
(2)培养24h后，加入不同浓度化合物继续培养12h；其中组1-组5中分别加入浓度为0.1μM、0.5μM、1μM、5μM、10μM的化合物3(20-O-VK)；NC组为空白组，加入等体积的培养基；C组为对照组，加入等体积的培养基；
(3)12h后于组1-组5、C组中分别加入10mM谷氨酸，NC组不作任何处理，继续培养24h；
(4)24h后，使用FITC-annexin V/PI双染细胞凋亡检测试剂盒(Dead Cell Apoptosis Kit with Annexin V Alexa Fluor TM 488 & Propidium Iodide(PI)，Invitrogen,USA)，使用Beckman流式细胞仪(CytoFLEX S型)按照常规流式凋亡检测。
检测结果如图4-6所示。结果显示，通过加入谷氨酸构建海马神经元损伤模型，经过C21甾体皂苷(5，10μM)给药处理后，通过流式检测仪检测发现细胞存活率明显上升，细胞凋亡率明显下降，说明C21甾体皂苷对谷氨酸诱导的海马神经元凋亡具有显著的保护活性，可抑制海马神经元细胞的凋亡，与C组的差异具有统计学意义(*P<0.05，**P<0.01，***P<0.001)。
实施例5 C21甾体皂苷对神经母细胞瘤细胞增殖影响实验
(1)取对数生长期的N2a-APP695细胞，将细胞以5000个/孔的密度种于96孔板，每组分别设置6个复孔；
(2)培养24h后，加入不同浓度化合物继续培养24h；其中组1-组5中分别加入浓度为0.1μM，1μM，2.5μM，5μM，10μM的化合物3(20-O-VK)；C组为对照组，加入等体积的培养基；
(3)24h后去除含有化合物的培养基后，每孔加入含10μL的CCK-8的DMEM培养基；
(4)于5％CO 2，37℃培养箱，孵育2h；
(5)在室温下振荡15s，用酶标仪450nm处测定各孔的吸光度(OD)。
本实施例中稳定表达人源淀粉样前体蛋白APP695基因的小鼠神经母细胞瘤细胞N2a(mouse Neuroblastoma-2a)，可稳定生成Aβ淀粉样蛋白，为体外AD筛选模型，由美国ATCC提供。
检测结果如图7所示。结果显示，C21甾体皂苷对N2a-APP695细胞的增殖活力没有显著影响。用CCK-8法测定了经C21甾体皂苷处理和未经药物处理的后N2a-APP695细胞活力，经过24h处理后，对照组和C21甾体皂苷处理组之间的细胞活力没有显著性差异(p>0.05)。这些结果表明，C21甾体皂苷对N2a-APP695细胞并没有明显的细胞毒性。
实施例6 C21甾体皂苷对神经母细胞瘤细胞相关蛋白表达的影响
(1)取对数生长期的N2a-APP695细胞，将细胞以20 5个/孔的密度种于6孔板中；
(2)培养24h后，加入不同浓度化合物继续培养相应时间；其中组1-组3中分别加入浓度为1μM、5μM、10μM的化合物3(20-O-VK)；C组为对照组，加入等体积的培养基；
(3)收集细胞，提取蛋白，进行Western blot实验；所用抗体分别为：beta Amyloid Polyclonal Antibody(CT695,Invitrogen,USA)、BACE1(#5606,CST,USA)、SQSTM1/p62(#5114S,CST,USA)、LC3B(#2775S,CST,USA)、Beclin 1(#3495T,CST,USA)及使用GAPDH(#5174S,CST,USA)。
首先检测C21甾体皂苷对full-APP及CTF的影响，结果如图8-10所示。结果显示，C21甾体皂苷对N2a-APP695细胞中CTF蛋白表达有抑制作用而不影响full-APP蛋白的表达。运用western blot法测定经C21甾体皂苷处理和未经药物处理的后N2a-APP695中full-APP和CTF蛋白的表达。经过24h处理，与对照组相比，5、10μM C21甾体皂苷处理组中的CTF蛋白表达量显著降低(p<0.01)而对full-APP蛋白无显著性影响(p>0.05)。这些结果表明，C21甾体皂苷在1、5、10μM浓度下可降低CTF蛋白的表达且呈浓度依赖性。
进一步地，对N2a-APP695细胞中BACE1蛋白的表达进行检测，结果显示(图11-12)，在C21甾体皂苷处理后N2a-APP695细胞24h后，BACE1蛋白表达未发生显著性变化(p>0.05)。这些结果表明，C21甾体皂苷可能不会通过阻断CTF生成的上游蛋白BACE1来降低CTF的表达量。
为了明确C21甾体皂苷对N2a-APP695细胞中抑制CTF蛋白抑的作用机制。运用Western blot法测定经C21甾体皂苷处理3、6、12、24h时，N2a-APP695细胞中自噬相关蛋白LC3BⅡ、自噬底物蛋白P62蛋白以及Beclin 1的表达，同时考察N2a-APP695细胞经C21甾体皂苷处理3、6、12、24h时，细胞中APP-full、CTF蛋白的表达。
结果如图13-17所示。LSD-t检验两两比较结果显示，与对照组3、6h相比，10μM20-O-VK组中LC3BⅡ/LC3BⅠ的蛋白表达明显增高(p<0.05)，提示给药3、6h时，20-O-VK对N2a-APP695细胞中CTF的清除可能通过增强自噬实现的(参见图13-14)。20-O-VK(10μM)给药3、6、12、24h后，APP-full蛋白无显著变化。给药后3、6、12h时，CTF蛋白有降低趋势且呈剂量依赖性且给药24h时，CTF蛋白显著降低(p<0.05)然而，给药24h时，LC3BⅡ/LC3BⅠ有降低趋势(参见图15-17)，说明在24小时CTF蛋白降低后，其自噬水平下降，提示CTF蛋白的下降可能与细胞内自噬相关。
为了进一步明确给药后N2a-APP695细胞内CTF蛋白的降低与自噬相关，考察了自噬相关蛋白P62和Beclin 1蛋白的表达，结果如图18-21所示。结果显示，自噬溶酶体底物 P62蛋白在给药后6h有降低趋势，给药后24h显著提升，而蛋白Beclin1给药后前3、6、12h都有提高趋势而给药24h，Beclin 1蛋白表达有降低趋势。自噬相关蛋白LC3BⅡ/LC3BⅠ、P62和Beclin 1的动态变化进一步说明20-O-VK降低N2a-APP695细胞中CTF蛋白可能是通过调控细胞自噬溶酶体来实现的。
综上可知，本发明提所提供的药物组合物中，其活性成分C21甾体皂苷一方面能够显著降低N2a-APP695细胞中β-淀粉样蛋白的生成，增加过量Aβ淀粉样蛋白的清除率；另一方面促进神经元细胞的增殖，对Aβ淀粉样蛋白过量累积导致的神经递质如谷氨酸诱导的神经元细胞毒性具有保护作用，从而综合发挥治疗神经退行性疾病的作用。因而在制备Aβ淀粉样蛋白形成抑制剂及神经保护剂，预防或治疗阿尔茨海默病、帕金森症、亨廷顿舞蹈病等神经退行性疾病的药物中具有良好的应用前景。
以上具体实施方式部分对本发明所涉及的分析方法进行了具体的介绍。应当注意的是，上述介绍仅是为了帮助本领域技术人员更好地理解本发明的方法及思路，而不是对相关内容的限制。在不脱离本发明原理的情况下，本领域技术人员还可以对本发明进行适当的调整或修改，上述调整和修改也应当属于本发明的保护范围。

Claims (10)

  1. 一种治疗预防和/或治疗神经退行性疾病的药物组合物，包含C21甾体皂苷、其药学上可接受的盐、其溶剂化物中的一种或多种，以及药学上可接受的载体，其特征在于，所述C21甾体皂苷具有如下式I所示结构：
    Figure PCTCN2022125749-appb-100001
    其中，R 1选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基、
    Figure PCTCN2022125749-appb-100002
    中的一种或多种；
    R 2选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基中的一种或多种；
    R 3选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基中的一种或多种；
    R 4选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基、
    Figure PCTCN2022125749-appb-100003
    Figure PCTCN2022125749-appb-100004
    中的一种或多种；
    式I结构化合物多元环内各相邻碳原子之间通过单键、双键或三键进行连接。
  2. 根据权利要求1所述的药物组合物，其特征在于，所述神经退行性疾病包括老年痴呆症、帕金森病、亨廷顿舞蹈症中的一种或多种。
  3. 根据权利要求2所述的药物组合物，其特征在于，所述老年痴呆症包括阿尔茨海默病、血管性痴呆病、路易体痴呆症以及额颞痴呆症中的一种或多种。
  4. 根据权利要求1所述的药物组合物，其特征在于，所述药学上可接受的载体包括填充剂、粘合剂、崩解剂、溶剂、防腐剂、润滑剂、矫味剂中的一种或多种。
  5. 根据权利要求1-4任一项所述的药物组合物，其特征在于，所述C21甾体皂苷选自如下结构所示化合物1-5中的一种或多种：
    Figure PCTCN2022125749-appb-100005
  6. C21甾体皂苷、其药学上可接受的盐或其溶剂化物中的一种或多种用于制备预防和/或治疗神经退行性疾病的产品中的应用，其特征在于，所述C21甾体皂苷具有如下式I所示结构：
    Figure PCTCN2022125749-appb-100006
    其中，R 1选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基、
    Figure PCTCN2022125749-appb-100007
    中的一种或多种；
    R 2选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基中的一种或多种；
    R 3选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基中的一种或多种；
    R 4选自H、O、羟基、任选取代的C 1-C 4烷基、任选取代的C 1-C 4烷氧基、
    Figure PCTCN2022125749-appb-100008
    Figure PCTCN2022125749-appb-100009
    中的一种或多种；
    式I结构化合物多元环内各相邻碳原子之间通过单键、双键或三键进行连接。
  7. 根据权利要求6所述的应用，其特征在于，所述神经退行性疾病包括老年痴呆症、帕金森病、亨廷顿舞蹈症中的一种或多种。
  8. 根据权利要求7所述的应用，其特征在于，所述老年痴呆症包括阿尔茨海默病、血管性痴呆病、路易体痴呆症以及额颞痴呆症中的一种或多种。
  9. 根据权利要求6所述的应用，其特征在于，所述产品包括药物、保健品、食品中的一种或多种。
  10. 根据权利要求6-9任一项所述的应用，其特征在于，所述C21甾体皂苷选自如下结构所示化合物1-5中的一种或多种：
    Figure PCTCN2022125749-appb-100010
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