技术领域Technical Field
本发明涉及医药技术领域，本发明涉及式(I)所示的甲基麦冬黄酮A的新医药用途，其具体为甲基麦冬黄酮A在制备治疗和/或预防心血管疾病的药物中的用途。The present invention relates to the field of medical technology, and in particular to a new medical use of methyl ophiopogon japonicus flavonoids A shown in formula (I), specifically to the use of methyl ophiopogon japonicus flavonoids A in the preparation of drugs for treating and/or preventing cardiovascular diseases.
背景技术Background Art
心血管疾病是我国导致居民死亡的第一病因，高于肿瘤及其他疾病，且患病率处于持续上升态势。根据《中国心血管健康与疾病报告2020》统计，我国现有心血管病患者人数达3.3亿，占总人口的约23％，其中冠心病1100万，心力衰竭890万，同时发病年龄逐渐年轻化。Cardiovascular disease is the leading cause of death among Chinese residents, higher than tumors and other diseases, and its prevalence rate is on a continuous rise. According to the "China Cardiovascular Health and Disease Report 2020", there are 330 million cardiovascular disease patients in my country, accounting for about 23% of the total population, including 11 million with coronary heart disease and 8.9 million with heart failure. At the same time, the age of onset is gradually getting younger.
近40年来，心血管疾病治疗药物有了很大进展，包括β受体阻滞剂、钙通道阻滞剂、利尿剂、肾素-血管紧张素-醛固酮系统药物、调脂药、血管扩张剂、窦房结通道阻滞剂、改善心肌代谢药及丹参片、速效救心丸、银杏叶制剂等中成药。但近年来，该领域治疗药物研发遇到瓶颈，除了在老药新用(如恩格列净)和少数的新作用靶点候选药物(Vericiguat和Omecamtiv Mecarbil)取得一定进展，整体研发明显落后于肿瘤药物和自身免疫性疾病药物，现有药物不能满足临床治疗的需要，迫切需要开发新的药物疗法。In the past 40 years, great progress has been made in the treatment of cardiovascular diseases, including beta-blockers, calcium channel blockers, diuretics, renin-angiotensin-aldosterone system drugs, lipid-regulating drugs, vasodilators, sinoatrial node channel blockers, drugs to improve myocardial metabolism, and Chinese patent medicines such as Danshen tablets, Suxiao Jiuxin pills, and Ginkgo biloba preparations. However, in recent years, the research and development of therapeutic drugs in this field has encountered bottlenecks. In addition to some progress in new uses of old drugs (such as empagliflozin) and a few new target candidate drugs (Vericiguat and Omecamtiv Mecarbil), the overall research and development is significantly behind that of tumor drugs and autoimmune disease drugs. Existing drugs cannot meet the needs of clinical treatment, and there is an urgent need to develop new drug therapies.
以中药与天然产物为资源发现和研发创新药物，一直是新药创制的重要途径，中国原创并得到国际认可的创新药物如青蒿素、石杉碱甲、双环醇、丁苯酞等都来源于中药。甲基麦冬黄酮A是中药麦冬中含有的成分，有研究显示其具有抗非酒精性脂肪肝及保肝活性，对Nrf2具有激动作用，其他药理学活性研究很少。在本发明中我们提出甲基麦冬黄酮A在抗心血管疾病药物方面的应用前景。Using traditional Chinese medicine and natural products as resources to discover and develop innovative drugs has always been an important way to create new drugs. Innovative drugs that are original in China and recognized internationally, such as artemisinin, huperzine A, bicyclol, butylphthalide, etc., are all derived from traditional Chinese medicine. Methyl ophiopogon japonicus flavonoid A is a component contained in the traditional Chinese medicine Ophiopogon japonicus. Studies have shown that it has anti-nonalcoholic fatty liver and liver protection activities, has an agonistic effect on Nrf2, and other pharmacological activities are rarely studied. In the present invention, we propose the application prospects of methyl ophiopogon japonicus flavonoid A in anti-cardiovascular disease drugs.
发明内容Summary of the invention
本发明要解决的技术问题在于提供甲基麦冬黄酮A、含有其的药物组合物在制备治疗和/或预防心血管疾病的药物中的应用。其特征在于，所述的药物组合物包含甲基麦冬黄酮A或其药学上可接受的盐和药学上可接受的载体或赋形剂；所述药物组合物包括片剂、胶囊剂、颗粒剂、丸剂、滴丸剂、预乳剂、微乳剂、混悬剂、糖浆剂、肠溶制剂或注射剂；所述心血管疾病包括冠心病、心肌缺血、心力衰竭、心肌梗死、心率失常、心绞痛、心肌病、心肌肥厚或高血压。The technical problem to be solved by the present invention is to provide the use of methyl ophiopogon japonicus flavonoids A and a pharmaceutical composition containing the same in the preparation of drugs for treating and/or preventing cardiovascular diseases. It is characterized in that the pharmaceutical composition comprises methyl ophiopogon japonicus flavonoids A or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient; the pharmaceutical composition includes tablets, capsules, granules, pills, drop pills, pre-emulsions, microemulsions, suspensions, syrups, enteric-coated preparations or injections; the cardiovascular diseases include coronary heart disease, myocardial ischemia, heart failure, myocardial infarction, arrhythmia, angina pectoris, cardiomyopathy, myocardial hypertrophy or hypertension.
为解决本发明的技术问题，本发明采用如下技术方案：In order to solve the technical problem of the present invention, the present invention adopts the following technical solution:
本发明技术方案的第一方面提供了甲基麦冬黄酮A在制备治疗和/或预防心血管疾病的药物中的应用，该类化合物结构如下：The first aspect of the technical solution of the present invention provides the use of methyl ophiopogon japonicus flavonoids A in the preparation of drugs for treating and/or preventing cardiovascular diseases. The structure of the compound is as follows:
所述心血管疾病包括冠心病、心肌缺血、心力衰竭、心肌梗死、心率失常、心绞痛、心肌病、高血压。The cardiovascular diseases include coronary heart disease, myocardial ischemia, heart failure, myocardial infarction, arrhythmia, angina pectoris, cardiomyopathy, and hypertension.
为验证本发明的技术方案，本发明提供了体内、体外相关实验。In order to verify the technical solution of the present invention, the present invention provides relevant in vivo and in vitro experiments.
本发明的体外实验之一为CCK8细胞增殖与活性检测实验。CCK8细胞增殖与活性检测实验是一种基于WST-8(2-(2-甲氧基-4硝基苯)-3-(4-硝基苯)-5-(2,4-二磺基苯)-2H-四唑单钠盐)的细胞活力检测方法，WST-8在电子耦合试剂存在的情况下，可以被线粒体内的一些脱氢酶还原生成橙黄色水溶性的甲臜染料。对于同样的细胞，生成甲臜物的数量与活细胞的数量成正比，颜色的深浅和细胞数目呈线性关系。用该方法来检测H9C2心肌细胞的存活率，考察甲基麦冬黄酮A对受损心肌细胞的保护作用。One of the in vitro experiments of the present invention is a CCK8 cell proliferation and activity detection experiment. The CCK8 cell proliferation and activity detection experiment is a cell viability detection method based on WST-8 (2-(2-methoxy-4-nitrobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt). In the presence of an electron coupling agent, WST-8 can be reduced by some dehydrogenases in the mitochondria to generate an orange-yellow water-soluble formazan dye. For the same cells, the amount of formazan generated is proportional to the number of living cells, and the depth of the color is linearly related to the number of cells. This method was used to detect the survival rate of H9C2 cardiomyocytes and investigate the protective effect of methyl ophiopogon japonicus flavonoid A on damaged cardiomyocytes.
本发明的体外实验之二为心肌细胞肥大实验。心室重构是心衰的基本病理机制，其主要特征包括病理性心肌肥大，心肌细胞凋亡，心脏成纤维细胞增殖与心肌间质纤维化。心肌细胞肥大是心室重构的起点，极易导致心肌缺血、心律失常的发生，最终可发展为心力衰竭。长时间高水平的β肾上腺素能受体刺激是诱发心肌细胞肥大的重要因素。从β肾上腺素能受体角度阐明药物对心肌细胞肥大的影响对开发有效药物具有重大意义。异丙肾上腺素(Isoproterenol，ISO)是强的β受体激动剂，用ISO作用于心肌H9C2细胞，制作心肌细胞肥大模型，通过分析软件分析单个心肌细胞表面积。利用该方法考察甲基麦冬黄酮A对心肌细胞肥大的防治作用。The second in vitro experiment of the present invention is a cardiomyocyte hypertrophy experiment. Ventricular remodeling is the basic pathological mechanism of heart failure, and its main characteristics include pathological myocardial hypertrophy, myocardial cell apoptosis, cardiac fibroblast proliferation and myocardial interstitial fibrosis. Myocardial cell hypertrophy is the starting point of ventricular remodeling, which can easily lead to myocardial ischemia and arrhythmia, and can eventually develop into heart failure. Long-term high-level β-adrenergic receptor stimulation is an important factor inducing myocardial cell hypertrophy. It is of great significance to develop effective drugs to clarify the effect of drugs on myocardial cell hypertrophy from the perspective of β-adrenergic receptors. Isoproterenol (ISO) is a strong β-receptor agonist. ISO is used to act on myocardial H9C2 cells to make a myocardial cell hypertrophy model, and the surface area of a single myocardial cell is analyzed by analysis software. The method is used to investigate the preventive and therapeutic effects of methyl ophiopogon japonicus flavonoids A on myocardial cell hypertrophy.
本发明的体内实验之一为利用ISO诱导的小鼠心肌缺血模型。ISO皮下注射诱导的心肌缺血模型，是冠状动脉粥样硬化性心脏病防治研究常用的心肌缺血模型，该模型制作简单，不需要特殊设备，可根据造模剂量的不同诱导心肌缺血损伤、心肌纤维化及心力衰竭动物模型，是一种无创且简便易行的经典动物模型，被广泛应用于药理及机制研究，在药物临床前研究评估及药物筛查中具有明显的优势。ISO是一个强的β受体兴奋剂，能通过加快心率、增强心肌收缩力等环节增加心肌耗氧量，造成心脏负荷过重缺血缺氧，心肌微循环障碍，冠状动脉痉挛、心肌梗死样变化、心肌坏死、甚至猝死。这些病理损伤可能与干扰线粒体能量代谢和呼吸链功能、诱导氧化应激、诱导心肌细胞凋亡、引起心机钙超载等有关。用该模型考察甲基麦冬黄酮A对心肌缺血的防治作用。One of the in vivo experiments of the present invention is to use ISO-induced mouse myocardial ischemia model. The myocardial ischemia model induced by subcutaneous injection of ISO is a commonly used myocardial ischemia model for the prevention and treatment of coronary atherosclerotic heart disease. The model is simple to make and does not require special equipment. It can induce myocardial ischemia damage, myocardial fibrosis and heart failure animal models according to different modeling doses. It is a non-invasive and simple and easy classic animal model, which is widely used in pharmacology and mechanism research and has obvious advantages in drug preclinical research evaluation and drug screening. ISO is a strong β-receptor stimulant that can increase myocardial oxygen consumption by accelerating heart rate, enhancing myocardial contractility, etc., causing heart overload, ischemia and hypoxia, myocardial microcirculation disorders, coronary artery spasm, myocardial infarction-like changes, myocardial necrosis, and even sudden death. These pathological injuries may be related to interference with mitochondrial energy metabolism and respiratory chain function, induction of oxidative stress, induction of myocardial cell apoptosis, and causing cardiac calcium overload. This model was used to investigate the preventive and therapeutic effects of methyl ophiopogon japonicus flavonoids A on myocardial ischemia.
本发明的体内实验之二为利用阿霉素(Doxorubicin，DOX)诱导的小鼠急性心脏毒性模型。阿霉素是临床治疗多种恶性肿瘤的一线化疗药物，在儿童肿瘤患者中的作用尤为突出。但阿霉素同时可产生严重的心脏毒性，是研究急性心脏毒性、亚慢性心脏毒性(心包炎-心包炎综合症)和慢性心脏毒性(左心室收缩功能障碍和心力衰竭)的常用损伤剂。阿霉素诱导的心脏毒性机制十分复杂，线粒体是其主要靶向器官，氧化应激是关键因素。用该模型考察甲基麦冬黄酮A对阿霉素引起急性心脏毒性的防治作用。The second in vivo experiment of the present invention is to use the acute cardiotoxicity model of mice induced by doxorubicin (DOX). Doxorubicin is a first-line chemotherapy drug for the clinical treatment of various malignant tumors, and its effect in children with tumors is particularly prominent. However, doxorubicin can also produce severe cardiotoxicity, and is a commonly used damaging agent for studying acute cardiotoxicity, subchronic cardiotoxicity (pericarditis-pericarditis syndrome) and chronic cardiotoxicity (left ventricular systolic dysfunction and heart failure). The mechanism of cardiotoxicity induced by doxorubicin is very complex, mitochondria are its main target organs, and oxidative stress is a key factor. This model was used to investigate the preventive and therapeutic effects of methyl ophiopogon japonicus flavonoids A on acute cardiotoxicity caused by doxorubicin.
本发明的体内实验之三为小鼠常压缺氧模型。该模型为非特异性缺氧，为筛选抗心脑缺血药的常用方法之一，能够考察整体动物在缺氧条件下的生存能力。用常压缺氧小鼠模型考察甲基麦冬黄酮A耐心脑缺氧的作用。The third in vivo experiment of the present invention is a mouse normobaric hypoxia model. This model is non-specific hypoxia and is one of the common methods for screening anti-cardiocerebral ischemia drugs. It can examine the survival ability of whole animals under hypoxic conditions. The normobaric hypoxia mouse model was used to examine the effect of methyl ophiopogon japonicus flavonoid A on patient brain hypoxia.
本发明利用上述实验方法评价了甲基麦冬黄酮A作为抗心血管疾病的药物的活性。The present invention uses the above experimental method to evaluate the activity of methyl ophiopogon japonicus flavonoids A as a drug for resisting cardiovascular diseases.
本发明技术方案的第二方面提供一种药物组合物在制备治疗和/或预防心血管疾病药物中的应用，所述的药物组合物包括含有第一方面所述的甲基麦冬黄酮A及其药学上可接受的盐和药学上可接受的载体或赋形剂。所述心血管疾病包括冠心病、心肌缺血、心力衰竭、心肌梗死、心率失常、心绞痛、心肌病、高血压。The second aspect of the technical solution of the present invention provides a use of a pharmaceutical composition in the preparation of a drug for treating and/or preventing cardiovascular diseases, wherein the pharmaceutical composition comprises the methyl ophiopogon japonicus flavonoid A and a pharmaceutically acceptable salt thereof as described in the first aspect and a pharmaceutically acceptable carrier or excipient. The cardiovascular diseases include coronary heart disease, myocardial ischemia, heart failure, myocardial infarction, arrhythmia, angina pectoris, cardiomyopathy, and hypertension.
该药物组合物可根据本领域公知的方法制备。可通过将本化合物与一种或多种药学上可接受的盐和药学上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型。本发明化合物在其药物组合物中的含量通常为0.1-95重量％。The pharmaceutical composition can be prepared according to methods known in the art. Any dosage form suitable for human or animal use can be prepared by combining the compound with one or more pharmaceutically acceptable salts and pharmaceutically acceptable solid or liquid excipients and/or adjuvants. The content of the compound of the present invention in its pharmaceutical composition is usually 0.1-95% by weight.
本化合物或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。The compound or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括o/w型、w/o型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w types, w/o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.
本化合物可以制成普通制剂、也制成是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。The compound can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
为了将本化合物制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；湿润剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。In order to prepare the present compound into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, isopropanol, etc.; the binder may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
为了将给药单元制成胶囊剂，可以将有效成分本化合物与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分本化合物先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本化合物片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本化合物的胶囊剂。In order to prepare the dosing unit into a capsule, the active ingredient of the present compound can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard capsule or a soft capsule. The active ingredient of the present compound can also be first prepared into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard capsule or a soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare tablets of the present compound can also be used to prepare capsules of the present compound.
为将本化合物制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、pH调剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；pH调剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。To prepare the compound into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent and an appropriate amount of a solubilizer, cosolvent, pH adjuster, and osmotic pressure regulator commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a support agent.
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其它添加剂。Furthermore, if necessary, colorants, preservatives, perfumes, flavoring agents or other additives may be added to the pharmaceutical preparations.
为达到用药目的，增强治疗效果，本药物或药物组合物可用任何公知的给药方法给药。In order to achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition can be administered by any known method of administration.
本化合物药物组合物的给药剂量依照所要预防或治疗疾病的性质和严重程度，患者或动物的个体情况，给药途径和剂型等可以有大范围的变化。一般来讲，本化合物的每天的合适剂量范围为0.001-150mg/Kg体重，优选为0.1-100mg/Kg体重，更优选为1-60mg/Kg体重，最优选为2-30mg/Kg体重。上述剂量可以一个剂量单位或分成几个剂量单位给药，这取决于医生的临床经验以及包括运用其它治疗手段的给药方案。The dosage of the pharmaceutical composition of the present compound can vary widely according to the nature and severity of the disease to be prevented or treated, the individual conditions of the patient or animal, the route of administration and the dosage form. Generally speaking, the suitable dosage range of the present compound per day is 0.001-150 mg/Kg body weight, preferably 0.1-100 mg/Kg body weight, more preferably 1-60 mg/Kg body weight, and most preferably 2-30 mg/Kg body weight. The above dosage can be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the doctor and the dosage regimen including the use of other treatment means.
本化合物或组合物可单独服用，或与其他治疗药物或对症药物合并使用。当本化合物与其它治疗药物存在协同作用时，应根据实际情况调整它的剂量。有益技术效果：The compound or composition can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound and other therapeutic drugs have synergistic effects, its dosage should be adjusted according to the actual situation. Beneficial technical effects:
1，本发明提供甲基麦冬黄酮A在治疗或预防心血管疾病药物方面的新用途。1. The present invention provides a new use of methyl ophiopogon japonicus flavonoids A in treating or preventing cardiovascular diseases.
2，实验证明本发明所述的化合物在药理上具有以下方面显著活性：对异丙肾上腺素(ISO)诱导的体外心肌细胞损伤具有显著的保护活性；对心肌细胞肥大具有显著抑制活性；对ISO诱导的心肌缺血小鼠具有明确的防治作用，能够降低血清心肌损伤生物标记物cTn-T和CK-MB水平，降低心力衰竭生物标记物/心肌肥厚生物标记物BNP和ANP水平，改善左心室舒张功能，提高左心室顺应性，改善心肌肥厚；抑制阿霉素诱导的心脏急性毒性，降低血清心肌损伤生物标记物CK-MB水平；显著提高小鼠在缺氧条件下的存活时间，提高心脑耐缺氧能力。甲基麦冬黄酮A 3mg/kg活性与钙通道阻滞剂地尔硫卓20mg/kg相当，心肌肥厚小鼠左心室顺应性的改善作用和对心衰血清生物标记物的降低作用优于地尔硫卓。甲基麦冬黄酮A 1mg/kg抗耐缺氧活性优于红景天600mg/kg。2. Experiments have shown that the compounds described in the present invention have significant pharmacological activity in the following aspects: significant protective activity against in vitro myocardial cell injury induced by isoproterenol (ISO); significant inhibitory activity against myocardial cell hypertrophy; clear preventive and therapeutic effects on ISO-induced myocardial ischemia mice, can reduce serum myocardial injury biomarkers cTn-T and CK-MB levels, reduce heart failure biomarkers/cardiac hypertrophy biomarkers BNP and ANP levels, improve left ventricular diastolic function, improve left ventricular compliance, and improve myocardial hypertrophy; inhibit doxorubicin-induced acute cardiac toxicity, reduce serum myocardial injury biomarker CK-MB levels; significantly increase the survival time of mice under hypoxia, and improve the heart and brain hypoxia tolerance. The activity of 3 mg/kg of methyl ophiopogon japonicus flavonoids A is equivalent to that of 20 mg/kg of calcium channel blocker diltiazem, and its effect on improving left ventricular compliance in myocardial hypertrophy mice and reducing serum biomarkers of heart failure is better than diltiazem. The anti-hypoxia activity of 1 mg/kg of methyl ophiopogon japonicus flavonoids A is better than that of 600 mg/kg of Rhodiola rosea.
综合以上体内、体外实验结果，甲基麦冬黄酮A具有潜在的抗心血管疾病药物的开发价值。Based on the above in vivo and in vitro experimental results, Methyl Ophiopogon japonicus Flavonoids A has potential development value as an anti-cardiovascular disease drug.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1.甲基麦冬黄酮A对异丙肾上腺素(ISO)引起心肌细胞肥大损伤改善作用的照片。Figure 1. Photographs of the improving effect of methyl ophiopogon japonicus flavonoids A on cardiomyocyte hypertrophy injury induced by isoproterenol (ISO).
图2.甲基麦冬黄酮A对异丙肾上腺素(ISO)引起心肌细胞肥大损伤抑制作用的统计结果。(###P<0.001与空白对照组比较；***P<0.001与模型组比较)。Figure 2. Statistical results of the inhibitory effect of methyl ophiopogon japonicus flavonoids A on myocardial cell hypertrophy injury induced by isoproterenol (ISO). (### P<0.001 compared with the blank control group;*** P<0.001 compared with the model group).
具体实施方式DETAILED DESCRIPTION
下面的实施例及药理活性实验用于进一步说明本发明，但这并不意味着对本发明的任何限制。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。The following examples and pharmacological activity experiments are used to further illustrate the present invention, but they do not mean any limitation of the present invention. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.
下述实施例中，部分物质相应的中文名称如下：In the following examples, the corresponding Chinese names of some substances are as follows:
Control：空白对照Control: blank control
Model：模型对照Model: Model comparison
ISO：异丙肾上腺素ISO: Isoproterenol
CK-MB：肌酸激酶-同工酶CK-MB: Creatine kinase-isoenzyme
cTn-T：肌钙蛋白TcTn-T: Troponin T
BNP：脑钠肽BNP: Brain Natriuretic Peptide
ANP：心钠肽ANP: Atrial natriuretic peptide
下述实施例中所述室温如本领域常规所述，一般指15～25℃。The room temperature described in the following examples is as commonly described in the art, generally referring to 15-25°C.
药理实验：甲基麦冬黄酮A的药理活性实验Pharmacological experiment: Pharmacological activity experiment of methyl ophiopogon japonicus flavonoid A
实验例1甲基麦冬黄酮A对异丙肾上腺素(ISO)损伤的H9C2心肌细胞保护作用活性测试Experimental Example 1 Test of the protective effect of methyl ophiopogon japonicus flavonoids A on H9C2 cardiomyocytes damaged by isoproterenol (ISO)
实验方法：Experimental methods:
对数生长期的大鼠H9C2心肌细胞，将8×104个/mL细胞接种于96孔板中，每孔100μL，分为空白对照组、ISO模型组、ISO+甲基麦冬黄酮A组(购自：乐美天医药，批号DST210602-025)。培养24小时后，ISO模型组和ISO+甲基麦冬黄酮A组分别加入1.1mM ISO，ISO+甲基麦冬黄酮A组同时加入10μM甲基麦冬黄酮A，空白对照组加入等体积DMSO，继续培养24h。吸出原有培养液，用PBS洗涤，每孔加入CCK8溶液(CCK8体积占DMEM培养液体积的10％)，细胞培养箱中孵育1h，置于酶标仪450nm处测定吸光度。计算细胞存活率。Rat H9C2 cardiomyocytes in the logarithmic growth phase were inoculated with 8×104 /mL cells in a 96-well plate, 100 μL per well, and divided into a blank control group, an ISO model group, and an ISO+methyl ophiopogon japonicus flavonoids A group (purchased from Lemeitian Pharmaceuticals, batch number DST210602-025). After culturing for 24 hours, 1.1 mM ISO was added to the ISO model group and the ISO+methyl ophiopogon japonicus flavonoids A group, and 10 μM methyl ophiopogon japonicus flavonoids A was added to the ISO+methyl ophiopogon japonicus flavonoids A group at the same time. An equal volume of DMSO was added to the blank control group and cultured for another 24 hours. The original culture medium was aspirated, washed with PBS, and CCK8 solution was added to each well (the volume of CCK8 accounted for 10% of the volume of DMEM culture medium), incubated in a cell culture incubator for 1 hour, and the absorbance was measured at 450 nm by an ELISA reader. The cell survival rate was calculated.
实验结果：Experimental results:
结果见表1。与空白对照组相比，ISO作用后可显著降低H9C2心肌细胞的存活率。与模型对照组比较，甲基麦冬黄酮A能够显著改善ISO引起的心肌细胞存活率降低现象，细胞存活率较模型组提高30.7％。The results are shown in Table 1. Compared with the blank control group, ISO can significantly reduce the survival rate of H9C2 cardiomyocytes. Compared with the model control group, methyl ophiopogon japonicus flavonoids A can significantly improve the phenomenon of ISO-induced decrease in cardiomyocyte survival rate, and the cell survival rate is increased by 30.7% compared with the model group.
表1甲基麦冬黄酮A对异丙肾上腺素(ISO)引起心肌细胞损伤的保护作用(n＝9)Table 1 Protective effect of flavonoids A on myocardial cell injury induced by isoproterenol (ISO) (n=9)
***P<0.001与空白对照组比较；#P<0.05与模型组比较。*** P<0.001 compared with blank control group;# P<0.05 compared with model group.
实施例2甲基麦冬黄酮A在H9C2心肌细胞肥大模型活性测试Example 2 Activity test of methyl ophiopogon japonicus flavonoids A in H9C2 cardiomyocyte hypertrophy model
实验方法：Experimental methods:
对数生长期的大鼠H9C2接种于96孔板中，每孔100μL，分为空白对照组、ISO模型组、ISO+甲基麦冬黄酮A 5μM组。培养24小时后，ISO模型组和ISO+甲基麦冬黄酮A组分别加入600μM ISO，ISO+甲基麦冬黄酮A组同时加入甲基麦冬黄酮A，空白对照组加入等体积DMSO，继续培养24h。弃掉培养液，用PBS洗3次。倒置显微镜下观察，每孔随机选取视野拍照。采用Image J软件测量视野内所有心肌细胞的表面积和，同时计数视野内细胞数，单个心肌细胞表面积＝视野内所有心肌细胞表面积和/视野内心肌细胞数。获得每一个心肌细胞表面积。Rat H9C2 in the logarithmic growth phase were inoculated in a 96-well plate, with 100 μL per well, and divided into a blank control group, an ISO model group, and an ISO+methyl ophiopogon japonicus flavonoids A 5 μM group. After 24 hours of culture, 600 μM ISO was added to the ISO model group and the ISO+methyl ophiopogon japonicus flavonoids A group, and methyl ophiopogon japonicus flavonoids A was added to the ISO+methyl ophiopogon japonicus flavonoids A group at the same time. An equal volume of DMSO was added to the blank control group and cultured for another 24 hours. The culture medium was discarded and washed three times with PBS. The cells were observed under an inverted microscope, and the field of view was randomly selected from each well for photography. The surface area and total of all cardiomyocytes in the field of view were measured using Image J software, and the number of cells in the field of view was counted at the same time. The surface area of a single cardiomyocyte = the surface area and total of all cardiomyocytes in the field of view/the number of cardiomyocytes in the field of view. The surface area of each cardiomyocyte was obtained.
实验结果：Experimental results:
如图1所示，ISO作用H9C2细胞24h，与空白对照组相比，模型组细胞数量明显减少，且心肌细胞体积显著增大，部分细胞膜边界不明显并发生融合，出现心肌细胞肥大现象，同时细胞出现死亡，漂浮细胞显著增多。甲基麦冬黄酮A5μM显著抑制ISO引起的心肌细胞肥大，同时改善细胞数下降和细胞漂浮情况，细胞膜边界与模型组相比也较清晰，细胞融合情况明显减少。单个心肌细胞表面积统计数据见表2，图1，模型组心肌细胞随机选取的视野内单个心肌细胞表面积均增大，与空白对照组比较有统计学差异。甲基麦冬黄酮A 5μM显著改善ISO引起的心肌细胞肥大损伤，单个心肌细胞表面积与模型组比较显著降低。As shown in Figure 1, ISO acted on H9C2 cells for 24 hours. Compared with the blank control group, the number of cells in the model group was significantly reduced, and the volume of myocardial cells increased significantly. The boundaries of some cell membranes were not obvious and fused, and myocardial cell hypertrophy occurred. At the same time, cell death occurred, and the number of floating cells increased significantly. Methyl ophiopogon japonicus flavonoids A 5μM significantly inhibited ISO-induced myocardial cell hypertrophy, and improved the decrease in cell number and cell floating. The cell membrane boundaries were also clearer than those in the model group, and cell fusion was significantly reduced. The statistical data of the surface area of single myocardial cells are shown in Table 2 and Figure 1. The surface area of single myocardial cells in the randomly selected field of view of myocardial cells in the model group increased, which was statistically different from the blank control group. Methyl ophiopogon japonicus flavonoids A 5μM significantly improved ISO-induced myocardial cell hypertrophy damage, and the surface area of single myocardial cells was significantly reduced compared with the model group.
表2甲基麦冬黄酮A对心肌细胞肥大的作用-心肌细胞表面积(μM2)(x±s，n＝3)Table 2 Effects of methyl ophiopogon japonicus flavonoids A on cardiomyocyte hypertrophy - cardiomyocyte surface area (μM2 ) (x±s, n＝3)
***P<0.01与空白对照组比较；###P<0.05与模型组比较。*** P<0.01 compared with blank control group;### P<0.05 compared with model group.
实验例3甲基麦冬黄酮A在小鼠心肌缺血模型活性测试Experimental Example 3 Activity test of methyl ophiopogon japonicus flavonoids A in mouse myocardial ischemia model
实验方法：Experimental methods:
雄性C57BL/6小鼠适应环境后随机分为5组，分别为空白对照组、ISO模型组、ISO+地尔硫卓20mg/kg阳性对照药组、ISO+甲基麦冬黄酮A 1mg/kg组、ISO+甲基麦冬黄酮A3mg/kg组。给药组小鼠均每天灌胃给药相应剂量的甲基麦冬黄酮A1次，地尔硫卓组每天腹腔注射给药1次，空白对照组及模型组小鼠给予相同量的溶剂，给药量均为10mL/kg，连续给药18天。给药第4～10天进行ISO造模，空白对照组皮下注射生理盐水，其余各组小鼠皮下注射ISO 40mg/kg(均在给药后2h造模)，共7次。实验过程中记录动物体重及动物状态。在给药的不同阶段对药效活性进行分析。Male C57BL/6 mice were randomly divided into 5 groups after acclimation, namely blank control group, ISO model group, ISO+diltiazem 20mg/kg positive control group, ISO+methyl ophiopogon japonicus flavonoids A 1mg/kg group, ISO+methyl ophiopogon japonicus flavonoids A 3mg/kg group. The mice in the drug-treated group were gavaged with the corresponding dose of methyl ophiopogon japonicus flavonoids A once a day, the diltiazem group was intraperitoneally injected once a day, and the blank control group and model group mice were given the same amount of solvent, the dosage was 10mL/kg, and the drug was administered continuously for 18 days. ISO modeling was performed on the 4th to 10th day of administration, the blank control group was subcutaneously injected with normal saline, and the mice in the other groups were subcutaneously injected with ISO 40mg/kg (all models were established 2h after administration), for a total of 7 times. The weight and state of the animals were recorded during the experiment. The pharmacodynamic activity was analyzed at different stages of administration.
超声心动图检测：给药第9天(ISO造模6次)，小鼠皮下注射ISO 4h后每组随机选取5只，麻醉后固定于37℃恒温加热板，剃除胸毛，于胸部涂少量耦合剂。采用Vevo770Imaging System小动物超声实时影像系统行超声心动图检查，所有动物心动图指标均连续测量3个心动周期，取平均值。在给药第17天(ISO停止损伤后7天)，同样方法每组随机取5只动物进行超声心动图检测，所有动物心动图指标均连续测量3个心动周期，取平均值。Echocardiography: On the 9th day of administration (ISO modeling 6 times), 5 mice were randomly selected from each group 4 hours after subcutaneous injection of ISO. After anesthesia, they were fixed on a 37°C constant temperature heating plate, chest hair was shaved, and a small amount of coupling agent was applied to the chest. Echocardiography was performed using the Vevo770 Imaging System small animal ultrasound real-time imaging system. All animal cardiographic indices were measured continuously for 3 cardiac cycles and the average value was taken. On the 17th day of administration (7 days after ISO injury was stopped), 5 animals were randomly selected from each group for echocardiography in the same way. All animal cardiographic indices were measured continuously for 3 cardiac cycles and the average value was taken.
心肌损伤生物标记物及心肌酶检测：在给药第10天(提前给药3天，然后与ISO一起给药连续7天)，每组随机取5只小鼠眼静脉丛取血，室温静置2h，3500rpm离心20min分离血清，按ELISA试剂盒说明书测定血清CK-MB、cTn-T含量。在给药第18天(ISO停止注射后第8天)，所有动物摘眼球取血，室温静置2h，3500rpm离心20min分离血清，按ELISA试剂盒说明书测定血清CK-MB、cTn-T含量。Myocardial injury biomarkers and myocardial enzyme detection: On the 10th day of administration (administered 3 days in advance, and then administered with ISO for 7 consecutive days), blood was randomly collected from the ophthalmic venous plexus of 5 mice in each group, left to stand at room temperature for 2 hours, centrifuged at 3500rpm for 20 minutes to separate serum, and the serum CK-MB and cTn-T levels were determined according to the instructions of the ELISA kit. On the 18th day of administration (8th day after ISO injection was stopped), blood was collected from all animals by removing the eyeballs, left to stand at room temperature for 2 hours, centrifuged at 3500rpm for 20 minutes to separate serum, and the serum CK-MB and cTn-T levels were determined according to the instructions of the ELISA kit.
心力衰竭生物标记物检测：在给药第18天(ISO停止注射后第8天)，所有动物摘眼球取血，室温静置2h，3500rpm离心20min分离血清，按ELISA试剂盒说明书测定血清BNP、ANP含量。Detection of heart failure biomarkers: On the 18th day of administration (8th day after stopping ISO injection), all animals had their eyeballs removed and blood was collected. The blood was left to stand at room temperature for 2 hours and centrifuged at 3500 rpm for 20 minutes to separate the serum. The serum BNP and ANP levels were determined according to the instructions of the ELISA kit.
心肌组织病理学分析：心脏经冰生理盐水清洗并用吸水纸拭干，横向切成两半，靠近心尖的一半固定于4％多聚甲醛溶液中，经梯度乙醇脱水，二甲苯透明，然后放入溶解的石蜡中浸透3次进行包埋，每次30min，包埋好后进行连续切片(厚度约为4μm)。常规苏木紫-伊红(H.E.)染色。在光学显微镜下观察心肌组织病理状态并照相。Myocardial tissue pathological analysis: The heart was washed with ice saline and dried with absorbent paper, cut into two halves transversely, and the half close to the apex was fixed in 4% paraformaldehyde solution, dehydrated with gradient ethanol, transparentized with xylene, and then immersed in dissolved paraffin for 3 times for embedding, each time for 30 minutes. After embedding, continuous sections (thickness of about 4μm) were made. Conventional hematoxylin-eosin (H.E.) staining was performed. The pathological state of myocardial tissue was observed under an optical microscope and photographed.
实验结果：Experimental results:
心肌损伤生物标记物cTn-T结果：Myocardial injury biomarker cTn-T results:
心肌坏死时，心肌内含有的一些蛋白质类物质会从心肌组织内释放出来，并出现在外周循环血中，可作为心肌损伤的判断性指标。肌钙蛋白(Tn)是心肌组织收缩的调节蛋白，心肌肌钙蛋白(cTn)与骨骼肌中的Tn在分子结构和免疫学上是不同的，因此是心肌所独有，是判断心肌坏死最特异和敏感的首选标志物。cTn有三个亚型，cTn-T，cTn-I，cTn-C。cTn-T对早期和晚期心肌坏死均具有很高的诊断价值。When myocardial necrosis occurs, some protein substances contained in the myocardium will be released from the myocardial tissue and appear in the peripheral circulating blood, which can be used as a diagnostic indicator of myocardial damage. Troponin (Tn) is a regulatory protein for myocardial tissue contraction. Cardiac troponin (cTn) is different from Tn in skeletal muscle in molecular structure and immunology, so it is unique to the myocardium and is the most specific and sensitive marker of choice for judging myocardial necrosis. There are three subtypes of cTn, cTn-T, cTn-I, and cTn-C. cTn-T has a high diagnostic value for both early and late myocardial necrosis.
预防给药阶段：甲基麦冬黄酮A连续给药10天，在给药第四天开始皮下注射ISO连续7天，模型组和空白对照组给予相应量的溶剂对照。结果见表3。模型组小鼠血清心肌损伤标志性蛋白cTn-T与空白对照组相比显著升高，甲基麦冬黄酮A 1mg/kg、3mg/kg剂量预防给药，均能显著降低血清cTn-T水平，与模型组比较有统计学差异。Preventive administration phase: methyl ophiopogon japonicus flavonoids A was administered for 10 consecutive days, and ISO was subcutaneously injected for 7 consecutive days starting from the fourth day of administration. The model group and the blank control group were given corresponding amounts of solvent control. The results are shown in Table 3. The serum myocardial injury marker protein cTn-T of the model group mice was significantly increased compared with the blank control group. Preventive administration of methyl ophiopogon japonicus flavonoids A at doses of 1 mg/kg and 3 mg/kg can significantly reduce the serum cTn-T level, which is statistically different from the model group.
表3甲基麦冬黄酮A预防给药对异丙肾上腺素(ISO)诱导心肌缺血小鼠血清心肌损伤生物标记物cTn-T的影响(n＝5)Table 3 Effect of preventive administration of methyl ophiopogon japonicus flavonoid A on serum myocardial injury biomarker cTn-T in isoproterenol (ISO)-induced myocardial ischemia mice (n=5)
***P<0.001与空白对照组比较；#P<0.05，##P<0.01，###P<0.001与模型组比较。*** P<0.001 compared with the blank control group;# P<0.05,## P<0.01,### P<0.001 compared with the model group.
ISO连续皮下注射7次后，停止注射，动物继续每天灌胃给予甲基麦冬黄酮A，进行治疗性给药8天。血清cTn-T结果见表4。ISO停止损伤小鼠7天后，心脏的损伤程度较损伤急性期有所恢复，但心脏损伤仍然存在，小鼠血清心肌损伤生物标记物cTn-T水平仍较空白对照组显著升高。甲基麦冬黄酮A3mg/kg剂量治疗给药，显著降低血清cTn-T水平，甲基麦冬黄酮A3mg/kg治疗活性与地尔硫卓20mg/kg相当。After 7 consecutive subcutaneous injections of ISO, the injection was stopped, and the animals continued to be gavaged with methyl ophiopogon japonicus flavonoids A every day for 8 days of therapeutic administration. The results of serum cTn-T are shown in Table 4. Seven days after ISO stopped injuring mice, the degree of heart damage recovered compared with the acute stage of injury, but heart damage still existed, and the level of cTn-T, a biomarker of myocardial injury in the serum of mice, was still significantly higher than that of the blank control group. The therapeutic administration of methyl ophiopogon japonicus flavonoids A at a dose of 3 mg/kg significantly reduced the serum cTn-T level, and the therapeutic activity of methyl ophiopogon japonicus flavonoids A at 3 mg/kg was equivalent to that of diltiazem 20 mg/kg.
表4甲基麦冬黄酮A治疗给药对异丙肾上腺素(ISO)诱导心肌缺血小鼠血清心肌损伤生物标记物cTn-T的影响(n＝6-8)Table 4 Effects of methyl ophiopogon japonicus flavonoids A on serum myocardial injury biomarker cTn-T in isoproterenol (ISO)-induced myocardial ischemia mice (n=6-8)
*P<0.05与空白对照组比较；#P<0.05，##P<0.01与模型组比较。* P<0.05 compared with blank control group;# P<0.05,## P<0.01 compared with model group.
心肌酶结果：Myocardial enzyme results:
血清肌酸激酶-同工酶(CK-MB)是判断心肌坏死的另一个临床特异性和敏感性指标。Serum creatine kinase-MB (CK-MB) is another clinically specific and sensitive indicator for myocardial necrosis.
ISO连续皮下注射7次后，停止注射，动物继续每天灌胃给予甲基麦冬黄酮A，进行治疗性给药8天。结果见表5。小鼠血清心肌损伤生物标记物CK-MB水平与空白对照组显著升高，同样提示心脏损伤在停止ISO刺激后8天仍然存在。甲基麦冬黄酮A1mg/kg、3mg/kg剂量治疗给药，均能显著降低血清CK-MB水平，甲基麦冬黄酮A 3mg/kg对心肌缺血的治疗活性与地尔硫卓20mg/kg相当。After 7 consecutive subcutaneous injections of ISO, the injection was stopped, and the animals continued to be gavaged with methyl ophiopogon japonicus flavonoids A every day for 8 days of therapeutic administration. The results are shown in Table 5. The level of CK-MB, a biomarker of myocardial injury in the serum of mice, was significantly increased compared with the blank control group, which also indicated that cardiac injury still existed 8 days after the cessation of ISO stimulation. Treatment with 1 mg/kg and 3 mg/kg doses of methyl ophiopogon japonicus flavonoids A significantly reduced serum CK-MB levels, and the therapeutic activity of 3 mg/kg of methyl ophiopogon japonicus flavonoids A on myocardial ischemia was equivalent to that of 20 mg/kg of diltiazem.
表5甲基麦冬黄酮A治疗给药对异丙肾上腺素(ISO)诱导心肌缺血小鼠血清心肌酶CK-MB含量的影响(n＝6-8)Table 5 Effects of methyl ophiopogon japonicus flavonoid A on the serum myocardial enzyme CK-MB content in mice with isoproterenol (ISO)-induced myocardial ischemia (n=6-8)
**P<0.01与空白对照组比较；#P<0.05，##P<0.01与模型组比较。** P<0.01 compared with blank control group;# P<0.05,## P<0.01 compared with model group.
心力衰竭生物标记物结果：Heart Failure Biomarker Results:
血清脑钠肽BNP是心力衰竭早期诊断的生物标记物，其水平降低也是药物治疗有效的有力证据。心钠肽ANP是另一个心力衰竭的血清敏感生物标记物，目前研究亦显示，BNP和ANP水平与心肌肥厚程度呈正相关，可作为心肌肥厚的生物标记物。Serum brain natriuretic peptide BNP is a biomarker for early diagnosis of heart failure, and its reduced level is also strong evidence of effective drug treatment. Atrial natriuretic peptide ANP is another serum sensitive biomarker for heart failure. Current studies have also shown that BNP and ANP levels are positively correlated with the degree of myocardial hypertrophy and can be used as biomarkers for myocardial hypertrophy.
在停止ISO刺激，动物继续每天灌胃给予甲基麦冬黄酮A进行治疗性给药8天后，利用ELISA试剂盒检测血清BNP和ANP水平，结果见表6。模型组动物血清BNP水平及ANP水平较空白对照组均显著升高，提示心脏损伤严重，有心衰可能，且存在心肌肥厚病征。甲基麦冬黄酮A1mg/kg，3mg/kg显著降低血清BNP和ANP水平，甲基麦冬黄酮A 1mg/kg亦能显著降低血清BNP水平，与模型组比较均有统计学差异。甲基麦冬黄酮A1mg/kg，3mg/kg对心力衰竭生物标记物的降低作用优于阳性对照药地尔硫卓20mg/kg。After stopping ISO stimulation, the animals continued to be given methyl ophiopogon japonicus flavonoids A by oral gavage for 8 days for therapeutic administration. The serum BNP and ANP levels were detected by ELISA kits. The results are shown in Table 6. The serum BNP and ANP levels of the animals in the model group were significantly higher than those in the blank control group, indicating that the heart was severely damaged, there was a possibility of heart failure, and there were signs of myocardial hypertrophy. Methyl ophiopogon japonicus flavonoids A 1mg/kg, 3mg/kg significantly reduced serum BNP and ANP levels, and methyl ophiopogon japonicus flavonoids A 1mg/kg also significantly reduced serum BNP levels, which were statistically different from the model group. Methyl ophiopogon japonicus flavonoids A 1mg/kg, 3mg/kg had a better effect on reducing heart failure biomarkers than the positive control drug diltiazem 20mg/kg.
表6甲基麦冬黄酮A治疗给药对异丙肾上腺素(ISO)诱导心肌缺血小鼠血清心力衰竭生物标记物BNP、ANP含量的影响(n＝6-8)Table 6 Effects of methyl ophiopogon japonicus flavonoid A on the levels of serum heart failure biomarkers BNP and ANP in isoproterenol (ISO)-induced myocardial ischemia mice (n=6-8)
***P<0.001与空白对照组比较；#P<0.05与模型组比较。*** P<0.001 compared with blank control group;# P<0.05 compared with model group.
超声心动图检查结果：Echocardiographic findings:
小动物超声心动图技术是一种操作简便、重复性好的评估小鼠心功能的技术。连续ISO给药导致的心肌损伤，主要是左心室心内膜下病变，增加左心室僵硬度，引起左心室舒张功能异常，降低左心室膨胀度。对获得的M型超声心动图进行测量和计算，结果见表7，表8。Small animal echocardiography is a simple and reproducible technique for evaluating cardiac function in mice. Myocardial damage caused by continuous ISO administration is mainly left ventricular subendocardial lesions, increased left ventricular stiffness, abnormal left ventricular diastolic function, and reduced left ventricular expansion. The obtained M-mode echocardiograms were measured and calculated, and the results are shown in Tables 7 and 8.
ISO 40mg/kg皮下注射小鼠每天一次，连续6天，在第六天ISO注射后4小时检测超声心动图，获得的主要结果见表7。连续ISO给药引起的心肌损伤导致显著的心肌肥厚现象，同时左心室舒张功能出现异常，表现在左心室收缩期后壁厚度显著增加，舒张期左心室内径和舒张期左心室容积均显著降低。甲基麦冬黄酮A3mg/kg能够显著降低左心室收缩期后壁厚度，改善心肌肥厚，同时显著升高舒张期左心室内径和舒张期左心室容量，改善左心室舒张期功能，与模型组比较有统计学差异。甲基麦冬黄酮A 3mg/kg在ISO损伤急性期对左心室舒张功能的改善活性优于地尔硫卓20mg/kg。ISO 40mg/kg was injected subcutaneously into mice once a day for 6 consecutive days. Echocardiography was performed 4 hours after ISO injection on the sixth day. The main results are shown in Table 7. Myocardial injury caused by continuous ISO administration led to significant myocardial hypertrophy and abnormal left ventricular diastolic function, which was manifested in a significant increase in the thickness of the left ventricular posterior wall during systole and a significant decrease in the left ventricular internal diameter during diastole and the left ventricular volume during diastole. Methyl ophiopogon japonicus flavonoids A 3mg/kg can significantly reduce the thickness of the left ventricular posterior wall during systole, improve myocardial hypertrophy, and significantly increase the left ventricular internal diameter during diastole and the left ventricular volume during diastole, and improve the left ventricular diastolic function, which was statistically different from the model group. Methyl ophiopogon japonicus flavonoids A 3mg/kg had better activity in improving left ventricular diastolic function in the acute stage of ISO injury than diltiazem 20mg/kg.
表7甲基麦冬黄酮A预防给药对异丙肾上腺素(ISO)诱导心肌缺血损伤小鼠M型超声心动图数据的影响(n＝5)Table 7 Effects of preventive administration of methyl ophiopogon japonicus flavonoids A on M-mode echocardiographic data of mice with myocardial ischemia induced by isoproterenol (ISO) (n=5)
*P<0.05，**P<0.01与空白对照组比较；#P<0.05，##P<0.01与模型组比较。* P<0.05,** P<0.01 compared with the blank control group;# P<0.05,## P<0.01 compared with the model group.
ISO停止注射后，甲基麦冬黄酮A继续治疗性给药7天，进一步检测超声心动图，重点对左心室舒张功能进行考察，结果见表7。ISO刺激肾上腺素能神经导致的心脏左心室舒张功能障碍在停止损伤7天后仍然存在，舒张期左心室内径和舒张期左心室容积较空白对照组降低。甲基麦冬黄酮A3mg/kg显著增加舒张期左心室内径和舒张期左心室容积，显著改善ISO引起的左心室顺应性降低。甲基麦冬黄酮A 3mg/kg治疗给药对左心室舒张功能的改善活性优于地尔硫卓20mg/kg。表8甲基麦冬黄酮A治疗给药对异丙肾上腺素(ISO)诱导心肌缺血损伤小鼠M型超声心动图数据的影响(n＝5)After the injection of ISO was stopped, methyl ophiopogon japonicus flavonoids A continued to be administered therapeutically for 7 days, and echocardiography was further tested, with a focus on the left ventricular diastolic function. The results are shown in Table 7. The left ventricular diastolic dysfunction caused by ISO stimulating the adrenergic nerves still existed 7 days after the cessation of injury, and the diastolic left ventricular internal diameter and diastolic left ventricular volume were lower than those of the blank control group. Methyl ophiopogon japonicus flavonoids A 3 mg/kg significantly increased the diastolic left ventricular internal diameter and diastolic left ventricular volume, and significantly improved the reduced left ventricular compliance caused by ISO. The therapeutic administration of methyl ophiopogon japonicus flavonoids A 3 mg/kg had a better activity in improving left ventricular diastolic function than diltiazem 20 mg/kg. Table 8 Effects of therapeutic administration of methyl ophiopogon japonicus flavonoids A on M-mode echocardiographic data of mice with isoproterenol (ISO)-induced myocardial ischemia injury (n=5)
#P<0.05与模型组比较。# P<0.05 compared with the model group.
实验例4甲基麦冬黄酮A在阿霉素诱导的小鼠心脏毒性模型活性测试Experimental Example 4 Activity test of methyl ophiopogon japonicus flavonoid A in adriamycin-induced mouse cardiotoxicity model
实验方法：Experimental methods:
雄性C57BL/6小鼠适应环境后随机分为5组，分别为空白对照组、阿霉素模型组、右丙亚胺100mg/kg阳性对照药组、阿霉素+甲基麦冬黄酮A 3mg/kg组。给药组小鼠每天灌胃给药相应剂量的甲基麦冬黄酮A1次，空白对照组及模型组小鼠给予相同量的溶剂，给药量均为10mL/kg，连续7天，右丙亚胺阳性对照组于造模前一小时给药，共两次。给药第4～5天，空白对照组皮下注射生理盐水，其余各组小鼠腹腔注射阿霉素10mg/kg。第7天，所有动物摘眼球取血，室温静置2h，3500rpm离心20min分离血清，全自动生化分析仪检测血清CK-MB含量。After acclimation, male C57BL/6 mice were randomly divided into 5 groups, namely, blank control group, doxorubicin model group, positive control group of 100 mg/kg dexrazoxane, and doxorubicin + 3 mg/kg methyl ophiopogon japonicus flavonoids A. The mice in the drug-treated group were gavaged with the corresponding dose of methyl ophiopogon japonicus flavonoids A once a day, and the mice in the blank control group and model group were given the same amount of solvent, with a dosage of 10 mL/kg, for 7 consecutive days. The positive control group of dexrazoxane was given one hour before modeling, for a total of two times. On the 4th and 5th days of administration, the blank control group was subcutaneously injected with normal saline, and the mice in the other groups were intraperitoneally injected with 10 mg/kg doxorubicin. On the 7th day, the eyeballs of all animals were removed for blood collection, and the blood was left to stand at room temperature for 2 hours, centrifuged at 3500 rpm for 20 minutes to separate the serum, and the serum CK-MB content was detected by an automatic biochemical analyzer.
实验结果：Experimental results:
血清肌酸激酶-同工酶(CK-MB)是判断心肌坏死的临床特异性和敏感性指标。结果见表9。阿霉素注射导致小鼠心肌损伤坏死，血清CK-MB水平较空白对照组显著升高。甲基麦冬黄酮A3mg/kg显著降低血清CK-MB水平，表现对阿霉素引起急性心肌毒性的保护作用。阳性对照药右丙亚胺为临床唯一特异性用于治疗阿霉素心脏毒性的药物，在该研究中亦表现良好药效。Serum creatine kinase-isoenzyme (CK-MB) is a clinically specific and sensitive indicator for judging myocardial necrosis. The results are shown in Table 9. Adriamycin injection caused myocardial damage and necrosis in mice, and the serum CK-MB level was significantly increased compared with the blank control group. Methyl ophiopogon japonicus flavonoids A 3mg/kg significantly reduced serum CK-MB levels, showing a protective effect against acute myocardial toxicity caused by adriamycin. The positive control drug dexrazoxane is the only clinically specific drug used to treat adriamycin cardiotoxicity, and it also showed good efficacy in this study.
表9甲基麦冬黄酮A治疗给药对异丙肾上腺素(ISO)诱导心肌损伤小鼠血清心肌酶CK-MB含量的影响(n＝9-10)Table 9 Effect of methyl ophiopogon japonicus flavonoids A on the serum myocardial enzyme CK-MB content in mice with isoproterenol (ISO)-induced myocardial injury (n=9-10)
***P<0.001与空白对照组比较；##P<0.01，###P<0.001与模型组比较。*** P<0.001 compared with blank control group;## P<0.01,### P<0.001 compared with model group.
实验例5甲基麦冬黄酮A在常压缺氧小鼠模型活性测试Experimental Example 5 Activity test of methyl ophiopogon japonicus flavonoid A in normobaric hypoxia mouse model
实验方法：Experimental methods:
SPF级雄性CD-1(ICR)小鼠适应环境后随机分为3组，分别为空白对照组、红景天600mg/kg阳性对照药组、甲基麦冬黄酮A1mg/kg组。给药组小鼠每天灌胃给药相应剂量的甲基麦冬黄酮A1次，空白对照组给予相同量的溶剂，给药量均为10mL/kg，连续4天，于末次给药1h后，小鼠进行常压耐缺氧实验。将小鼠放进250ml的密封性好的磨口玻璃瓶内，每瓶1只小鼠，瓶内放置15g钠石灰。将小鼠放入瓶内后立即用封口膜密封盖紧，以小鼠挣扎抽搐后突然瘫软，且胸部不再起伏，呼吸停止为死亡依据，记录各组小鼠从密封至死亡的时间。SPF male CD-1 (ICR) mice were randomly divided into 3 groups after acclimation, namely, blank control group, positive control group of Rhodiola rosea 600mg/kg, and 1mg/kg group of methyl ophiopogon japonicus flavonoids A. The mice in the drug-treated group were gavaged with the corresponding dose of methyl ophiopogon japonicus flavonoids A once a day, and the blank control group was given the same amount of solvent, with the dosage of 10mL/kg for 4 consecutive days. One hour after the last administration, the mice were subjected to a normal pressure hypoxia tolerance experiment. The mice were placed in a 250ml ground-mouth glass bottle with good sealing performance, with 1 mouse per bottle and 15g of soda lime placed in the bottle. After the mice were placed in the bottle, they were immediately sealed with a sealing film. The mice were considered dead when they suddenly became limp after struggling and twitching, and their chests no longer rose and fell, and breathing stopped. The time from sealing to death of each group of mice was recorded.
实验结果：Experimental results:
小鼠存活时间结果见表10。甲基麦冬黄酮A1mg/kg提前给药4天，能够显著延长小鼠在常压缺氧情况下的存活时间，存活时间较空白对照组动物延长10min(14.49％)，表现显著的耐心脑缺氧活性，增强动物在缺氧条件下的生存能力。甲基麦冬黄酮A1mg/kg抗耐缺氧活性优于红景天600mg/kg。The results of mouse survival time are shown in Table 10. Methyl ophiopogon japonicus flavonoids A 1mg/kg administered 4 days in advance can significantly prolong the survival time of mice under normal pressure hypoxia, and the survival time is extended by 10min (14.49%) compared with the blank control group, showing significant anti-brain hypoxia activity and enhancing the survival ability of animals under hypoxia. Methyl ophiopogon japonicus flavonoids A 1mg/kg anti-hypoxia activity is better than Rhodiola rosea 600mg/kg.
表10甲基麦冬黄酮A在常压缺氧模型对小鼠生存时间的影响(n＝10)Table 10 Effect of methyl ophiopogon japonicus flavonoids A on the survival time of mice in the normobaric hypoxia model (n=10)
*P<0.01与空白对照组比较。* P<0.01 compared with the blank control group.
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