WO2016127787A1 - 绿原酸在制备治疗心肌病的药物中的用途 - Google Patents

绿原酸在制备治疗心肌病的药物中的用途 Download PDF

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WO2016127787A1
WO2016127787A1 PCT/CN2016/072121 CN2016072121W WO2016127787A1 WO 2016127787 A1 WO2016127787 A1 WO 2016127787A1 CN 2016072121 W CN2016072121 W CN 2016072121W WO 2016127787 A1 WO2016127787 A1 WO 2016127787A1
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chlorogenic acid
cardiomyopathy
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rats
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张洁
张梦甜
黄望
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四川九章生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem

Definitions

  • the invention relates to the use of chlorogenic acid in the preparation of a medicament for treating cardiomyopathy, and belongs to the field of medicine.
  • Chlorogenic acid CGA also known as caffeic acid, is a phenolic acid consisting of caffeic acid CA and quinic acid QA. Its chemical name is 3-o-caffeoyl quinine. Acid (3-o-caffeoylquinic acid CGA). Chlorogenic acid is a phenylpropanoid synthesized by plants in the process of aerobic respiration through the pentose phosphate pathway intermediate. Chlorogenic acid has been used in many fields such as food, health care products, cosmetics and pharmaceuticals.
  • cardiovascular protection Because it is widely found in a variety of common vegetables and fruits, it has a variety of biological activities, such as: cardiovascular protection, anti-oxidation, anti-ultraviolet and anti-radiation effects, anti-mutagenic and anti-cancer effects, antibacterial effects, anti- Viral action, lipid-lowering and hypoglycemic effects, immunomodulatory effects, etc. It has a wide range of applications in the fields of pharmaceutical, chemical and food.
  • Cardiomyopathy is defined as a cardiomyopathy of unknown cause, divided into primary cardiomyopathy and secondary cardiomyopathy.
  • Primary cardiomyopathy is divided into three types according to etiology and pathological cardiomyopathy: 1 dilated cardiomyopathy; 2 hypertrophic cardiomyopathy; 3 restricted cardiomyopathy. At present, no research has been reported on the treatment of cardiomyopathy with chlorogenic acid.
  • the above object of the present invention is achieved by the following technical solution: a use of chlorogenic acid for the preparation of a medicament for treating cardiomyopathy.
  • the cardiomyopathy is preferably dilated cardiomyopathy and hypertrophic cardiomyopathy.
  • chlorogenic acid as a drug, has the effect of up-regulating the myocardial-specific expression of the structural gene ACTC1 and its corresponding protein. Further, as a drug, chlorogenic acid has the effect of down-regulating the caspase-3 gene and the corresponding protein, and the Caspase family plays a very important role in mediating apoptosis, wherein caspase-3 is a key executive molecule. It functions in many ways of apoptotic signaling. Its appearance and activation suggest the opening of the apoptotic pathway, indicating the apoptosis of the cells.
  • the Bcl-2 gene ie, B-cell lymphoma/leukemia-2 gene
  • the Bcl-2 gene is a proto-oncogene that has an inhibitory effect on apoptosis, and its activation can inhibit the programmed apoptosis process to some extent.
  • Applicants have learned that improving the pathological changes of cardiomyopathy (dilated cardiomyopathy and hypertrophic cardiomyopathy) can effectively improve cardiac function and significantly reduce ventricular burden. Up-regulates the myocardial-specific expression of the structural gene ACTC1 and its corresponding protein, Down-regulation of caspase-3 gene and its corresponding protein, up-regulation of Bcl-2 gene and corresponding protein, provide a theoretical basis for its clinical treatment of cereal cardiomyopathy (dilated cardiomyopathy and hypertrophic cardiomyopathy). Through the above mechanism of action, chlorogenic acid has a remarkable effect in the treatment of cardiomyopathy, especially dilated cardiomyopathy and hypertrophic cardiomyopathy.
  • Another object of the present invention is to provide a medicament for treating cardiomyopathy, which comprises chlorogenic acid, a pharmaceutically acceptable adjuvant or an auxiliary ingredient.
  • the adjuvant or auxiliary ingredients include, but are not limited to, mannitol, sodium hydrogen sulfite, starch, dextrin powder, absolute ethanol, water for injection, powdered sugar, lactose, hypromellose, magnesium stearate, sucrose, Povidone K30.
  • the drug may be an oral preparation and an injection preparation.
  • the preparation of the preparation contains 1-10 mg of chlorogenic acid per unit preparation, and the clinical use dose is 1-100 mg/kg.
  • Another object of the present invention is to provide a combination drug for treating cardiomyopathy, which comprises chlorogenic acid and a medicament for treating cardiomyopathy.
  • the medicament for treating cardiomyopathy includes, but is not limited to, propranolol, aminoacylamine, metoprolol, bisoprolol, verapamil, diltiazem, amiodarone, diisopropylamine .
  • chlorogenic acid is beneficial to improve cardiac function and obviously reduce ventricular load
  • chlorogenic acid is beneficial to healthy contraction of myocardial, beneficial to inhibiting apoptosis of cardiomyocytes
  • beneficial to improving morphology of myocardial cells beneficial to Recovery of the ventricular index.
  • VW/rat weight a rat ventricular weight index according to Example 1 of the present invention (the ordinate is a ventricular weight index in units of mg/g).
  • Example 2 is a measurement of a rat myocardial apoptosis index according to Example 1 of the present invention (the abscissa is the percentage of myocardial apoptosis, and the unit is %).
  • FIG. 3 is a diagram showing the expression of ACTC1, caspase-3 and Bcl-2 genes by RT-PCR according to Example 2 of the present invention.
  • Figure 4 is a diagram showing the expression of ACTC1, caspase-3 and Bcl-2 proteins by Western-blot according to Example 2 of the present invention.
  • Fig. 5 is a graph showing the results of measurement of rat ventricular mass index VW1 (VW/rat weight) according to Example 3 of the present invention (the ordinate is the ventricular weight index in units of mg/g).
  • Figure 6 is a graph showing the percentage of myocardial apoptosis in each group of rats according to Example 4 of the present invention.
  • Example 1 In vivo pharmacodynamic study of chlorogenic acid in the treatment of dilated cardiomyopathy (DCM)
  • LVEDd left ventricular end diastolic diameter
  • LVESd left ventricular end systolic diameter
  • EF left ventricular ejection fraction
  • each group of rats was taken about 4.0 mL of whole blood, anticoagulated with disodium edetate, left to stand for 30 min, centrifuged at 2000 g for 10 min, and separated in plasma at -70 ° C. Saved inside. Blood potassium and serum creatinine were detected by conventional methods, and BNP was directly detected by ELISA kit.
  • the rats in each group were weighed and sacrificed by the method of vertebral dislocation.
  • the heart was quickly opened and the heart was removed.
  • the surrounding large blood vessels were cut and washed in ice isotonic saline.
  • the TUNEL apoptosis detection kit was used to detect the apoptosis of cardiomyocytes.
  • the cardiac specimens taken out in 2.3.3 were patched back to the original state, washed with pre-cooled PBS, and the middle section of the heart was cut into 2 to 3 mm thick tissue. It was placed in a vial containing 10% neutral buffered formalin, fixed in a refrigerator at 4 ° C overnight (not more than 24 h), and then subjected to steps of dehydration, impregnation, and embedding to form a paraffin block. After routine sectioning of the paraffin block, the apoptotic cell nucleus was labeled in situ by TUNEL method, and the procedure was strictly in accordance with the instructions of the kit.
  • CMAI cardiomyocyte apoptosis index
  • ventricular weight index VW1 VW/rat weight
  • Figure 1 The results of ventricular weight index VW1 (VW/rat weight) are shown in Figure 1.
  • the VW1 values of the DCM groups in each group were significantly increased, and there was a significant difference between the two (p ⁇ 0.01). ), the DCM group showed significant pathological phenomena of ventricular dilatation.
  • the VW1 value of each treatment group was lower than that of the DCM group.
  • the H-LYS group had the most significant effect on the down-regulation of VW1, and there was a significant difference between the two groups and the DCM model group (p ⁇ 0.05).
  • Fig. 1 compared with the NC group, *p ⁇ 0.05, **p ⁇ 0.01 compared with the HCM group, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with Metoprotol, ⁇ p ⁇ 0.05
  • a rat model of dilated cardiomyopathy was successfully established by using furazolidone.
  • Metoprolol was used as a positive control drug, and three groups of high, medium and low doses of chlorogenic acid were set up, which were negative in normal rats.
  • rats with dilated cardiomyopathy were treated by intraperitoneal injection.
  • Echocardiographic test results show that high, medium and low doses of chlorogenic acid can effectively reduce LVEDd (mm) and LVESd (mm), LVEF (%) values compared with the model untreated group (DCM).
  • the high and medium dose chlorogenic acid treatment group can reduce the ventricular mass index more effectively (p ⁇ 0.05).
  • (4) Detection of apoptotic cells The experimental results showed that the percentage of apoptotic cells in the high, medium and low dose chlorogenic acid treatment group was significantly lower than that in the DCM group, and the degree of reduction was compared with the positive drug Metoprolol treatment group. Close, no statistical difference (p>0.05).
  • Example 2 Effect of chlorogenic acid on the expression of ACTC1, caspase-3, Bcl-2 genes and their corresponding proteins in cardiomyocytes of rats with dilated cardiomyopathy.
  • DCM dilated cardiomyopathy
  • RT-PCR was used to detect the expression of ACTC1, caspase-3 and Bcl-2 genes in cardiomyocytes of each group.
  • RNAprep Pure Culture Cell/Bacterial Total RNA Extraction Kit (centrifugal column type) is used to extract total RNA from cells, and RNase contamination should be prevented and avoided during the whole extraction process.
  • the centrifuge tubes used are all enzyme-free, and gloves are replaced in time. All operations are performed on a clean bench. The operation process is carried out according to the instructions.
  • the specific extraction steps are as follows:
  • the patient was sacrificed by the force of the vertebral body, and the heart was quickly opened to remove the heart.
  • the heart of each mouse was evenly shredded. After that, 20 mg of the sample was weighed from each heart fragment, and the remaining samples were frozen in a -70 ° C refrigerator for later use;
  • the adsorption column After separately washing the adsorption column with the deproteinized solution and the rinsing liquid, the adsorption column is placed in the collection tube, and the residual liquid above is sufficiently evaporated;
  • the corresponding first strand cDNA is synthesized using the cDNA first strand synthesis kit and the above extracted RNA solution.
  • the specific reverse transcription process follows the instructions and is briefly described as follows:
  • EvaGreen fluorescent dyes bind to double-stranded DNA to produce strong fluorescence. By detecting the final fluorescence intensity, we can get the total amount of DNA generated by the reaction.
  • the system used RNase-free water instead of cDNA products group as NTC control. Each sample was set to a relative value of the ⁇ -actin primer set for relative quantification.
  • 3.1 RT-PCR was used to detect the expression of ACTC1, caspase-3 and Bcl-2 genes in cardiomyocytes of each group.
  • This example detects the expression of ACTC1, Caspase-3 and Bcl-2 genes and their corresponding proteins in myocardial tissue when LYS is used to treat dilated cardiomyopathy from both the gene and protein layers.
  • the ACTC1 gene is a cardiomyocyte. A gene specifically expressed has an important effect on the healthy contraction of the myocardium and the like.
  • the Csapase-3 and Bcl-2 genes and corresponding proteins are closely related to the pathogenesis and apoptosis of dilated cardiomyopathy, reflecting the pathological state of dilated cardiomyopathy.
  • chlorogenic acid treatment of dilated cardiomyopathy can significantly reduce the expression of apoptosis-associated Caspase-3 gene and its protein, promote the expression of Bcl-2 gene and its protein, and is very active in inhibiting myocardial apoptosis.
  • chlorogenic acid can significantly up-regulate the expression of the ACTC1 gene and its protein specifically expressed in the myocardium, which is conducive to the maintenance of normal contractile function of the myocardium. This effect is significantly different from the positive drug Metoprolol.
  • This study provides a theoretical basis for the clinical treatment of dilated cardiomyopathy with chlorogenic acid.
  • HCM hypertrophic cardiomyopathy
  • mice in each group were treated with intraperitoneal administration.
  • the specific dosage regimen is shown in Table 5:
  • LVAWs left ventricular anterior wall thickness
  • LVPWs systolic left ventricular posterior wall thickness
  • LVPWd diastolic left ventricular anterior wall thickness
  • the obtained group of heart tissues were dehydrated, embedded, sectioned and HE stained, and then observed under a high power microscope.
  • the experimental results are shown in Table 6: (1) Compared with the NC group, the HCM group had p ⁇ 0.01; the hypertrophic cardiomyopathy model mice had significantly thicker ventricular wall than the wild mice. (2) Compared with the HCM group, the Metoprolol treatment group showed a significant decrease in the data of each group, and the ventricular wall was significantly thinner. There was a significant difference between the two groups (p ⁇ 0.01). Compared with the NC group, there was no significant difference. The difference is p>0.05.
  • ventricular weight index VW1 VW/rat weight
  • Figure 5 The results of ventricular weight index VW1 (VW/rat weight) are shown in Figure 5.
  • the VW1 of the HCM group is significantly higher, which is a typical phenomenon of cardiac hypertrophy, and is very significant between the two groups.
  • the difference (p ⁇ 0.01) showed a significant decrease in VW1 in the LYS-treated and Metoprolol-treated groups.
  • mice in the HCM group showed significantly increased myocardial cells, deformed morphology, and disordered arrangement.
  • the myocardial cells of the mice treated with Metoprolol also had a certain degree of enlargement, but the basic arrangement was neat and compact, which was significantly improved compared with the HCM group.
  • transgenic hypertrophic cardiomyopathy mice were used as experimental subjects, and Metoprolol was used as a positive control drug.
  • the experimental results showed that the LYS treatment group can significantly improve the ventricular wall thickness of hypertrophic cardiomyopathy, significantly improve myocardial cell morphology, and make the ventricle The index has been significantly reduced, and its therapeutic effect is comparable to that of positive drugs.
  • Chlorogenic acid responds well to the treatment of hypertrophic cardiomyopathy.
  • each group of rats was administered by intraperitoneal injection.
  • the specific dosage regimen is shown in Table 7:
  • LVEDd left ventricular end diastolic diameter
  • LVESd left ventricular end systolic diameter
  • EF% left ventricular ejection fraction
  • the rats in each group were taken about 4.0 mL of whole blood, EDTA-2Na was anticoagulated, and after standing for 30 min, it was centrifuged at 2000 g/min for 10 min, and the plasma was separated and stored in a refrigerator at -70 °C. Blood potassium and serum creatinine were detected by conventional methods, and BNP was directly detected by ELISA kit.
  • the TUNEL apoptosis detection kit was used to detect the apoptosis of cardiomyocytes.
  • the cardiac specimens taken out in 2.3.3 were patched back to the original state, washed with pre-cooled PBS, and the middle section of the heart was cut into 2 to 3 mm thick tissue. It was placed in a vial containing 10% neutral buffered formalin, fixed in a refrigerator at 4 ° C overnight (not more than 24 h), and then subjected to steps of dehydration, impregnation, and embedding to form a paraffin block. After routine sectioning of the paraffin block, the apoptotic cell nucleus was labeled in situ by TUNEL method, and the procedure was strictly in accordance with the instructions of the kit.
  • CMAI cardiomyocyte apoptosis index
  • a p ⁇ 0.05 compared with the NC group
  • b p ⁇ 0.05 compared with the DCM group
  • c p ⁇ 0.05 compared with the Metoprolol group
  • d p ⁇ 0.05 with the chlorogenic acid group.
  • a rat model of dilated cardiomyopathy was successfully established by using furazolidone.
  • a combined administration group was designed, and all the test results were treated with Metoprolol alone.
  • the results of the group and the chlorogenic acid alone treatment group were comprehensively compared.
  • the above results showed that chlorogenic acid can effectively improve cardiac function, significantly reduce ventricular load, and partial treatment after medium and high dose intraperitoneal injection of chlorogenic acid.
  • the effect was better than that of the positive control drug Metoprolol treatment group, which showed that it had a better therapeutic effect on dilated cardiomyopathy.
  • the chlorogenic acid raw material used in the present embodiment is obtained by extracting and purifying from honeysuckle, and the purity is 99.42%.
  • the above prescription was completely dissolved in water for injection, filtered, and then finely filtered with a 0.22 ⁇ m sterile microfiltration membrane to adjust the pH. After the conventional operation of the sterile powder injection, 1000 ml of 2 ml powder injections were prepared. Contains 50mg of chlorogenic acid.
  • the chlorogenic acid used in the present example was obtained by extraction and purification from burdock, and the purity was 98.41%.
  • Chlorogenic acid with a purity of 98.41% (main drug) 1g Starch (diluent) 50g Dextrin powder (adhesive) Moderate amount Anhydrous ethanol (wetting agent) Moderate amount
  • the chlorogenic acid used in the present example was extracted and purified from honeysuckle, and the purity was 99.17%.
  • Chlorogenic acid with a purity of 99.17% 100g Powdered sugar (filler) 100g Lactose (filler) 200g Hypromellose (adhesive) 50g Magnesium stearate (lubricant) 50g
  • a chlorogenic acid tablet is prepared by a wet granule tableting method.
  • (1) Prepare an aqueous solution of hypromellose according to the prescription; (2) mix the prescribed amount of chlorogenic acid, powdered sugar and lactose, add the hypromellose aqueous solution, stir well and make it soft. (3) the prepared soft material according to the conventional wet granulation operation procedure, sieving, drying and granulating to obtain granules of uniform size; (4) mixing the prepared granules with magnesium stearate uniformly After compression, a total of 1000 tablets were prepared, each containing 100 mg of chlorogenic acid.
  • the chlorogenic acid used in the present example was obtained by extraction and purification from Eucommia ulmoides leaves, and the purity was 99.35%.
  • each capsule contains 50mg of chlorogenic acid.
  • the chlorogenic acid used in the present example was extracted and purified from Eucommia ulmoides leaves, and the purity was 99.66%.
  • Chlorogenic acid with a purity of 99.66% 200g Mannitol (diluent) 100g Sucrose (diluent) 400g Povidone K30 (adhesive) Moderate amount
  • Povidone K30 was taken and water for injection was added to prepare a solution. After the prescription amount of chlorogenic acid, mannitol and lactose were uniformly mixed, the povidone K30 solution was added to prepare a soft material. According to the conventional preparation process of the granules, the granules are obtained after sieving, drying and granulating the soft materials. The granules were dispensed under aseptic conditions to prepare 400 bags of granules, each bag containing 500 mg of chlorogenic acid.
  • the chlorogenic acid raw material used in the present example is obtained by extracting and purifying the burdock leaves, and the purity is 98.97%.

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Abstract

本发明提供一种绿原酸在制备治疗心肌病的药物中的用途，以及含有绿原酸和药学上可接受的辅料的药物组合物。本发明还提供一种包含绿原酸和治疗心肌病药物的联合药物组合物。

Description

绿原酸在制备治疗心肌病的药物中的用途 技术领域
本发明涉及绿原酸在制备治疗心肌病的药物中的用途，属于药物领域。
背景技术
绿原酸(chlorogenic acid CGA)又名咖啡鞣酸，是由咖啡酸(caffeic acid CA)和奎尼酸(quinic acid QA)组成的缩酚酸，其化学名为3-o-咖啡酰奎尼酸(3-o-caffeoylquinic acid CGA)。绿原酸是植物在进行有氧呼吸的过程中，经磷酸戊糖途径中间产物合成的一种苯丙素类物质。绿原酸已经被开放应用于食品，保健品，化妆品和药品等多个领域。由于它广泛的存在于常见的各种蔬菜水果中，具有多种生物活性，如:心血管保护作用、抗氧化作用、抗紫外及抗辐射作用、抗诱变及抗癌作用、抗菌作用、抗病毒作用、降脂降糖作用、免疫调节作用等。在医药化工和食品等领域都具有广泛的应用。
心肌病定义为原因不明的心肌疾病，分为原发性心肌病和继发性心肌病。原发性心肌病按病因和病理心肌病被分为三型：①扩张型心肌病；②肥厚型心肌病；③限制型心肌病。目前，未见报道针对绿原酸在治疗心肌病方面的研究。
发明内容
针对上述问题，本发明的目的在于提供一种绿原酸的新用途。
本发明的上述目的是通过如下技术方案实现的：一种绿原酸在制备治疗心肌病的药物中的用途。
在本发明中，所述心肌病优选为扩张型心肌病和肥厚型心肌病。
申请人研究发现，绿原酸作为药物，具有上调心肌特异性表达结构基因ACTC1及对应蛋白的作用。进一步的，绿原酸作为药物，具有下调caspase-3基因及对应蛋白的作用，而Caspase家族在介导细胞凋亡的过程中起着非常重要的作用，其中caspase-3为关键的执行分子，它在凋亡信号传导的许多途径中发挥功能。它的出现和激活提示着凋亡途径的开启，预示着细胞的凋亡。
申请人还研究发现，绿原酸作为药物，具有上调Bcl-2基因及对应蛋白的作用。Bcl-2基因(即B细胞淋巴瘤/白血病-2基因)是一种原癌基因，它具有抑制凋亡的作用，对其的激活从一定程度上可以抑制程序性的凋亡过程。
申请人研究得知，改善心肌病(扩张型心肌病和肥厚型心肌病)的病理变化，有效的改善心功能，明显的减轻心室负荷。上调心肌特异性表达结构基因ACTC1及对应蛋白， 下调caspase-3基因及其对应蛋白，上调Bcl-2基因及对应蛋白，为其临床治疗谷心肌病(扩张型心肌病和肥厚型心肌病)提供了理论依据。通过上述作用机理，绿原酸在治疗心肌病，尤其是扩张型心肌病和肥厚型心肌病具有显著的效果。
本发明的另一目的是提供一种用于治疗心肌病的药物，所述的药物包括绿原酸，药学上可接受的辅料或辅助性成分。所述辅料或辅助性成分包括但不限于甘露醇、亚硫酸氢钠、淀粉、糊精粉、无水乙醇、注射用水、糖粉、乳糖、羟丙甲纤维素、硬脂酸镁、蔗糖、聚维酮K30。
进一步的，在本发明中，所述的药物可以是口服制剂和注射制剂。
其中，所述的制剂的每单位制剂含绿原酸1-1000mg，临床使用剂量为：1-100mg/kg。
本发明的另一目的是提供一种治疗心肌病的联合用药物，所述联合用药物包括绿原酸和治疗心肌病的药物。
进一步的，所述治疗心肌病的药物包括但不限于心得安、氨酰心安、美托洛尔、比索洛尔、异搏定、硫氮卓酮、乙胺碘呋酮、双异丙比胺。
本发明的有益效果在于：绿原酸有益于改善心功能，明显减轻心室负荷；绿原酸有益于心肌的健康收缩，有益于抑制心肌细胞的凋亡；有益于改善心肌细胞的形态，有益于心室指数的恢复。
附图说明
图1为本发明实施例1大鼠心室重量指数VW1(VW/大鼠体重)的测定结果(纵坐标为心室重量指数，单位为mg/g)。
图2为本发明实施例1大鼠心肌凋亡指数的测定(横坐标为心肌凋亡百分率，单位为％)。
图3为本发明实施例2的RT-PCR检测ACTC1、caspase-3和Bcl-2基因的表达情况。
图4为本发明实施例2的Wester-blot检测ACTC1、caspase-3和Bcl-2蛋白的表达情况。
图5为本发明实施例3的大鼠心室重量指数VW1(VW/大鼠体重)的测定结果(纵坐标为心室重量指数，单位为mg/g)。
图6为本发明实施例4各组大鼠的心肌凋亡百分比。
具体实施方式
实例1：绿原酸治疗扩张型心肌病(DCM)的体内药效学试验研究
1.实验材料
1.1动物
出生两周，母乳喂养的Wistar大鼠，雌雄各半，共110只。
1.2实验药物及仪器
呋喃唑酮，10％水合氯醛，PHLIPsonos7500彩色超声检测仪，TUNEL凋亡检测试剂盒，BNP的ELISA检测试剂盒，万分之一的电子天平,乙二胺四乙酸二钠。
2.实验方法
2.1大鼠扩张型心肌病(DCM)模型的建立和实验分组
取2周龄的Wistar大鼠115只，雌雄各半，随机挑出12只大鼠，作为实验的正常对照组，喂以正常的鼠饲料。同时，其余103只作为造模大鼠，开始喂添加有呋喃唑酮的鼠饲料，呋喃唑酮的剂量按0.2mg/g体重给予，每周按体重调整一次用药剂量，正常对照组的大鼠和造模大鼠均喂养8周。
8周后，剔除死亡的4只大鼠，剩余的99只大鼠，经超声心动图证实患有扩张型心肌病(DCM)的有78只，随机舍弃3只后，将剩余的75只患有DCM的模型大鼠随机平均分为5组，分别命名为：模型对照组(DCM组，n＝15)、美托洛尔治疗组(Metoprolol组，n＝15)、绿原酸低剂量治疗组(L-LYS组，n＝15)、绿原酸中剂量治疗组(M-LYS组，n＝15)和绿原酸高剂量治疗组(H-LYS组，n＝15)，连同正常对照组(NC组，n＝12)，共6个实验组，共87只。
2.2分组给药实验
本实验采用腹腔注射给药的方式对各组大鼠进行给药治疗，其具体的给药方案如表1所示：
表1.实验分组、给药方式及给药量(注：所有给药组的给药体积相同)
Figure PCTCN2016072121-appb-000001
2.3观察指标及测定
2.3.1超声检测仪评估心脏结构和功能(实验第90天)
给药90天后，将各组大鼠禁食12h、禁饮2h后，用10％水合氯醛麻醉后，使用 PHLIPsonos7500彩色超声检测仪进行心动图检查：测量左室舒张末期内径(LVEDd)、左室收缩末期内径(LVESd)、左室射血分数(EF)。
2.3.2血浆生化和BNP检测
待超声心动图检测完毕后，将各组大鼠分别取全血4.0mL左右，用乙二胺四乙酸二钠抗凝，静置30min后，2000g离心10min，分离血浆，于-70℃的冰箱内保存。血钾和血肌酐按常规方法检测，BNP用ELISA试剂盒直接检测。
2.3.3心室重量指数检测
超声心动图检测和取血完毕后，将各组大鼠称重后，采用劲椎脱臼法处死，迅速打开胸腔摘取心脏，肉眼观察后，剪去周围大血管，在冰等渗盐水中洗净血液，滤纸吸干水后，沿房室交界处去除左右心房，用电子天平(精确度0.0001g)称取心室重量(ventricular weight，VW)，计算心室重量指数VW1(VW/大鼠体重)。
2.3.4TUNEL法检测心肌细胞凋亡指数
使用TUNEL凋亡检测试剂盒检测心肌细胞的凋亡情况，将2.3.3中所取出的心脏标本拼凑恢复原状，用预冷的PBS冲洗，心脏中段横断面切取2～3mm厚的组织1块，放入装有10％中性缓冲福尔马林的小瓶中，入4℃冰箱固定过夜(不超过24h)，再经过脱水、浸渍和包埋等步骤，制成石蜡块。石蜡块常规切片后，应用TUNEL法原位标记凋亡细胞核，操作严格按照试剂盒的说明书进性。征程的心肌细胞核呈蓝色，而凋亡的心肌细胞核呈深浅不一的棕褐色。心肌细胞凋亡指数(CMAI)的计算方法：凋亡的心肌细胞数/心肌细胞总数×100％。
3.实验结果
3.1超声心动图检查结果
表2超声心动图检测结果
Figure PCTCN2016072121-appb-000002
实验结果如表2所示：(1)DCM组与NC组大鼠相比，各项数据具有非常显著差异，p<0.01；造模效果良好。(2)Metoprolol治疗组与DCM组相比，LVEDd(mm)和LVESd(mm) 均有了明显的降低，具有非常显著性差异p<0.01，与NC组相比，其仍具有显著性差异p<0.05，LVEF(％)也有明显的升高，与DCM组相比，有非常显著性差异p<0.01，与NC组相比，也具有非常显著性差异，p<0.01；(3)LYS高、中、低三个剂量的给药组，LVEDd(mm)和LVESd(mm)均有了一定程度的下降，其中，L-LYS组下降幅度较小，而M-LYS和H-LYS组的下降幅度较大，与DCM组表现出非常显著性差异p<0.01，与NC组间无显著性差异；LVEF(％)值在3个剂量的治疗组中，均有一定程度的升高，其中，M-LYS和H-LYS组与DCM组相比，表现出非常显著性差异p<0.01，而与NC组相比，仍表现出显著性差异p<0.05；与Metoprolol相比，M-LYS和H-LYS组的LVEDd(mm)、LVESd(mm)和LVEF(％)均没有表现出显著性差异。
3.2血浆生化和BNP检测结果
表3血浆生化和BNP检测实验结果
Figure PCTCN2016072121-appb-000003
实验结果如表3所示：各组的血钾和血肌酐水平均没有统计学差异(p>0.05)；与NC组相比，DCM组的BNP值有了明显的升高，具有非常显著性差异p<0.01，而Metoprolol和LYS的3个剂量治疗组，与DCM组相比，BNP水平显著下降，各组数据均有显著性差异，其中M-LYS和H-LYS组的BNP值较DCM组有非常显著性差异P<0.01，表明其降低BNP的程度较大。
3.3心室重量指数检测
心室重量指数VW1(VW/大鼠体重)的测定结果如图1所示：与NC组相比各组的DCM组的VW1值有显著增高，二者之间有极显著性差异(p<0.01)，DCM组表现出明显的心室扩张病理现象。各治疗组的VW1值比DCM组均有了一定程度的下降，其中H-LYS组对VW1的下调作用最为显著，其与DCM模型组之间有显著性差异p<0.05。图1中与NC组相比，*p<0.05,**p<0.01与HCM组相比，△p<0.05，△△p<0.01；与Metoprotol相比，×p<0.05
3.4TUNEL法检测心肌细胞凋亡指数
用TUNEL法检测心肌细胞凋亡的实验中，结果如图2所示，各治疗组与DMC组相比，凋亡指数均有明显的恢复，各组与DMC相比均有非常显著性差异p<0.01。
4统计学处理
连续型变量形式的实验数据以x±sd表示，2组数据间的比较用两样本t检验。采用SPSS13.0统计软件，P<0.05为有统计学意义。
5，实验结果
本实施例中，采用呋喃唑酮的方法成功建立了大鼠扩张型心肌病模型，以Metoprolol作为阳性对照药物，设置了高、中、低3个剂量绿原酸的治疗组，以正常大鼠作为阴性对照组，通过腹腔注射的方式对扩张型心肌病大鼠进行治疗。(1)超声心动图检测实验结果显示，与模型不治疗组(DCM)相比，高、中、低剂量的绿原酸能有效降低LVEDd(mm)和LVESd(mm)，LVEF(％)值也得到较大的恢复，该部分治疗效果与阳性药物Metoprolol相近，且中、高剂量的绿原酸治疗组(M-LYS组和H-LYS组)与Metoprolol治疗相比，各组数据之间无显著性差异。(2)血浆生化的结果显示，与DCM组相比，各治疗组均能有效的显著降低血浆BNP水平，且高、中、低剂量的绿原酸治疗组，与阳性药物Metoprolol相比，治疗效果相当，无显著性差异。(3)心室重量指数检测结果显示，与DCM组相比，高、中、低剂量的绿原酸治疗组，能够显著的恢复心室重量指数，其与DCM组相比，均有非常显著性差异(p<0.01),而与阳性药物Metoprolol治疗组相比，高、中剂量绿原酸治疗组能更有效的降低心室重量指数(p<0.05)。(4)凋亡细胞的检测实验结果显示，高、中、低剂量绿原酸治疗组的大鼠心肌细胞，其凋亡百分比，较DCM组，明显降低，且降低程度与阳性药物Metoprolol治疗组接近，无统计学差异(p>0.05)。
以上实验结果显示，绿原酸可以有效的改善心功能，明显的减轻心室负荷，且中、高剂量腹腔注射绿原酸后的部分治疗效果，优于阳性对照药物Metoprolol治疗组，显示其对扩张型心肌病具有较好的治疗效果。
实例2：绿原酸对扩张型心肌病大鼠模型心肌细胞中的ACTC1、caspase-3、Bcl-2基因及其各自对应蛋白的表达情况的影响。
1.实验材料
1.1动物
出生两周，母乳喂养的Wistar大鼠，雌雄各半，共80只。
1.2实验药物及仪器
呋喃唑酮，10％水合氯醛，PHLIPsonos7500彩色超声检测仪，PCR仪，总RNA提取试剂盒，cDNA第一链合成试剂盒，电泳仪，凝胶成像仪。
2.实验方法
2.1大鼠扩张型心肌病(DCM)模型的建立和实验分组
取2周龄的Wistar大鼠60只，雌雄各半，随机挑出10只大鼠，作为实验的正常对照组，喂以正常的鼠饲料。同时，其余50只作为造模大鼠，开始喂添加有呋喃唑酮的鼠饲料，呋喃唑酮的剂量按0.2mg/g体重给予，每周按体重调整一次用药剂量，正常对照组的大鼠和造模大鼠均喂养8周。
8周后，剔除死亡的2只大鼠，剩余的48只大鼠，经超声心动图证实患有扩张型心肌病(DCM)的有38只，将38只患有DCM的模型大鼠随机舍弃2只后，随机平均分为3组，分别命名为：模型对照组(DCM组，n＝12)、美托洛尔治疗组(Metoprolol组，n＝12)、绿原酸治疗组(LYS组，n＝12)连同正常对照组(NC组，n＝10)，共4个实验组，共46只。
2.2分组给药实验
本实验采用灌胃给药的方式对各组大鼠进行给药治疗，其具体的给药方案如表4所示：
表4.实验分组、给药方式及给药量(注：所有给药组的给药体积相同)
Figure PCTCN2016072121-appb-000004
2.3检测实验
2.3.1RT-PCR法检测各组大鼠心肌细胞中的ACTC1、caspase-3和Bcl-2基因的表达情况。
(1)心肌细胞总RNA的提取
利用RNAprep Pure培养细胞/细菌总RNA提取试剂盒(离心柱型)提取细胞内的总RNA，在整个提取过程当中应该预防和避免RNase污染，所用的离心管等均做无酶处理，及时更换手套，所有操作均在超净台上进行。操作过程按说明书进行，具体提取步骤简述如下：
1.60天实验结束后，采用劲椎脱臼法处死，迅速打开胸腔摘取心脏，分组编号。每只老鼠的心脏被均匀的剪碎，之后，从每份心脏碎片中称取20mg样品，其余的样品冻存于-70℃冰箱内，备用；
2.向每份心肌组织中加入300μl的预先加有β-巯基乙醇的裂解液RL，用电动匀浆器彻底的 研磨组织。向匀浆液中加入600μl的RNase-free ddH2O和10μl的蛋白酶K,混匀后56℃处理20min；
3.将上述液体在12,000rpm条件下离心5min，小心吸取上清液使用；
4.向上清液中缓慢加入0.5倍体积的无水乙醇，混匀后转移至吸附柱CR3中，12,000rpm离心1min，弃去废液，留吸附柱；
5.加入去蛋白液RW1至吸附柱中，去除蛋白，之后离心1min，弃去废液；
6.加入DNase I工作液，去除柱体上的DNA；
7.用去蛋白液和漂洗液分别清洗吸附柱后，将吸附柱放于收集管中，充分挥干上面的残留液体；
8.向吸附柱中加入60μlRNase free ddH2O，放置2min后，12,000rpm离心2min，即得mRNA样品；
(2)mRNA的逆转录
利用cDNA第一链合成试剂盒和上述提取得到的RNA溶液合成对应的第一链cDNA。具体逆转录过程按照说明书操作，简述如下：
1.取200μl的无酶离心管置于冰浴上，并于其中加入下列溶液：
Figure PCTCN2016072121-appb-000005
2.离心后，将离心管放置于PCR仪中，70℃孵育5min后，简短收集液体后，迅速将其转移到冰上冷却2min后补加如下试剂：
Figure PCTCN2016072121-appb-000006
3.将离心管轻轻混匀并简短离心后，设置PCR仪42℃孵育50min，之后95℃加热5min。最后向得到的cDNA溶液中加入30ul RNase-Free ddH2O稀释至50ul，记得第一链cDNA。
4.将得到的产物置于－20℃条件下保存。
(3)RT-PCR定量
EvaGreen荧光染料与双链的DNA结合会产生很强的荧光，通过检测最终的荧光强度，我们可以得到反应生成DNA的总量。在试管中加入荧光染料、(1)(2)步中所合成的cDNA产物、ACTC1引物(上游引物5’-ATCTCACGTTCAGCTGTGGTCA-3’，下游引物5’-ACCACCGGCATCGTGTTGGAT-3’),caspase-3引物(上游引物5’-CAAGTCGATGGACTCTGGAA-3’，下游引物5’-GTACCATTGCGAGCTGACAT-3’)，Bcl-2引物(上游引物5；-CCGGGAGAACAGGGTATGATAA-3’，下游引物5’-CCCACTCGTAGCCCCTCTG-3’)，分组混合均匀后进行RT-PCR检测本体系使用RNase-free water代替cDNA products组为NTC对照。每个样品均设β-actin引物组为相对值，进行相对定量。
2.3.2ACTC1，caspase-3和Bcl-2蛋白表达的分析
(1)Western Blot检测
①将之前冻存的生物样本取出，每份样品切取80mg左右的心肌组织，加适量PBS匀浆，弃去上清，加入5倍体积的细胞裂解液，冰上放置20min，12000rpm4℃离心1h，收集上清液；
②BCA法定量样品的蛋白浓度；
③把蛋白样品制成相同的浓度，加入样品缓冲液，沸水煮5min；
④按常规方法进行电泳；
⑤取下PVDF膜，TBS浸泡10min，5％脱脂奶粉封闭1h，TBS洗脱2次，加入一抗(1:300)，孵育2h后，TBST洗脱2次，再加入二抗(1:4000)1h，TBST洗脱2次，之后用Western-blue染液显色后观察结果。
3.实验结果
3.1 RT-PCR法检测各组大鼠心肌细胞中的ACTC1、caspase-3和Bcl-2基因的表达情况
RT-PCR实验结果如图3所示：与NC组相比，DCM组的ACTC1、Caspase-3和Bcl-2mRNA的表达均明显下调。LYS治疗组的ACTC1mRNA水平显著上调，Caspase-3mRNA也有明显下调，此外，Bcl-2mRNA表达也有明显的上调。与阳性药物Metoprolol治疗组相比，其ACTC1基因表达更显著的增高。
3.2Western Blot电泳试验结果
电泳试验结果如图4所示：与NC组相比，DCM组的ACTC1、Caspase-3和Bcl-2蛋白的表达均显著降低。LYS治疗组的ACTC1有显著的提高，Caspase-3蛋白表达有一定程度的减弱，另外Bcl-2蛋白表达也得到较大的恢复。与阳性药物Metoprolol治疗组相比，其ACTC1蛋白的表达显著增高。该实验结果，与基因层面ACTC1、Caspase-3和Bcl-2对应mRNA的表达水平结果基本一致。
4统计学处理
连续型变量形式的实验数据以±sd表示，2组数据间的比较用两样本t检验。采用SPSS13.0统计软件，P<0.05为有统计学意义。
5.结论
该实施例从基因和蛋白两个层检测了LYS用于治疗扩张型心肌病时，心肌组织中ACTC1、Caspase-3和Bcl-2基因及其对应蛋白的表达情况，其中，ACTC1基因是心肌细胞特异性表达的基因，对于心肌的健康收缩等具有重要作用。而Csapase-3和Bcl-2基因及对应蛋白与扩张型心肌病的病程发展、细胞凋亡等密切相关，反映着扩张型心肌病的病理状态。实施例的结果显示，绿原酸治疗扩张型心肌病可以显著的降低凋亡相关Caspase-3基因及其蛋白的表达，促进Bcl-2基因及其蛋白的表达，对抑制心肌凋亡具有非常积极的作用。且绿原酸能够显著的上调心肌特异性表达的ACTC1基因及其蛋白的表达，有利于心肌的正常收缩功能维持，这一作用，与阳性药物Metoprolol相比，具有显著性差异。此项研究对于绿原酸临床治疗扩张型心肌病提供了理论依据。
实施例3绿原酸对肥厚型心肌病的治疗效果的体内药效学研究
1.实验材料
1.1动物
cTnTR92Q转基因肥厚型心肌病(HCM)模型C57BL/6J小鼠
1.2实验药物及仪器
10％水合氯醛，PHLIPsonos7500彩色超声检测仪，万分之一的电子天平，高倍显微镜。
2.实验方法
2.1小鼠的分组
将cTnTR92Q转基因肥厚型心肌病模型C57BL/6J小鼠32只，随机分为4组，每组8只小鼠。分别设置为模型对照组(HCM组，n＝8)、美托洛尔治疗组(Metoprolol组，n＝8)、绿原酸治疗组(L-LYS组，n＝8)。同时，取野生型的C57BL/6J小鼠8只作为作为实验的正常对照 组(NC组，n＝8)。
2.2实验的给药方案设计
本实验采用腹腔注射的给药方式对各组小鼠进行给药治疗，其具体的给药方案如表5所示：
表5.实验给药方案(注：所有给药组的给药体积相同)
Figure PCTCN2016072121-appb-000007
2.3观察指标及测定
2.3.1超声检测仪评估心脏结构和功能(实验第90天)
给药90天后，将各组大鼠禁食12h、禁饮2h后，用10％水合氯醛麻醉后，使用心脏超声仪器检测，对各组小鼠收缩期左室前壁厚度(LVAWs)、舒张期左室前壁厚度(LVAWd)、收缩期左室后壁厚度(LVPWs)、舒张期左室前壁厚度(LVPWd)进行检测。
2.3.2心室重量指数检测
超声心动图检测和取血完毕后，将各组大鼠称重后，采用劲椎脱臼法处死，迅速打开胸腔摘取心脏，肉眼观察后，剪去周围大血管，在冰等渗盐水中洗净血液，滤纸吸干水后，沿房室交界处去除左右心房，用电子天平(精确度0.0001g)称取心室重量(ventricular weight，VW)，计算心室重量指数VW1(VW/大鼠体重)
2.3.3心肌组织观察
心室重量指数称重完毕后，将所得到的各组心脏组织进行脱水、包埋、切片和HE染色，然后置于高倍显微镜下观察。
3.实验结果
3.1心脏超声仪器检测结果
表6心脏超声仪器检测结果
Figure PCTCN2016072121-appb-000008
实验结果如表6所示：(1)HCM组与NC组大鼠相比，p<0.01；肥厚型心肌病模型小鼠的心室壁比野生小鼠明显厚肥许多。(2)Metoprolol治疗组与HCM组相比，各组数据均有了明显的下降，心室壁明显变薄，两组之间具有非常显著性差异p<0.01，与NC组相比，没有显著性差异p>0.05。(3)LYS治疗组中，各组数据也均有了一定程度的下降，与HCM组相比，表现出非常显著性差异p<0.01，与NC组间无显著性差异；与Metoprolol组相比，二者的治疗效果没有表现出统计学意义(p>0.05)。
3.2心室重量指数检测
心室重量指数VW1(VW/大鼠体重)的测定结果如图5所示：与NC组相比，HCM组的VW1明显较高，是典型的心肌肥大的现象，两组之间具有非常显著性差异(p<0.01)，LYS治疗组与Metoprolol治疗组的VW1均有较大幅度的下降。图5中与NC组相比，*p<0.05,**p<0.01；与HCM组相比，△p<0.05，△△p<0.01；与Metoprotol相比，×p<0.05，××p<0.01。
3.3心肌组织显微观察结果
HE染色之后，对各组小鼠的心肌组织进行观察后发现：与野生型的NC组相比，HCM组的小鼠其心肌细胞明显增大，形态变形，且排列紊乱。LYS治疗组合Metoprolol治疗组的小鼠，其心肌细胞也有一定程度的变大，但是基本排列整齐，致密，较HCM组有明显的改善。
4.统计学处理
连续型变量形式的实验数据以±sd表示，2组数据间的比较用两样本t检验。采用SPSS13.0统计软件，P<0.05为有统计学意义。
5.实验结果
本实施例以转基因的肥厚型心肌病小鼠作为实验对象，以Metoprolol作为阳性对照药物，实验结果显示LYS治疗组可以明显的改善肥厚型心肌病的心室壁厚度，显著改善心肌细胞形态，使心室指数有明显下调，且其治疗效果与阳性药物相当。绿原酸在治疗肥厚型心肌病中，反应出良好的药效。
实施例4绿原酸联合美托洛尔治疗大鼠扩张型心肌病的体内药效学考察
1.实验材料
1.1动物
出生两周，母乳喂养的Wistar大鼠，雌雄各半，共58只。
1.2实验药物及仪器
呋喃唑酮，10％水合氯醛，PHLIPsonos7500彩色超声检测仪，TUNEL凋亡检测试剂盒，BNP的ELISA检测试剂盒，万分之一的电子天平,乙二胺四乙酸二钠。2.实验方法
2.1大鼠扩张型心肌病(DCM)模型的建立和实验分组
取2周龄的Wistar大鼠58只，随机挑出10只大鼠，作为实验的正常对照组，喂以正常的鼠饲料。同时，其余48只作为造模大鼠，开始喂添加有呋喃唑酮的鼠饲料，呋喃唑酮的剂量按0.2mg/g体重给予，每周按体重调整一次用药剂量，正常对照组的大鼠和造模大鼠均喂养8周。
8周后，剔除死亡的5只大鼠，剩余的43只大鼠，经超声心动图证实患有扩张型心肌病(DCM)的有43只，随机舍弃3只后，将剩余的40只患有DCM的模型大鼠随机平均分为4组，分别命名为：模型对照组(DCM组，n＝10)、美托洛尔治疗组(Metoprolol组，n＝10)、绿原酸低剂量治疗组(LYS组，n＝10)、绿原酸联合美托洛尔治疗组(LM组，n＝10)，连同正常对照组(NC组，n＝10)，共5个实验组，共50只。
2.2分组给药实验
本实验采用腹腔注射给药的方式对各组大鼠进行给药治疗，其具体的给药方案如表7所示：
表7.实验分组、给药方式及给药量(注：所有给药组的给药体积相同)
Figure PCTCN2016072121-appb-000009
2.3观察指标及测定
2.3.1超声检测仪评估心脏结构和功能(实验第90天)
给药90天后，将各组大鼠禁食12h、禁饮2h后，用10％水合氯醛麻醉后，使用 PHLIPsonos7500彩色超声检测仪进行心动图检查：测量左室舒张末期内径(LVEDd)、左室收缩末期内径(LVESd)、左室射血分数(EF％)。
2.3.2血浆生化和BNP检测
待超声心动图检测完毕后，将各组大鼠分别取全血4.0mL左右，EDTA-2Na抗凝，静置30min后，2000g/min离心10min，分离血浆，于-70℃的冰箱内保存。血钾和血肌酐按常规方法检测，BNP用ELISA试剂盒直接检测。
2.3.3TUNEL法检测心肌细胞凋亡指数
使用TUNEL凋亡检测试剂盒检测心肌细胞的凋亡情况，将2.3.3中所取出的心脏标本拼凑恢复原状，用预冷的PBS冲洗，心脏中段横断面切取2～3mm厚的组织1块，放入装有10％中性缓冲福尔马林的小瓶中，入4℃冰箱固定过夜(不超过24h)，再经过脱水、浸渍和包埋等步骤，制成石蜡块。石蜡块常规切片后，应用TUNEL法原位标记凋亡细胞核，操作严格按照试剂盒的说明书进性。征程的心肌细胞核呈蓝色，而凋亡的心肌细胞核呈深浅不一的棕褐色。心肌细胞凋亡指数(CMAI)的计算方法：凋亡的心肌细胞数/心肌细胞总数×100％。
3.实验结果
3.1超声心动图检查结果
表8超声心动图检测结果(±sd)
Figure PCTCN2016072121-appb-000010
与NC组相比a:p<0.05；与DCM组相比b:p<0.05；与Metoprolol组相比c:p<0.05；与绿原酸组先比d:p<0.05。
实验结果如表8所示：(1)DCM组与NC组大鼠相比，各项数据两两比较均具有显著差异(p<0.05)提示造模效果良好；(2)Metoprolol治疗组、绿原酸治疗组及联合给药组的各组数据，分别与DCM组的各组数据相比，LVEDd(mm)和LVESd(mm)均有了明显的降低，而EF(％)也均有一定程度的回升。其中，绿原酸单独治疗组的各组数据与NC组相比，不存在统计学差异。Metoprolol治疗组的各组数据与NC组相比，尚有显著性差异 (p<0.05)。(3)联合给药(LM组)的各组数据比单独使用Metoprolol或单独使用绿原酸均更加的接近NC组的各组数据，其EF(％)组的数据也恢复到临床EF(％)正常值(50％以上)的水平，显示出联合用药相比与其它各种给药方式的治疗优越性。
3.2血浆生化和BNP检测结果
表3血浆生化和BNP检测实验结果(±sd)
Figure PCTCN2016072121-appb-000011
实验结果如表9所示：各组的血钾和血肌酐水平，两两相比之后均没有统计学差异(p>0.05)；与NC组相比，DCM组的BNP值有了明显的升高，具有显著性差异p<0.05，提示BNP含量增加是扩张型心肌病的病理特征之一。Metoprolol组、LYS组和LM组的BNP值，较DCM组均有了一定程度的下降，其分别与NC组相比，均没有表现出统计学差异(P>0.05)。其中，联合给药组的下降趋势最为明显，其BNP的结果也最接近正常值。
3.3TUNEL法检测心肌细胞凋亡指数
用TUNEL法检测心肌细胞凋亡的实验中，结果如图6所示，各治疗组与DMC组相比，凋亡指数均有明显的恢复，分别与DMC相比均有显著性差异p<0.05，其中联合给药组凋亡指数的恢复程度最为显著，表现出更为明显的抑制心肌细胞凋亡的疗效。
4统计学处理
连续型变量形式的实验数据以±sd表示，2组数据间的比较用两样本t检验。采用SPSS13.0统计软件，P<0.05为有统计学意义。
5，实验结果
本实施例中，采用呋喃唑酮的方法成功建立了大鼠扩张型心肌病模型，为了考察绿原酸与Metoprolol联合用药的用药效果，特设计了联合给药组，并将所有试验结果与Metoprolol单独治疗组以及绿原酸单独治疗组的结果进行全面的比较，以上实验结果显示，绿原酸可以有效的改善心功能，明显的减轻心室负荷，且中、高剂量腹腔注射绿原酸后的部分治疗效果，优于阳性对照药物Metoprolol治疗组，显示其对扩张型心肌病具有较好的治疗效果。结果显 示，联合给药组在所有给药治疗组中，能更为有效的改善扩张型心肌病的超声心动各指数(左室舒张末期内径、左室收缩末期内径、左室射血分数)，调节模型大鼠的血清BNP含量，此外，联合用药在抑制心肌细胞的凋亡方面也显示出更为突出的效果。综上，绿原酸可以联合治疗心肌病的有效药物(Metoprolol)有效的治疗扩张型心肌病，而不限于此类心肌病的治疗。
实施例5：用绿原酸制备冻干粉针剂
1.绿原酸的提取：
本实施例中所用的绿原酸原料药，系由金银花中提取、纯化得到，纯度为99.42％。
2.绿原酸冻干粉针剂的制备
2.1处方：
纯度为99.42％的绿原酸(主药) 50g
甘露醇(支撑剂) 60g
亚硫酸氢钠(抗氧化剂) 5g
将以上处方完全溶解于注射用水中，过滤后，再用0.22μm的除菌微孔滤膜精滤，调节pH后，按照无菌粉针剂的常规操作共制成2ml粉针剂1000支，每支含绿原酸50mg。
实施例6：用绿原酸制备丸剂
1.绿原酸的提取
本实施例中所使用的绿原酸，系由牛蒡子中提取、纯化得到的，纯度为98.41％。
2.绿原酸丸剂的制备
2.1处方
纯度为98.41％的绿原酸(主药) 1g
淀粉(稀释剂) 50g
糊精粉(粘合剂) 适量
无水乙醇(润湿剂) 适量
2.2.制法：
取适量的聚维酮K30，用无水乙醇配制成溶液，再取处方量的绿原酸和淀粉，采用等量稀释法混合均匀之后，加入糊精粉的乙醇溶液中，充分搅拌后制得软材，采用搓丸法制得绿原酸丸剂1000粒，每粒丸剂含绿原酸1mg。
实施例7：用绿原酸制备口服溶液剂
1.绿原酸的提取：本实施例中所使用的绿原酸，系由杜仲叶中提取、纯化得到，纯度为99.39％。
1.绿原酸口服溶液剂的制备
2.1处方
纯度为99.39％的绿原酸(主药) 20g
亚硫酸氢钠(抗氧化剂) 10g
注射用水(溶剂) 10L
2.2制法
取处方量的绿原酸和亚硫酸氢钠，溶解于10L注射用水中，按照口服液的常规制备工艺，过滤后，无菌灌装成100支口服液，每支口服液为100mL，含绿原酸200mg。
实施例8：用绿原酸制备片剂
1.绿原酸的提取：
本实施例中所使用的绿原酸，系由金银花中提取、纯化得到，纯度为99.17％。
2.绿原酸片剂的制备
2.1处方：
纯度为99.17％的绿原酸(主药) 100g
糖粉(填充剂) 100g
乳糖(填充剂) 200g
羟丙甲纤维素(粘合剂) 50g
硬脂酸镁(润滑剂) 50g
2.2制法：
本实施例采用制湿颗粒压片法制备绿原酸片剂。(1)按处方量取羟丙甲纤维素制成水溶液；(2)取处方量的绿原酸、糖粉和乳糖混合均匀后，加入羟丙甲纤维素水溶液，充分搅拌均匀后制成软材；(3)将制备好的软材按常规的湿法制粒的操作规程，过筛、干燥和整粒后得到大小均一的颗粒；(4)将制得的颗粒与硬脂酸镁混合均匀后压片，共制成1000片剂，每片含绿原酸100mg。
实施例9：用绿原酸制备胶囊剂
1.绿原酸的提取：
本实施例中所使用的绿原酸，系由杜仲叶中提取、纯化得到的，纯度为99.35％。
2.绿原酸胶囊剂的制备：
2.1处方：
纯度为99.35％的绿原酸 100g
糖粉 200g
2.2制法：
取处方量的绿原酸和淀粉，混合均匀，加入80％乙醇溶液制成软材，干燥，整粒后按照胶囊剂的常规制备工艺制备2000粒胶囊，每粒胶囊含绿原酸50mg。
实施例10：用绿原酸制备颗粒剂
1.绿原酸的提取
本实施例中所使用的绿原酸，系由杜仲叶中提取、纯化得到的，纯度为99.66％。
2.绿原酸颗粒剂的制备
2.1处方：
纯度为99.66％的绿原酸(主药) 200g
甘露醇(稀释剂) 100g
蔗糖(稀释剂) 400g
聚维酮K30(粘合剂) 适量
2.2制法：
取聚维酮K30，加入注射用水，制成溶液。取处方量的绿原酸、甘露醇和乳糖混合均匀之后，加入聚维酮K30溶液，制成软材。按照颗粒剂的常规制备工艺，对软材进行过筛、干燥和整粒之后，得到颗粒剂。在无菌条件下分装颗粒剂，制备400袋颗粒剂，每袋颗粒剂含绿原酸500mg。
实施例11：用绿原酸制备散剂
1.绿原酸的提取：本实施例所用的绿原酸原料药，系由牛蒡叶中提取、纯化得到的，纯度为98.97％。
2.绿原酸散剂的制备：
2.1处方
纯度为98.97％的绿原酸1000g；
2.2制法
取处方量绿原酸过筛后，按照散剂的常规制备工艺，无菌分装成含1000瓶/袋散剂，每瓶/袋散剂含绿原酸1000mg。

Claims (10)

  1. 一种绿原酸在制备治疗心肌病的药物中的用途。
  2. 如权利要求1所述的用途，其中所述心肌病优选为扩张型心肌病和肥厚型心肌病。
  3. 如权利要求1或2所述的用途，所述药物上调心肌特异性表达结构基因ACTC1及对应蛋白。
  4. 如权利要求1-3任一项所述的用途，所述药物下调caspase-3基因及对应蛋白。
  5. 如权利要求1-3任一项所述的用途，所述药物上调Bcl-2基因及对应蛋白。
  6. 一种用于治疗心肌病的药物，所述的药物包括绿原酸，药学上可接受的辅料或辅助性成分。
  7. 如权利要求6所述药物，所述的药物是口服制剂和注射制剂。
  8. 如权利要求7所述药物，,每单位制剂含绿原酸1-1000mg，临床使用剂量为1-100mg/kg。
  9. 一种治疗心肌病的联合用药物，所述联合用药物包括绿原酸和治疗心肌病的药物。
  10. 如权利要求9所述的联合用药物，其中治疗心肌病的药物选自心得安、氨酰心安、美托洛尔、比索洛尔、异搏定、硫氮卓酮、乙胺碘呋酮、双异丙比胺中的一种或几种。
PCT/CN2016/072121 2015-02-13 2016-01-26 绿原酸在制备治疗心肌病的药物中的用途 WO2016127787A1 (zh)

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CN115631871A (zh) * 2022-12-22 2023-01-20 北京大学第三医院（北京大学第三临床医学院） 一种药物相互作用等级的确定方法和装置

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CN109419840A (zh) * 2017-08-22 2019-03-05 北京中医药大学 金银花的新用途
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