技术领域technical field
本发明属于生物化学中的多肽药物技术领域，涉及由僵蚕(Bombyx Batryticatus)中分离纯化的一种抗血小板聚集多肽，尤其涉及该多肽的制备方法以及作为抗血栓药物的应用。The invention belongs to the technical field of polypeptide drugs in biochemistry, and relates to an anti-platelet aggregation polypeptide isolated and purified from Bombyx Batryticatus, in particular to a preparation method of the polypeptide and an application as an antithrombotic drug.
背景技术Background technique
心脑血管疾病是一大类包括急性心肌梗死、缺血性心脏病、心脏瓣膜病、周围性血管疾病、心率不齐，高血压以及脑卒中等的疾病，是严重威胁人类身体健康的重要疾病之一。而其中重要的病因是血栓的形成。诱导血管内血栓形成的因素有很多种，血小板聚集性过高是促使心血管疾病发病的重要因素。随着人口的老龄化，其发病率在快速增高。因此，快速研究开发抗血栓药物具有重要意义。Cardiovascular and cerebrovascular diseases are a large category of diseases including acute myocardial infarction, ischemic heart disease, valvular heart disease, peripheral vascular disease, arrhythmia, hypertension and stroke, and are important diseases that seriously threaten human health. one. And one of the important etiology is the formation of thrombus. There are many factors that induce intravascular thrombosis, and high platelet aggregation is an important factor that promotes the onset of cardiovascular diseases. With the aging of the population, its incidence is increasing rapidly. Therefore, rapid research and development of antithrombotic drugs is of great significance.
以抗血小板聚集为靶点的抗血栓药物具有较好的专一性，一直受到广泛关注。抗血小板聚集药物在临床上的使用最为广泛，在血栓性疾病的治疗中发挥重要作用。但常用的一线药物还是有一些严重的副作用，如阿司匹林的胃肠反应、出血以及出现抵抗的问题；噻氯匹定和氯吡格雷的导致粒细胞减少的问题。近年来，国外大量文献报道了由天然动植物，微生物诸如蜈蚣、大豆和食用真菌中分离纯化的具有抗血小板聚集的蛋白质和多肽。从天然生物中发现和鉴定新的抗血小板聚集多肽仍然是抗血栓研究的一个重要方向。Antithrombotic drugs targeting antiplatelet aggregation have good specificity and have been widely concerned. Antiplatelet aggregation drugs are the most widely used clinically and play an important role in the treatment of thrombotic diseases. However, the commonly used first-line drugs still have some serious side effects, such as gastrointestinal reactions, bleeding, and resistance to aspirin; ticlopidine and clopidogrel can cause neutropenia. In recent years, a large number of foreign literatures have reported proteins and polypeptides with anti-platelet aggregation properties isolated and purified from natural animals and plants, microorganisms such as centipedes, soybeans and edible fungi. Discovering and identifying new anti-platelet aggregation polypeptides from natural organisms is still an important direction of anti-thrombotic research.
僵蚕为传统中药，是蚕蛾科昆虫家蚕(Bombyx mori)4-5龄的幼虫感染(或人工接种)白僵菌(Beauveria bassiana)而致死的干燥体。药典载：味咸辛，性平，归肝、肺、胃经，具有息风止痉、祛风止痛、祛风热、化痰镇咳、活络通经、解毒散结等功效。临床广泛用于惊风抽搐，咽喉肿痛，颌下淋巴结炎，面神经麻痹，皮肤瘙痒，热症哮喘，乳部癖块等病症。现代药理学研究表明：僵蚕含有脂肪4.4％、蛋白质67％，有10多种氨基酸和镁、钙、锌、铁等20多种元素，还有多种生物活性物质。僵蚕所含的蛋白质，有促肾上腺皮质作用，全品具有抗惊厥、抗凝、降血糖和一定的抗肿瘤作用。Bombyx mori is a traditional Chinese medicine, which is the dead body of the 4-5 instar larvae of Bombyx mori infected (or artificially inoculated) with Beauveria bassiana. According to the Pharmacopoeia: salty and pungent, neutral in nature, it belongs to the liver, lung, and stomach meridian. It is widely used clinically for convulsions and convulsions, sore throat, submandibular lymphadenitis, facial paralysis, skin itching, fever asthma, breast lumps and other diseases. Modern pharmacological research shows that silkworm contains 4.4% fat, 67% protein, more than 10 kinds of amino acids, more than 20 kinds of elements such as magnesium, calcium, zinc and iron, and many biologically active substances. The protein contained in silkworm has adrenal cortex-stimulating effect, and the whole product has anticonvulsant, anticoagulant, hypoglycemic and certain antitumor effects.
本发明从僵蚕中分离纯化出一个由八个氨基酸组成的多肽，通过体外兔血小板聚集实验来测定其抗血小板聚集活性。实验证实，该多肽对胶原诱导的血小板聚集有显著的抑制作用。同时应用小鼠急性肺血栓栓塞模型实验。结果表明，该多肽对小鼠死亡或偏瘫症状有较好的保护作用。该多肽有望发展成新型抗血栓药物。The present invention isolates and purifies a polypeptide composed of eight amino acids from Bombyx mori, and measures its anti-platelet aggregation activity through an in vitro rabbit platelet aggregation experiment. Experiments have confirmed that the polypeptide has a significant inhibitory effect on collagen-induced platelet aggregation. At the same time, the model experiment of acute pulmonary thromboembolism in mice was applied. The results show that the polypeptide has a better protective effect on the death or hemiplegia symptoms of mice. The polypeptide is expected to be developed into a new type of antithrombotic drug.
发明内容Contents of the invention
本发明涉及一种对兔血小板聚集具有抑制活性的多肽，该多肽是僵蚕(BombyxBatryticatus)来源的。The present invention relates to a polypeptide with inhibitory activity on rabbit platelet aggregation, which is derived from Bombyx Batryticatus.
本发明所提供的具有抗血小板聚集活性的僵蚕多肽是利用Edman降解法测得其氨基酸序列的，序列为Asp-Pro-Asp-Ala-Asp-IIe-Leu-Gln。The amino acid sequence of the Bombyx mori polypeptide with anti-platelet aggregation activity obtained by Edman degradation method is Asp-Pro-Asp-Ala-Asp-IIe-Leu-Gln.
本发明所提供的具有抗血小板聚集活性的僵蚕多肽，经质谱测定其分子量为885Da。The Bombyx mori polypeptide provided by the present invention has anti-platelet aggregation activity, and its molecular weight is 885Da as determined by mass spectrometry.
本发明还涉及获得该多肽的方法。它主要包含下述步骤：The invention also relates to methods for obtaining the polypeptide. It mainly includes the following steps:
[1]取生僵蚕粉碎匀浆，有机溶剂回流提取，收集上清。[1] Raw silkworms were crushed and homogenized, the organic solvent was refluxed for extraction, and the supernatant was collected.
[2]上清液去掉有机溶剂后浓缩、冻干，缓冲液重新溶解，Sephadex G-50凝胶过滤层析，得到活性峰B；[2] The supernatant was concentrated after removing the organic solvent, freeze-dried, redissolved in the buffer, and subjected to gel filtration chromatography on Sephadex G-50 to obtain the active peak B;
[3]峰B部分再经过Source30Q阴离子交换色谱后，得到活性峰B3；[3] After the part of peak B is subjected to Source30Q anion exchange chromatography, the active peak B3 is obtained;
[4]峰B3部分最后通过C8柱RP-HPLC纯化，得到活性峰B3-6，收集冻干，命名为BB octapeptide。[4] The part of peak B3 was finally purified by C8 column RP-HPLC to obtain the active peak B3-6, which was collected and lyophilized and named as BB octapeptide.
本发明所提供的多肽，经测试具有抗血小板聚集活性：在给药剂量100μg/ml至1000μg/ml范围内可抑制胶原诱导的血小板聚集，且呈现剂量依赖关系。The polypeptide provided by the present invention has been tested to have anti-platelet aggregation activity: it can inhibit collagen-induced platelet aggregation in a dose range of 100 μg/ml to 1000 μg/ml, and presents a dose-dependent relationship.
本发明所提供的多肽，经测试对小鼠急性肺血栓栓塞模型具有较好的保护作用：在连续4日尾静脉给药，给药剂量分别10，30，50mg/kg时，对小鼠死亡或偏瘫症状有较好的保护作用，且呈现剂量依赖关系。The polypeptide provided by the present invention has a good protective effect on the acute pulmonary thromboembolism model in mice after testing: when administered through the tail vein for 4 consecutive days, when the dosage is 10, 30, and 50 mg/kg, the mice die Or hemiplegia symptoms have a better protective effect, and presents a dose-dependent relationship.
附图说明Description of drawings
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明发明范围。The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.
图1为僵蚕回流提取后上清液Sephadex G-50凝胶过滤层析图：活性峰为峰B。Figure 1 is the gel filtration chromatogram of the supernatant Sephadex G-50 after the silkworm reflux extraction: the active peak is peak B.
图2为峰B部分Source30Q阴离子交换色谱图：活性峰为峰B3。Figure 2 is the Source30Q anion exchange chromatogram of peak B part: the active peak is peak B3.
图3为峰B3部分RP-HPLC色谱图：活性峰为峰B3-6，即BB octapeptide。Fig. 3 is the RP-HPLC chromatogram of peak B3 part: active peak is peak B3-6, namely BB octapeptide.
图4为BB octapeptide的化学结构式。Figure 4 is the chemical structural formula of BB octapeptide.
具体实施方式Detailed ways
实施例1：BB octapeptide的制备Embodiment 1: Preparation of BB octapeptide
(1)取白僵蚕，浸泡清洗后，粉碎匀浆，按照W/V1∶6比例添加60％乙醇。回流3次，时间分别为1h、40min、20min，合并3次回流液体，回流温度80℃。(1) Take white silkworm, soak and wash it, pulverize and homogenate, and add 60% ethanol according to the ratio of W/V 1:6. Reflux 3 times, the time is 1h, 40min, 20min respectively, and combine the reflux liquid for 3 times, and the reflux temperature is 80°C.
(2)将回流液用微孔滤膜除去颗粒，并将所得滤液减压浓缩，然后离心10000×g30min，取上清。将上清液减压浓缩和冻干。(2) Use a microporous membrane to remove particles from the reflux liquid, concentrate the obtained filtrate under reduced pressure, then centrifuge at 10000×g for 30 min, and take the supernatant. The supernatant was concentrated under reduced pressure and lyophilized.
(3)将冻干后的僵蚕提取粉末溶解于少量20mM PBS PH7.6，Sephadex G-50凝胶过滤层析。洗脱液20mM PBS PH7.6，流速0.3ml/min，收集单位3ml。得到4个峰，收集第3个峰，即峰B，冻干，如图1所示。(3) Dissolve the lyophilized Bombyx mori extract powder in a small amount of 20mM PBS pH7.6, and perform gel filtration chromatography on Sephadex G-50. The eluent is 20mM PBS pH7.6, the flow rate is 0.3ml/min, and the collection unit is 3ml. 4 peaks were obtained, and the third peak, peak B, was collected and freeze-dried, as shown in Figure 1.
(4)峰B溶解于少量10mM Tris-HCl，PH7.6，Source30Q阴离子交换层析。洗脱液A：10mM Tris-HCl，PH7.6；B：10mM Tris-HCl，PH7.6+1M NaCl。流速2ml/min。洗脱程序：0-25ml，0％B；25-125ml，0％B-50％B。得到5个峰，收集第3个峰，即峰B3，冻干，如图2所示。(4) Peak B was dissolved in a small amount of 10mM Tris-HCl, pH7.6, Source30Q anion exchange chromatography. Eluent A: 10mM Tris-HCl, pH7.6; B: 10mM Tris-HCl, pH7.6+1M NaCl. Flow rate 2ml/min. Elution program: 0-25ml, 0%B; 25-125ml, 0%B-50%B. Five peaks were obtained, and the third peak, peak B3, was collected and freeze-dried, as shown in FIG. 2 .
(5)峰B3溶解于少量50％乙腈+0.1％三氟乙酸。C8柱RP-HPLC纯化，洗脱液A：水+0.1％三氟乙酸；B：乙腈+0.1％三氟乙酸。流速0.7ml/min。洗脱程序：0-5ml，0％B；5-55ml，0％B-60％B。得到7个峰，收集第6个峰，即峰B3-6，即BB octapeptide，冻干，如图3所示。(5) Peak B3 was dissolved in a small amount of 50% acetonitrile + 0.1% trifluoroacetic acid.C8 column RP-HPLC purification, eluent A: water + 0.1% trifluoroacetic acid; B: acetonitrile + 0.1% trifluoroacetic acid. Flow rate 0.7ml/min. Elution program: 0-5ml, 0%B; 5-55ml, 0%B-60%B. Seven peaks were obtained, and the sixth peak, ie, peak B3-6, BB octapeptide, was collected and lyophilized, as shown in FIG. 3 .
(6)BB octapeptide利用Edman降解法测得其氨基酸序列为Asp-Pro-Asp-Ala-Asp-IIe-Leu-Gln。其化学结构式如图4所示。经质谱测定其分子量为885Da。(6) The amino acid sequence of BB octapeptide determined by Edman degradation method was Asp-Pro-Asp-Ala-Asp-IIe-Leu-Gln. Its chemical structural formula is shown in Figure 4. Its molecular weight was determined to be 885Da by mass spectrometry.
实施例2：BB octapeptide的体外抗血小板聚集活性Example 2: In vitro anti-platelet aggregation activity of BB octapeptide
(1)将雄性家兔麻醉，颈总动脉取血置于含有lml的3.2％枸橼酸钠的15ml离心管中，(每管终体积约为10ml，保证兔血和抗凝剂体积比为9∶1)，离心120×g10min，20℃，得到上层液即富血小板血浆。余下血样以1600×g10min离心，得到贫血小板血浆。(1) male rabbit is anesthetized, common carotid artery blood is taken and placed in the 15ml centrifuge tube containing 3.2% sodium citrate of 1ml, (every tube final volume is about 10ml, guarantees that rabbit blood and anticoagulant volume ratio are 9:1), centrifuged at 120×g for 10 min at 20° C. to obtain the supernatant, namely platelet-rich plasma. The remaining blood samples were centrifuged at 1600×g for 10 min to obtain platelet-poor plasma.
(2)吸取300μl贫血小板血浆置于血小板聚集仪测试区调零，再吸取270μl富血小板血浆置于预热槽中，分别加入生理盐水配置的不同浓度梯度的BB octapeptide30μl(100，300，600，900，1000μg/ml)和测试珠，预热37℃，1min后置于测试区，加入诱导剂胶原30μl(终浓度2μg/ml)，测定5min内血小板最大聚集率。其中，空白对照为生理盐水。通过以下公式计算血小板聚集抑制率：[(X-Y)/X]×100％。X代表生理盐水组血小板最大聚集率；Y代表各浓度多肽组血小板最大聚集率。(2) Take 300 μl of platelet-poor plasma and place it in the test area of the platelet aggregation meter to adjust to zero, then draw 270 μl of platelet-rich plasma and place it in the preheating tank, add 30 μl of BB octapeptide with different concentration gradients prepared in normal saline (100, 300, 600, 900, 1000 μg/ml) and test beads, preheated at 37°C, placed in the test area after 1 min, added 30 μl of inducer collagen (final concentration 2 μg/ml), and measured the maximum aggregation rate of platelets within 5 min. Wherein, the blank control was physiological saline. The platelet aggregation inhibition rate was calculated by the following formula: [(X-Y)/X]×100%. X represents the maximum aggregation rate of platelets in the normal saline group; Y represents the maximum aggregation rate of platelets in the polypeptide groups of various concentrations.
(3)该多肽经血小板聚集实验，BB octapeptide能显著抑制胶原诱导的兔血小板聚集，呈现剂量依赖关系，其结果如下：(3) According to the platelet aggregation test of the polypeptide, BB octapeptide can significantly inhibit collagen-induced platelet aggregation in rabbits, showing a dose-dependent relationship. The results are as follows:
BB octapeptide对胶原诱导的兔血小板聚集的抑制作用Inhibitory effect of BB octapeptide on collagen-induced platelet aggregation in rabbits
从以上结果可以看出，BB octapeptide对胶原诱导的兔血小板聚集有抑制作用，呈现剂量依赖关系。From the above results, it can be seen that BB octapeptide has an inhibitory effect on collagen-induced platelet aggregation in rabbits, showing a dose-dependent relationship.
实施例3：BB octapeptide的小鼠急性肺血栓栓塞模型的保护作用Example 3: Protective effect of BB octapeptide in mouse acute pulmonary thromboembolism model
(1)取健康ICR小鼠36只，体重20-25g，分为4组，每组9只。BB octapeptide以生理盐水稀释成不同剂量梯度10，30和50mg/kg，阴性对照为生理盐水。以上各组均尾静脉给药，每日1次，连续4日，给药容积为10ml/kg。(1) Take 36 healthy ICR mice, weighing 20-25 g, and divide them into 4 groups with 9 mice in each group. BB octapeptide was diluted with normal saline into different dose gradients of 10, 30 and 50 mg/kg, and the negative control was normal saline. All the above groups were given tail vein administration, once a day, for 4 consecutive days, and the administration volume was 10ml/kg.
(2)末次给药后15min，尾静脉注射胶原12mg/kg和肾上腺素1mg/kg混合诱导剂，形成急性肺血栓栓塞，记录注射混合诱导剂后15min内各组死亡或者偏瘫(30s内失去正直反射)的小鼠的数量。通过以下公式计算急性肺血栓栓塞保护率：[1-(X/Y)]×100％。X代表各组死亡或者偏瘫的小鼠的数量；Y代表各组的小鼠总数量。(2) 15 minutes after the last administration, a mixed inducer of collagen 12 mg/kg and epinephrine 1 mg/kg was injected into the tail vein to form acute pulmonary thromboembolism, and the death or hemiplegia of each group within 15 minutes after the injection of the mixed inducer was recorded (loss of integrity within 30 s) reflex) the number of mice. The protection rate of acute pulmonary thromboembolism was calculated by the following formula: [1-(X/Y)]×100%. X represents the number of dead or hemiplegic mice in each group; Y represents the total number of mice in each group.
(3)该多肽经小鼠急性肺血栓栓塞模型实验，BB octapeptide对小鼠死亡或偏瘫症状有较较好的保护作用，且呈现剂量依赖关系，其结果如下：(3) The peptide was tested in a mouse model of acute pulmonary thromboembolism, and BB octapeptide had a relatively good protective effect on mouse death or hemiplegia symptoms, and it showed a dose-dependent relationship. The results are as follows:
BB octapeptide对小鼠急性肺血栓栓塞模型的保护作用Protective effect of BB octapeptide on acute pulmonary thromboembolism model in mice
从以上结果可以看出，剂量为30和50mg/kg时，BB octapeptide对小鼠死亡或偏瘫症状的保护率分别为33.3％和44.4％。BB octapeptide对小鼠急性肺血栓栓塞模型有较好的保护作用，呈现剂量依赖关系。From the above results, it can be seen that when the dose is 30 and 50 mg/kg, the protection rates of BB octapeptide on the death or hemiplegia symptoms of mice are 33.3% and 44.4%, respectively. BB octapeptide has a better protective effect on the acute pulmonary thromboembolism model in mice, showing a dose-dependent relationship.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310069320.8ACN103113456B (en) | 2013-03-05 | 2013-03-05 | Stiff silkworm polypeptide with antiplatelet aggregation activity as well as preparation method and application of stiff silkworm polypeptide |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310069320.8ACN103113456B (en) | 2013-03-05 | 2013-03-05 | Stiff silkworm polypeptide with antiplatelet aggregation activity as well as preparation method and application of stiff silkworm polypeptide |
| Publication Number | Publication Date |
|---|---|
| CN103113456A CN103113456A (en) | 2013-05-22 |
| CN103113456Btrue CN103113456B (en) | 2014-07-16 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310069320.8AExpired - Fee RelatedCN103113456B (en) | 2013-03-05 | 2013-03-05 | Stiff silkworm polypeptide with antiplatelet aggregation activity as well as preparation method and application of stiff silkworm polypeptide |
| Country | Link |
|---|---|
| CN (1) | CN103113456B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107033220B (en)* | 2016-10-17 | 2019-12-24 | 昆明医科大学第一附属医院 | A platelet aggregation-stimulating polypeptide KM13 |
| CA3078625C (en) | 2017-10-09 | 2023-01-17 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| CN109125355B (en)* | 2018-08-03 | 2022-07-08 | 昆明医科大学第一附属医院 | Antiplatelet and antithrombotic pharmacological application of Xinmailong |
| CN109521132A (en)* | 2018-11-16 | 2019-03-26 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of method for building up of bombyx batryticatus HPLC finger-print |
| EP3938742A1 (en) | 2019-03-14 | 2022-01-19 | Terumo BCT Biotechnologies, LLC | Multi-part lyophilization container and method of use |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0628571A1 (en)* | 1992-09-07 | 1994-12-14 | Nippon Steel Corporation | Novel peptide, and antithrombotic agent, anticoagulant for extracorporeal circulation, cell fusion inhibitor, cancer metastasis inhibitor, protective for platelet preparation for transfusion and pack of platelet preparation for transfusion |
| CN101239178A (en)* | 2008-03-13 | 2008-08-13 | 中国药科大学 | Antithrombotic application of a kind of peptide compound |
| CN101921326A (en)* | 2010-04-27 | 2010-12-22 | 中国药科大学 | A recombinant antithrombotic peptide mutant, preparation method and use |
| CN102020699A (en)* | 2009-09-10 | 2011-04-20 | 秦引林 | Anti-platelet aggregation peptide and application thereof |
| CN102060908A (en)* | 2010-09-17 | 2011-05-18 | 中国药科大学 | Novel synthetic antithrombotic polypeptide, and preparation method and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992007870A1 (en)* | 1990-11-02 | 1992-05-14 | Genentech, Inc. | Platelet aggregation inhibitors |
| ATE284896T1 (en)* | 2000-02-23 | 2005-01-15 | Zealand Pharma As | NEW ANTIARRYTHMIC PEPTIDES |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0628571A1 (en)* | 1992-09-07 | 1994-12-14 | Nippon Steel Corporation | Novel peptide, and antithrombotic agent, anticoagulant for extracorporeal circulation, cell fusion inhibitor, cancer metastasis inhibitor, protective for platelet preparation for transfusion and pack of platelet preparation for transfusion |
| CN101239178A (en)* | 2008-03-13 | 2008-08-13 | 中国药科大学 | Antithrombotic application of a kind of peptide compound |
| CN102020699A (en)* | 2009-09-10 | 2011-04-20 | 秦引林 | Anti-platelet aggregation peptide and application thereof |
| CN101921326A (en)* | 2010-04-27 | 2010-12-22 | 中国药科大学 | A recombinant antithrombotic peptide mutant, preparation method and use |
| CN102060908A (en)* | 2010-09-17 | 2011-05-18 | 中国药科大学 | Novel synthetic antithrombotic polypeptide, and preparation method and application thereof |
| Title |
|---|
| Cyclic RGD Peptide Analogues as Antiplatelet Antithrombotics;Peter L.Barker et.al.,;《Journal of Medicinal Chemistry》;19921231;第35卷(第11期);全文* |
| JP特表平6-502407A 1994.03.17 |
| Peter L.Barker et.al.,.Cyclic RGD Peptide Analogues as Antiplatelet Antithrombotics.《Journal of Medicinal Chemistry》.1992,第35卷(第11期),全文. |
| 僵蚕抗凝作用的研究进展;雷田香等;《湖南中医药大学学报》;20070630;第27卷(第3期);全文* |
| 僵蚕抗凝成分对血小板聚集的抑制效应;彭延古等;《血栓与止血学》;20071231;第13卷(第2期);全文* |
| 彭延古等.僵蚕抗凝成分对血小板聚集的抑制效应.《血栓与止血学》.2007,第13卷(第2期),全文. |
| 雷田香等.僵蚕抗凝作用的研究进展.《湖南中医药大学学报》.2007,第27卷(第3期),全文. |
| Publication number | Publication date |
|---|---|
| CN103113456A (en) | 2013-05-22 |
| Publication | Publication Date | Title |
|---|---|---|
| CN103113456B (en) | Stiff silkworm polypeptide with antiplatelet aggregation activity as well as preparation method and application of stiff silkworm polypeptide | |
| EP1927363B1 (en) | An extract for preventing or treating thrombotic diseases | |
| CN111233972B (en) | Anti-inflammatory tripeptide, extraction and separation method thereof and application of anti-inflammatory tripeptide in memory improvement | |
| CN111153963B (en) | Anti-inflammatory pentapeptide, extraction and separation method thereof and application of anti-inflammatory pentapeptide in memory improvement | |
| CN104004806A (en) | Lumbricus polypeptide having anticoagulant and thrombolytic effects, and enzymatic hydrolysis preparation method and application thereof | |
| CN111253466B (en) | Anti-inflammatory tetrapeptide, extraction and separation method thereof and application of anti-inflammatory tetrapeptide in preparation of memory improving medicines | |
| CN109527270A (en) | Leech specific nutrient induces the extracting method of liquid and natural hirudin | |
| JP7097037B2 (en) | Hill log and its use | |
| CN116903724B (en) | A leech-derived polypeptide with both analgesic and antithrombotic functions, its precursor protein, and its application | |
| CN107898782A (en) | A kind of ginkgo diterpenoid-lactone composition | |
| CN103387610B (en) | Method for separating and purifying cobra neurotoxin protein through dual-ion exchange chromatography, and preparation of cobra neurotoxin protein | |
| EP0506820A1 (en) | Neutrophil chemoattractants | |
| CN103145800B (en) | Centipede zymolyte anti-thrombus polypeptide | |
| CN109438556B (en) | Active peptide, recombinant vector, recombinant cell, anti-inflammatory composition, and preparation method and application thereof | |
| CN104672306B (en) | Preparation method and application of a polypeptide with antithrombotic activity | |
| CN111956781A (en) | Application of polypeptide in medicine for treating eye inflammation | |
| CN102311484A (en) | Hexapeptide for inhabiting angiotensin transferase and preparation method thereof | |
| CN108676074A (en) | A kind of active peptides of hepatic cell growth promotion | |
| CN110540585B (en) | Scorpion toxin active polypeptide HsTx1 with function of improving cerebral ischemia and hypoxia, and preparation method and application thereof | |
| CN103919799A (en) | Application of portulaca oleracea polysaccharide in preparation of drug for preventing or treating hepatic fibrosis | |
| CN105861474B (en) | Sipunculus nudus active protease with antithrombotic effect and preparation method and application thereof | |
| CN104800294B (en) | The extracting method of anti-tumor Chinese medicine extract and application | |
| CN116041427B (en) | ACE (angiotensin converting enzyme) inhibitory peptide derived from two miscellaneous cereals as well as preparation method and application thereof | |
| CN113730549B (en) | Application of milk-derived active peptide CCL-1S in preparation of sleep-promoting product | |
| CN119112967A (en) | Application of fermented ginseng hydrolysate in the preparation of products for improving inflammatory bowel disease |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee | Granted publication date:20140716 Termination date:20160305 |