WO2021204170A1 - 一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用 - Google Patents

一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用 Download PDF

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WO2021204170A1
WO2021204170A1 PCT/CN2021/085866 CN2021085866W WO2021204170A1 WO 2021204170 A1 WO2021204170 A1 WO 2021204170A1 CN 2021085866 W CN2021085866 W CN 2021085866W WO 2021204170 A1 WO2021204170 A1 WO 2021204170A1
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active peptide
turtle
soil
enzymatic hydrolysis
eluent
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PCT/CN2021/085866
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English (en)
French (fr)
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王少平
代龙
张加余
杨爱琳
姜珊
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滨州医学院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the present invention relates to the technical field of active peptides. More specifically, it relates to an active peptide with the effect of lowering blood lipids derived from Trionyx sinensis and its preparation method and application.
  • Hyperlipidemia is a common cardiovascular disease at present. With the improvement of people's living standards, high-calorie and high-fat diets continue to increase the incidence of hyperlipidemia. There are many complications of hyperlipidemia, such as fatty liver, atherosclerosis, diabetes, etc. Therefore, the prevention and treatment of hyperlipidemia is one of the current research hotspots.
  • the beetle is a traditional Chinese medicine, which has the effect of breaking blood and removing blood stasis, renewing the muscles and bones. It is used for bruises, muscle fractures, blood stasis amenorrhea, postpartum stasis to prevent abdominal pain and addiction to lumps. Modern studies have concluded that the soil turtle has the effect of treating blood and cerebrovascular diseases, such as atherosclerosis, hyperlipidemia, and cerebral thrombosis.
  • Proteins are mostly substances with a molecular weight of 100K or greater. After oral administration, they are mostly absorbed into the human body through gastrointestinal proteases such as pepsin and trypsin, which are absorbed into the human body and exert pharmacological effects. Therefore, protein has disadvantages such as low absorption efficiency and allergies.
  • the invention uses pepsin and trypsin to perform biomimetic enzymatic hydrolysis of the soil turtle in vitro to simulate the digestion of proteins in the body, so that the macromolecular protein can be cleaved into polypeptides in vitro, and the absorption efficiency is increased.
  • the decomposed peptides are separated by ultrafiltration, nanofiltration, macroporous resin, and gel molecular sieve chromatography to obtain peptide monomers with the function of promoting blood circulation and removing blood stasis.
  • the peptide monomers are of high purity, safe and effective; eliminating the use of foreign proteins, etc. Immunogenic reaction reduces the incidence of adverse reactions.
  • the first object of the present invention is to provide an active peptide derived from Trionyx sinensis.
  • the second object of the present invention is to provide a method for preparing the above-mentioned active peptide
  • the third object of the present invention is to provide the application of the above-mentioned active peptides in the preparation of drugs or health care products for preventing or treating blood lipids.
  • the present invention provides an active peptide derived from Trionyx sinensis, and the amino acid sequence of the active peptide is LAPAPGTL, as shown in SEQ ID NO:1.
  • the active peptide may also be an active peptide having 80% or more homology with the amino acid sequence of the above-mentioned active peptide, and the function of the active peptide and the above-mentioned active peptide is the same or similar.
  • the amino acid sequence of the active peptide may be a sequence having 85%, 90%, 95%, or 97% homology with the amino acid sequence of the above-mentioned active peptide.
  • the present invention also provides polynucleotides encoding the above-mentioned active peptides.
  • nucleotide sequence of the above-mentioned active peptide is ATGCTGGCACCAGCGCCGGGTACCCTG, as shown in SEQ ID NO: 2 in the sequence table.
  • the present invention provides a method for preparing the above-mentioned active peptide derived from Trionyx sinensis, which includes the following steps:
  • step (3) Use the DA201-C resin column to perform adsorption chromatography on the bionic enzymatic hydrolysate of the soil turtle prepared in step (2), and the ratio of the weight of DA201-C resin to the volume of the bionic enzymatic hydrolysate of the soil turtle is 5:1-10:1 After the adsorption is completed, 25% ethanol is used for elution; the eluent A is prepared;
  • step (3) Use a dextran G25 gel column to perform molecular sieve chromatography on the eluent A prepared in step (3).
  • the amount of eluent A added for each time is 0.5-2ml, and it is eluted with deionized water.
  • the eluent B prepared in step (4) is separated by RP-HPLC.
  • the chromatographic conditions used are chromatographic column: waters ZOBRX-300SB C18 chromatographic column, 150mm X 4.6um, the detector is UV detection Detector, detection wavelength: 220nm, 280nm, mobile phase: A phase 0.1% trifluoroacetic acid deionized aqueous solution (TFA), B phase 0.1% trifluoroacetic acid (TFA) acetonitrile solution; use gradient elution method for analysis, gradient sequence: 0-55min 90%A-30%A; 55-60min 30%A-60%A; 60-75min 60%A-90%A;
  • Each component sample separated by RP-HPLC is screened for lowering blood lipid activity, and the amino acid sequence of the most active component sample is analyzed to obtain.
  • the enzyme activity of the pepsin is not less than 1500 U/g
  • the enzyme activity of trypsin is not less than 2500 U/mg
  • the casein conversion power of trypsin is not less than 30.0.
  • the sample separated by RP-HPLC is analyzed by LC-TOF-MS to obtain the active peptide mass spectrum, and the active peptide structure is obtained as LAPAPGTL, named LL-8, and the relative molecular mass is 738Da .
  • the present invention provides the application of the active peptide in the preparation of drugs or health products for preventing or treating blood lipid reduction. Furthermore, the present invention further relates to a medicine or a health product, which contains the active peptide LAPAPGTL of the present invention.
  • the medicines and health products of the present invention can be made into various forms common in the art, including but not limited to powders, tablets (including various coated tablets, sustained-release or controlled-release tablets), and tablets suitable for oral administration. Preparations, capsules (including soft capsules and hard capsules), granules, pills, dispersible powders, aqueous or oily suspensions, aqueous or oily solutions, emulsions, elixirs, syrups, etc., suitable for gastrointestinal Sterile aqueous or oily injections or freeze-dried powder injections for intravenous, subcutaneous or intramuscular injection for external administration.
  • the medicines and health products of the present invention may further contain various conventional auxiliary materials and/or other active ingredients.
  • Suitable excipients include but are not limited to excipients, lubricants, binders, disintegrating agents, water-soluble polymers, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antiseptics Oxygen and so on.
  • the active peptide of the present invention is preferably prepared as an injection or powder injection, wherein the solvent is sterile water; the preservative is chlorobutanol; one or two of procaine hydrochloride combination.
  • the invention uses pepsin and trypsin to perform biomimetic enzymatic hydrolysis of the soil turtle in vitro to simulate the digestion of proteins in the body, so that the macromolecular protein can be cleaved into polypeptides in vitro, and the absorption efficiency is increased.
  • the decomposed peptides are separated by adsorption chromatography, gel molecular sieve chromatography, RP-HPLC, LC-TOF-MS, etc., to obtain peptide monomers with the function of reducing blood lipids.
  • the peptide monomers have high purity, safety and effectiveness; Immunogenic reactions caused by proteins, etc., reduce the incidence of adverse reactions.
  • Figure 1 shows the G25 gel chromatographic separation diagram.
  • Figure 2 shows a thrmeo semi-preparative HPLC chromatogram.
  • Figure 3 shows the LC-TOF-MS first-order mass spectrum.
  • Figure 4 shows the LC-TOF-MS secondary mass spectrum.
  • DA201-C resin Take DA201-C resin, wash it with absolute ethanol and deionized water, and put it into a glass chromatography column.
  • the biomimetic enzymatic hydrolysate of the soil turtle is added to the resin column from the upper end.
  • the weight of the DA201-C resin is equal to the volume of the bionic enzymatic hydrolysate of the soil turtle. The ratio is 10:1.
  • 25% ethanol is used for elution to prepare eluent A.
  • the eluent B is separated by RP-HPLC.
  • the chromatographic conditions used are chromatographic column: waters ZOBRX-300SB C18 chromatographic column, 150mm X 4.6um, detector is ultraviolet detector, detection wavelength: 220nm, 280nm, flow Phase: A phase 0.1% trifluoroacetic acid deionized aqueous solution (TFA), B phase 0.1% trifluoroacetic acid (TFA) acetonitrile solution; use gradient elution method for analysis, gradient sequence: 0-55min 90%A-30%A ; 55-60min 30%A-60%A; 60-75min 60%A-90%A.
  • Each component sample separated by RP-HPLC was screened for lowering blood lipid activity, and 2 components with lowering blood lipid activity were screened out, named LL-8 and LM-9 respectively.
  • the component samples LL-8 and LM-9 were analyzed by LC-TOF-MS, and the active peptide mass spectrum was obtained, and the active peptide structure was obtained as LAPAPGTL and LHRIGAVPM.
  • Example 2 Based on the amino acid sequences of the LL-8 and LM-9 active peptides identified in Example 1, the experimental samples were obtained by solid-phase synthesis, and the lipid-lowering activity was verified. Solid-phase synthetic peptide company: Shanghai Jier Biotechnology Co., Ltd.
  • each mouse in other groups per day 50g of high-fat feed was given.
  • 4.0 mL of high-fat emulsion was given to each rat at 9:00 am every day. 45 days after giving high-fat feed and high-fat emulsion 45 days later.
  • the LM9 high and low dose group can significantly reduce the content of TG, TC and LDL in the serum of hyperlipidemia rats, and there is a significant difference Compared with the LM9 high and low dose group and the model group, the LL8 high and low dose group can significantly reduce the four items of serum lipids in the model group (P ⁇ 0.01), indicating that LL8 has a better function of reducing blood lipids than LM9.
  • Example 1 Based on the amino acid sequence of the LL-8 active peptide identified in Example 1, the experimental samples were obtained by solid-phase synthesis, and the safety experiment was verified. Solid-phase synthetic peptide company: Shanghai Jier Biotechnology Co., Ltd.
  • Healthy SD rats, 250 ⁇ 300g, 40 rats, adaptively reared for 7 days, and then set to blank group, saline group, LL-8 group, LL-8 group were given 20.0g/200g high-dose drugs, administration Method: subcutaneous injection; the normal saline group was also given the same volume of normal saline by subcutaneous injection for 12 days. The diet and water intake of the rats were measured every day, and the rats' food intake was measured every 3 days. Weight, body temperature. At the same time, the weight of immune organs such as thymus and spleen of rats in each group was measured to measure the safety of LL-8.
  • LL-8 did not cause significant changes in the body temperature of normal rats, which also shows that LL-8 does not cause abnormal immune responses in rats, and LL-8 does not cause normal organs atrophy in rats. At the same time, it will not cause the rat immune organs to proliferate, so LL-8 is safe under injection administration conditions.

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Abstract

本发明公开一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用。本发明所述的活性肽氨基酸序列为LAPAPGTL。本发明提供的活性肽具有降血脂功能，可以开发成药物或保健品。

Description

一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用 技术领域
本发明涉及活性肽技术领域。更具体地，涉及一种中华冀土鳖虫来源的具有降血脂作用的活性肽及其制备方法和应用。
背景技术
高血脂是目前常见的心血管疾病，随着人民生活水平的提高，高热量与高脂肪饮食使得高血脂的发病率持续增高。高血脂存在很多并发症，例如脂肪肝、动脉粥样硬化、糖尿病等，因此对高血脂的防治与治疗是目前研究的热点之一。
土鳖虫为传统中药，具有破血逐瘀，续筋接骨之功效，用于跌打损伤，筋伤骨折，血瘀经闭，产后瘀阻腹痛，瘾瘕痞块。现代研究得出土鳖虫具有治疗血脑血管疾病的作用，例如动脉粥样硬化、高血脂、脑血栓等。
目前对于中药动物药研究是中药基础研究的一大热点，而且取得了很大的进展，例如在水蛭中发现具有防凝血的水蛭素，在地龙体内发现具有溶栓作用的蚓激酶，这些研究发现中药动物药发挥药理作用的物质基本为蛋白质，对土鳖虫蛋白质进行研究得出土鳖虫蛋白具有促进血栓溶解、扩张血管、消除自由基的作用。
蛋白质多为100K或更大分子量的物质，口服后多经过胃蛋白酶、胰蛋白酶等胃肠道蛋白酶作用成小分子多肽被吸收进入人体，发挥药理作用。所以蛋白质存在吸收效率低、过敏等缺点。
本发明采用胃蛋白酶和胰蛋白酶在体外对土鳖虫进行仿生酶解来模拟蛋白质在体内的消化，这样使得大分子蛋白在体外既可以被裂解为多肽，增加吸收效率。分解后的多肽采用超滤、纳滤、大孔树脂、凝胶分子筛色谱进行分离，得到具有活血化瘀功能的多肽单体，多肽单体纯度高，安全有效；消除了服用异蛋白等引起的免疫原反应，减小了不良反应发生率。
发明内容
本发明的第一个目的在于提供一种中华冀土鳖虫来源的活性肽。
本发明的第二个目的在于提供上述活性肽制备方法
本发明的第三个目的在于提供上述活性肽在制备预防或治疗降血脂的药物或保健品中的应用。
为达到上述目的，本发明采用下述技术方案：
第一方面，本发明提供一种中华冀土鳖虫来源的活性肽，所述活性肽的氨基酸序列为LAPAPGTL，如SEQ ID NO:1所示。
可选的，所述活性肽还可以是与上述活性肽的氨基酸序列具有80％及以上同源性的活性肽，该活性肽和上述活性肽的功能相同或相似。例如，所述活性肽的氨基酸序列可以是与上述活性肽的氨基酸序列具有85％，90％，95％或97％同源性的序列。
本发明还提供了编码上述活性肽的多核苷酸。
在已知活性肽的氨基酸序列的情况下，本领域技术人员可以根据对于活性肽表达的需要，基于密码子的简并性原则和不同物种对于密码子的使用偏好性，设计具有不同核苷酸序列的活性肽的编码基因。
在本发明中，示例性的，上述活性肽的核苷酸序列为ATGCTGGCACCAGCGCCGGGTACCCTG，如序列表SEQ ID NO:2所示。
第二方面，本发明提供了一种上述中华冀土鳖虫来源的活性肽的制备方法，包括如下步骤：
(1)将中华冀土鳖鲜虫体加入8-10倍重量份的去离子水或者蒸馏水进行剪切，制成土鳖虫浆液，剪切时间为10-20min，剪切速率为5000转/分钟，剪切温度为室温；
(2)将土鳖虫浆液加热至100℃杀菌15min，然后放冷至37℃，调节浆液pH值至2.5，加入胃蛋白酶进行酶解，酶解时间为45min-80min，酶解温度为37℃；然后再调节浆液的pH值至8.5，加入胰蛋白酶进行酶解，酶解时间为180min-300min，酶解温度为37℃，制得土鳖虫仿生酶解液；其中，按土鳖虫浆液重量计，胃蛋白酶的加入量为1.5％-2.5％，胰蛋白酶的加入量为1.5％-2.5％；
(3)用DA201-C树脂柱对步骤(2)制得的土鳖虫仿生酶解液进行吸附层析，DA201-C树脂重量与土鳖虫仿生酶解液体积比例为5:1-10:1，吸附完毕后采用25％乙醇进行洗脱；制得洗脱液A；
(4)用葡聚糖G25凝胶柱对步骤(3)制得的洗脱液A进行分子筛层析，每次洗脱液A的加入量为0.5-2ml，用去离子水洗脱，制得洗脱液B；
(5)将步骤(4)制得的洗脱液B经过RP-HPLC进行组分分离，采用的色谱条件为色谱柱：waters ZOBRX-300SB C18色谱柱，150mm X 4.6um，检测器为紫外检测器，检测波长：220nm、280nm，流动相：A相0.1％三氟乙酸去离子水溶液(TFA)，B相0.1％三氟乙酸(TFA)乙腈溶液；采用梯度洗脱方法进行分析，梯度顺序：0-55min 90％A-30％A；55-60min 30％A-60％A；60-75min 60％A-90％A；
(6)经RP-HPLC分离后的各组分样品进行降血脂活性筛选，对活性最强的组分样品进行氨基酸序列分析，即得。
上述方法中，优选的，所述胃蛋白酶的酶活力不低于1500U/g，胰蛋白酶的酶活力不低于2500U/mg，胰蛋白酶的酪蛋白转化力不低于30.0。
在本发明的具体实施方式中，经RP-HPLC分离后的样品采用LC-TOF-MS进行分析，得到活性肽质谱，得出活性肽结构为LAPAPGTL，命名为LL-8，相对分子质量为738Da。
第三方面，本发明提供所述活性肽在制备预防或治疗降血脂的药物或保健品中的应用。进而，本发明进一步涉及一种药物或一种保健品，其中含有本发明的活性肽LAPAPGTL。
本发明的药物和保健品可以制成本领域常见的各种形式，包括但不限于，适于口服的散剂、片剂(包括各种包衣片剂、缓释或控释片剂)、锭剂、胶囊剂(包括软胶囊和硬胶囊)、颗粒剂、丸剂、可分散粉末、水性或油性混悬剂、水性或油性溶液剂、乳剂、酏剂、糖浆剂等等，适于经胃肠外给药的静脉内、皮下或肌内注射用无菌水性或油性的注射剂或冻干粉针剂等等。
本发明的药物和保健品中可以进一步含有常规的各种辅料和/或其他活性成分。合适的辅料包括但不限于赋形剂、润滑剂、粘合剂、崩解剂、水溶性聚合物、无机盐、溶剂、溶解助剂、悬浮剂、等渗剂、缓冲液、防腐剂、抗氧剂等等。
根据本发明的具体实施方案，本发明的活性肽优选被制成注射剂或粉针剂，其中溶剂为无菌用水；防腐剂为三氯叔丁醇；盐酸普鲁卡因的一种或两种的组合。
本发明的有益效果如下：
本发明采用胃蛋白酶和胰蛋白酶在体外对土鳖虫进行仿生酶解来模拟蛋白质在体内的消化，这样使得大分子蛋白在体外既可以被裂解为多肽，增加吸收效率。分解后的多肽采用吸附层析、凝胶分子筛色谱、RP-HPLC、LC-TOF-MS等进行分离，得到具有降血脂功能的多肽单体，多肽单体纯度高，安全有效；消除了服用异蛋白等引起的免疫原反应，减小了不良反应发生率。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出G25凝胶色谱分离图。
图2示出thrmeo半制备HPLC色谱图。
图3示出LC-TOF-MS一级质谱图。
图4示出LC-TOF-MS二级质谱图。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例1 活性肽制备
(1)土鳖虫药材处理
中华冀土鳖鲜虫体加入10倍重量份的去离子水或者蒸馏水，然后进行剪切15min，剪切速率为5000转/分钟，剪切温度为室温，制成土鳖虫浆液。
(2)蛋白酶酶解
将土鳖虫浆液加热至100℃杀菌15min，然后放冷至37℃，采用0.1mol/L HCl调整溶液pH至2.5，加入1.5％重量份的胃蛋白酶置恒温酶解仪进行酶解，酶解时间为60min，酶解温度为37℃。然后再采用2.0％氢氧化钠溶液调整pH至8.5，加1.5％重量份的胰蛋白酶置于恒温酶解仪进行酶解，酶解时间为180min，酶解温度为37℃，制得土鳖虫仿生酶解液。
(3)吸附层析
取DA201-C树脂，采用无水乙醇、去离子水洗涤后装入玻璃层析柱内，土鳖虫仿生酶解液由上端加入树脂柱内，DA201-C树脂重量与土鳖虫仿生酶解液体积比例为10:1，吸附完毕后采用25％乙醇进行洗脱，制得洗脱液A。
(4)分子筛层析
取葡聚糖G25，无水乙醇、去离子水洗涤完毕后，装入玻璃柱内，然后将土鳖虫25％乙醇洗脱液加入，每次加入量0.5ml，去离子水洗脱，制得洗脱液B。
(5)RP-HPLC分离
将洗脱液B经过RP-HPLC进行组分分离，采用的色谱条件为色谱柱：waters ZOBRX-300SB C18色谱柱，150mm X 4.6um，检测器为紫外检测器，检测波长：220nm、280nm，流动相：A相0.1％三氟乙酸去离子水溶液(TFA)，B相0.1％三氟乙酸(TFA)乙腈溶液；采用梯度洗脱方法进行分析，梯度顺序：0-55min 90％A—30％A；55-60min30％A-60％A；60-75min 60％A-90％A。
f，LC-TOF-MS分离
经RP-HPLC分离后的各组分样品进行降血脂活性筛选，筛选出2个具有降血脂活性的组分，分别命名为LL-8、LM-9。
对于组分样品LL-8、LM-9采用LC-TOF-MS进行分析，得到活性肽质谱，得出活性肽结构为LAPAPGTL，LHRIGAVPM。
实施例2
基于实施例1鉴定得到的LL-8和LM-9活性肽的氨基酸序列，通过固相合成方法获得实验样品，进行降血脂活性验证。固相合成肽公司：上海吉尔生物技术有限公司。
SD大鼠80只，雄性，适应性饲养7天，然后分为九组，即空白组，模型组、血栓通胶囊组、土鳖虫药材组、LL-8高剂量组(0.5g/kg)、LL-8低剂量组(0.25g/kg)、LM-9高剂量组(0.5g/kg)，LM-9低剂量组(0.25g/kg)、除空白组以外，其他组每只老鼠每天定量给予50g高脂饲料，同时除空白组大鼠，每天上午9:00给予每只大鼠高脂乳剂4.0mL，高脂乳剂饲养方式：灌胃口服，空白组大鼠持续给予普通饲料，连续45天，给予高脂饲料、高脂乳剂45天后。先将5.0mLEP管采用预选配置后的肝素钠溶液润洗后，备用。给药15天后，于第16天末次给药后皮下注射给药5.0％戊巴本妥钠溶液进行麻醉，待大鼠被完全麻醉后，先在腹主动脉取血3.0mL，注入未加抗凝剂的玻璃硅化管内。离心；取血清；采用ELISA酶联检测方法检测各组血液内血脂四项(LDL、HDL、TG、TC)含量。
表1 调节血脂实验结果
Figure PCTCN2021085866-appb-000001
Figure PCTCN2021085866-appb-000002
观察除空白组、模型组以外的其他实验组血脂四项数据，所有组别的血脂四项数据和空白组进行对比，都具有显著性或者极显著性差异(P<0.05、P<0.01)，说明造模良好，给药45天后，在持续给予高脂饲料的同时，与模型组，LM9高低剂量组可明显降低高脂血症大鼠血清内TG、TC和LDL的含量，存在显著性差异，而与LM9高低剂量组，模型组相比，LL8高低剂量组则可更加明显降低模型组大鼠血清中血脂四项(P<0.01)，说明LL8较LM9具有更好地降低血脂功能。
实施例3
基于实施例1鉴定得到的LL-8活性肽的氨基酸序列，通过固相合成方法获得实验样品，进行安全性实验验证。固相合成肽公司：上海吉尔生物技术有限公司。
健康SD大鼠，250～300g，40只，适应性饲养7天，然后设置为空白组、生理盐水组、LL-8组，LL-8组分别给予20.0g/200g的大剂量药物，给药方式：皮下注射给药；生理盐水组同样采用皮下注射给药方式给予等体积的生理盐水，持续给药12天，每天测定大鼠的饮食量、饮水量，同时每隔3天测定大鼠的体重、体温。同时测定各组大鼠胸腺，脾脏等免疫器官重量，衡量LL-8的安全性。
表2 短期安全性评价各组大鼠体温变化结果
测定次数 空白组(℃) 生理盐水组(℃) LL-8组(℃)
1 38.32±0.1432 38.12±0.4351 38.36±0.2145
2 38.17±0.3521 38.97±0.3912 39.08±0.0011
3 38.53±0.4721 38.16±0.0013 38.54±0.1132
4 38.34±0.3361 38.21±0.3328 37.90±0.7921
5 38.04±0.1453 38.45±0.4128 38.71±0.1348
表3 各组大鼠器官重量结果
组别 肝重(g) 肾重(g) 脑重(g) 脾脏重(g) 胸腺重(g)
空白组 15.364±1.4428 3.6479±0.3637 1.8096±0.0911 0.6827±0.0149 0.2496±0.0394
生理盐水组 15.793±3.3575 3.6458±0.2549 1.9007±0.0762 0.6024±0.0771 0.2245±0.0339
LL-8组 15.821±0.5968 3.6225±0.1913 1.9916±0.0733 0.7690±0.0217 0.2774±0.0419
表4 免疫器官指数实验结果
组别 脾脏指数(mg/g) 胸腺指数(mg/g)
空白组 1.8512±0.4219 0.4071±0.0743
生理盐水组 1.7084±0.8575 0.3982±0.0537
LL-8组 1.9831±0.8701 0.4361±0.0757
以上实验结果表明：LL-8并未造成正常大鼠体温的明显变化，这也说明LL-8不会引起大鼠的异常免疫反应，同时LL-8也不会引起大鼠正常器官的萎缩，同时也不会使大鼠免疫器官出现增殖，因此LL-8在注射给药条件下是安全的。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
Figure PCTCN2021085866-appb-000003

Claims (8)

  1. 一种中华冀土鳖虫来源的活性肽，其特征在于，所述活性肽的氨基酸序列为LAPAPGTL。
  2. 一种多核苷酸，其特征在于，编码权利要求1所述的活性肽。
  3. 如权利要求1所述的中华冀土鳖虫来源的活性肽的制备方法，其特征在于，该方法包括如下步骤：
    (1)将中华冀土鳖鲜虫体加入8-10倍重量份的去离子水或者蒸馏水进行剪切，制成土鳖虫浆液，剪切时间为10-20min，剪切速率为5000转/分钟，剪切温度为室温；
    (2)将土鳖虫浆液加热至100℃杀菌15min，然后放冷至37℃，调节浆液pH值至2.5，加入胃蛋白酶进行酶解，酶解时间为45min-80min，酶解温度为37℃；然后再调节浆液的pH值至8.5，加入胰蛋白酶进行酶解，酶解时间为180min-300min，酶解温度为37℃，制得土鳖虫仿生酶解液；其中，按土鳖虫浆液重量计，胃蛋白酶的加入量为1.5％-2.5％，胰蛋白酶的加入量为1.5％-2.5％；
    (3)用DA201-C树脂柱对步骤(2)制得的土鳖虫仿生酶解液进行吸附层析，DA201-C树脂重量与土鳖虫仿生酶解液体积比例为5:1-10:1，吸附完毕后采用25％乙醇进行洗脱；制得洗脱液A；
    (4)用葡聚糖G25凝胶柱对步骤(3)制得的洗脱液A进行分子筛层析，每次洗脱液A的加入量为0.5-2ml，用去离子水洗脱，制得洗脱液B；
    (5)将步骤(4)制得的洗脱液B经过RP-HPLC进行组分分离，采用的色谱条件为色谱柱：waters ZOBRX-300SB C18色谱柱，150mm X 4.6um，检测器为紫外检测器，检测波长：220nm、280nm，流动相：A相0.1％三氟乙酸去离子水溶液，B相0.1％三氟乙酸乙腈溶液；采用梯度洗脱方法进行分析，梯度顺序：0-55min 90％A-30％A；55-60min 30％A-60％A；60-75min 60％A-90％A；
    (6)经RP-HPLC分离后的各组分样品进行降血脂活性筛选，对活性最强的组分样品进行氨基酸序列分析，即得。
  4. 根据权利要求3所述的制备方法，其特征在于，所述胃蛋白酶的酶活力不低于1500U/g，胰蛋白酶的酶活力不低于2500U/mg，胰蛋白酶的酪蛋白转化力不低于30.0。
  5. 权利要求1所述的中华冀土鳖虫来源的活性肽在制备预防或治疗降血脂的药物或保健品中的应用。
  6. 一种药物，其特征在于，包含权利要求1所述的中华冀土鳖虫来源的活性肽。
  7. 根据权利要求6所述的药物，其特征在于，所述药物为注射剂或粉针剂。
  8. 一种保健品，其特征在于，包含权利要求1所述的中华冀土鳖虫来源的活性肽。
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