WO2013023442A1 - 甲鱼肽在制药中的应用 - Google Patents

甲鱼肽在制药中的应用 Download PDF

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WO2013023442A1
WO2013023442A1 PCT/CN2012/001096 CN2012001096W WO2013023442A1 WO 2013023442 A1 WO2013023442 A1 WO 2013023442A1 CN 2012001096 W CN2012001096 W CN 2012001096W WO 2013023442 A1 WO2013023442 A1 WO 2013023442A1
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peptide
turtle
soft
tumor
solution
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PCT/CN2012/001096
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English (en)
French (fr)
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钟虹光
卢建中
易敏之
马莉
刘根云
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江中药业股份有限公司
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Publication of WO2013023442A1 publication Critical patent/WO2013023442A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • A61K35/586—Turtles; Tortoises, e.g. terrapins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates

Definitions

  • the present invention relates to the use of turtle peptides, and in particular to the use of agriculture in the field of tumor suppressing drugs. Background technique
  • Tumors are divided into benign and malignant tumors.
  • pf refers to malignant tumors.
  • the number of people dying from cancer in the world is as many as millions of people every year. Since 1997, cancer has become the first cause of death in China. Nearly 1.3 million people die of cancer every year.
  • the methods of inhibiting the growth and metastasis of cancer cells include surgery, chemotherapy and radiotherapy, which generally cause more damage to the body and easily reduce the immunity of humans.
  • the object of the present invention is to provide a new application of turtle peptide, that is, a new application in pharmacy; another object of the present invention is to provide a method for inhibiting growth and metastasis of cancer cells, having high nutritional value and absorption rate. High, no harm to the body's health drugs or health foods.
  • the turtle peptide of the present invention is a small molecular peptide having a molecular weight distribution within a lOOOODalton, wherein the molecular weight range of 140-100 Odalton is 50%.
  • the present invention also relates to the use of turtle peptide as a medicament for the preparation of a medicament for the prevention and treatment of neoplastic diseases.
  • the invention relates to the use of a turtle peptide having a molecular weight distribution of 100OODalton as a medicine for preparing a tumor disease prevention and treatment.
  • turtle peptide having a molecular weight distribution within 2000 Dalt O n is useful as a medicament for preparing a tumor disease prevention and treatment.
  • the invention relates to the application of the turtle peptide having a molecular weight distribution in the range of 300 to 700Dalt O n in the preparation of a medicament for preventing and treating tumor diseases.
  • the turtle peptide of the present invention is prepared from the whole of the genus Trio ⁇ iyx sinensis Wiegmann.
  • test drug is the turtle liquid oral powder dry powder, lg turtle peptide / thousand powder, provided by Jiangzhong Pharmaceutical Co., Ltd., the clinical dose is 4g turtle peptide / d, L00ml / d.
  • mice [0017] 1. 3. 2 dose design: the recommended intake of the turtle liquid oral liquid per day, the daily intake is: 4. 0g / 60kg body weight. From this, it is estimated that the daily intake of mice is: low dose group, 3. 33g crude drug / kg body weight; middle dose group, 6. 67g crude drug / kg body weight; sputum dose group, 13. 34g crude drug / kg body weight, respectively It is equivalent to 5, 10 and 20 times the daily intake of people. 2 ⁇ /10g The amount of the gastric gavage of the mice was 0. 2ml/10g, the amount of the gavage was 0. 2ml/10g, the amount of the gavage was 0. 2ml/10g Weight calculation. 1 time per 3 times for 30 consecutive days. All groups of mice were fed with normal feed and were free to eat and drink. After 30 days, each subgroup was tested for adjuvant inhibition of tumor function.
  • Tumor growth inhibition rate % (control mean tumor weight - treatment group flat tumor weight) ⁇ control group average tumor weight X 100%.
  • the low, medium and high doses of the turtle liquid oral solution were administered as follows: 3.33g crude drug/kg body weight, 6.67g crude drug/kg body weight, 13.34g crude drug/kg body weight, low, medium and
  • the sputum concentration of the squid peptide liquid was administered once a day for 30 days. 24 hours after the last administration, the animals were sacrificed by cervical dislocation, and the body weight and tumor weight were respectively weighed. The tumor growth inhibition rate (%) was calculated.
  • Tumor growth inhibition rate % (average tumor weight of the control group - mean tumor weight of the treatment group) ⁇ The average tumor weight of the control group was X 100%.
  • mice were sacrificed 24 hours after the last administration, press ⁇ mL / Only intraperitoneal injection of calf serum Hank's solution, light Lightly massage the abdomen 20 times to fully wash out the peritoneal macrophages, then cut a small opening into the abdominal wall, and use a plastic suction tube to absorb 2 ml of the abdominal cavity wash in a test tube (or a syringe), and use a 1 ml sampler to absorb the peritoneal lotion.
  • the mixture was filled with 0.5 ml of 1% chicken red blood cell suspension in a test tube, mixed, with a syringe (with a large needle) to absorb 0. 5ml mixture, added to the agar ring of the slide, placed in the incubator 37 Incubate at °C for 15 ⁇ 20min. After incubating the bundle, quickly wash off the unattached cells with physiological saline, fix lrain in methanol solution, stain the Giemsa solution for 15min, rinse with distilled water, dry, and count the macrophage under oil microscope. The cells were counted 100 times per tablet. The percentage of phagocytosis and the phagocytic index were calculated according to the following formula, and the phagocytic ability of mouse macrophages was expressed by the percentage of phagocytosis or the phagocytic index.
  • % of phagocytosis (the number of phagocytic chicken red blood cells I counts the number of macrophages) X 100%
  • Phagocytic index number of erythrocytes in the phagocytized chicken I The number of macrophages that phagocytose chicken red blood cells.
  • the venous vein was injected from the tail vein, and the saline solution was diluted 3-4 times Indian ink 0. lml/lOg body weight, respectively, from the intraocular vein of the mouse at 2 min and 10 min after the ink was injected.
  • the plexus was taken 20 ⁇ , and immediately added to 2 mL of O. l% Na2C03 solution, and mixed, and the optical density value (0D) was measured at a distance of 600 nm, and 0.1% of Na2C03 solution was used as a blank control.
  • the mice were sacrificed, the liver and spleen were taken, and the filter paper was dried.
  • the phagocytic index indicates the ability of the mouse to clear the carbon.
  • mice were sacrificed by cervical dislocation, the spleen was aseptically taken, placed in a small dish containing appropriate amount of sterile Hank's solution, the spleen was ground, and a single cell suspension was prepared and filtered through a 200 mesh screen.
  • the target cells (YAC21 cells) were subcultured 24 h before the test and washed 3 times with Hank's solution before application. The cells were adjusted with RPMI 1640 complete medium; the agronomic rate was 4 X 10 5 / mL. 100 PL of YAC21 cells and spleen cells (effective target ratio 50: 1) were added to U-shaped 96-well culture plates, and YAC21 cells were naturally released.
  • Kongjia YAC21 cells and culture medium are 100, each of the above three parallel holes, in 37 ° C, 5% CO 2 incubator Raising 4h, then the 96-well plate was centrifuged at 1500r ⁇ min 1 5min, the supernatant in each well 100 ⁇ L opposed flat bottom 96 well culture plate, while the LDH substrate solution was added 10 (f ⁇ L, the reaction 8min, per well The HC1 30 L of lraol *L 1 was added and the optical density value was measured at 490 nm.
  • 3 ⁇ 4 s indicates that the difference between the model control group and the turtle group was compared by one-way analysis of variance, and P ⁇ 0.05 was judged to be significant.
  • the medium- and high-dose of the squid peptide oral liquid had a strong inhibitory effect on the mouse sarcoma S18 (j, the inhibition rates were 30.86% and 46.91%, respectively (P ⁇ 0.05), see Table 1.
  • the medium and high doses of the squid peptide oral solution had a strong inhibitory effect on the transplanted Lewis lung cancer in mice, and the inhibition rates were 39.6% and 50.0% (P ⁇ 0.05), respectively.
  • the medium and high doses of the turtle liquid oral solution have a strong effect on the conversion rate of spleen lymphocytes in mouse transplanted tumor LS180 mice and Lewis lung cancer mice, as shown in Table 3.
  • turtle fish oral liquid complex powder recommended daily exposure 4 amount 4. 0g raw / 60kg weight gain 5, 10 and 30 times set low, medium and high three dose groups, namely 3. 33g crude drug, 6. 67g crude drug, 13. 34g crude drug/kg body weight, another transplanted tumor LS180 mouse, Lewis lung cancer mouse model control group.
  • the relevant indicators were tested after 30 days of oral administration of the corresponding drugs.
  • the experimental results were judged to be significant by P ⁇ 0.05.
  • the animal experiment showed that the medium and high dose of the turtle liquid oral solution had a strong inhibitory effect on the mouse sarcoma S180, and the inhibition rates were 30.86% and 46.91%, respectively.
  • the turtle liquid oral liquid, high dose to small The mouse transplanted tumor has a strong inhibitory effect on Lewis lung cancer, and the inhibition rates are 39.6% and 50.0%, respectively.
  • the medium and high doses of the turtle liquid oral solution have a strong effect on the spleen lymphocyte transformation rate of mouse transplanted tumor LS180 mice and Lewis lung cancer mice; the squid peptide oral liquid and sputum dose groups have significantly improved mice.
  • a soft-shell peptide of molecular weight 140-10000 can inhibit the growth and metastasis of tumors, and can be used for preparing foods or products that help inhibit the growth and metastasis of tumors, and has a good application prospect.
  • the 5% ratio of the ratio of the raw material of the soft-shelled turtle to the flavor of the soft-shelled turtle is 0. 513 ⁇ 4.
  • the enzyme solution was heated to 100 ° C, and the enzyme was inactivated for 30 min;
  • Low molecular weight protein hydrolysates (including peptides and amino acids) are soluble in trichloroacetic acid solution; high molecular weight proteins are easily precipitated in ⁇ : chloroacetic acid solution.
  • the precipitated protein substance is separated by centrifugation, and the content of the acid-soluble protein in the centrifuged supernatant is determined.
  • the content of the acid-soluble protein in the clear and the subtracted free amino acid content is the content of the oligopeptide.
  • Sample preparation Weigh 20 ⁇ 30mg sample to the nearest
  • oligopeptides The content of oligopeptides is calculated according to formula (1) -
  • the oligopeptide content of the soft-shelled turtle was determined, and the results are shown in Table 7.
  • Acetonitrile chromatographically pure; trifluoroacetic acid: analytically pure; water: super-aqueous or double distilled water.
  • cytochrome C cytochrome C
  • aprotinin MW6500
  • bacitase bacitracin, Li [450]
  • ethionine-ethine-tyrosine Acid monoarginine MW451
  • ethionine-alanine-ethylamine MW189
  • High performance liquid chromatograph equipped with UV detector and chromatographic workstation or integrator with
  • the column efficiency of the gel column that is, the number of theoretical plates ( ⁇ ) according to the tripeptide standard (ethine-alanine-I-lysine)
  • the peak calculation is not less than 5000, and the partition coefficient (Kd) of the oligopeptide should be between 0 and 1.
  • the sample solution prepared in 3. 6 was analyzed under the above chromatographic strip. Then, using GPC data processing software, the chromatographic data of the sample is substituted into the calibration curve equation to calculate the relative molecular mass of the peptide in the sample and its distribution range.
  • the relative molecular mass range is calculated by the peak area normalization method
  • the molecular weight distribution range of the soft-shelled peptides of turtles is shown in Table 8.
  • the enzyme is added to the ratio of the weight of the raw turtle, and the alkaline protease is added at a constant temperature of 50 °C. After lh, the obtained enzymatic hydrolysate was heated to 85 ° C, and the enzyme was sterilized for 20 min;
  • the enzymatic hydrolysate was centrifuged, and the supernatant was purified by a tubular membrane having a molecular weight cut off of 0.50 Daltons.
  • the inlet pressure was 1.2 bar
  • the outlet pressure was 1 bar
  • the enzymatic hydrolysis temperature was 20 ° C. 2 ⁇ peptide ⁇
  • the film was concentrated, spray drying to obtain 9. 2kg of turtle peptide finished product.
  • the molecular weight of the turtle peptide was mainly concentrated in 140-2000, accounting for more than 50%.
  • the molecular weight of the turtle peptide was mainly concentrated below 10,000, accounting for more than 99%.
  • the enzymatic hydrolysate was centrifuged and filtered, and the supernatant was purified by a roll-type ultrafiltration membrane having a molecular weight cutoff of 100,000 Daltons.
  • the inlet pressure was 2. 8 bar
  • the outlet pressure was 2 bar
  • the temperature of the enzyme solution was 30 C.
  • the film was concentrated, and spray dried to obtain 12. 4 kg of soft-shelled peptides.
  • the molecular weight of the soft-shell peptide of the soft-shelled turtle is concentrated at 300-700, accounting for more than 30%.

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Abstract

本发明的目的在于提供了甲鱼肽在制备防治肿瘤疾病的药中的应用，本发明甲鱼低聚肽是以甲鱼为主要原料，分子量分布在10000Dalton以内的小分子肽，其中140-1000分子量范围占50%以上。

Description

甲鱼肽在制药中的应用 技术领域
[0001 ] 本发明涉及甲鱼肽的用途，尤其是涉农在抑制肿瘤药物领域中的用途。 背景技术
[0002] 肿瘤分为良性肿瘤和恶性肿瘤，一般 pf说的癌即指恶性肿瘤，世界每年死于癌症 的人数多达数百万人，而自 1997年以来，癌症成为中国人的第一死因，每年有近 130万人死 于癌症，现在抑制癌细胞的生长与转移的方法 要有手术，化疗和放疗，一般对身体的伤害 较大，容易使人的免疫力下降。
[0003] 申请号 201010103157. 9专利文献中 开了甲鱼小分子多肽的制备方法；《中国食 品学报》2008, 02：徐怀德等"甲鱼蛋白酶解产 体外 ACE抑制和抗氧化活性研究"公开了体 外实验证实甲鱼蛋白酶解物 ACE抑制和抗氧化活性的作用；现有技术和专利中均未述及甲 鱼低聚肽关于辅助抑制肿瘤细胞生长或转移应用的报导，也没有关于分子量在 1000以下 的甲鱼低聚肽在治疗和预防抗肿瘤方面的报 il:。 发明内容
[0004] 本发明的目的是提供甲鱼肽的新用申，即在制药中的新应用；本发明的另一目的 是提供一种能抑制癌细胞生长和转移，有较高 营养价值,吸收率高，不妨害人体身体健康 的药物或保健食品。
[0005] 本发明甲鱼肽是以甲鱼为主要原料 f分子量分布在 lOOOODalton以内的小分子 肽，其中 140-lOOODalton分子量范围占 50% 上。
[0006] 本发明还涉及甲鱼肽在作为制备防治肿瘤疾病的药中的应用。
[0007] 涉及分子量分布在 lOOOODalton以^的甲鱼肽在作为制备防治肿瘤疾病的药中 的应用。
[0008] 涉及分子量分布在 2000DaltOn以内的甲鱼肽在作为制备防治肿瘤疾病的药中的 应用。
[0009] 涉及分子量分布在 140〜 lOOODal n范围以内的甲鱼肽在作为制备防治肿瘤疾 病的药中的应用。
[0010]
涉及分子量分布在 300〜 700DaltOn范围 |以内的甲鱼肽在作为制备防治肿瘤疾病的药 中的应用。
[001 1 ] 本发明的甲鱼肽是鳖科动物鳖 Trio^iyx sinensis Wiegmann的全体为原料制备 的。
[0012] 为了更好地理解本发明的实质，下面 用分子量在 140〜 10000Dalton的甲鱼低 聚肽的药理实验及结果来说明其在制药领域 新用途。
[0013] 分子量小于 lOOOODalton的甲鱼肽 制肿瘤的生长和转移的功效研究。
[0014] 1. 材料与方法 1. 1 样品来源及处理：受试药为甲鱼肽口服液干粉， lg甲鱼肽 /千粉，由江中药业股份 有限公司提供，人临床服用量为 4g甲鱼肽 /d， L00ml/d。
[0015] 1. 2 实验动物及环境：清洁级健康昆明种小鼠，雌雄各半，体重 18_22g，江西中医 学院实验动物中心提供，许可证号： SCXK (赣） 2006 - 0001。 动物饲养于江西中医学院动 物室，环境许可证： SYXK (赣） 2004-0001 ,饲养 境温度 21〜 23°C，相对湿度 50〜 60%。
[0016] 1. 3 实验方法
1. 3. 1 动物分组：根据体重随机分为 4组，每组 10只。设立空白对照组、甲鱼肽口服液 低、中、高剂量组。
[0017] 1. 3. 2剂量设计：甲鱼肽口服液每人，日推荐摄入量为：4. 0g/60kg体重。由此推 算出小鼠每日摄入量为：低剂量组, 3. 33g生药 /kg体重；中剂量组， 6. 67g生药 /kg体重； 髙剂量组， 13. 34g生药 /kg体重,分别相当于人每日摄入量的 5、 10和 20倍。将样品以蒸镏 水配制成相应浓度（0. 1665g干粉 /ml、0. 3335 g干粉 /ml、0. 667g干粉 /ml )的灌胃液进行 实验，小鼠灌胃量按 0. 2ml/10g体重计算。 每 3灌胃 1次,连续 30天。 各组小鼠均饲以普 通饲料，自由进食、饮水。 30天后对各亚组小 进行辅助抑制肿瘤功能检验。
[0018] 1. 3. 3 甲鱼肽对小鼠肉瘤 S180 的抑甯作用：将腹腔接种小鼠肉瘤 S180细胞后 8 天的昆明小鼠颈椎脱臼处死，无菌条件下取出 J ^水，用生理盐水 1 : 2稀释,给每只小鼠腋窝 皮下接种瘤液 0. 2ml 接种后次日，甲鱼肽口服液低、中、高剂量给分别按 3. 33g生药 /kg体 重、 6. 67g生药 /kg体重、 13. 34g生药 /kg体重灌胃低、中、高浓度的甲鱼肽药液，每天 1次， 连续给药 30天。末次给药后 24小时,颈椎脱 处死动物，分别称体重、瘤重，计算肿瘤生长 抑制率 (％)，计算公式为：
肿瘤生长抑制率％ = (对照均组平均瘤重―治疗组平瘤重） ÷对照组平均瘤重 X 100%。
[0019] 1. 3. 4 甲鱼肽对小鼠移植性肿瘤 L iS 肺癌的抑瘤作用：用小鼠移植性肿瘤 Lewis 肺癌生长良好的瘤源，无菌条件下取荷:窗动物的实体瘤组织，用剪刀剪成小碎块，用 套管针移植或用玻璃匀浆器加无菌生理盐水磨成匀浆，计数每 ml 匀浆液中的瘤细胞数，给 每只动物皮下接种 2 X 106细胞。接种后 24小时分组给药，甲鱼肽口服液低、中、高剂量给 分别按 3. 33g生药 /kg体重、 6. 67g生药 /kg体重、 13. 34g生药 /kg体重灌胃低、中、髙浓度 的甲鱼肽药液，每天 1次，连续给药 30天。末次给药后 24小时,颈椎脱臼处死动物，分别称 体重、瘤重，计算肿瘤生长抑制率（％)，计算公 ii为：
肿瘤生长抑制率％ = (对照组平均瘤重 -治疗组平均瘤重） ÷对照组平均瘤重 X 100%。
[0020] 1. 3. 5 甲鱼肽对 S180小鼠、 Lewis 肺癌小鼠脾淋巴细胞转化的影响：接种瘤液 与给药同 1. 3. 3和 1. 3. 4。 末次给药后 24小 取脾脏，分别在筛网上研磨、离心、计数单个 细胞，配成终浓度，颈椎脱臼处死动物。 无菌条件下取出脾为 2. 5 X 106个细胞 /ml，分别加 入 96 孔板中，同时设空白对照孔。 置 37°C 5% C02 培养箱中培养 72 小时，培养结束前 6 小时向每孔加入 [¾]- TdR，终浓度为 1 μ Ci/孔,多头收集器将细胞收集于玻璃纤维滤纸上， 烤干，放入加有 4ml 闪烁液的测量瓶中，液闪仅上测定每个样品的 cpm值。
[0021 ] 1. 3. 6 甲鱼肽对 S180小鼠腹腔巨噬细胞吞噬鸡红细胞的影响：接种瘤液与给药 同 1. 3. 3 末次给药后 24小时处死小鼠， 按 ^ mL/只腹腔注射含小牛血清 Hank' s液，轻 轻按摩腹部 20次，以充分洗出腹腔巨噬细胞，然后将腹壁剪开一个小口，用胶头吸管吸取 腹腔洗液 2ml于试管内（或用注射器)，用 1ml加样器吸取腹腔洗液 0. 5ml加入盛有 0. 5ml 1% 鸡血红细胞悬液的试管内，混匀，用注射器 (装大针头）吸取 0. 5ml混合液，加入玻片的琼脂 圈内，放置孵箱内 37°C孵育 15〜 20min，孵育 束后迅速用生理盐水将未贴壁细胞冲掉,于 甲醇液中固定 lrain，姬姆萨液染色 15min，用蒸馏水冲洗干净，晾干，油镜下计数巨噬细胞， 每张片计数 100个，按下式计算吞噬百分率和吞噬指数，以吞噬百分率或吞噬指数表示小 鼠巨噬细胞的吞噬能力。
[0022] 吞噬百分率％ = (吞噬鸡红细胞的巨 数 I计数巨噬细胞数） X 100%
吞噬指数=被吞噬鸡红细胞数 I吞噬鸡红细胞的巨噬细胞数。
[0023] 1. 3. 7 甲鱼肽对 Lewis 肺癌小^碳粒廓清实验的影响：接种瘤液与给药同 1. 3. 4。
[0024] 小鼠末次给药后，从尾静脉注入用,理盐水稀释 3-4 倍印度墨汁 0. lml/lOg体 重，分别于注入墨汁后 2 min和 10 min时分别从小鼠眼内眦静脉丛取血 20 μ ί,并立即加 到 2 mLO. l%Na2C03溶液中，混匀，在 600 nm 长处测光密度值（0D)，以 0. l%Na2C03溶液 作为空白对照。 将小鼠处死，取肝脏和脾脏， ϊ滤纸擦干，
分别称重，计算胸腺 I体重和脾脏 I体重 < 以吞噬指数表示小鼠碳廓清的能力。
[0025] 1. 3. 7 甲鱼肽对 Lewis肺癌小鼠 M:细胞活性的影响（乳酸脱氢酶测定法）： 接种瘤液与给药同 1. 3. 4。小鼠末次给药后，颈椎脱臼处死小鼠，无菌取脾，置于盛有适量 无菌 Hank' s液的小平皿中，研磨脾脏， 制^单细胞悬液，经 200 目筛网过滤，用 Hank' s 液洗 2 次，每次离心 10min (1000r · πήη - *) ,弃上清液将细胞桨弹起， 加入 015mL 灭 菌水 20s ,裂解红细胞后再加入 0. 5mL 2 Hank' s 液及 8mLHank， s 液，离心 lOmin (lOOOr 'min " ') ，用 lmL 10 %小牛血清 RPMI 1640 完全培养液重悬，用 1 %乙酸稀释后计 数，台盼蓝染色计数活细胞数（均在 95 %以— ) ，用 RPMI 1640 完全培养液调整细胞浓度 为 2 X 107个 / mL 。 试验前 24h将靶细胞（ YAC21 细胞） 传代培养，应用前以 Hank' s 液洗 3 次，用 RPMI 1640 完全培养液调整细胞;农度为 4 X 105个 / mL 。 取 YAC21 细胞和 脾细胞各 100 P L (效靶比 50 ： 1) 加入 U型 96孔培养板中， YAC21 细胞自然释放孔加 YAC21 细胞和培养液各 100 ,上述各项设 3 个平行孔， 于 37 °C、5 %C02培养箱中培 养 4h ,然后将 96 孔培养板以 1500r · min 1离心 5min ,每孔吸取上清液 100 μ L 置平 底 96孔培养板中， 同时加入 LDH基质液 10(f μ L , 反应 8min， 每孔加入 lraol *L 1的 HC1 30 L ,在醇标仪 490nm处测定光密度值。
[0026] 1 . 3 . 8 4 统 计 [¾" 法 ： 实 验 数 据 以
¾s表示，采用单因素方差分析，比较模型对照组、甲鱼伏组之间的差异， P〈0. 05判断为差 异具有显著性。
[0027] 2 结果
2. 1甲鱼肽口服液对小鼠肉瘤 S180 的抑 |g作用：
甲鱼肽口服液中、高剂量对小鼠肉瘤 S18(j 有较强的抑制作用，抑制率分别为 30. 86% 和 46. 91% (P〈0. 05)，见表 1。
[0028] 表 1 甲鱼肽口服液对小鼠肉瘤 Sl 的抑瘤作用的影响（ )
Figure imgf000005_0001
与模型对照组比较， *Ρ<0. 05；
2. 2甲鱼肽口服液对小鼠移植性肿瘤 Le ^s肺癌的抑瘤作用
甲鱼肽口服液中、高剂量对小鼠移植性肿 Lewis肺癌 有较强的抑制作用，抑制率分 别为 39. 6%和 50. 0% (P〈0. 05)，见表 2。
[0029] 表 2 甲鱼肽口服液对小鼠移植性肿癣 Lewis肺癌的抑瘤作用的影响（ )
Figure imgf000005_0002
与模型对照组比较， *P<0. 05；
2. 3 甲鱼肽口服液对移植性肿瘤 LS180小 i鼠、 Lewis肺癌小鼠脾淋巴细胞转化率的影 响
甲鱼肽口服液中、高剂量对小鼠移植性肿瘤 LS180小鼠、 Lewis 肺癌小鼠脾淋巴细胞 转化率均有较强的提高作用，见表 3。
[0030] 表 3 甲鱼肽口服液对小鼠脾淋巴细 转化率的影响（ s )
觸 l
綳 …：曜：'： ; 纖圃 顯
m x 哪 漏
id ： 機, so^e ae m m a
13.34
10 : Ι7ϊ難 o
与模型对照组比较， *P<0.05 ;
2.4 甲鱼肽口服液对小鼠腹腔巨噬细胞 噬功能的影响：与模型对照组比较， 甲鱼 肽口服液中、髙剂量组均有显著提高小鼠腹腔 έ噬细胞吞噬百分率的作用（Ρ<0.05) 。实验 结果见表 4。
[0031] 表 4 甲鱼肽口服液对腹腔巨噬细胞吞噬功能的影响（ ¾^ )
Figure imgf000006_0001
与模型对照组比较， *Ρ〈0.05；
2.5 甲鱼肽口服液对小鼠碳粒廓清功能^]影响：与模型对照组比较， 甲鱼肽口服液 中、高剂量组均有显著提高吞噬指数的作用（Ρ< 0.05) 。 实验结果见表 5。
[0032] 表 5 甲鱼肽口服液对小鼠碳粒廓清功能的影响（ s )
Figure imgf000006_0002
与模型对照组比较， *P〈0.05；
2.6 甲鱼肽口服液对小鼠 NK细胞活性的 响：与模型对照组比较， 甲鱼肽口服液低、 中、高剂量组小鼠 N 细胞活性无显著性差异 (/io.05)结果见表 6。
[0033] 表 6 甲鱼肽口服液对小鼠 NK细胞活 I性的影响（ )
Figure imgf000007_0001
[0034] 3. 结论：
根据甲鱼肽口服液复合粉人群推荐日摄 4量 4. 0g生 /60kg体重扩大 5、 10和 30倍设 置低、中、高三个剂量组，即 3. 33g生药、 6. 67g生药、 13. 34g生药 /kg体重，另设移植性肿瘤 LS180小鼠、 Lewis肺癌小鼠模型对照组。灌胃 予相应药物 30天后进行检测相关指标。实 验结果以 P〈0. 05判断为差异具有显著性。 经动物实验研究表明：甲鱼肽口服液中、高剂量 对小鼠肉瘤 S180 有较强的抑制作用，抑制率分别为 30. 86%和 46. 91%；甲鱼肽口服液中、 高剂量对小鼠移植性肿瘤 Lewis肺癌 有较强 ½抑制作用，抑制率分别为 39. 6%和 50. 0%。 甲鱼肽口服液中、高剂量对小鼠移植性肿瘤 LS180小鼠、 Lewis 肺癌小鼠脾淋巴细胞转化 率均有较强的提高作用；甲鱼肽口服液中、髙剂量组均有显著提高小鼠腹腔巨噬细胞吞噬 百分率和小鼠碳粒廓清能力的作用；甲鱼肽口服液低、中、高剂量组小鼠 NK细胞活性无显 著性差异，认为甲鱼肽口服液具有辅助抑制肿艏功能。
[0035] 经过实验发现，分子量 140-10000的甲鱼低聚肽能抑制肿瘤的生长和转移，可用 于制备辅助抑制肿瘤的生长和转移的食品或 品，有很好的应用前景。 具体实施方式
[0036] 实施例 1
一、甲鱼低聚肽制法：
1. 以中华鳖为原料，选用鲜活甲鱼 100k ，去除内脏和油脂，搅碎，加入 1000L纯水， 100° C煎煮 2h ;
2.冷却至 60° C，按原料甲鱼的重量的 29f的比率加入碱性蛋白酶 2kg，70°C恒温酶解 3h,再分别按原料甲鱼重量的 0. 5%的比率加 风味蛋白酶 0. 51¾，55°0恒温酶解0. 511，将 得到的酶解液升温至 100° C,灭酶 30min；
3.将酶解液离心后过滤，上清液经过膜截 分子量为 10万道尔顿的中空超滤膜超滤， 进口压力为 1. 8-2. 5bar，出口压力 1. 5- 1. 8bar 酶解液温度为 20-30° C进行超滤分离，薄 膜浓缩，喷雾干燥即得到 14. 6kg粉末甲鱼低聚 i肽，加入 345升纯水，配制成甲鱼肽口服液。
[0037] 二、甲鱼低聚肽含量测定：
1. 方法提要
低分子量的蛋白质水解物 (包含肽类及游 氨基酸）可溶于三氯乙酸溶液；高分子量的 蛋白质在^:氯乙酸溶液中易沉淀。 样品经三 乙酸溶液溶解后，离心分离出沉淀蛋白质物 质，测定出离心清液中的酸溶蛋白质含量，清、^中的酸溶蛋白质含量减去游离氨基酸含量 即为低聚肽的含量。 [0038] 2. 分析步骤
2. 1 酸溶蛋白质含量的测定
称取 2g (精确至 lmg)样品，加入 10mL15% .三氯乙酸溶液，混合均匀，静置 10min。 将样 品溶液在 4000rpm下离心 lOmin后，取全部清¾，按 GB/T 5009. 5规定的方法测定清液中的 酸溶蛋白质，蛋白质换算系数为 6. 25。 检验结 根据样品的干燥失重，折算为干基。
[0039] 2. 2游离氨基酸含量的测定
样品前处理：称取 20〜 30mg样品，精确至|| 0. OOOlg，用 3 %磺基水杨酸溶液溶解均匀。 将样品溶液转移至 50ml容量瓶中，定容。 将样品溶液在转速为 4000r/min离心机上离心 5min得清液，再用 0. 45 μ m微孔滤膜过滤清液，将滤液转移至 50ml容量瓶中，定容后作为仪 器检测用样品。 其余操作同 GB 12292水果、 ^菜汁游离氨基酸含量的测定规定的方法。
[0040] 2. 3 结果的表述
低聚肽的含量 按式（1 )计算-
P^ P^ A ( 1 )
式中 -
4- 一样品中低聚肽含量（以干基计)，％；
-样品中酸溶蛋白质含量（以干基计† ；
A— -样品中游离氨基酸含量（以干基计 J. %。
[0041 ] 2. 4测定结果
测定甲鱼低聚肽含量，结果见表 7。
[0042] 表 7肽粉中低聚肽的含量测定结果
Figure imgf000008_0001
[0043] 3.相对分子质量小于 1000的肽所占 t匕例（髙效凝胶过滤色谱法）
3. 1方法提要
采用高效凝胶过滤色谱法测定。 即以多 ίΐ)饪填料为固定相,依据样品组分分子体积大 小的差别进行分离，在肽键的紫外吸收波长 2^nm条件下检测，使用凝胶色谱测定相对分 子质量分布的专用数据处理软件 (即 GPC软件) I对色谱图及其数据进行处理，计算得到低聚 肽的相对分子质量大小及分布范围。
[0044] 3. 2试剂
乙腈：色谱纯；三氟醋酸：分析纯；水：超 水或二次蒸馏水。
[0045] 相对分子质量校正曲线所用标准品：细胞色素 C (cyyochrome, MW12500)；抑酞酶 (aprotinin, MW6500)；杆菌酶（bacitracin,丽【450)；乙氨酸—乙氨酸一酪氨酸一精氨酸 (MW451 )；乙氨酸一乙氨酸一乙氨酸（MW189)。
[0046] 3. 3仪器和设备
高效液相色谱仪：配有紫外检测器和含有 |GPC数据处理软件的色谱工作站或积分仪； 流动相真空抽滤脱气装置；超声波振荡器；分^天平：感量 0. 0001g„
[0047] 3. 4色谱条件与系统适应性实验
色谱柱： TSKgel G2000 SWXL 300mmX 7. {|醒或性能与此相近的同类型其他适用于测 定蛋白质和多肽的凝胶柱：流动相：乙腈：水：三氟乙酸, 45: 55 :0. 1 (体积比）检测波长： UV220nm；流速：0. 5ml/min；柱温 :30°C；进样体积 : 10 μ 1。
[0048] 为使色谱系统符合检测要求，规定在上述色谱条件下，凝胶色谱柱的柱效即理论 塔板数（Ν)按三肽标准品（乙氨酸一乙氨酸一 i氨酸）峰计算不低于 5000，低聚肽的分配系 数（Kd)应在 0〜 1之间。
[0049] 3. 5相对分子质量校正曲线制作
分别用流动相配制成 0. 1% (W/V)的上述 同相对分子质量的肽标准品溶液，用孔径为 0. 2-0. 5 μ πι聚四氟乙烯或尼龙过滤膜过滤后分别进样，得到系列标准品的色谱图。 以相对 分子质量的对数（lgMW)对保留时间作图或作 性回归得到相对分子质量校正曲线及其方
[0050] 3. 6样品制备
称取样品 20. Omg于 10mL容量瓶中，用流 相定容至刻度，超声振荡 lOmin，使样品充分 溶解混匀，用孔径为 0. 2-0. 5 μ !η聚四氟乙烯 ^尼龙过滤膜过滤后，上机进样。
[0051 ] 3. 7相对分子质量的计算
将 3. 6 制备的样品溶液在上述色谱条 下分析。 然后用 GPC数据处理软件，将样品 的色谱数据代入校正曲线方程中进行计算，即 |ϊ得到样品中肽的相对分子质量及其分布范 围。用峰面积归一化法计算相对分子质量范围 |在 1000以下的肽的峰面积相对百分比之和。
[0052] 3. 8测定结果
甲鱼低聚肽分子量分布范围测定结果见表 8
表 8甲鱼低聚肽结果
Figure imgf000009_0001
以上结果表明，甲鱼低聚肽分子量主要集 在 140-1000，占 70%以上
[0053] 实施例 2
甲鱼肽制法：
一、甲鱼低聚肽的制备方法，其步骤如下：
1. 以中华鳖为原料，选用鲜活甲鱼 100 ，搅碎，煎煮 lh；
2. 冷却至 50° C,按原料甲鱼的重量的 ％的比率加入碱性蛋白酶，50°C恒温酶解 lh, 再分别按原料甲鱼重量的 0. 15%的风味蛋白 ，50°C恒温酶解 lh，将得到的酶解液升温至 85° C，灭酶 20min ;
3. 将酶解液离心，上清液经过膜截 分子量为 0. 5万道尔顿的管式滤膜精制分 离，进口压力为 1. 2bar，出口压力 lbar,酶解 温度为 20° C进行超滤分离，薄膜浓縮，喷 雾干燥即得到 9. 2kg甲鱼肽成品。
[0054] 二、甲鱼肽含量测定：
按照实施例 1的方法，甲鱼肽分子量主要集中在 140-2000，占 50%以上。
[0055] 实施例 3
―、 甲鱼肽的制备方法，其步骤如下： 1. 以中华鳖为原料，选用鲜活甲鱼 lOOkf ，搅碎，煎煮 4h ;
2. 冷却至 40 ° C，按原料甲鱼的重量的 5%的比率加入碱性蛋白酶， 40°C恒温酶解 10h，或加入原料甲鱼重量的 2%的中性蛋白 .40°C恒温酶解 4h，将得到的酶解液升温至 120° C，灭酶 60min ;浓缩，喷雾千燥即得。
[0056] 二、甲鱼肽含量测定：
按照实施例 1的方法，甲鱼肽分子量主要集中在 10000以下，占 99%以上。
[0057] 实施例 4
一、 甲鱼低聚肽的制备方法，其 骤如下：
1. 以中华鳖为原料，选用鲜活甲鱼 100kg,去除内脏和油脂，搅碎,煎煮 1. 5-3h；
2.冷却至 50-62° C，按原料甲鱼的重量的 0. 5- 3%的比率加入碱性蛋白酶, 60'C恒温酶 解 2_4h，再分别按原料甲鱼重量的 0. 25-1% 风味蛋白酶， 60Ό恒温酶解 0. 3-2h,将得到 的酶解液升温至 110° C,灭酶 40min ;
3.将酶解液离心后过滤，上清液经过膜截留分子量为 10万道尔顿的卷式超滤膜精制 分离，进口压力为 2. 8bar,出口压力 2bar,酶 液温度为 30 C进行超滤分离，薄膜浓縮, 喷雾干燥即得到 12. 4kg甲鱼低聚肽成品。
[0058] 二、甲鱼低聚肽含量测定：
按照实施例 1的方法，甲鱼低聚肽分子量 要集中在 300-700，占 30%以上

Claims

WO 2013/023442 权 利 要 求 书 PCT/CN2012/001096
1. 甲鱼肽在制备防治肿瘤疾病的药中的申用。
2.根据权利要求 1所述的甲鱼肽，其特征! ¾于：甲鱼肽分子量分布在 lOOOODalton以 内。
3.根据权利要求 2所述的甲鱼肽,其特征在于：甲鱼肽分子量分布在 2000DaltOn以 内。
4. 根据权利要求 3所述的甲鱼肽，其時征在于：甲鱼肽分子量分布在 140〜 lOOODalton范围以内。
5.根据权利要求 4所述的甲鱼肽，其特征 于：甲鱼肽分子量分布在 300 ~ 700Dalton 范围以内。
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CN103845720A (zh) * 2012-11-29 2014-06-11 江中药业股份有限公司 一种防治辐射损伤或化学药物治疗损伤的组合物及其制备方法
CN103845721A (zh) * 2012-11-29 2014-06-11 江中药业股份有限公司 一种防治辐射损伤或化学药物治疗损伤的组合物及其制备方法
CN109438558B (zh) * 2018-10-31 2021-02-05 深圳凯联健康生物科技有限公司 活性肽、重组载体、重组细胞、药物组合物及其制备方法和应用
CN109467590B (zh) * 2018-10-31 2021-02-05 深圳凯联健康生物科技有限公司 活性肽、重组载体、重组细胞、药物组合物及其制备方法和应用
CN109364238A (zh) * 2018-12-03 2019-02-22 佛山市肽硒其生物科技有限公司 一种龟肽组合物及其制品

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