WO2009140833A1 - 霞水母来源有免疫增强活性的胶原蛋白肽及制备和应用 - Google Patents

霞水母来源有免疫增强活性的胶原蛋白肽及制备和应用 Download PDF

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WO2009140833A1
WO2009140833A1 PCT/CN2008/072397 CN2008072397W WO2009140833A1 WO 2009140833 A1 WO2009140833 A1 WO 2009140833A1 CN 2008072397 W CN2008072397 W CN 2008072397W WO 2009140833 A1 WO2009140833 A1 WO 2009140833A1
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collagen peptide
acid
jellyfish
peptide according
preparing
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PCT/CN2008/072397
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English (en)
French (fr)
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汤鲁宏
张本田
邓超
林丹
王琪
陈伟
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张丽丽
江南大学
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Priority to JP2011509839A priority Critical patent/JP2011520927A/ja
Priority to US12/993,819 priority patent/US8450100B2/en
Publication of WO2009140833A1 publication Critical patent/WO2009140833A1/zh

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17Amino acids, peptides or proteins
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17Amino acids, peptides or proteins
    • A23L33/18Peptides; Protein hydrolysates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • Collagen peptide with immunopotentiating activity derived from Xia jellyfish, preparation and application thereof
  • the invention relates to a collagen peptide with immune enhancement activity derived from Xia jellyfish and a preparation process thereof, and belongs to the field of natural biological active substances.
  • Cyanea nozaki i is a large marine plankton, belonging to the Phylum Cnidaria, Class Scyphomedusae, Order Phylum cnidaria, and Xia jellyfish.
  • Family Cyanea There are four species of Cyanea nozaki Kishinouye, Cyanea capillata, Cyanea ferruginea Eschscholtz, and CyEmea purpurea Kishinouye.
  • White Xia jellyfish has the largest number and the widest distribution range. Xia jellyfish is usually bait with small zooplankton. Its gonads are developed, its reproductive ability is strong, and its growth rate is extremely fast. At present, the annual output of Xia jellyfish in China has reached 10 million tons, which constitutes a new natural marine biological resource.
  • Marine-derived polysaccharides, proteoglycans and collagen peptides are important natural marine food resources, and often have significant biological activities, such as sea cucumber polysaccharides, seaweed polysaccharides, etc., which have been reported to have significant biological activity.
  • Research reports on the separation and extraction of collagen peptides from Xia jellyfish are rare at home and abroad. So far, there have been no reports on the isolation and extraction of collagen peptides with immunopotentiating activity. Summary of the invention
  • the invention discloses a collagen peptide derived from Xia jellyfish and having immunopotentiating activity, and a preparation process and application thereof. More uniquely, it uses a new type of natural marine biological resource, Xia Shuimu (Cyim ⁇ 'O- as a raw material for the preparation of collagen peptides, to achieve the purpose of development and utilization of Xia jellyfish.
  • the invention operates to obtain a high-purity collagen peptide product in an extremely high yield, which is essential for the comprehensive utilization of the natural jellyfish, a natural marine living resource.
  • a collagen peptide derived from Xia jellyfish derived from an immunopotentiating activity characterized in that it has a white or yellowish appearance, an odorless, slightly bitter powder, and a seawater jellyfish (C) w ⁇ / ⁇ ⁇ H ⁇ ⁇ ⁇ ), Cyweflilla (Cy wefl capillata), Cywefl ferruginea Escfec/iote or Purple Jellyfish (Cyanea purpurea S ⁇ w ⁇ ) freshly caught or isolated from the marinated pickled products, non-toxic, with significant immune-enhancing activity, containing 80-90% protein, 10 ⁇ 20% of sugar, the average molecular weight is between 1,000 and 3,000.
  • the monosaccharide contains glucose as the main component.
  • the glycine in the amino acid accounts for more than 16%, and the combination of proline and hydroxyproline accounts for more than 18%.
  • the solubilization treatment refers to the use of a melting agent to melt the Xia Shui mother into water to form a liquid.
  • the type of the hydrolyzing agent and the temperature used in the hydrolysis are the most important for the hydrolysis yield.
  • the effective solvent is a common alkali (such as sodium, potassium, calcium, barium hydroxide or carbonate, ammonia, etc.)
  • the concentration is 0.1 ⁇ 10molL - common acid (such as hydrochloric acid, phosphoric acid, sulfuric acid, etc.
  • Acid or acid acetic acid, malic acid, lactic acid, citric acid and other organic acids, concentration of 0.1 ⁇ 10mol L - ⁇ or various proteases (such as pepsin, trypsin, trypsin, acid protease, alkaline protease, neutral protease
  • proteases such as pepsin, trypsin, trypsin, acid protease, alkaline protease, neutral protease
  • papain, bromelain, collagenase, elastase, etc. melted at a pH of 0.5 to 14.0, at a temperature of 10 to 120 ° C, and at an operating pressure of 0.0001 to 0.5 MPa.
  • sodium is a solvent
  • the pH should be in the range of 7.0 - 14.0.
  • the pH should be in the range of 0.5-6.8.
  • the pH should be The optimum pH range of the enzyme is selected; when the acid-base is used as the solvent, the melting temperature should be in the range of 10 - 120 ° C, preferably in the range of 0 - 60 ° C.
  • hydrolysis should be carried out at an optimum temperature of the protease.
  • the soaking should be carried out at a certain value within the range of pH adjustment of 2-12, preferably in water of 6.5 ⁇ 6.8, and the soaking temperature is in the range of 0 ⁇ 98 °C, preferably 0 ⁇ 15°. Within the range of C, the soaking operation is carried out until the conductivity of the soaking water is lower than 3 ( ⁇ Scm- 1 stops. Centrifugal separation is carried out at 0 ⁇ 120 °C, preferably at 0 ⁇ 10 °C, the speed of the centrifuge Between 4000 and 12000 rpm, preferably between 6000 and 8000 rpm, the centrifugation time is 10 to 60 minutes.
  • the drying method of the liquid may be freeze drying or spray drying.
  • the Xia jellyfish active collagen peptide of the invention has been confirmed by preliminary research, has significant immunopotentiating activity, and can be widely used as a novel bioactive material and health food raw material for food, health care products, skin care and beauty products, medical supplies, etc. In the field, it is used for preparing medicines, health products, skin care and beauty products with immune enhancement functions.
  • the above melt treatment can also be carried out under the operating pressure of 0.001 MPa.
  • a clear and transparent liquid which is a collagen peptide solution
  • the collagen peptide solution is concentrated at 40 ° C, O.OOlMPa to After 1/2 of the original volume, it was freeze-dried to obtain 1000 g of a collagen peptide powder solid having a purity-enhancing activity of 95% or more.
  • Example three The above melt treatment can also be carried out under the operating pressure of 0.1 MPa.
  • Example three The above melt treatment can also be carried out under the operating pressure of 0.1 MPa.
  • a sample of collagen peptide powder (e.g., prepared according to the method of Example 2) was used to determine the protein content, the determination of the total sugar content, the determination of the amino acid composition, and the determination of the monosaccharide composition. The results showed that the sample contained 88% protein, 10.5% sugar, and the average molecular weight was 1274.
  • the monosaccharide contained is mainly glucose, and the composition of the amino acids contained in Table 1 is shown in Table 1.
  • mice Administration dose One-week weight gain Growth rate Number of deaths Blank control 10 0.5ml/only 3.374g 14.79% 0
  • the experimental group 10 7500mg/kg 3.299g 14.55% 0
  • the activity of the mice decreased within 1 hour after administration, and gradually resumed normal activities after 4 hours.
  • the food intake increased gradually within 7 days, and the appetite, spirit and hair color of the mice were normal.
  • the neck was sacrificed and the main organ lesions and poisoning were not observed in the eyes, liver, spleen, lung and kidney.
  • the observation period was weighed every day for 7 days, and the weight of the mice in the test group increased continuously.
  • the results of the study showed that in the case of large doses of the test sample, no mice died and no significant toxicity was observed. It indicates that it is non-toxic or has little toxicity, and its maximum safe dose is above 7500mg/Kg.
  • Test group 25 0.26 soil 0.04
  • Hemolysin is a method that reflects the status of humoral immune function. If the hemolysin production increases after administration, the absorbance value of red blood cell hemolysis increases, indicating that the humoral immunity of the body is enhanced after administration. From the results in Table 4, the difference between the high-dose group and the blank control group was significant (P ⁇ 0.05). There was no difference between the low-dose group and the middle-dose group and the control group (P>0.05), indicating that the high dose was high. The test sample has a significant effect on the production of serum hemolysin (IgM), showing a better humoral immune enhancement.
  • Delayed hypersensitivity reaction is a method to reflect the status of cellular immune function. It can be seen from Table 5 that the difference in weight between the left and right auricles of the experimental group is increased compared with the control group, and the difference between the middle dose group and the blank control group. There was a significant difference (P ⁇ 0.01), and there was no significant difference between the low-dose group and the high-dose group (P > 0.05). It indicates that the sample can significantly improve the cellular immune function of mice in a certain concentration range.
  • Test group 25 32.58 soil 1.16 2.25 soil 1.00
  • the phagocytic function of phagocytic cells is a method to reflect the status of non-specific immune function. It can be seen from Table 6 that the percentage of phagocytosis is significantly different between the middle dose group and the high dose group compared with the control group (P ⁇ 0.05), the middle dose group and the high dose group. The phagocytic index was significantly different from the control group (P ⁇ 0.01). There was no difference between the low dose group and the control group (P>0.05), indicating that a certain dose of the test sample can activate phagocytic activity and increase mononuclear phagocytic cells. System phagocytosis.
  • the results of the cellular immune function, the humoral immune function, the mononuclear-macrophage function, and the NK cell activity are positive in two aspects, and the test sample can be judged to have an enhanced immunity function.
  • This experiment is positive in the three aspects of cellular immune function, humoral immune function and monocyte-macrophage function in experimental mice, so it can be determined that the test sample has immunopotentiating effect.

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Description

说明书
霞水母来源有免疫增强活性的胶原蛋白肽及制备和应用 技术领域
本发明涉及一种霞水母来源的具有免疫增强活性的胶原蛋白肽及其制备工艺,属天然 生物活性物质领域。
霞水母(Cyanea nozaki i) , 俗称麻蜇, 是一种大型海洋浮游生物, 属刺胞动物门 (Phylum Cnidaria) , 钵水母纲 (Class Scyphomedusae) , 旗口水母目 (Order Phylum cnidaria), 霞水母科(Family Cyanea)。 我国沿海已发现的有白色霞水母(Cyanea nozaki Kishinouye)、 发形霞水母 (Cyanea capillata)、 綜色霞水母 (Cyanea ferruginea Eschscholtz) , 禾口紫色霞水母(CyEmea purpurea Kishinouye) 4个种, 其中以白色霞水母 数量最多、分布范围最广。霞水母通常以小型浮游动物为饵,其生殖腺发达,繁殖能力强, 生长速度极快, 目前我国霞水母的年产量已经高达千万吨,构成了一种新的天然海洋生物 资源。
海洋来源的多糖、蛋白聚糖和胶原蛋白肽是十分重要的天然海洋食品资源,常常具有 显著的生物活性, 如海参多糖、海藻多糖等均有许多的研究报道, 证实其具有显著的生物 活性。 以霞水母为原料, 从中分离提取制备胶原蛋白肽的研究报道国内外尚很少见, 迄今 未见有从中分离提取具有免疫增强活性的胶原蛋白肽的报道。 发明内容
本发明发明了一种霞水母来源的具有免疫增强活性的胶原蛋白肽及其制备工艺和应 用。 较为独特的是, 它以一种新型的天然海洋生物资源-霞水母 (Cyim^ ^^^'O-作为胶原 蛋白肽的制备原料进行加工,实现了对霞水母进行开发利用的目的。按照本发明进行操作, 能够以极高的产率获得高纯度胶原蛋白肽产品。这对于霞水母这一天然海洋生物资源的综 合利用而言是至关重要的。
一种霞水母来源的具有免疫增强活性的胶原蛋白肽, 其特征在于外观为白色或淡黄 色, 无嗅, 略带苦味的粉末, 自霞水母科海洋生物白色霞水母 (C)w^ /^^H^ }^)、 发开霞水母 (Cy wefl capillata) , 棕色霞水母 (Cy wefl ferruginea Escfec/iote)或紫色霞水母 (Cyanea purpurea S^^w }^)的新鲜捕捞品或其经过三矾腌制的腌制品中分离提取得到, 无毒, 具有显著的免疫增强活性, 其中含有 80~90%的蛋白质, 10~20%的糖, 平均分子量 在 1,000~3,000之间, 所含单糖以葡萄糖为主, 所含氨基酸中甘氨酸占 16%以上, 脯氨酸 与羟脯氨酸之和占 18%以上。
上述胶原蛋白肽的制备工艺, 该工艺由下列步骤组成:
(1) 将霞水母用水浸泡;
(2) 对浸泡后的霞水母进行溶融处理, 然后离心分离, 得到无色或淡黄色的上清液;
(3) 将上清液直接干燥或经过均质、 脱色、 脱盐处理后干燥, 得到白色或浅黄色的粉 末, 即为胶原蛋白肽。
所述的溶融处理指采用溶融剂将霞水母融化于水中形成液体。
具体来说,首先,我们发现新鲜的或经过三矾腌制处理的市售麻蜇可以在经过脱盐处 理之后用作胶原蛋白肽的制备原料。其中所含的胶原蛋白肽对水浸泡处理是稳定的,在我 们所采取的浸泡工艺条件下, 既不会因受到浸泡而被降解、被提取到水中流失, 也不会因 受过浸泡而影响其在后续的水解处理中的分散性,即经过三矾腌制处理的市售麻蜇与刚捕 捞上岸的新鲜的霞水母具有同等的可加工性,均可用作制备高纯度高生物活性胶原蛋白肽 的原料。 由此奠定了以霞水母为原料加工制备高纯度高生物活性胶原蛋白肽的基础。
其次, 在对温度、 压力等水解条件以及氢氧化钠、 盐酸、 胶原蛋白酶等各种常用的溶 融剂进行了系统的考察和对比后, 我们发现, 在适当的水解条件下, 霞水母中所含有的胶 原蛋白不经过变性处理, 便可直接被水解, 分散到水中, 得到澄清透明, 所含杂质很少的 水解分散液。
我们发现, 在浸泡溶剂、 浸泡时间、 水解剂等水解工艺参数中, 水解剂的种类和水解 时所采用的温度对于水解得率而言是最为重要的。其中, 有效的溶融剂为常见的碱 (如钠、 钾、 钙、 钡的氢氧化物或碳酸盐、 氨水等) , 浓度为 0.1~10molL— 常见的酸 (如盐酸、 磷 酸、 硫酸等无机酸或甲酸、 醋酸、 苹果酸、 乳酸、 柠檬酸等有机酸, 浓度为 0.1~10molL— ^ 或各种蛋白酶 (如胃蛋白酶、 胰蛋白酶、 胰糜蛋白酶、 酸性蛋白酶、 碱性蛋白酶、 中性蛋 白酶、 木瓜蛋白酶、 菠萝蛋白酶、 胶原蛋白酶、 弹性蛋白酶等)中的一种或若干种, 溶融 处理 pH 0.5~14.0, 温度 10~120°C, 操作压力 0.0001~0.5 MPa条件下进行。 当选用氢氧化 钠为溶融剂时, pH值应当在 7.0 - 14.0范围之内, 当选用盐酸为溶融剂时, pH值应当在 0.5-6.8范围之内, 当选用蛋白酶类为溶融剂时, pH值应当在所选酶的最适 pH范围之内; 当选用酸碱为溶融剂时, 融溶温度应当在 10 - 120°C范围内, 最好是在 0 - 60°C的范围 内,当选用蛋白酶类为溶融剂时,应当在蛋白酶类的最适温度下进行水解。其他各项参数, 浸泡应在 pH调节为 2-12范围内某一定值, 最好是在 6.5~6.8 的水中进行, 浸泡温度在 0~98°C 范围内, 最好是在 0~15°C 的范围内, 浸泡操作进行至浸泡水的电导率低于 3(^Scm— 1停止。 离心分离在 0~120°C, 最好是在 0 ~ 10°C条件下进行, 离心机的转速在 4000~12000rpm之间, 最好是在 6000 ~ 8000rpm之间, 离心时间为 10~60min。 液体的干 燥方式, 可以是冷冻干燥, 也可以是喷雾干燥。
本发明发明的霞水母活性胶原蛋白肽经初步的研究确认, 具有显著的免疫增强活性, 可作为新型的生物活性材料和保健食品原料被广泛应用于食品、保健品、护肤美容用品和 医药用品等领域, 用于制备具有免疫增强功能的药品、 保健品、 护肤美容用品。
下面的实例将具体说明本发明的操作方法, 但不能作为对本发明的限定。 实例一
取 1000g三矾腌制处理的市售麻蜇, 浙水后, 室温下浸没于 2-5倍量的去离子水中浸 泡,并每 2-3小时换一次水,直至浸泡水中氯化钠的含量低于 1.5ppm (氯离子检测呈阴性), 浸泡水的电导率低于 3(^Scm— 1为止。 浸泡后的脱盐霞水母添加活力为 4000 Units的胰蛋 白酶 10g, 在 30-60°C下保温溶融 4-24 小时, 得到一悬浮液。 然后离心 (4°C, 8000rpm, 30min), 弃去不溶性残渣, 得到澄清透明的液体, 即为胶原蛋白肽液; 胶原蛋白肽液置旋 转蒸发仪上, 40°C, O.OOlMPa下浓缩至原体积的 1/5后喷雾干燥 (进风温度 185°C, 出风温 度 85°C), 得到 20g纯度达到 90%以上的具有显著的免疫增强活性的胶原蛋白肽粉固体。
上述溶融处理也可在操作压力 0.001 MPa条件下进行。
实例二
取 100kg新鲜霞水母, 3~10°C下浸没于 2-5倍量的去离子水中浸泡, 并每 2-3小时 换一次水, 直至浸泡水的电导率低于 3(^Scm— 1为止。浸泡后的脱盐霞水母添加 500g复合 酶制剂 (由市售胰蛋白酶、木瓜蛋白酶、胶原蛋白酶和中性蛋白酶按照 1:1:1:1的配比组成), 在 35~50°C下保温处理 8-16小时,然后在 4°C, 8000rpm下离心 30min。弃去不溶性残渣, 得到澄清透明的液体, 即为胶原蛋白肽液; 胶原蛋白肽液在 40°C, O.OOlMPa下浓缩至原 体积的 1/2后冷冻干燥,得到 1000g纯度达到 95%以上的具有显著的免疫增强活性的胶原 蛋白肽粉固体。
上述溶融处理也可在操作压力 0.1 MPa条件下进行。 实例三
对一份胶原蛋白肽粉样品(如:按照实施例 2方法制备得到)进行了蛋白含量的测定, 总糖含量的测定, 氨基酸组成的测定以及单糖组成的测定。 测定结果表明, 该样品中含 88%的蛋白质, 10.5%的糖, 平均分子量为 1274。 所含单糖以葡萄糖为主, 所含氨基酸的 组成见表 1。
供试样品的氨基酸组成
氨基酸 含量 (mg/g) %
天冬氨酸 6.36229 8.66
谷氨酸 9.64957 13.14
丝氨酸 3.4011 4.63
组氨酸 0.1257 0.17
甘氨酸 12.9557 17.64
苏氨酸 2.57489 3.51
精氨酸 5.43067 7.39
丙氨酸 4.59921 6.26
酪氨酸 1.02847 1.40
胱氨酸 0.961188 1.31
缬氨酸 2.43407 3.31
蛋氨酸 0.306633 0.42
苯丙氨酸 1.31228 1.79
异亮氨酸 1.69354 2.31
亮氨酸 2.90096 3.95
赖氨酸 3.48776 4.75
脯氨酸 8.69692 11.84
羟脯氨酸 5.5269 7.52
总氨基酸 73.447851
实例四
以健康昆明种小白鼠 (雌雄各半, 4周龄, 体重 18-22g, 由无锡市惠山江南实验动物 场提供【批准号: SCXK (苏) 2002-0006】)为实验动物, 对所得到的胶原蛋白肽粉(如: 按 照实施例 2方法制备得到) 进行急性毒性试验, 所得结果见表 2和表 3。
表 2 供试样品对小鼠体重的影响
天数 /组别 空白对照 试验组
Id 22.806g 22.667
2d 22.985g 23.075
3d 24.300g 23.635
4d 24.688g 23.766
5d 25.146g 24.566
6d 26.084g 25.066
7d 26.180g 25.966 急性毒性实验结果
组别 小鼠数量 给药剂量 一周体重增长 增长率 死亡数 空白对照 10 0.5ml/只 3.374g 14.79% 0
试验组 10 7500mg/kg 3.299g 14.55% 0 通过观察, 给药后 lh内小鼠活动均减少, 4 h后逐渐恢复正常活动。 试验组 7 d内进 食量逐渐增加, 且小鼠食欲、 精神、 毛色均正常; 7d后, 脱颈处死, 肉眼尸检未见心、 肝、 脾、 肺、 肾等主要脏器病变和中毒现象。 观察期 7 d内每天称重, 试验组小鼠的体重 不断增加。研究结果表明, 在供试样品大剂量灌胃的情况下, 未出现小鼠死亡, 也未有明 显毒性反应。 说明其无毒性或毒性很小, 其最大安全剂量在 7500mg/Kg以上。 实例五
以健康昆明种小白鼠 (雌雄各半, 4周龄, 体重 18-22g, 由无锡市惠山江南实验动物 场提供【批准号: SCXK (苏) 2002-0006】)为实验动物, 对所得到的胶原蛋白肽(如: 按照 实施例 2方法制备得到) 进行免疫增强测试, 所得结果见表 4~6。
1小鼠血清溶血素测定
表 4供试样品对小鼠血清溶血素的影响
组别 给药剂量 (mg/kg) 溶血素含量
空白对照组 0.25土 0.03
试验组 25 0.26土 0.04
50 0.28土 0.05
100 0.40土 0.11* 方差分析, P<0.05; 与对照组比较 *P<0.05
溶血素 (IgM)是反映体液免疫功能状况的一种方法, 如果用药后溶血素生成增多, 则 出现红细胞溶血时的吸光度值增高,表明用药后机体的体液免疫作用增强。由表 4结果可 知, 高剂量组与空白对照組比较, 差异有显著性 (P<0.05), 低剂量组、 中剂量组与对照 组比较无差异性(P>0.05), 这说明, 高剂量供试样品对血清溶血素 (IgM)的生成有十分明 显的影响, 表现出较好的体液免疫增强作用。
2小鼠迟发型超敏反应 (DTH) 测定
表 5 供试样品对小鼠迟发型超敏反应的影响 组别 给药剂量 (mg/kg) 左右耳廓片重量之差 (mg) 空白对照组 3.40土 1.01
试验组 25 6.55土 1.62
50 11.05+5.35**
100 4.00土 2.12
方差分析, P<0.01 ; 与对照组比较 **P<0.01
迟发型超敏反应是反映细胞免疫功能状况的一种方法, 由表 5可知, 试验组的小鼠左 右耳廓片重量之差与对照组比较均有增加,中剂量组与空白对照組比较差异有高度显著性 (P<0.01), 其中低剂量组和高剂量组与对照组比较无显著性差异 (P >0.05)。 表明供试样 品在一定浓度范围内能显著提高小鼠细胞免疫功能。
3小鼠吞噬细胞吞噬功能测定
表 6 供试样品对小鼠吞噬细胞吞噬功能的影响
组别 给药剂量(mg/kg) 吞噬百分率 (%) 吞噬指数
空白对照 ― 33.83+1.05 2.23土 0.17
试验组 25 32.58土 1.16 2.25土 1.00
50 46.15+2.12* 5.52+0.56**
100 58.50土 2.04* 6.30+1.06**
方差分析, P<0.01 ; 与对照组比较 *P<0.05; **P<0.01
吞噬细胞吞噬功能是反映非特异性免疫功能状况的一种方法, 由表 6可知, 中剂量组 与高剂量组与对照组比较吞噬百分率有显著性差异 (P<0.05), 中剂量组与高剂量组与对 照组比较吞噬指数有高度显著性差异(P<0.01 ),低剂量组与对照组比较无差异(P>0.05), 说明一定剂量的供试样品能激活吞噬活性, 提高单核吞噬细胞系统的吞噬功能。
免疫增强实验结果表明一定剂量的供试样品对小鼠体液免疫功能,细胞免疫功能和单 核一巨噬细胞功能都有一定的增强作用。
根据功能学评价检验方法, 在细胞免疫功能、 体液免疫功能、 单核一巨噬细胞功能、 NK 细胞活性四个方面任两个方面结果阳性, 可判定该受试样品具有增强免疫力功能作 用。本实验在实验小鼠细胞免疫功能、体液免疫功能、单核一巨噬细胞功能三个方面结果 阳性, 因此可判定供试样品具有免疫增强作用。

Claims

权利要求书
1. 一种霞水母来源的具有免疫增强活性的胶原蛋白肽, 其特征在于外观为白色或淡黄 色, 无嗅, 略带苦味的粉末, 自霞水母科海洋生物白色霞水母 (Cyimefl nozaki Kishinouye) >发形霞水母 (Cyimeo capillata) >綜色霞水母 (Cyimeo/err Vzefl Eschscholtz) 或紫色霞水母 (C}¾mefl purpurea Kishinouye)的新鲜捕捞品或其经过三矾腌制的腌制品 中分离提取得到,无毒,具有显著的免疫增强活性,其中含有 80~90%的蛋白质, 10~20% 的糖, 平均分子量在 1,000~3,000之间, 所含单糖以葡萄糖为主, 所含氨基酸中甘氨 酸占 16%以上, 脯氨酸与羟脯氨酸之和占 18%以上。
2. 权利要求 1所述的胶原蛋白肽的制备工艺, 该工艺由下列步骤组成:
(1) 将霞水母用水浸泡;
(2) 对浸泡后的霞水母进行溶融处理, 然后离心分离, 得到无色或淡黄色的上清液;
(3) 将上清液直接干燥或经过均质、 脱色、 脱盐处理后干燥, 得到白色或浅黄色的粉 末, 即为胶原蛋白肽。
3. 如权利要求 2所述的胶原蛋白肽的制备工艺, 其特征在于将霞水母在 pH调节为 2-12 范围内某一定值的水中浸泡至浸泡水的电导率低于 3(^Scm— 1为止,浸泡温度在 0~98°C 范围内。
4. 如权利要求 2所述的胶原蛋白肽的制备工艺, 其特征在于所述的溶融处理指采用溶融 剂将霞水母融化于水中形成液体。
5. 如权利要求 4所述的胶原蛋白肽的制备工艺, 其特征在于所述的溶融剂为碱、 酸或各 种蛋白酶中的一种或几种混合。
6. 如权利要求 5所述的胶原蛋白肽的制备工艺, 其特征在于所述的碱为钠、 钾、 钙、 钡 的氢氧化物或碳酸盐、 氨水, 浓度为 0.1~10molL— 所述的酸为盐酸、 磷酸、 硫酸、 甲酸、 醋酸、 苹果酸、 乳酸或柠檬酸, 浓度为 0.1~10molL— 所述的蛋白酶为胃蛋白 酶、 胰蛋白酶、 胰糜蛋白酶、 酸性蛋白酶、 碱性蛋白酶、 中性蛋白酶、 木瓜蛋白酶、 菠萝蛋白酶、 胶原蛋白酶或弹性蛋白酶中的一种或若干种。
7. 如权利要求 4 所述的胶原蛋白肽的制备工艺, 其特征在于所述的溶融处理在 pH 0.5-14.0 , 温度 10~120°C, 操作压力 0.0001~0.5 MPa条件下进行。
8. 如权利要求 2所述的胶原蛋白肽的制备工艺, 其特征在于所述的干燥为冷冻干燥或喷 雾干燥。 权利要求 1所述的胶原蛋白肽在具有免疫增强功能的药品、 保健品、 护肤美容用 制备中的应用。
PCT/CN2008/072397 2008-05-20 2008-09-17 霞水母来源有免疫增强活性的胶原蛋白肽及制备和应用 WO2009140833A1 (zh)

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