WO2008049367A1 - Utilisation d'un extrait de protéine de graine de plante et composition pharmaceutique renfermant ledit extrait - Google Patents

Utilisation d'un extrait de protéine de graine de plante et composition pharmaceutique renfermant ledit extrait Download PDF

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Publication number
WO2008049367A1
WO2008049367A1 PCT/CN2007/070914 CN2007070914W WO2008049367A1 WO 2008049367 A1 WO2008049367 A1 WO 2008049367A1 CN 2007070914 W CN2007070914 W CN 2007070914W WO 2008049367 A1 WO2008049367 A1 WO 2008049367A1
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Prior art keywords
protein
plant seed
extract
drug
protein extract
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PCT/CN2007/070914
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English (en)
French (fr)
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Gengxi Hu
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Zhejiang Wolwo Biotech Co., Ltd.
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Publication of WO2008049367A1 publication Critical patent/WO2008049367A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines

Definitions

  • the invention belongs to the field of biopharmaceutical technology, and particularly relates to a plant seed protein extract used as a protein drug stabilizer and a use thereof. Background technique
  • Seeds are a period in the life history of seed plants at the highest stage of plant kingdom evolution and are the beginning of the next generation of independent plants.
  • the seeds are prepared for transmission from the structure and physiology, and sufficient nutrients are stored for the seedlings to become autotrophs.
  • the protein plays an important role in seed formation, development and seedling formation. It provides nutrients for seed growth and development, and also regulates various physiological and biochemical reactions and metabolic processes of seeds.
  • Seed proteins can be classified into storage proteins, protease inhibitors, and lectins (Study on Seed Proteins and Proteomes, Botanical Bulletin 2005, 22 (3 ); 257-266). Practice has shown that mature seeds are still viable for years to decades under normal temperature and dry conditions, so plant seeds should have mechanisms to sustain their lives for a long time.
  • Protein is one of the most basic substances in life. It is the material basis of life activities. Life activities are almost always realized by proteins. Some proteins are structural substances in living organisms, and some proteins are functional substances in living organisms. Many proteins, such as insulin, interferons, immunoglobulins, etc., can be used as medicines for the treatment of diseases.
  • protein molecules Due to the large molecular weight and complex structure of proteins, protein molecules are very unstable, susceptible to physical or chemical factors, and lose their biological activity. Especially when the concentration of the biologically active protein contained in the protein preparation is low, protein degradation or protein adsorption by the tube wall is prone to occur, thereby affecting the efficacy of the protein preparation.
  • One of the keys to the development of proteinaceous pharmaceutical preparations is to address the stability of such drugs.
  • One way to address the stability of proteinaceous drugs is to add protein stabilizers to low concentrations of protein drugs to protect proteins from degradation.
  • These protein stabilizers are typically proteins of animal origin, such as human serum albumin.
  • animal proteins presents a serious potential risk that animal proteins are contaminated by animal viruses that are not completely killed. For example, cases of public hazards in human serum albumin products due to HIV contamination have been reported.
  • animal proteins are generally only competitively preventing biologically active proteins from being physically adsorbed by the tube wall or by physical, chemical and biological factors in the environment.
  • the present invention provides a protein preparation stabilizer using a plant seed protein extract as a raw material and a main active ingredient.
  • the first aspect of the invention relates to the use of a plant seed protein extract as a stabilizer for a protein drug.
  • the plant seed protein extract refers to a mixture containing protein components extracted from plant seeds by a conventional extraction method in the prior art. There are many ways to obtain such plant seed proteins, and the extraction method varies depending on the variety of plant seeds.
  • the plant seed protein extract is prepared by the following method:
  • step (C) Aseptically treating the resulting solution of step (b) to obtain the plant seed protein extract.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a plant seed protein extract, a therapeutically effective amount of an active protein drug, and a pharmaceutically acceptable carrier, wherein the concentration of the plant seed protein extract It is 0.01 ⁇ g/ml ⁇ 50 mg/ml.
  • the plant seed protein extract of the present invention is of plant origin, thereby avoiding the risk of animal protein contamination of animal proteins which are prone to animal-derived pathogenic microorganisms such as hepatitis virus or AIDS virus.
  • the plant seed protein extract of the present invention exhibits a more excellent stabilizing effect in comparison with protein extracts derived from other tissues of plants. Further, the present invention utilizes a plant seed protein extract as a protein stabilizer, thereby enabling appropriate quality control at the time of formulating a protein preparation.
  • the first aspect of the invention provides the use of a plant seed protein extract for the preparation of a stabilizer for a protein drug.
  • Plant seed as used herein has the meaning commonly recognized or accepted by those skilled in the art.
  • Plant seed sources suitable for use in the present invention may include, for example, but are not limited to, Anacardium; Arachis, Asparagus, Oats (Wwa), and Watermelon (C/tra ⁇ ).
  • the plant seeds can be, for example, corn, wheat, rice, sorghum, pumpkin, gourd, lettuce, onion, tomato, beet, peanut, soybean Seeds of rapeseed, clover, tea, coffee, cocoa, etc., especially the mature seeds of these plants.
  • plant seeds as used herein also includes the addition of plant seeds. (Such as drying, grinding;), or the product obtained after material, such as a mature plant seed ground into powder after drying.
  • plant seed protein extract refers to a mixture containing protein components extracted from plant seeds by various extraction methods of the prior art. These extraction methods generally include, but are not limited to, extraction with an aqueous solution or an organic solvent (e.g., an alcohol, etc.; When the extraction is carried out using an aqueous solvent (e.g., water, physiological saline, phosphoric acid buffer, Tris buffer, alkaline buffer;), the pH of the aqueous solvent is preferably maintained in the range of pH 4-11. In order to remove unnecessary impurities (such as lipids), it is also possible to perform pretreatment steps such as grinding, drying, degreasing, etc., on the plant seeds during or before the extraction. However, it should be noted that the preparation of plant seed protein extracts should ensure that the proteins therein are not degraded, destroyed or inactivated.
  • an organic solvent e.g., an alcohol, etc.
  • an aqueous solvent e.g., water, physiological saline, phosphoric acid buffer, Tri
  • the plant seed protein extract is obtained by the following steps:
  • the plant seeds are degreased. For easy extraction, the plant seeds can be ground into powder. From the standpoint of source and cost, a preferred solution uses flour milled from wheat seeds.
  • the degreasing agent is preferably an organic solvent such as acetone or diethyl ether.
  • plant seeds can be continuously soaked in acetone for degreasing 3 times, 0.1 to 24 hours each time. After the degreased acetone is colorless, the degreased solid is naturally dried to an acetone-free taste and weighed. Laboratory operations should be performed in a fume hood, and a pilot-concentrated vacuum extraction tank is used.
  • the degreased plant seeds are contacted with the extract for at least 60 hours.
  • the extraction is preferably carried out at a low temperature (preferably about 2 to 8 ° C).
  • the extract is preferably an aqueous extract, preferably physiological saline.
  • the extraction operation should be carried out for 0.1 to 72 hours, preferably for 1 to 24 hours.
  • both the degreased and dried plant seeds and physiological saline are 1:2 to 1:50 (ie, each gram of the plant seed is degreased and dried and extracted with 2-50 ml of physiological saline solution) (preferably 1: 5 ⁇ 1: 15, more preferably 1: 10) (W/V)
  • Proportional extraction in order to ensure that the active ingredient is not deactivated, the extraction should be at a low temperature of 1-16 ° C, more preferably 2-8 ° C carry out.
  • the mixture was stirred intermittently for 72 hours (each stirring time was 8 hours, and after standing overnight, magnetic stirring was again performed for 8 hours, and thus repeated).
  • a preservative may be added to the extract, and the preservative may be selected from, but not limited to, phenol, thimerosal, and the like.
  • the solution obtained in the step (b) is subjected to sterilization treatment to obtain the plant seed protein extract.
  • the physiological saline solution leaching solution is first filtered with a common filter paper to obtain a crude filtrate.
  • the obtained crude filtrate is finely filtered with a microporous membrane having a pore diameter of not more than ⁇ to obtain a desired plant seed protein extract in the form of a solution.
  • the tested plant seed protein extract was stored at a low temperature of 4 °C.
  • the plant seed protein extract may also be lyophilized, concentrated, or the like according to a conventional technique.
  • plant seed protein extract does not only refer to plant seed protein extracts, but also includes plant seed protein extracts in other forms (eg, solid form;), as long as the extract remains The desired biological activity (ie, stabilizer action) is retained.
  • the plant seed protein extract of the present invention exhibits an excellent protective effect of highly stable proteinaceous drugs (also referred to herein as "proteinaceous drugs", “biologically active proteins”) in pharmaceutical preparations or pharmaceutical compositions. While not wishing to be bound by any theory, the inventors believe that this effect is obtained by the presence of protease inhibitors that prevent protein degradation or molecules that help proteins maintain their spatial structure, such as molecular chaperones, in plant seed protein extracts.
  • the present invention relates to the preparation of a protein pharmaceutical composition (or protein pharmaceutical preparation) by combining the plant seed protein extract obtained above with a protein drug.
  • the protein pharmaceutical composition comprises a plant seed protein extract prepared by the above method, a therapeutically effective amount of a protein drug, and optionally a pharmaceutically acceptable carrier, wherein the plant seed protein extract is L ⁇ 10 ⁇ / ⁇
  • the concentration of the composition is from 0.01 ⁇ g / ml to 50 mg / ml, preferably 0. l ⁇ 10mg / ml.
  • the protein pharmaceutical composition of the present invention may contain a plurality of protein extracts from different plant seeds to achieve further stabilization of the drug protein.
  • the protein drug (or biologically active protein) in the protein pharmaceutical composition of the present invention may be orally administered Administration of protein drugs, sublingual and buccal mucosal administration of protein drugs, rectal and colonic administration of protein drugs, transdermal administration of protein drugs, inhalation of protein drugs, including, for example but not limited to, interleukin IL- 1.
  • a "pharmaceutically acceptable carrier” should be compatible with the biologically active protein of the pharmaceutical composition of the present invention, i.e., can be blended therewith without substantially reducing the effectiveness of the pharmaceutical composition in treating the disease.
  • Specific examples of some substances which can be used as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, such as carboxymethyl fibers Sodium, ethyl cellulose and methyl cellulose; western yellow gum powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as
  • the mature wheat (seed;) was air-dried, ground into a powder, and continuously degreased three times with acetone for 4 hours, until the degreased acetone was colorless, and the degreased solid was dried and weighed.
  • the degreasing process should be done in a fume hood or vacuum concentration extraction tank for safety.
  • the extraction should be done at a low temperature of 2-8 °C.
  • the detailed steps are as follows: the degreased wheat flour and the physiological saline obtained in the step 1) are extracted in a ratio of 1:10 (W/V) (that is, each 1 gram of the degreased wheat flour is extracted with 10 ml of physiological saline solution;), 4 The mixture was stirred magnetically at °C for 72 hours (each stirring time was 8 hours, and after standing overnight, magnetic stirring was again performed for 8 hours, and thus repeated).
  • the physiological saline solution leaching solution was filtered through a common filter paper to obtain a crude filtrate.
  • the crude filtrate obtained in the step 3) is finely filtered with a microporous membrane having a pore diameter of not more than ⁇ , and the obtained filtrate is a flour extract, and the total protein concentration is measured (the protein content is determined by BCA protein assay;).
  • Example 2 Effect of wheat seed protein extract on the stability of allergen preparations
  • the house dust mite allergen extract was prepared by the following method:
  • the dust mites were cultivated in a medium (2 parts of laboratory animal feed, 2 parts of dry yeast, 1 part of dried fish meal, and a medium humidity of 16%) to a density of 300-500/g.
  • the mixture was suspended and saturated with a saturated NaCl solution to collect dust particles.
  • the obtained household dust mites were washed with physiological saline suspension, dried, and stored at -20 °C until use. Weigh the worm body, grind the liquid body with liquid nitrogen, immerse it in acetone for 3 times, 4 hours each time, until the acetone after degreasing is colorless, then dry the degreased solids to dryness without acetone. weight.
  • Both the house dust mites and physiological saline were soaked in 1:25 (W/V) (that is, each 1 gram of degreased house dust mites were extracted with 25 ml of physiological saline solution;), intermittent magnetic stirring at 4 ° C 72 Hours (; each stirring time is 8 hours, after standing overnight, magnetic stirring again for 8 hours, so repeated;).
  • the physiological saline solution leaching solution was filtered through a common filter paper to obtain a crude filtrate.
  • the obtained crude filtrate is sterilized by a microporous membrane having a pore diameter of not more than ⁇ , and the obtained filtrate is an extract of the house dust mite allergen, and the total protein concentration is determined (the protein content is determined by BCA protein assay). ;), and stored at 4 ° C low temperature.
  • the house dust mite allergen extract and the wheat seed protein extract were formulated into different mixed liquids according to the formulation of Table 1.
  • the mixture was filtered and sterilized, placed at 25 ° C for stability test, and samples were taken at 3, 6, 9, 12, and 18 months for determination of allergen activity.
  • the activity at 0 months was 100%, and the values measured at other times were compared with the values at 0 months.
  • the activity changes at different time points were determined to determine the activity of wheat protein stabilizers on allergens (reflecting stability). ) Impact.
  • control 1 means that pepsin and papain were added to the wheat seed protein extract, and after hydrolysis of the wheat seed protein extract for 6 hours, the enzyme was inactivated by 10CTC for 2 hours and then added to the allergen extract; It is a protein extracted from the stem of wheat by the method of Example 1, and the concentration of wheat stem protein in the allergen preparation is 20 mg/ml.
  • Example 3 Preparation of a mixture of wheat seed protein extract and allergen extract
  • Example 1 The wheat seed protein extract obtained in Example 1 and the house dust mite allergen extract obtained in Example 2 were diluted with physiological saline, and then an equal volume of glycerin was added to prepare a mixed allergen preparation. Diluted into a concentration of 1: 100 (V / V) to 1: 100000000 (V / V) according to the concentration required for treatment; sub-packaging, potting; filtration sterilization, that is, sublingual Agent
  • the mature soybeans are dried, ground into powder, and continuously degreased with acetone for 5 times for 4 hours, until the degreased acetone is colorless, and the degreased solid is naturally dried to an acetone-free taste and weighed.
  • Laboratory operations should be performed in a fume hood, and a vacuum concentration extraction tank is used in the pilot test.
  • the extraction is done at 4 °C.
  • the detailed steps are as follows: the degreased soybean powder obtained in the step 1) and the physiological saline are extracted in a ratio of 1:10 (W/V) (that is, each 1 g of the degreased soybean powder is extracted with 10 ml of physiological saline solution; , intermittent magnetic stirring at 4 ° C for 72 hours (; each stirring time is 8 hours, after standing overnight, magnetic stirring again for 8 hours, so repeated;).
  • the physiological saline solution leaching solution was filtered through a common filter paper to obtain a crude filtrate.
  • Interferon (IFN a _2b) and soy protein extract were formulated into different mixtures according to the formulation of Table 3.
  • a propellant was added to the mixture and sprayed according to a conventional method. After sterilization, the test was carried out at 4 ° C for stability test, and samples were taken at 2, 4, 6, 8, and 10 months for activity measurement. The measurement was carried out according to the SFDA-regulated standard method for the biological activity of interferon "cytopathic inhibition method" (People's Republic of China Pharmacopoeia 2005 edition, three appendices, pages 56 to 57). The activity at 0 months was 100%, and the values measured at other times were compared with the values at 0 months.
  • Control 1 in Table 4 means that pepsin and papain were added to the soy protein extract, and after hydrolysis of the soy protein extract for 6 hours, 10CTC inactivated the enzyme for 2 hours and then added to the interferon; Control 2 used Example 4 Method The protein extracted from the stem of soybean plants, in the interferon pharmaceutical preparation, the soybean stem protein concentration was 20 mg/ml.
  • the interferon and soy protein extract were formulated into different mixtures according to the formulation of Table 3.
  • Control 1 refers to the addition of pepsin and papain to the soy protein extract, after hydrolysis of the soy protein extract for 6 hours, 10CTC inactivates the enzyme for 2 hours and then adds interferon to make a suppository;
  • Control 2 is the use of Example 4.
  • Example 6 Preparation of Corn Seed Protein Extract
  • the mature corn seeds were air-dried, ground into powder, and continuously degreased with ether for 3 times, each time for 4 hours, until the degreased diethyl ether was colorless, and the degreased solid was dried and weighed.
  • the degreasing process should be done in a fume hood or in a vacuum concentrating extraction tank for safety.
  • the degreased corn flour and deionized water obtained in the step 1) are dispersed in a ratio of 1:6 (W/V) (that is, each 1 gram of the degreased corn flour is extracted with 6 ml of deionized water;), at room temperature
  • the pH was adjusted to 9 to 10 with a 1 mol/L NaOH solution under stirring (pH adjustment should be completed within 15 min), extraction was carried out for 15 to 60 min, and centrifuged at 13,000 g for 10 min, and the supernatant was retained.
  • the pH was adjusted to 3.5 to 4.5 with a lmol/L HCl solution to precipitate a protein. After centrifugation at 13,000 g for 15 min, the precipitate was dispersed with deionized water and the pH was adjusted to 6.5-7.5.
  • the solution obtained in the step 3) is finely filtered with a microporous membrane having a pore diameter of not more than ⁇ , and the obtained filtrate is a corn seed protein extract, and the total protein concentration is determined (BCA method, specifically, BCA protein assay) .
  • BCA method specifically, BCA protein assay
  • An oral insulin preparation was prepared according to the formulation of Table 6, and the preparation was orally administered to diabetic mice.
  • Insulin activity was determined according to the SFDA-specified insulin bioassay (People's Republic of China Pharmacopoeia 2005 Edition, Part 2, Appendix 104). The activity at 0 months was 100%, and the values measured at other times were compared with the values at 0 months. The activity changes at different time points were determined to determine the biological activity of corn seed protein extracts on insulin (reflecting stability). The effect of the determination is shown in Table 7.
  • Control 1 refers to the addition of pepsin and papain to the corn seed protein extract.
  • Example 8 Effect of corn seed protein extract on biological activity stability of recombinant human epidermal growth factor external solution
  • the recombinant human epidermal growth factor and the corn seed protein extract were formulated into different mixed liquids according to the formulation of Table 8, and the external solution was prepared after sterilization.
  • the measurement was carried out according to the SFDA-regulated method for measuring the biological activity of recombinant human epidermal growth factor, "cell proliferation method/MTT colorimetry" (Phase of the People's Republic of China, 2005 edition, three appendices, 60 pages).
  • the activity at 0 week was taken as 100%, and the values measured at other times were compared with the values at 0 weeks.
  • the activity changes at different time points were determined, and the soybean protein extract was used for the external solution of recombinant human growth factor.
  • the effect of biological activity reflecting stability).
  • the measurement results are shown in Table 9.
  • Control 1 means that pepsin and papain were added to the corn seed protein extract, and the corn seed protein extract was hydrolyzed for 6 hours, and then the enzyme was inactivated at 100 ° C for 2 hours and then added to the external solution of recombinant human epidermal growth factor;
  • the protein extracted from the stem of the corn plant by the method of Example 6 was used, and the concentration of the corn stem protein was 20 mg/ml in the external solution of the recombinant human epidermal growth factor.
  • IFN a -2b 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg Soy protein extract O g 0.01 ⁇ g 1 ⁇ g 100 ⁇ g lmg 5mg lOmg 20mg 30mg 50mg Normal saline added to 1ml Lml lml lml lml lml lml lml lmlmlmlmlmlml
  • Insulin 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU Corn Protein Extract o u g 0.01 ⁇ g l ⁇ g 100 ⁇ g lmg 5mg lOmg 20mg 30mg 50mg Refined Plant with 10% Ve 0.32g 0.32g

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Description

楠物种子蛋白提取物的用途及其鉬合物
技术领域
本发明属于生物药物技术领域，具体涉及用作蛋白质药物稳定剂的植物种子蛋白 提取物及其用途。 背景技术
种子是植物界演化最高阶段的种子植物生活史中的一个时期，是下一代独立植株 的开始。 种子从结构和生理上已做好了传播的准备， 也储藏了足够的养分以供幼苗长 成为自养体。 其中的蛋白质在种子形成、 发育直至成苗的过程中扮演着及其重要的角 色， 它为种子生长发育提供养料， 还调控着种子的各种生理生化反应和代谢过程。 种 子蛋白可分为储藏蛋白、 蛋白酶抑制剂和凝集素等 （种子蛋白质与蛋白质组的研究， 植物学通报 2005， 22 (3 )； 257〜266)。 实践证明， 成熟的种子在常温、 干燥的条件 下保存数年至数十年仍然具有活力， 因此植物种子应该具有相应的机制来长期维持其 生命。
蛋白质是生命的最基本物质之一， 是生命活动的物质基础， 生命活动几乎都是通 过蛋白质实现的， 有的蛋白质在生物体内是结构物质， 有的蛋白质在生物体内是功能 物质。 许多蛋白质， 如胰岛素、 干扰素、 免疫球蛋白等等， 都可以作为治疗疾病的药 物。
由于蛋白质的分子量大， 结构复杂， 蛋白质分子很不稳定， 易受物理或化学因素 的影响而变性， 丧失其生物活性。 尤其当蛋白类药物制剂中所含生物活性蛋白浓度较 低时， 易出现蛋白降解或蛋白被管壁吸附等现象， 从而影响蛋白质制剂的药效。 蛋白 质类药物制剂的研制关键之一是解决这类药物的稳定性问题。解决蛋白质类药物的稳 定性的方法之一是在低浓度的蛋白药物中加入蛋白稳定剂， 从而保护蛋白不被降解。 这些蛋白稳定剂通常是动物来源的蛋白， 例如人血清白蛋白。 然而， 采用动物蛋白存 在着严重的潜在风险， 即动物蛋白被未完全杀灭的动物病毒污染。 例如， 人血清白蛋 白制品中因为爱滋病毒的污染而发生公共危害的案例时有所闻。 另外， 动物蛋白一般 仅仅是竞争性地防止生物活性蛋白被管壁物理性地吸附， 或被环境中的物理、 化学和 生物因子损伤。
本领域中迫切需要提供一种安全、 廉价、 高效的新型蛋白质制剂稳定剂。 发明内容
为实现上述目的， 本发明提供了一种以植物种子蛋白提取物作为原料和主 要有效成分的蛋白质制剂稳定剂。
具体而言， 本发明第一方面涉及植物种子蛋白提取物作为蛋白质药物的稳 定剂的用途。 植物种子蛋白提取物是指用现有技术中的常规提取方法从植物种子提 取出来的含有蛋白组分的混合物。 获得这样的植物种子蛋白质的方法可以有很多， 且 提取方法随各种植物种子的变化而异。在一个较佳的实施方案中， 所述植物种子蛋 白提取物用以下方法制得：
(a)对植物种子进行脱脂处理；
(bM吏经脱脂处理的植物种子与提取缓冲液接触；
(C)无菌处理步骤 (b)所得的溶液， 获得所述植物种子蛋白提取物。
本发明另一方面提供了一种药物组合物， 其特征在于， 它含有植物种子蛋 白提取物、 治疗有效量的活性蛋白质药物以及药学上可接受的载体， 其中所述 植物种子蛋白提取物的浓度为 0.01 μ g/ml〜50mg/ml。
本发明的植物种子蛋白提取物是植物来源， 从而避免了动物蛋白所具有的 易发生动物源性病源微生物， 例如肝炎病毒或爱滋病病毒， 对动物蛋白污染的 风险。
与来自植物其它组织的蛋白提取物进行对比， 本发明的植物种子蛋白提取物 显示出更为优异的稳定效果。 另外， 本发明通过植物种子蛋白提取物作为蛋白 稳定剂， 从而能够在配制蛋白质制剂的时候实现合适的质量控制。 本发明的其 他目的和优点可以从下文中明显得出。 具体实施方式
具体而言， 本发明第一方面提供了植物种子蛋白提取物在制备蛋白质药物 用的稳定剂的用途。
本文所用的术语 "植物种子"具有本领域技术人员通常认可或接受的含义。 适用于本发明的植物种子来源可以包括，例如但不局限于，腰果属 (Anacardium；)、 落花生属 (Arachis)、 天门冬属 (Asparagus)、 燕麦属 ( wwa)、 西瓜属 (C/tra〃w 、
Figure imgf000004_0001
、 椰子属 (Coco 、 咖啡属 (Q ea)、 黄瓜 属 (C c mis)、 |¾ jH j¾ (Cucurbita) , 贯众属 (Cyrtomiwn)、 古月萝卜属 (Ζ)α 、 油棕 属 (Elaeis)、 草莓属 (Fragaria)、 大显属 (Glycine)、 向日葵属 (Helianthus)、 Heterocallis、 大麦属 (Horafe謹)、 天仙子属 (Hycwe_ya 、 萬宦属 ( actwca)、 亚麻 属 (Linum)、 黑麦草属 ( O//MW)、 lm3- (Lupinus) ,
Figure imgf000005_0001
, 苹果 属 (Malus)、 木薯属 (Ma«z zot)、 苜蓿属 (Mecft'cago)、 烟草属 (N/cot/a«a)、 木犀榄属 {Oled) , 禾舀属 (( r_yza)、 紫萁属 (( 扁謂 cfa)、 泰属 (Panieum)、 Pannesetum , 湾梨属 (Persea) ,菜豆属 (Ph olus)、黄连木属 (i¾tac w'aj、豌豆属 (/¾扁)、 (Pyrus) , 李属 (Primus)、 萝卜属 (Raphanus)、 黑麦属 (Seca/e)、 千里光属 (Se"ec/o)、 白芥属 (Sinapis) , 茄属 (So 謹 m)、 高梁属 (Sorg w )、 可可树属 (J¾eobro w5 、 胡卢巴属 (Trigonella) , 小麦属 (Triticum)、 野豆花属（Mc/a)、 葡萄属（Mt/ 、 豇豆属（Mgna) 和玉米属 (Ze ^等可食用的物种种类。 在较佳的实施方案中， 所述植物种子例如 可以是玉米、 小麦、 水稻、 高粱、 南瓜、 葫芦、 莴苣、 洋葱、 西红柿、 甜菜、 花生、 大豆、 油菜、 三叶苜蓿、 茶叶、 咖啡、 可可等的种子， 尤其是这些植物 的成熟种子。 本文所用的术语 "植物种子"还包括了对植物种子经过加工 (如干 燥、 研磨;)后获得的产物或材料， 如成熟的植物种子干燥后磨成的粉。
术语 "植物种子蛋白提取物"指用现有技术中的各种提取方法从植物种子 提取出来的含有蛋白组分的混合物。 这些提取方法通常包括， 但不限于， 用水 性溶液或有机溶剂 (如醇等;)提取等。 在采用水性溶剂 (如水、 生理盐水、 磷酸缓 冲液、 Tris缓冲液、碱性缓冲液；)进行提取时，该水性溶剂的 pH优选维持在 pH 4-11 范围内。 为了除去不必要的杂质 (如脂类；)， 还可在提取过程中或提取前， 可视需 要对植物种子进行研磨、 干燥、 脱脂等预处理步骤。 然而， 应当注意的是， 在 制备植物种子蛋白提取物应当确保其中的蛋白不被降解、 破坏或失活。
在本发明的一个较佳实施方案中， 所述植物种子蛋白提取物是用以下步骤 制得的：
(1)脱脂、 干燥： 将植物种子进行脱脂处理。 为便于提取， 可先将植物种子 研磨成粉。 从来源和成本的角度考虑， 一个优选的方案采用由小麦种子研磨而 成的面粉。 所述脱脂剂优选丙酮、 乙醚等有机溶剂。 例如， 可连续用丙酮浸泡 植物种子进行脱脂 3次， 每次 0.1〜24小时。 至脱脂后的丙酮为无色后， 将脱脂 后的固体物自然干燥至无丙酮味后称重。 实验室操作应在通风橱内完成， 中试 采用真空浓缩提取罐。
(2)提取： 使经脱脂处理的植物种子与提取液接触至少 60小时。 所述提取 操作宜在低温下 (优选大约 2〜8°C)进行。 所述提取液宜为水性提取液， 优选生 理盐水。 提取操作应进行 0. 1〜72小时， 优选进行 1〜24小时。 例如， 将经脱 脂干燥后的植物种子和生理盐水二者以 1 : 2〜1： 50 (即每 1克植物种子脱脂干 燥后用 2-50毫升生理盐水溶液提取） （较佳的为 1： 5〜1： 15，更佳的为 1： 10) (W/V) 比例提取， 为保证有效成分不失活， 提取宜在 1-16°C、 更佳为 2-8°C的低温下完 成。 间歇磁力搅拌 72小时 (每次搅拌时间为 8小时， 静置过夜后再次磁力搅拌 8 小时， 如此反复）。 另夕卜， 在所述提取液中还可加入防腐剂， 所述防腐剂可选自， 但不限于， 苯酚、 硫柳汞等。
(3)除菌：将步骤 (b)所得的溶液进行除菌处理， 获得所述的植物种子蛋白提 取物。 在一个优选的实施方案中， 在结束步骤 (b)的搅拌后， 先用普通滤纸过滤 生理盐水溶液浸出液， 得到粗滤液。 然后， 再将得到的粗滤液用孔径不大于 ΐμηι 的微孔滤膜进行精滤， 得到所需的溶液形式的植物种子蛋白提取物。 将检测合 格的植物种子蛋白提取液于 4°C低温保存。也可根据常规技术将所述植物种子蛋 白提取物进行冻干、 浓缩等。 就此意义而言， 本发明所用的术语 "植物种子蛋 白提取物"不仅仅指植物种子蛋白提取液， 其也包括了其他形式 (如固体形式;) 的植物种子蛋白提取物， 只要该提取物仍然保留了所需的生物活性 (即稳定剂作 用)。
本发明的植物种子蛋白提取物表现出了使蛋白类药物 (本文中也称为 "蛋白 质药物"、 "生物活性蛋白"；)在药物制剂或药物组合物中高度稳定的优异保护效 果。 虽然不希望拘泥于任何理论， 发明者相信该效果的获得是由于植物种子蛋 白提取物中存在着防止蛋白降解的蛋白酶抑制剂或帮助蛋白质维持其空间结构 的分子， 例如分子伴侣等。
因此， 本发明另一方面涉及将上述获得的植物种子蛋白提取物与蛋白类药 物配制在一起获得蛋白药物组合物 (或蛋白药物制剂）。 具体而言， 所述蛋白药 物组合物含有用上述方法制得的植物种子蛋白提取物、 治疗有效量的蛋白类药 物以及任选的药学上可接受的载体， 其中所述植物种子蛋白提取物在药物组合 物中的浓度为 0.01 μ g/ml〜50mg/ml， 优选 0. l〜10mg/ml。
另外， 可以预见的是， 本发明的蛋白药物组合物中可以含有多种来自不同 植物种子的蛋白提取物， 以实现进一步稳定药物蛋白的效果。
在本发明的蛋白药物组合物中的蛋白类药物 (或生物活性蛋白） 可以是口服 给药蛋白质药物、 舌下及颊粘膜给药蛋白质药物、 直肠及结肠给药蛋白质药物、 透皮给药蛋白质药物、 吸入给药蛋白质药物， 具体包括， 例如但不局限于， 白 细胞介素 IL-1、 IL-2、 IL-3、 IL-4、 IL-5、 IL-6、 IL-7、 IL-8、 IL-9、 IL-10、 IL-11、 IL-12、 IL-13、 IL-14、 IL-15、 IL-16、 IL-17、 IL-18和它们的所有的亚型如 IL-loc 和 IL-Ιβ, 肿瘤坏死因子 (TNF；)、 转化生长因子 (TGF-β和 -o 、 I型和 II型干扰素 (IFN- l , IFN- 2, (IFN- o)、 IFN-β, IFN-γ), 迁移抑制因子 （MIF)、 c-kit配体、 粒细胞巨噬细胞集落剌激因子 (； GM-CSF；)、 单核细胞巨噬细胞集落剌激因子 (M-CSF), 粒细胞集落剌激因子 (G-CSF)、 趋化因子如 IL-8、 RANTES, 前列腺 素、 粘附因子如可溶性 ICAM1、 脱唾液酸血清类粘蛋白 (； ASOR；)、 运铁蛋白、 脱 唾液酸糖蛋白、 干细胞因子和促红细胞生成素 （EPO) 等。
本文所用的 "药学上可接受的载体"应当与本发明药物组合物中的生物活 性蛋白相容， 即能与其共混而不会在通常情况下大幅度降低药物组合物在治疗 疾病方面的效果。 可作为药学上可接受的载体或其组分的一些物质的具体例子 是糖类， 如乳糖、 葡萄糖和蔗糖； 淀粉， 如玉米淀粉和土豆淀粉； 纤维素及其 衍生物， 如羧甲基纤维素钠、 乙基纤维素和甲基纤维素； 西黄蓍胶粉末； 麦芽； 明胶； 滑石； 固体润滑剂， 如硬脂酸和硬脂酸镁； 硫酸钙； 植物油， 如花生油、 棉籽油、 芝麻油、 橄榄油、 玉米油和可可油； 多元醇， 如丙二醇、 甘油、 山梨 糖醇、 甘露糖醇和聚乙二醇； 海藻酸； 乳化剂， 如吐温； 润湿剂， 如月桂基硫 酸钠； 着色剂； 调味剂； 压片剂、 稳定剂； 抗氧化剂； 防腐剂； 无热原水； 等 渗盐溶液； 和磷酸盐缓冲液等， 其中较佳的载体选自生理盐水、 甘油和磷酸盐 缓冲盐水。
下面将结合实施例进一步详细地描述本发明。 然而， 应当理解， 下面的实 施例仅仅是为了起说明作用， 这并不意味着本发明的适用范围仅仅限于所述的 特定生物活性蛋白。 实施例 1小麦种子蛋白提取物的制备
1)脱脂、 干燥
将成熟的小麦 (种子;)晾干， 研磨成粉， 连续用丙酮浸泡脱脂 3 次， 每次 4 小时， 至脱脂后的丙酮为无色后， 将脱脂后的固体物干燥后称重。 脱脂过程宜 在通风橱或真空浓缩提取罐内完成， 以策安全。
2)提取 为保证有效成分不失活， 提取应在 2— 8°C低温下完成。 详细步骤为： 将步 骤 1)获得的脱脂后的小麦粉和生理盐水二者以 1 : 10(W/V)比例提取 (即每 1克脱 脂后的小麦粉用 10毫升生理盐水溶液提取；)， 4°C间歇磁力搅拌 72小时 (每次搅 拌时间为 8小时， 静置过夜后再次磁力搅拌 8小时， 如此反复)。
3)去渣
结束搅拌后将生理盐水溶液浸出液用普通滤纸过滤， 得到粗滤液。
4)精滤
将步骤 3)得到的粗滤液用孔径不大于 Ιμηι的微孔滤膜进行精滤， 得到的滤 液为面粉提取液， 对其进行总蛋白浓度测定 (；用 BCA蛋白测定法测定蛋白含量；)。 实施例 2小麦种子蛋白提取物对变应原制剂稳定性的影响
首先， 用以下方法制备户尘螨变应原提取物：
1)清洗、 研磨、 脱脂、 干燥
在培养基 (2份实验室动物饲料， 2份干酵母， 1 份干鱼粉， 培养基湿度为 16%)中培养户尘螨使其密度达到 300— 500只 /克。 用饱和 NaCl溶液悬浮分离， 收集户尘螨螨体。 将获得的户尘螨螨体以生理盐水悬浮清洗， 晾干后， 置于 -20 °C保存备用。 称取虫体， 对虫体进行液氮研磨， 连续用丙酮浸泡脱脂 3 次， 每 次 4 小时， 至脱脂后的丙酮为无色后， 将脱脂后的固体物自然干燥至无丙酮味 后称重。
2)提取
将户尘螨虫体和生理盐水二者以 1 : 25(W/V)浸泡提取 (即每 1克脱脂后的户 尘螨虫体用 25毫升生理盐水溶液提取；)， 4°C间歇磁力搅拌 72小时 (；每次搅拌时 间为 8小时， 静置过夜后再次磁力搅拌 8小时， 如此反复；)。
3)去渣
结束搅拌后将生理盐水溶液浸出液用普通滤纸过滤， 得到粗滤液。
4)精滤
将得到的粗滤液用孔径不大于 Ιμηι的微孔滤膜进行除菌过滤， 得到的滤液 为户尘螨变应原提取液， 对其进行总蛋白浓度测定 (；用 BCA蛋白测定法测定蛋白 含量；)， 并于 4°C低温保存。
然后， 将户尘螨变应原提取物与小麦种子蛋白提取物按照表 1 的配方， 配 制成不同的混合液。 将混合液过滤除菌之后放置在 25°C进行稳定性试验， 于 3、 6、 9、 12、 18 个月取样品进行变应原活性的测定。 以 0个月时的活性为 100%， 其他时间测定 的数值与 0个月时的数值对比进行比值分析， 检测不同时间点的活性变化， 判 断小麦蛋白稳定剂对变应原活性 （反映稳定性） 的影响。
本实施例中不同配方的变应原制剂的稳定性考察测定结果见表 2。表 2中对 照 1 是指在小麦种子蛋白提取物中加入胃蛋白酶和木瓜蛋白酶， 水解小麦种子 蛋白提取物 6小时后， 10CTC使酶失活 2小时然后再加入变应原提取物中； 对照 2是采用实施例 1的方法从小麦的茎中提取的蛋白，在变应原制剂中小麦茎蛋白 的浓度为 20mg/ml。 实施例 3小麦种子蛋白提取物与变应原提取液混合液的制备
1) 将实施例 1获得的小麦种子蛋白提取物和实施例 2获得的户尘螨变应原 提取物用生理盐水稀释， 然后加入等体积甘油制成混合变应原制剂。 按照治疗所需浓度， 稀释成 1 : 100(V/V)至 1 : 100000000(V/V)范围内 的多个浓度的制剂； 分装、 灌封； 过滤除菌， 即得舌下含服剂；
2) 取上述原液按常规制剂方法制得片剂或胶囊剂。 实施例 4大豆蛋白提取物的制备
1 ) 脱脂、 干燥
将成熟的大豆晾干， 研磨成粉， 连续用丙酮浸泡脱脂 5次， 每次 4小时， 至脱脂后的丙酮为无色后， 将脱脂后的固体物自然干燥至无丙酮味后称重。 实 验室操作应在通风橱内完成， 中试采用真空浓缩提取罐。
2)提取
为保证有效成分不失活， 提取在 4°C完成。 详细步骤为： 将步骤 1)获得的 脱脂后的大豆粉末和生理盐水二者以 1 : 10(W/V)比例提取 (即每 1克脱脂后的大 豆粉用 10毫升生理盐水溶液提取;)，4°C间歇磁力搅拌 72小时 (；每次搅拌时间为 8 小时， 静置过夜后再次磁力搅拌 8小时， 如此反复；)。
3)去渣
结束搅拌后将生理盐水溶液浸出液用普通滤纸过滤， 得到粗滤液。
4)精滤 将步骤 3)得到的粗滤液用孔径不大于 Ιμηι的微孔滤膜进行精滤， 得到的滤 液为大豆蛋白提取液， 对其进行总蛋白浓度测定 (； BCA法， 具体是采用 Pierce公 司的 BCA蛋白测定试剂盒；)。 实施例 5大豆蛋白提取物对干扰素生物学活性稳定性影响
将干扰素 (IFN a _2b)与大豆蛋白提取物按照表 3的配方， 配制成不同的混合 液。 向混合液中加入抛射剂并按常规方法制成喷剂， 除菌之后放置在 4°C进行稳 定性试验， 于 2、 4、 6、 8、 10个月取样品进行活性测定。 测定按 SFDA规定的 干扰素生物学活性标准方法 "细胞病变抑制法"进行（中华人民共和国药典 2005 年版 三部 附录 56〜57页）。 以 0个月时的活性为 100%， 其他时间测定的数值 与 0个月时的数值对比进行比值分析， 检测不同时间点的活性变化， 判断大豆 蛋白对干扰素生物学活性（反映稳定性） 的影响。 测定结果见表 4。 表 4中对照 1是指在大豆蛋白提取物中加入胃蛋白酶和木瓜蛋白酶， 水解大豆蛋白提取物 6 小时后 10CTC使酶失活 2小时然后再加入干扰素中；对照 2是采用实施例 4的方 法从大豆植株茎中提取的蛋白， 在干扰素药物制剂中， 大豆茎蛋白浓度为 20mg/ml。
将干扰素与大豆蛋白提取物按照表 3 的配方， 配制成不同的混合液。 向混 合液中加入栓剂常用基质如吐温 -61并按常规方法制成栓剂， 放置在 4°C进行稳 定性试验， 于 2、 4、 6、 8、 10个月取样品， 溶于 lml生理盐水， 进行活性测定， 测定结果见表 5。 对照 1是指在大豆蛋白提取物中加入胃蛋白酶和木瓜蛋白酶， 水解大豆蛋白提取物 6小时后 10CTC使酶失活 2小时然后再加入干扰素中制成栓 剂； 对照 2是采用实施例 4的方法从大豆植株茎中提取的蛋白， 在干扰素药物 制剂中， 大豆茎蛋白浓度为 20mg/ml。 实施例 6玉米种子蛋白提取物的制备
1)脱脂、 干燥
将成熟的玉米种子晾干， 研磨成粉， 连续用乙醚浸泡脱脂 3次， 每次 4小 时， 至脱脂后的乙醚为无色后， 将脱脂后的固体物干燥后称重。 脱脂过程宜在 通风橱或真空浓缩提取罐内完成， 以策安全。
2)提取 将步骤 1)获得的脱脂后的玉米粉和去离子水二者以 1 : 6(W/V)比例分散 (即 每 1 克脱脂后的玉米粉用 6 毫升去离子水溶液提取；)， 在室温下边搅拌边用 Imol/LNaOH溶液将 pH调至 9〜10 (pH调节应在 15min内完成）， 提取 15〜 60min， 13000g离心 10min， 保留上清。
3)沉淀
用 lmol/L HCl 溶液调整 pH为 3.5〜4.5， 沉淀出蛋白。 13000g离心 15min， 沉淀用去离子水分散并调节 pH为 6.5〜7.5。
4)精滤
将步骤 3)得到的溶液用孔径不大于 Ιμιη的微孔滤膜进行精滤，得到的滤液为玉米 种子蛋白提取液，对其进行总蛋白浓度测定 (BCA法，具体是采用 BCA蛋白测定法)。 实施例 7 玉米种子蛋白提取物对胰岛素口服制剂活性的影响
按照表 6的配方配制胰岛素口服制剂， 然后将制剂通过口服喂食给糖尿病 小鼠。按照 SFDA规定的胰岛素生物测定法（中华人民共和国药典 2005年版 二 部 附录 104页） 测定胰岛素活性。 以 0个月时的活性为 100%， 其他时间测定 的数值与 0个月时的数值对比进行比值分析， 检测不同时间点的活性变化， 判 断玉米种子蛋白提取物对胰岛素生物学活性 （反映稳定性） 的影响， 测定结果 见表 7。对照 1是指在玉米种子蛋白提取物中加入胃蛋白酶和木瓜蛋白酶， 水解 玉米种子蛋白提取物 6小时后 10CTC使酶失活 2小时然后再加入胰岛素中；对照 2是采用实施例 6的方法从玉米植株茎中提取的蛋白， 在胰岛素药物制剂中， 玉 米茎蛋白浓度为 20mg/ml。 实施例 8玉米种子蛋白提取物对重组人表皮生长因子外用溶液生物学活性 稳定性影响
将重组人表皮生长因子与玉米种子蛋白提取物按照表 8 的配方， 配制成不 同的混合液， 除菌后制成外用溶液。
测定按 SFDA规定的重组人表皮生长因子生物学活性测定方法 "细胞增殖 法 /MTT比色法"进行 （中华人民共和国药典 2005年版 三部 附录 60页）。 以 0周时的活性为 100%，其他时间测定的数值与 0周时的数值对比进行比值分析， 检测不同时间点的活性变化， 判断大豆蛋白提取物对重组人生长因子外用溶液 生物学活性（反映稳定性） 的影响。 测定结果见表 9。 对照 1是指在玉米种子蛋 白提取物中加入胃蛋白酶和木瓜蛋白酶，水解玉米种子蛋白提取物 6小时后 100 °C使酶失活 2小时然后再加入重组人表皮生长因子外用溶液中； 对照 2是采用 实施例 6 的方法从玉米植株茎中提取的蛋白， 在重组人表皮生长因子外用溶液 中， 玉米茎蛋白浓度为 20mg/ml。
表 1 户尘螨变应原提取物与小麦种子蛋白提取物的不同配制方法
配方 1# 2# 3# 4# 5# 6# 8# 9# 10# 变应原提取物 1.2mg 1.2mg 1.2mg 1.2mg 1.2mg 1.2mg 1.2mg 1.2mg 1.2mg 1.2mg 小麦种子蛋白提取
Oug 10 μ g lmg lOOmg lOOOmg 5000mg lOOOOmg 20000mg 30000mg 50000mg 物
甘油 500ml 500ml 500ml 500ml 500ml 500ml 500ml 500ml 500ml 500ml 硫柳汞 lOOmg lOOmg lOOmg lOOmg lOOmg lOOmg lOOmg lOOmg lOOmg lOOmg 生理盐水加至 1000ml 1000ml 1000ml 1000ml 1000ml 1000ml 1000ml 1000ml 1000ml 1000ml 表 2 小麦种子蛋白提取物对户尘螨变应原提取物活性的影响
Figure imgf000013_0001
IFNa-2b与大豆蛋白提取物的不同配制万法 配方 1# 2# 3# 4# 5# 6# 7# 8# 9# 10#
IFN a -2b 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 0.5mg 大豆蛋白提取物 O g 0.01 μ g 1 μ g 100 μ g lmg 5mg lOmg 20mg 30mg 50mg 生理盐水加至 1ml lml lml lml lml lml lml lml lml lml
表 4大豆蛋白提取物对干扰素 （喷剂） 活性的影响
Figure imgf000014_0001
表 5 大豆蛋白提取物对干扰素 （栓剂） 活性的影响
Figure imgf000015_0001
表 6 胰岛素口服制剂的配方 （每一颗胶囊的含量)
配方 1# 2# 3# 4# 5# 6# 7# 8# 9# 10#
Insulin 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU 玉米蛋白提取物 o u g 0.01 μ g l^ g 100 μ g lmg 5mg lOmg 20mg 30mg 50mg 含 10%Ve的精制植 0.32g 0.32g
0.32g 0.32g 0.32g 0.32g 0.32g 0.32g 0.32g 0.32g
50mg 50mg 50mg 49.9mg 49mg 45mg 40mg 30mg 20mg Omg
Figure imgf000016_0001
表 8 重组人表皮生长因子 （rEGF) 外用溶液与玉米种子蛋白提取物的不同配制方法
配方 1# 2# 3# 4# 5# 6# 7# 8# 9# 10# rEGF 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU 50IU 甘油 100 μ 1 100 μ 1 100 μ 1 100 μ 1 100 μ 1 100 μ 1 100 μ 1 100 μ 1 100 μ 1 100 μ 1 甘露醇 10 μ 1 10 μ 1 10 μ 1 10 μ 1 10 μ 1 10 μ 1 10 μ 1 10 μ 1 10 μ 1 10 μ 1 大豆蛋白提取物 oug 0.01 μ g l^g 100 μ g lmg 5mg lOmg 20mg 30mg 50mg 生理盐水加至 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml
表 9玉米种子蛋白提取物对 rEGF活性的影响
Figure imgf000017_0001
从表 2、 4、 5、 7、 9的结果可以看出， 在蛋白质制剂中加入植物种子蛋白提取物 可以提高药物制剂的稳定性，这种提高药物制剂稳定性的能力随着加入的植物种子蛋 白提取物的浓度的增加而有一定程度的提高。 尽管本发明描述了具体的例子， 但是有一点对于本领域技术人员来说是明显的， 即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。 因此， 所附 权利要求覆盖了所有这些在本发明范围内的变动。

Claims

权利要求
1. 植物种子蛋白提取物在制备蛋白质药物中作为稳定剂的用途。
2. 如权利要求 1所述的用途， 其特征在于， 所述植物种子为可食用植物种子。
3. 如权利要求 1所述的用途， 其特征在于， 所述蛋白质药物为口服给药蛋白质 药物、 舌下及颊粘膜给药蛋白质药物、 直肠及结肠给药蛋白质药物、 透皮给药蛋白质 药物、 吸入给药蛋白质药物。
4. 如权利要求 1所述的用途， 其特征在于， 所述植物种子蛋白提取物用不导致 蛋白质严重降解或失活的去离子水或蛋白质提取缓冲液提取获得。
5. —种药物组合物， 其特征在于， 它含有植物种子蛋白提取物、 治疗有效量的 活性蛋白质药物以及药学上可接受的载体。
6. 如权利要求 5所述的药物组合物， 其特征在于， 所述植物种子蛋白提取物的 蛋白浓度为 0.01 μ g/ml〜50mg/ml。
7. 如权利要求 5所述的药物组合物， 其特征在于， 所述植物种子为可食用植物 种子。
8. 如权利要求 5所述的药物组合物， 其特征在于， 所述药物组合物为口服给药 蛋白质药物、 舌下及颊粘膜给药蛋白质药物、 直肠及结肠给药蛋白质药物、 透皮给药 蛋白质药物、 吸入给药蛋白质药物。
9. 如权利要求 5所述的药物组合物， 其特征在于， 所述植物种子蛋白提取物用 不导致蛋白质严重降解或失活的去离子水或蛋白质提取缓冲液提取获得。
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CN1103258A (zh) * 1993-12-04 1995-06-07 金学陵 植物蛋白及其制备方法
CN1913785A (zh) * 2004-01-30 2007-02-14 巴斯福股份公司 稳定酶制剂

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CN1103258A (zh) * 1993-12-04 1995-06-07 金学陵 植物蛋白及其制备方法
CN1913785A (zh) * 2004-01-30 2007-02-14 巴斯福股份公司 稳定酶制剂

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