WO2020108629A1 - 一种多肽rdp1及其提纯方法与应用 - Google Patents

一种多肽rdp1及其提纯方法与应用 Download PDF

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WO2020108629A1
WO2020108629A1 PCT/CN2019/122077 CN2019122077W WO2020108629A1 WO 2020108629 A1 WO2020108629 A1 WO 2020108629A1 CN 2019122077 W CN2019122077 W CN 2019122077W WO 2020108629 A1 WO2020108629 A1 WO 2020108629A1
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rdp1
polypeptide
hyperuricemia
application
purifying
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WO2020108629A8 (zh
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杨新旺
孙俊
孟步亮
刘乃心
王滢
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西安棣加生物科技有限公司
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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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • 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 invention belongs to the field of biomedicine, and specifically relates to a polypeptide RDP1 and its purification method and application.
  • Gout has become China's second largest metabolic disease after diabetes. Gout is caused by the accumulation of uric acid in the blood and crystallizing in joints or soft tissues. The pre-disease symptom is hyperuricemia. Hyperuricemia is generally caused by too much uric acid production or too little excretion, and xanthine, as a key enzyme in uric acid production, is most commonly used as a drug target for anti-hyperuricemia.
  • Anti-hyperuricemia is the main goal of anti-gout in the non-acute phase, but the clinical anti-hyperuricemia drugs have certain defects, including low activity, high manufacturing cost, large side effects and difficult to reverse. Therefore, there is an urgent need to explore or develop new antihyperuricemia drugs with few adverse reactions and economical and practical.
  • the first object of the present invention is to provide a polypeptide RDP1.
  • the amino acid sequence included in the polypeptide RDP1 is shown in SEQ No. 1.
  • the second object of the present invention is to provide a method for purifying the polypeptide RDP1, which comprises the following steps:
  • step (3) Perform the first high-performance liquid chromatography reverse phase chromatography on the separated product of step (2), and collect the active components;
  • the active component is subjected to the second high-performance liquid chromatography reverse phase chromatography to obtain the purified polypeptide RDP1.
  • the third object of the present invention is to provide an application of the polypeptide RDP1 against hyperuricemia, including oral application.
  • the fourth object of the present invention is to provide a medicine containing the polypeptide RDP1.
  • the fifth object of the present invention is to provide an application of the polypeptide RDP1 in a drug against hyperuricemia.
  • FIG. 1 is a Sephadex G50 molecular sieve diagram of the anti-hyperuricemia active polypeptide RDP1 of the present invention; in the figure, the arrow indicates the absorption peak of RDP1.
  • FIG. 2 is a HPLC reversed-phase C18 column chromatogram of the active anti-hyperuricemia polypeptide RDP1.
  • the arrow indicates the absorption peak of RDP1.
  • FIG. 3 is the second HPLC reversed-phase C18 column chromatogram of the active anti-hyperuricemia polypeptide RDP1.
  • the arrow indicates the absorption peak of RDP1.
  • Fig. 4 is a mass spectrum of the active anti-hyperuricemia polypeptide RDP1 of the present invention.
  • Fig. 5 is a primary structure diagram of the anti-hyperuricemia active polypeptide RDP1 of the present invention.
  • Fig. 6 is a graph showing the results of uric acid lowering of the anti-hyperuricemia active polypeptide RDP1 of the present invention.
  • *** represents P ⁇ 0.001 (t test).
  • Fig. 7 is a graph showing the results of reducing creatinine of the anti-hyperuricemia active polypeptide RDP1 of the present invention.
  • * represents P ⁇ 0.05
  • ** represents P ⁇ 0.01
  • *** represents P ⁇ 0.001 (t test).
  • FIG. 8 is a graph of HE staining results of the anti-hyperuricemia active polypeptide RDP1 of the present invention.
  • FIG. 9 is the result of in vitro inhibition of xanthine oxidase activity of the anti-hyperuricemia active polypeptide RDP1.
  • ** represents P ⁇ 0.01
  • *** represents P ⁇ 0.001 (t test).
  • FIG. 10 is a graph showing the results of inhibiting xanthine oxidase activity in rat serum after administration of anti-hyperuricemia active polypeptide RDP1 of the present invention.
  • * represents P ⁇ 0.05
  • ** represents P ⁇ 0.01 (t test).
  • polypeptide RDP1 of the present invention is derived from rice extract, and the amino acid sequence contained in the polypeptide RDP1 is shown in SEQ No. 1.
  • the invention also provides a method for purifying the polypeptide RDP1 from rice extract, which includes the following steps:
  • step (3) The separation product obtained in step (2) is pre-balanced with ultrapure water (containing 0.1% trifluoroacetic acid) in a Hypersil ODS2 5mm column, the experimental instrument is Waters 1525 high-pressure liquid system, and the flow rate is 1mL/min Under conditions, elute with acetonitrile (containing 0.1% trifluoroacetic acid) under a linear gradient (0-100%, 100min), and collect the active product with a monitoring wavelength of 220nm;
  • step (3) The active product of step (3) is freeze-dried in vacuum and then dissolved in deionized water, and then the process of step (3) is repeated to obtain a purified RDP1 polypeptide.
  • the polypeptide RDP1 of the present invention can also be obtained by artificial synthesis.
  • there are many methods for preparing biologically active peptides including protective chemical synthesis, hydrolysis, recombinant DNA technology, etc. These methods are all suitable for preparing the polypeptide RDP1 of the present invention.
  • the polypeptide RDP1 of the present invention has a simple structure and has anti-hyperuricemia activity.
  • the purified active polypeptide RDP1 shows strong anti-hyperuricemia activity in an animal model experiment of hyperuricemia induced by potassium oxazinate It is revealed that the polypeptide RDP1 of the present invention has great application prospect in anti-hyperuricemia.
  • the application of the polypeptide RDP1 of the present invention may be oral, and the oral application is that the dosage of the polypeptide RDP1 given daily ranges from about 10 to 1000 micrograms per kilogram of body weight. Preferably about 10 micrograms per kilogram of body weight or about 100 micrograms per kilogram of body weight.
  • Example 1 Isolation, purification and identification of anti-hyperuricemia active polypeptide RDP1
  • the rice was soaked in deionized water overnight. After filtration, the supernatant was freeze-dried in vacuum and stored at -80° until use.
  • the lyophilized powder of the obtained rice extract was dissolved in deionized water, and 1 mL of Sephadex G50 (GE Healthcare, ultra Thin) column (length 40cm, inner diameter width 1.5cm), eluted with the same buffer, the flow rate is 3mL/10min, collected once every 10min. Detect the absorbance of each tube, the detection wavelength is 280nm, and combine the samples under the absorption peak indicated by the arrow in Figure 1.
  • the obtained sample was loaded on a Hypersil ODS25mm column (Elite product, size 4.6mm ⁇ 300mm) pre-equilibrated with ultrapure water (containing 0.1% trifluoroacetic acid) in advance, and the experimental instrument was Waters1525 high-pressure liquid system at Under the condition of 1mL/min, elute with acetonitrile (containing 0.1% trifluoroacetic acid) under the condition of linear gradient (0 ⁇ 100%, 100min), the monitoring wavelength is 220nm, and the obtained separation and purification pattern is shown in Figure 2. Shown: The peak indicated by the arrow is RDP1. The peak indicated by the arrow in Figure 2 was collected, dissolved in deionized water after freeze-drying in vacuum, and then the first HPLC process was repeated. The resulting separation and purification pattern was shown in Figure 3, and the arrow indicated the peak of purified RDP1.
  • the molecular weight of the sample was determined by mass spectrometry, and the result was 785.91 Da ( Figure 4). 1 ⁇ L of the sample was mixed with 1 ⁇ L of ⁇ -cyano-4-hydroxyoctanoic acid (5 mg/mL, dissolved in 50% ACN, 0.1% TFA), and punctate crystallization was performed on the sample plate. The AutoFlex Speed MALDI TOF/TOF mass spectrometer was used to analyze the positive crystalline samples.
  • the sample was dissolved in 25mmol/LNH 4 HCO 3 , reduced with dithiothreitol at 37°C for 1 h, blocked with iodoacetamide for 30 min, and then the sample was combined with ⁇ -cyano-4- Hydroxyoctanoic acid was mixed and subjected to tandem mass spectrometry on the same equipment.
  • the anti-hyperuricemia activity of the polypeptide RDP1 was detected using an animal model of hyperuricemia induced by potassium oxazidate.
  • SPF grade SD rats weighing 100-150g were selected for experiments. The rats were randomly divided into 6 groups of 5 rats. These 6 groups were blank control, negative control, positive control and 3 RDP1 treatment groups (10, 100 , 1000 ⁇ g/kg).
  • the blank control was given an equal amount of normal saline by gavage every day, and the remaining groups were given gavage of potassium oxazidate (450mg/kg) and adenine (100mg/kg) by gavage for 1 hour, and then given by intragastric administration: the negative control group was given
  • the normal control group was given 10 mg/kg allopurinol, and the three RDP1 treatment groups were given 10, 100, and 1000 ⁇ g/kg RDP1, respectively, for a total of 7 days.
  • the renal protective activity of peptide RDP1 is shown in Figure 8: the negative control group (model group) can see the brush border disappear and the renal tubules atrophy compared with the blank group; while the positive control group and RDP1 group and the negative control group Than can see that the pathological changes of the kidneys have been significantly alleviated.
  • the test was divided into a negative control, a positive control and three RDP1 treatment groups, which were tested for xanthine oxidase activity inhibition in vivo and in vitro; at the same time, a 50 mmol/LTris-HCl pH 8 buffer was prepared for use.
  • Negative control 128 ⁇ L 2mmol/L xanthine solution + 16 ⁇ L 0.52mU/mL xanthine oxidase solution + 928 ⁇ L buffer + 32 ⁇ L buffer.
  • RDP1 treatment groups 128 ⁇ L 2 mmol/L xanthine solution + 16 ⁇ L 0.52 mU/mL xanthine oxidase solution + 928 ⁇ L buffer + 32 ⁇ L 1/10/1000 ⁇ g/kg RDP1 solution.
  • Negative control 128 ⁇ L of 2 mmol/L xanthine solution + 16 ⁇ L of 0.52 mU/mL xanthine oxidase solution + 928 ⁇ L buffer + 32 ⁇ L of negative control rat serum.
  • Three RDP1 treatment groups in vivo 32 ⁇ L 1/10/1000 ⁇ g/kg rat serum + 928 ⁇ L buffer.
  • Xanthine oxidase inhibition rate (%) (negative control OD 292nm value-positive control or treatment group OD 292nm value)/negative control OD 292nm value ⁇ 100%.
  • the peptide RDP1 inhibits xanthine oxidase activity in vitro as shown in Figure 9: the positive control has the highest xanthine oxidase inhibition rate, followed by the RDP1 treatment groups, which are significantly higher than the negative control.
  • FIG. 10 The inhibition of xanthine oxidase activity in vivo is shown in Figure 10: the highest inhibition rate of xanthine oxidase in the 1000 ⁇ g/kg RDP1 group in serum, followed by the 100 ⁇ g/kg RDP1 group, the positive control group and the 10 ⁇ g/kg RDP1 group.
  • the present invention relates to an anti-hyperuricemia active polypeptide RDP1 which has beneficial features such as simple structure, high activity, and simple administration method.

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Abstract

本发明公开了一种抗高尿酸血症多肽RDP1，所述多肽RDP1包含的氨基酸序列如SEQ No.1所示。本发明还公开了所述多肽RDP1的提纯方法和应用。本发明所述抗高尿酸血症肽RDP1可减轻高尿酸血症，缓解肾损伤，具有广阔的应用前景。

Description

一种多肽RDP1及其提纯方法与应用 技术领域
本发明属于生物医药领域，具体涉及一种多肽RDP1及其提纯方法与应用。
背景技术
根据2017年《中国痛风现状报告***》，中国痛风患者已经超过8000万人，痛风已成为中国仅次于糖尿病的第二大代谢类疾病。痛风因血液中尿酸堆积并结晶在关节或软组织中而发病，其疾病前期症状为高尿酸血症。高尿酸血症一般是由尿酸产生过多或排出过少引起，而黄嘌呤作为尿酸产生中的关键酶，被最常用作抗高尿酸血症的药物靶点。抗高尿酸血症是非急性期抗痛风的主要目标，但临床上现有的抗高尿酸血症类药物都存在一定缺陷，包括活性低、制作成本高、副作用大且难以逆转等。因此，急需探索或研发出不良反应少、经济实用的新型抗高尿酸血症药物。
已报道的具有抗高尿酸血症活性的物质主要是小分子化合物，但大部分难以储存和生产。近几十年来，许多研究表明：含有5～10个氨基酸的短肽更易于吸收，而且往往更易于检测出特异的活性。研究人员发现：短肽通常具有高活性、高稳定性和特异性，而且也更经济且容易大量生产；因此，短肽具有广泛的应用价值，这引起了全世界研究人员的高度关注。一些多肽药物已在临床上被广泛使用，如艾塞那肽，胰岛素等。同时，还发现了大量具有其他活性的生物肽，如抗菌肽、镇痛肽等，但具有抗高尿酸血症活性的多肽研究和发现仍处于起步阶段。
为此，我们研发一种抗高尿酸血症的活性多肽，它可以有效缓解高尿酸 血症，且具有较高的稳定性及肾保护活性。
发明内容
本发明的第一目的在于提供一种多肽RDP1，所述多肽RDP1包含的氨基酸序列如SEQ No.1所示。
本发明的第二目的在于提供一种所述多肽RDP1的提纯方法，其特征在于，包括以下步骤：
(1)大米用去离子水浸泡过夜，取过滤后上清液真空冷冻干燥，获得大米提取物冻干粉，-80℃保存备用；
(2)将所述大米提取物冻干粉溶解于去离子水中，使用Sephadex G50柱子层析分离；
(3)将步骤(2)的分离产物进行第一次高效液相色谱反相层析，并收集活性组分；
(4)将所述活性组分进行第二次高效液相色谱反相层析，得到纯化的多肽RDP1。
本发明的第三目的在于提供一种所述多肽RDP1抗高尿酸血症的应用，包括口服应用。
本发明的第四目的在于提供一种含有所述多肽RDP1的药物。
本发明的第五目的在于提供一种所述多肽RDP1在抗高尿酸血症的药物中的应用。
附图说明
图1为本发明抗高尿酸血症活性多肽RDP1的Sephadex G50分子筛图；图中，箭头所指为RDP1吸收峰。
图2为本发明抗高尿酸血症活性多肽RDP1的HPLC反相C18柱层析图。
图中，箭头所指为RDP1吸收峰。
图3为本发明抗高尿酸血症活性多肽RDP1的第2次HPLC反相C18柱层析图。
图中，箭头所指为RDP1吸收峰。
图4为本发明抗高尿酸血症活性多肽RDP1的质谱图。
图5为本发明抗高尿酸血症活性多肽RDP1的一级结构图。
图6为本发明抗高尿酸血症活性多肽RDP1的降尿酸结果图。图中，***代表P<0.001(t检验)。
图7为本发明抗高尿酸血症活性多肽RDP1的降肌酐结果图。图中，*代表P<0.05，**代表P<0.01,***代表P<0.001(t检验)。
图8为本发明抗高尿酸血症活性多肽RDP1的HE染色结果图。
图9为本发明抗高尿酸血症活性多肽RDP1体外的抑制黄嘌呤氧化酶活性结果。图中，**代表P<0.01,***代表P<0.001(t检验)。
图10为本发明抗高尿酸血症活性多肽RDP1给药后大鼠血清的体内的抑制黄嘌呤氧化酶活性结果图。图中，*代表P<0.05，**代表P<0.01(t检验)。
具体实施方式
下面结合附图对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所做的任何变换或替换，均属于本发明的保护范围。
本发明所述多肽RDP1，来源于大米提取物，所述多肽RDP1包含的氨基酸序列如SEQ No.1所示。
本发明还提供一种从大米提取物中提纯所述多肽RDP1的提纯方法，包括以下步骤：
(1)大米用去离子水浸泡过夜，取过滤后上清液真空冷冻干燥，获得大米提取物冻干粉，-80℃保存备用；
(2)将所述大米提取物冻干粉溶解于去离子水中，使用Sephadex G50柱子层析分离，具体为将所述大米提取物冻干粉，溶解于去离子水中，取1mL上预先用20mmol/L Tris-HCl缓冲液(pH＝7.8，含0.1mol/LNaCl)平衡24h的Sephadex G50柱子(长度40cm，内径宽度1.5cm)，用同样的缓冲液进行洗脱，流速为3mL/10min，每10min收集1次。
(3)将步骤(2)得到的分离产物预先用超纯水(含0.1％的三氟乙酸)平衡好的Hypersil ODS2 5mm柱子，实验仪器为Waters 1525高压液相***，在流速为1mL/min的条件下，用乙腈(含0.1％的三氟乙酸)在线性梯度(0-100％，100min)条件下进行洗脱，并收集活性产物，监测波长为220nm；
(4)将步骤(3)的活性产物真空冷冻干燥后再溶于去离子水，然后重复步骤(3)过程，得到纯化的RDP1多肽。
除了从大米提取物中获得外，本发明所述的多肽RDP1还可通过人工合成的方法得到。目前生物活性肽的制备方法有很多种，包括保护化学合成法、水解法、重组DNA技术等，这些方法均适用于制备本发明所述的多肽RDP1。
本发明所述多肽RDP1结构简单，具有抗高尿酸血症的活性，纯化得到的活性多肽RDP1在氧嗪酸钾诱发的高尿酸血症动物模型实验中显示了较强的抗高尿酸血症活性，揭示本发明所述的多肽RDP1在抗高尿酸血症方面具有巨大的应用前景。
本发明的所述多肽RDP1的应用可以为口服应用，所述的口服应用为每 日给与的多肽RDP1剂量范围为每千克体重约10微克到1000微克。优选每千克体重约10微克或每千克体重约100微克。
下面将结合具体实施例，对本发明进行进一步的说明。
实施例1：抗高尿酸血症活性多肽RDP1分离纯化和鉴定
1、分离纯化
大米用去离子水浸泡过夜，过滤后上清液真空冷冻干燥，-80°保存备用。将获得大米提取物冻干粉，溶解于去离子水中，取1mL上预先用20mmol/L Tris-HCl缓冲液(pH7.8，含0.1mol/L NaCl)平衡24h的Sephadex G50(GE Healthcare，超细)柱子(长度40cm，内径宽度1.5cm)，用同样的缓冲液进行洗脱，流速为3mL/10min，每10min收集1次。检测每管吸光度，检测波长为280nm，将图1箭头所指吸收峰下样品合并。
将所得样品上样于预先用超纯水(含0.1％的三氟乙酸)平衡好的Hypersil ODS25mm柱子(伊利特产品，尺寸为4.6mm×300mm)，实验仪器为Waters1525高压液相***，在流速为1mL/min的条件下，用乙腈(含0.1％的三氟乙酸)在线性梯度(0～100％，100min)条件下进行洗脱，监测波长为220nm，所得的分离纯化图谱如图2所示：箭头所指峰为RDP1。收集图2箭头所指峰，真空冷冻干燥后溶于去离子水，然后重复第1次HPLC过程，所得的分离纯化图谱如图3所示，箭头所指为纯化的RDP1所在峰。
2、氨基酸序列的测定
用质谱法测定样品的分子量，结果为785.91Da(图4)。将1μL样品与1μLα-氰基-4-羟基辛酸(5mg/mL，溶于50％ACN，0.1％TFA)混合，并在样品板上进行点状结晶。用AutoFlex Speed MALDI TOF/TOF质谱仪对正结晶样品进行分析。
为了确定氨基酸序列，将样品溶于25mmol/LNH 4HCO 3中，在37℃下用二硫代苏糖醇还原1h，用碘乙酰胺阻断30min，然后将样品与α-氰基-4-羟基辛酸混合，并在同一设备上进行串联质谱分析。
结果如图5所示：抗高尿酸血症活性多肽RDP1的氨基酸序列为AAAAGAKAR，同SEQ No.1所示。
实施例2：多肽RDP1的抗高尿酸血症活性测定
(1)血清尿酸和肌酐含量测定
利用氧嗪酸钾诱导的高尿酸血症动物模型检测多肽RDP1的抗高尿酸血症活性。选体重100～150g的SPF级SD大白鼠进行实验，将大鼠随机分成6组，每组5只，这6组分别为空白对照、阴性对照、阳性对照以及3个RDP1处理组(10、100、1000μg/kg)。空白对照每天灌胃给等量生理盐水，其余各组每天灌胃给氧嗪酸钾(450mg/kg)和腺嘌呤(100mg/kg)1h后，再分别灌胃给药：阴性对照组给等量生理盐水，阳性对照组给10mg/kg别嘌呤醇，3个RDP1处理组分别给10、100、1000μg/kg RDP1，一共给药7天。第7天给药(生理盐水、别嘌呤醇、RDP1)结束1h后，给大鼠腹腔注射1％的戊巴比妥钠(0.3mL/100g)麻醉大鼠，然后于大鼠股动脉处取全血。将全血于室温下，6000r/min，离心5min后以获取血清。将部分大鼠血清使用试剂盒进行尿酸、肌酐含量检测，结果如图6、7所示：阳性对照组和RDP1处理各组的血清尿酸和肌酐含量明显低于阴性对照组。其余血清用于体内黄嘌呤氧化酶抑制活性检测。
取血后，取大鼠肾脏固定于4％甲醛中24～48h，组织通过梯度乙醇脱水，二甲苯透明。使用石蜡包埋法，切片5μm，进行HE染色。结果通过正置显微镜放大200倍观察。多肽RDP1的肾保护活性如图8所示：阴性对照组(模型组) 大鼠肾脏与空白组相比可以看到刷状缘消失，肾小管萎缩；而阳性对照组和RDP1组与阴性对照组比可以看到肾脏病理改变得到了明显的缓解。
(2)黄嘌呤氧化酶抑制率测定
试验分为阴性对照、阳性对照和3个RDP1处理组，分别进行体内外黄嘌呤氧化酶活性抑制测定；同时配制50mmol/LTris-HCl pH 8的缓冲液，备用。
①体外实验：
阴性对照：128μL 2mmol/L黄嘌呤溶液+16μL 0.52mU/mL黄嘌呤氧化酶溶液+928μL缓冲液+32μL缓冲液。
阳性对照：128μL 2mmol/L黄嘌呤溶液+16μL 0.52mU/mL黄嘌呤氧化酶溶液+928μL缓冲液+32μL 1mg/mL别嘌呤醇溶液。
3个RDP1处理组：128μL 2mmol/L黄嘌呤溶液+16μL 0.52mU/mL黄嘌呤氧化酶溶液+928μL缓冲液+32μL 1/10/1000μg/kg RDP1溶液。
②体内实验：
阴性对照：128μL 2mmol/L黄嘌呤溶液+16μL 0.52mU/mL黄嘌呤氧化酶溶液+928μL缓冲液+32μL阴性对照组大鼠血清。
阳性对照：128μL 2mmol/L黄嘌呤溶液+16μL 0.52mU/mL黄嘌呤氧化酶溶液+928μL缓冲液+32μL 1mg/mL阳性对照组大鼠血清。
体内3个RDP1处理组：32μL 1/10/1000μg/kg大鼠血清++928μL缓冲液。
将各试验组37℃孵育15min，加入48μL 1mol/L HCL终止反应，在292nm处测定吸光度。抑制活性计算公式如下：
黄嘌呤氧化酶抑制率(％)＝(阴性对照OD 292nm值-阳性对照或者处理组OD 292nm值)/阴性对照OD 292nm值×100％。
多肽RDP1体外抑制黄嘌呤氧化酶活性如图9所示：阳性对照的黄嘌呤氧化酶抑制率最高，其次为RDP1各处理组，均明显高于阴性对照。
体内抑制黄嘌呤氧化酶活性如图10所示：血清中1000μg/kg RDP1组的黄嘌呤氧化酶抑制率最高，其次为100μg/kg RDP1组、阳性对照组和10μg/kg RDP1组。
综上所述，本发明涉及抗高尿酸血症活性多肽RDP1具有结构简单、活性高、给药方式简单等有益特点。

Claims (15)

  1. 一种抗高尿酸血症活性多肽RDP1，其特征在于，所述多肽RDP1包含的氨基酸序列如SEQ No.1所示。
  2. 一种权利要求1所述多肽RDP1的提纯方法，其特征在于，包括以下步骤：
    (1)大米用去离子水浸泡过夜，取过滤后上清液真空冷冻干燥，获得大米提取物冻干粉，-80℃保存备用；
    (2) (2)将所述大米提取物冻干粉溶解于去离子水中，使用Sephadex G50柱子层析分离；
    (3) (3)将步骤(2)的分离产物进行第一次高效液相色谱反相层析，并收集活性组分；
    (4) (4)将所述活性组分进行第二次高效液相色谱反相层析，得到纯化的多肽RDP1。
  3. 根据权利要求2所述多肽RDP1的提纯方法，其特征在于，所述步骤(2)具体为将所述大米提取物冻干粉，溶解于去离子水中，取1mL上预先用20mmol/L Tris-HCl缓冲液平衡24h的Sephadex G50柱子，用同样的缓冲液进行洗脱，流速为3mL/10min，每10min收集1次。
  4. 根据权利要求3所述多肽RDP1的提纯方法，其特征在于，所述Tris-HCl缓冲液pH 7.8，含0.1mol/L NaCl。
  5. 根据权利要求3所述多肽RDP1的提纯方法，其特征在于，所述Sephadex G50柱子长40cm，内径1.5cm。
  6. 根据权利要求2所述多肽RDP1的提纯方法，其特征在于，所述步骤(3)具体为将步骤(2)得到的分离产物预先用含0.1％三氟乙酸的超纯水平衡后的Hypersil ODS2 5mm柱子，实验仪器为Waters 1525高压液 相***，在流速为1mL/min的条件下，用含0.1％三氟乙酸的乙腈在线性梯度条件下进行洗脱，并收集活性产物，监测波长为220nm；所述步骤(4)具体为将步骤(3)的活性产物真空冷冻干燥后再溶于去离子水，然后重复步骤(3)，得到纯化的RDP1多肽。
  7. 根据权利要求6所述所述多肽RDP1的提纯方法，其特征在于，所述线性梯度为0～100％，100min。
  8. 一种权利要求1所述多肽RDP1在抗高尿酸血症的应用。
  9. 一种含有权利要求1所述多肽RDP1的抗高尿酸血症组合物。
  10. 如权利要求8所述多肽RDP1在抗高尿酸血症的应用，其特征在于，所述应用为口服应用。
  11. 如权利要求10所述多肽RDP1在抗高尿酸血症的应用，其特征在于，所述的口服应用为每日给与的多肽RDP1剂量范围为每千克体重约10微克到1000微克。
  12. 如权利要求11所述多肽RDP1在抗高尿酸血症的应用，其特征在于，所述的口服应用为每日给与的多肽RDP1剂量为每千克体重约10微克。
  13. 如权利要求11所述多肽RDP1在抗高尿酸血症的应用，其特征在于，所述的口服应用为每日给与的多肽RDP1剂量为每千克体重约100微克。
  14. 如权利要求11所述多肽RDP1在抗高尿酸血症的应用，其特征在于，所述的口服应用为每日给与的多肽RDP1剂量为每千克体重约1000微克。
  15. 一种权利要求1所述多肽RDP1在抗高尿酸血症的药物中的应用。
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