WO2022048634A1 - 一种抗炎护肾的蛤蜊肽的制备方法和应用 - Google Patents

一种抗炎护肾的蛤蜊肽的制备方法和应用 Download PDF

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WO2022048634A1
WO2022048634A1 PCT/CN2021/116427 CN2021116427W WO2022048634A1 WO 2022048634 A1 WO2022048634 A1 WO 2022048634A1 CN 2021116427 W CN2021116427 W CN 2021116427W WO 2022048634 A1 WO2022048634 A1 WO 2022048634A1
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clam
inflammatory
kidney
peptide
protecting
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PCT/CN2021/116427
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English (en)
French (fr)
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邹圣灿
任慧慧
刘金丽
刘廷廷
王尚龙
曹廷锋
宗建成
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琛蓝(美国)营养制品股份有限公司
青岛琛蓝生物科技有限公司
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Priority to US17/533,444 priority Critical patent/US20220089662A1/en
Publication of WO2022048634A1 publication Critical patent/WO2022048634A1/zh

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    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a preparation method and application of a clam peptide for anti-inflammatory and kidney protection.
  • inflammatory response is a moderate or abnormal systemic response of the body's inflammatory immune-related cells according to changes in the internal and external environment.
  • the occurrence of various diseases is related to the inflammatory response. Therefore, the inflammatory response is the basic pathological feature of various diseases, which can occur in various tissues and organs, and is also the main link of drug intervention.
  • Inflammatory reactions generally lead to changes in the body content of important inflammatory factors such as IL-6, IL-8, TNF- ⁇ and CRP.
  • common anti-inflammatory drugs mainly include penicillin, cephalosporin, amoxicillin, etc. Long-term use of such drugs will cause the body to develop drug resistance and cause damage to liver, kidney and other functions. Therefore, the development of food-derived anti-inflammatory active substances with less toxic and side effects has become an urgent need for the majority of patients.
  • Renal function refers to the function of the kidneys to excrete metabolic wastes in the body and maintain the balance of sodium, potassium, and calcium in the body. Serum creatinine, urea/urea nitrogen, and uric acid are common indicators of renal function. Renal damage is mainly manifested as kidney deficiency, renal insufficiency, renal failure and impaired renal function. In severe cases, it can develop into uremia, which is life-threatening.
  • the drugs for protecting kidney function on the market are mainly Chinese herbal medicines, which play the role of remission and protection.
  • Clams are common low-value shellfish widely distributed in the coastal areas of my country. They have the characteristics of high protein, low fat, high trace elements, and low sugar. They are high-quality marine products. They are not only rich in nutrition, but also have high dietary and medicinal value. Clam peptides are marine-derived small-molecule bioactive peptides, which are safe, easily absorbed, and enhance immunity. Marine-derived clam active peptides have become a research hotspot. The research on clam active peptides mostly focuses on biological functions such as lowering blood pressure, blood lipids, and anti-aging. There are not many studies on other aspects of clam active peptides, and there is no clam peptide in alleviating inflammation caused by hypertension and protecting kidney function. related reports.
  • the present invention provides a preparation method and application of an anti-inflammatory and kidney-protecting clam peptide.
  • the present invention adopts Hongdao clam to prepare a clam peptide, which is used for relieving the body's inflammatory response and renal function damage caused by hypertension, and is further applied to inflammation and kidney damage from other diseases.
  • a preparation method of a clam peptide for anti-inflammatory and kidney protection the specific steps are as follows:
  • the whole meat of the red island clam is taken, washed and crushed to obtain a slurry, 0.1-0.3% of the composite protease by weight of the slurry is added, enzymatic hydrolysis, centrifugation, membrane separation and purification, and spray drying are used to obtain the clam peptide powder.
  • the composite protease includes neutral protease, alkaline protease, and flavored protease, and the weight ratio of neutral protease:alkaline protease:flavored protease is 2:1:1.
  • the weight ratio of clam meat:water during the enzymatic hydrolysis is 1:1-1:3.
  • the clam peptide is enzymatically hydrolyzed at a temperature of 50-60° C. for 4-6 hours.
  • the centrifugation speed of the clam peptide in the production process is 16000 r/min.
  • the clam peptidase hydrolyzed solution is filtered through a microfiltration-ultrafiltration-nanofiltration membrane to intercept the enzymatic hydrolysis solution with a molecular weight below 2KDa.
  • the clam peptide in the present invention has been proved to have a significant inhibitory effect on inflammatory factors through experiments.
  • the clam peptide of the present invention has been proved to have the effect of significantly reducing serum creatinine, uric acid and blood urea nitrogen in renal function indicators.
  • the present invention expands the application of the clam peptide in medical care, has the advantages of high safety and is easily absorbed by the human body, and provides a new choice of anti-inflammatory and kidney-protecting food-derived medicine for the majority of patients.
  • the clam peptide production method provided by the technical solution has the advantages of simple process, mild conditions, short cycle, no addition of any inorganic or organic solvent, high yield, and is more suitable for industrial production.
  • the prepared clam peptide has pure color, off-white, no fishy smell, no other peculiar smell, and good taste and flavor.
  • the active peptides of clams are small in molecular weight, mainly tetrapeptides to hexapeptides, and are easily absorbed by the human body.
  • Figure 1 shows the effect of clam peptide on rat serum IL-8
  • Figure 2 shows the effect of clam peptide on TNF- ⁇ in rat serum
  • Figure 3 shows the effect of clam peptide on hs-CRP in rat serum
  • Figure 4 is the effect of clam peptide on SCr in rat serum
  • Figure 5 is the effect of clam peptide on SUA in rat serum
  • Figure 6 shows the effect of clam peptide on BUN in rat serum.
  • Embodiment 1 of the present invention discloses a preparation method and application of an anti-inflammatory and kidney-protecting clam peptide, and the adopted technical scheme is as follows:
  • Embodiment 2 of the present invention discloses a preparation method and application of an anti-inflammatory and kidney-protecting clam peptide, and the adopted technical scheme is as follows:
  • Embodiment 3 of the present invention discloses a preparation method and application of an anti-inflammatory and kidney-protecting clam peptide, and the adopted technical scheme is as follows:
  • the spontaneously hypertensive model rats used were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • Spontaneous hypertension model rats male, 40, about 12 weeks old, weighing about 270 g, were adaptively raised for one week.
  • the rats were randomly divided into 5 groups, namely model control group (MC), positive drug control group (AC), clam peptide low-dose group (CP1), medium-dose group (CP2), and high-dose group (CP3) (low-dose group).
  • the dose group was 50 mg/kg, the middle dose group was 100 mg/kg, and the high dose group was 200 mg/kg), 8 in each group.
  • the AC group was given 10 mg/kg captopril by gavage, and the MC group was given the same volume of distilled water by gavage at the same time. It was administered once a day for 4 consecutive weeks. Serum was taken to measure IL-8, TNF- ⁇ and hs-CRP, the data obtained were analyzed and processed by SPSS software, and variance analysis was used. indicates a very significant difference. The statistics of the data results are shown in Table 1.
  • inflammatory factor MC AC CP1 CP2 CP3 IL-8 (pg/ml) 181.20 ⁇ 27.72 122.00 ⁇ 12.15** 143.30 ⁇ 17.70** 158.40 ⁇ 18.52 89.08 ⁇ 16.27** TNF- ⁇ (pg/ml) 148.70 ⁇ 52.12 87.41 ⁇ 19.19** 119.80 ⁇ 16.18 139.20 ⁇ 8.87 84.43 ⁇ 18.95** hs-CRP( ⁇ g/L) 2.16 ⁇ 0.36 1.53 ⁇ 0.41* 1.83 ⁇ 0.36 1.75 ⁇ 0.49 1.43 ⁇ 0.55**
  • FIG. 1 shows that the clam peptide group can reduce the level of IL-8.
  • the clam peptide high-dose group and the positive drug group were The content of IL-8 decreased to 89.08 ⁇ 16.27 pg/ml (p ⁇ 0.01) and 122 ⁇ 12.15 pg/ml (p ⁇ 0.01), which was statistically significant.
  • Figure 2 shows that the serum TNF- ⁇ content of the high-dose clam peptide group was reduced by nearly 43% compared with the model group, with a very significant difference, reaching an effect equivalent to that of the positive drug.
  • Figure 3 reflects the effect of clam peptide on hs-CRP in rat serum.
  • hs-CRP gradually decreased, which was higher than that of the model control group (2.16 ⁇ 0.36 ⁇ g/L).
  • the dose of clam peptide gavage group was reduced to 1.43 ⁇ 0.55 ⁇ g/L, a very significant difference, close to the level of the positive drug group.
  • the clam peptide can effectively reduce the levels of inflammatory factors such as IL-8, TNF- ⁇ and hs-CRP in the serum of rats, has a good anti-inflammatory effect, and can be used in anti-inflammatory related drugs or health products.
  • the spontaneously hypertensive model rats used were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • Spontaneous hypertension model rats male, 40, about 12 weeks old, weighing about 270 g, were adaptively raised for one week.
  • the rats were randomly divided into 5 groups, namely model control group (MC), positive drug control group (AC), clam peptide low-dose group (CP1), medium-dose group (CP2), and high-dose group (CP3) (low-dose group).
  • the dose group was 50 mg/kg, the middle dose group was 100 mg/kg, and the high dose group was 200 mg/kg), 8 in each group.
  • the AC group was given 10 mg/kg captopril by gavage, and the MC group was given the same volume of distilled water by gavage at the same time. It was administered once a day for 4 consecutive weeks.
  • Serum was taken to measure SCr, SUA and BUN, and the data obtained were analyzed and processed by SPSS software. Using variance analysis, the results are shown as the index mean ⁇ standard deviation, p ⁇ 0.05 indicates a significant difference, p ⁇ 0.01 indicates a very significant difference. The statistics of the data results are shown in Table 2.
  • Renal function index MC AC CP1 CP2 CP3 SCr( ⁇ mol/L) 687.10 ⁇ 111.20 421.50 ⁇ 101.20** 508.90 ⁇ 128.90* 527.60 ⁇ 66.78** 330.50 ⁇ 75.89** SUA(mg/L) 283.50 ⁇ 56.66 211.60 ⁇ 39.89** 220.90 ⁇ 37.27* 245.90 ⁇ 39.17 169.90 ⁇ 30.11** BUN(mmol/L) 17.06 ⁇ 3.70 10.56 ⁇ 3.17** 12.43 ⁇ 2.76** 13.69 ⁇ 2.29 8.10 ⁇ 2.43**
  • Figure 4 shows that different doses of clam peptides have the effect of reducing serum SCr in rats, and the high-dose group has the most significant effect, which can be reduced by 51.9% compared with the model control group.
  • Figure 5 shows that the clam peptide has the effect of reducing SUA. Compared with the model control group (283.50 ⁇ 56.66mg/L), the high-dose group reduced it to 169.90 ⁇ 30.11mg/L, with a very significant difference.
  • Figure 6 shows that the clam peptide has a good effect on the clearance of BUN in serum, and the high-dose group of clam peptide has the most significant effect. Compared with the model control, the clearance rate of BUN can reach 52.5%.
  • 1SBP continued to decrease during the test period; 2Compared with 0 days, there was a statistical difference after taking 21 days (3 weeks) (p ⁇ 0.05), and there was a significant difference after taking 30 days (1 month) ( p ⁇ 0.01), there was a very significant difference (p ⁇ 0.001) after taking 60 days (2 months); 3 After taking 81 days (about 3 months), SBP decreased to normal levels, and the follow-up was 14 days (2 weeks) SBP remained at a normal level; 4After taking 14 days (2 weeks), blood pressure decreased from grade 1 hypertension to high-normal blood pressure, and after taking 81 days (about 3 months), blood pressure decreased from high-normal blood pressure to normal blood pressure.
  • the blood pressure was graded and compared, and it was concluded that after 14 days of administration, grade 2 hypertension turned into grade 1 hypertension, and some normal high blood pressure turned into normal blood pressure; compared with graded blood pressure on days 1-7 and 15-21 of the trial diet There was no statistical difference (p>0.05); compared with the graded blood pressure on the 8-14th and 22-44 days of the trial diet (p ⁇ 0.05); compared with the graded blood pressure on the 45-90th day of the trial diet and the 14th day of follow-up The comparison was significantly different (p ⁇ 0.01). During the trial period, the blood pressure of 2 of them has been in a state of fluctuation. In order to maintain the authenticity and integrity of the data, the data was not excluded from the analysis. Therefore, the blood pressure rebounded within 68-90 days of the trial. Diet and physical condition are related.
  • clam peptide can significantly reduce blood pressure (systolic and diastolic blood pressure) in hypertensive patients, has a good antihypertensive effect, and can be used in antihypertensive related drugs or health products.
  • clam peptide can reduce the contents of AST, CK-Mb(M), CREA, ET-1, ACEI, IL-1 ⁇ , IL-17A, TNF- ⁇ and MDA in hypertensive patients, and increase the contents of ACE2, NO and MDA.
  • the content of IL-10 suggests that taking clam peptide can help improve the heart function, liver function and renal function of hypertensive patients, and on the other hand, it suggests that the antihypertensive mechanism of clam peptide may be related to the regulation of antihypertensive factor and vascular endothelial factor content, reducing blood pressure and reducing blood pressure. It is related to inflammatory response and the improvement of antioxidant capacity.
  • Clam peptides can be used in medicines or health care products related to alleviating inflammatory responses and renal function damage caused by hypertension or other diseases.

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Abstract

一种抗炎护肾的蛤蜊肽的制备方法和应用，取红岛蛤蜊整肉，洗净绞碎得到浆液，加入占浆液重量0.1-0.3%的复合蛋白酶，酶解、离心、膜分离纯化、喷雾干燥得到蛤蜊肽粉，可用于缓解高血压引发的机体炎症反应和肾功能损伤。

Description

一种抗炎护肾的蛤蜊肽的制备方法和应用 技术领域
本发明涉及生物医药技术领域，更具体的说是涉及一种抗炎护肾的蛤蜊肽的制备方法和应用。
背景技术
在现实生活中，炎症反应是机体炎症免疫相关细胞依据内外环境变化所表现出的适度或异常的系统反应。各种疾病的发生都与炎症反应相关，因此炎症反应是多种疾病的基本病理特点，可发生于各个组织器官，也是药物干预的主要环节。炎症反应一般会引发IL-6、IL-8、TNF-α以及CRP等重要炎症因子体内含量的变化。目前常见消炎药物主要有青霉素、头孢、阿莫西林等，长期服用此类药物会导致机体产生抗药性，对肝、肾等功能造成损伤。因此，开发食源性的毒副作用较小的抗炎活性物质已成为广大患者的迫切需求。
一些病症不仅会引发相关炎症反应，同时也会影响肾功能，两者互为因果，互相影响，其中高血压就是一种能双重影响炎症和肾功能的病症。肾功能是指肾脏可以排泄体内代谢废物，维持机体钠、钾、钙等平衡的功能。血清中肌酐、尿素/尿素氮、尿酸等是表征肾功能的常见指标。肾功能损害主要表现为肾虚、肾功能不全、肾功能衰竭和肾功能受损，严重时可发展为尿毒症，危及生命。目前市面上保护肾功能药物主要为中草药类，起到缓解和保护的作用，暂无可逆转肾功能损坏的特效药。
蛤蜊是广泛分布于我国沿海的常见低值贝类，具有高蛋白、低脂肪、高微量元素、低糖等特点，是优质海洋产品，不仅营养丰富，还具有很高的食疗药用价值。蛤蜊肽属海洋源的小分子生物活性肽，安全性高，易被吸收，具有增强免疫力，海洋源的蛤蜊活性肽目前已成为研究的热点。对于蛤蜊活性肽的研究多集中于降血压、降血脂、延缓衰老等生物功能上，对于蛤蜊活 性肽的其他方面研究尚且不多，也没有蛤蜊肽在缓解高血压引起的炎症与保护肾功能方面的相关报道。
因此，结合上述问题，提供一种能够应用于抗炎药物和保护肾功能药物的蛤蜊肽的制备方法，是本领域技术人员亟需解决的问题。
发明内容
鉴于此，本发明提供了一种抗炎护肾的蛤蜊肽的制备方法和应用，本发明采用红岛蛤蜊制备蛤蜊肽，用于缓解高血压引发的机体炎症反应和肾功能损伤，并进一步应用到其他疾病引起的炎症和肾损伤中。
为了实现上述目的，本发明采用如下技术方案：
一种抗炎护肾的蛤蜊肽的制备方法，具体步骤如下：
取红岛蛤蜊整肉，洗净搅碎得到浆液，加入占浆液重量0.1～0.3％的复合蛋白酶，酶解、离心、膜分离纯化、喷雾干燥得到蛤蜊肽粉。
优选的，所述复合蛋白酶包括中性蛋白酶、碱性蛋白酶、风味蛋白酶，中性蛋白酶：碱性蛋白酶：风味蛋白酶的重量比为2:1:1。
优选的，所述酶解时蛤蜊肉：水的重量比为1:1～1:3。
优选的，所述酶解时，蛤蜊肽在50～60℃温度下酶解4～6h。
优选的，所述离心时，蛤蜊肽在生产工艺中的离心转速为16000r/min。
优选的，所述膜分离纯化时，蛤蜊肽酶解液经过微滤-超滤-纳滤膜过滤，截取2KDa分子量以下的酶解液。
一种抗炎护肾的蛤蜊肽在制备抗炎药物和保护肾功能药物中的应用。
经由上述技术方案可知，与现有技术相比，本发明的有益效果如下：
1、本发明中所述蛤蜊肽通过试验证实具有显著的抑制炎症因子的作用。
2、本发明所述蛤蜊肽通过试验证实具有显著降低肾功能指标中血清肌酐、尿酸和尿素氮的作用。
3、本发明拓展了蛤蜊肽在医疗保健方面的用途，具有安全性高、易被人体吸收的优点，为广大患者提供了一种抗炎、护肾的食源性药物新选择。
本技术方案提供的蛤蜊肽生产方法工艺简单，条件温和，周期短，不添加任何无机或有机溶剂，产率高，更适合工业化生产。制备得到的蛤蜊肽，颜色纯正，类白色，无腥臭味，无其他异味，口感和风味俱佳。并且蛤蜊活性肽分子量小，以四肽～六肽为主，易被人体吸收。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为蛤蜊肽对大鼠血清IL-8的影响；
图2为蛤蜊肽对大鼠血清中TNF-α的影响；
图3为蛤蜊肽对大鼠血清中hs-CRP的影响；
图4为蛤蜊肽对大鼠血清中SCr的影响；
图5为蛤蜊肽对大鼠血清中SUA的影响；
图6为蛤蜊肽对大鼠血清中BUN的影响。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本发明实施例1公开了一种抗炎护肾的蛤蜊肽的制备方法和应用，采用的技术方案如下：
取红岛蛤蜊整肉，洗净沥水后匀浆，向蛤蜊肉浆中加入一定量的去离子水，使得最终蛤蜊肉:水＝1:2的重量比。设置酶解温度为50℃，加入占蛤蜊肉 浆重量0.13％的复合蛋白酶(配方为中性蛋白酶：碱性蛋白酶：风味蛋白酶＝2:1:1)，酶解4h，16000r/min离心，上清液经微滤-超滤-纳滤膜过滤后，喷雾干燥得蛤蜊肽。
实施例2
本发明实施例2公开了一种抗炎护肾的蛤蜊肽的制备方法和应用，采用的技术方案如下：
取红岛蛤蜊整肉，洗净沥水后匀浆，向蛤蜊肉浆中加入一定量的去离子水，使得最终蛤蜊肉:水＝1:3的重量比。设置酶解温度为50℃，加入占蛤蜊肉浆重量0.13％的复合蛋白酶(配方为中性蛋白酶：碱性蛋白酶：风味蛋白酶＝2:1:1)，酶解6h，16000r/min离心，上清液经微滤-超滤-纳滤膜过滤后，喷雾干燥得蛤蜊肽。
实施例3：
本发明实施例3公开了一种抗炎护肾的蛤蜊肽的制备方法和应用，采用的技术方案如下：
取红岛蛤蜊整肉，洗净沥水后匀浆，向蛤蜊肉浆中加入一定量的去离子水，使得最终蛤蜊肉:水＝1:2的重量比。设置酶解温度为55℃，加入占蛤蜊肉浆重量0.2％的复合蛋白酶(配方为中性蛋白酶：碱性蛋白酶：风味蛋白酶＝2:1:1)，酶解4h，16000r/min离心，上清液经微滤-超滤-纳滤膜过滤后，喷雾干燥得蛤蜊肽。
自发性高血压模型大鼠给药后抗炎症测试
所用自发性高血压模型大鼠购买于北京维通利华实验动物技术有限公司。
自发性高血压模型大鼠，雄性，40只，12周龄左右，体重约270g，适应性饲养一周。将大鼠随机分为5组，分别为模型对照组(MC)、阳性药物对照组(AC)、蛤蜊肽低剂量组(CP1)、中剂量组(CP2)、高剂量组(CP3)(低剂量组50mg/kg，中剂量组100mg/kg，高剂量组200mg/kg),每组8只。AC组给予10mg/kg的卡托普利灌胃，MC组同时给予等体积的蒸馏水灌胃。每天给药一次，连续灌胃4周。取血清测定IL-8、TNF-α以及hs-CRP，所得数据 采用SPSS软件进行分析处理，采用方差分析，结果显示为指标平均值±标准偏差，p＜0.05表示有显著性差异，p＜0.01表示有极显著差异。数据结果统计如表1所示。
表1蛤蜊肽对高血压大鼠IL-8，TNF-α，hs-CRP水平的影响
炎症因子 MC AC CP1 CP2 CP3
IL-8(pg/ml) 181.20±27.72 122.00±12.15** 143.30±17.70** 158.40±18.52 89.08±16.27**
TNF-α(pg/ml) 148.70±52.12 87.41±19.19** 119.80±16.18 139.20±8.87 84.43±18.95**
hs-CRP(μg/L) 2.16±0.36 1.53±0.41* 1.83±0.36 1.75±0.49 1.43±0.55**
注：与模型组比较，*：p＜0.05；**：p＜0.01
根据图1所示，说明蛤蜊肽组均能降低IL-8的水平，与模型组(IL-8含量为181.2±27.72pg/m1)相比，蛤蜊肽高剂量组和阳性药组分别将血清中IL-8含量降低到89.08±16.27pg/ml(p＜0.01)和122±12.15pg/ml(p＜0.01)，在统计学上具有极显著差异。图2说明高剂量蛤蜊肽组血清中TNF-α含量与模型组相比降低了近43％，具有极显著差异性，达到了与阳性药物相当的效果。图3反应了蛤蜊肽对大鼠血清中hs-CRP的影响，随蛤蜊肽剂量的增大，hs-CRP出现了逐渐降低的趋势，与模型对照组(2.16±0.36μg/L)相比高剂量蛤蜊肽灌胃组降低到1.43±0.55μg/L，有极显著差异，接近阳性药物组水平。
以上结果表明，蛤蜊肽可以有效降低大鼠血清中IL-8、TNF-α和hs-CRP这些炎症因子的水平，具有良好的抗炎效果，可应用于抗炎相关药物或保健品中。
自发性高血压模型大鼠给药后保护肾功能测试
所用自发性高血压模型大鼠购买于北京维通利华实验动物技术有限公司。
自发性高血压模型大鼠，雄性，40只，12周龄左右，体重约270g，适应性饲养一周。将大鼠随机分为5组，分别为模型对照组(MC)、阳性药物对照组(AC)、蛤蜊肽低剂量组(CP1)、中剂量组(CP2)、高剂量组(CP3)(低剂量组50mg/kg，中剂量组100mg/kg，高剂量组200mg/kg)，每组8只。AC组给予10mg/kg的卡托普利灌胃，MC组同时给予等体积的蒸馏水灌胃。每天给药一次，连续灌胃4周。取血清测定SCr、SUA和BUN，所得数据采用SPSS 软件进行分析处理。采用方差分析，结果显示为指标平均值±标准偏差，p＜0.05表示有显著性差异，p＜0.01表示有极显著差异。数据结果统计如表2所示。
表2蛤蜊肽对高血压大鼠SCr，SUA，BUN水平的影响
肾功指标 MC AC CP1 CP2 CP3
SCr(μmol/L) 687.10±111.20 421.50±101.20** 508.90±128.90* 527.60±66.78** 330.50±75.89**
SUA(mg/L) 283.50±56.66 211.60±39.89** 220.90±37.27* 245.90±39.17 169.90±30.11**
BUN(mmol/L) 17.06±3.70 10.56±3.17** 12.43±2.76** 13.69±2.29 8.10±2.43**
注：与模型组比较，*：p＜0.05；**：p＜0.01
根据图4，说明不同剂量蛤蜊肽均有降低大鼠血清SCr的作用，以高剂量组效果最为显著，与模型对照组比较，可降低51.9％。图5说明蛤蜊肽有降低SUA的作用，与模型对照(283.50±56.66mg/L)组比较，高剂量组将其降低到169.90±30.11mg/L，具极显著性差异。图6可知，蛤蜊肽具对血清中BUN的清除有良好的效果，以蛤蜊肽高剂量组效果最为显著，与模型对照相比，BUN的清除率可达52.5％。
以上结果表明，蛤蜊肽灌胃组中对影响肾功能SCr、SUA和BUN指标均有显著的降低，对大鼠肾脏起到一定的保护作用，说明蛤蜊肽具有保护肾功能的活性，可用于肾功能保护相关的药物或保健品中。
高血压患者试食测试
选取高血压患者10位，10位患者在服用原降压药不变的基础上，加用蛤蜊肽胶囊(选用实施例1的成品制备，300mg/粒)，每日2次，早晚各1次，每次3粒，早饭/晚饭后半小时，9：00/21：00前，试服期间可根据自身身体状况及血压变化选择停服原降压药。干预治疗90天(3个月)，研究期间患者每天测量血压，每两周/每月进行一次杜氏高血压生活质量和36项简单健康调查评分，并于治疗前、30天(一个月)以及90天(3个月)进行生理生化和安全性评价指标检测，观察比较患者指标变化，评价蛤蜊肽有效性和安全性。所得数据采用SPSS分析。
相关数据结果统计如下所示：
表3 SBP(收缩压)(
Figure PCTCN2021116427-appb-000001
mmHg，n＝10)
Figure PCTCN2021116427-appb-000002
Figure PCTCN2021116427-appb-000003
注：与0d比较，*p＜0.05，**p＜0.01，***p＜0.001
通过表3可知，①SBP在试验期间持续下降；②与0天相比，服用21天(3周)后有统计学差异(p＜0.05)，服用30天(1个月)后有显著差异(p＜0.01)，服用60天(2个月)后有极显著差异(p＜0.001)；③服用81天(约3个月)后，SBP降至正常水平，且随访14天(2周)SBP仍维持在正常水平；④服用14天(2周)后，血压由1级高血压降为正常高值血压，服用81天(约3个月)后，血压由正常高值血压降为正常血压。
表4 DBP(舒张压)(
Figure PCTCN2021116427-appb-000004
mmHg，n＝10)
Figure PCTCN2021116427-appb-000005
注：与0d比较，*p＜0.05，**p＜0.01，***p＜0.001
通过表4可知，①DBP在试验期间持续下降；②与0天相比，服用21天(3周)后有统计学差异(p＜0.05)，服用37天(5周)后有显著差异(p＜0.01)，服用60天(2个月)后有极显著差异(p＜0.001)；③服用14天(2周)后DBP降至正常水平，且随访14天(2周)DBP仍维持在正常水平；④服用14天(2周)后，血压由正常高值血压降为正常血压。
对血压进行分级对比，得出服用14天后，2级高血压转为1级高血压，部分正常高值血压转为正常血压；0天与试食第1-7、15-21天分级血压比较均没有统计学差异(p>0.05)；与试食第8-14、22-44天分级血压比较有统计学差异(p<0.05)；与试食第45-90天及随访14天分级血压比较有显著差异(p<0.01)。试服期间，其中2人血压一直处于波动状态，为保持数据的真实性和完整性，因此数据分析时并未剔除，所以试服68-90天出现血压回升的现象，推测和试服人员期间饮食和身体状况有关。
表5血压分级人数占有率
Figure PCTCN2021116427-appb-000006
以上结果表明，蛤蜊肽可以显著降低高血压患者血压(收缩压和舒张压)，具有良好的降压作用，可应用于降压相关药物或保健品中。
表6杜氏高血压生活质量评分比较(
Figure PCTCN2021116427-appb-000007
分)
Figure PCTCN2021116427-appb-000008
Figure PCTCN2021116427-appb-000009
注：与0d比较，*p＜0.05，**p＜0.01
通过表6可知，与0天相比，服用蛤蜊肽后杜氏高血压生活质量表总分均增加，且服用90天(3个月)有统计学差异(p<0.05)；在生理状况、躯体化症状、性功能失调、睡眠状况、生气或活力、焦虑、压抑、强迫状况、人际关系敏感、工作状态及敌对等11个维度的评分均增加；服用30天(1个月)，在敌对维度有统计学差异(p<0.05)；服用60天(2个月)，在生理状况和性功能失调维度有统计学差异(p<0.05)；服用90天(3个月)，在躯体化症状、性功能失调、生气或活力、焦虑、人际关系敏感等5个维度均有统计学差异(p<0.05)，在生理状况及敌对维度有显著差异(p<0.01)。
表7 36项简单健康调查评分比较(
Figure PCTCN2021116427-appb-000010
分)
Figure PCTCN2021116427-appb-000011
Figure PCTCN2021116427-appb-000012
注：与0d比较，*p＜0.05
通过表7可知，与0天相比，服用蛤蜊肽后36项简单健康调查表总分增加，且服用30天(1个月)有统计学差异(p<0.05)；在生理机能、生理职能、躯体疼痛、一般健康状况、精力、情感职能、精神健康及健康变化等8个维度的评分均增加；服用14天(2周)，在精力维度有统计学差异(p<0.05)；服用90天(3个月)，在躯体疼痛和一般健康状况维度有统计学差异(p<0.05)；社会功能维度呈现先升高再降低的趋势，但无统计学差异(p＞0.05)。
以上结果表明，服用蛤蜊肽对高血压患者的生活质量及身体状况有显著的改善作用。
表8心功、肝功、肾功相关影响因子
Figure PCTCN2021116427-appb-000013
注：与0d比较，**p＜0.01，***p＜0.001
通过表8可知，与0天相比，服用蛤蜊肽可以降低高血压患者体内谷草转氨酶、磷酸肌酸激酶同工酶和肌酐含量；服用30天(1个月)对谷草转氨酶有显著差异(p<0.01)，对肌酐有极显著差异(p<0.001)；服用90天(3个月)对磷酸肌酸激酶同工酶和肌酐有极显著差异(p<0.001)。
表9降血压因子
Figure PCTCN2021116427-appb-000014
注：与0d比较，*p＜0.05
通过表9可知，与0天相比，服用蛤蜊肽可以降低高血压患者体内内皮素和血管紧张素Ⅰ转化酶含量，提高血管紧张素转化酶2含量；服用30天(1个月)对内皮素有统计学差异(p<0.05)；服用90天(3个月)对血管紧张素转化酶2有统计学差异(p<0.05)。
表10血管内皮因子
Figure PCTCN2021116427-appb-000015
通过表10可知，与0天相比，服用蛤蜊肽90天(3个月)可以提高高血压患者体内一氧化氮的含量，但无统计学差异(p＞0.05)。
表11炎症因子
Figure PCTCN2021116427-appb-000016
Figure PCTCN2021116427-appb-000017
通过表11可知，与0天相比，服用蛤蜊肽可以降低高血压患者体内白介素1β、白介素17A和肿瘤坏死因子α的含量，同时提高白介素10的含量，但均无统计学差异(p＞0.05)。
表12氧化应激因子
Figure PCTCN2021116427-appb-000018
注：与0d比较，**p＜0.01
通过表12可知，与0天相比，服用蛤蜊肽可以降低高血压患者体内丙二醛的含量，且服用90天(3个月)有显著差异(p<0.01)。
以上结果表明，服用蛤蜊肽可以降低高血压患者体内AST、CK-Mb(M)、CREA、ET-1、ACEⅠ、IL-1β、IL-17A、TNF-α和MDA含量，提高ACE2、NO和IL-10含量，一方面提示服用蛤蜊肽有助于改善高血压患者的心功、肝功和肾功，另一方面提示蛤蜊肽降压机制可能与调节降压因子和血管内皮因子含量、减轻炎症反应以及提高抗氧化能力有关，蛤蜊肽可应用于缓解高血压或其他疾病引发的炎症反应及肾功能损伤相关药物或保健品中。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (7)

  1. 一种抗炎护肾的蛤蜊肽的制备方法，其特征在于，具体步骤如下：
    取红岛蛤蜊整肉，洗净搅碎得到浆液，加入占浆液重量0.1～0.3％的复合蛋白酶，酶解、离心、膜分离纯化、喷雾干燥得到蛤蜊肽粉。
  2. 根据权利要求1所述的一种抗炎护肾的蛤蜊肽的制备方法，其特征在于，所述复合蛋白酶包括中性蛋白酶、碱性蛋白酶、风味蛋白酶，中性蛋白酶：碱性蛋白酶：风味蛋白酶的重量比为2:1:1。
  3. 根据权利要求1所述的一种抗炎护肾的蛤蜊肽的制备方法，其特征在于，所述酶解时蛤蜊肉：水的重量比为1:1～1:3。
  4. 根据权利要求1所述的一种抗炎护肾的蛤蜊肽的制备方法，其特征在于，所述酶解时，蛤蜊肽在50～60℃温度下酶解4～6h。
  5. 根据权利要求1所述的一种抗炎护肾的蛤蜊肽的制备方法，其特征在于，所述离心时，蛤蜊肽在生产工艺中的离心转速为16000r/min。
  6. 根据权利要求1所述的一种抗炎护肾的蛤蜊肽的制备方法，其特征在于，所述膜分离纯化时，蛤蜊肽酶解液经过微滤-超滤-纳滤膜过滤，截取2KDa分子量以下的酶解液。
  7. 一种抗炎护肾的蛤蜊肽在制备抗炎药物和保护肾功能药物中的应用。
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