阅读说明：本技术 具有抗氧化应激损伤的脉红螺多肽及其制备方法与应用 (Rapana venosa polypeptide with function of resisting oxidative stress damage as well as preparation method and application of rapana venosa polypeptide ) 是由 张姗姗 刘可春 李晓彬 张轩铭 张云 侯海荣 夏青 孙晨 于 2020-04-20 设计创作，主要内容包括：本发明涉及一种具有抗氧化应激损伤的脉红螺多肽及其制备方法与应用。一种具有氧化应激损伤功能的脉红螺多肽复合物,氨基酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.6所示。本发明还公开了上述多肽作为药效成分在制备治疗氧化应激损伤引起的疾病的药物或者作为保健成分在制备抗氧化保健品中的应用。本发明所述5种多肽化合物均可以单独通过清除体内ROS的产生,减少斑马鱼体内巨噬细胞聚集,抑制血管中血管紧张素转化酶的生成以及抑制炎症细胞因子白细胞介素1(IL-11)的生成,修复由氧化应激导致的机体损伤,具有广阔的市场前景。(The invention relates to a rhodospirillum polypeptide with oxidative stress damage resistance and a preparation method and application thereof.A rhodospirillum polypeptide compound with oxidative stress damage resistance function has an amino acid sequence shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.5 or SEQ ID No. 6. the invention also discloses the application of the polypeptide as a drug effect component in the preparation of a drug for treating diseases caused by oxidative stress damage or as a health-care component in the preparation of an antioxidant health-care product.)

1. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 1.

2. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 2.

3. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 3.

4. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID No. 5.

5. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 6.

6. A polypeptide combination with antioxidant stress injury function comprises polypeptide combination composed of amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

7. A method for extracting the polypeptide with the function of resisting oxidative stress damage is characterized by comprising the following steps:

(1) removing shells of the rapana venosa, taking all soft tissue parts, grinding, adding an acid solution with the pH value of 1.0-4.0, adding pepsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, adjusting the pH value to 7.0-9.0, adding trypsin and chymotrypsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, centrifuging, taking supernatant, concentrating and freeze-drying to obtain the rapana venosa polypeptide extract;

(2) redissolving the rapana venosa polypeptide extract prepared in the step (1) by using a buffer salt solution, carrying out column separation by using Sephadex G25, eluting 5 column volumes by using a buffer salt solution with the pH value of 6.0-8.0 as an eluent, collecting fractions of column volumes of 3 rd-5 th, freeze-drying, dissolving dry powder saline, separating by using Sephadex L H-20, collecting a sample by using the saline as the eluent at the speed of 10m L/45 min, collecting one part every 45min, combining 14 th-20 th parts of active section eluent, and concentrating to prepare a polypeptide active section crude extract;

(3) dissolving the crude extract of the active polypeptide segment prepared in the step (2) by using ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, filtering the solution by using a 4.5 mu m microporous membrane, and separating the solution by using a Welch HI L IC Amide column, wherein the binary mobile phase comprises Acetonitrile (ACN) and ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, the volume ratio of the Acetonitrile (ACN) to the ammonium acetate buffer solution is 85:15, and the flow rate is 0.8 ml/min^-1Collecting eluent with an absorption peak with in-vitro DPPH free radical scavenging activity at 210nm, identifying and determining amino acid composition, and freeze-drying to obtain the polypeptide with the function of resisting oxidative stress damage.

8. The method according to claim 7, wherein in the step (1), the pepsin enzymolysis pH value is 2.0-3.0, and the trypsin and chymotrypsin enzymolysis pH value is 7.2-8.0; further preferably, in the step (1), the pH regulator is hydrochloric acid and sodium hydroxide;

preferably, in the step (1), the enzyme activity ratio of trypsin to chymotrypsin is 1: (0.2-5);

preferably, in the step (2), the buffer salt system is a phosphate buffer system, and the pH value is 6.8-7.2;

preferably, in the step (3), the identification and determination of the amino acid composition adopt L C-MS protein identification technology.

9. The use of a combination according to any one or more of claims 1 to 5, and of a combination with a polypeptide having an amino acid sequence as shown in SEQ ID No.4, as a pharmaceutical active ingredient in the manufacture of a medicament for the treatment of oxidative stress injury.

10. The use of any one or combination of two or more of claims 1 to 5, and the combination with a polypeptide having an amino acid sequence as shown in SEQ ID No.4 as an active ingredient in the preparation of antioxidant health food.

Technical Field

The invention relates to a rapana venosa polypeptide with antioxidant stress injury and a preparation method and application thereof, belonging to the technical field of functional polypeptides.

Background

Oxidative stress refers to the condition that a large amount of Reactive Oxygen Species (ROS) and other oxidizing substances are generated in vivo due to endogenous (endogenous reactive oxygen species generated by various metabolic reactions) or exogenous (environmental factors, drugs and body aging) causes imbalance of the redox balance of the body, so that excessive ROS are accumulated in the body, and the excessive ROS in the body can reduce the oxidation resistance of the body per se, generate lipid peroxidation, damage and even apoptosis of cell DNA, promote the generation of inflammatory factors, hinder the metabolism of nutrient substances and damage the functions of tissues, thereby causing diseases. Modern researches show that oxidative stress is crucial to the occurrence and development of chronic non-infectious diseases such as cardiovascular and cerebrovascular diseases, neurological diseases, inflammatory diseases and the like, with the continuous improvement of living standard of people, diseases caused by oxidative stress damage become main diseases affecting the health of people, and with the continuous aggravation of the aging situation of China, the probability of suffering from oxidative stress damage related diseases is higher for the aged population due to the reduction of body functions, and great economic burden is caused to families and lives, so that the early intervention on the oxidative stress state of the organism is performed, the oxidative reduction imbalance state of the organism is improved, the occurrence and development of diseases are reduced, and the research hotspot of the current medicine research and development and the large health industry is formed.

The marine organisms are of various and large numbers, and are derived from active substances of marine organisms, such as peptides and polysaccharidesTerpenoids and the like have good activities of resisting inflammation, oxidation, bacteria and virus^[1]. With the advance of the national policy of "sea-forcing," the development of marine organisms has been more and more emphasized in recent years. The marine organisms have great difference from terrestrial organisms in living environments, and active substances derived from the marine organisms often have novel structures and unique biological activities, so that more possibilities are provided for lead compounds required by the research and development of new drugs.

For example, chinese patent document CN109180781A (application No. 201810915407.5) discloses a polypeptide having a function of repairing oxidative damage, and a preparation method and an application thereof. A polypeptide with function of repairing oxidative damage has an amino acid sequence shown in SEQ ID NO. 1. The invention also discloses the application of the polypeptide as a drug effect component in preparing a drug for treating diseases caused by oxidative damage or as a health-care component in preparing an antioxidant health-care product. The invention discloses a polypeptide compound containing 10 amino acid residues extracted from a Chinese osbeckia for the first time, and the detection shows that the polypeptide compound can be used for inhibiting the generation of angiotensin converting enzyme in blood vessels, inhibiting the increase of blood sugar and repairing oxidative stress injury caused by peroxide by eliminating the generation of ROS in vivo, and can be used for developing a medicament and an antioxidant health-care product for subsequently treating diseases caused by oxidative injury.

The venorula (Raoana venosa) genus Mollusca (molusca), Gastropoda (Gastropoda), gilles (Prosobranchia), Neogastropoda (Neogastropoda), osteanaceae (Muricidae), is a large marine animal of great economic importance. The method is mainly distributed in yellow sea, Bohai sea and east sea in China, Japanese coastal sea, Korean peninsula and other areas. The soft part of the rapana venosa consists of three parts, namely a head part, a foot part and an internal organ ball, so that the rapana venosa has the advantages of fleshy and compact meat quality, delicious taste and high nutritional value. To date, most of the research on the rapana venosa has focused on its biology, such as genome, nutrition, reproductive characteristics, etc., or its nutritional components, such as crude proteins, polysaccharides, crude lipids, etc. Currently, there are few reports relating to the active ingredient of the rapana venosa.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a polypeptide with the function of resisting oxidative stress damage and a preparation method and application thereof.

The technical scheme of the invention is as follows:

the polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 1.

SEQ ID NO.1：Met-Val-Leu-Leu-Gly-Val-Leu-Met-Gly MVLLGLVLMG。

The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 2.

SEQ ID NO.2：Ala-Arg-Leu-Gly-Leu-Ala-Thr-Leu ARLGLATL

The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 3.

SEQ ID NO.3：Leu-Leu-Thr-Arg-Ala-Gly-Leu LLTRAGL

The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID No. 5.

SEQ ID NO.5：Lys-Ser-Thr-Glu-Leu-Leu-Ile KSTELLI

The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 6.

SEQ ID NO.6：Phe-Gly-Ile-Asn-Leu-Ile-Gln FGINLIQ

A polypeptide combination with antioxidant stress injury function comprises polypeptide combination composed of amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

A method for extracting the polypeptide with the function of resisting oxidative stress damage comprises the following steps:

(1) removing shells of the rapana venosa, taking all soft tissue parts, grinding, adding an acid solution with the pH value of 1.0-4.0, adding pepsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, adjusting the pH value to 7.0-9.0, adding trypsin and chymotrypsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, centrifuging, taking supernatant, concentrating and freeze-drying to obtain the rapana venosa polypeptide extract;

(2) redissolving the rapana venosa polypeptide extract prepared in the step (1) by using a buffer salt solution, carrying out column separation by using Sephadex G25, eluting 5 column volumes by using a buffer salt solution with the pH value of 6.0-8.0 as an eluent, collecting fractions of column volumes of 3 rd-5 th, freeze-drying, dissolving dry powder saline, separating by using Sephadex L H-20, collecting a sample by using the saline as the eluent at the speed of 10m L/45 min, collecting one part every 45min, combining 14 th-20 th parts of active section eluent, and concentrating to prepare a polypeptide active section crude extract;

(3) dissolving the crude extract of the active polypeptide segment prepared in the step (2) by using ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, filtering the solution by using a 4.5 mu m microporous membrane, and separating the solution by using a Welch HI L IC Amide column, wherein the binary mobile phase comprises Acetonitrile (ACN) and ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, the volume ratio of the Acetonitrile (ACN) to the ammonium acetate buffer solution is 85:15, and the flow rate is 0.8 ml/min^-1Collecting eluent with an absorption peak with in-vitro DPPH free radical scavenging activity at 210nm, identifying and determining amino acid composition, and freeze-drying to obtain the polypeptide with the function of resisting oxidative stress damage.

Preferably, in the step (1), the enzymolysis pH value of pepsin is 2.0-3.0, and the enzymolysis pH value of trypsin and chymotrypsin is 7.2-8.0; further preferably, in the step (1), the pH regulator is hydrochloric acid and sodium hydroxide.

Preferably, in the step (1), the enzyme activity ratio of trypsin to chymotrypsin is 1: (0.2-5).

According to the present invention, in the step (2), the buffer salt system is a phosphate buffer system, and the pH value is 6.8 to 7.2.

Preferably, in the step (3), L C-MS protein identification technology is adopted for identifying and determining amino acid composition.

The application of one or the combination of more than two of the polypeptides with the function of resisting oxidative stress damage as a drug effect component in preparing the drugs for treating oxidative stress damage diseases.

The application of one or the combination of more than two of the polypeptides with the function of resisting oxidative stress damage as an effective component in preparing antioxidant health-care food.

Advantageous effects

The invention discloses 5 anti-oxidative stress active peptides extracted from rapana venosa for the first time, and the detection shows that the 5 polypeptide compounds can independently remove the generation of ROS in vivo, reduce the macrophage aggregation in zebra fish bodies, inhibit the generation of angiotensin converting enzyme in blood vessels and the generation of inflammatory cytokine interleukin 1(I L-11), repair the body damage caused by oxidative stress, and can be used for developing the medicines and anti-oxidative health-care products for preventing diseases caused by oxidative stress damage in the subsequent process, thereby having wide market prospect.

Drawings

FIG. 1 is a photograph of the raw Rapana venosa used in the example;

in the figure: A. the overall appearance of the venosa; soft tissue of the Bolus omaculatus;

FIG. 2 is a graph of the Molecular Weight (MW) distribution from the active segment of Rapana venosa as determined by gel permeation chromatography;

FIG. 3 is a graph showing the MS/MS mass spectrum results of the amino acid sequence of the active peptide;

wherein: FIG. 3-1 is a graph showing the result of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 1;

FIG. 3-2 is a graph showing the result of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 2;

FIGS. 3-3 are graphs showing the results of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 3;

FIGS. 3 to 4 are graphs showing the results of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 4;

FIGS. 3 to 5 are graphs showing the results of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 5;

FIGS. 3 to 6 are graphs showing the results of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 6;

FIG. 4 is a graph showing the results of activity detection of fractions eluted by the Rapana venosa Sephadex L H-20;

FIG. 5 shows the detection result of HI L IC chromatographic column of crude extract of active segment of Rapana venosa;

FIG. 6 is a graph showing the results of the HI L IC column detection of samples of each fraction section in comparative example 1;

in the figure, A is HI L IC chromatogram of 8 th to 13 th samples, B is HI L IC chromatogram of 21 st to 29 th samples;

FIG. 7 is a graph of the HI L IC column assay results for a sample of the cut fraction from comparative example 2;

FIG. 8 is a graph showing the repairing effect of each sample on the in vivo oxidative damage of zebra fish;

FIG. 9 is a graph of the anti-inflammatory effect of each sample on zebrafish in vivo;

in the figure: blank control; b, model group; c positive control group; group D example 1; e example 2-1; f example 2-2; g examples 2-3; examples 2-4; i examples 2 to 5; j examples 2-6;

FIG. 10 is a graph showing the effect of the docking of each sample with ACE enzyme based on molecular docking;

wherein: FIG. 10-1 is a 3D diagram of the molecular docking of example 2-1 and example 2-4 with ACE enzyme;

FIG. 10-2 is a 2D diagram of the molecular docking of example 2-1 and example 2-4 with ACE enzyme;

FIG. 11 is a graph showing the effect of docking I L-11 on each sample based on molecular docking technology;

FIG. 11-1 is a 3D diagram of the molecular docking of example 2-1 and example 2-4 with I L-11;

FIG. 11-2 is a 2D diagram of the molecular docking of example 2-1 and example 2-4 with I L-11;

Detailed Description

The technical solutions of the present invention are further described below with reference to the following embodiments and the drawings of the specification, but the scope of the present invention is not limited thereto.

Source of biological material