CN111423495B - Red snail polypeptide with anti-oxidative stress injury and its preparation method and application - Google Patents

Abstract

The invention relates to a rapana venosa polypeptide with antioxidant stress injury and a preparation method and application thereof. A Rapana venosa polypeptide compound with oxidative stress injury function has amino acid sequence shown in 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 preparing a drug for treating diseases caused by oxidative stress damage or as a health-care component in preparing an antioxidant health-care product. The 5 polypeptide compounds can independently eliminate the generation of ROS in vivo, reduce macrophage aggregation in zebra fish bodies, inhibit the generation of angiotensin converting enzyme in blood vessels and the generation of inflammatory cytokine interleukin 1(IL-11), repair body injuries caused by oxidative stress and have wide market prospects.

Description

Red snail polypeptide with anti-oxidative stress injury and its preparation method and application

technical field

The invention relates to a red snail polypeptide with anti-oxidative stress damage, a preparation method and application thereof, and belongs to the technical field of functional polypeptides.

Background technique

Oxidative stress refers to the production of a large number of reactive oxygen species (ROS) in the body due to endogenous (endogenous reactive oxygen species generated by various metabolic reactions) or exogenous (environmental factors, drugs, and aging of the body). ) and other oxidizing substances, make the body's redox balance ability out of balance, and then lead to the accumulation of too much ROS in the body, and too much ROS in the body will reduce the body's own antioxidant capacity, produce lipid peroxidation, and cause DNA damage to cells. Apoptosis promotes the production of inflammatory factors, hinders the metabolism of nutrients, and damages tissue function, thereby leading to the occurrence of diseases. Modern research shows that oxidative stress is very important to the occurrence and development of chronic non-communicable diseases such as cardiovascular and cerebrovascular diseases, neurological diseases, inflammatory diseases, etc. With the continuous improvement of people's living standards, diseases caused by oxidative stress damage It has become a major disease affecting people's health, and as my country's aging situation continues to intensify, the elderly population has a higher probability of suffering from oxidative stress injury-related diseases due to the decline of physical function, causing a great economic burden on families and life. Therefore, early intervention on the body's oxidative stress state to improve the body's redox imbalance and reduce the occurrence and development of diseases has become a research hotspot in today's pharmaceutical R&D and general health industry.

There are a wide variety of marine organisms and a huge number. Active substances from marine organisms, such as peptides, polysaccharides, and terpenoids, have good anti-inflammatory, antioxidant, antibacterial, and antiviral activities ^[1] . With the advancement of the national policy of "maritime power", more and more attention has been paid to the development of marine life in recent years. The living environment of marine organisms is very different from that of terrestrial organisms. Active substances derived from marine organisms often have novel structures and unique biological activities, which can provide more possibilities for the lead compounds required for the development of new drugs.

For example, Chinese patent document CN109180781A (application number 201810915407.5) discloses a polypeptide with the function of repairing oxidative damage and its preparation method and application. A polypeptide with the function of repairing oxidative damage, the amino acid sequence is shown in SEQ ID NO.1. The present invention also discloses the application of the above-mentioned polypeptides as medicinal components in the preparation of medicines for treating diseases caused by oxidative damage or as health-care components in the preparation of antioxidant health-care products. The present invention discloses for the first time a polypeptide compound containing 10 amino acid residues extracted from Snail snails. Through detection, it is found that the polypeptide compound can suppress the generation of angiotensin-converting enzyme in blood vessels by removing the production of ROS in the body, and inhibit the increase of blood sugar. It can increase and repair the oxidative stress damage caused by peroxides, and can carry out the development of drugs and antioxidant health care products for the subsequent treatment of diseases caused by oxidative damage.

Raoana venosa belongs to Mollusca, Gastropoda, Prosobranchia, Neogastropoda, Muricidae. Large marine animals of economic value. It is mainly distributed in my country's Yellow Sea, Bohai Sea and East China Sea, as well as the coast of Japan, the Korean Peninsula and other regions. The soft body of the red snail is composed of three parts: the head, the foot and the viscera. The flesh is thick and firm, delicious in taste and high in nutritional value. So far, most of the researches on the red snail have mainly focused on its biology, such as genome, nutrition, reproductive characteristics, etc., or its nutritional components, such as crude protein, polysaccharide, crude lipid, etc. At present, there are few reports related to the active components of Paihong snail.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a polypeptide with anti-oxidative stress damage function and a preparation method and application thereof.

The technical scheme of the present invention is as follows:

The polypeptide with anti-oxidative stress damage function, the amino acid sequence is shown in SEQ ID NO.1.

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

The polypeptide with anti-oxidative stress damage function, the amino acid sequence is shown in SEQ ID NO.2.

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

The polypeptide with anti-oxidative stress damage function, the amino acid sequence is shown in SEQ ID NO.3.

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

The polypeptide with anti-oxidative stress damage function, the amino acid sequence is shown in SEQ ID NO.5.

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

The polypeptide with anti-oxidative stress damage function, the amino acid sequence is shown in SEQ ID NO.6.

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

A polypeptide combination with anti-oxidative stress damage function, consisting of amino acid sequences such as 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 together constitute a combination of polypeptides.

A method for extracting the above-mentioned polypeptide with anti-oxidative stress damage function, the steps are as follows:

(1) After shelling, take all the soft tissue parts, grind them, then add an acid solution 1 to 10 times the weight of the soft tissue, the pH of the acid solution is 1.0 to 4.0, and then add 5 to 20% of the weight of the soft tissue of pepsin, oscillating for 1-5 hours at 35-40 °C, then adjust the pH value to 7.0-9.0, add 5-20% trypsin and chymotrypsin by weight of soft tissue, and shake at 35-40 °C for enzymatic hydrolysis 1～5h, centrifuge, take the supernatant, then concentrate and freeze-dry to obtain the red snail polypeptide extract;

(2) Redissolving the red snail polypeptide extract prepared in step (1) with a buffered salt solution, using Sephadex G25 for column separation, and using a buffered salt solution with a pH value of 6.0 to 8.0 as an eluent to elute 5 column volumes, collect the 3rd to 5th column volume fractions, freeze-dry, dissolve dry powder in brine, separate with Sephadex LH-20, use brine as eluent, collect samples at a rate of 10mL/45min, and collect a portion every 45min , the 14th to 20th active segment eluates were combined and concentrated to obtain the crude extract of polypeptide active segment;

(3) The crude extract of polypeptide active segment obtained in step (2) was dissolved in ammonium acetate buffer with a concentration of 10 mM and pH 5.8-6.2, filtered through a 4.5 μm microporous membrane, and then separated by a Welch HILIC Amide column. The primary mobile phase is acetonitrile (ACN) and ammonium acetate buffer with a concentration of 10 mM and pH 5.8 to 6.2. The volume ratio of acetonitrile (ACN) to ammonium acetate buffer is 85:15, and the flow rate is 0.8 ml·min ^-1 . The eluate of the absorption peak with in vitro DPPH free radical scavenging activity at 210 nm was identified and determined for amino acid composition, and lyophilized to obtain a polypeptide with anti-oxidative stress damage function.

Preferably according to the present invention, in the step (1), the pH value of the pepsin enzymolysis is 2.0-3.0, and the pH value of the trypsin and chymotrypsin enzymolysis is 7.2-8.0; further preferably, in the step (1), The pH adjusters are hydrochloric acid and sodium hydroxide.

Preferably according to the present invention, in the step (1), the enzymatic activity ratio of the trypsin and chymotrypsin is 1:(0.2-5).

Preferably according to the present invention, in the step (2), the buffer salt system is a phosphate buffer system with a pH value of 6.8-7.2.

Preferably according to the present invention, in the step (3), the identification and determination of the amino acid composition adopts the LC-MS protein identification technology.

Application of one of the above polypeptides with anti-oxidative stress injury function or a combination of two or more of them as medicinal components in the preparation of a medicine for treating oxidative stress injury diseases.

Application of one of the above polypeptides with anti-oxidative stress damage function or a combination of two or more as active ingredients in the preparation of anti-oxidative health food.

beneficial effect

The present invention discloses for the first time five anti-oxidative stress active peptides extracted from the red snail. Through detection, it is found that the above five polypeptide compounds can individually eliminate the production of ROS in the body, reduce the aggregation of macrophages in zebrafish, inhibit the The production of angiotensin-converting enzyme in the blood vessels and the inhibition of the production of the inflammatory cytokine interleukin 1 (IL-11), repair the body damage caused by oxidative stress, and can be used for subsequent prevention of diseases caused by oxidative stress damage. The development of antioxidant health care products has broad market prospects.

Description of drawings

Photo of the raw material of the red snail used in the embodiment of Fig. 1;

In the figure: A, the overall appearance of the red snail; B soft tissue of the red snail;

Fig. 2 adopts gel permeation chromatography to measure the molecular weight (MW) distribution result figure of the active section of Congmaihongluo;

Figure 3 MS/MS mass spectrometry result of amino acid sequence of active peptide;

Wherein: Figure 3-1 is the MS/MS mass spectrum result of the amino acid sequence shown in SEQ ID NO.1;

Figure 3-2 is the MS/MS mass spectrum result of the amino acid sequence shown in SEQ ID NO.2;

Figure 3-3 is the MS/MS mass spectrum result of the amino acid sequence shown in SEQ ID NO.3;

Figure 3-4 is the MS/MS mass spectrum result of the amino acid sequence shown in SEQ ID NO.4;

Figure 3-5 is the MS/MS mass spectrum result of the amino acid sequence shown in SEQ ID NO.5;

Figure 3-6 is the MS/MS mass spectrum result of the amino acid sequence shown in SEQ ID NO.6;

Fig. 4 is a curve diagram of the detection results of the fractional activity of each fraction eluted by Sephadex LH-20;

Fig. 5 HILIC chromatographic column detection result of crude extract of active segment of Paihong snail;

The HILIC chromatographic column detection result diagram of each fractional sample in Fig. 6 comparative example 1;

In the figure: A HILIC chromatogram of the 8th to 13th samples; B HILIC chromatogram of the 21st to 29th samples;

The HILIC chromatographic column detection result diagram of Fig. 7 comparative example 2 middle-distillate fractional samples;

Figure 8 shows the effect of each sample on the repair of oxidative damage in zebrafish;

Figure 9 shows the anti-inflammatory effect of each sample on zebrafish in vivo;

In the figure: A blank control; B model group; C positive control group; D example 1 group; E example 2-1; F example 2-2; G example 2-3; H example 2-4; I embodiment 2-5; J embodiment 2-6;

Figure 10 shows the effect of docking each sample with ACE enzyme based on molecular docking technology;

Wherein: Figure 10-1 is a 3D diagram of the molecular docking of Example 2-1 and Example 2-4 with ACE enzyme;

Figure 10-2 is a 2D diagram of the molecular docking of Example 2-1 and Example 2-4 with the ACE enzyme;

Figure 11 shows the docking effect of each sample and IL-11 based on molecular docking technology;

Wherein: Figure 11-1 is a 3D diagram of the molecular docking of Example 2-1 and Example 2-4 with IL-11;

Figure 11-2 is a 2D diagram of the molecular docking of Example 2-1 and Example 2-4 with IL-11;

Detailed ways

The technical solutions of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the protection scope of the present invention is not limited thereto.

source of biological material

The pulse red snail described in the embodiment was purchased from Shandong Jinan Seafood Market, a common commercially available product, as shown in Figure 1.

Detection method

Active component molecular weight distribution detection method

The molecular weight (MW) distribution of the active segment from Rhodotorula sinensis was determined by gel permeation chromatography using a TSK-gel G2000 SW _XL column (7.8 mm x 250 mm) (TOSOH, Yamaguchi, Japan) (Figure 2). The mobile phase consisted of 0.1 mol·L ⁻¹ phosphate buffer (pH 6.7) and 0.1 mol·L ⁻¹ Na ₂ SO ₄ , and the flow rate was set at 0.2 mL·min ⁻¹ .

Using ribonuclease (13700Da), aprotinin hydrochloride (6511Da), angiotensin II (1046Da), HHL (430Da) and L-serine (105Da) as controls, draw the elution volume-molecular weight curve as In Mw= 17.50～0.089T (R ² =0.9785, Mw is molecular weight, T is elution volume).

Active ingredient amino acid composition detection method

The lyophilized active peptide to be detected was dissolved in 6 mol·L ^-1 HCl (1 mg peptide/mL HCl), and hydrolyzed in a drying oven at 110° C. for 24 hours. The filtered hydrolyzed samples were evaporated by rotary evaporator at 45°C. The residue was dissolved in distilled water and lyophilized. Then, samples and mixture amino acid standards were derivatized with AQC and determined by RP- _HPLC18 . The amino acid composition of the sample fractions was identified and quantified from a standard curve of mixed amino acids (Table 1). All samples were assayed in triplicate.

Sequence identification of active peptides by nano-LC-LTQ-Orbitrap-MS/MS

Amino acid sequences of active peptides were identified using an EASY-Nlc1000 chromatography system (Thermo Finnigan, Bremen, Germany), a LTQ OrbitrapVelos Pro mass spectrometer (Thermo Finnigan, Bremen, Germany). The purified peptide was dissolved in ultrapure water at a concentration of 0.1 mg·mL ⁻¹ containing 0.1% trifluoroacetic acid. 2 μL of sample was then injected into a trap column (100 μm×20 mm, RP-C18, thermo Inc.) for preconcentration. The pre-concentrated sample was then automatically fed into an analytical column (75 μm×150 mm, RP-C18, thermo Inc.). Using 0.1% (v/v) formic acid in ultrapure water as eluent, analysis time: 60min, detection mode: positive ion mode, spray voltage: 1.8kV, ion transmission capillary temperature: 250 ℃, standard calibration solution before use Calibration, precursor ion scanning range: 350-1800m/z, mass spectrometry scanning mode is information-dependent acquisition mode (IDA, Information Dependent Analysis), and the 10 strongest fragments are collected after each full scan ( MS2 scan), fragmentation method: collision-induced dissociation (CID, collision-induced dissociation), normalization energy 35%, q value 0.25, activation time: 30ms, dynamic exclusion time: 30s. MS1 has a resolution of 60,000 at M/Z 400 and MS2 has a unit mass resolution in the ion trap. The primary mass spectrometer was acquired in profile mode, and the secondary mass spectrometer was acquired in centroid mode to reduce the data file size. Mascot 2.3 software (Matrix Science, USA) was used for data analysis. The database is the Moth snail family database, the enzyme is trypsin, and the maximum allowable missed cleavage site is 2. Fixed modification: Carbamidomethyl (C); Variable modification: Acetyl (Protein N-term), Deamidated (NQ), Dioxidation (W), Oxidation (M); MS tolerance is ±30ppm, MSMS tolerance is ±0.15 Da. The NCBInr database was used for peptide identification. Only identified peptides with expected values below 0.05 were considered. The BIOPEP database was used to find previously identified amino acid sequences with antioxidant properties. The MS/MS mass spectrometry results of the amino acid sequence of the active peptide are shown in Figure 3.

Example 1

The method for extracting a polypeptide with anti-oxidative stress damage function is as follows:

(1) After removing the shell of the red snail, take all the soft tissue parts, grind them, and adopt the semi-biomimetic preparation technology of multiple digestive tract enzymes, that is, add 5 times the acid aqueous solution (pH value 2.2) to the red snail tissue, and press the enzyme substrate. The ratio of 8% was added with pepsin, and 37.6°C was shaken and extracted for 2 hours. Then, the pH value of the reaction system was adjusted to 7.8, and trypsin and chymotrypsin were added according to the enzyme-substrate ratio of 8%. The enzyme activity ratio of trypsin and chymotrypsin was 1:1, shaking and extracting at 37.6°C for 2 hours, the reaction solution was centrifuged and the supernatant was taken, concentrated, freeze-dried, and the dry powder was stored at low temperature to obtain the red snail polypeptide extract;

(2) Reconstitute the polypeptide extract obtained in step (1) with a buffered saline solution, and perform column separation with Sephadex G25, and use phosphate buffer at pH 6.8 as the eluent to elute for 5 column volumes , collect the 3rd to 5th column volume fractions, freeze-dry, dissolve the dry powder in saline, separate with Sephadex LH-20, use saline as the eluent, collect samples at a rate of 10mL/45min, collect a portion every 45min, combine with in vitro DPPH For free radical scavenging activity, the 14th to 20th fractions of the active segment eluates were combined (Fig. 4), and concentrated to obtain a crude extract of the polypeptide active segment. The molecular weight distribution of the active peptide segment of P. <3000Da area;

(3) The crude extract of polypeptide active segment obtained in step (2) was dissolved in ammonium acetate buffer with a concentration of 10 mM and pH 5.8-6.2, filtered through a 4.5 μm microporous membrane, and then separated by a Welch HILIC Amide column. The primary mobile phase is acetonitrile (ACN) and ammonium acetate buffer with a concentration of 10mM and pH 5.8-6.2. The volume ratio of acetonitrile (ACN) and ammonium acetate buffer is 85:15, and the flow rate is 0.8ml·min ^-1 . In vitro DPPH free radical scavenging activity, collect the eluate with the active absorption peak at 210nm (Figure 5), freeze-dried to prepare the polypeptide with anti-oxidative stress damage function, and determine its amino acid by LC-MS protein identification technology composition.

After testing, the amino acid sequences of polypeptides with anti-oxidative stress damage function are shown in SEQ ID NO. 1-6:

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

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

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

SEQ ID NO. 4: Gly-Thr-Ser-Phe-Thr-Thr-Thr-Ala-Glu-Arg GYSFTTTAER

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

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

Example 2-1

Adopt the Fmoc solid-phase synthesis method described by Liu Zhennan et al. (Liu Zhennan, Huang Qiang. Fmoc solid-phase synthesis method. Journal of Guangxi University for Nationalities, 1999, 5(2): 110-112), the artificially synthesized amino acid sequence is as shown in SEQ ID NO.1 shown polypeptides.

Example 2-2

The Fmoc solid-phase synthesis method described by Liu Zhennan et al. was adopted (Liu Zhennan, Huang Qiang. Fmoc solid-phase synthesis method. Journal of Guangxi University for Nationalities, 1999, 5(2): 110-112), the artificially synthesized amino acid sequence is as shown in SEQ ID NO.2 shown polypeptides.

Example 2-3

The Fmoc solid-phase synthesis method described by Liu Zhennan et al. was adopted (Liu Zhennan, Huang Qiang. Fmoc solid-phase synthesis method. Journal of Guangxi University for Nationalities, 1999, 5(2): 110-112), the artificially synthesized amino acid sequence is as shown in SEQ ID NO.3 shown polypeptides.

Example 2-4

The Fmoc solid-phase synthesis method described by Liu Zhennan et al. was adopted (Liu Zhennan, Huang Qiang. Fmoc solid-phase synthesis method. Journal of Guangxi University for Nationalities, 1999, 5(2): 110-112), the artificially synthesized amino acid sequence is as shown in SEQ ID NO.4 shown polypeptides.

Example 2-5

The Fmoc solid-phase synthesis method described by Liu Zhennan et al. was adopted (Liu Zhennan, Huang Qiang. Fmoc solid-phase synthesis method. Journal of Guangxi University for Nationalities, 1999, 5(2): 110-112), the artificially synthesized amino acid sequence is as shown in SEQ ID NO.5 shown polypeptides.

Examples 2-6

The Fmoc solid-phase synthesis method described by Liu Zhennan et al. was adopted (Liu Zhennan, Huang Qiang. Fmoc solid-phase synthesis method. Journal of Guangxi University for Nationalities, 1999, 5(2): 110-112), the artificially synthesized amino acid sequence is as shown in SEQ ID NO.6 shown polypeptides.

Comparative Example 1-1

The method is as described in Example 1, except that the collected eluates in step (2) are concentrated in the 8th to 13th parts (A) to obtain the corresponding polypeptide mixture samples. According to the purification method described in step (3), under the condition of the same retention time, no corresponding chromatographic peak was found (Fig. 6). It indicated that the active polypeptide only existed in the fractions of 585-900 min. Therefore, the sample prepared in step (2) was used as the test sample for the next step of activity evaluation experiment.

Comparative Example 1-2

The method is as described in Example 1, except that the collected eluates in step (2) are concentrated in the 21st to 29th parts (B) to obtain the corresponding polypeptide mixture samples. According to the purification method described in step (3), under the condition of the same retention time, no corresponding chromatographic peak was found (Fig. 6). It indicated that the active polypeptide only existed in the fractions of 585-900 min. Therefore, the sample prepared in step (2) was used as the test sample for the next step of activity evaluation experiment.

Comparative Example 2

The method as described in Example 1, the difference is that the experimental raw materials described in step (1) are all the soft tissues of the snail. According to the purification method described in step (3), under the condition of the same retention time, no corresponding chromatographic peak was found (Fig. 7). It indicated that the active polypeptide was not contained in the extract of all soft tissues of Snail snail. Therefore, the sample prepared in step (2) was used as the test sample for the next step of activity evaluation experiment.

Experimental example

DPPH free radical scavenging activity

The sample polypeptides were tested for DPPH free radical scavenging activity according to the method described by Lee et al.

A 200 μl fraction was added to a tube containing 200 μl of a 0.15 mmol·L ⁻¹ DPPH solution in ethanol, and the mixture was vortexed for a few seconds. Then, the mixture was incubated in the dark at 37°C for 12 hours. Ultrapure water was used as a control. Absorbance was measured at 517 nm and assayed in triplicate. The DPPH free radical scavenging activity of the peptide fraction was calculated as follows:

DPPH free radical scavenging activity (%)=(1-As/Ac)×100

where As is the absorbance of the sample and Ac is the absorbance of the control. While the DPPH free radical scavenging activity of active peptides is expressed as the half inhibitory concentration (IC50), _IC50 is defined as the concentration of peptide required to inhibit 50% of free radical formation. The DPPH radical scavenging rate IC ₅₀ of the examples and comparative examples is shown in Table 2.

Determination of Antioxidant Activity in vivo

In vivo antioxidant detection of polypeptide samples was performed by using the transgenic zebrafish line Tg(krt4:NTR-hKikGR)cy17.

Transgenic zebrafish embryos developing at 24 hpf were dispensed into 24-well cell culture plates (10 embryos/well) and incubated with 2 mL of 10 mM metronidazole (MTZ, dissolved in zebrafish culture water) and polypeptide samples at a dose of 100 μg· mL ^-1 , 24 hours after drug treatment at 28°C. Zebrafish treated with fish water without metronidazole and peptides were used as vehicle controls. Zebrafish treated with metronidazole without peptides were used as negative controls. Peptide was replaced with Vitmin C as a positive control. Perform at least three parallel repetitions for each set. After incubation, zebrafish embryos were anesthetized with tricaine (0.16%, w/v), and then the zebrafish embryos were observed for fluorescence and imaged using a FSX100 Bio Imaging Navigator instrument. The number of fluorescent spots was assessed by using imagepro-plus software. The in vivo antioxidant activity of the peptide samples was calculated as follows:

Antioxidant activity (%)=(FS _s -FS _nc )/(FS _vc -FS _nc )×100

where FS _s is the fluorescent spot of the sample (polypeptide sample), FS _nc is the fluorescent spot of the negative control, and FS _vc is the fluorescent spot (vitamin C). The in vivo antioxidant evaluation results of Examples 1 and 2 and Comparative Examples 1 and 2 are shown in FIG. 8 .

Determination of anti-inflammatory activity in vivo

In vivo anti-inflammatory detection of polypeptide samples was performed by using macrophage fluorescent TG zebrafish (zlyz: EGFP).

The healthy zebrafish that developed for 72h were randomly distributed into 24-well plates, 10 per well, and the volume of each well was 2 ml. A blank group, a model group, a positive control group and an administration group were set. The blank group was only treated with fish culture water, and the model group was In order to add copper sulfate after treatment, the positive control group was treated by adding ibuprofen solution with a concentration of 100 μg/ml and copper sulfate successively, and the treatment group was treated by adding sample solutions of different concentrations and copper sulfate successively. The model group, the positive control group and the treatment group were treated with drugs for a certain period of time, and then an appropriate amount of copper sulfate solution was added to each experimental group to make the final concentration of the copper sulfate solution in the system to reach 20 μg/ml, and then covered and sealed. Incubate in a light box at 28 °C for 1 h, then wash off copper sulfate and drug residues on the surface of zebrafish juveniles with fish culture water, add a small amount of tricaine, anesthetize the juveniles, and observe each juvenile under a stereoscopic fluorescent stereo microscope The changes of macrophages were recorded at 4 times of zebrafish larvae, and the images were processed and analyzed by Image pro-plus software, and the number of neutrophils migrating to the midline of the notochord was counted. The in vivo anti-inflammatory evaluation results of Examples 1 and 2 and Comparative Examples 1 and 2 are shown in FIG. 9 .

ACE inhibitory activity based on molecular docking technology

Using the ChemDraw module and Chem3D pro module in ChemBioOffice2014, the 3D structures of 6 pure peptides were drawn, and the peptides were used as ligands. Using ACE as a molecular docking target for antihypertensive activity. The tertiary structure of ACE comes from the Protein Data Bank (PDB) database. Add valence and apply forcefoild to the ligand, remove water, clean protein, add valence and apply forcefoild to the receptor After that, set the active site and docking radius, and use the CDocker module in Discovery Studio2016 software for molecular docking. According to CDocker energy and CDocker interaction energy values, the potential antihypertensive activities of 6 pure peptides were discussed.

IF-1α inhibitory activity based on molecular docking technology

Using the ChemDraw module and Chem3D pro module in ChemBioOffice2014, the 3D structures of 6 pure peptides were drawn, and the peptides were used as ligands. Interleukin 1 (IL-11) as an anti-inflammatory target for molecular docking. The tertiary structures of IL-11 were obtained from the Protein Data Bank (PDB) database, respectively. Add valence, apply forcefoild to the ligand, remove water, clean protein, add valence, and apply forcefoild to the receptor After processing, the active site and docking radius were set, and the CDocker module in the Discovery Studio 2016 software was used for molecular docking. According to the CDocker energy and CDocker interaction energy values, the potential anti-inflammatory activities of 6 pure peptides were investigated.

Determination of cellular oxidative damage repair activity in vitro

The cellular oxidative damage repair activity of polypeptide samples was determined using H ₂ O ₂ -induced macrophage RAW264.7 oxidative damage repair model.

The RAW264.7 cells cultured to the logarithmic phase were inoculated into 96-well plates (the cell density was 2×104 cells/mL), and 190 μL were inoculated in each well. The sample group, negative control group and injury control group were established, except for the negative control group. In addition to DMSO treatment, the other groups were treated with 10 μL H ₂ O ₂ for 4 h, then the sample group was added with 1 μL of the sample to be tested, and the damaged control group was added with 1 μL DMSO, and then placed in a 37 °C, 5% CO ₂ incubator for 48 h, and then determined. Examples of the effects of samples on injury model cell viability, intracellular oxidation level and antioxidant enzymes. The experimental results are shown in Table 3.

Statistical Analysis

All tests were repeated three times and results were expressed as mean ± standard deviation. SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All data were produced by Origin 9.0 (Origin Lab, Northampton, MA, USA). One-way analysis of variance (ANOVA) was used to analyze differences. P values less than 0.05 were considered statistically significant. The Pearson correlation coefficient was used to assess the correlation between content and activity. The linear regression equation was calculated by linear regression analysis.

Experimental results

The amino acid composition of the active peptide complex from the red snail is shown in Table 1, and the evaluation results of the in vitro DPPH free radical scavenging experiments of the Examples and Comparative Examples are shown in Table 2. Table 3 shows the evaluation results of in vitro cell oxidative damage repair activity of Examples and Comparative Examples. The evaluation results of in vivo antioxidant activity of Examples and Comparative Examples are shown in Figure 8, the evaluation results of in vivo anti-inflammatory activities of Examples and Comparative Examples are shown in Figure 9, and the ACE inhibitory effects of Examples and Comparative Examples are shown in Figure 10. Figure 11 shows the inhibitory effect of IF-1α.

It can be seen from Table 1 that the total amino acid content of the red snail active peptide complex was 925.93 mg/g, and a total of 17 amino acids were detected, among which, serine (Ser), arginine (Arg) and threonine (Thr) were The three amino acids with the highest content in the snail account for 51.45% of the total amino acid.

Table 1 Identification of amino acid composition of active segment of Paihong snail

It can be seen from Table 2 that each sample of the embodiment has strong in vitro DPPH free radical scavenging experimental activity, and the sample obtained in the comparative example does not have in vitro antioxidant activity.

)Table 2 Results of in vitro antioxidant activity evaluation of the samples of Examples and Comparative Examples (IC ₅₀ value, n=3,

)

It can be seen from Table 3 that each sample in the example can improve the degree of cellular oxidative damage, which is significantly different from that of the model group, and each sample in the comparative example cannot improve the cellular oxidative damage.

)Table 3 Results of in vitro cell oxidative damage repair evaluation of the samples of the example (IC ₅₀ value, n=3,

)

It can be seen from Figure 8 that each sample in the examples can significantly reduce the apoptosis of zebrafish skin fluorescent cells caused by metronidazole and improve the oxidative damage of zebrafish. Compared with the model group, the zebrafish skin of each sample in the comparative example There is no significant difference in the number of fluorescent cells, and the experimental results further show that the polypeptide of the characteristic peptide sequence prepared by the above method has significant antioxidant activity. The zebrafish-based anti-inflammatory activity evaluation results of each sample are shown in Figure 9. In the examples, each sample can significantly reduce the migration of zebrafish macrophages caused by copper sulfate, and compared with the model group, each sample in the comparative example There was no significant difference in the migration rate of zebrafish macrophages, indicating that each polypeptide has significant anti-inflammatory activity. Combined with the molecular docking experiment, it was further confirmed that the characteristic peptide of the present invention has an inhibitory effect on the inflammation-related receptor IF-1α (Figure 11), and at the same time can inhibit the function of angiotensin-converting enzyme (Figure 10), which has potential Antihypertensive activity.

Based on the above experimental results, it can be seen that the polypeptide of the characteristic peptide sequence prepared by the above method has significant in vivo and in vitro antioxidant activity and in vivo anti-inflammatory activity, and can inhibit the production of angiotensin-converting enzyme and inhibit the cellular inflammatory factor IF- The production of 1α has the potential to resist oxidative stress damage, and can be used as a medicinal ingredient for the preparation of oxidative stress damage repair drugs and antioxidant health care products.

sequence listing

<110> Institute of Biology, Shandong Academy of Sciences

<120> Pulse red snail polypeptide with anti-oxidative stress injury and its preparation method and application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Rapana venosa

<400> 1

Met Val Leu Leu Gly Leu Val Leu Met Gly

1 5 10

<210> 2

<211> 8

<212> PRT

<213> Rapana venosa

<400> 2

Ala Arg Leu Gly Leu Ala Thr Leu

1 5

<210> 3

<211> 7

<212> PRT

<213> Rapana venosa

<400> 3

Leu Leu Thr Arg Ala Gly Leu

1 5

<210> 4

<211> 10

<212> PRT

<213> Rapana venosa

<400> 4

Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Rapana venosa

<400> 5

Lys Ser Thr Glu Leu Leu Ile

1 5

<210> 6

<211> 7

<212> PRT

<213> Rapana venosa

<400> 6

Phe Gly Ile Asn Leu Ile Gln

1 5

Claims (14)

1. A polypeptide with anti-oxidative stress injury function, the amino acid sequence of which is shown in SEQ ID NO.1. 2. A polypeptide with anti-oxidative stress injury function, the amino acid sequence is shown in SEQ ID NO.2. 3. A polypeptide with anti-oxidative stress injury function, the amino acid sequence is shown in SEQ ID NO.3. 4. A polypeptide with anti-oxidative stress injury function, the amino acid sequence of which is shown in SEQ ID NO.5. 5. A polypeptide with anti-oxidative stress injury function, the amino acid sequence is shown in SEQ ID NO.6. 6. A polypeptide combination with anti-oxidative stress damage function, comprising amino acid sequences such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. NO.6 together constitute a combination of polypeptides. 7. A method for extracting the polypeptide with anti-oxidative stress damage function described in any one of claims 1-5, wherein the steps are as follows: (1) After shelling, take all the soft tissue parts, grind them, then add an acid solution with a weight of 1 to 10 times the weight of the soft tissue, the pH of the acid solution is 1.0 to 4.0, and then add 5 to 20% of the weight of the soft tissue of pepsin, oscillating for 1-5 hours at 35-40 °C, then adjust the pH to 7.0-9.0, add 5-20% trypsin and chymotrypsin by weight of soft tissue, and shake at 35-40 °C for enzymatic hydrolysis 1～5h, centrifuge, take the supernatant, then concentrate and freeze-dry to obtain the red snail polypeptide extract; (2) Redissolving the red snail polypeptide extract obtained in step (1) with a buffered salt solution, and performing column separation with Sephadex G25, using a buffered salt solution with a pH value of 6.0 to 8.0 as an eluent, and eluting 5 column volumes, collect the 3rd to 5th column volume fractions, freeze-dry, dissolve dry powder in brine, separate with Sephadex LH-20, use brine as eluent, collect samples at a rate of 10mL/45min, and collect a portion every 45min , the 14th to 20th active segment eluates were combined and concentrated to obtain the crude extract of polypeptide active segment; (3) The crude extract of polypeptide active segment obtained in step (2) was dissolved in ammonium acetate buffer with a concentration of 10 mM and pH 5.8-6.2, filtered through a 4.5 μm microporous membrane, and then separated by a Welch HILIC Amide column. The primary mobile phase is acetonitrile (ACN) and ammonium acetate buffer with a concentration of 10 mM and pH 5.8 to 6.2. The volume ratio of acetonitrile (ACN) to ammonium acetate buffer is 85:15, and the flow rate is 0.8 ml·min ^-1 . The eluate of the absorption peak with in vitro DPPH free radical scavenging activity at 210 nm was identified and determined for amino acid composition, and lyophilized to obtain a polypeptide with anti-oxidative stress damage function. The method according to claim 7, wherein in the step (1), the pH value of pepsin enzymolysis is 2.0-3.0, and the pH value of trypsin and chymotrypsin enzymolysis is 7.2-8.0; 9. The method of claim 8, wherein, in the step (1), the pH regulator is hydrochloric acid and sodium hydroxide. The method according to claim 7, characterized in that, in the step (1), the enzyme activity ratio of the trypsin to chymotrypsin is 1:(0.2-5). 11. The method of claim 7, wherein in the step (2), the buffer salt system is a phosphate buffer system with a pH value of 6.8-7.2. 12 . The method of claim 7 , wherein, in the step (3), LC-MS protein identification technology is used to identify and determine the amino acid composition. 13 . 13. Use of any one of claims 1-5 or the combination of more than two of them, and the combination with a polypeptide whose amino acid sequence is shown in SEQ ID NO. 4 as a medicinal component in the preparation of a medicine for treating oxidative stress injury diseases. 14. The application of any one of claims 1-5 or the combination of more than two, and the combination with the polypeptide whose amino acid sequence is shown in SEQ ID NO. 4 as an active ingredient in the preparation of antioxidant health food.

Images