CN111004305B - Agaricus blazei small peptide and its preparation method and application - Google Patents
Agaricus blazei small peptide and its preparation method and application Download PDFInfo
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- CN111004305B CN111004305B CN201911342554.9A CN201911342554A CN111004305B CN 111004305 B CN111004305 B CN 111004305B CN 201911342554 A CN201911342554 A CN 201911342554A CN 111004305 B CN111004305 B CN 111004305B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
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- C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- 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
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- 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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Abstract
本发明涉及一种姬松茸小肽及其制备方法和应用，属于天然活性产物技术领域。该姬松茸小肽包括如SEQ ID No.1所示氨基酸序列的肽段。该姬松茸小肽通过酶解和色谱柱纯化得到，其主要肽段序列为RQR(精氨酸‑谷氨酰胺‑精氨酸)；由动物实验中可证明本发明的姬松茸小肽可提高Morris水迷宫中小鼠的学习记忆能力，并且检测小鼠血清中生化指标显示姬松茸小肽可显著改善小鼠体内氧化应激水平，CAT，T‑AOC值明显提高，ROS，MDA含量下降，即该姬松茸小肽能够改善动物记忆障碍、提高学习能力，有明显抗衰老作用。以上实验表明本发明的姬松茸小肽具有较好的改善记忆障碍、提高学习能力，抗衰老作用，且具有肽段短，溶解性好，吸收好的优势，具有广泛的应用前景。The invention relates to a small peptide of Agaricus blazei, a preparation method and application thereof, and belongs to the technical field of natural active products. The small peptide of Agaricus blazei includes a peptide segment of the amino acid sequence shown in SEQ ID No.1. The Agaricus blazei small peptide is obtained by enzymatic hydrolysis and chromatographic column purification, and its main peptide sequence is RQR (arginine-glutamine-arginine). The learning and memory ability of mice in Morris water maze, and the detection of biochemical indicators in the serum of mice showed that Agaricus blazei peptides could significantly improve the oxidative stress level in mice, the CAT and T-AOC values were significantly increased, and the ROS and MDA contents were decreased, namely The small peptide of Agaricus blazei can improve animal memory impairment, improve learning ability, and has obvious anti-aging effect. The above experiments show that the small peptide of Agaricus blazei of the present invention has better effects of improving memory impairment, improving learning ability, and anti-aging, and has the advantages of short peptide segment, good solubility and good absorption, and has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of natural active products, in particular to an agaricus blazei murill small peptide and a preparation method and application thereof.
Background
Aging of human cells and tissues refers to the phenomenon of degenerative changes occurring in structure and function, in which the nervous system gradually declines during the process, thereby causing behavior disorder, and decline in learning and memory abilities. The increased degree of oxidative stress in the body is a significant cause of aging, and the degree of oxidative stress depends on the balance of the oxidative and antioxidant systems. Therefore, the antioxidant capacity of the organism is improved, and the aging process can be delayed. Natural products contain a large amount of antioxidant active substances, and the prevention of aging and related diseases has become a hot spot in the field of modern biological research.
Fungi are used as an important source of natural products, and the research on active substances of the fungi has shown a wide development prospect. Agaricus blazei Murill (Agaricus blazei Murill), also known as Pleurotus citrinopileatus and Agaricus blazei Murill, is a large fungus with strong almond flavor, and belongs to the genus Agaricus (black umbrella) of the family Agaricales (black umbrella) of the class Hymenomycetes of Basidiomycotina. Agaricus blazei is not only a popular food but also a rare fungus for anticancer, anti-infectious, etc. in the local. The fruiting body of Agaricus blazei is rich in various bioactive substances such as protein, saccharide, sterol, flavone, etc., and has wide bioactivity, such as antitumor, blood sugar lowering, antiinflammatory, and immunity enhancing effects. The agaricus blazei murill protein has obvious antioxidation and can improve the activity of acetylcholinesterase and the like. The protein is partially hydrolyzed by protease to obtain polypeptide with bioactivity, and compared with macromolecular protein, the small molecular biological polypeptide has the advantages of safety, stability, easy water dissolution, easy absorption and the like. However, at present, no small molecular peptide derived from agaricus blazei murill is confirmed to have a good application prospect.
Disclosure of Invention
In view of the above, there is a need for providing a small peptide of agaricus blazei murill belonging to small molecular peptide fragments, which can improve memory disorder and learning ability of animals and has significant anti-aging effect.
An Agaricus blazei murill small peptide, which comprises a peptide segment of an amino acid sequence shown as SEQ ID No. 1.
The agaricus blazei murill small peptide can improve the memory disorder of animals, improve the learning ability and has obvious anti-aging effect; in a cell experiment, the agaricus blazei small peptide can be proved to have a remarkable protection effect on D-gal induced NIH/3T3 senescent cells, reduce cell damage, keep the original shape of the cells and reduce senescence, and a specific mechanism can relieve oxidation reaction through regulating an Nrf2/ARE signal channel to generate an anti-senescence effect. And has the advantages of short peptide segment, good solubility and good absorption.
In one embodiment, the content of the peptide fragment of the amino acid sequence shown in SEQ ID No.1 is not less than 98% by peak area normalization of HPLC.
The invention also discloses a preparation method of the agaricus blazei murill small peptide, which comprises the following steps:
extraction: collecting fruiting body of Agaricus blazei Murill, homogenizing, centrifuging to obtain supernatant, and precipitating soluble protein in the supernatant;
and (3) dialysis: dissolving the precipitate with water, and dialyzing with dialysis membrane to obtain dialyzed protein;
enzymolysis: carrying out enzymolysis on the dialyzed protein by using trypsin to obtain a crude polypeptide fraction;
and (3) purification: taking the crude polypeptide fraction, performing chromatography by using a DEAE-32 ion exchange chromatographic column as a stationary phase and 0-0.5M/L sodium chloride solution as a mobile phase, and sequentially obtaining four components of DE1, DE2, DE3 and DE4 according to the elution order; taking a DE2 component, performing chromatography by taking a CM-52 ion exchange chromatographic column as a stationary phase and 0-0.5M/L sodium chloride solution as a mobile phase, and sequentially obtaining two components of CM1 and CM2 according to the elution order; taking a CM1 component, purifying and desalting by Sephadex G25 to obtain a unimodal component, purifying and desalting a G25 component by Superdex 30, sequentially obtaining two components S1 and S2 according to the elution sequence, and obtaining the agaricus blazei murrill small peptide (ABp) by S2.
By the preparation method, active small peptides in the agaricus blazei murill are extracted, and the peptide fragments are separated and purified by chromatography, so that the small molecular peptide fragments with high purity and good activity are finally obtained.
In one embodiment, in the extracting step, the ratio of the agaricus blazei murill fruiting body: adding distilled water into the mixture at a material-liquid ratio of 3g:4-6mL, homogenizing on ice, centrifuging for 2-20min under the conditions of 4000 and 6000r/min, taking supernatant, and precipitating soluble protein in the supernatant for 8-14h at 0-4 ℃ under the condition of ammonium sulfate with the mass percentage concentration of 75-85%.
In one embodiment, in the extraction step, the precipitate is taken, deionized water is added for dissolution, and a dialysis membrane with a molecular weight cut-off of 30kDa is used for dialysis at 0-4 ℃ for 12-36h to obtain dialyzed protein.
In one embodiment, in the step of enzymatic hydrolysis, the ratio of dialyzed protein: 1g of water: adding water into 15-25mL of feed-liquid ratio, adjusting pH to 2-3 with hydrochloric acid, adding trypsin according to the amount of 2500-.
In one embodiment, in the purification step, the crude polypeptide fraction is applied to a DEAE-32 ion exchange chromatography column, and eluted with 0.2M/L sodium chloride solution for 1 column volume, and the elution peak of 0.1 to 0.6 column volume is collected, i.e., the DE2 component;
loading the DE2 component to a CM-52 ion exchange chromatographic column, eluting 1 column volume by using 0.2M/L sodium chloride solution, and collecting an elution peak with the column volume of 0.2-0.6, namely a CM1 component;
and (3) loading the CM1 component to a Sephadex G25 chromatographic column, and eluting 1 column volume by using deionized water to obtain a unimodal component, namely a G25 component.
And (3) loading the G25 component to a Superdex 30 chromatographic column, eluting by using deionized water for 1 column volume, and collecting an elution peak S2 with the column volume of 0.6-0.7 to obtain the agaricus blazei murrill small peptide.
In one embodiment, in the purification step, when DEAE-32 ion exchange chromatography is used for separation and purification, the flow rate of eluent is 0.5mL/min, and the detection wavelength is 220 nm; when the CM-52 ion exchange chromatographic column is used for separation and purification, the flow rate of the eluent is 0.5mL/min, and the detection wavelength is 220 nm; when the Sephadex G25 chromatographic column is used for purification, the flow rate of eluent is 1mL/min, and the detection wavelength is 220 nm; during Superdex 30 column purification, the flow rate of the eluent was 1mL/min and the detection wavelength was 220 nm.
The invention also discloses application of the agaricus blazei murill small peptide in preparing a medicine for regulating the Nrf2/ARE signal pathway.
In one embodiment, the medicament for modulating the Nrf2/ARE signaling pathway is used for improving memory impairment, improving learning ability and/or anti-aging.
Compared with the prior art, the invention has the following beneficial effects:
the agaricus blazei small peptide is obtained by enzymolysis and chromatographic column purification, and the main peptide segment sequence of the agaricus blazei small peptide is RQR (arginine-glutamine-arginine); animal experiments can prove that the agaricus blazei small peptide can improve the learning and memory capacity of a mouse in a Morris water maze, has significant difference compared with a model group, and tests on biochemical indexes in the serum of the mouse show that the agaricus blazei small peptide can significantly improve the in-vivo oxidative stress level of the mouse, the CAT and T-AOC values are significantly improved, and the ROS and MDA contents are reduced, namely the agaricus blazei small peptide can improve the animal memory disorder and improve the learning capacity, and has obvious anti-aging effect; in a cell experiment, the agaricus blazei small peptide can be proved to have a remarkable protection effect on D-gal induced NIH/3T3 senescent cells, reduce cell damage, keep the original shape of the cells and reduce senescence, and a specific mechanism can relieve oxidation reaction through regulating an Nrf2/ARE signal channel to generate an anti-senescence effect.
The experiments show that the agaricus blazei murill small peptide has the effects of improving dysmnesia, improving learning ability and resisting aging, has the advantages of short peptide segment, good solubility and good absorption, and has wide application prospect.
Drawings
FIG. 1 is a DEAE-32 column chromatography elution chart in example 1;
FIG. 2 is a CM-52 column chromatography elution diagram of example 1;
FIG. 3 is the Sephadex G25 column chromatography elution diagram of example 1;
FIG. 4 is a Superdex 30 column chromatography elution diagram of example 1;
FIG. 5 is a diagram of HPLC chromatography in example 1;
FIG. 6 is a MS chromatography chart in example 1;
FIG. 7 shows the results of the β -galactosidase activity assay in example 3 (x 40);
wherein: con is blank group; mod: a model group; pir is a positive drug group; ABp-L, Agaricus blazei small peptide low dose group; ABp-M Agaricus blazei small peptide middle dose group; ABp-H, high dose group of Agaricus blazei small peptide;
FIG. 8 shows the results of the expression amounts of the 3Western blot proteins in examples;
wherein: con is blank group; mod: a model group; pir is a positive drug group; ABp-L, Agaricus blazei small peptide low dose group; ABp-M Agaricus blazei small peptide middle dose group; ABp-H high dose group of small peptides of Agaricus blazei.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The starting materials used in the following examples are all commercially available unless otherwise specified.
Example 1
Firstly, preparing the agaricus blazei murill small peptide.
1. And (4) extracting.
Homogenizing powdery fruiting body of Agaricus blazei Murill (about 3000g) in 5000mL distilled water on ice, centrifuging at 5000r/min for 15min to obtain supernatant, and adding ammonium sulfate ((NH)4)2SO4To a concentration of 75-85 wt%, precipitating the soluble protein in the supernatant overnight at 4 deg.C, and centrifuging to obtain precipitate A.
2. And (6) dialyzing.
Dissolving the precipitate in deionized water, dialyzing with 30kDa molecular weight cut-off dialysis membrane at 4 deg.C for 24 hr, and freeze drying to obtain dialyzed protein powder.
3. And (4) enzymolysis.
Adding distilled water into the dialyzed protein powder according to a ratio of 1:20(g/mL), adjusting the pH value to 2.5 by using 0.1mol/L hydrochloric acid (HCl), adding a trypsin hydrolysate according to an enzyme adding amount of 3000u/mg, uniformly mixing, placing in a 37 ℃ constant-temperature water bath for hydrolysis for 4h, inactivating enzyme at 100 ℃ for 10min after the enzymolysis is finished, and initially extracting a crude polypeptide fraction after freeze-drying.
4. And (5) purifying.
The crude polypeptide fraction was isolated using an AKTA Purifier protein chromatograph.
(1)Preswollen DEAE-32。
Presvollen DEAE-32 packing was pretreated and charged into 5mL Bio-Rad. Three Column Volumes (CV) were then equilibrated with 1.5mM Tris-aminomethane/hydrochloric acid (Tris-HCl) buffer at a flow rate of 0.5 mL/min.
The crude polypeptide fraction was applied to the column in Tris-HCl buffer, filtered using a 0.45 μ M filter and eluted continuously in Tris-HCl with a gradient of 0.2-0.5M sodium chloride (NaCl) at a flow rate of 0.5 mL/min. And the column effluent was collected using a fraction collector. In the elution mode, the detection wavelength is 220nm, the eluates are collected, and the effluents are combined according to the absorption peaks and lyophilized.
The mobile phase gradient elution profile of the elution procedure described above is shown in the table below.
TABLE 1 gradient mobile phase
| Time | Mobile phase (sodium chloride concentration) |
| 0-10min | 0.2mol/L |
| 10-20min | 0.5mol/L |
The chromatogram of the elution process is shown in fig. 1, wherein the abscissa of fig. 1 represents the elution volume and the ordinate represents the absorbance, and it can be seen from the figure that four absorption peaks of DE1, DE2, DE3 and DE4 are obtained by elution in sequence along with the elution process. The activity determination shows that the optimal activity absorption peak is DE2, the elution volume of the DE2 absorption peak is 1.5-3ml, and the column volume is 0.3-0.6.
(2) Cellulose CM-52 chromatography.
The Cellulose CM-52 column was set up and equilibrated using the same method as described above, and the active fraction (DE2) was further applied to the CM-52 column and washed with a gradient of 0-0.5M NaCl solution at a flow rate of 0.5mL/min and a detection wavelength of 220 nm.
The mobile phase gradient elution profile of the elution procedure described above is shown in the table below.
TABLE 2 gradient mobile phase
| Time | Mobile phase (sodium chloride concentration) |
| 0-10min | 0.2mol/L |
| 10-20min | 0.5mol/L |
The chromatogram of the elution process is shown in fig. 2, wherein the abscissa of fig. 2 represents the elution volume and the ordinate represents the absorbance, and it can be seen from the figure that two absorption peaks of CM1 and CM2 are obtained by elution in sequence along with the elution process. The activity determination shows that the optimal activity absorption peak is CM1, the elution volume of CM1 absorption peak is 1-3ml, and the column volume is converted to 0.2-0.6.
(3) Sephadex G25 chromatography.
The CM1 component was lyophilized to a powder and dissolved in deionized water, passed through Sephadex G25, eluted with deionized water for 1 CV at a flow rate of 1.0mL/min and a detection wavelength of 220nm to give a single peak, named G25.
The chromatogram of the elution process is shown in FIG. 3, wherein the abscissa of FIG. 3 represents the elution volume and the ordinate represents the absorbance, and it can be seen that G25 is obtained by Superdex 30 purification and desalination.
(4) Superdex 30 chromatography.
The G25 component is lyophilized into powder and dissolved in deionized water, the powder is purified by Superdex 30, 1 CV is eluted by the deionized water with the flow rate of 1.0mL/min, the detection wavelength is 220nm, and finally two absorption peaks S1 and S2 are obtained. The activity determination shows that the optimal absorption peak of the activity is S2.
The chromatogram of the elution process is shown in FIG. 4, wherein the abscissa of FIG. 4 represents the elution volume and the ordinate represents the absorbance, and it can be seen from the figure that two absorption peaks S1 and S2 are obtained by sequentially eluting through Superdex 30. Activity determination shows that the optimal active absorption peak is named as S2 ABp, the elution volume of the S2 absorption peak is 20.2-20.5, and the corresponding column volume is 0.6-0.7.
And II, identifying the structure of the agaricus blazei murill small peptide.
1. And (4) MS secondary chromatographic identification.
The molecular weight of ABp (Agaricus blazei small peptide) was identified by MS secondary chromatography, and its mass spectrum is shown in FIG. 5, in which ABp has a molecular weight of 458.
2. Peptide fragment sequence
The ABp (agaricus blazei small peptide) is taken and compared with a database to obtain a peptide fragment RQR of which the peptide fragment sequence of ABp is an amino acid sequence shown in SEQ ID No. 1.
3. And (4) measuring the content.
The content of the compound is identified by HPLC high performance liquid chromatography and calculated by an area homogenization method.
The detection conditions are as follows: mobile phase: 90% acetonitrile; flow rate: 0.7 mL/min; column temperature: room temperature; detection wavelength: 220 nm.
The liquid chromatogram is shown in FIG. 6, and the content is calculated to be more than or equal to 98%.
Example 2
And (4) activity screening.
Firstly, a method is provided.
The activity of each component in the process of preparing the agaricus blazei murill small peptide is measured according to the following method, as shown in the table below.
Determination of DPPH radical scavenging Capacity:
(1) diluting Agaricus blazei Murill polypeptide solution to appropriate concentration.
(2) Weighing 7.88mg of DPPH, dissolving in a 100mL volumetric flask with absolute ethyl alcohol, and shaking uniformly for later use.
(3) Respectively adding 100 μ L of sample into 96-well plate, adding 100 μ L of DPPH, making 3 parallel samples of sample group, control group (Axo), and blank group (Ao), shaking thoroughly, wrapping with tinfoil, shading, and standing in 25 deg.C incubator for 30 min. The absorbance value (Ax) was measured at 517 nm. The DPPH clearance calculation formula is:
and II, obtaining a result.
The activity results are shown in the table below.
TABLE 3 Activity screening results
The above results show that as the extraction, separation and purification process proceeds, the higher the purity of the obtained product is, the better the activity is, and ABp is the most preferable.
Example 3
The agaricus blazei murill small peptide improves the protection effect and mechanism research of the aged mice induced by D-gal.
Firstly, a method is provided.
1. Grouping and administration of laboratory animals
Male ICR mice of 6w age were selected and fed for 7 days, randomized into 4 groups of 13 mice each: blank control group (Con), model group (Mod), agaricus blazei small peptide group (ABp), and positive drug (piracetam (Pir)). Except for the Con group, mice in other groups were subcutaneously injected with D-gal 300mg/kg at a fixed time daily, mice in the Con group were injected with the same dose of physiological saline, the Pir group was simultaneously gavaged with piracetam 800mg/kg daily, and the ABp group was gavaged with Agaricus blazei Murill 400mg/kg daily. The Con group and the Mod group were administered with distilled water in equal amounts for 42 days by intragastric administration.
2. Behavioral experiments
Experiment administration at 36d the Morris water maze experiment was performed to examine the spatial learning and memory ability of mice. Training acquired memory: the mice in each group were placed in a pool at a fixed point, the time (latency) for each mouse to reach the stealth platform within 120s was recorded, and the training was performed for 6d, while the first 2d was the practice. 7d withdrawal of the platform the number of swimming shuttles of the mouse in the central zone, the middle ring and the target quadrant within 120s, respectively, represents the ability of the mouse to learn memory.
3. Biochemical index detection
After 24h of the last administration, blood is taken from eyeballs, serum is separated by centrifugation (10000r/min) at 4 ℃, the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), Malondialdehyde (MDA), active oxygen (ROS) and indexes in the serum are detected, and the specific operation flow is carried out according to the kit instruction.
4. Action and mechanism research of agaricus blazei small peptide for improving NIH/3T3 senescent cells
4.1 screening and group administration of D-gal concentration
D-Gal concentrations were 50, 100, 200, 300, 500mM for use. Setting normal control group and D-gal group at 1.07X 10 concentration4The MTT assay was performed by adding one/mL cell suspension to a 96-well plate. The ratio of the absorbance value of the damaged group to the normal control group is the cell viability value.
Grouping experiments: blank control group (Con), model group (Mod), D-gal + 5. mu.M ABp group (ABp-L), D-gal + 10. mu.M ABp group (ABp-M), D-gal + 20. mu.M ABp group (ABp-H). Each group was cultured for 24h, after which 20. mu.L of MTT (5mg/mL) was added to each well, after 4h the supernatant was aspirated, 150. mu.L of dimethyl sulfoxide was added, shaken well, left at 37 ℃ for 10min, and absorbance was measured at 490nm with an enzyme reader. Cell viability was calculated as the ratio of absorbance values of the remaining groups to the normal control group.
4.2 Biochemical index detection
After the drug treatment according to 4.1 is finished, the culture medium supernatant is sucked, and the activities of SOD, MDA, CAT, ROS, T-AOC, IL-1 beta, IL-6 and TNF-alpha in the cell supernatant are measured according to the kit instruction.
4.3 beta-galactosidase staining
NIH/3T3 cell concentration was adjusted to 2X 105Inoculating into 6-well plate, and culturing overnight in 37 deg.C incubator; the assay was determined with reference to kit instructions, and the next day beta-galactosidase expressing cells that turned dark blue were observed under an optical microscope and photographed.
4.4 Western Blot
NIH/3T3 cells were lysed and protein extracted by adding an appropriate volume of RIPA lysate and the concentration was determined by the Bradford method. Separating the protein by using SDS (sodium dodecyl sulfate) gel with the concentration of 12%, and then performing membrane transfer treatment; after the membrane transfer is finished, membranes are cut according to the size of a target band, the target band is blocked by 5% skimmed milk powder for 1h, and Nrf2, HO-1, Keap1, NF-kappa Bp65 and TLR4 (1: 500) are incubated for one-time overnight dilution. The next day, incubation of lgG secondary antibody (1:2000 diluted) for 1h at constant temperature of 37 deg.C, washing with TBS-T solution for 3 times, developing with luminous liquid, developing with film processor to expose target protein band, scanning film with scanner and storing picture.
And II, a statistical method.
SPSS 19.0 statistical software is adopted to process each group of data, the result is expressed by mean +/-standard deviation (x +/-s), the significance of the result is judged by selecting t test, and the difference is statistically significant when P is less than 0.05.
And thirdly, experimental results.
1. Mouse Morris Water maze results
The following table is presented in the evaluation of memory function in the Morris water maze test.
TABLE 4 Morris Water maze experiment bench latency period (
n＝13)
Note: con is blank group; mod: a model group; pir is a positive drug group; ABp Agaricus blazei small peptide group.
*p＜0.05，**p is less than 0.01vs. model group;#p＜0.05，##p < 0.01vs. blank set
As can be seen from the above results, the Mod group showed a significant increase in the latency to the plateau during periods 4-5 d (p < 0.05) compared to the Con group. However, a significant decrease in latency was observed in ABp-treated mice compared to the Mod group (p < 0.05).
The results of the space exploration experiment of 7d are shown in the following table.
TABLE 5 Morris Water maze Experimental space exploration (
n＝13)
Note: con is blank group; mod: a model group; pir is a positive drug group; ABp Agaricus blazei small peptide group
*p＜0.05，**p is less than 0.01vs. model group;#p＜0.05，##p < 0.01vs. blank set
As can be seen from the above experiments, in the 7d space exploration experiment, the time taken for the Mod group to find the platform increases (p < 0.05), and the number of times of crossing the central loop and the middle loop decreases. In ABp group, mice showed increased residence time in the central and middle loops and increased crossing times (p < 0.05) compared to the Mod group, which resulted in shorter latency.
2. Biochemical index detection
The results of the biochemical index measurements are shown in the following table.
TABLE 6 Biochemical index detection
n＝10)
Con is blank group; mod: a model group; pir is a positive drug group; ABp Agaricus blazei small peptide group
*p＜0.05，**p is less than 0.01vs. model group;#p＜0.05，##p < 0.01vs. blank set
As can be seen from the above results, the serum MDA content (P < 0.05), ROS content (P < 0.01), and CAT and T-AOC content (P < 0.01) were increased in the mice of the Mod group as compared with the Con group. Compared with the Mod group, the ABp mice have reduced MDA and ROS contents in blood serum (P is less than 0.05 or P is less than 0.01), and CAT and T-AOC activities are obviously improved (P is less than 0.01).
3. Research result of improving effect of agaricus blazei murill small peptide on NIH/3T3 senescent cells
3.1D-gal injury NIH/3T3 cell viability assay results.
The results are shown in the following table.
TABLE 7D-gal injury NIH/3T3 cell viability assay results
From the above results, it was found that the cell viability was 66.21% at a D-Gal concentration of 300mM and a damage time of 24 hours, and the degree of damage was appropriate, and the damage condition was determined for this next experiment.
3.2 beta-galactosidase Activity assay results
The results of the activity assay of Senescence-associated β -galactosidase (SA- β -gal) are shown in FIG. 7, where cells stained dark blue are senescent cells positively expressing SA- β -gal. As seen by observation under an optical microscope, the aged cells in the D-gal treated group were significantly increased, larger in size and irregular in morphology, compared with the Con group, while the ABp treated group was significantly decreased in number of cells staining positive for SA- β -gal, and very few and dose-dependent in number of aged cells, compared with the Mod group. The results suggest that ABp delays D-gal-induced cell aging.
3.3 Western-blot to detect the influence of Keap1, Nrf2 and HO-1 protein expression
The expression of each group of Keap1, Nrf2 and HO-1 was observed from the protein level by using the Western blot method. The results are shown in FIG. 8, in which A is a Western blot gel graph, B is a graph showing comparison of the expression levels of Keap1 protein, C is a graph showing comparison of the expression levels of Nrf2 protein, and D is a graph showing comparison of the expression levels of HO-1 protein.
As can be seen from the results, the expression level of Keap1 was increased in cells of the Mod group (P < 0.05), and the expression levels of Nrf2 and HO-1 proteins were decreased (P < 0.05) as compared with the Con group. Compared with the Mod group, the expression level of the Keap1 in ABp groups is remarkably reduced (P < 0.05), the expression level of the Nrf2 and HO-1 protein is increased (P < 0.05), and ABp-L, ABp-M and ABp-H groups are dose-dependent.
In conclusion, the extraction process of the agaricus blazei small peptide is optimized, and the obtained ABp peptide segment sequence is RQR (more than or equal to 98%); animal experiments can prove that the agaricus blazei small peptide can improve the learning and memory capacity of a mouse in a Morris water maze, has significant difference compared with a model group, and tests on biochemical indexes in the serum of the mouse show that the agaricus blazei small peptide can significantly improve the in-vivo oxidative stress level of the mouse, the CAT and T-AOC values are significantly improved, the ROS and MDA contents are reduced, namely the agaricus blazei small peptide can improve the animal memory disorder and the learning capacity, and has obvious anti-aging effect; in a cell experiment, the agaricus blazei small peptide can be proved to have a remarkable protection effect on D-gal induced NIH/3T3 senescent cells, reduce cell damage, keep the original shape of the cells and reduce senescence, and a specific mechanism can relieve oxidation reaction through regulating an Nrf2/ARE signal channel to generate an anti-senescence effect.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> university of North China
<120> Agaricus blazei murill small peptide, preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Arg Gln Arg
1
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