CN111253466A - Anti-inflammatory tetrapeptide, extraction and separation method thereof and application of anti-inflammatory tetrapeptide in preparation of memory improving medicines - Google Patents
Anti-inflammatory tetrapeptide, extraction and separation method thereof and application of anti-inflammatory tetrapeptide in preparation of memory improving medicines Download PDFInfo
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Abstract
The invention discloses an anti-inflammatory tetrapeptide, an extraction and separation method thereof and application thereof in preparing a memory improving drug, wherein the amino acid sequence of the anti-inflammatory tetrapeptide is Gly-Val-Tyr-Tyr, and the extraction and separation method comprises the following steps: alkali-dissolving and acid-precipitating to extract walnut protein from walnut meal, taking the walnut protein, adding deionized water for mixing, performing enzymolysis, and taking supernatant to obtain walnut protein hydrolysate; collecting the permeate by using a 3kDa molecular weight ultrafiltration membrane; the low molecular weight component of the walnut protein enzymolysis liquid is loaded on a gel filtration chromatographic column, is eluted by deionized water, the detection wavelength is 220nm, the low molecular weight component is collected and combined into a plurality of elution peak components, and the component with stronger anti-inflammatory activity is selected for mass spectrum detection, so that the anti-inflammatory tetrapeptide disclosed by the invention is proved to contain. The anti-inflammatory tetrapeptide has better inhibitory activity on BV-2 cell inflammatory reaction stimulated by LPS, and can be used for preparing memory improving medicaments or memory improving health care products.
Description
Technical Field
The invention belongs to the field of active peptides, and particularly relates to an anti-inflammatory tetrapeptide, an extraction and separation method thereof and application thereof in preparation of a memory improving drug.
Background
Inflammatory responses refer to complex biological responses of the microcirculatory system caused by noxious irritation, infection, or trauma. Typically, inflammation is initiated by a series of soluble mediators (complement, chemokines, cytokines, reactive oxygen species released by inflammation-associated cells, etc.) that then eliminate the lesion. However, overexpression or long-term inflammation is a potential cause of many diseases, including cancer, rheumatoid arthritis, chronic asthma, multiple sclerosis, obesity, autoimmune diseases, diabetes, inflammatory bowel disease, and cardiovascular diseases, among others. In the brain, neuroinflammation is considered to be a pathogenesis of neurodegenerative diseases.
Microglia are macrophage-like cells that play a key role in the inflammatory response of the Central Nervous System (CNS). Excessive activation of microglia releases a variety of proinflammatory mediators including Nitric Oxide (NO), prostaglandin E2(PGE2) And proinflammatory cytokines such as interleukin-1 β (IL-1 β), interleukin-6 (IL-6) and tumor necrosis factor- α (TNF- α) as well as other potentially neurotoxic compoundsSo as to achieve the purpose. Activated pro-inflammatory transcription factors can enhance the formation of cytokines and chemokines, leading to the generation and development of inflammatory responses. Conversely, the process will produce more ROS. This vicious circle exacerbates cellular damage by mediating mitochondrial dysfunction and ultimately has deleterious effects on neurons. This suggests that oxidative stress has an important role in the inflammatory response.
BV-2 is an immortalized mouse microglia that is widely used to study inflammation-related neurodegenerative diseases due to its sustained, stable, and excessive production of inflammatory factors upon activation. Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria, and can promote the release of inflammatory factors by mediating various cellular responses, and is commonly used to induce inflammation in vitro and in vivo.
Studies have shown that intraperitoneal injection of LPS in mice promotes the progression of the inflammatory response to create a model of learning and memory dysfunction. In addition, more and more studies report a role of oxidative stress in the development of inflammatory responses. The production of ROS increases the expression of inflammatory mediators in BV-2 microglia, thereby accelerating neuronal damage and death. It has been reported that excessive production of ROS in microglia can cause inflammation and cause damage to the nervous system. As a major source of ROS in cells, mitochondrial function changes are also involved.
Thus, to screen for anti-inflammatory peptides, LPS was used to stimulate BV-2 cells to create an inflammation model to determine whether the peptides could inhibit the production and development of an inflammatory response.
Disclosure of Invention
The primary object of the present invention is to provide an anti-inflammatory tetrapeptide.
Another object of the present invention is to provide a method for extracting and separating the tetrapeptide.
It is a further object of the present invention to provide the use of the above tetrapeptides.
The purpose of the invention is realized by the following technical scheme:
the amino acid sequence of the tetrapeptide is Gly-Val-Tyr (GVYY).
The tetrapeptides described above can be chemically synthesized using known techniques. For example, the tetrapeptide of the invention is synthesized by a solid phase synthesizer, dichloro resin is swelled and washed, Fmoc protecting group is removed, amino acid is added for condensation reaction, and the process of removing-protecting-condensing is repeated until all amino acid is connected.
In addition, the anti-inflammatory tetrapeptide can also be separated from walnut protein hydrolysate by separation means such as ultrafiltration and sephadex chromatography, and the like, and specifically comprises the following steps:
(1) extracting walnut protein from walnut meal by adopting an alkali-soluble acid-precipitation method, and specifically comprises the following steps of pulverizing walnut meal, adding water to dissolve the pulverized walnut meal (the material ratio is 1:12), adjusting the pH value of the solution to 8.0-9.0, stirring for 2-3h, centrifuging (8000-10000rpm, 10-15min, 4 ℃), adjusting the pH value of supernatant to 4.0-5.0, stirring for 2-3h, centrifuging again (8000-10000rpm, 10-15min, 4 ℃), dissolving precipitate, dialyzing and desalting for more than 2d (1: 5 of residue and water), and drying in vacuum to obtain walnut protein powder;
(2) mixing walnut protein powder with deionized water, performing enzymolysis at 50-60 deg.C for 12-16h with compound plant hydrolase and pancreatin, wherein the addition amount of the compound plant hydrolase and pancreatin is 0.5-1.0% (w/w, based on the mass of the walnut protein powder); then inactivating enzyme, centrifuging, and taking supernatant to obtain walnut protein hydrolysate; collecting the permeate by using a 3kDa molecular weight ultrafiltration membrane to obtain a low molecular weight component (MW <3kDa) of the walnut protein enzymolysis liquid;
the enzyme deactivation is carried out for 10-15min at the temperature of 95 ℃;
(3) loading the low molecular weight component of the walnut protein enzymolysis liquid onto a gel filtration chromatographic column Sephadex G-15, eluting with deionized water at the flow rate of 1.0-2.0mL/min, collecting and combining into a plurality of elution peak components, selecting a component with strong anti-inflammatory activity for mass spectrum detection, and confirming that the low molecular weight component contains the anti-inflammatory tetrapeptide;
the anti-inflammatory tetrapeptide GVYY can inhibit cell inflammatory reaction induced by LPS and relieve cell oxidative stress reaction induced by LPS, so that the anti-inflammatory tetrapeptide GVYY has a potential function of relieving brain neuroinflammation, and can be used in memory improving medicines and health care products.
The inhibition of the inflammatory response of cells induced by LPS comprises the reduction of the production of proinflammatory mediators and proinflammatory cytokines in cells induced by LPS;
the proinflammatory mediators include NO and PGE2；
The proinflammatory cytokines include IL-6, IL-1 β and TNF- α.
The relieving of LPS-induced cellular oxidative stress reaction refers to reducing the production amount of LPS-induced intracellular active oxygen and reversing the reduction of LPS-induced mitochondrial membrane potential.
The cell is a microglial cell;
the cell is a BV-2 cell.
Compared with the prior art, the invention has the following advantages and effects:
1. the anti-inflammatory tetrapeptide has clear structure, can be prepared by adopting a solid-phase chemical synthesis method, and can also be obtained by separating and purifying walnut protein zymolyte.
2. The anti-inflammatory tetrapeptide has good inhibitory activity on BV-2 cell inflammatory reaction stimulated by LPS, has potential efficacy of relieving cerebral neuroinflammation, can be used for preparing memory improving medicines or memory improving health care products, and can also be compounded with other health care products or food additives for use.
Drawings
FIG. 1 is the elution profile of Sephadex G-15 gel filtration chromatography.
FIG. 2 is a secondary mass spectrum of the tetrapeptide GVYY.
FIG. 3 is a graph of the effect of GVYY on LPS-induced ROS and MMP in BV-2 cells; wherein the content of the first and second substances,#representative vs. normal control, p<0.05；*Representation and model set comparison, p<0.05。
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
BV-2 cell culture
Adding Fetal Bovine Serum (FBS) to DMEM medium in an amount of 10% (v/v), and adding penicillin 100U/mL and 100. mu.LMixed double antibody composed of g/mL streptomycin. The cell culture chamber provides the environment for the growth of BV-2 cells, and the conditions in the chamber are set at 37 ℃ and contain 5% CO2. And timely carrying out liquid change and passage according to the cell growth condition.
Example 2
Synthesis of Gly-Val-Tyr-Tyr polypeptide by solid phase synthesis method
And swelling and washing the dichloro resin, removing the Fmoc protecting group, adding amino acid for condensation reaction, and repeating the processes of removing, protecting and condensing until all the amino acid is connected. Cutting the resin to obtain a polypeptide Gly-Val-Tyr-Tyr crude product, and purifying by using a reverse phase high performance liquid chromatography to obtain a polypeptide pure product (> 95%).
Example 3
Method for separating and purifying Gly-Val-Tyr-Tyr polypeptide
(1) Preparing walnut protein powder: extracting walnut protein from walnut dregs by an alkali dissolution and acid precipitation method. The specific process comprises the following steps of pulverizing walnut meal, adding water to dissolve the walnut meal (the material ratio is 1:12), adjusting the pH value of the solution to 8.5, stirring for 2h → centrifuging (8000rpm, 15min, 4 ℃), adjusting the pH value of the supernatant to 4.5, stirring for 2h → centrifuging (8000rpm, 15min, 4 ℃), dissolving the precipitate, dialyzing and desalting for 2d (slag and water is 1:5), drying → walnut protein powder.
(2) Preparing a walnut protein zymolyte: taking 1 part of walnut protein powder and 8 parts of deionized water according to the weight, uniformly mixing, and carrying out enzymolysis for 12 hours at 55 ℃ by using two proteases (composite plant hydrolase and pancreatin), wherein the enzyme adding amounts of the composite plant hydrolase and the pancreatin are 1.0 percent and 1.0 percent (w/w) respectively. Inactivating enzyme at 95 deg.C for 15min, centrifuging, and collecting supernatant to obtain walnut protein hydrolysate; and collecting the permeate by using a 3kDa molecular weight ultrafiltration membrane to obtain the low molecular weight components of the walnut protein enzymolysis liquid. Collecting low molecular weight component (MW <3kDa) of walnut protein hydrolysate, freeze drying, and storing at-20 deg.C for use.
(3) Separation and purification of the anti-inflammatory peptide: separating and purifying with gel filtration chromatographic column Sephadex G-15, eluting with deionized water at flow rate of 1.5mL/min, detecting wavelength of 220nm, eluting curve shown in figure 1, selecting components G3 and G6 with strong antiinflammatory activity, collecting, and freeze drying to obtain powder. Obtaining the target polypeptide. The secondary mass spectrogram of the target polypeptide is shown in figure 2, and the sequence is Gly-Val-Tyr-Tyr.
TABLE 1 different elution peak fractions for LPS-induced BV-2 intracellular NO and PGE2Influence of
#Representative vs. normal control, p<0.05，*Representation and model set comparison, p<0.05。
LPS-activated microglia can overproduce pro-inflammatory mediators (NO and PGE)2) Accelerating the inflammatory reaction process, ultimately leading to cell death. Thus, NO and PGE2Is used to evaluate the inhibition of LPS-stimulated BV-2 cell inflammation by the sample. Subjecting the walnut hydrolysate to Sephadex G-15 gel filtration chromatography to obtain a low molecular weight fraction (MW)<3kDa) into 6 main components (G1-G6). The results of the measurement are shown in Table 1, and NO and PGE in the cell culture medium after LPS treatment2The content is remarkably increased and is 101.73 +/-7.98 mu M and 205.58 +/-23.58 pg/mL respectively. However, G3 and G6 treatment significantly inhibited NO and PGE in BV-2 cells due to LPS2Generation of (p)<0.05). Therefore, the anti-inflammatory peptides of the specific amino acid sequences contained in G4 and G6 were analyzed by UPLC-ESI-Q-TOF-MS/MS to subsequently obtain the anti-inflammatory tetrapeptides GVYY of the present invention.
Example 4
Effect of tetrapeptide GVYY on LPS-induced levels of pro-inflammatory mediators and cytokines in BV-2 cells
The study used a co-culture cell model in which both LPS, an inducer, and GVYY, an anti-inflammatory tetrapeptide, were added to BV-2 cells to evaluate the protective effect of GVYY on LPS-induced inflammatory injury of BV-2 microglia. Cells were plated at 2X 105one/mL density was seeded in 12-well plates for 24 hours of adherent growth followed by the addition of GVYY and LPS (0.1. mu.g/mL) to treat BV-2 cells for 24 hours. The normal control group was treated with the same amount of DMEM medium without GYY and LPS. When the cell treatment is finished, collecting supernatant and determining oxygen in the supernatant according to the instruction of using the kitNitric Oxide (NO), prostaglandin E2(PGE2) Interleukin-1 β (IL-1 β), interleukin-6 (IL-6) and tumor necrosis factor- α (TNF- α).
TABLE 2 Effect of GVGY on LPS-induced NO and PGE2 production in BV-2 cells
# represents p <0.05 compared to the normal control group, and # represents p <0.05 compared to the model group.
LPS-activated microglia can overproduce pro-inflammatory mediators (NO and PGE)2) Accelerating the inflammatory reaction process, ultimately leading to cell death. Thus, NO and PGE2The amount of GVYY produced was used to evaluate the inhibition of LPS-stimulated BV-2 cell inflammation by GVYY. The results of the measurement are shown in Table 2, and NO and PGE in the cell culture medium after LPS treatment2The content is remarkably increased and is 84.41 +/-7.98 mu M and 235.46 +/-23.56 pg/mL respectively. However, GVGY treatment significantly inhibited LPS-induced NO and PGE in BV-2 cells2Generation of (p)<0.05) to 53.37 + -4.75 μ M and 107.83 + -10.58 pg/mL, respectively.
TABLE 3 Effect of GVYY on LPS-induced IL-1 β -6 and TNF- α production in BV-2 cells
# represents p <0.05 compared to the normal control group, and # represents p <0.05 compared to the model group.
In this study, the inhibition of LPS-stimulated BV-2 cell proinflammatory cytokine outbreaks by GVYY was assessed by measuring the levels of IL-6, IL-1 β and TNF- α production in this study. the results of the assay are shown in Table 3, a sudden increase in IL-6, IL-1 β and TNF- α levels was observed in LPS-treated BV-2 cells, however, TNF- α production in BV-2 cells protected by the addition of GVYY decreased significantly from 4000.53 + -357.75 pg/mL to 1300.18 + -159.51.
Example 5
Effect of tetrapeptide GVYY on LPS-induced active oxygen and mitochondrial membrane potential in BV-2 cells
Intracellular ROS levels were determined using fluorescence probe method (DCFH-DA). Cells were plated at 2X 105one/mL density was seeded in 96-well plates for 24 hours of adherent growth followed by addition of GVYY and LPS to treat BV-2 cells for 24 hours. After completion of the BV-2 cell sample treatment, the BV-2 cells were incubated with 10. mu.M DCFH-DA probe for 30 minutes in an incubator and then washed with PBS. In the presence of ROS, DCFH can be converted to Dichlorofluorescein (DCF) with strong green fluorescence. Fluorescence intensity was measured using a multi-template reader (Ex 488nm, Em 525 nm).
Intracellular Mitochondrial Membrane Potential (MMP) was measured using the JC-1 fluorescence probe method. JC-1 fluorescence probe method is a measurement method which is very sensitive to mitochondrial membrane potential change, and when cells have higher mitochondrial membrane potential, the fluorescence probe forms red fluorescence aggregates (J-aggregates) in a mitochondrial matrix. On the contrary, JC-1 exists in a monomer (J-monomer) form and has green fluorescence per se. Cells were plated at 2X 105one/mL density was seeded in 96-well plates for 24 hours of adherent growth followed by addition of GVYY and LPS to treat BV-2 cells for 24 hours. After BV-2 cells were cultured for sample treatment, the cells were incubated with JC-1 working solution at 37 ℃ for 30 min. Cell samples were then collected and washed twice with PBS, then resuspended and analyzed with a fluorescence spectrophotometer. The change in the ratio of the fluorescence intensities of Δ Ψ m monomers and aggregates was calculated.
There is increasing evidence that oxidative stress can promote the progression of the inflammatory response, and inhibition of intracellular oxidative stress will therefore contribute to anti-inflammation. As shown in FIG. 3, GVYY ameliorates ROS production by LPS stimulation. Simultaneously, GVYY also significantly reversed LPS-induced MMP loss in BV-2 cells. After BV-2 cells are treated by LPS, the content of intracellular ROS is increased by 2.24 times compared with the control group, and GVYY can reduce the generation of intracellular ROS to 137.10% + -5.24% of the control group (p < 0.05). Furthermore, LPS stimulation caused loss of MMPs in BV-2 cells (68.48% ± 3.34%), GVYY could significantly reverse the intracellular decrease in MMPs due to LPS stimulation (79.99% ± 4.00%).
In conclusion, the tetrapeptide GVYY of the invention can obviously inhibit inflammatory reaction in BV-2 cells caused by LPS induction, and simultaneously, the relief of oxidative stress by GVYY also contributes to the anti-inflammatory activity of GVYY.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. An anti-inflammatory tetrapeptide is characterized in that the amino acid sequence is Gly-Val-Tyr-Tyr.
2. The method for extracting and separating an anti-inflammatory tetrapeptide according to claim 1, comprising the steps of:
(1) pulverizing walnut dregs, adding water to dissolve, adjusting the pH value of the solution to 8.0-9.0, stirring for 2-3h, centrifuging, adjusting the pH value of supernatant to 4.0-5.0, stirring for 2-3h, centrifuging again, dissolving precipitate, dialyzing and desalting for more than 2d, and vacuum drying to obtain walnut protein powder;
the centrifugation is carried out for 10-15min at 8000-;
(2) mixing walnut protein powder with deionized water, performing enzymolysis at 50-60 deg.C for 12-16h with compound plant hydrolase and pancreatin, wherein the addition amount of the compound plant hydrolase and pancreatin is 0.5-1.0% (w/w, based on the mass of the walnut protein powder); then inactivating enzyme, centrifuging, and taking supernatant to obtain walnut protein hydrolysate; collecting the permeate by using a 3kDa molecular weight ultrafiltration membrane to obtain low molecular weight components of the walnut protein enzymolysis liquid;
(3) loading the low molecular weight component of the walnut protein enzymolysis liquid onto a gel filtration chromatographic column Sephadex G-15, eluting with deionized water at the flow rate of 1.0-2.0mL/min, collecting and combining into a plurality of elution peak components, selecting a component with strong anti-inflammatory activity for mass spectrum detection, and confirming that the low molecular weight component contains the anti-inflammatory tetrapeptide;
the anti-inflammatory activity refers to the effect on the amount of pro-inflammatory mediator production in cells induced by LPS.
3. Use of the anti-inflammatory tetrapeptide according to claim 1 for the preparation of a medicament and a health product for improving memory.
4. Use according to claim 3, characterized in that: the memory improvement refers to the alleviation of brain neuroinflammation.
5. Use according to claim 4, characterized in that: the relieving of the brain neuroinflammation refers to inhibiting cell inflammatory reaction induced by LPS and relieving cell oxidative stress reaction induced by LPS.
6. Use according to claim 5, characterized in that: the inhibition of the inflammatory response of cells induced by LPS comprises the reduction of the production of proinflammatory mediators and proinflammatory cytokines in cells induced by LPS.
7. Use according to claim 6, characterized in that:
the proinflammatory mediators include NO and PGE2；
The proinflammatory cytokines include IL-6, IL-1 β and TNF- α.
8. Use according to claim 5, characterized in that: the relieving of LPS-induced cellular oxidative stress reaction refers to reducing the production amount of LPS-induced intracellular active oxygen and reversing the reduction of LPS-induced mitochondrial membrane potential.
9. Use according to claim 5, characterized in that: the cells are microglia.
10. Use according to claim 5, characterized in that: the cell is a BV-2 cell.
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