CN111153963A - Anti-inflammatory pentapeptide, extraction and separation method thereof and application of anti-inflammatory pentapeptide in memory improvement - Google Patents
Anti-inflammatory pentapeptide, extraction and separation method thereof and application of anti-inflammatory pentapeptide in memory improvement Download PDFInfo
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Abstract
The invention discloses an anti-inflammatory pentapeptide, an extraction and separation method thereof and application thereof in improving memory, wherein the amino acid sequence of the anti-inflammatory pentapeptide is Ala-Pro-Thr-Leu-Trp, 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 pentapeptide is proved to contain. The anti-inflammatory pentapeptide 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 pentapeptide, an extraction and separation method thereof and application thereof in memory improvement.
Background
Microglia are resident macrophages of the Central Nervous System (CNS), playing a key role in innate immune regulation and neuronal homeostasis. However, in the brain neuroinflammatory state, activated microglia induce the release of pro-inflammatory 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), etc. they play a key role in neurodegenerative diseases IL-1 β has an important role in controlling innate and adaptive immune responses, it can enhance the expression of other proinflammatory cytokines such as TNF-a and IL-6. overproduction of IL-1 β also stimulates microglia to produce and release Reactive Oxygen Species (ROS), while ROS induces the expression of proinflammatory genes by activating the NF-. kappa.B pathway.
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 learning and memory dysfunction models can be created by intraperitoneal injection of LPS in mice, thereby promoting the progression of the inflammatory response. 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.
Disclosure of Invention
The primary object of the present invention is to provide an anti-inflammatory pentapeptide.
The invention also aims to provide the method for extracting and separating the anti-inflammatory pentapeptide.
It is a further object of the present invention to provide the use of the above anti-inflammatory pentapeptide.
The purpose of the invention is realized by the following technical scheme:
a pentapeptide has an amino acid sequence of Ala-Pro-Thr-Leu-Trp (APTLW).
The pentapeptide can be chemically synthesized by adopting the prior art. For example, the pentapeptide 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 pentapeptide 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 anti-inflammatory pentapeptide contains the anti-inflammatory pentapeptide.
The anti-inflammatory pentapeptide APTLW can inhibit cell inflammatory reaction induced by LPS and relieve cell oxidative stress reaction induced by LPS, so that the anti-inflammatory pentapeptide APTLW has a potential function of relieving brain neuroinflammation, and can be used in medicines and health care products for improving memory.
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 pentapeptide has a clear structure, can be prepared by adopting a solid-phase chemical synthesis method, and can also be obtained by separating and purifying a walnut protein zymolyte.
2. The anti-inflammatory pentapeptide 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.
Figure 2 is a secondary mass spectrum of the pentapeptide APTLW.
FIG. 3 is the effect of APTLW on LPS-induced intracellular 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
Fetal Bovine Serum (FBS) was added to DMEM medium in an amount of 10% (v/v), followed by the addition of a mixed double antibody consisting of 100U/mL penicillin and 100. mu.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
Solid phase synthesis method for synthesizing APTLW polypeptide
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. And cutting the resin to obtain a crude polypeptide Ala-Pro-Thr-Leu-Trp product, and purifying by using a reversed phase high performance liquid chromatography to obtain a pure polypeptide product (> 95%).
Example 3
Separation and purification method of APTLW 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 spectrum of the target polypeptide is shown in FIG. 2, and the sequence is APTLW.
TABLE 1 LPS-induced intracellular NO and PGE from each elution fraction2Influence of
# 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 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). Thus, the anti-inflammatory peptides of the specific amino acid sequences contained in G4 and G6 were analyzed by UPLC-ESI-Q-TOF-MS/MS, followed by the anti-inflammatory pentapeptide APTLW of the invention.
Example 4
Effect of pentapeptide APTLW on LPS-induced levels of pro-inflammatory mediators and pro-inflammatory cytokines in BV-2 cells
In the study, an inducer LPS and anti-inflammatory pentapeptide APTLW are added into BV-2 cells at the same time, namely a co-culture cell model is adopted to evaluate the protective effect of APTLW on LPS-induced BV-2 microglia cell inflammatory injury. Cells were plated at 2X 105one/mL density was seeded in 12-well plates for 24 hours of adherent growth followed by APTLW and LPS (0.1. mu.g/mL) to treat BV-2 cells for 24 hours. In the normal control group, APTLW and LPS were not added, and only the same amount of DMEM medium was added. When the cell treatment is finished, collecting supernatant and determining Nitric Oxide (NO) and prostaglandin E according to the instruction of the kit2(PGE2) Interleukin-1 β (IL-1 β), interleukin-6 (IL-6) and tumor necrosis factor- α (TNF- α).
TABLE 2 Effect of APTLW 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 PGE2Was used to evaluate the inhibitory effect of APTLW on LPS-stimulated BV-2 cell inflammation. 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, APTLW treatment significantly inhibited NO and PGE in BV-2 cells due to LPS2Generation of (p)<0.05) to 55.77 ± 3.95 μ M and 114.68 ± 9.76pg/mL, respectively.
TABLE 3 Effect of APTLW 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 APTLW was assessed by measuring the levels of IL-6, IL-1 β and TNF- α. 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 addition of APTLW decreased significantly from 4000.53 + -357.75 pg/mL to 900.93 + -178.83. at the same time, IL-1 β and IL-6 production in model cells was significantly reduced using APTLW compared to LPS group (p < 0.05).
Example 5
Effect of pentapeptide APTLW on LPS-induced BV-2 intracellular reactive oxygen species and mitochondrial membrane potential
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 APTLW and LPS treatment of BV-2 cells for 24 hours. After the BV-2 cell sample is treated, the BV-2 cells are treatedThe probe was incubated with 10. mu.M DCFH-DA 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 APTLW and LPS treatment of 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. The results of the assay are shown in FIG. 3, and APTLW relieves ROS production by LPS stimulation. At the same time, APTLW 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 APTLW can reduce the generation of intracellular ROS to 143.25% + -4.74% of the control group (p < 0.05). Furthermore, LPS stimulation caused loss of MMPs in BV-2 cells (68.48% ± 3.34%), and APTLW could significantly reverse the intracellular decrease in MMPs due to LPS stimulation (93.38% ± 2.35%).
In conclusion, the pentapeptide APTLW can obviously inhibit the inflammatory response in BV-2 cells caused by LPS induction, and the relief of oxidative stress by the APTLW also contributes to the anti-inflammatory activity of the APTLW.
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 pentapeptide characterized by the amino acid sequence Ala-Pro-Thr-Leu-Trp.
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 pentapeptide;
the anti-inflammatory activity refers to the effect on the amount of pro-inflammatory mediator production in cells induced by LPS.
3. The use of the anti-inflammatory pentapeptide of claim 1 in the manufacture of a medicament or 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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