CN116554271B

CN116554271B - Ginseng peptide

Info

Publication number: CN116554271B
Application number: CN202310814858.0A
Authority: CN
Inventors: 郭轶; 徐力; 朱玉花
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2023-07-05
Filing date: 2023-07-05
Publication date: 2023-09-01
Anticipated expiration: 2043-07-05
Also published as: CN116554271A

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a ginseng peptide, wherein the amino acid sequence of the ginseng peptide is shown in any one of the following (1) - (4): the amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 1; (2) the amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 2; (3) the amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 3; the amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 4. The ginseng peptide has good antioxidant capacity, and can relieve Abeta _1‑42 Oxidative damage of induced nerve cells and inhibition of aβ _1‑42 Is effective in delaying nematode paralysis and improving AD symptoms.

Description

Ginseng peptide

Technical Field

The invention relates to the technical field of medicines, in particular to ginseng peptide.

Background

The body produces various free radicals and Reactive Oxygen Species (ROS) during normal metabolism, which are involved in physiological processes such as gene transcription, signal transduction, and apoptosis. These small amounts of ROS do not cause damage to the body, but rather coordinate with the antioxidant system in the body, thereby maintaining a balance between pro-oxidants and antioxidants. Among them, antioxidant enzymes (SOD, CAT and glutathione peroxidase) are the most important antioxidant systems of the body, which cooperate with some non-enzymatic antioxidant factors (e.g. melatonin) to combat ROS. However, once the level of ROS exceeds the antioxidant capacity of the cells, oxidative stress occurs, resulting in the occurrence of various diseases.

Since neurons need to consume large amounts of oxygen and the antioxidant levels are relatively low, they are more susceptible to direct damage from oxidative stress. Oxidative stress can promote aβ aggregation and tau protein phosphorylation, forming amyloid plaques and neurofibrillary tangles, leading to neuronal cell death and further oxidative stress. Thus, using antioxidants, maintaining redox homeostasis within neuronal cells is also a means of treating disease. Because of the side effects of synthetic antioxidants, the search for natural antioxidants has become a recent research focus. Among them, bioactive peptides have attracted attention because of their high biosafety, low immunogenicity, easy synthesis, and antioxidant function.

The ginseng peptide is a peptide segment with biological function obtained from ginseng or ginseng protein decomposition product. During the hydrolysis of ginseng protein to form peptide, peptide bond is degraded, which increases dissociable group, thereby increasing the hydrophilicity and the amount of net charge; degradation of peptide bonds also alters their molecular structure, exposing hydrophobic groups within the protein. In addition, the number of amino acids and the relative molecular mass of the peptide are changed, and the change improves the solubility, the stability, the emulsifying property and the rheology of the ginseng peptide, and in addition, compared with the amino acid, the ginseng peptide has lower osmotic pressure and high biological safety, and can be used as an antioxidant and a treatment or auxiliary treatment means to delay the development of diseases.

Disclosure of Invention

The invention aims to provide a ginseng peptide, wherein four ginseng peptide monomers have good antioxidant capacity and can relieve Abeta _1-42 Oxidative damage of induced nerve cells and inhibition of aβ _1-42 Is effective in delaying nematode paralysis and improving AD symptoms.

In order to achieve the above object, the present invention provides a ginseng peptide, the amino acid sequence of which is as shown in any one of the following (1) to (4):

(1) The amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 1;

(2) The amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 2;

(3) The amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 3;

(4) The amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 4.

The invention prepares the ginseng peptide mixture by utilizing an enzymolysis mode, and separates and identifies four ginseng peptide monomers by a separation and purification and high resolution liquid chromatography-mass spectrometry technology. The amino acid sequence of the ginseng peptide is ITGYAP, LTGYAP, ITGYPA, LTGYPA, and the four ginseng peptide monomers have good antioxidant effect.

The invention passes through Abeta _1-42 Oligomer stimulation to build nervesCell oxidative damage model, detection of cell viability using thiazole blue (MTT), fluorescence microscopy and flow cytometry to detect content of reactive oxygen species (Reactive oxygen species, ROS) in nerve cells, fluorescence microscopy to detect apoptosis, demonstrating that ginsenoside monomer ITGYAP, LTGYAP, ITGYPA, LTGYPA can inhibit Abeta _1-42 Oligomer-induced neuronal cell damage.

The invention uses the transgenic caenorhabditis elegans CL4176 as an animal model to detect the panaxadiol monomer pair A beta _1-42 Induced nematode paralysis, longevity, and effects of aβ in vivo.

The invention uses molecular docking simulation technology to prove ITGYAP, LTGYAP, ITGYPA, LTGYPA and Abeta _1-42 Interaction between them. Specifically, a method for preparing a small molecular peptide by hydrolyzing a large molecular weight protein in a system by means of enzymolysis has been widely used, a ginseng peptide mixture is prepared by using alkaline protease, and then a ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA, LTGYPA is prepared by sequentially performing ion exchange chromatography, gel filtration chromatography and a high resolution liquid chromatography-mass spectrometry technology. Utilization of Abeta _1-42 The oligomer-stimulated cells construct an AD cell model. The ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA, LTGYPA was found to be non-toxic to cells when acting alone on the cells. The ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA, LTGYPA pretreated cells 4 h can obviously inhibit Abeta _1-42 Oligomer-induced Ca ²⁺ Internal flow, maintaining stable mitochondrial membrane potential, improving mitochondrial function injury, reducing intracellular ROS level, and inhibiting apoptosis.

Transgenic caenorhabditis elegans CL4176 is used as an AD animal model, the culture temperature of the nematode is increased to 23 ℃, and Abeta is induced _1-42 Expression of the gene causes the nematode to manifest symptoms of AD. Through administration of the ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA, LTGYPA, the four ginseng peptide monomers are found to delay paralysis of nematodes, reduce paralysis rate, prolong nematode life, reduce ROS level in the nematode, improve egg laying rate of the nematodes and reduce deposition quantity of Abeta plaque in the nematode.

Therefore, the invention adopts the ginseng peptide, ginseng peptide ITGYAP,LTGYAP, ITGYPA, LTGYPA A beta inhibition is achieved _1-42 Is effective in relieving Abeta _1-42 Is a neurotoxicity of (a).

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

FIG. 1 is a schematic diagram of separation and purification of ginseng peptide; (A) DEAE Sephadex A-25 ion exchange chromatography of GFREH. (B) Detecting the scavenging capacity of the FractionI, II and III on the hydroxy free radicals, and taking VC as a positive control group; (C) Detecting the scavenging capacity of the FractionI, II and III to superoxide anions, and taking VC as a positive control group; (D) Sephadex G-10 gel filtration chromatography for Fraction II; (E) Detecting the scavenging capacity of the Fraction 1 and Fraction 2 on the hydroxyl free radicals, and taking VC as a positive control group; (F) Detecting the scavenging capacity of superoxide anions by using Fraction 1 and Fraction 2, and taking VC as a positive control group;

FIG. 2 is a schematic diagram of structural identification of ginseng peptide monomers; (A) The total ion peak diagram of the Fraction II with highest antioxidant activity is detected by an HRLC-MS system; (B) primary mass spectrum of the ginseng peptide monomer; (C) a secondary mass spectrum of the ginseng peptide monomer; (D) the amino acid sequence of the ginseng peptide monomer;

FIG. 3 is an in vitro antioxidation assay of a ginseng peptide monomer; (A) The clearance rate of the four ginseng peptide monomers to hydroxyl radicals; (B) The clearance rate of the four ginseng peptide monomers to superoxide anions;

FIG. 4 is a graph of monomer pair A.beta.of ginseng peptide _1-42 A graph of in vitro aggregation; (A) No ginseng peptide monomer group is added, along with Abeta _1-42 An increase in concentration, a change in fluorescence intensity; (B) Following addition of ITGYAP, Aβ _1-42 An increase in concentration, a change in fluorescence intensity; (C) Following addition of LTGYAP, Aβ _1-42 An increase in concentration, a change in fluorescence intensity; (D) Following addition of ITGYPA, Aβ _1-42 An increase in concentration, a change in fluorescence intensity; (E) Following addition of LTGYPA, Aβ _1-42 An increase in concentration, a change in fluorescence intensity;

FIG. 5 is a graph of monomer pair Aβ of ginseng peptide _1-42 Oligomer-induced SH-SYAction pattern of 5Y lesions; (A) With Abeta _1-42 An increase in the concentration of drug administered, a change in cell viability; (B) cytotoxicity of the four ginseng peptide monomers when administered alone; (C) Aβ _1-42 At a concentration of 5 μm, the cell viability of ITGYAP ginseng peptide monomer at different dosing concentrations varied; (D) Aβ _1-42 At 5 μm, the cell viability of LTGYAP ginseng peptide monomer at different dosing concentrations varied; (E) Aβ _1-42 At a concentration of 5 μm, the cell viability of ITGYPA ginseng peptide monomer at different dosing concentrations varied; (F) Aβ _1-42 At a concentration of 5 μm, the cell viability of the LTGYPA panaxadiol monomer was varied at different dosing concentrations;

FIG. 6 is a graph of monomer pair Aβ of ginseng peptide _1-42 Oligomer-induced cell Ca ²⁺ A map of the internal flow; (a) green fluorescence intensity in cells for different dosing states; (B) Quantification bar graph of green fluorescence intensity in cells after administration of ITGYAP ginseng peptide monomer; (C) Quantification bar graph of green fluorescence intensity in cells after administration of LTGYAP ginseng peptide monomer; (D) Quantification bar graph of green fluorescence intensity in cells after administration of ITGYPA ginseng peptide monomer; (E) Quantification bar graph of green fluorescence intensity in cells after administration of LTGYPA ginseng peptide monomer;

FIG. 7 is a graph of monomer pair A.beta.of ginseng peptide _1-42 An influence diagram of oligomer-induced mitochondrial membrane potential of cells; (A) Red fluorescence and green fluorescence intensity change patterns in cells after administration of ITGYAP ginseng peptide monomers at different concentrations; (B) Red fluorescence and green fluorescence intensity change patterns in cells after administration of LTGYAP ginseng peptide monomers; (C) Red fluorescence and green fluorescence intensity change patterns in cells after administration of ITGYPA ginseng peptide monomer; (D) Red fluorescence and green fluorescence intensity change patterns in cells after administration of LTGYPA ginseng peptide monomers; (E) Quantitative analysis bar graphs of red fluorescence and green fluorescence intensity changes in cells after administration of ITGYAP ginseng peptide monomers at different concentrations; (F) Quantitative analysis of red fluorescence and green fluorescence intensity changes in cells after administration of LTGYAP ginseng peptide monomers; (G) Quantitative analysis of red fluorescence and green fluorescence intensity changes in cells after administration of ITGYPA ginseng peptide monomer; (H) Fine after administration of LTGYPA ginseng peptide monomerQuantitative analysis bar graphs of intracellular red fluorescence and green fluorescence intensity changes;

FIG. 8 is a graph of monomer pair A.beta.of ginseng peptide _1-42 A graph of the effect of oligomer-induced intracellular ROS levels; (A) Analyzing green fluorescence intensity in cells after different administration treatments; (B) Analysis of ROS content in SH-SY5Y cells using a flow cytometer; (C) The quantitative analysis of the intracellular ROS content by ITGYAP ginseng peptide monomers with different administration concentrations is carried out; (D) The quantitative analysis of the intracellular ROS content by the LTGYAP ginseng peptide monomers with different administration concentrations is carried out; (E) The quantitative analysis of the intracellular ROS content by ITGYPA ginseng peptide monomers with different administration concentrations is carried out; (F) The quantitative analysis of the intracellular ROS content by the LTGYPA ginseng peptide monomers with different administration concentrations is carried out;

FIG. 9 is a graph of monomer pair Aβ of ginseng peptide _1-42 A graph of the effect of oligomer-induced apoptosis; (a) analysis of fluorescence intensity of cells after different dosing treatments; (B) Performing fluorescence quantitative analysis on ITGYAP ginseng peptide monomer treated cells with different administration concentrations; (C) Fluorescent quantitative analysis of LTGYAP ginseng peptide monomer treated cells with different administration concentrations; (D) Performing fluorescence quantitative analysis on ITGYPA ginseng peptide monomer treated cells with different administration concentrations; (E) Fluorescent quantitative analysis of LTGYPA ginseng peptide monomer treated cells with different administration concentrations;

FIG. 10 is a graph showing the effect of different ginseng peptide monomers on nematode paralysis;

FIG. 11 is a graph showing the effect of different ginseng peptide monomers on ROS levels in a nematode;

FIG. 12 is a graph showing the effect of different ginseng peptide monomers on nematode longevity;

FIG. 13 is a graph showing the effect of ginseng peptide monomer on egg laying rate of insects; (A) A histogram of daily egg number change of nematodes after different dosing treatments; (B) A histogram of total egg yield change of nematodes after different dosing treatments;

FIG. 14 is a graph showing the effect of ginseng peptide monomer on Abeta deposition in nematodes; (A) Plaque changes in aβ deposition in nematodes treated with different doses; (B) Fluorescent intensity quantitative bar graphs for different ginseng peptide monomer treatments;

FIG. 15 is a molecular docking simulation of ginseng peptide monomer and Abeta _1-42 Mutually of (2) acting; (A) ITGYAP-A beta _1-42 Molecular docking schematic of (2); (B) ITGYAP-A beta _1-42 A molecular docking space structure diagram of (2); (C) LTGYAP-A beta _1-42 Molecular docking schematic of (2); (D) LTGYAP-A beta _1-42 A molecular docking space structure diagram of (2); (E) ITGYPA-Abeta _1-42 Molecular docking schematic of (2); (F) ITGYPA-Abeta _1-42 A molecular docking space structure diagram of (2); (G) LTGYPA-Abeta _1-42 Molecular docking schematic of (2); (H) LTGYPA-Abeta _1-42 Molecular docking space structure diagram of (2).

Detailed Description

Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

Example one preparation of Ginseng peptide monomer

(1) Crushing and enzymolysis

Pulverizing Ginseng radix fibrous root into powder with pulverizer, and sieving with 30 mesh sieve. A certain amount of ginseng powder was weighed into a conical flask, distilled water was added at a ratio of 1:10 (m/v), and pH was adjusted to 8.0 with NaOH of 1. 1M. Then placing the sample into a water bath kettle with the temperature of 50 ℃ for preheating for 10 min, adding alkaline protease according to the proportion of 1:50 (v/v), and then placing the conical flask into an oscillating water bath kettle with the temperature of 50 ℃ for enzymolysis reaction. In the reaction process, the pH value of the enzymolysis liquid can be reduced along with the prolongation of the enzymolysis time, so that a certain amount of NaOH solution of 1M is added into the enzymolysis liquid every 20 min, the pH value of the solution is kept at 8.0 until the pH value is not changed, and the enzymolysis reaction is finished. Heating the conical flask in a water bath at 95 ℃ for 10 min to inactivate enzymes, cooling enzymolysis liquid, adjusting the pH to 7.0 by using HCl of 1M, centrifuging at 7000 rpm for 15 min, collecting supernatant, sequentially filtering with filter paper, 1 mu m, 0.45 mu m and 0.22 mu m filter membrane, and then freeze-drying the filtrate to obtain a ginseng peptide mixture (GFREH).

(2) Chromatography

Ion exchange chromatography is a method of separating substances to be separated according to the difference in charge at a certain pH. Separation conditions: the ion exchange column had a diameter of 2.0. 2.0 cm and a length of 20. 20 cm, the column was DEAE Sephadex A-25, and the eluate was 10 mM Tris-HCl buffer (pH=9.0). And (3) dissolving GFREH with ultrapure water, eluting the balance column material with buffer solution until the baseline is stable, loading the sample, eluting with 0-5M NaCl solution at the eluting speed of 1 mL/min and the protein detection wavelength of 220 nm, collecting the eluent when the eluting peak appears, freeze-drying the eluent, and detecting the in-vitro antioxidant activity. As shown in FIG. 1A-C, GFREH was subjected to ion exchange chromatography to obtain three components, wherein the antioxidant activity of Fraction II was the highest, and Fraction II was collected for the next experiment.

And separating the Fraction II by adopting a gel filtration chromatography method. Separation conditions: the gel chromatographic column has a diameter of 2.0 cm, a length of 100 cm, a chromatographic column of Sephadex G-10, high purity water as eluent, an elution flow rate of 0.5 mL/min and a protein detection wavelength of 220 nm. Preparing a group of Fraction II with highest antioxidant activity into an aqueous solution for loading, eluting with high-purity water, collecting eluent when an elution peak appears, freeze-drying the eluent, and detecting the in-vitro antioxidant activity. As shown in D-F in FIG. 1, fraction II was subjected to gel filtration chromatography to obtain two components, wherein Fraction 1 had the highest antioxidant activity, and Fraction 1 was collected for the next experiment.

And (3) carrying out structural identification on the component with highest antioxidant activity after ion chromatography by using a high-resolution liquid chromatography mass spectrometer (HRLC-MS) Agilent1290-Bruker micrOTOF QII. The column was an Agilent reverse C18 column (2.7 μm,2.1×150 mm), the eluent was ultrapure water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, the elution gradient was from 15% acetonitrile to 90% acetonitrile, the elution flow rate was 0.7 mL/min, the elution time was 6 min, an electrospray ion source (ESI) was used, the positive ion detection mode, the ion source temperature was 200 ℃, the collision energy was 10 ev, and the scan range was 50-1500 m/z. FIG. 2A-D shows the total ion peak diagram of the flow of Fraction 1 through the liquid phase into the mass spectrum; a primary mass spectrum of the ginseng peptide monomer; a secondary mass spectrum of the ginseng peptide monomer; amino acid sequence diagram of ginseng peptide monomer.

The ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA and LTGYPA have the scavenging ability for hydroxyl radicals and superoxide anion radicals. In fig. 3, a shows that the clearance rate of the four ginseng peptide monomers to hydroxyl radicals is about 80%, and B shows that the clearance rate of the four ginseng peptide monomers to superoxide anions is about 95%.

Example two detection of Abeta by thioflavin T staining _1-42 In vitro aggregation of (C)

Taking proper amount of Abeta _1-42 Is removed by nitrogen, and Abeta is treated with PBS solution _1-42 The peptide was completely dissolved and formulated as 5, 10 and 15 μm solutions, incubated 96 h at 37 ℃ before the aβ was incubated _1-42 The solution was mixed with thioflavin T (final concentration 50 μm) and fluorescence measured (excitation wavelength 440 nm, emission wavelength 480 nm) to determine the optimum aβ _1-42 Concentration of the solution. Then Aβ is added _1-42 Dissolving with Ginseng radix peptide monomer at ratio of 15:0, 15:1 and 15:5 (μM) in PBS buffer, incubating in 37deg.C constant temperature shaker for 96 h, adding thioflavin T solution, and performing fluorescence measurement.

As shown in fig. 4 a, as aβ compared with the control group _1-42 Concentration increase, fluorescence intensity increase, when Abeta _1-42 When the concentration of (2) is 15 mu M, the difference between the fluorescence intensity and the control group is obvious, so that 15 mu M is selected as the subsequent experiment concentration. As shown in FIGS. 4B-E, A.beta.without the addition of the ginseng peptide monomer compared to CTRL group _1-42 The fluorescence intensity of the group is obviously enhanced, while the addition of the ginseng peptide monomer obviously reduces the fluorescence intensity, and the more obvious the fluorescence intensity reduction effect is (B-E in figure 4) along with the increase of the concentration of the ginseng peptide monomer, which proves that the ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA, LTGYPA can obviously inhibit Abeta in vitro _1-42 A kind of electronic device aggregation.

Examples Triginseng peptide monomer ITGYAP, LTGYAP, ITGYPA and LTGYPA vs. Abeta _1-42 Influence of induced neuronal cell injury

Cells were plated at 5X 10 per well ³ Inoculating into 96-well plate containing complete culture medium, culturing in carbon dioxide incubator at 37deg.C for 24 h, discarding culture medium, and respectively adding Abeta with different concentrations (1, 5, 10, 15 μm) _1-42 The oligomer-treated cells 24 h were then added to 10. Mu.L (5 mg/mL) of MTT solution per well and incubated in a 37℃cell incubator at 4 h. The supernatant was discarded, 150 μl of dimethyl sulfoxide (DMSO) solution was added to each well, absorbance was measured at 492 nm using a microplate reader, and cell viability was calculated. Cells were treated with different concentrations of the ginseng peptide monomers (0.1, 1, 10 and 100 μm) according to the above method, respectively, and toxicity of the ginseng peptide monomers to cells was examined. Cells 4 h were then pretreated with different concentrations of the peptide monomers according to the method described above, followed by 5. Mu.M of Abeta _1-42 Cell viability was determined by oligomer treatment of cells 24 h.

Cell viability calculation:

；

as shown in fig. 5 a, with aβ _1-42 Increased drug administration concentration, SH-SY5Y cells are subjected to Abeta _1-42 The damaging effects of the oligomers, the cell viability decreased in a dose-dependent manner when Aβ _1-42 At drug administration concentrations of 1, 5, 10, 15 μm, cell viability was 79.30% ± 2.57, 50.54% ± 3.00, 37.02% ± 3.18 and 30.89% ± 3.42, respectively (n=5). When the administration concentration is 5 mu M, the cell survival rate is close to the IC50 value, so that Abeta in the subsequent cell experiment _1-42 The optimal modeling concentration of (2) is 5 mu M. Shown in fig. 5B, the ginseng peptide monomer was cytotoxic when administered alone. Administration of Abeta alone _1-42 The cell viability of the oligomer was only 50% of that of the CTRL group, while the inhibition by Abeta was significant after pretreatment with ITGYAP, LTGYAP, ITGYPA and LTGYPA at different concentrations of 4 h _1-42 Apoptosis induced by oligomer increases cell survival rate, and cell survival rate increases with increasing concentration of ginseng peptide monomer. When the administration concentration of the ginseng peptide monomer was 40. Mu.M, the cell viability was 82.05.+ -. 2.14, 84.62.+ -. 1.96, 86.26.+ -. 2.53 and 83.89.+ -. 1.61, respectively (C-F in FIG. 5). The results indicate that the ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA and LTGYPA pair Abeta _1-42 Oligomer-induced SH-SY5Y cell damage has neuroprotective effect.

Example four Ginseng radixPeptide monomer pair nerve cell Ca ²⁺ Influence of internal flows

SH-SY5Y cells were grown at 3X 10 ⁴ Inoculating 24/well density into 24-well plate, culturing 24 h, discarding culture medium, pretreating cells with Ginseng radix peptide monomer of different concentrations (10, 20, 40 μm) 4 h, discarding Ginseng radix peptide solution, adding 5μm Abeta _1-42 Culturing is continued for 24 h, the culture medium is discarded, fluo-4 AM probe with a final concentration of 5 mu M is added to each well, the culture is incubated for 30 min at 37 ℃ in a dark place, finally the excess probe is removed by washing 3 times with PBS, photographing is immediately carried out by a fluorescence microscope, and quantitative analysis is carried out by using imageJ.

As shown in fig. 6 a, CTRL group cells were not dosed with aβ _1-42 The oligomer has good cell state and weaker green fluorescence. At the administration of Abeta _1-42 In the oligo group, cells showed strong fluorescence, indicating that cells were subjected to Aβ _1-42 Induced damage of oligomer, destruction of cell membrane, and Ca ²⁺ Influx, intracellular Ca ²⁺ Overload. After the cells are pretreated by the ginseng peptide monomers ITGYAP, LTGYAP, ITGYPA and LTGYPA with different concentrations (10, 20 and 40 mu M) for 4 h, the green fluorescence intensity in the cells is weakened to different degrees, which shows that the ginseng peptide monomers ITGYAP, LTGYAP, ITGYPA and the intracellular Ca after ITGYAP treatment ²⁺ Is remarkably reduced in content, and the ginseng peptide monomer can inhibit the action of A beta _1-42 Oligomer-induced cell damage and intracellular Ca ²⁺ Overload. We then quantified the green fluorescence intensity in SH-SY5Y cells using imageJ software (B-E in FIG. 6). The above results demonstrate that the ginseng peptide monomer can reverse the reaction of A beta _1-42 Oligomer-induced neuronal cell injury and cellular Ca ²⁺ And (3) internal flow.

Example influence of pentapeptide monomer on mitochondrial membrane potential of nerve cells

SH-SY5Y cells were grown at 3X 10 ⁴ Inoculating 24/well density into 24-well plate, culturing 24 h, discarding culture medium, pretreating cells with Ginseng radix peptide monomer of different concentrations (10, 20, 40 μm) 4 h, discarding Ginseng radix peptide solution, adding 5μm Abeta _1-42 Continuing to culture 24 h according to Biyundian reagentThe cassette assay showed that JC-1 probe was added to each well at a final concentration of 10 μg and stained by incubation at 37 ℃.20 After min, wash 3 times with PBS, immediately photograph with fluorescence microscope, and quantitate with ImageJ.

As shown in FIGS. 7A-D, CTRL group cells were treated with Abeta _1-42 The oligomer is treated, the cell state is good, the mitochondrial membrane potential is normal, JC-1 exists in a polymer form, and stronger red fluorescence is formed. At the administration of Abeta _1-42 In the oligomer group, the red fluorescence was reduced and the green fluorescence was significantly increased, indicating that JC-1 exists in monomeric form, the mitochondrial membrane potential was reduced when the cells were subjected to Abeta _1-42 When the oligomer is damaged by induction, mitochondria are destroyed, and mitochondrial membrane potential is lowered. After the cells are pretreated by the ginseng peptide monomers ITGYAP, LTGYAP, ITGYPA and LTGYPA with different concentrations (10, 20 and 40 mu M) for 4 h, the red fluorescence intensity in the cells is enhanced, the green fluorescence intensity is obviously weakened, which indicates that the mitochondrial membrane potential of the cells is obviously recovered after the ginseng peptide monomers ITGYAP, LTGYAP, ITGYPA and LTGYPA are pretreated for 4 h, and the ginseng peptide monomers can inhibit the fluorescent light emitted by Abeta _1-42 Oligomer-induced cell damage and mitochondrial membrane potential decline. The red and green fluorescence intensities within SH-SY5Y cells were then quantitatively analyzed using imageJ software (E-H in FIG. 7). The above results demonstrate that the ginseng peptide monomer can effectively inhibit Abeta _1-42 Oligomer-induced decrease in mitochondrial membrane potential in SH-SY5Y cells.

Example Effect of hexaginseng peptide monomer on ROS level in nerve cells

SH-SY5Y cells were grown at 3X 10 ⁴ Inoculating 24/well density into 24-well plate, culturing 24 h, discarding culture medium, pretreating cells with Ginseng radix peptide monomer of different concentrations (10, 20, 40 μm) 4 h, discarding Ginseng radix peptide solution, adding 5μm Abeta _1-42 Culturing for 24 h, adding DCFH-DA probe with final concentration of 10 mu M into each hole according to detection instruction of ROS kit, incubating and dyeing for 15 min at 37 ℃, washing for 3 times by PBS, immediately photographing by a fluorescence microscope, and collecting cells for fluorescence quantification by a flow cytometer.

As shown in fig. 8 a, CTRL group cells were not treated with the drug, and cellsThere was no strong green fluorescence, indicating normal cell status and relatively low intracellular ROS content. Administration of Abeta alone _1-42 After the oligomer, the cells exhibited strong green fluorescence, indicating that DCFH-DA entering the cells was oxidized by intracellular ROS to DCF with green fluorescence, and that A.beta.was administered alone compared to CTRL group _1-42 The intracellular ROS content of the oligomer is significantly increased. After the cells are pretreated by the ginseng peptide monomers ITGYAP, LTGYAP, ITGYPA and LTGYPA with different concentrations (10, 20 and 40 mu M) for 4 h, the green fluorescence intensity in the cells is obviously weakened, which indicates that the ginseng peptide monomers can obviously inhibit the fluorescent light from Abeta _1-42 Excess accumulation of ROS in neural cells induced by oligomers. Then, the ROS content in SH-SY5Y cells was analyzed and quantified using a flow cytometer (B-F in FIG. 8, results consistent with fluorescence microscopy images. The above results indicate that the panaxadiol monomer can effectively inhibit Aβ _1-42 Oligomer-induced excessive accumulation of ROS within SH-SY5Y cells.

Example heptapanaxpeptide monomer pair A beta _1-42 Influence of induced neuronal cell injury

SH-SY5Y cells were grown at 2X 10 ⁵ Density of individual/wells was inoculated into six-well plates and cultured in a carbon dioxide incubator at 37 ℃. After 24 and h, pretreatment is carried out on the ginseng peptide monomer with different concentrations (10, 20 and 40 mu M) for 4 to h, and then Abeta with the concentration of 5 mu M is added _1-42 Treatment 24 h. To each well, hoechst 33342 dye was added, incubated in an incubator at 37℃for 20 min, then washed 3 times with PBS, and the nuclear morphology was observed by fluorescence under fluorescence microscopy and quantified with ImageJ.

As shown in fig. 9 a, CTRL group cells did not use aβ _1-42 The oligomer treatment shows uniform blue color of cells, which indicates that the cell state is good; in use of Abeta _1-42 In the oligomer-treated group, intense particle-block fluorescence appeared in the cells, indicating apoptosis. And particle block fluorescence in the cells is obviously reduced after the cells are pretreated by the ginseng peptide monomers ITGYAP, LTGYAP, ITGYPA and the LTGYPA with different concentrations (10, 20 and 40 mu M) for 4 h. The results of the image J fluorescence quantification (B-E in FIG. 9) showed that Abeta compared to CTRL group _1-42 The fluorescence intensity of the oligomer is obviously enhanced, and the fluorescence intensity can be obviously reduced when ITGYAP, LTGYAP, ITGYPA and LTGYPA with different concentrations are administered. These results indicate that the ginseng peptide monomer can reduce Abeta _1-42 The oligomer stimulates SH-SY5Y nerve cells, inhibits apoptosis caused by the stimulation, and has a protective effect on nerve cells.

Example influence of the monomer of the octagen peptide on the paralysis rate of the insects

Transgenic nematodes CL4176 in L3 phase after synchronization are transferred to NGM culture mediums of a control group and an administration group respectively, and the paralyzed numbers of the nematodes are counted after 36 h culture at 23 ℃. The nematodes can perform rolling movements and are regarded as normal; when the nematode body is touched, only the head can swing and can not do rolling motion any more and can not move, and paralysis is considered; nematode body stiffness, touch without stress is regarded as death.

As shown in fig. 10, the rate of paralysis of nematodes was 50% or more on both days 4 and 5 in the untreated control group. In contrast, aβ _1-42 The induced paralysis rate is reduced along with the increase of the concentration of the peptide monomers, the highest concentration of the ginseng peptide monomers is 200 mu M, and the paralysis rate of nematodes is obviously reduced. Compared to the control group, 200 μm ITGYAP, LTGYAP, ITGYPA and LTGYPA treated CL4176 nematodes reduced physical paralysis rates by 23.67% and 26%, 30% and 33.33%, 15% and 18.55%, 30% and 30% on days 4 and 5, respectively. These results indicate that the ginseng peptide monomer can reduce the Abeta in the CL4176 nematode body _1-42 Toxicity is induced, thereby reducing nematode paralysis.

Example influence of the monomer of the nonapanaxadiol on ROS levels in the body of insects

The synchronized L3 transgenic nematodes CL4176 are divided into a control group and an administration group, after 36 h are cultured at 16 ℃, the nematodes of each group are transferred to 23 ℃ and then are cultured for 36 h, then the nematodes are collected, the E.coli on the surfaces of the nematodes are removed by washing 3 times with M9 buffer, the nematodes are transferred to a black 96-well plate containing M9 buffer, DCFH-DA dye is added to make the final concentration of the nematodes 50 mu M, and after 40 min, the fluorescence intensity of the nematodes is detected by a fluorescent enzyme-labeled instrument.

As shown in FIG. 11, with the unused ginseng peptide monomerTreated expression of Abeta _1-42 The treatment with 200 μm ITGYAP, LTGYAP, ITGYPA and LTGYPA reduced ROS content in nematodes by 21.32%, 20.7%, 19.22% and 20.47%, respectively, compared to control nematodes of the group. These results indicate that the ginseng peptide monomer can reduce Abeta _1-42 Induced ROS content in CL4176 nematodes.

Example Effect of Ten Ginseng peptide monomers on nematode longevity

Transgenic nematodes CL4176 in L3 phase after synchronization are divided into a control group and a dosing group, the culture medium is replaced every two days in a 23 ℃ incubator, the nematode survival condition is observed, and the number of nematode survival, death and loss is recorded until all nematodes die, and the experiment is ended.

As shown in fig. 12, the survival curves for all nematodes dosed ITGYAP, LTGYAP, ITGYPA and LTGYPA were significantly shifted to the right compared to the CTRL group. Statistical results show that ITGYAP of 10 mu M, 100 mu M and 200 mu M respectively prolongs the average service life of nematodes by 19.59%, 42.40% and 50.00%; different concentrations of the ginseng peptide monomer LTGYAP respectively prolong the average life span of nematodes by 13.54%, 25.42% and 41.63%; the ginseng peptide monomer ITGYPA respectively prolongs the average life span of nematodes by 38.77%, 42.33% and 50.14%; the average life of the nematode is prolonged by 21.61%, 24.03% and 36.24% respectively by the ginseng peptide monomer LTGYPA, and the experimental results show that ITGYAP, LTGYAP, ITGYPA and LTGYPA can obviously prolong the life of the nematode.

Example influence of undecylenic peptide monomer on egg laying number of insects

The synchronized L3 transgenic nematodes CL4176 were transferred to NGM medium in control and dosing groups, 5 nematodes per plate, 3 per group in parallel, and the nematodes were transferred daily to the new NGM plate until the nematodes entered the spawning period, and the number of nematode eggs on the original plate was recorded until the nematodes stopped spawning.

As shown in fig. 13, when 200 μm of ITGYAP, LTGYAP, ITGYPA and LTGYPA were administered respectively, the daily egg yield of nematodes increased to various degrees, and the total egg yield was counted, and the four peptides increased egg yields by 191, 277, 187 and 216 respectively, compared to the CTRL group.

Example effect of dodecapeptide monomer on aβ deposition in insects

The synchronized L3 transgenic nematodes CL4176 are divided into a control group and an administration group, and then placed into a 23 ℃ incubator for 120 h, and then washed 3 times with M9 buffer, and centrifuged at 1000 rpm for 1 min to settle the nematodes. Fixed overnight in a refrigerator at 4℃with 4% paraformaldehyde/PBS (pH 7.4), followed by 3 washes with pre-chilled PBS buffer. At 37deg.C, nematodes were allowed to permeate in permeation solution (containing 1% Triton X-100, 5% beta-mercaptoethanol, 125 mM Tris,pH 7.4) for 24 h, and PBST buffer was washed 3 times. Then stained with 0.125% thioflavin T/50% ethanol solution for 2 min and rinsed with a gradient of 50%, 75% and 100% ethanol. Observing the deposition condition of the Abeta at the head of the nematode under a laser confocal microscope, quantitatively analyzing green fluorescence by using imageJ software,

as shown in fig. 14, the CTRL group was not fed with the ginsenosides, the nematode heads had significant aβ plaques, while the nematode heads of the respective groups fed with ginsenosides ITGYAP, LTGYAP, ITGYPA and LTGYPA had significantly reduced deposition of aβ plaques. The results of the ImageJ software analysis also show that the ginseng peptide monomer can significantly inhibit the deposition of the nematode head aβ plaque.

Example thirteen molecular docking analysis

Molecular docking is a powerful computational method that has been used to predict the interaction energy between a receptor and a ligand. The 3D structure of the ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA and LTGYPA was completed by Chemdraw 20.0 software, Aβ _1-42 The crystal structure of the protein was downloaded from database RSCB Protein Data Bank (PDB, PDB ID:1 IYT). Ginseng peptide monomer ITGYAP, LTGYAP, ITGYPA and LTGYPA and Abeta _1-42 Molecular docking of (c) was done by AutoDock vina1.5.7 software, then using Pymol 2.4.0 to get a complex structure, finally visualizing the image by Discovery Studio 2019.

As shown in FIG. 15, ITGYAP-Abeta _1-42 、LTGYAP- Aβ _1-42 、ITGYPA- Aβ _1-42 And LTGYPA-Abeta _1-42 The results of molecular docking of (C) are shown in FIG. 15A-H, and the binding energies are-5.5, -5.7, -5.2 Kcal/mol, respectively. ITGYAP is shown in FIGS. 15A and BBinding to Abeta by formation of two intermolecular hydrogen bonds with Gln15/Lys16 and hydrophobic interaction with Val18/Phe19 _1-42 Molecular attachment. FIGS. 15C and D show that LTGYAP binds to Abeta through hydrogen bonding with Ala21/Glu22/Gly25/Ser26 and hydrophobic interaction with His13/Lys16/Leu17 _1-42 Molecular attachment. FIGS. 15E and F show that ITGYPA is bound to Abeta by hydrogen bonding with Leu17/Ala21/Glu22 and hydrophobic interaction with Val18/Leu34 _1-42 The molecules are bound to each other. FIG. 15G and H show that LTGYPA binds to Abeta through hydrogen bonding with Leu17/Ala21/Glu22 and hydrophobic interaction with Val18/Leu34 _1-42 Molecular attachment.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.

Nucleotide or amino acid sequence table of instruction book

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Claims

1. A ginseng peptide, characterized in that the amino acid sequence of the peptide is as shown in any one of the following (1) to (4):

(1) The amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 1;

(2) The amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 2;

(3) The amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 3;

(4) The amino acid sequence of the ginseng peptide is shown as SEQ ID NO. 4.