阅读说明：本技术 一种具有抑制人结肠癌细胞（ht-29细胞）增殖作用的鸡源胶原肽及其制备与应用 (Chicken collagen peptide with effect of inhibiting proliferation of human colon cancer cells (HT-29 cells), and preparation and application thereof ) 是由 黄雅钦 邓贵雅 郭尚伟 于 2021-08-31 设计创作，主要内容包括：一种具有抑制人结肠癌细胞(HT-29细胞)增殖作用的鸡源胶原肽及其制备与应用。本发明涉及一种具有抑制人结肠癌细胞(HT-29细胞)增殖的鸡源III型胶原蛋白肽GSpGPpGPSGPAGDR,其氨基酸序列为Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg。多肽GSpGPpGPSGPAGDR具有抑制HT-29细胞增殖的功能,并且具有补充人体必须氨基酸的能力及营养功能,用其制备出的相关食品、保健品或药物用于治疗结肠炎或结肠癌的食品或药物具有潜在的应用价值以及广阔的应用前景。(Chicken collagen peptide with effect of inhibiting proliferation of human colon cancer cell (HT-29 cell), and its preparation method and application are provided. The invention relates to a chicken source type III collagen peptide GSpGPpGPSGPAGDR capable of inhibiting the proliferation of a human colon cancer cell (HT-29 cell), and the amino acid sequence of the chicken source type III collagen peptide GSpGPpGPSGPAGDR is Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg. The polypeptide GSpGPpGGPSGPADDR has the function of inhibiting HT-29 cell proliferation and has the capability of supplementing essential amino acids to human bodies and a nutritional function, and related food, health-care products or medicaments prepared by the polypeptide GSPpGGPSGPADDR have potential application value and wide application prospect when being used as food or medicaments for treating colitis or colon cancer.)

1. A collagen peptide, characterized by: the polypeptide is GSpGPpGGPSGPAGDR; the amino acid sequence of the polypeptide is Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg, wherein p and Hyp represent hydroxyproline.

2. A food, health product or pharmaceutical product containing the collagen peptide according to claim 1.

3. The food, health product or pharmaceutical product according to claim 2, which is supplemented with a dietetic or a pharmaceutically acceptable carrier or adjuvant.

4. Use of a collagen peptide according to claim 1 in the manufacture of a food, health care product or medicament for the prevention and/or treatment of colitis or colon cancer related diseases.

5. Use according to claim 4, characterized in that: the food, the health care product and the medicine for preventing and treating the diseases related to the colitis or the colon cancer take the collagen peptide as an active ingredient, and a carrier or an auxiliary material which is acceptable in food or pharmacology is added.

Technical Field

The invention relates to the field of proteins, and in particular relates to a chicken III type collagen peptide GSPpGGPSGPAGDR with the effect of inhibiting the proliferation of human colon cancer cells (HT-29 cells) and application thereof.

Background

The colon cancer is one of malignant tumors with high morbidity and mortality all over the world, has high annual incidence rate in the world, is second to the lung cancer and the stomach cancer, and seriously threatens the health of human beings. The main causes of colon cancer development are the lack of high fat diet and cellulose intake. In recent years, with the increasing quality and level of life of people, the living habits and dietary structures of people have changed greatly. The incidence of colon cancer in China is on the trend of rising year by year, and how to effectively control and treat colon cancer is a more popular subject.

Bioactive peptides are natural polypeptides. The activity of the compound is changed along with different amino acid compositions and different amino acid arrangement sequences, so the compound has physiological activities of resisting oxidation, resisting bacteria, resisting cancer, reducing blood pressure, improving human immunity and the like. The bioactive peptide has the nutritional characteristics of easy digestion and absorption, has great development potential in the aspects of medicines, health products, food base materials and the like, and has become the current international anticancer medicine and the product with anticancer activity.

Disclosure of Invention

The invention aims to provide a chicken III type collagen peptide GSpGPpGGPSGPAGDR which has the effect of inhibiting HT-29 cell proliferation (p is hydroxyproline), and has good application prospect as a food, a health product and a medicine lead compound for preventing and treating diseases related to colitis or colon cancer.

In order to achieve the purpose, the collagen peptide GSpGPpGGPSGPADDR is an effective component for inhibiting the proliferation of HT-29 cells.

The collagen peptide GSpGPpGPSGPAGDR is derived from chicken type III collagen. The collagen peptide GSpGPpGGPSGPADDR is an active ingredient of food, health care products and pharmaceutical lead compounds for preventing and treating diseases related to colitis or colon cancer, and can be added with carriers or auxiliary materials acceptable in food or pharmacology.

The amino acid sequence of the collagen peptide GSpGPpGGPSGPADDR with the effect of inhibiting HT-29 cell proliferation is Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg. The single-chain linear structure has the molecular weight of 1337.30, is white powder, is easy to dissolve in water, and has an inhibitory effect on HT-29 cell proliferation.

Compared with the prior art, the invention has the following advantages:

the invention obtains and determines the activity and the efficacy of the collagen peptide GSpGPpGGPSGPAGDR from the chicken source III type collagen, has the functions of inhibiting HT-29 cell proliferation, being easily absorbed by human bodies, and supplementing the necessary amino acid capacity and the nutritional function of the human bodies, and has good application prospect as food, health care products and medicine lead compounds for preventing and treating diseases related to colitis or colon cancer.

Drawings

FIG. 1 is a primary mass spectrum of the collagen peptide GSpGPpGGPSGPAGDR;

FIG. 2 is a secondary mass spectrum of the collagen peptide GSpGPpGGPSGPAGDR;

FIG. 3 shows the results of experiments on the inhibition of HT-29 cell proliferation by the collagen peptide GSpGPpGGPSGPADDR.

Detailed Description

The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited thereto.

Example 1 Synthesis of collagen peptide GSpGPpGGPSGPADDR

The invention utilizes a polypeptide synthesizer to synthesize the collagen peptide by a solid-phase synthesis method:

0.05mmol of Fmoc-Ala-Wang resin (functional group content 0.33mmol/g) was weighed into a solid phase reactor, swollen overnight with 8mL of DCM and the solvent was removed under reduced pressure. Adding 8mL of 200mL/L piperidine solution, reacting for 5min at room temperature, and draining; then 8mL of 200mL/L piperidine solution is added, the reaction is carried out for 20min at room temperature, and then the solution is drained; washing 3 times with 8mL of DMF in succession is carried out for each time, lmin. Accurately measuring 0.2mmol of amino acid solution with alpha-amino protected by Fmoc, 0.2mmol of HBTU solution and 0.195mmol of HOBT solution, adding into a solid phase reactor, reacting for 5min, adding 0.4mmol of DIEA, and reacting at room temperature under nitrogen bubble oscillation for 2 h. The column was washed 3 times with 8mL of DMF each time for 1 min. Adding 8mL of 200mL/L piperidine solution, reacting for 5min at room temperature, and draining; then 8mL of 200mL/L piperidine solution is added, the reaction is carried out for 20min at room temperature, and then the solution is drained; the column was washed 3 times with 8mL of DMF each time for 1 min. The collagen peptide GSpGPpGGPSGPADDR is synthesized successively from the carbon end to the nitrogen end of the peptide chain one by one according to the above steps. After the synthesis is finished, the resin connected with the polypeptide chain is washed by DMF and DCM in sequence, and then the resin is dried in a vacuum drying oven for standby.

After the resin was dried, the resin was transferred to a heart-shaped flask, magnetons were added, 10mL of a cleavage solution for removing the resin (TFA in the cleavage solution: purified water: thioanisole: phenol: dithioglycol in a ratio of 82.5:5:5:5:2.5) was slowly added in an ice-water bath, and the reaction was carried out for 2 hours in an ice-water bath. After the reaction is finished, the reaction solution and the resin are transferred to a filter, the filter is pumped by a water pump, the obtained filtrate is placed in a round-bottom flask, and the round-bottom flask is dried under nitrogen flow. After the sample in the round-bottom flask is blown to be viscous, removing a nitrogen tube, pouring about 20mL of 4 ℃ ethyl glacial ether into the round-bottom flask, mixing and fully scattering, then centrifuging for 15min at 8000r/min at 4 ℃ in a refrigerated centrifuge, discarding the supernatant, scattering in 20mL of 4 ℃ ethyl glacial ether, and centrifuging; repeating the operation for 3 times, and vacuum drying the precipitate to obtain collagen peptide.

And purifying and identifying the collagen peptide by using a reverse high performance liquid chromatography. Semi-preparative column using C18, mobile phase:

phase a (aqueous phase): deionized water (containing 0.1% TFA (m/v));

phase B (organic phase): 80% aqueous acetonitrile (containing 0.1% TFA (m/v));

the flow rate is 6 mL/min; elution gradient: phase a decreased from 55% to 10% at 1% change per minute, and phase B increased from 45% to 90% at 1% change per minute. Collecting eluate for 6-8min, removing organic phase acetonitrile in the eluate with rotary evaporator, freezing the residual peptide water solution in refrigerator, and removing water with freeze dryer to obtain fluffy solid powder as pure collagen peptide product. FIG. 1 is a primary mass spectrum of the compound, and a signal in the primary mass spectrum is a signal of a parent collagen peptide fragment. Fig. 2 is a secondary mass spectrum of a collagen peptide, which includes mass-to-charge ratios and abundances of various fragment ion information of the collagen peptide.

Example 2 detection of the Activity of the collagen peptide GSpGPpGGPSGPADDR to inhibit HT-29 cell proliferation

HT-29 cells were plated in flat bottom 96 well cell culture plates at 100. mu.L/well containing 5X 10⁴Making the cells adhere to the wall, and incubatingAfter 24h of culture, the collagen peptide is diluted by DMEM complete culture solution, then the diluted medicine is added into a 96-well cell culture plate, three multiple wells are arranged for collagen peptide concentration, and only 100 mu L of cell culture solution is added into a control well. After the medicine is added, the total volume of each hole is 100 mu L, and the concentration of the collagen peptide is 600 mu g/mL. After dosing, cells were cultured for an additional 24 hours.

If not specifically stated, the growth of the cells is detected by the CCK-8 method after the cells are treated by the collagen peptide prepared by the invention for 24 hours. After the cell culture was completed, the liquid in the wells was aspirated. mu.L of DMEM medium containing 10% CCK-8 was added to each well and incubated at 37 ℃ for 1-4 h. The absorbance of the wells at 450nm was measured using a microplate reader. Cell viability (%) = dosing well OD value/control well OD value × 100%. The experiment was repeated 3 times.

Statistical analysis was performed on each group using the number of cells in the control group without drug as 100. Collagen peptide has inhibitory effect on HT-29 cell growth. The collagen peptide had an inhibitory effect on the growth of HT-29 cells at a concentration of 600. mu.g/mL, compared to the non-dosed control group, at which the cell survival rate was 75.50%.

Example 3 detection of the Activity of the collagen peptide GSpGPpGGPSGPADDR to inhibit HT-29 cell proliferation

HT-29 cells were plated in flat bottom 96 well cell culture plates at 100. mu.L/well containing 5X 10⁴And (3) attaching the cells, incubating and culturing for 24h, diluting collagen peptide by using a DMEM complete culture solution, adding the diluted medicine into a 96-well cell culture plate, setting three multiple wells for collagen peptide concentration, and adding only 100 mu L of cell culture solution into a control well. After the medicine is added, the total volume of each hole is 100 mu L, and the final concentration of the collagen peptide is 300 mu g/mL respectively. After dosing, cells were cultured for an additional 24 hours.

If not specifically stated, the growth of the cells is detected by the CCK-8 method after the cells are treated by the collagen peptide prepared by the invention for 24 hours. After the cell culture was completed, the liquid in the wells was aspirated. mu.L of DMEM medium containing 10% CCK-8 was added to each well and incubated at 37 ℃ for 1-4 h. The absorbance of the wells at 450nm was measured using a microplate reader. Cell viability (%) = dosing well OD value/control well OD value × 100%. The experiment was repeated 3 times.

Statistical analysis was performed on each group using the number of cells in the control group without drug as 100. Collagen peptide has inhibitory effect on HT-29 cell growth. The collagen peptide had an inhibitory effect on the growth of HT-29 cells at a concentration of 300. mu.g/mL, compared to the non-dosed control group, at which the cell survival rate was 80.45%.

Example 4 detection of Activity of the collagen peptide GSpGPpGGPSGPADDR for inhibiting HT-29 cell proliferation

HT-29 cells were plated in flat bottom 96 well cell culture plates at 100. mu.L/well containing 5X 10⁴And (3) attaching the cells, incubating and culturing for 24h, diluting collagen peptide by using a DMEM complete culture solution, adding the diluted medicine into a 96-well cell culture plate, setting three multiple wells for collagen peptide concentration, and adding only 100 mu L of cell culture solution into a control well. After the medicine is added, the total volume of each hole is 100 mu L, and the final concentration of the collagen peptide is 200 mu g/mL respectively. After dosing, cells were cultured for an additional 24 hours.

If not specifically stated, the growth of the cells is detected by the CCK-8 method after the cells are treated by the collagen peptide prepared by the invention for 24 hours. After the cell culture was completed, the liquid in the wells was aspirated. mu.L of DMEM medium containing 10% CCK-8 was added to each well and incubated at 37 ℃ for 1-4 h. The absorbance of the wells at 450nm was measured using a microplate reader. Cell viability (%) = dosing well OD value/control well OD value × 100%. The experiment was repeated 3 times.

Statistical analysis was performed on each group using the number of cells in the control group without drug as 100. Collagen peptide has inhibitory effect on HT-29 cell growth. The collagen peptide had an inhibitory effect on the growth of HT-29 cells at a concentration of 200. mu.g/mL, compared to the non-dosed control group, at which the cell survival rate was 83.97%.

The foregoing embodiments illustrate and describe the principles and general features of the present invention and its advantages. It will be understood by those skilled in the art that the present invention is not limited by the embodiments described above, which are given by way of illustration of the principles of the invention and are not to be taken as limiting the scope of the invention in any way, and that various changes and modifications may be made therein without departing from the scope of the invention as defined by the appended claims.