CN112940093A - Small peptide for promoting osteoblast proliferation - Google Patents
Small peptide for promoting osteoblast proliferation Download PDFInfo
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- CN112940093A CN112940093A CN202110180152.4A CN202110180152A CN112940093A CN 112940093 A CN112940093 A CN 112940093A CN 202110180152 A CN202110180152 A CN 202110180152A CN 112940093 A CN112940093 A CN 112940093A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Abstract
The invention provides a small peptide for promoting osteoblast proliferation, and belongs to the technical field of food processing. A small peptide having pro-proliferative activity on osteoblasts, having the following amino acid sequence: GQTPLFPR. The small peptide is derived from soybean protein, has good safety, has remarkable effect of promoting osteoblast proliferation, can remarkably promote osteoblast differentiation, and is expected to be used for preparing medicaments or functional foods for promoting osteoblast proliferation and resisting osteoporosis. The small peptide is convenient to synthesize, can be industrially produced, and has good application prospect in the fields of food, medicine, cosmetics and the like.
Description
Technical Field
The invention relates to a small peptide for promoting osteoblast proliferation, and belongs to the technical field of food processing.
Background
Osteoporosis is a metabolic disease characterized by reduced bone mass, decreased bone strength and toughness due to bone tissue degradation and microstructural destruction, and has a high incidence rate, and particularly with the advent of the aging age, osteoporosis and complications thereof such as fracture and the like increasingly become social problems endangering the health of the public.
At present, the drugs for treating osteoporosis mainly act on bone absorption of osteoclasts, but the drugs for promoting bone formation by targeting osteoblast proliferation are few, and the current anti-osteoporosis drugs in the market, such as bisphosphonates, selective estrogen receptor modulators and calcitonin, have serious side effects. Therefore, the development of natural product components for resisting osteoporosis with high efficiency, safety and small side effect is increasingly concerned.
Disclosure of Invention
The object of the present invention is to provide a small peptide having a proliferative activity on osteoblasts.
The purpose of the invention is realized by adopting the following technical scheme:
a small peptide with proliferation promoting activity on osteoblasts has an amino acid sequence shown as SEQ ID NO. 1.
The invention also provides a modified peptide of the small peptide, which is used for modifying the small peptide for operation, and the N end, the C end or the middle residue of the small peptide is connected with amino acid, polypeptide, protein or PEG.
In the present invention, modifications to the small peptide include: formylating or acetylating the N-terminal of the small peptide, or connecting fatty acid, hydrazinonicotinamide, diethylenetriaminepentaacetic acid, myristic acid, palmitic acid or succinamide or PEG at the N-terminal; or amidating the C end of the small peptide, or connecting p-nitroaniline or 7-amino-4-methylcoumarin to the C end; or glycosylation, phosphorylation, methylation, acetylation, nitration, sulfonation or PEG modification is carried out on the small peptide intermediate residue or the small peptide intermediate residue is coupled with protein.
The invention also provides application of the small peptide in preparing products for promoting osteoblast proliferation and resisting osteoporosis.
The invention also provides application of the small peptide in preparing medicines for promoting osteoblast proliferation and resisting osteoporosis.
The invention also provides a food of the small peptide, which has the functions of promoting osteoblast proliferation and resisting osteoporosis.
Has the advantages that: the small peptide B is derived from soybean protein, has good safety, has obvious effect of promoting osteoblast proliferation, can obviously promote osteoblast differentiation, and is expected to be used for preparing medicaments or functional foods for promoting osteoblast proliferation and resisting osteoporosis. The small peptide is convenient to synthesize, can be industrially produced, and has good application prospect in the fields of food, medicine, cosmetics and the like.
Drawings
FIG. 1 Effect of papain enzymatic product concentration on osteoblast proliferation viability with the abscissa being the concentration of the papain enzymatic product solution. Indicates a significant difference (P < 0.05) from the control group, and indicates a significant difference (P < 0.01) from the control group
FIG. 2 Effect of the fractions of the ultrafiltration product on the proliferative activity of osteoblasts. Different letters indicate significant differences (P < 0.05). The same applies below.
FIG. 3 Sephadex G-15 separation chromatogram.
Figure 4 bone cell proliferation-promoting activity of each active component.
Fig. 5 effect of small peptide B on osteoblast proliferation viability, wherein 10, 25, 50 and 100 represent intervention concentrations of small peptide B in μ M.
Fig. 6 shows the effect of small peptide B on osteoblast ALP (alkaline phosphatase) viability, GQTPLFPR is a well to which small peptide B was added. D7 and D10 indicate that the culture time before intervention of the small peptide is 7 days and 10 days respectively.
Detailed Description
Test materials: soy protein isolate, shanghai source leaf biotechnology limited; dimethyl sulfoxide (DMSO), Sigma, usa; thiazole blue (MTT), papain, beijing solibao science and technology ltd; MC3T3-E1 cell line, Shanghai Zhongyao cell bank; fetal Bovine Serum (FBS), α -MEM medium, EDTA-pancreatin (0.05%), penicilin-Streptomycin (100 ×), PBS phosphate buffer, Gibco, usa; alkaline phosphatase Alcalase 2.4L, Novoversson Biotechnology Ltd, Denmark; sephadex G-15, GE (China) medical group; other reagents are all made in China and are purchased from chemical reagents of national drug group, Inc.
Instruments and equipment: SpectraMax M2e microplate reader, Molecular Devices, USA; protein purification system, Shanghai West Analyzer Co., Ltd; model 8400 ultrafiltration cups, Millipore, USA; HY-1 vortex mixer, Shanghai apparatus, electrosciences instruments, Inc.; micro vertical electrophoresis system, chemiluminescence gel imaging system, Bio-rad, usa; a freeze dryer, Labconco, USA; JY 92-II ultrasonic cell crusher, Ningbo New technology ultrasonic Equipment Co.
Determination of proliferative Activity of osteoblasts:
(1) setting a sample adding group: osteoblasts (MC3T3-E1) were digested and collected to give a mixture containing 5X 10 cells/ml4The suspension of each cell was added to 100. mu.L of the suspension in each well of a 96-well plate, and the mixture was left at 37 ℃ under CO2The cells are attached to the wall after 24h of culture in an incubator. And removing the culture solution after the cells adhere to the wall, adding 100 mu L of samples to be detected into each hole, and setting 6 samples to be detected in parallel. Exposing the cells to CO2After further culturing in the incubator for 24 hours, 10. mu.L of 5 mg/mL solution was added to each well-1After continuously culturing for 4 hours, the MTT solution and the culture solution are sucked out, 50 mu L of DMSO is added, and then the MTT solution and the culture solution are vibrated in a constant temperature oscillator at 37 ℃ for 20min, and then the absorbance is measured by using a microplate reader at a single wavelength of 570 nm.
(2) In addition, a blank group and a control group were set. The control group replaces the sample to be detected with the same volume of alpha-MEM culture solution, and the other groups are the same as the sample-adding group. The blank was added to 100. mu.L of PBS buffer per well,in CO2After 48 hours of incubation in an incubator, 10. mu.L of 5 mg/mL solution was added to each well-1After further culturing for 4 hours, the MTT solution and PBS buffer solution were aspirated and 50. mu.L of DMSO was added, followed by oscillation in a 37 ℃ constant temperature oscillator for 20min and then absorbance was measured at a single wavelength of 570nm using a microplate reader.
(3) Osteoblast proliferation Activity (OD)Sample adding group-ODBlank group)/(ODControl group-ODBlank group)×100％。
Example 1 method for preparing an active peptide that promotes osteoblast proliferation
A method for preparing an active peptide for promoting osteoblast proliferation, comprising the steps of:
(1) enzymolysis of papain
Weighing soybean protein isolate (Shanghai-derived leaf Biotechnology Co., Ltd., product No. S30914), dissolving in ultrapure water to obtain a protein solution with a mass percentage concentration of 4.5%, adding NaOH solution, and adjusting pH to 7.0. Adding papain into the protein solution according to the proportion of adding 3000U papain into each gram of protein, placing on a rotary mixing machine, performing enzymolysis reaction for 5h at 55 ℃, and detecting that the hydrolysis degree of the soybean protein isolate is 11.08 +/-0.31%. After the reaction is finished, heating to 85 ℃, keeping the temperature for 20min to inactivate the papain, cooling to room temperature, centrifuging for 15min under the condition of 3000 Xg, taking supernatant, dialyzing to remove salt, freeze-drying to obtain a papain enzymolysis product, and storing at-20 ℃.
The papain enzymolysis product is prepared into 50, 100, 150, 200 and 250 mu g/mL by adopting alpha-MEM culture solution-1The solution of (1). According to the method for measuring the proliferation activity of osteoblasts, papain enzymolysis product solutions with different concentrations are used as samples to be detected, and the proliferation activity of osteoblasts after intervention of each sample is detected. As shown in FIG. 1, the concentration of the papain enzymolysis product solution was 100. mu.g.mL-1And 150. mu.g.mL-1Then, the osteoblast proliferation activity was enhanced as compared with that of the control group (P)<0.05) when the concentration is 200. mu.g.mL-1And 250. mu.g.mL-1The time phase comparison control group hasVery significant difference (P)<0.01), but there was no significant difference (P) between the two doses>0.05). When the concentration of the papain enzymolysis product solution is 200 mug.mL-1The osteoblast proliferation activity was 118.24. + -. 2.73%.
(2) Separation with ultrafiltration membrane
Dissolving the papain enzymolysis product obtained in the step (1) in deionized water to prepare 10 mu g/mL-1The solution of (4) was filtered through a 0.45-. mu.m cellulose membrane to remove insoluble substances. Separating the filtrate by adopting a 10kDa ultrafiltration membrane to respectively obtain a trapped fluid A and a permeate A. Separating the trapped fluid A by adopting a 30kDa ultrafiltration membrane to obtain a trapped fluid B and a permeate B. The conditions of ultrafiltration were: the temperature was 4 ℃ and the pressure was 0.2 MPa. Respectively freeze-drying the permeate A (containing less than 10kDa component), the retentate B (containing greater than 30kDa component) and the permeate B (containing 10-30kDa component) to obtain less than 10kDa component, greater than 30kDa component and 10-30kDa component.
Respectively preparing the components with less than 10kDa, more than 30kDa and 10-30kDa into 200 μ g/mL with alpha-MEM culture solution-1The solution of (1) is used as a sample to be tested, and the osteoblast proliferation activity is detected according to the osteoblast proliferation activity measuring method. The results are shown in FIG. 2. Compared with the control group, the component with the molecular weight of more than 30kDa has no obvious proliferation promoting effect on osteoblast (P)>0.05), 10-30kDa component and less than 10kDa component have significant proliferation promoting effect, wherein the less than 10kDa component has high proliferation promoting activity, and osteoblast proliferation activity reaches 120.45 + -2.28%, and has significant difference (P) with other groups<0.05). It is known that the smaller the molecular weight of the papain enzymatic hydrolysate, the higher the activity of the papain enzymatic. Thus, permeate a (less than 10kDa fraction) was selected for subsequent enzymatic hydrolysis experiments.
(3) Enzymolysis with alkaline protease
Dissolving the component less than 10kDa obtained in the step (2) in ultrapure water to prepare a protein solution with the mass percentage concentration of 5%, then adjusting the pH to 8.0 by using a NaOH solution, adding 3000U of alkaline protease (Danish Novicin biotechnology, Inc.) according to the amount of the alkaline protease added to each gram of protein, performing enzymolysis for 0.5h at 55 ℃, heating to 85 ℃, keeping the temperature for 20min to inactivate the alkaline protease, cooling to room temperature, centrifuging for 15min at 3000 Xg, taking supernatant, dialyzing to remove salt, freeze-drying to obtain an alkaline protease enzymolysis product, and storing at-20 ℃.
Preparing alkaline protease enzymolysis product into 200 mug/mL by adopting alpha-MEM culture solution-1The solution of (1) is used as a sample to be tested, and the osteoblast proliferation activity is detected according to the osteoblast proliferation activity measuring method. As a result: the proliferation activity of the alkaline protease enzymolysis product on osteoblasts reaches 123.02 +/-2.69%.
(4) Sephadex G-15 separation
And (4) separating the alkaline protease enzymolysis product obtained in the step (3) by using Sephadex G-15. Firstly, swelling Sephadex G-15 dry powder, then loading the powder into a chromatographic column with the diameter of 15mm multiplied by 600mm, dissolving an alkaline protease enzymolysis product in ultrapure water to prepare a solution with the concentration of 2 percent (mass percentage concentration), filtering the solution by a filter membrane with the diameter of 0.22 mu m to remove particles, wherein the loading amount is 3 percent of the volume of the column, and the separation process is carried out at the temperature of 4 ℃. Using ultrapure water at a flow rate of 0.6 mL/min-1Eluting at flow rate, detecting light absorption value at 220nm with ultraviolet detector, collecting 1 tube (about 2mL) of eluate every 5min with automatic collector, and recording ultraviolet detection result with recorder. According to the recorded curve, the eluates under the same elution peak are combined and freeze-dried.
As shown in FIG. 3, after Sephadex G-15 separation, 4 elution peaks were obtained, which were designated as active components F1 (retention time 40-55min), F2 (retention time 70-90min), F3 (retention time 95-115min) and F4 (retention time 115-130min), respectively. F1, F2, F3 and F4 were each freeze-dried and prepared into 200. mu.g/mL using an alpha-MEM culture medium-1The solution of (1) is used as a sample to be tested, and the osteoblast proliferation activity is detected according to the osteoblast proliferation activity measuring method. As shown in FIG. 4, F1, F2, F3 and F4 all had a proliferative activity on osteoblasts (P) as compared with the control group<0.05), wherein the osteoblast proliferation activity is up to 125.80 ± 2.94% after F3 intervention.
(5) Analysis of amino acid content and structural identification of peptides and their effect on osteoblast proliferation viability
And (4) carrying out amino acid analysis on the active component F3 obtained in the step (4), and determining the amino acid content by referring to GB 5009.124-2016. 0.2g of active ingredient F3 was weighed into a hydrolysis tube, and 10mL of 6 mol. L was added-1The tube is sealed after hydrochloric acid is obtained, the tube is hydrolyzed in a baking oven at 110 ℃ for 24 hours, and then the content of amino acid in a sample is determined by using a sulfonic acid type cationic resin column by using a full-automatic amino acid analyzer.
The Shanghai Boyuan Biotechnology Limited company is entrusted to complete the structural identification of the small peptide in the active component F3. The active component F3 was structurally characterized by tandem mass spectrometry (ESI-TOF MS/MS). Mobile phase A: an aqueous solution containing 2% (by volume) acetonitrile and 0.1% (by volume) formic acid; mobile phase B: acetonitrile, formic acid and water in a volume ratio of 98: 0.1: 1.9 the solvent. The mass spectrum was obtained using a TripleTOF 5600 system (AB SCIEX) in combination with a nanoliter spray III ion source at a spray voltage of 2.5kV, an atomization pressure of 5PSI, an air curtain pressure of 30PSI, and a heater temperature of 150 ℃.
The active component F3 contains small peptide B. The sequence of small peptide B (SEQ ID NO:1) is: GQTPLFPR. The small peptide B (purity more than 95%) is synthesized by chemical solid phase method, and the experiment is finished by Jiangsu Jinsrie biological science and technology limited company.
The small peptide B prepared by a chemical solid-phase synthesis method is prepared into 10, 25, 50 and 100 mu M solutions by respectively adopting alpha-MEM culture solutions and is used as a sample to be detected, and the osteoblast proliferation activity is detected according to an osteoblast proliferation activity determination method so as to investigate whether the small peptide B has the effect of promoting osteoblast proliferation. As shown in fig. 5, the osteoblast proliferation activity gradually increases with the increase of the concentration of the small peptide B, and the proliferation activity of the small peptide B on osteoblasts reaches 129.11 ± 3.12% at a concentration of 100 μ M, so that the small peptide B has a significant effect of promoting osteoblast proliferation.
Example 7 Effect of Small peptide B on osteoblast ALP (alkaline phosphatase) Activity
The effect of small peptide B on osteoblast ALP viability was examined using the alkaline phosphatase kit. The specific method comprises the following steps: osteoblasts MC3T3-E1 were digested and then prepared in alpha-MEM medium at a concentration of 1X 105Cell suspension per mL, seeded in 6-well plates, 2mL per well. After MC3T3-E1 cells were grown to confluence, the medium was removed, cultured in mineralization induction medium (ODM from Sigma, USA supplemented with ascorbic acid at a final concentration of 50. mu.g/mL and 4 mM. beta. -glycerophosphate) for 7, 10 days for induction of differentiation, and then the serum-free medium was replaced while small peptide B at a final concentration of 20. mu.M was added for treatment for 48h, 3 replicates. Meanwhile, a control group is set, and small peptide B is not intervened, and the others are not changed. ALP viability was determined according to the alkaline phosphatase kit instructions; the total intracellular protein content was measured by BCA assay to correct ALP activity. As shown in FIG. 6, the activity of ALP was 13.68. + -. 0.30U/g in the control group and 27.44. + -. 0.73U/g in the well to which the small peptide B was added at the differentiation time of 7 days; the activity of ALP in the control group was 19.45. + -. 1.30U/g and that in the well to which the small peptide B was added was 53.38. + -. 2.08U/g at 10 days of differentiation. The experimental results show that the ALP activity of osteoblasts intervened by small peptide B is obviously higher than that of a control group (P) on the 7 th day and the 10 th day of differentiation<0.05), indicating that small peptide B can significantly promote osteoblast differentiation.
SEQUENCE LISTING
<110> university of financial institution of Nanjing
<120> Small peptide promoting osteoblast proliferation
<130> 202102072
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> PRT
<213> Soybean
<400> 1
Gly Gln Thr Pro Leu Phe Pro Arg
1 5
Claims (6)
1. A small peptide for promoting osteoblast proliferation has an amino acid sequence shown as SEQ ID NO. 1.
2. The modified peptide of the small peptide of claim 1, wherein the osteogenic active peptide is modified by attaching an amino acid, polypeptide, protein or PEG to the N-terminal, C-terminal or middle residue of the osteogenic active peptide.
3. The modified peptide of claim 2, wherein the modification to the small peptide comprises: formylating or acetylating the N-terminal of the small peptide, or connecting fatty acid, hydrazinonicotinamide, diethylenetriaminepentaacetic acid, myristic acid, palmitic acid or succinamide or PEG at the N-terminal; or amidating the C end of the small peptide, or connecting p-nitroaniline or 7-amino-4-methylcoumarin to the C end; or glycosylation, phosphorylation, methylation, acetylation, nitration, sulfonation or PEG modification is carried out on the small peptide intermediate residue or the small peptide intermediate residue is coupled with protein.
4. Use of the small peptide according to claim 1 and the modified peptide of the small peptide according to claim 2 in preparation of products for promoting osteoblast proliferation and resisting osteoporosis.
5. Use of the small peptide according to claim 1 or a modified peptide of the small peptide according to claim 2 for the preparation of a medicament for promoting osteoblast proliferation and resisting osteoporosis.
6. A food having osteoblast proliferation promoting and anti-osteoporosis effects, which contains the small peptide according to claim 1 or a modified peptide of the small peptide according to claim 2.
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| CN114480264A (en) * | 2022-03-03 | 2022-05-13 | 烟台大学 | Application of soybean peptide in promoting osteogenesis activity |
| CN115006382A (en) * | 2022-06-15 | 2022-09-06 | 广州中医药大学第一附属医院 | Application of myristic acid in preparation of medicine for resisting senile osteoporosis |
| CN116082454A (en) * | 2022-09-09 | 2023-05-09 | 大连工业大学 | Polypeptide with bone mineral density regulating activity and application thereof |
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| CN107375261A (en) * | 2017-06-27 | 2017-11-24 | 东北林业大学 | Application of the pinostrobin in osteoporosis is prevented and treated |
| CN110627897A (en) * | 2019-10-12 | 2019-12-31 | 中国科学院理化技术研究所 | Active peptide for promoting osteoblast proliferation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107375261A (en) * | 2017-06-27 | 2017-11-24 | 东北林业大学 | Application of the pinostrobin in osteoporosis is prevented and treated |
| CN110627897A (en) * | 2019-10-12 | 2019-12-31 | 中国科学院理化技术研究所 | Active peptide for promoting osteoblast proliferation and application thereof |
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| CN114480264A (en) * | 2022-03-03 | 2022-05-13 | 烟台大学 | Application of soybean peptide in promoting osteogenesis activity |
| CN114480264B (en) * | 2022-03-03 | 2024-01-30 | 烟台大学 | Application of soybean peptide in promotion of osteogenesis activity |
| CN115006382A (en) * | 2022-06-15 | 2022-09-06 | 广州中医药大学第一附属医院 | Application of myristic acid in preparation of medicine for resisting senile osteoporosis |
| CN116082454A (en) * | 2022-09-09 | 2023-05-09 | 大连工业大学 | Polypeptide with bone mineral density regulating activity and application thereof |
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