Use of ergothioneine in preparation of collagen peptide intestinal absorption promoter

Abstract

The use of ergothioneine in preparation of a collagen peptide intestinal absorption promoter, a collagen peptide intestinal absorption promoter and the use of ergothioneine in promoting intestinal absorption of collagen peptide. In the present invention, animal experiments prove that the combined supplementation of ergothioneine and collagen peptide can obviously promote the intestinal bioavailability of collagen peptide, and cell experiments show that ergothioneine can up-regulate the expression of peptide transporter protein Pept1 in the intestine, thus providing mechanistic support for the promotion of intestinal absorption of collagen peptide by the combined supplementation of ergothioneine.

Description

Application of ergothioneine in the preparation of collagen peptide intestinal absorption enhancer Technical Field

[0001] The present invention relates to the technical field of food and health products, and in particular to application of ergothioneine in preparing a collagen peptide intestinal absorption accelerator, a collagen peptide intestinal absorption accelerator, and application of ergothioneine in promoting intestinal absorption of collagen peptide. Background Art

[0002] Collagen is one of the main macromolecules that make up the extracellular matrix. It is mainly found in the skin, joints, muscles, etc., and has been widely used in various food and supplement products. However, collagen is a type of macromolecule with a relative molecular weight of up to 300,000 Daltons. It cannot be directly absorbed by the human body after consumption. It needs to be digested and broken down into small molecular peptides or free amino acids before it can be absorbed by the human intestine and exert its effects. The above conversion process results in a very low absorption rate of direct collagen supplementation in the human body. In order to improve the absorption rate of collagen, most collagen peptides are introduced on the market as a source of collagen supplementation. Although reducing the molecular weight can increase the intestinal absorption rate of collagen peptides, there are still large individual differences.

[0003] When collagen peptides in food and supplement products are ingested into the human digestive system, they must pass through the gastrointestinal tract and cross the intestinal epithelial barrier. During this process, the peptides need to resist the effects of digestive enzymes and reach the target organs in an effective form to exert their various biological effects. When peptides are broken down into individual amino acids, they may lose the ability to interact with some receptors and biomolecules, thereby reducing their biological efficacy. Therefore, improving the intestinal bioavailability of collagen peptides and ensuring that they exert their due efficacy are current technical needs.

[0004] The primary transporter for small peptides in the intestine is peptide transporter 1 (Pept1), which can transport peptide molecules consisting of 2-5 peptides, particularly dipeptides and tripeptides. Pept1 is primarily expressed on the apical microvilli of intestinal epithelial cells and is responsible for the transport and absorption of small peptides in the intestine. Lack of Pept1 activity or low expression can impair the absorption of biological peptides.

[0005] Ergothioneine (EGT) is a highly effective antioxidant that effectively scavenges free radicals and reduces oxidative stress. Although it cannot be synthesized by the human body, it has a specific transporter in mammals: novel organic cation transporter 1 (OCTN-1). This transporter is highly expressed in most mammalian tissues and organs, particularly the eyes, liver, kidneys, heart, and skin. EGT is currently used in skincare products to promote its anti-aging effects. Summary of the Invention

[0006] The present invention aims to provide an application of ergothioneine in preparing a collagen peptide intestinal absorption accelerator, a collagen peptide intestinal absorption accelerator, and an application of ergothioneine in promoting intestinal absorption of collagen peptide.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides the use of ergothioneine in the preparation of a collagen peptide intestinal absorption enhancer.

[0008] Specifically, in the application provided by the present invention, the ergothioneine upregulates the expression of the peptide transporter Peptl in the intestine, thereby promoting the absorption of the collagen peptide in the intestine.

[0009] A second aspect of the present invention provides a collagen peptide intestinal absorption enhancer, which comprises ergothioneine and collagen peptide, wherein the ergothioneine promotes the absorption of the collagen peptide in the intestine.

[0010] Optionally, the collagen peptide intestinal absorption enhancer provided by the present invention comprises a first dose and a second dose, wherein the first dose comprises ergothioneine and the second dose comprises collagen peptide.

[0011] Optionally, in the collagen peptide intestinal absorption enhancer provided by the present invention, the ergothioneine and the collagen peptide are mixed uniformly.

[0012] Optionally, the collagen peptide intestinal absorption enhancer provided by the present invention is an oral preparation.

[0013] Optionally, in the collagen peptide intestinal absorption enhancer provided by the present invention, more than 85% of the collagen peptides have a molecular weight distribution of 200 to 1000 Daltons; and the collagen peptides are derived from collagen peptide powder prepared by enzymatic hydrolysis of animal collagen.

[0014] Optionally, in the collagen peptide intestinal absorption enhancer provided by the present invention, the mass ratio of ergothioneine to collagen peptide is 0.05:100 to 1:100.

[0015] Preferably, in the collagen peptide intestinal absorption enhancer provided by the present invention, the mass ratio of ergothioneine to collagen peptide is 0.05:100 to 0.5:100.

[0016] More preferably, in the collagen peptide intestinal absorption enhancer provided by the present invention, the mass ratio of ergothioneine to collagen peptide is 0.3:100 to 0.5:100.

[0017] Optionally, in the collagen peptide intestinal absorption enhancer provided by the present invention, the oral dosage of ergothioneine is 1 to 50 mg / day, and the oral dosage of collagen peptide is 1 to 20,000 mg / day.

[0018] A third aspect of the present invention provides an application of ergothioneine in promoting intestinal absorption of collagen peptides. This application can be for therapeutic purposes or for non-therapeutic purposes.

[0019] Compared with the prior art, the present invention confirms through animal experiments that the combined supplementation of ergothioneine and collagen peptide can significantly promote the intestinal bioavailability of collagen peptide, and cell experiments show that ergothioneine can upregulate the expression of peptide transporter Pept1 in the intestine, which also confirms from a mechanistic level that supplementation of ergothioneine can effectively promote the absorption of collagen peptide in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is a drug-time curve of the tripeptide Gly-Pro-Hyp concentration in the plasma of rats in each experimental group in Example 1;

[0021] FIG2 is a histogram of the AUC of the tripeptide Gly-Pro-Hyp concentration in the plasma of rats in each experimental group in Example 1;

[0022] FIG3 is a drug-time curve of the dipeptide Pro-Hyp concentration in the plasma of rats in each experimental group in Example 1;

[0023] FIG4 is a bar graph showing the AUC of the dipeptide Pro-Hyp concentration in the plasma of rats in each experimental group in Example 1;

[0024] FIG5 is a bar graph showing the protein expression of Pept1 in Caco-2 cells in each experimental group in Example 2. DETAILED DESCRIPTION

[0025] To make the objectives, technical solutions, and advantages of the present invention more clearly apparent, various embodiments of the present invention will be described in detail below. However, those skilled in the art will appreciate that, in various embodiments of the present invention, many technical details are provided to help readers better understand the present application. However, even without these technical details and various variations and modifications based on the following embodiments, the technical solutions claimed in the present application can still be implemented.

[0026] All raw materials in the embodiments of the present invention are commercially available products:

[0027] Ergothioneine: Appearance: white powder, purity: >99%;

[0028] Collagen peptide: Properties: white powder, molecular weight characteristics: 200-1000 Daltons.

[0029] Example 1 Experiment on combined administration of EGT and collagen peptide

[0030] 1. Drug administration and blood sampling in rats

[0031] Eight-week-old male Sprague-Dawley rats were randomly divided into five groups, each consisting of six rats. All six groups received oral administration of EGT and collagen peptides. Groups A, B, and C received three dose ratios of EGT and collagen peptides: 0.1 mg / kg EGT and 200 mg / kg collagen peptide (Group A), 1 mg / kg EGT and 500 mg / kg collagen peptide (Group B), and 10 mg / kg EGT and 1000 mg / kg collagen peptide (Group C). Groups A1 and B1 received collagen peptides alone: ​​200 mg / kg collagen peptide (Group A1) and 1000 mg / kg collagen peptide (Group C1). A blank control group received water alone. Approximately 120 μL of blood samples were collected at 0 minute before administration and at 15, 30, 45, 60, 120, and 360 minutes after administration. After each blood draw, the rats were immediately flushed with sodium chloride injection solution to prevent blood clotting. Plasma was separated and stored frozen at -20°C.

[0032] 2. LC-MS / MS method for the determination of tripeptide and dipeptide concentrations in rat plasma

[0033] The concentrations of the characteristic tripeptide Gly-Pro-Hyp and dipeptide Pro-Hyp of collagen in rat plasma were determined by LC-MS / MS. The instrumentation was an Agilent 1260 HPLC system coupled with an Agilent 6460 triple quadrupole mass spectrometer. Separation was performed using an Agilent Zorbax (1.8 μm, 2.0 mm) reversed-phase column. Column temperature was 30°C. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The flow rate was 0.25 mL / min. An initial composition of 100% A was maintained for 2 minutes, followed by a linear increase from 0% B to 90% B over 2 minutes and a 1-minute hold. After 1 minute, the gradient was returned to its initial state and then maintained for 2 minutes, for a total run time of 8 minutes.

[0034] 3. Test Results

[0035] FIG1 is a drug-time curve of the concentration of the tripeptide Gly-Pro-Hyp (characteristic peptide structure of collagen: glycyl-prolyl-hydroxyproline) in the plasma of rats in each experimental group;

[0036] FIG2 is a histogram of the AUC of the tripeptide Gly-Pro-Hyp concentration in the plasma of rats in each experimental group, where * indicates p < 0.05 compared with the blank control group (control); & indicates p < 0.05 compared with C1;

[0037] FIG3 is a drug-time curve of the concentration of dipeptide Pro-Hyp (another characteristic peptide structure of collagen: prolyl-hydroxyproline) in the plasma of rats in each experimental group;

[0038] FIG4 is a bar graph showing the AUC of the dipeptide Pro-Hyp concentration in the plasma of rats in each experimental group, where * indicates p < 0.05 compared with the blank control group (control); % indicates p < 0.05 compared with A1; and & indicates p < 0.05 compared with C1.

[0039] As can be seen from Figure 1 to Figure 4, the group (A1 group and C1 group) that only supplements collagen peptides, the level of the characteristic tripeptide Gly-Pro-Hyp and dipeptide Pro-Hyp of collagen in blood plasma increases. The group (A group, B group and C group) that jointly supplements thioneine and collagen peptides, the level of the characteristic tripeptide Gly-Pro-Hyp and dipeptide Pro-Hyp of collagen in blood plasma all has more obvious improvement. And, the group (A group and C group) that jointly supplements thioneine and collagen peptides, relative to the group (A1 group and C1 group) that only supplements collagen peptides with the same dosage, the raising of the characteristic tripeptide Gly-Pro-Hyp and dipeptide Pro-Hyp level of collagen in blood plasma has significant difference. The above experimental results show that, jointly supplementing thioneine and collagen peptides has synergistic effect, and thioneine can promote the intestinal bioavailability of collagen peptides.

[0040] Example 2 Pept1 protein expression experiment in Caco-2 cell model

[0041] 1. Caco-2 cell model establishment and drug administration

[0042] The Caco-2 cell model is a human cloned colon adenocarcinoma cell line with a structure and function similar to differentiated intestinal epithelial cells. It has structures such as microvilli and contains enzyme systems related to the small intestinal brush border epithelium. It can be used to conduct experiments simulating intestinal transit in vivo.

[0043] Caco-2 cells were plated at 1.0 × 10 cells per well. 4 Cells were seeded into 96-well cell culture plates and cultured in a CO2 incubator (37°C, 5%) in DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acid solution, and antibacterial antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin). After 24 hours of culture with the addition of 500 μM indomethacin, the culture medium in the 96-well plates was removed and 200 μL of fresh culture medium containing varying concentrations of EGT was added. The culture medium was replaced daily and continued in the incubator for 3 days to serve as the treatment group. A model group and a control blank group were also established. The model group was cultured with only 500 μM indomethacin; the blank group was cultured with 200 μL of fresh culture medium, and the culture medium was replaced daily.

[0044] 2. Western blot detection of Pept1 protein content

[0045] The Caco-2 cells in each experimental group were washed twice with PBS, stored in RIPA buffer containing protease inhibitors, and lysed at 4°C for 30 minutes. The lysate was centrifuged at 12,000 × g for 30 minutes at 4°C to determine the protein concentration. The expression of Pept1 protein was detected by Western blot using bovine serum albumin as the standard.

[0046] 3. Test Results

[0047] FIG5 is a bar graph of the protein expression of Pept1 in Caco-2 cells in each experimental group, where * indicates p < 0.05, ** indicates p < 0.01 compared with the blank control group (control); # indicates p < 0.05 compared with the model group (Model).

[0048] As shown in Figure 5, compared with the blank group, the expression level of Pept1 protein in the model group cells was significantly reduced, indicating that the model was successfully established. Compared with the model group, the experimental groups cultured with different concentrations of EGT increased the expression of Pept1 protein (among which, the increase in Pept1 protein expression in the 1% EGT group and the 10% EGT group was significant), indicating that EGT can upregulate the expression of Pept1, an important small molecule peptide transporter in the intestine, and explain the possible mechanism by which EGT can promote the intestinal absorption of collagen peptides.

[0049] Example 3 Experimental study on the dosage ratio of combined administration

[0050] 1. Collagen peptide absorption improvement rate detection

[0051] The same experimental procedures as described in the sections "Dosing and blood sampling in rats" and "Detection of tripeptide and dipeptide concentrations in rat plasma by LC-MS / MS" in Example 1 were followed to determine the concentrations of the tripeptide Gly-Pro-Hyp and the dipeptide Pro-Hyp in the plasma of rats in dose experimental groups 1 to 6. The following formula was used to calculate the collagen peptide absorption enhancement rate (%) for each of the dose experimental groups 1 to 6:

[0052] Among them, M1 is the tripeptide concentration value in the plasma of rats co-administered with EGT + collagen peptide, and M0 is the tripeptide concentration value in the plasma of rats administered with collagen peptide; N1 is the dipeptide concentration value in the plasma of rats co-administered with EGT + collagen peptide, and N0 is the dipeptide concentration value in the plasma of rats administered with collagen peptide.

[0053] 2. Test Results and Discussion

[0054] Table 1 shows the dosage and collagen peptide absorption improvement rate of dosage experimental groups 1 to 6:

[0055] Table 1 Grouping and test results of combined drug administration dosage exploration experiment

[0056] From the test results of Table 1, it can be seen that in the dose experimental groups 1 to 6, the combined administration of EGT and collagen peptide showed a significant improvement in the absorption rate of collagen peptide relative to the administration of collagen peptide alone; in particular, the collagen peptide absorption improvement rate of the dose experimental groups 4 to 5 even exceeded 30%; while in the dose experimental group 6 with a relatively higher EGT dose, the collagen peptide absorption improvement rate showed a downward trend. Therefore, in the collagen peptide intestinal absorption enhancer of the present invention, the mass ratio of ergothioneine to collagen peptide is 0.05:100 to 1:100; preferably 0.05:100 to 0.5:100; more preferably 0.3:100 to 0.5:100, thereby achieving the effect of promoting the intestinal absorption and utilization of collagen peptide with a smaller amount of EGT.

[0057] In summary, above-mentioned Examples 1 to 3 confirm through animal experiments that the combined supplementation of ergothioneine and collagen peptide can significantly promote the intestinal bioavailability of collagen peptide, and cell experiments show that ergothioneine can up-regulate the expression of peptide transporter Pept1 in the intestine, and confirm from the mechanism level that supplementation of ergothioneine can effectively promote the absorption of collagen peptide in the intestine; and provide a better dosage ratio of EGT and collagen peptide co-administration through a combined administration dosage exploration experiment, which can achieve a significant improvement in the intestinal absorption availability of collagen peptide with a less EGT dosage.

[0058] Based on Examples 1 to 3, embodiments of the present invention provide the use of ergothioneine in preparing a collagen peptide intestinal absorption enhancer, a collagen peptide intestinal absorption enhancer, and the use of ergothioneine in promoting intestinal absorption of collagen peptides.

[0059] According to a first aspect of the present invention, some embodiments of the present invention provide the use of ergothioneine in preparing a collagen peptide intestinal absorption enhancer. Specifically, the ergothioneine upregulates the expression of the peptide transporter Pept1 in the intestine, thereby promoting the absorption of the collagen peptide in the intestine.

[0060] According to the second aspect of the present invention, some embodiments of the present invention also provide a collagen peptide intestinal absorption enhancer, which comprises ergothioneine and collagen peptide, and the ergothioneine promotes the absorption of the collagen peptide in the intestine.

[0061] In some embodiments of the present invention, the collagen peptide intestinal absorption enhancer comprises a first dose and a second dose, wherein the first dose comprises ergothioneine and the second dose comprises collagen peptide.

[0062] In some embodiments of the present invention, ergothioneine and collagen peptide are uniformly mixed in the collagen peptide intestinal absorption enhancer.

[0063] In some embodiments of the present invention, the collagen peptide intestinal absorption enhancer is an oral preparation.

[0064] In some embodiments of the present invention, more than 85% of the collagen peptides in the collagen peptide intestinal absorption enhancer have a molecular weight distribution of 200 to 1000 Daltons; and the collagen peptides are derived from collagen peptide powder prepared by enzymatic hydrolysis of animal collagen.

[0065] In some embodiments of the present invention, the mass ratio of ergothioneine to collagen peptide is 0.05:100 to 1:100.

[0066] In another embodiment of the present invention, the mass ratio of ergothioneine to collagen peptide is 0.05:100 to 0.5:100.

[0067] In still another embodiment of the present invention, the mass ratio of ergothioneine to collagen peptide is 0.3:100 to 0.5:100.

[0068] In some embodiments of the present invention, the oral dosage of the collagen peptide intestinal absorption enhancer is: an oral dosage of ergothioneine of 1 to 50 mg / day, and an oral dosage of collagen peptide of 1 to 20,000 mg / day.

[0069] According to a third aspect of the present invention, some embodiments of the present invention also provide an application of ergothioneine to promote intestinal absorption of collagen peptides; this application can be an application for therapeutic purposes or an application for non-therapeutic purposes, and by jointly supplementing ergothioneine and collagen peptides, the intestinal bioavailability of collagen peptides is improved and the efficacy of collagen peptides is promoted.

[0070] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. Application of ergothioneine in the preparation of collagen peptide intestinal absorption enhancer.

2. The use according to claim 1, characterized in that: The ergothioneine upregulates the expression of the peptide transporter Pept1 in the intestine, thereby promoting the absorption of the collagen peptide in the intestine.

3. A collagen peptide intestinal absorption enhancer, characterized in that: The invention comprises ergothioneine and collagen peptide, wherein the ergothioneine promotes the absorption of the collagen peptide in the intestinal tract.

4. The collagen peptide intestinal absorption enhancer according to claim 3, characterized in that: The collagen peptide intestinal absorption enhancer comprises a first dose and a second dose, wherein the first dose comprises the ergothioneine, and the second dose comprises the collagen peptide.

5. The collagen peptide intestinal absorption enhancer according to claim 3, characterized in that: In the collagen peptide intestinal absorption accelerator, the ergothioneine and the collagen peptide are mixed uniformly.

6. The collagen peptide intestinal absorption enhancer according to claim 3, characterized in that: The collagen peptide intestinal absorption accelerator is an oral preparation.

7. The collagen peptide intestinal absorption enhancer according to claim 3, characterized in that: In the collagen peptide intestinal absorption promoter, more than 85% of the collagen peptides have a molecular weight distribution of 200 to 1000 Daltons; and the collagen peptides are derived from collagen peptide powder prepared by enzymatic hydrolysis of animal collagen.

8. The collagen peptide intestinal absorption enhancer according to any one of claim 3, characterized in that: The mass ratio of the ergothioneine to the collagen peptide is 0.05:100 to 1:100; preferably 0.05:100 to 0.5:100; more preferably 0.3:100 to 0.5:

100.

9. The collagen peptide intestinal absorption enhancer according to any one of claims 3 to 8, characterized in that: The oral dosage of the ergothioneine is 1 to 50 mg / day, and the oral dosage of the collagen peptide is 1 to 20000 mg / day.

10. The use of ergothioneine to promote intestinal absorption of collagen peptides, preferably, the use is for non-therapeutic purposes.

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