Method for extracting cyclic dipeptide from mussels, extracted cyclic dipeptide and application thereof

Abstract

The invention discloses a method for extracting cyclic dipeptide from mussels, the extracted cyclic dipeptide and application of the cyclic dipeptide, and belongs to the technical field of food science and engineering. According to the method disclosed by the invention, the problems that the content of cyclic dipeptide in a natural matrix is low, the separation efficiency is poor and a high-purity monomer is difficult to obtain in the prior art are solved through a synergistic purification strategy of double-enzyme step-by-step enzymolysis, organic solvent extraction and binary / ternary silica gel column gradient elution. The method is stable in process, good in reproducibility and high in extraction purity. Cell experiments prove that the obtained cyclic dipeptide (especially cyclic (proline-leucine)) has remarkable antioxidant and anti-aging activity, and a technical support and an experimental basis are provided for developing marine-derived functional foods and health-care products.

Description

Technical Field

[0001] This invention relates to the fields of food science and engineering technology, and in particular to a method for extracting cyclic dipeptides from mussels, the extracted cyclic dipeptides, and their applications. Background Technology

[0002] Cyclic dipeptides, also known as 2,5-diketopiperazine, are the smallest cyclic peptide structures formed by the cyclization of two amino acids through peptide bonds. These compounds form the core active backbone of many natural products and are widely found in microorganisms, marine organisms, and terrestrial plants and animals. Due to their unique rigid structure and conformational constraints, cyclic dipeptides typically exhibit superior chemical stability and biological activity compared to their linear precursors. Numerous studies have demonstrated that cyclic dipeptides possess a variety of significant pharmacological activities, including antibacterial, antitumor, immunomodulatory, and neuroprotective effects. Of particular note is their potent antioxidant potential, which has attracted considerable attention in recent years due to their ability to effectively scavenge free radicals and inhibit lipid peroxidation, showing broad application prospects in delaying cellular aging and preventing related diseases. Therefore, the discovery of novel, highly efficient, and safe cyclic dipeptide compounds from natural resources has become a hot topic in drug development and the development of functional food additives.

[0003] Mussels, bivalve mollusks, are widely distributed along coastal areas worldwide and are an important marine economic species and traditional food source. Since ancient times, mussels have been used in traditional medicine in many countries to treat arthritis, rheumatism, and other diseases related to oxidative stress. Modern nutritional studies have confirmed that mussels are rich in protein, various essential amino acids, unsaturated fatty acids (such as EPA and DHA), and trace elements, making them extremely nutritious. More importantly, as filter feeders, mussels have evolved a powerful antioxidant defense system to cope with the harsh environmental stresses of the intertidal zone, such as high oxygen levels and strong ultraviolet radiation. Their bodies typically contain uniquely structured and highly active secondary metabolites. This makes mussels a highly promising treasure trove of marine bioactive substances, awaiting in-depth development and utilization.

[0004] Although the bioactivity of cyclic dipeptides is widely recognized, and mussels have attracted attention as a source of active substances, existing technologies still have significant gaps and shortcomings. Currently, most research on developing antioxidant active ingredients from mussels focuses on protein hydrolysates or large-molecule linear oligopeptides. These studies have confirmed that crude mussel extracts or peptide mixtures possess certain antioxidant capabilities. However, these preparations are complex in composition, with unclear active monomers, resulting in limited efficacy, difficulty in elucidating the mechanism of action, and challenges in product quality control. To date, no research has been able to directionally isolate and identify cyclic dipeptide monomers with well-defined chemical structures from mussels and systematically verify their antioxidant and anti-aging activities. This lack of research on specific highly active small molecules (such as cyclic dipeptides) severely restricts the in-depth development of high-value-added mussel products. Therefore, there is an urgent need in this field for a method to specifically discover, prepare, and definitively verify the biological efficacy of cyclic dipeptide monomers from mussel resources, in order to solve key problems such as unclear active ingredient targeting, low efficacy levels, and weak application specificity in existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting cyclic dipeptides from mussels, the extracted cyclic dipeptides, and their applications, thereby addressing the problems existing in the prior art. This invention establishes for the first time a method for efficiently separating high-purity cyclic dipeptide monomers from mussels, solving the problems of low separation efficiency and difficulty in obtaining monomers in existing technologies. Cell experiments have confirmed that the obtained cyclic dipeptides (especially the cyclic (proline-leucine)) possess significant antioxidant and anti-aging activities, providing technical support and experimental basis for the development of marine-derived functional foods and health products.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for extracting cyclic dipeptides from mussels, comprising the following steps:

[0008] (1) Mussel meat is hydrolyzed by alkaline protease and flavor enzyme in sequence, and the hydrolysate is prepared into mussel peptide powder.

[0009] (2) Extract the mussel peptide powder obtained in step (1) with a mixed solvent of ethyl acetate and n-butanol. Extract the residue twice with ethyl acetate. Combine the organic phases and concentrate to obtain a lipid-soluble crude extract.

[0010] (3) The lipid-soluble crude extract obtained in step (2) is subjected to gradient elution by column chromatography. The eluent is a binary mixed solvent of ethyl acetate and n-butanol. The elution fraction rich in cyclic dipeptides is collected.

[0011] (4) The fraction obtained in step (3) is subjected to gradient elution by column chromatography again. The eluent is a ternary solvent system consisting of n-hexane, ethyl acetate and n-butanol. After elution, the target cyclic dipeptide is collected.

[0012] Optionally, the volume ratio of ethyl acetate to n-butanol in the mixed solvent in step (2) is 2:1.

[0013] Optionally, the elution fraction rich in cyclic dipeptides in step (3) is obtained by elution with pure ethyl acetate or by a binary mixed solvent of ethyl acetate and n-butanol in a volume ratio of 9:1.

[0014] Optionally, the gradient elution procedure for the ternary solvent system in step (4) includes:

[0015] First, elute with 100% hexane (by volume);

[0016] Then elute with a solvent containing n-hexane and ethyl acetate in a volume ratio of 50:50.

[0017] Optionally, the enzymatic hydrolysis conditions of the alkaline protease in step (1) are pH 9.0 and 50℃ water bath for 2 h; the enzymatic hydrolysis conditions of the flavor protease are pH 6.5 and 50℃ water bath for 1 h.

[0018] The present invention also provides a cyclic dipeptide extracted from mussels, the cyclic dipeptide comprising at least one of cyclic (proline-leucine), cyclic (phenylalanine-proline), cyclic (L-proline-L-valine), cyclic (proline-alanine), and cyclic (proline-tyrosine).

[0019] The present invention also provides the application of the cyclic dipeptide described above in the preparation of products with antioxidant and / or anti-aging functions.

[0020] Optionally, the products include antioxidants, functional foods, dietary supplements, health products, and cosmetics.

[0021] The present invention also provides a product having antioxidant and / or anti-aging functions, wherein the active ingredient includes the cyclic dipeptide described above.

[0022] Optionally, the products include antioxidants, functional foods, dietary supplements, health products, and cosmetics.

[0023] The present invention discloses the following technical effects:

[0024] This invention establishes for the first time a complete technical route for the systematic isolation and purification of cyclic dipeptide monomers and their compositions from mussels. Through a synergistic purification strategy of "two-enzyme stepwise enzymatic hydrolysis—organic solvent extraction—binary / ternary silica gel column gradient elution," it overcomes the difficulties of low cyclic dipeptide content in natural matrices, poor separation efficiency, and difficulty in obtaining high-purity monomers in existing technologies. This method is stable, reproducible, and can efficiently enrich and accurately separate key cyclic dipeptide components, represented by the cyclic (proline-leucine) group, with a purity exceeding 90%, filling the technological gap in the preparation of specific cyclic dipeptide monomers from mussel resources.

[0025] This invention has clearly demonstrated through cell experiments that the extracted cyclic dipeptides (especially the cyclic (proline-leucine) monomers) possess significant antioxidant and anti-aging bioactivity. Experiments show that this component can effectively improve cell survival rate under oxidative stress, inhibit the formation of lipid peroxidation product MDA, alleviate ferroptosis, and reduce cell senescence, providing solid experimental evidence for its application in functional foods, health products, and cosmetics. This invention not only expands the types of marine-derived natural antioxidants but also opens up new technical pathways for the high-value utilization of mussel processing by-products, enhancing the added value and economic benefits of the entire mussel industry. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a process flow diagram for preparing the cyclic dipeptide monomer and composition in Example 1;

[0028] Figure 2 The images shown are thin-layer chromatography (TLC) images of mussel cyclic dipeptides at different purification stages in Example 2. A represents the TLC results of different fractions of the lipid-soluble crude extract after elution with an ethyl acetate / n-butanol gradient, with 1-12 corresponding to gradient stages 1-12 in Table 1. B represents the TLC results of different fractions of the preliminarily purified cyclic dipeptide fraction after elution with a hexane-ethyl acetate-methanol ternary gradient system, with 1-6 corresponding to stages 1-6 in Table 2. C represents the TLC results of the high-purity cyclic dipeptide composition and the cyclic (proline-leucine) monomer; sample 1 is the high-purity cyclic dipeptide composition, sample 2 is a non-peptide lipid-soluble control sample, and sample 3 is the cyclic (proline-leucine) monomer.

[0029] Figure 3The LC-MS total ion chromatograms of the high-purity cyclic dipeptide composition (A) and the cyclic (proline-leucine) monomer (B) in Example 2 are shown.

[0030] Figure 4 This is the secondary mass spectrum of the cyclic (proline-leucine) monomer in Example 2;

[0031] Figure 5 Bar chart showing the effect of cyclic (proline-leucine) monomer and cyclic dipeptide composition on H2O2-induced NIH / 3T3 cell survival (A) and MDA content (B);

[0032] Figure 6 A bar chart showing the effect of cyclic (proline-leucine) monomer and cyclic dipeptide combination on H2O2-induced ferroptosis in NIH / 3T3 cells;

[0033] Figure 7 This is a bar chart showing the effect of a combination of cyclic (proline-leucine) monomers and cyclic dipeptides on the fluorescence of H2O2-induced senescence in NIH / 3T3 cells. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1: Preparation of mussel-derived cyclic dipeptide composition

[0040] The process flow diagram for preparing the cyclic dipeptide composition in this embodiment is shown below. Figure 1 .

[0041] 1. Preparation of mussel peptide powder

[0042] Fresh mussel meat was shelled, cleaned, minced, and homogenized with water. After adjusting the pH to 9.0, 1.8% (w / w) alkaline protease was added, and the mixture was hydrolyzed in a 50°C water bath for 2 hours. Subsequently, the pH was adjusted to 6.5, and 0.5% (w / w) flavor enzyme was added to continue hydrolysis at 50°C for 1 hour. After inactivating the enzyme in a boiling water bath for 15 minutes, the mixture was rapidly cooled in ice water, centrifuged at 8000 r / min for 20 minutes, and the supernatant was collected and spray-dried.

[0043] 2. Extraction of crude mussel cyclic dipeptide

[0044] Take 100g of spray-dried mussel peptide powder, add 50mL of ethyl acetate:n-butanol (2:1, v / v) mixed solvent, vortex to mix, and then sonicate under ice bath conditions for 30 min. Centrifuge at 1000 rpm for 10 min and collect the supernatant. Extract the residue twice with 50 mL of ethyl acetate, combine all supernatants, filter, and concentrate by rotary evaporation to obtain a lipid-soluble crude extract.

[0045] 3. Isolation and purification of mussel cyclic dipeptide mixture

[0046] (1) First silica gel column chromatography (preliminary purification)

[0047] The obtained lipid-soluble crude extract was dissolved in a small amount of ethyl acetate and loaded onto a silica gel column (200-300 mesh) pre-equilibrated with ethyl acetate. Gradient elution was performed sequentially with ethyl acetate / n-butanol volume ratios of 100:0, 90:10, 80:20...up to 0:100, as shown in Table 1. Thin-layer chromatography (TLC) analysis revealed that only the fractions obtained from elution with pure ethyl acetate and 10% n-butanol showed positive spots for ninhydrin after hydrolysis with hydrochloric acid fumigation, while no color was observed before hydrolysis (plate B showed purplish-red spots not seen on plate A), indicating the possible presence of cyclic peptides in the sample. Figure 2 The fractions obtained from the two elution stages (gradient stages 1 and 2, i.e., pure ethyl acetate and 10% n-butanol) were combined to obtain the preliminarily purified cyclic dipeptide component.

[0048] Table 1 Elution gradient of the first silica gel column chromatography

[0049]

[0050] (2) Second silica gel column chromatography (fine purification)

[0051] The preliminarily purified cyclic dipeptide fraction was concentrated and subjected to a second silica gel column chromatography. A ternary gradient elution system of hexane-ethyl acetate-methanol was used. The elution program was as follows: first, 100% hexane; then hexane / ethyl acetate (50:50, v / v); followed by 100% ethyl acetate; then ethyl acetate / n-butanol (98:2, 95:5, 90:10, v / v) gradient elution, as shown in Table 2. The fraction eluted with pure hexane was collected and rotary dried to obtain the cyclic (proline-leucine) monomer. The fraction appearing under the hexane / ethyl acetate (50:50, v / v) elution conditions was rotary dried to obtain a high-purity cyclic dipeptide composition. Figure 2 B).

[0052] Table 2 Elution gradient of the second silica gel column chromatography

[0053]

[0054] Example 2: Identification of components in a cyclic dipeptide composition

[0055] The mussel peptide powder, crude extract of mussel cyclic dipeptide, preliminarily purified cyclic dipeptide components, and high-purity cyclic dipeptide composition from Example 1 were analyzed by TLC and LC-MS.

[0056] (1) Thin-layer chromatography detection

[0057] The analyte was spotted onto three silica gel G plates (100mm × 100mm, plate A, plate B, and plate C), using chloroform / methanol (9:1 v / v) as the developing solvent. After development, the solvent was evaporated. Plate A was allowed to stand, while plates B and C were suspended above a sealed ground glass jar (acid- and heat-resistant) containing approximately 1.5 mL of concentrated hydrochloric acid at the bottom. The plates were then heated in an oven at 110°C for 1 hour for hydrolysis before removal. Plates A and B were sprayed with 0.2% ninhydrin-acetone reagent and heated in an oven for 5 minutes for color development. Based on the color development results, if plate B showed purple-red or yellow spots, while plate A showed no spots at the corresponding positions, it indicated that the analyte might contain a cyclic peptide.

[0058] The non-peptide lipid-soluble reference standard, the high-purity cyclic dipeptide composition from Example 1, and the cyclic (proline-leucine) monomer were analyzed by TLC. The results are as follows: Figure 2 As shown in C, non-peptide lipid-soluble reference standards did not develop color in this TLC method, while cyclic (proline-leucine) monomers and cyclic dipeptide compositions both produced cyclic peptide spots after HCl hydrolysis.

[0059] (2) LC-MS detection

[0060] Chromatographic conditions: C18 column, water-acetonitrile gradient elution. A C18 column (2.1 mm × 150 mm, 1.7 μm) was used. The injection volume was 10 μL, the flow rate was 0.3 mL / min, and the mobile phase was water (A)-acetonitrile (B). The gradient elution program was 0 min 50% B; 20 min 90% B; 21 min 90% B; 22 min 50% B. The column oven was set to 40℃.

[0061] Mass spectrometry conditions: Electrospray ionization (ESI) source in positive ion mode, full scan. Ion transmission tube temperature 300 ℃, desolvation temperature 250 ℃, scan range 200-1200 m / z, spray voltage 3500 V, maximum injection time 100 ms; collision-induced dissociation (CID) mode was used for secondary mass spectrometry analysis, with a collision energy of 30 eV.

[0062] Forty cyclic dipeptide molecules were identified in the high-purity cyclic dipeptide composition. The total ion chromatogram is shown below. Figure 3 A. Specific components include various cyclic dipeptides such as cyclic (proline-leucine), cyclic (phenylalanine-proline), cyclic (L-proline-L-valine), cyclic (proline-alanine), and cyclic (proline-tyrosine), as shown in Table 3. The total ion chromatogram of cyclic (proline-leucine) is shown in [reference needed]. Figure 3 B has a m / z of 245.1398 [M+H]. +The monomer was confirmed to be a cyclic (proline-leucine) compound by high-resolution mass spectrometry and secondary mass spectrometry fragment ion analysis (m / z 120.1 and 85.0). Secondary identification results are shown below. Figure 4 Table 4 shows a comparison of the peak areas of the purified ring (proline-leucine). Based on the calculation of the relative content, it is proven that the purity of the ring (proline-leucine) is above 90%.

[0063] Table 3. LC-MS Detection Results of High-Purity Cyclic Dipeptide Composition from Mussels

[0064]

[0065] Table 4. Peak area detection results of mussel ring (proline-leucine) by LC-MS

[0066]

[0067]

[0068]

[0069] Example 3 Evaluation of the antioxidant and anti-aging activities of cyclic dipeptide monomers and compositions

[0070] 1. Cell Culture

[0071] Mouse embryonic fibroblasts (NIH / 3T3) were selected as the experimental cell model. Cells were cultured in complete culture medium prepared with DMEM basal medium, 10% fetal bovine serum (FBS), and a 1% penicillin-streptomycin-neomycin antibiotic mixture. Cells were cultured at 37°C in a 5% CO2 incubator, with the complete culture medium replaced every other day. When cell confluence reached 80%–90%, cells were passaged at a ratio of 1:6. All experiments used cells cultured to passages 10–30.

[0072] 2. Cytotoxicity test

[0073] The potential toxicity of cyclic dipeptide samples to NIH / 3T3 cells was assessed using the CCK-8 assay. The specific steps are as follows:

[0074] NIH / 3T3 cells in logarithmic growth phase were used at a density of 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 100 μL in each well of a 96-well plate. The cells were incubated at 37°C and 5% CO2 for 24 h to allow for full cell adhesion.

[0075] The original culture medium was discarded, and the experimental groups were respectively added with complete culture medium containing different concentrations of cyclic dipeptide samples (cyclic (proline-leucine) monomers and high-purity cyclic dipeptide compositions prepared in Example 1). The final sample concentrations were set to 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / mL. A separate blank control group was set up with complete culture medium without cyclic dipeptide samples.

[0076] After culturing for another 24 hours, 10 μL of CCK-8 solution was added to each well, and incubation was continued for another hour under controlled conditions. The absorbance of each well was then measured at 450 nm using a microplate reader. Cell viability was calculated using the formula (OD value of experimental group / OD value of blank control group) × 100%. Based on the results, a safe and effective concentration (e.g., 5 μg / mL) was determined for subsequent experiments.

[0077] 3. Assay of cellular antioxidant activity

[0078] 3.1 Construction of the oxidative damage model and sample processing

[0079] NIH / 3T3 cells were randomly divided into the following four groups:

[0080] Normal control group: No oxidative stress or sample treatment was performed.

[0081] Model group: treated with only 400 μM hydrogen peroxide (H2O2) for 4 h.

[0082] Sample pretreatment groups: including cyclic (proline-leucine) monomer pretreatment group and cyclic dipeptide composition pretreatment group.

[0083] All cells were spaced at 1×10⁻⁶ per well. 5 Samples were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 hours until adherence. The pretreatment group was then replaced with complete culture medium containing 5 μg / mL of the corresponding cyclic dipeptide sample, and pretreated for another 24 hours. Afterward, except for the normal control group, both the model group and the pretreatment group were replaced with culture medium containing 400 μM H2O2 to induce oxidative stress for 4 hours.

[0084] 3.2 Cell viability detection

[0085] After oxidative stress induction, cell viability was assessed using the CCK-8 assay (procedure as in "2. Cytotoxicity Assay"). Results are as follows: Figure 5 As shown in Figure A, the cell survival rate in the model group decreased to approximately 63%. However, the cell survival rate of cells pretreated with a 5 μg / mL cyclic (proline-leucine) monomer and cyclic dipeptide combination significantly increased to over 84%, indicating that both have a significant protective effect against H2O2-induced cellular oxidative damage.

[0086] 3.3 Detection of lipid peroxidation level

[0087] The antioxidant activity of cells was characterized using the malondialdehyde (MDA) assay. The principle of MDA is that some fatty acids, upon oxidation, gradually decompose into a series of complex compounds, including MDA. Therefore, the level of lipid oxidation can be detected by measuring the level of MDA; hence, MDA measurement is widely used as an indicator of lipid oxidation.

[0088] Intracellular lipid peroxidation was assessed using a malondialdehyde (MDA) assay kit. Cell grouping and treatment were the same as in section "3.1". After oxidative stress induction, cells were lysed and reacted according to the kit instructions, and the absorbance at 532 nm was measured to calculate the MDA content.

[0089] The results are as follows Figure 5 As shown in Figure B, the intracellular MDA content in the model group was significantly increased compared to the normal control group. However, compared to the model group, the intracellular MDA content in both the 5 μg / mL cyclic dipeptide composition and the cyclic (proline-leucine) monomer treatment group was significantly reduced, with the cyclic (proline-leucine) monomer treatment group even recovering to levels close to the normal control group. This demonstrates that both have good anti-lipid peroxidation capabilities, and the cyclic (proline-leucine) monomer is more effective than the cyclic dipeptide composition.

[0090] 4. Assay for Cellular Anti-aging Activity

[0091] 4.1 Inhibition of ferroptosis

[0092] A cell ferroptosis model was established using the ferroptosis inducer RSL3.

[0093] NIH / 3T3 cells were distributed at a rate of 1×10⁶ cells per well. 5 Individual samples were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 h. The experimental groups were pretreated with 5 μg / mL of either a cyclic (proline-leucine) monomer or a cyclic dipeptide combination for 24 h. Subsequently, all groups (except the normal control group) were stimulated with 2 μM RSL3 for 12 h to induce ferroptosis.

[0094] After induction, lipid peroxidation levels were detected using the Lipid Peroxidation Probe - BDP 581 / 591 C11 reagent. Results are as follows: Figure 6 As shown, compared with the RSL3 model group, the lipid peroxidation fluorescence signal of cells pretreated with cyclic dipeptide samples was significantly weakened, indicating that the cyclic (proline-leucine) monomer and the cyclic dipeptide combination have significant inhibitory activity on RSL3-induced ferroptosis in NIH / 3T3 cells, and the effect of the cyclic (proline-leucine) monomer is more obvious.

[0095] 4.2 Inhibitory effect on cell senescence

[0096] A cell senescence model was established using D-galactose induction.

[0097] Cell seeding was the same as in "4.1". The experimental groups were pretreated with 5 μg / mL of either a cyclic (proline-leucine) monomer or a cyclic dipeptide combination for 24 h. Subsequently, all groups (except the normal control group) were stimulated with 100 mM D-galactose for another 12 h to induce cell senescence.

[0098] After induction, the CellEvent™ Senescence Green Detection Kit was used for detection, strictly following the instructions. The green fluorescence signal (representing aging-related β-galactosidase activity) was observed and quantified under a fluorescence microscope. Results are as follows: Figure 7 As shown, compared with the D-galactose model group, the green fluorescence signal of the cyclic (proline-leucine) monomer and cyclic dipeptide combination treatment groups was significantly reduced, proving that both can effectively reduce the degree of cell aging and have a clear anti-aging effect. Moreover, the effect of the cyclic (proline-leucine) monomer is more obvious than that of the cyclic dipeptide combination.

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method of extracting cyclic dipeptides from mussels, characterized in that, It comprises the following steps: (1) The mussel meat is sequentially subjected to alkaline protease and flavor enzyme hydrolysis, and the hydrolyzate is prepared into mussel peptide powder; (2) The mussel peptide powder obtained in step (1) is extracted with a mixed solvent of ethyl acetate and n-butanol, the residue is extracted twice with ethyl acetate, and the organic phase is concentrated to obtain a fat-soluble crude extract; (3) The fat-soluble crude extract obtained in step (2) is subjected to gradient elution by column chromatography, and the eluent is a binary mixed solvent of ethyl acetate and n-butanol, and the elution fraction rich in cyclic dipeptides is collected; (4) The fraction obtained in step (3) is subjected to gradient elution by column chromatography again, and the eluent is a ternary solvent system composed of n-hexane, ethyl acetate and n-butanol, and the target cyclic dipeptide is collected after elution.

2. The method of claim 1, wherein, The volume ratio of ethyl acetate to n-butanol in the mixed solvent in step (2) is 2:

1.

3. The method of claim 1, wherein, The elution fraction rich in cyclic dipeptides in step (3) is obtained by elution with pure ethyl acetate or a binary mixed solvent of ethyl acetate and n-butanol with a volume ratio of 9:

1.

4. The method of claim 1, wherein, The gradient elution program of the ternary solvent system in step (4) comprises: First elute with n-hexane with a volume fraction of 100%; Then elute with a solvent with a volume ratio of n-hexane to ethyl acetate of 50:

50.

5. The method of claim 1, wherein, The alkaline protease in step (1) is subjected to hydrolysis at a pH value of 9.0 in a 50℃ water bath for 2 hours; the flavor protease is subjected to hydrolysis at a pH value of 6.5 in a 50℃ water bath for 1 hour.

6. A cyclic dipeptide extracted from mussels, characterized in that, The cyclic dipeptide comprises at least one of cyclic (proline-leucine), cyclic (phenylalanine-proline), cyclic (L-proline-L-valine), cyclic (proline-alanine) and cyclic (proline-tyrosine).

7. Use of the cyclic dipeptide of claim 6 in the preparation of a product with antioxidant and / or anti-aging function.

8. Use according to claim 7, characterized in that, The product includes antioxidants, functional foods, dietary supplements, health products and cosmetics.

9. A product having an antioxidant and / or anti-aging function, characterized by, The active ingredient includes the cyclic dipeptide of claim 6.

10. Use according to claim 9, characterized in that, The product includes antioxidants, functional foods, dietary supplements, health products and cosmetics.

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