An antioxidative, hypoglycemic and tyrosinase-inhibiting multifunctional peptide derived from bovine serum albumin and preparation and application thereof

Abstract

This invention discloses a multifunctional peptide derived from bovine serum albumin, possessing antioxidant, hypoglycemic, and tyrosinase-inhibiting properties, as well as its preparation and applications, belonging to the fields of food, biopharmaceutical, and cosmetic technologies. This invention screened and verified a novel multifunctional peptide, SFLYEYSRR, with a defined sequence. SFLYEYSRR exhibits a maximum ABTS scavenging rate of 89.93%, a maximum DPPH free radical scavenging rate of 35.48%, a maximum hydroxyl free radical scavenging rate of 46.49%, an α-glucosidase inhibition rate of 64.36%, and a maximum tyrosinase inhibition activity of 48.33%, overcoming the limitations of existing single-functional peptides in application.

Description

Technical Field

[0001] This invention relates to a multifunctional peptide derived from bovine serum albumin that has antioxidant, hypoglycemic, and tyrosinase-inhibiting properties, as well as its preparation and application, belonging to the fields of food, biopharmaceutical, and cosmetic technologies. Background Technology

[0002] In modern society, unhealthy lifestyles, environmental pollution, aging, and various diseases lead to an excess of free radicals in the body, triggering oxidative stress. Oxidative stress is an important pathophysiological mechanism in the occurrence and development of many chronic diseases, including but not limited to cardiovascular diseases, neurodegenerative diseases, diabetes, cancer, and skin aging. For a long time, people have been actively searching for natural or synthetic antioxidants that can effectively scavenge free radicals and reduce oxidative damage in order to prevent and treat these diseases closely related to oxidative stress.

[0003] Diabetes, especially type 2 diabetes, has become an increasingly serious public health challenge worldwide. Its main characteristic is disordered glucose metabolism, leading to persistently elevated blood glucose levels. Long-term hyperglycemia not only damages the function of pancreatic beta cells but also triggers systemic complications such as diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, cardiovascular and cerebrovascular diseases, and delayed wound healing, severely impacting patients' quality of life and imposing a significant economic burden. Currently, diabetes treatments mainly include lifestyle interventions, oral hypoglycemic agents, and insulin injections; however, these methods often have limitations such as poor efficacy, numerous side effects, and poor adherence. Therefore, developing safe, effective, and low-side-effect-prone new strategies for lowering blood glucose, especially natural active ingredients that can intervene in glucose metabolism from multiple aspects, has significant clinical and research value.

[0004] Furthermore, in the field of skin health and beauty, abnormal pigment metabolism (such as melasma and post-inflammatory hyperpigmentation) is a common concern. Tyrosinase is a key rate-limiting enzyme in the melanin biosynthesis pathway, and its overactivation is the core mechanism leading to excessive pigmentation. Existing tyrosinase inhibitors (such as hydroquinone), while effective, are often accompanied by safety issues such as skin irritation and cytotoxicity. Therefore, the development of novel, highly effective, and safe natural tyrosinase inhibitors has urgent market demand and application value.

[0005] Whey protein is a major byproduct of cheese production and is a mixture of various proteins, including α-lactalbumin, β-lactoglobulin, and bovine serum albumin, among others. Through enzymatic hydrolysis or microbial fermentation, various peptides with potential physiological activity, such as antioxidant peptides, antimicrobial peptides, and antihypertensive peptides, can be released from whey protein, demonstrating significant application potential in the food, health product, and pharmaceutical fields.

[0006] Given the above background, there is an urgent need to develop a novel whey protein peptide with synergistic antioxidant, hypoglycemic, and tyrosinase inhibitory activities. This peptide should not only possess significant free radical scavenging capabilities and protect cells from oxidative damage, but also effectively regulate blood glucose levels, improve insulin resistance, and fundamentally prevent or alleviate chronic diseases associated with oxidative stress and hyperglycemia. Summary of the Invention

[0007] [Technical Issues]

[0008] Existing technologies mostly focus on improving the hydrolysis degree or general biological activity (such as antioxidation) of bioactive peptides, lacking targeted release strategies for multifunctional peptides with antioxidant, hypoglycemic, and tyrosinase inhibitory activities.

[0009] [Technical Solution]

[0010] To address the aforementioned problems, this invention first provides a multifunctional peptide SFLYEYSRR derived from bovine serum albumin in whey protein, which has antioxidant, hypoglycemic, and tyrosinase inhibitory activities. The amino acid sequence of the multifunctional peptide SFLYEYSRR is Ser-Phe-Leu-Tyr-Glu-Tyr-Ser-Arg-Arg.

[0011] This invention also provides the application of the multifunctional peptide SFLYEYSRR in the preparation of food or health products.

[0012] In one embodiment, the food product includes dairy products, fruit and vegetable products, meat products, or soy products.

[0013] This invention also provides the application of the multifunctional peptide SFLYEYSRR in the preparation of antioxidant products.

[0014] In one embodiment, the product includes pharmaceuticals or daily chemical products.

[0015] In one embodiment, the daily chemical products include skin care products or cosmetics.

[0016] In one embodiment, the dosage form of the drug includes tablets, capsules, injections, liposome nanoparticles, sustained-release formulations, or dispersible tablets.

[0017] In one embodiment, the pharmaceutical product comprises one or more of the following: resistant dextrin, mixed fruit and vegetable powder, fructooligosaccharides, probiotic freeze-dried powder, maltodextrin, citric acid, malic acid, steviol glycosides, chromium picolinate, silicon dioxide, and flavoring.

[0018] In one embodiment, the daily chemical products include skin care products or cosmetics.

[0019] In one embodiment, the dosage form of the cosmetic includes solutions, emulsions, creams, gels, powders, aerosols, wax-based agents, or suspensions.

[0020] In one embodiment, the cosmetic includes serum, lotion, cream, or soluble essence powder.

[0021] In one embodiment, the cosmetic comprises one or more of the following: glycerin, dipropylene glycol, niacinamide, acetylglucosamine, xanthan gum, ascorbyl glucoside, caprylic / capric triglyceride, polydimethylsiloxane, cetearyl oleate, sorbitan oleate, tocopheryl acetate, ceramide, phenoxyethanol, and ethylhexylglycerin.

[0022] The present invention also provides the use of the multifunctional peptide SFLYEYSRR in the preparation of pharmaceuticals that help maintain healthy blood glucose levels.

[0023] This invention also provides the application of the multifunctional peptide SFLYEYSRR in the preparation of daily-use products for whitening and fading dark spots.

[0024] In one embodiment, the daily-use product includes skincare or cosmetic products.

[0025] Beneficial effects:

[0026] This invention screened and verified a novel multifunctional peptide, SFLYEYSRR, with a defined sequence. The SFLYEYSRR peptide exhibits a maximum ABTS scavenging rate of 89.93%, a maximum DPPH radical scavenging rate of 35.48%, a maximum hydroxyl radical scavenging rate of 46.49%, an α-glucosidase inhibition rate of 64.36%, and a maximum tyrosinase inhibition activity of 48.33%. This overcomes the limitations of existing single-functional peptides in application. Attached Figure Description

[0027] Figure 1 The in vitro hypoglycemic activity (α-glucosidase inhibition rate) of the pure peptide SFLYEYSRR.

[0028] Figure 2 The in vitro antioxidant activity of the pure peptide SFLYEYSRR is evaluated. (A) Scavenging rate of DPPH; (B) Scavenging rate of ABTS; (C) Scavenging ability of hydroxyl radicals.

[0029] Figure 3 The in vitro tyrosinase inhibitory activity of the pure peptide SFLYEYSRR was measured.

[0030] Figure 4 Molecular docking simulation of the pure peptide SFLYEYSRR (Kelch domain of Keap1, a key regulatory protein for antioxidants). Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below, but the scope of protection of the present invention is not limited to the scope described in the embodiments.

[0032] Example 1: In vitro hypoglycemic activity of pure peptide SFLYEYSRR

[0033] The peptide SFLYEYSRR was synthesized using a solid-phase method. Solutions of SFLYEYSRR at concentrations of 0.1, 0.5, 1, 2.5, and 5 mg / mL were prepared using PB buffer (0.1 mM, pH 6.8). 40 μL of PB buffer, 20 μL of samples at different concentrations (PB was used as a blank), and 10 μL of α-glucosidase (1 U / mL) were added to each well of a 96-well plate and incubated at 37°C for 15 min. Then, 50 μL of PNPG (1 mM) was added, mixed, and incubated at 37°C in the dark for 20 min. After incubation, 100 μL of NaCO3 (1 M) was added to each well of the 96-well plate. The absorbance was measured at 405 nm.

[0034] α-glucosidase inhibition rate (%) = (A0 - A1) / A0 × 100%; where A1 is the absorbance value of the experimental group and A0 is the absorbance value of the control group.

[0035] Conclusion: From Figure 1 The inhibition rate of the pure peptide SFLYEYSRR against α-glucosidase was concentration-dependent within the concentration range of 0.1-5 mg / mL, with the inhibition rate increasing with increasing peptide concentration. The inhibition rate reached its highest level (64.36%) at a concentration of 5 mg / mL. These results indicate that the pure peptide SFLYEYSRR possesses definite in vitro hypoglycemic activity.

[0036] Example 2: In vitro antioxidant activity of pure peptide SFLYEYSRR

[0037] The peptide SFLYEYSRR was synthesized artificially in a solid phase. Aqueous solutions of SFLYEYSRR at different concentrations were prepared. The in vitro antioxidant activity of the peptide SFLYEYSRR was detected by DPPH radical scavenging rate, ABTS radical scavenging rate and hydroxyl radical scavenging ability.

[0038] Test method:

[0039] (1) DPPH free radical scavenging rate of pure peptide SFLYEYSRR

[0040] 0.5 mL of peptide SFLYEYSRR at concentrations of 0.1, 0.5, 1, 2.5, and 5 mg / mL (dissolved in pure water) was thoroughly mixed with 0.5 mL of DPPH solution (0.1 mM, dissolved in anhydrous ethanol) and incubated at 37°C in the dark for 30 min. The absorbance was then measured at 517 nm. Pure water was used as a blank control instead of the sample solution.

[0041] DPPH free radical scavenging rate (%) = (A0 - A1) / A0 × 100%; where A1 is the absorbance value of the experimental group and A0 is the absorbance value of the control group.

[0042] like Figure 2 As shown in (A), the pure peptide SFLYEYSRR exhibits a clear scavenging ability against DPPH free radicals in a concentration-dependent manner. At a concentration of 5 mg / mL, the scavenging rate reaches 35.48%.

[0043] (2) ABTS free radical scavenging rate of pure peptide SFLYEYSRR

[0044] The ABTS stock solution was diluted with PBS (50 mM, pH 7.4) to an absorbance of 0.7 ± 0.02 at 734 nm to obtain the ABTS working solution. 10 µL of 0.1, 0.5, 1, 2.5, and 5 mg / mL peptide SFLYEYSRR was thoroughly mixed with 990 µL of ABTS working solution and incubated at room temperature in the dark for 6 min. The absorbance was measured at 734 nm. Pure water was used as a blank control instead of the sample solution.

[0045] ABTS free radical scavenging rate (%) = (A0 - A1) / A0 × 100%; where A1 is the absorbance value of the experimental group and A0 is the absorbance value of the blank group.

[0046] like Figure 2 As shown in (B), the pure peptide has a more significant scavenging effect on ABTS free radicals, which is also concentration-dependent. At a concentration of 5 mg / mL, the scavenging rate reaches 89.93%.

[0047] (3) Hydroxyl radical scavenging ability of pure peptide SFLYEYSRR

[0048] 0.5 mL of 0.5, 1, and 2.5 mg / mL peptide SFLYEYSRR (dissolved in pure water) was thoroughly mixed with 0.5 mL of FeSO4 solution (9 mM) and 0.5 mL of H2O2 (8.8 mM); the mixture was incubated at 37°C in the dark for 10 min; then 0.5 mL of salicylic acid-ethanol solution (9 mM) was added; the mixture was incubated at 37°C in the dark for another 30 min; finally, the absorbance was measured at 510 nm. Pure water was used instead of the sample solution, and the remaining procedures were exactly the same as described above, serving as a blank control.

[0049] Hydroxyl radical scavenging capacity (%) = (A0 - A1) / A0 × 100%; where A1 is the absorbance value of the experimental group and A0 is the absorbance value of the blank group.

[0050] like Figure 2 As shown in (C), the scavenging ability of this pure peptide against hydroxyl radicals exhibits a linear concentration-dependent relationship. Within the tested concentration range, the scavenging rate peaks at 2.5 mg / mL, reaching 46.49%.

[0051] Conclusion: From Figure 2 It was found that, within the tested concentration range, the pure peptide SFLYEYSRR exhibited certain scavenging effects against DPPH radicals, ABTS radicals, and hydroxyl radicals, with DPPH radical scavenging activity, ABTS radical scavenging activity, and hydroxyl radical scavenging capacity reaching 35.48%, 89.93%, and 46.49%, respectively. Its antioxidant activity may be attributed to its amino acid composition, with the phenolic hydroxyl group of tyrosine (Y) potentially being an important antioxidant group.

[0052] Example 3: In vitro tyrosinase inhibitory activity of the pure peptide SFLYEYSRR

[0053] The artificially synthesized peptide SFLYEYSRR was used to prepare phosphate-buffered saline (PBS) solutions of SFLYEYSRR at different concentrations. The in vitro tyrosinase inhibitory activity of the peptide SFLYEYSRR was detected by measuring the tyrosinase inhibition rate.

[0054] The purified peptide SFLYEYSRR was prepared into solutions of 1, 2.5, and 5 mg / mL using phosphate-buffered saline (PBS, 50 mM, pH 6.8) for use as experimental samples. A 1 mM kojic acid solution was also prepared as a positive control sample.

[0055] The specific experimental steps are as follows:

[0056] Sample group: In a 96-well plate, add 100 μL of sample solutions of different concentrations dissolved in PBS (or PBS buffer as a control) and 50 μL of tyrosinase solution dissolved in PBS (enzyme activity 400 U / mL) sequentially, and incubate at 37°C in the dark for 10 min. Then, add 200 μL of levodopa solution (0.5 mM, dissolved in 50 mM PBS, pH 6.8) to each well, mix well, and continue incubating at 37°C in the dark for 15 min.

[0057] Sample blank group: Same as the sample group, except that PBS buffer is used instead of tyrosinase solution.

[0058] Enzyme reaction control group: Same as the sample group, except that PBS buffer is used instead of the sample solution.

[0059] Reagent blank group: Same as the sample group, except that PBS buffer is used instead of sample solution and tyrosinase solution.

[0060] Immediately after the reaction, the absorbance (OD) value of each well was measured at a wavelength of 475 nm using an ELISA reader.

[0061] The formula for calculating the tyrosinase inhibition rate (%) is: R = [1 - (OD)] D - OD C ) / (OD B - OD A ) ] ×100%. The experiment consisted of four groups to calculate the inhibition rate: OD A (Reagent blank): Buffer solution + levodopa, OD B (Enzyme reaction control): Buffer (in place of sample) + Tyrosinase + L-DOPA, OD C (Sample blank): Sample + buffer (instead of enzyme) + L-DOPA, OD D (Sample group): Sample + Tyrosinase + Levodopa.

[0062] Conclusion: From Figure 3 The results showed that the inhibitory activity of the pure peptide SFLYEYSRR against tyrosinase in the concentration range of 1-5 mg / mL was significantly concentration-dependent, with the inhibition rate increasing with increasing peptide concentration. At the highest tested concentration of 5 mg / mL, the inhibition rate reached 48.33%, exceeding that of 1 mM kojic acid (positive control, 47.35%). These results indicate that the pure peptide SFLYEYSRR possesses certain in vitro tyrosinase inhibitory activity.

[0063] Example 4: Molecular docking simulation of pure peptide SFLYEYSRR (based on the key antioxidant target Keap1)

[0064] To further explore the molecular mechanism of its antioxidant activity, molecular docking technology was used to simulate the interaction between the pure peptide SFLYEYSRR and the Kelch domain of Keap1, a key regulatory protein for antioxidant activity. The docking results showed that the peptide could stably embed into the Kelch domain pocket of the Keap1 protein, and the two exhibited a strong binding affinity.

[0065] The specific quantitative binding parameters are as follows: the binding free energy (CDOCKER_ENERGY) is -150.283 kcal / mol, and the interaction energy (CDOCKER_INTERACTION_ENERGY) is -101.01 kcal / mol, indicating that the two are very stable in combination.

[0066] Table 1. Energy for docking of peptide SFLYEYSRR with Keap1

[0067]

[0068] Analysis from the mode of action (e.g.) Figure 4 As shown in the figure), the SFLYEYSRR peptide mainly binds to the active pocket of Keap1 through a series of intricate non-covalent interactions: (1) Hydrogen bond network: Multiple amino acid residues in the peptide chain (such as arginine ARG, serine SER, etc.) form multiple hydrogen bonds with key amino acids in the Kelch domain (such as tyrosine TYR, etc.) (green dashed lines in the figure). These hydrogen bonds are the key to determining the binding specificity and directionality. (2) Hydrophobic interaction and van der Waals force: The hydrophobic part of the peptide chain is in close contact with the hydrophobic region in the pocket, generating extensive van der Waals force, which provides the main driving force and stability for binding. (3) Electrostatic interaction: The positively charged arginine (ARG) residues in the peptide may form favorable electrostatic attraction with the negatively charged amino acid environment in the pocket.

[0069] Technical Effects and Mechanism Explanation: The high negative binding energy and detailed interaction patterns described above indicate that the pure peptide SFLYEYSRR can competitively bind to the Kelch domain of Keap1 with high affinity. Under physiological conditions, this may interfere with the normal binding of Keap1 to the transcription factor Nrf2, thereby preventing Nrf2 from being ubiquitinated and degraded, and subsequently translocating to the cell nucleus to initiate the expression of a series of downstream antioxidant enzymes (such as HO-1, NQO1, etc.).

[0070] Example 5: Preparation of an anti-glycation brightening serum containing the multifunctional peptide SFLYEYSRR

[0071] Accurately weigh all raw materials required for the aqueous phase, including deionized water, glycerol, dipropylene glycol, nicotinamide, acetylglucosamine, and xanthan gum. Then, slowly stir and heat to 75-80°C until completely dissolved and homogeneous. Accurately weigh all raw materials required for the oil / emulsion phase, including caprylic / capric triglycerides, polydimethylsiloxane, cetearyl oleate / sorbitan oleate, tocopheryl acetate, and ceramide NP. Place the oil phase components in another container and heat to 75-80°C to melt and mix thoroughly. Once both the aqueous and oil phases have reached the correct temperature and are homogeneous, slowly pour the oil phase into the aqueous phase under high-speed shear (approximately 3000 rpm), continuing homogenization for 5-8 minutes until a homogeneous and stable emulsion is formed. Dissolve the multifunctional peptide SFLYEYSRR and ascorbate glucoside (AA2G) together in a small amount of deionized water to prepare an active ingredient premix. Once the emulsion has cooled to below 40°C, the active ingredient premix and phenoxyethanol / ethylhexylglycerin preservative are slowly and gradually added to the matrix while maintaining gentle and continuous stirring. Finally, the pH of the system is adjusted to 5.5-6.0 with citric acid solution, and after continuous stirring and cooling to room temperature (approximately 30°C), the mixture is discharged and filled.

[0072] The essence contains the following components: deionized water (balance), glycerin (1.0%-10.0%), dipropylene glycol (1.0%-5.0%), niacinamide (1.0%-5.0%), acetylglucosamine (1.0%-3.0%), xanthan gum (0.1%-2.0%), multifunctional peptide SFLYEYSRR (0.1%-7.0%), ascorbyl glucoside (AA2G) (0.1%-3.0%), caprylic / capric triglycerides (2.0%-15.0%), polydimethylsiloxane (1.0%-10.0%), cetearyl oleate / sorbitan oleate (1.0%-5.0%), tocopheryl acetate (0.1%-3.0%), ceramide NP (0.1%-3.0%), and phenoxyethanol / ethylhexylglycerin (0.5%-1.2%). All percentages refer to mass percentages.

[0073] Example 6: Preparation of a blood glucose management solid beverage containing the multifunctional peptide SFLYEYSRR and probiotics

[0074] The multifunctional peptide SFLYEYSRR powder was premixed with maltodextrin, resistant dextrin, mixed fruit and vegetable powder, fructooligosaccharides, citric acid, malic acid, steviol glycosides, chromium picolinate, silicon dioxide, and mixed berry flavoring. The premixing employed an incremental, equal-volume method: first, SFLYEYSRR peptide powder was mixed with an equal volume of maltodextrin for 5 minutes. Then, trace amounts of chromium picolinate, steviol glycosides, citric acid, and malic acid were added sequentially, mixing for 5-10 minutes after each addition to ensure uniform distribution. Subsequently, resistant dextrin, mixed fruit and vegetable powder, fructooligosaccharides, and the remaining maltodextrin were added and mixed for 10 minutes. Finally, under conditions of ambient temperature below 25℃ and relative humidity ≤45%, the lyophilized probiotic powder was slowly added to the premix and mixed for 20-30 minutes until the color and texture were completely homogeneous. The uniformly mixed powder was then sieved through a 60-mesh sieve to obtain a homogeneous product with good flowability. The sieved powder is then sealed in an automatic powder packaging machine with nitrogen filling in an environment with a relative humidity of no more than 45%. High-barrier packaging materials such as aluminum foil bags are used to prevent moisture and oxidation, and to maximize the activity of probiotics.

[0075] In the powder, the multifunctional peptide SFLYEYSRR accounts for 0.1%-5% of the total mass of the mixture (added according to the reconstitution ratio), resistant dextrin accounts for 2.0%-50.0%, mixed fruit and vegetable powder (sugar-free type) accounts for 1.0%-30.0%, fructooligosaccharides (prebiotics) account for 1.0%-15.0%, and probiotic freeze-dried powder (optionally Bifidobacterium lactis BL-04 and Lactobacillus acidophilus NCFM, viable count ≥1 × 10⁻⁶) 11 The composition includes: 1.0%-10.0% (CFU / g), maltodextrin as the balance, 0.1%-3.0% citric acid, 0.2%-1.5% malic acid, 0.01%-0.1% steviol glycosides (substitute for sucralose), 0.01%-0.1% chromium pyridinecarboxylate, 0.1%-1.5% silicon dioxide, and 0.1%-1.0% mixed berry flavoring. All percentages refer to mass percentages.

[0076] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Application of the multifunctional peptide SFLYEYSRR in food preparation.

2. The application as described in claim 1, characterized in that, The food products include dairy products, fruit and vegetable products, meat products, or soy products.

3. Application of the multifunctional peptide SFLYEYSRR in the preparation of antioxidant health products.

4. Application of the multifunctional peptide SFLYEYSRR in the preparation of skin care products.

5. Application of the multifunctional peptide SFLYEYSRR in the preparation of cosmetics.

6. The application as described in claim 5, characterized in that, The dosage forms of the cosmetics include solutions, emulsions, creams, gels, powders, aerosols, wax-based agents, or suspensions.

7. The application as described in claim 6, characterized in that, The cosmetics include serums, lotions, creams, or soluble essence powders; the cosmetics contain one or more of the following: glycerin, dipropylene glycol, niacinamide, acetylglucosamine, xanthan gum, ascorbyl glucoside, caprylic / capric triglyceride, polydimethylsiloxane, cetearyl oleate, sorbitan oleate, tocopheryl acetate, ceramide, phenoxyethanol, and ethylhexylglycerin.

8. Application of the multifunctional peptide SFLYEYSRR in the preparation of health supplements that help maintain healthy blood sugar levels.

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