Peony seed meal-derived tyrosinase inhibitory peptide and preparation method thereof

Abstract

The present invention provides a tyrosinase inhibitory peptide derived from peony seed meal protein, wherein the main components of the tyrosinase inhibitory peptide are SFAPRFD, HYGR, SPGRLP, TGFR, LGFR and SHPHRLP. The present invention also provides a method for preparing the tyrosinase inhibitory peptide from the peony seed meal protein.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a tyrosinase inhibitory peptide derived from peony seed meal protein and a preparation method thereof. Background Art

[0002] Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that catalyzes the conversion of monophenol (tyrosine) or o-diphenol (L-DOPA) to o-quinone, which forms melanin through spontaneous reactions such as cyclization, decarboxylation, and oxidative polymerization (Shen et al, Journal of Enzyme Inhibition and Medicinal Chemistry. 2019, 34(1): 1633-1640). As a complex polyphenol biopolymer, melanin is widely distributed in bacteria, fungi, plants, and animals, and its concentration and distribution largely determine the color of mammalian skin and hair (Kim et al, Cellular and Molecular Life Sciences. 2005, 62(15): 1707-1723). Although melanin can play a photoprotective role in the skin, excessive accumulation of melanin in the skin can lead to skin problems such as spots (Chang, International Journal of Molecular Sciences. 2009, 10(6): 2440-2475). In addition, tyrosinase-catalyzed oxidative browning of polyphenols is a common phenomenon in post-harvest agricultural products such as mushrooms, significantly reducing their commercial value. Therefore, the search for potential tyrosinase inhibitors is of great value in the fields of food, medicine, and beauty.

[0003] Commonly used tyrosinase inhibitors include ascorbic acid, hydroquinone, kojic acid, arbutin, sulfites, etc. 5. These inhibitors have been greatly limited in their application due to some adverse side effects. For example, arbutin and hydroquinone have been reported to cause contact dermatitis and exogenous ochronosis, respectively (Numata et al, Contact Dermatitis. 2016, 75(3): 187-188); benzoquinone is cytotoxic to hepatocytes and melanocytes and may also be carcinogenic (Smith et al, Environmental Health Perspectives. 1989, 82: 23-29); kojic acid has also been shown to be carcinogenic (Burnett et al, International Journal of Toxicology. 2010, 29: 244S-273S). Therefore, obtaining tyrosinase inhibitors with good application value from natural sources has become an important approach.

[0004] Peony is a famous ornamental flower in my country and an important medicinal plant. It has rich germplasm resources and a good momentum of industrial development. Oil-producing peony is one of the important directions of peony industry development. At present, oil-producing peony varieties represented by "Fengdan" and "Ziban" have been included in my country's woody oil system, and peony seed oil has also been included in the national new food raw material catalog. The national standard "Peony Seed Oil" has been officially promulgated and implemented, and the oil-producing peony industry has ushered in huge opportunities. With the rapid development of the oil-producing peony industry, the planting area of oil-producing peonies in my country has reached 106,700 hectares, the annual output of peony seeds is 240,000 tons, and the processing capacity of peony seed oil can reach 20,000 tons (Jiang Xia et al., Biomass Chemical Engineering. 2022, 56(02): 60-68). It is expected that the annual by-product of oil extraction, peony seed meal, will exceed 100,000 tons. In contrast to the booming peony seed oil industry, there is currently little research on the deep processing of peony seed meal, and resource utilization is poor. It is usually discarded as waste, causing huge environmental pressure, or used as feed, resulting in serious waste of resources (Deng et al, Journal of Food Science. 2018, 83(12): 2943-2953).

[0005] Protein is the most abundant substance in peony seed meal, typically accounting for around 30%. Peony seed meal protein is rich in amino acids and has a balanced composition, making it a good source of plant protein (Deng et al., Industrial Crops and Products. 2022, 187:115333). Therefore, enhancing the efficient utilization of peony seed meal protein will become a key approach to its deep processing and the development of high-value products.

[0006] Therefore, the present invention provides a tyrosinase inhibitory peptide derived from peony seed meal protein and a preparation method thereof. Summary of the Invention

[0007] The invention provides a tyrosinase inhibitory peptide derived from peony seed meal protein, characterized in that the tyrosinase inhibitory peptide is a polypeptide mixture, whose main components include: SFAPRFD, HYGR, SPGRLP, TGFR, LGFR, SHPHRLP, NPFGR, FIR, SFRIR, ELGF, PPFRLAEIRA, IPPR, LHARF, IGGRGR, LFR, SFR, HFPIR, PHNPR, SARAAFN, PGSSSIMVR, LFD, AGSSMIR, NPR, KGFL, LDWYKGPT, LFEG, NLGFR, KLF, QSDFRRN, SLRDYRGP, NASFPR, VNDPF, EMNPNF, GDLL, PGKP, IPIR, SPGQRNLKKR, SIDLEWLR and LLDM; more preferably, SFAPRFD, HYGR, SPGRLP, TGFR, LGFR, SHPHRLP, NPFGR, FIR, SFRIR, ELGF and PPFRLAEIRA; most preferably, SFAPRFD, HYGR, SPGRLP, TGFR, LGFR and SHPHRLP.

[0008] According to one embodiment of the present invention, the docking energies of SFAPRFD, HYGR, SPGRLP, TGFR, LGFR and SHPHRLP with tyrosinase (PDB accession number: 2y9x) are -7.7, -7.6, -7.5, -7.3, -7.2 and -7.2 kcal / mol, respectively, which are significantly less than the docking energy of the positive control kojic acid: -5.6 kcal / mol.

[0009] According to one embodiment of the present invention, the in vitro inhibition IC values of SFAPRFD, HYGR, SPGRLP, TGFR, LGFR and SHPHRLP on tyrosinase are 50 The values were 1.10±0.05, 0.96±0.02, 1.58±0.07, 1.78±0.05, 1.69±0.08 and 1.82±0.04 mM, which were significantly lower than the in vitro inhibition IC of the positive control arbutin on tyrosinase. 50 Value 5.22±0.12mM.

[0010] According to one embodiment of the present invention, the tyrosinase inhibitory peptide can be used as a health product and a skin care product for improving pigmentation.

[0011] The present invention also provides a method for preparing a tyrosinase inhibitory peptide from peony seed meal protein. The method comprises: hydrolyzing the peony seed meal protein for 4 hours using a protease at a substrate concentration of 5% and an enzyme / substrate ratio of 5000 U / g. After the hydrolysis is completed, the reaction mixture is kept in a boiling water bath for 5 minutes to terminate the reaction. The supernatant is collected by centrifugation, and the supernatant is dried to obtain a solid, which is the tyrosinase inhibitory peptide.

[0012] According to one embodiment of the present invention, the peony seed meal protein comes from peony seed meal after peony seed oil production, and the peony varieties include (but are not limited to) "Fengdan" peony or "Ziban" peony.

[0013] According to one embodiment of the present invention, the protein used to prepare the tyrosinase inhibitory peptide can be selected from neutral proteases, composite proteases, trypsin, pepsin, alkaline proteases, flavor proteases and papain, preferably, neutral proteases and composite proteases, most preferably neutral proteases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Hydrolysis degree and tyrosinase inhibitory activity of peony seed meal protein. DETAILED DESCRIPTION

[0015] The present invention is described in detail by the following examples.

[0016] Example 1 Preparation of Peony Seed Meal Protein Hydrolysate

[0017] Peony seed meal protein hydrolysates were prepared using trypsin, pepsin, a combined protease, alkaline protease, neutral protease, flavor protease, and papain. The hydrolysis conditions were as follows: hydrolysis was performed for 4 hours at a substrate concentration of 5% and an enzyme / substrate ratio of 5,000 U / g, under the optimal temperature and pH conditions for each protease (Table 1). After hydrolysis, the reaction mixture was placed in a boiling water bath for 5 minutes to terminate the reaction and then centrifuged at 10,000 × g for 10 minutes. The supernatant was collected and the degree of hydrolysis and tyrosinase inhibitory activity were determined.

[0018] Table 1 Optimum temperature and pH of protease

[0019]

[0020] Determination of hydrolysis degree: The OPA method was used to determine the degree of protein hydrolysis [5]. The specific method is as follows: 25 ml of 100 mM sodium tetraborate solution, 10 ml of 5% SDS solution, 13.9 ml of water, 1 ml of OPA solution, and 0.1 ml of β-mercaptoethanol were mixed to prepare an OPA assay solution. 50 μl of the test sample was mixed with 2 ml of the OPA assay solution and reacted at room temperature for 8 minutes. After the reaction was completed, the absorbance of the reactant was measured at 340 nm. A standard curve was established using glycine-glycine dipeptide as a standard, and the amino content in the reaction mixture before and after hydrolysis was determined based on the standard curve. The degree of protein hydrolysis D was determined based on the following formula: H :

[0021]

[0022] Where: C1, free amino group content in protein before hydrolysis, mol / L;

[0023] C2, free amino group concentration in the reaction mixture after hydrolysis, mol / L;

[0024] C0, total amino content (determined by Kjeldahl method)

[0025] Determination of tyrosinase inhibitory activity: Peony seed meal hydrolyzate was diluted to 1 mg / ml with 0.05 M phosphate buffer (pH 6.8). 450 μl of the diluted hydrolyzate was incubated with 300 μl of tyrosinase (500 U / ml) at 25°C for 5 min. Then, the reaction was initiated by adding 150 μl of substrate L-dopa (0.5 mM) and incubated at 25°C for another 5 min. After the reaction was completed, the absorbance was measured at 475 nm using a spectrophotometer. 0.05 M phosphate buffer (pH 6.8) was used as a control, and the inhibition percentage was calculated according to the following formula:

[0026]

[0027] Where: A1 is the absorbance value of the hydrolyzate;

[0028] A2 is the absorbance value of the control.

[0029] from Figure 1 As can be seen from the results, with the exception of trypsin and papain, the remaining five proteases showed good hydrolysis effects on peony seed meal protein, with hydrolysis degrees exceeding 20%. Alkaline protease had the highest hydrolysis degree (32.5%), followed by central protease (29.6%). In terms of tyrosinase inhibitory activity, neutral protease and composite protease exhibited the highest inhibitory activities, reaching 59.7% and 47.2%, respectively. Therefore, based on the comprehensive consideration of hydrolysis degree and tyrosinase inhibitory activity, neutral protease hydrolysates were selected for subsequent studies.

[0030] Example 2 Identification of polypeptide sequences in peony seed meal protein hydrolysate

[0031] The peptide sequences in peony seed meal hydrolysate were analyzed by LC-MS / MS. The specific method is as follows: the sample was treated with reductive alkylation and then analyzed by LC-MS / MS under the following conditions:

[0032] Pre-column: 300μm id×5mm, packed with Acclaim PepMap RPLC C18, 5μm,

[0033] Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm,

[0034] Mobile phase A: 0.1% formic acid;

[0035] Mobile phase B: 0.1% formic acid, 80% ACN;

[0036] Gradient: 0, 4% mobile phase B; 0-2 min, 4%-8% mobile phase B; 2-45 min, 8%-28% mobile phase B; 45-55 min, 28%-40% mobile phase B; 55-56 min, 40%-95% mobile phase B; 56-66 min, 95% mobile phase B.

[0037] Flow rate: 600nL / min.

[0038] A total of 594 peptide sequences were obtained through identification, among which the proteins with the largest number of peptides were ProteinYcf2 (A0A7D7G2K5), Protein TIC 214 (A0A2S1P3P9), DNA-directed RNApolymerasesubunit beta" (A0A4D6SW21), DNA-directed RNA polymerase subunit bet (A0A7D7JSH5) and Photosystem IP700 chlorophyll a apoprotein A1 (A0A4D6SW30) produced 108, 61, 54, 42, and 34 peptides, respectively. The top 20 peptide sequences by abundance were AGLI, KIGGIGTV, TSRILR, KNAESR, HGSIDL, NPR, FNVK, SSNINRLIVS, FHDIM[+15.995]YG, QNALVP, LIVGA, LISEISR, VIPQ, SERVLSF, PVALSI, LVRVE, PVDTSEGINVG, QGLPK, KDLPGE, and LEPFQ, indicating high levels of these peptides in the hydrolysis mixture. Of the 594 peptides, 12 were found to have alkylation modifications at cysteine residues, and 11 were found to have oxidative modifications at methionine residues. These 23 modified peptides were discarded, and the remaining 571 peptides were evaluated for bioactivity and water solubility.

[0039] Example 3 Evaluation of biological activity and water solubility of peony seed meal polypeptides

[0040] Biological activity evaluation: The biological activity of the polypeptide sequences identified in Example 2 was evaluated using the PeptideRanker program (http: / / distilldeep.ucd.ie / PeptideRanker / ), with a score of 0.5 as the activity threshold.

[0041] Evaluation of Water Solubility: The molecular weight, isoelectric point, net charge at pH 7, and water solubility of the polypeptide sequence identified in Example 2 were calculated using the Peptide Property Calculator (http: / / www.innovagen.com / ~proteomics-tools).

[0042] 571 peony seed meal polypeptides were scored using the PeptideRanker program. The results showed that 193 polypeptides had scores greater than the threshold of 0.5, indicating that these polypeptides had potential biological activity. Among them, there were 42 tripeptides, 47 tetrapeptides, 33 pentapeptides, 26 hexapeptides, and 45 polypeptide sequences longer than hexapeptides. Tripeptides to pentapeptides accounted for 63.2% of the total active peptides, indicating that peony seed meal polypeptides are mainly small peptides and have good absorbability, which is consistent with the high hydrolysis degree result determined above during neutral protease hydrolysis.

[0043] Water solubility is the basis for drug transport and distribution in the body and is an important indicator to consider when peptides are used in biology, food, cosmetics and other fields. Therefore, this study further predicted the water solubility of the above 193 oligopeptides, and a total of 39 water-soluble oligopeptides were obtained (Table 2). Based on the amino acid sequences of these oligopeptides, a total of 20 water-soluble oligopeptides contain the basic amino acid arginine (R) at the C-terminus, indicating that the presence of the C-terminal basic amino acid has a significant impact on the water solubility of peptides.

[0044] Analysis of the physicochemical properties of these 39 oligopeptides revealed that they have low molecular weights. With the exception of SIDLEWLR, PPFRLAEIRA, and SPGQRNLKKR, their molecular weights are all less than 1000 Da, enabling rapid absorption in the gastrointestinal tract and further facilitating the development of their biological activities. Most of these peptides are basic, with two peptides, SFAPRFD and LDWYKGPT, being neutral. Eight peptides, including LFD, VNDPF, EMNPNF, ELGF, GDLL, SIDLEWLR, LFEG, and LLDM, are acidic, reflecting the ratio of basic to acidic amino acids they contain.

[0045] Based on this, the above 39 aqueous oligopeptides with good biological activity were further analyzed.

[0046] Table 2 Predicted activity and physicochemical properties of peony seed meal-derived peptides

[0047]

[0048] Note: A0A2S1P3P9: Protein TIC 214; A0A4D6SW30: Photosystem IP700chlorophyll a apoproteinA1; A0A0C4K370: CO; A0A7D7G2K5: ProteinYcf2; A0A6G5RTH0: Repressor ofgene silencing 1; A0A6M3Z5A7: ELF3; A0A7H1RHJ4: Sucrose synthase; A0A4D6SW21: DNA-directed RNA polymerase subunitbeta"; J9PFG8: Flavanone 3'-hydroxylase; A0A7D7JSH5: DNA-directed RNA polymerase subunitbeta; A0A172MK00: Glyceraldehyde-3-phosphate dehydrogenase(Fragment);A0A7G5CEJ5: Squamosa-binding protein-like 10; A0A172MJW4: Elongation factor 1alpha (Fragment); A0A343J2C0: Ethylenereceptor; A0A1W6BRX1: Fatty aciddesaturase; H6UFA0: Heatshockprotein 70; A0A7D7K233: NAD(P)H-quinone oxidoreductase chain4, chloroplastic.

[0049] Example 4 Toxicity and ADMET Evaluation of Peony Seed Meal Polypeptides

[0050] Toxicity evaluation: The potential toxicity of the peony seed meal polypeptides screened in Example 3 was predicted using the ToxinPred program (http: / / crdd.osdd.net / raghava / / toxinpred / ).

[0051] ADMET evaluation. The peony seed meal peptides screened in Example 3 were evaluated online using the admetSAR (http: / / lmmd.ecust.edu.cn / admetsar2 / ) program. Human intestinal absorption (HIA) was selected to determine absorption characteristics, and blood-brain barrier (BBB) penetration and cytochrome P450 (CYP 450) 2D6 interaction were selected to analyze their distribution characteristics. The skin sensitization of these peptides was also evaluated.

[0052] Table 3 ADMET analysis and activity prediction of peony seed meal-derived peptides

[0053]

[0054]

[0055] Example 5 Analysis of the binding energy between peony seed meal polypeptide and tyrosinase

[0056] Molecular docking studies were performed using Autodock vina software. The crystal structure of tyrosinase (PDB: 2y9x) was obtained from the RCSB Protein Data Bank (PDB). Water molecules were removed from the crystal structure and the structure was optimized before docking. Kojic acid was used as a positive control, and the inhibitory activity of various peptides against tyrosinase was compared based on binding energy.

[0057] Table 4 Binding energy of peptides and tyrosinase

[0058]

[0059]

[0060] According to Table 4, among the 39 peptides screened for potential activity, 38 of them had docking energies less than or equal to the positive control kojic acid, indicating that this method is an excellent method for preparing tyrosine from peony seed meal protein.

[0061] Example 6 In vitro inhibition of tyrosinase by peony seed meal polypeptide

[0062] The in vitro inhibition IC values of SFAPRFD, HYGR, SPGRLP, TGFR, LGFR and SHPHRLP on tyrosinase were evaluated according to the method of “Determination of tyrosinase inhibitory activity” described in Example 1. 50 Value, where IC 50 The concentration of peptide sample that inhibits 50% of tyrosinase activity was calculated using GraphPad Prism 5. 50 Arbutin was used as a positive control.

[0063] Among the peptide sequences predicted to have tyrosinase inhibitory activity, the in vitro inhibitory activities of the top six peptides with the smallest docking energy, SFAPRFD, HYGR, SPGRLP, TGFR, LGFR, and SHPHRLP, on tyrosinase were determined. As shown in Table 5, the in vitro inhibitory IC values of SFAPRFD, HYGR, SPGRLP, TGFR, LGFR, and SHPHRLP on tyrosinase were 50The values were 1.10±0.05, 0.96±0.02, 1.58±0.07, 1.78±0.05, 1.69±0.08 and 1.82±0.04 mM, respectively, which were significantly lower than the in vitro inhibition IC of the positive control arbutin on tyrosinase. 50 The value was 5.22±0.12 mM, indicating that they have excellent tyrosinase inhibitory activity and thus have broad application prospects in food, medicine, and cosmetics.

[0064] Table 5 In vitro inhibition of tyrosinase by peptides

[0065]

[0066]

Claims

1. A tyrosinase inhibitory peptide derived from peony seed meal protein, characterized in that: The tyrosinase inhibitory peptide is one or a combination of multiple of SFAPRFD, HYGR, SPGRLP, TGFR, LGFR and SHPHRLP.

2. The tyrosinase inhibitory peptide derived from peony seed meal protein according to claim 1, characterized in that Among them, the docking energies of SFAPRFD, HYGR, SPGRLP, TGFR, LGFR and SHPHRLP with tyrosinase are -7.7, -7.6, -7.5, -7.3, -7.2 and -7.2 kcal / mol, respectively.

3. The tyrosinase inhibitory peptide derived from peony seed meal protein according to claim 1, characterized in that Among them, the IC values of SFAPRFD, HYGR, SPGRLP, TGFR, LGFR and SHPHRLP on tyrosinase in vitro were 50 The values were 1.10±0.05, 0.96±0.02, 1.58±0.07, 1.78±0.05, 1.69±0.08 and 1.82±0.04 mM, respectively.

4. Use of the tyrosinase inhibitory peptide derived from peony seed meal protein according to any one of claims 1 to 3 in the preparation of a skin care product for improving pigmentation.

5. A method for preparing the tyrosinase inhibitory peptide according to claim 1 from peony seed meal protein, characterized in that: The method comprises: using a neutral protease to hydrolyze peony seed meal protein for 3-5 hours at a substrate concentration of 4-6% and an enzyme / substrate ratio of 4000-6000 U / g; after the hydrolysis is completed, keeping the reaction mixture in a boiling water bath to terminate the reaction; collecting the supernatant by centrifugation, and drying the supernatant to obtain a solid, which is a tyrosinase inhibitory peptide.

6. The method according to claim 5, characterized in that The peony seed meal protein comes from peony seed meal after peony seed oil production, and the peony variety is selected from "Fengdan" peony or "Purple Spot" peony.

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