Several walnut pentapeptides and analogues thereof as well as preparation method, application and composition thereof

Abstract

The invention relates to walnut pentapeptide and analogues thereof as well as a preparation method, application and composition thereof, belongs to the technical field of organic synthesis, and aims to solve the problems of high toxicity, poor stability and insufficient water solubility of existing tyrosinase inhibitors such as kojic acid and the limitation of difficulty in large-scale application of natural walnut pentapeptide due to low content. The pentapeptide compound is prepared by screening a high-affinity structure by taking tyrosinase as a target spot and combining a solid-phase synthesis method by taking 2-CTC resin as a carrier and taking Oxyma / DIC as a condensing agent, the water solubility is remarkably improved to Log P =-3.21, the tyrosinase inhibitory activity IC50 is 83.32 + / -0.92 mu g / mL, which is superior to that of kojic acid, and the cytotoxicity is small. Experiments show that the compound has DPPH free radical scavenging capacity IC50 of 113.95 + / -3.92 mu g / mL, can effectively inhibit browning of fresh-cut fruits and vegetables, and is suitable for the fields of food preservatives, tyrosinase inhibitors and medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis. More specifically, the present invention relates to several walnut pentapeptide compounds and their analogs, as well as their preparation methods, applications and compositions. Background Art

[0002] Tyrosinase is a copper-containing oxidase with dual catalytic functions, which is widely present in microorganisms, animals, plants and the human body. In different organisms, the distribution positions of tyrosinase are different and play different roles. For example, in fruits and vegetables, tyrosinase regulates the browning process and quality flavor of fruits and vegetables; in insects, it is related to their defense and immune functions; in microorganisms, it regulates the sensitivity of microorganisms to ultraviolet radiation; in mammals, most of it exists in melanocytes and is the key rate-limiting enzyme for regulating melanin production. Abnormal secretion of melanin will cause the occurrence of some diseases, such as melanoma, one of the malignant tumors, Parkinson's disease and chloasma. Therefore, tyrosinase has become an important target for the development of fruit and vegetable anti-browning agents, antibacterial agents, malignant tumor drugs, skin whitening agents and agricultural insecticides, and has very important potential application values in industries such as pharmaceuticals, agriculture, food and cosmetics. Well-known tyrosinase inhibitors widely used in the market, such as kojic acid and arbutin, etc., but they have high toxicity, instability and serious side effects at the same time. Among them, kojic acid is listed as a class 3 carcinogen in the WHO carcinogen list and has been banned many times.

[0003] In addition, the existing inhibitors generally have high hydrophobicity (Log P values are mostly greater than 0), resulting in poor water solubility and limiting their applicability in food or pharmaceutical preparations. Developing safe, effective, stable and low-toxic tyrosinase inhibitors has become a technical problem to be solved urgently.

[0004] Walnut (Juglans regia L.), also known as English walnut and Persian walnut, has the reputations of "longevity fruit" and "son of longevity". As an important genus plant in the Juglandaceae family, it is mainly distributed in the northwest, southwest, south China, east China and other regions of China. Currently, its annual output reaches 5 million tons, ranking first in the world. Traditional Chinese medicine believes that walnuts are warm in nature and sweet in taste, and can be used as a tonic. In traditional Chinese medicine, it is called walnut kernel. Li Shizhen recorded in "Compendium of Materia Medica": "Walnut kernels can tonify qi and nourish blood, moisten dryness and resolve phlegm, benefit the life gate, regulate the triple energizer, warm the lungs and moisten the intestines, and treat deficiency-cold cough, heavy pain in the waist and feet, abdominal hernia pain, bloody dysentery and intestinal wind." Modern medical research believes that walnut kernels have multiple effects such as anti-aging, antioxidant, blood pressure lowering, antibacterial and anti-tumor. Walnut green peel, called Qinglongyi in traditional Chinese medicine, is used for external treatment of dermatitis and tinea diseases such as bacillary dysentery and stomach diseases. Modern pharmacological research shows that it has antibacterial, antioxidant, anti-cancer and other activities. The inner septum of walnut fruit, called Fuxinmu in traditional Chinese medicine, has the effects of promoting diuresis and clearing heat, and fixing color and arresting sweating. It is often used in diseases such as summer heat diarrhea, frequent urination and hematuria. Modern medical theory confirms that it has anti-inflammatory, antioxidant, anti-tumor, antibacterial and sedative-hypnotic effects. Walnuts are rich in trace elements such as vitamins, chromium, zinc, selenium, as well as various micro-components beneficial to the human body such as squalene, flavonoids and carotenoids. It also contains 18 kinds of amino acids, 8 of which are essential amino acids for the human body. Walnuts do not contain cholesterol, taste good and can be eaten raw, and are an excellent tonic. "Shennong's Herbal Classic" classifies walnuts as superior traditional Chinese medicines that can prolong life and improve qi and lighten the body after long-term consumption, and they belong to medicinal plants with homology of medicine and food. In terms of the protein contained in walnuts, the proportion of the 8 essential amino acids contained in it is similar to the proportion required by the human body, so walnut protein can be considered a high-quality protein.

[0005] CTLEW is a pentapeptide isolated from walnut (Juglans regia L.), which has diverse biological activities and relatively low cytotoxicity. However, the low content of CTLEW in natural walnuts limits the further research on the structure-activity relationship and biological activities of this compound, as well as its industrial application. Therefore, if tyrosinase is used as the target, a series of pentapeptide compounds and their analogs can be designed by combining Autodock 4.3 software with computer-aided drug design technology. And using 2-CTC resin as the carrier and Oxyma / DIC as the condensing agent, CTLEW and its analogs with diverse biological activities and relatively low cytotoxicity can be obtained through solid-phase synthesis. This not only has important value for expanding the library of CTLEW and its analogs, but also lays a foundation for the discovery of new anti-tyrosinase compounds, and can also promote the further development and utilization of walnuts. Summary of the Invention

[0006] The object of the present invention is to provide walnut pentapeptides and their analogues, their preparation methods, applications and compositions, and to solve at least the problems of high toxicity, instability and poor water solubility existing in the existing tyrosinase inhibitors (such as kojic acid), as well as the technical problem that the content of pentapeptide compounds from natural sources is extremely low and it is difficult to meet the research or application requirements.

[0007] To achieve the object of the present invention, a walnut pentapeptide compound is provided, which is characterized by having the following chemical structure: 。

[0008] The analogue of the walnut pentapeptide compound provided by the present invention has any one of the following chemical structures: 。

[0009] A preparation method of the pentapeptide compound provided by the present invention is characterized by including: a) Using tyrosinase as the target, performing molecular docking with Autodock 4.3 software to design a compound with the structure of general formula A; b) Using 2-CTC resin as the carrier and Oxyma / DIC as the condensing agent, synthesizing the target compound by solid-phase synthesis.

[0010] Specifically, the synthesis route is as follows: An application of the pentapeptide compound and its analogue provided by the present invention in preparing a food preservative.

[0011] Preferably, the preservative includes but is not limited to being used for inhibiting the enzymatic browning of fruits and vegetables.

[0012] Preferably, the fruits and vegetables include but are not limited to potatoes and apples.

[0013] An application of the pentapeptide compound and its analogue provided by the present invention in preparing a tyrosinase inhibitor.

[0014] A composition provided by the present invention contains the pentapeptide compound or its analogue, and a pharmaceutically or foodologically acceptable carrier.

[0015] The composition is a food preservative composition or a tyrosinase inhibitor composition.

[0016] The carrier includes, but is not limited to: deionized water, phosphate buffer (pH 6.5), food-grade ethanol (concentration ≤ 30%), glycerol, sodium alginate solution (1 - 2% w / v), β-cyclodextrin, corn starch, lactose, microcrystalline cellulose, food-grade silica, chitosan (1 - 3% acetic acid solution), sodium carboxymethyl cellulose (CMC-Na), sunflower oil-based emulsion, polyvinyl alcohol (PVA), gelatin, ascorbic acid (antioxidant), Tween 80 (emulsifier), potassium sorbate (preservative).

[0017] Preferably, the dosage form of the composition is solution, powder or coating.

[0018] The use of the analog of the walnut pentapeptide compound provided by the present invention as a preparation of an antioxidant.

[0019] The present invention has at least the following beneficial effects: 1. The present invention provides a pentapeptide compound with a novel structure, wide substrate applicability, simple operation, few by-products, easy separation and purification, and is applicable to large-scale preparation, showing promising application prospects.

[0020] 2. The pentapeptide compound provided by the present invention has excellent anti-tyrosinase activity. The results of the enzyme kinetics of the compound on tyrosinase show that the pentapeptide compound CTLEW binds strongly to tyrosinase. The anti-browning experiment of fresh-cut fruits and vegetables shows that the pentapeptide compound CTLEW has a stronger anti-browning effect than kojic acid on both potato slices and apple slices. Therefore, it is suitable for use as a food preservative or a tyrosinase inhibitor, and can also be formulated into a composition for use as a food preservative or a tyrosinase inhibitor.

[0021] Other advantages, objectives and features of the present invention will be partially reflected by the following description, and partially understood by those skilled in the art through the research and practice of the present invention. Description of the Drawings

[0022] Figure 1 It is the structural formula of the pentapeptide compound CTLEW described in the present invention; Figure 2 It is the HPLC purity analysis of the pentapeptide compound CTLEW described in the present invention; Figure 3 It is the flow chart of the preparation method of the pentapeptide compound CTLEW described in the present invention; Figure 4 It is the cytotoxicity of the pentapeptide compound CTLEW and its analogs of the present invention under 40 μmol / L human normal lung epithelial cells; Figure 5Determination of the inhibition type and inhibition constant of the pentapeptide compound CTLEW of the present invention on tyrosinase; wherein, A is the Lineweaver-Burk curve of CTLEW; B is the secondary plot of 1 / Vm against different concentrations of inhibitor I-j. Figure 6 Anti-browning effect of the pentapeptide compound CTLEW of the present invention on freshly cut potato slices stored at room temperature for 6 days; wherein A is the change in ΔE value; B is the change in L* value; C is a photograph. Figure 7 Anti-browning effect of the pentapeptide compound CTLEW of the present invention on freshly cut apple slices stored at room temperature for 8 days; wherein, A is the change in ΔE value; B is the change in L* value; C is a photograph. Detailed implementation manners

[0023] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it according to the description in the specification.

[0024] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation schemes are all conventional methods, and the reagents and materials, unless otherwise specified, can all be obtained from commercial channels.

[0025] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the existence or addition of one or more other elements or their combinations.

[0026] In the present invention: Amino acid sequence of CTLVW: Cys (C) - Thr (T) - Leu (L) - Val (V) - Trp (W); Full name: Cysteine - Threonine - Leucine - Valine - Tryptophan.

[0027] Amino acid sequence of CLW: Cys (C) - Leu (L) - Trp (W); Full name: Cysteine - Leucine - Tryptophan (tripeptide).

[0028] Amino acid sequence of CFLWW: Cys (C) - Phe (F) - Leu (L) - Trp (W) - Trp (W); Full name: Cysteine - Phenylalanine - Leucine - Tryptophan - Tryptophan.

[0029] Amino acid sequence of CVLFW: Cys (C) - Val (V) - Leu (L) - Phe (F) - Trp (W); Full name: Cysteine - Valine - Leucine - Phenylalanine - Tryptophan Amino acid sequence of CRLWW: Cys (C) - Arg (R) - Leu (L) - Trp (W) - Trp (W) Full name: Cysteine - Arginine - Leucine - Tryptophan - Tryptophan Amino acid sequence of CILRW: Cys (C) - Ile (I) - Leu (L) - Arg (R) - Trp (W) Full name: Cysteine - Isoleucine - Leucine - Arginine - Tryptophan Amino acid sequence of CRLFW: Cys (C) - Arg (R) - Leu (L) - Phe (F) - Trp (W) Full name: Cysteine - Arginine - Leucine - Phenylalanine - Tryptophan Amino acid sequence of CWLIW: Cys (C) - Trp (W) - Leu (L) - Ile (I) - Trp (W) Full name: Cysteine - Tryptophan - Leucine - Isoleucine - Tryptophan Amino acid sequence of CILFW: Cys (C) - Ile (I) - Leu (L) - Phe (F) - Trp (W) Full name: Cysteine - Isoleucine - Leucine - Phenylalanine - Tryptophan Amino acid sequence of CTLEW: Cys (C) - Thr (T) - Leu (L) - Glu (E) - Trp (W) Full name: Cysteine - Threonine - Leucine - Glutamic acid - Tryptophan Amino acid sequence of CLEW: Cys (C) - Leu (L) - Glu (E) - Trp (W) Full name: Cysteine - Leucine - Glutamic acid - Tryptophan (tetrapeptide) Example 1 As Figure 1 shown, the present invention provides a pentapeptide compound having the structural formula of General Formula A as Figure 1 shown in the text.

[0030] .

[0031] Example 2 As Figure 2 shown, the present invention provides a method for preparing a pentapeptide compound CTLEW and its analogs, comprising the following steps: Step 1: Using tyrosinase as a target, a series of CTLEW (pentapeptide) analogs are designed by combining Autodock 4.3 software with computer - aided drug design technology.

[0032] Step 2: Using 2-CTC resin as the carrier and Oxyma / DIC as the condensing agent, the target compound was obtained through solid-phase synthesis.

[0033] The synthetic route is as Figure 3 : The specific structural formula of the target compound CTLEW is shown as follows: .

[0034] Specifically, the detailed steps for designing the pentapeptide compound CTLEW based on tyrosinase as the target using Autodock 4.3 software in Step 1 are as follows: Step 1: Target protein preparation Obtain the crystal structure and download the crystal structure file (.pdb) of tyrosinase (such as human TYR, PDB ID: for example 3NM8) from the Protein Data Bank (PDB). Verify the structural integrity to ensure that there are no deletions in the key active sites (such as the copper ion binding domain).

[0035] Preprocess the protein structure, use software such as PyMOL or Chimera to remove the crystallization water molecules and irrelevant ligands (such as sulfate ions). Add hydrogen atoms and optimize the side chain conformation (tool: AutoDockTools). Calculate the Gasteiger charges and save as the.pdbqt format.

[0036] Step 2: CTLEW analogue design Construct the pentapeptide backbone. Based on the CTLEW sequence (Cys-Thr-Leu-Glu-Trp), use a chemical drawing tool (such as ChemDraw) to draw the two-dimensional structure. Convert it to a three-dimensional structure (tool: Open Babel or Avogadro) and optimize the geometric configuration.

[0037] Amino acid modification strategies: Side chain replacement: For example, replace Cys with Penicillamine (containing a mercapto group but with a more stable structure). Terminal modification: Introduce a hydrophobic group (such as benzyloxycarbonyl) at the N-terminus or add a hydrophilic chain (such as polyethylene glycol) at the C-terminus. Conformational restriction: Introduce a cyclopropyl group to restrict the conformation of flexible residues (such as Leu).

[0038] Generate an analogue library, use combinatorial chemistry methods to generate 5 - 10 modified variants and save them in the.mol2 or.pdb format.

[0039] Step 3: Molecular docking analysis Set the parameters of Autodock 4.3 and define the docking box (Grid Box): covering the active pocket of tyrosinase, and the box size is recommended to be ≥22×22×22 Å. Set the parameters of the genetic algorithm: population size (150), number of iterations (27000), and energy evaluation (100 times).

[0040] Perform docking calculations, dock each analogue with the target separately, and generate 10 - 20 conformations / compounds. Record the lowest binding energy and key interactions (such as hydrogen bonds, π-π stacking).

[0041] Result visualization and screening, use PyMOL to analyze the docking poses, and preferentially retain the compounds that form coordination bonds with copper ions (such as Cys / His residues). Binding energy threshold: Usually select candidate molecules with ≤ -7.0 kcal / mol.

[0042] Step Four: Optimization of candidate compounds Predict pharmacokinetics, use ADMET Predictor to evaluate the water solubility (LogP) and metabolic stability (CYP450 inhibition) of candidates. Eliminate molecules with LogP > 5 or metabolic half-life < 1 h.

[0043] The specific steps of the solid-phase peptide synthesis experiment in Step 2 are as follows: I. Resin pretreatment Swelling: Weigh 10.0 g of 2-CTC resin (loading capacity 0.8 mmol / g) into a reaction column, add 50 mL of dichloromethane (DCM), and stir magnetically (200 rpm) for 30 minutes (at room temperature).

[0044] Washing: After swelling with DCM, wash three times with DMF (3×50 mL each time, stir for 2 minutes each time), and filter by suction until the resin is in a semi-dry state.

[0045] II. Initial amino acid loading (Fmoc-Cys(Trt)-OH) Prepare the solution: Dissolve 18.1 g (32 mmol) of Fmoc-Cys(Trt)-OH (MW 566.7) in 30 mL of DMF.

[0046] Activation: Add 5.6 mL (32 mmol) of DIEA and stir at room temperature for 15 minutes for activation.

[0047] Coupling: Transfer the activation solution to the resin column and react at 25°C for 2 hours (under nitrogen protection).

[0048] Detection: Take a small amount of resin for Kaiser test detection until the free amino group shows negative (blue).

[0049] III. Fmoc Deprotection Deprotection solution: 20% piperidine / DMF solution (prepared freshly before use) Operation procedure: Add 50 mL of the deprotection solution, stir and react for 5 minutes, then filter by suction; repeat adding 50 mL of fresh deprotection solution, stir and react for 15 minutes; wash with DMF 6 times (6×50 mL), stir for 1 minute each time.

[0050] IV. Amino Acid Coupling (Fmoc-Thr(tBu)-OH) Dissolve Fmoc-Thr(tBu)-OH (12.72 g, 32 mmol) and Oxyma (4.48 g, 32 mmol) in 30 mL of DMF, cool to 4 °C in an ice bath, add dropwise DIC (5.0 mL, 32 mmol) and activate for 5 minutes, then transfer to a resin column and react for 2 hours (25 °C). Ninhydrin detection shows that the coupling efficiency > 99%.

[0051] V. Recycling Operation Repeat steps III to IV in the order of the sequence Trp(Boc)-Glu(OtBu)-Leu-Thr(tBu)-Cys(Trt). Note: For tryptophan coupling, the operation needs to be carried out in the dark (wrap the reaction column with aluminum foil); since the side chain of glutamic acid is relatively large, extend the reaction time to 3 hours; after each step, contract the resin with ethanol / ether (1:1).

[0052] VI. Cleavage and Deprotection Preparation of cleavage solution: 95 mL of TFA + 2.5 mL of H2O + 2.5 mL of TIS (pre-cooled in an ice bath).

[0053] Operation: Wash the resin with DCM and then dry it under vacuum for 30 minutes; add 50 mL of the pre-cooled cleavage solution, stir and react in an ice bath for 2 hours; filter to collect the filtrate, wash the resin with 5 mL of fresh TFA twice; combine the filtrates, concentrate to about 10 mL by nitrogen blowing; add 40 mL of pre-cooled ether (-20 °C), precipitate in an ice bath for 1 hour; centrifuge (4 °C, 8000 rpm, 10 minutes), discard the supernatant; wash the precipitate with cold ether three times and dry it under vacuum to obtain the crude peptide.

[0054] Results and Characterization HPLC purity: The purity of CTLEW-Pen is 92.5% (retention time 9.78 minutes, C18 column, gradient elution with acetonitrile / water).

[0055] The mass spectrometry and nuclear magnetic resonance characterization of the pentapeptide compound CTLEW are as follows: White powder, mp. 190.2 - 193.4 °C, MS(ESI) m / z: calculated for C29 H 42 N6O9S[M-H] + :649.27, FOUND:649.25. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 10.87 (d, J J = 2.4 Hz,1H, Ar-NH), 8.51 (d, J J = 8.0 Hz, 1H, CO-NH-), 8.14 (d, J J = 7.4 Hz, 1H, CO-NH-),8.02 (dd, J J = 15.7, 8.1 Hz, 2H, CO-NH-×2), 7.51 (d, J J = 7.9 Hz, 1H, ArH), 7.32(d, J J = 8.1 Hz, 1H, ArH), 7.15 (d, J J = 2.4 Hz, 1H, ArH), 7.05 (t, J J = 7.5 Hz, 1H,ArH), 6.97 (t, J J = 7.4 Hz, 1H, ArH), 5.03 (s, 1H,-OH), 4.43 (q, J J = 7.0 Hz, 1H),4.37 – 4.21 (m, 3H), 4.01 (dt, J J = 20.7, 5.6 Hz, 2H), 3.14 (dd, J J = 14.7, 5.6 Hz,2H), 3.04 (dd, J J = 14.8, 7.7 Hz, 1H), 2.96 (dd, J J = 14.3, 5.9 Hz, 1H), 2.86 (dd, J J = 14.2, 5.0 Hz, 1H), 2.31 – 2.16 (m, 2H), 1.88 (ddt, J J = 15.5, 10.8, 5.9 Hz,1H), 1.73 (dtd, J J = 14.1, 9.3, 6.2 Hz, 1H), 1.60 (dp, J J = 13.4, 6.7 Hz, 1H), 1.43(dd, J= 9.6, 5.7 Hz, 2H), 1.07 (d, J = 6.3 Hz, 3H, -CH3), 0.84 (d, J = 6.6 Hz, 3H, -CH3), 0.80 (d, J = 6.5 Hz, 3H, -CH3). 13 C-NMR (126 MHz, DMSO- d 6 ) δ 174.10, 173.26, 171.83, 171.06, 169.55, 169.47, 136.09, 127.23, 123.67, 120.94, 118.42, 118.20, 111.41, 109.55, 66.58, 58.71, 54.00, 53.18, 51.67, 51.06, 40.71, 30.08, 27.59, 27.07, 24.09, 23.20, 21.49, 19.73. According to the above method, analogs of the pentapeptide compound CTLEW were synthesized, including: The structural formula and characterization of CLEW are as follows: White powder, mp. 183.9 - 185.3 °C, MS(ESI) m / z: calculated for C 25 H 35 N5O7S [M+H] + : 550.23, FOUND: 550.15. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 10.85 (d, J = 2.5 Hz, 1H, Ar-NH), 8.50 (d, J = 7.7 Hz, 1H, CO-NH-), 8.16 – 8.07 (m, 2H, CO-NH-×2), 7.51(dd, J = 7.9, 3.5 Hz, 1H, ArH), 7.33 (dd, J = 8.2, 2.1 Hz, 1H, ArH), 7.14 (t, J = 3.1 Hz, 1H, ArH), 7.06 (ddd, J = 8.2, 6.9, 1.3 Hz, 1H, ArH), 6.97 (td,J = 7.4, 6.8, 1.1 Hz, 1H, ArH), 4.44 (td, J = 7.5, 5.5 Hz, 1H), 4.38 – 4.27 (m, 2H), 3.98 (t, J = 5.3 Hz, 1H), 3.09 – 3.03 (m, 1H), 2.99 – 2.83 (m, 2H), 2.30 – 2.18 (m, 2H), 1.91 (s, 2H), 1.75 (qd, J = 8.8, 8.3, 5.4 Hz, 1H), 1.62 (dq, J = 13.1, 6.6 Hz, 1H), 1.50 – 1.39 (m, 2H), 0.87 (d, J = 6.6 Hz, 3H, -CH3), 0.84 (d, J = 6.6 Hz, 3H, -CH3). 13 C-NMR (126 MHz, DMSO- d 6 ) δ 174.50, 173.57, 172.48, 172.07, 171.44, 136.53, 127.66, 124.06, 121.38, 118.86, 118.63, 111.84, 110.02, 54.29, 53.50, 52.13, 51.87, 40.82, 30.55, 28.18, 27.47, 26.06, 24.54, 23.55, 21.77. The structural formula and characterization of CLW are as follows White powder, mp. 211.2 - 212.7 °C, MS(ESI) m / z: calculated for C 20 H 28 N4O4S [M + H] + : 421.18, FOUND: 421.10. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 10.85 (d, J = 2.4 Hz, 1H, Ar-NH), 8.54 (d, J = 7.9 Hz, 1H, CO-NH-), 8.35 (d, J= 7.6 Hz, 1H, CO-NH-), 7.51(d, J = 7.9 Hz, 1H, ArH), 7.33 (d, J = 8.1 Hz, 1H, ArH), 7.15 (d, J = 2.3 Hz, 1H,ArH), 7.06 (t, J = 7.5 Hz, 1H, ArH), 6.97 (t, J = 7.5 Hz, 1H, ArH), 4.48 (td, J =7.6, 5.4 Hz, 1H), 4.38 (q, J = 7.7 Hz, 1H), 4.01 (t, J = 5.4 Hz, 1H), 3.19 – 3.03(m, 2H), 2.91 (ddd, J = 69.0, 14.4, 5.3 Hz, 2H), 1.64 (dp, J = 13.4, 6.7 Hz, 1H),1.46 (t, J = 7.3 Hz, 2H), 0.89 (d, J = 6.6 Hz, 3H, -CH3), 0.86 (d, J = 6.5Hz, 3H, -CH3). 13 C-NMR (126 MHz, DMSO- d 6 ) δ 173.10, 171.74, 166.70, 136.09,127.24, 123.61, 120.94, 118.41, 118.19, 111.39, 109.69, 53.78, 53.02, 51.42,40.60, 27.02, 25.46,24.07, 23.13, 21.43. The structural formula and characterization of CTLVW are as follows White powder, mp. 193.9 - 195.6 ℃, MS(ESI) m / z: calculated for C 29 H 44 N6O7S [M+H] + :621.30, FOUND:621.40. 1 H-NMR (500 MHz, DMSO- d 6) δ 10.83 (d, J J = 2.5 Hz, 1H, Ar-NH), 8.48 (d, J J = 8.1 Hz, 1H, CO-NH-), 8.20 (d, J J = 7.4 Hz, 1H, CO-NH-), 8.03(d, J J = 8.2 Hz, 1H, CO-NH-), 7.76 (d, J J = 9.0 Hz, 1H, CO-NH-), 7.51 (d, J J = 7.9 Hz,1H, ArH), 7.32 (d, J J = 8.1 Hz, 1H, ArH), 7.13 (d, J J = 2.4 Hz, 1H, ArH), 7.05 (t, J J = 7.6 Hz, 1H, ArH), 6.97 (t, J J = 7.4 Hz, 1H, ArH), 5.01 – 4.92 (m, 1H,-OH),4.45 (q, J J = 7.0 Hz, 1H), 4.36 (td, J J = 8.8, 5.7 Hz, 1H), 4.27 (dd, J J = 8.1, 4.8Hz, 1H), 4.21 (dd, J J = 9.0, 6.8 Hz, 1H), 4.04 (t, J J = 5.4 Hz, 1H), 3.99 (q, J J = 6.3Hz, 1H), 3.12 – 3.00 (m, 2H), 2.96 – 2.82 (m, 2H), 2.00 – 1.90 (m, 1H), 1.60(dp, J J = 19.3, 6.6 Hz, 1H), 1.44 (dq, J J = 12.6, 7.9, 6.3 Hz, 2H), 1.07 (d, J J = 6.3Hz, 3H, -CH3), 0.88 – 0.75 (m, 12H, -CH3×4). 13 C-NMR (126 MHz, DMSO- d 6) δ173.14, 171.70, 170.87, 169.39, 167.52,136.10, 127.21, 123.56, 120.95,118.41, 118.18, 111.40, 109.68, 66.58, 58.63, 57.33, 53.96, 52.95, 51.23,40.70, 30.88, 27.13, 25.90, 24.12, 23.20, 21.53,19.68, 19.13, 18.05. The structural formula and characterization of CFLWW are as follows White powder, mp. 204.5 - 206.6 °C, MS(ESI) m / z: calculated for C 40 H 47 N7O6S [M + H] + : 754.33, FOUND: 754.40. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 10.85 (d, J = 2.5 Hz, 1H,Ar-NH), 10.80 (d, J = 2.4 Hz, 1H, Ar-NH), 8.53 (d, J = 8.1 Hz, 1H, CO-NH-), 8.31(d, J = 8.3 Hz, 1H, CO-NH-), 8.19 (d, J = 7.5 Hz, 1H, CO-NH-), 7.92 (d, J = 7.9 Hz,1H, CO-NH-), 7.56 (d, J = 7.9 Hz, 1H, ArH), 7.50 (d, J = 7.9 Hz, 1H, ArH), 7.33(d, J = 8.1 Hz, 1H, ArH), 7.30 (d, J = 8.1 Hz, 1H, ArH), 7.26 (d, J = 7.5 Hz, 2H,ArH×2), 7.21 (t, J= 7.4 Hz, 2H, ArH×2), 7.17 – 7.12 (m, 2H, ArH), 7.11 (d, J =2.4 Hz, 1H, ArH), 7.08 – 7.01 (m, 2H, ArH×2), 6.96 (t, J = 7.4 Hz, 2H, ArH×2), 4.59 (dddd, J = 14.3, 11.2, 8.0, 4.4 Hz, 2H), 4.49 (q, J = 6.9 Hz, 1H), 4.32(q, J = 7.7 Hz, 1H), 3.86 (t, J = 5.0 Hz, 1H), 3.16 – 3.12 (m, 2H), 3.08 – 3.00(m, 2H), 2.98 (dd, J = 8.7, 6.0 Hz, 2H), 2.84 – 2.70 (m, 2H), 1.91 (s, 1H),1.54 (dt, J = 13.6, 6.8 Hz, 1H), 1.39 (t, J = 7.3 Hz, 2H), 0.83 (d, J = 6.6 Hz,3H, -CH3), 0.78 (d, J = 6.5 Hz, 3H, -CH3). 13 C-NMR (126 MHz, DMSO- d 6 ) δ 173.06,172.03, 171.61, 171.26, 170.75, 137.64, 136.07, 136.03, 129.14, 128.10,127.46, 127.30, 126.36, 123.63, 123.51, 120.92, 120.82, 118.39, 118.21,111.38, 111.24, 109.84, 109.55, 54.17, 53.77, 53.12, 51.55, 40.84, 36.90,28.15, 27.22,26.03, 24.10, 23.12, 21.46. The structural formula and characterization of CVLFW are as follows White powder, mp. 202.4 - 206.5 °C, MS(ESI) m / z: calculated for C 34 H 46 N6O6S[M+H] + : 667.32, FOUND: 667.40. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 10.85 (d, J = 2.5 Hz, 1H, Ar-NH), 8.39 (d, J = 8.4 Hz, 1H, CO-NH-), 8.24 (d, J = 7.6 Hz, 1H, CO-NH-), 8.09 (d, J = 8.4 Hz, 1H, CO-NH-), 7.85 (d, J = 8.1 Hz, 1H, CO-NH-), 7.52 (d, J = 7.9 Hz, 1H, ArH), 7.33 (d, J = 8.1 Hz, 1H, ArH), 7.19 (d, J = 5.5 Hz, 4H, ArH×2), 7.17 – 7.13 (m, 2H, ArH), 7.09 – 7.02 (m, 1H, ArH), 6.97 (t, J = 7.3 Hz, 1H, ArH), 4.57 (td, J = 8.6, 4.5 Hz, 1H), 4.48 (td, J = 7.3, 5.6 Hz, 1H), 4.29 (td, J = 9.2, 5.2 Hz, 1H), 4.16 (dd, J = 8.4, 6.9 Hz, 1H), 4.01 (t, J = 5.5Hz, 1H), 3.16 (dd, J = 14.6, 5.8 Hz, 2H), 3.08 – 2.99 (m, 2H), 2.91 (dd, J = 14.2, 5.9 Hz, 1H), 2.85 – 2.76 (m, 2H), 1.91 (d, J = 6.6 Hz, 2H), 1.57 – 1.47(m, 1H), 1.35 (ddq, J= 14.2, 9.7, 4.8 Hz, 2H), 0.84 – 0.74 (m, 12H, -CH3×4). 13 C-NMR (126 MHz, DMSO- d 6 ) δ 173.51, 171.96, 171.25, 170.74, 167.64, 137.94, 136.55, 129.63, 128.38, 127.70, 126.62, 124.06, 121.38, 118.83, 118.64, 111.83, 109.99, 58.59, 54.21, 53.73, 53.45, 51.43, 41.30, 37.99, 30.80, 27.66, 26.27, 24.50, 23.52, 21.91, 19.65, 18.55. The structural formula and characterization of CRLWW are as follows White powder, mp. 179.1 - 183.0 °C, MS(ESI) m / z: calculated for C 37 H 50 N 10 O6S[M + H] + : 763.36, FOUND: 763.40. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 11.43 (d, J = 2.5 Hz, 1H, Ar-NH), 11.40 (d, J = 2.4 Hz, 1H, Ar-NH), 9.22 (d, J = 7.6 Hz, 1H, CO-NH-), 8.72 (d, J = 7.2 Hz, 1H, CO-NH-), 8.67 (d, J = 8.4 Hz, 1H, CO-NH-), 8.52 (d, J = 8.0 Hz, 1H, CO-NH-), 8.14 (t, J = 7.3 Hz, 1H, ArH), 8.10 (d, J= 7.9 Hz, 1H, ArH), 7.93 (s, 1H, =NH), 7.91 (d, J = 7.7 Hz, 2H, ArH×2), 7.71 (dd, J = 7.6, 2.4 Hz, 2H, ArH×2), 7.65 (td, J = 7.5, 3.7 Hz, 2H, ArH×2), 7.55 (td, J = 7.4, 2.3 Hz, 2H, ArH×2), 5.16 (td, J = 8.2, 4.8 Hz, 1H), 5.06 (q, J = 6.8 Hz, 1H), 4.88 (q, J = 7.9 Hz, 2H), 4.60 (t, J = 5.3 Hz, 1H), 3.79 – 3.72 (m, 2H), 3.69 (dd, J = 13.6, 5.8 Hz, 2H), 3.55 (dt, J = 15.9, 8.2 Hz, 2H), 3.09 (d, J = 1.7 Hz, 2H), 2.24 (dt, J = 11.1, 5.0 Hz, 1H), 2.15 – 2.03 (m, 4H), 1.94 (t, J = 7.4 Hz, 2H), 1.40 (d, J = 6.6 Hz, 3H, -CH3), 1.35 (d, J = 6.4 Hz, 3H, -CH3). 13 C-NMR (126 MHz, DMSO- d 6 ) δ 172.48, 172.14, 171.76, 171.12, 167.30, 157.27, 136.52, 127.84, 124.10, 121.36, 118.86, 118.70, 111.82, 111.72, 110.29, 110.01, 54.11, 53.75, 53.67, 53.07, 51.55, 41.34, 40.84, 29.29, 28.13, 27.63, 25.91, 25.45, 24.50, 23.55, 21.83. The structural formula and characterization of CILRW are as follows White powder, mp. 207.8 - 209.5 °C, MS(ESI) m / z: calculated for C 32 H 51 N9O6S[M + H] + : 690.37, FOUND: 690.40 [M + 2H] 2+ : 345.69, FOUND: 345.90. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 10.84 (d, J = 2.6 Hz, 1H, Ar-NH), 8.42 (d, J = 8.5 Hz, 1H, CO-NH-), 8.20 (d, J = 8.2 Hz, 1H, CO-NH-), 8.11 (d, J = 7.4 Hz, 1H, CO-NH-), 7.87 (d, J = 8.0 Hz, 1H, =NH), 7.65 (d, J = 5.7 Hz, 1H, CO-NH-), 7.51 (d, J = 7.9 Hz, 1H, ArH), 7.32 (dd, J = 8.0, 2.2 Hz, 1H, ArH), 7.12 (d, J = 2.4 Hz, 1H, ArH), 7.05(dd, J = 9.1, 6.0 Hz, 1H, ArH), 6.96 (t, J = 7.4 Hz, 1H, ArH), 4.45 (q, J = 6.7 Hz, 1H), 4.37 – 4.27 (m, 2H), 4.20 (q, J = 9.6, 7.9 Hz, 1H), 3.91 (t, J = 5.4 Hz, 1H), 3.15 (dd, J = 14.7, 5.9 Hz, 2H), 3.06 (t, J= 7.7 Hz, 2H), 2.90 – 2.79 (m, 2H), 1.91 (s, 1H), 1.76 – 1.63 (m, 2H), 1.54 – 1.38 (m, 6H), 1.11 – 1.03 (m, 1H), 0.86 – 0.81 (m, 6H, -CH3×2), 0.79 (d, J = 6.4 Hz, 6H, -CH3×2). 13 C-NMR (126MHz, DMSO- d 6 ) δ 173.57, 172.49, 172.15, 171.54, 171.08, 157.24, 136.53, 127.72, 124.04, 121.36, 118.81, 116.49, 111.81, 110.01, 57.70, 54.56, 53.52, 52.28, 51.45, 41.01, 40.92, 36.91, 29.85, 27.63, 26.89, 25.22, 24.79, 24.54, 23.58, 21.82, 21.53, 15.71, 11.34. The structural formula and characterization of CRLFW are as follows White powder, mp. 188.6 - 191.5 °C, MS(ESI) m / z: calculated for C 35 H 49 N9O6S[M + H] + : 724.35, FOUND: 724.40. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 10.88 (t, J = 3.4 Hz, 1H, Ar-NH), 8.34 (d, J = 7.3 Hz, 1H), 8.28 (d, J = 7.6 Hz, 1H, CO-NH-), 8.14 (d, J = 8.3 Hz, 1H, CO-NH-), 8.09 (d, J = 8.4 Hz, 1H, CO-NH-), 7.99 (d, J = 8.3 Hz, 1H, =NH), 7.93 (d, J= 8.3 Hz, 1H, CO-NH-), 7.52 (d, J = 7.9 Hz, 1H, ArH), 7.33 (d, J =8.1 Hz, 1H, ArH), 7.21 (t, J = 4.7 Hz, 4H, ArH), 7.18 – 7.14 (m, 2H, ArH),7.06 (td, J = 7.6, 3.0 Hz, 1H, ArH), 6.97 (td, J = 7.5, 3.3 Hz, 1H, ArH), 4.55(qd, J = 9.4, 4.4 Hz, 1H), 4.47 (q, J = 6.9 Hz, 1H), 4.27 (ddd, J = 19.9, 14.2,7.8 Hz, 2H), 4.06 (dt, J = 51.2, 6.1 Hz, 1H), 3.17 (dd, J = 14.8, 5.8 Hz, 1H),3.05 (td, J = 13.7, 12.0, 6.2 Hz, 3H), 2.97 (dt, J = 21.5, 5.9 Hz, 2H), 2.90 –2.72 (m, 2H), 1.68 – 1.58 (m, 1H), 1.47 (dtt, J = 24.4, 12.6, 6.4 Hz, 3H), 1.33(q, J = 7.7 Hz, 2H), 0.84 – 0.79 (m, 3H, -CH3), 0.76 (t, J = 7.2 Hz, 3H, -CH3). 13 C-NMR (126 MHz, DMSO- d 6) δ 173.61, 172.09, 171.46, 171.07, 170.82, 167.18, 138.86, 136.97, 130.44, 128.43, 127.70, 126.68, 124.13, 121.39, 118.85, 111.85, 109.96, 54.01, 53.62, 53.08, 51.33, 41.40, 40.85, 37.95, 27.58, 24.55, 24.47, 23.58, 23.49, 21.95, 21.87. The structural formula and characterization of CILFW are as follows: White powder, mp. 212.2 - 214.6 °C, MS(ESI) m / z: calculated for C 35 H 48 N6O6S [M+H] + : 681.34, FOUND: 681.40. 1 H-NMR (500 MHz, DMSO- d 6 ) δ 10.87 (s, 1H, Ar-NH), 8.52 (dd, J J = 62.1, 8.6 Hz, 1H, CO-NH-), 8.32 (dd, J J = 32.2, 7.6 Hz, 1H, CO-NH-), 8.18 (dd, J J = 38.4, 8.5 Hz, 1H, CO-NH-), 7.87 (t, J J = 7.0 Hz, 1H, CO-NH-), 7.52(dd, J J = 8.0, 4.0 Hz, 1H, ArH), 7.32 (d, J J = 7.9 Hz, 1H, ArH), 7.18 (d, J J = 5.1 Hz, 4H, ArH), 7.14 (dd, J J = 9.8, 4.6 Hz, 2H, ArH), 7.08 – 7.03 (m, 1H, ArH), 6.97(q, J J = 7.5 Hz, 1H, ArH), 4.60 (dtd, J= 24.9, 8.5, 4.6 Hz, 1H), 4.48 (p, J = 7.1Hz, 1H), 4.35 – 4.27 (m, 1H),4.20 (dt, J = 32.9, 8.2 Hz, 1H), 4.06 (dt, J = 37.8,6.0 Hz, 1H), 3.15 (dd, J = 14.6, 6.1 Hz, 2H), 3.08 – 3.01 (m, 2H), 2.99 – 2.86(m, 2H), 2.77 (ddd, J = 14.4, 9.5, 5.9 Hz, 1H), 1.64 (q, J = 7.5 Hz, 1H), 1.49(ddt, J = 20.9, 14.2, 7.3 Hz, 1H), 1.34 (qq, J = 13.9, 8.8, 7.1 Hz, 2H), 1.03(dt, J = 24.6, 7.1 Hz, 1H), 0.83 – 0.70 (m, 12H, -CH3×4). 13 C-NMR (126 MHz, DMSO- d 6 ) δ 173.11, 171.48, 170.93, 170.40, 170.21, 137.49, 136.12, 129.24, 127.96, 127.27, 126.21, 123.67, 120.97, 118.43, 118.23, 111.43, 109.55, 57.34, 53.61, 53.09, 51.49, 50.97, 40.92, 36.81, 27.31, 24.34, 24.14, 23.07, 21.63, 15.20, 10.99. Example 3 I. Determination of the inhibition of tyrosinase activity by CTLEW and its analogs 2 mmol / L L-DOPA solution: Accurately weigh 0.19735 g of L-DOPA, dissolve it with phosphate buffer solution, transfer it to a 50 mL volumetric flask, make up the volume to the mark, and shake well.

[0056] Tyrosinase solution at 125 U / mL: Take 25 KU of tyrosinase, dissolve it with phosphate buffer solution, place it in a 25 mL volumetric flask, make up the volume, shake well to obtain an enzyme solution at 1000 U / mL, dilute it to 125 U / mL, and place it in a refrigerator at -20 °C for use. Thaw the tyrosinase solution at room temperature for 2 h before use.

[0057] Preparation of kojic acid solution: Weigh 1 mg of kojic acid precisely, and use phosphate buffer solution to prepare test solutions at 1000, 500, 250, 125, 62.5, 31.25, 15.625 μg / mL by the method of two-fold dilution.

[0058] Preparation of polypeptide sample: Weigh 1 mg of polypeptide sample precisely, and use 5% DMSO / phosphate buffer solution to prepare test solutions at 1000, 500, 250, 125, 62.5, 31.25, 15.625 μg / mL by the method of two-fold dilution.

[0059] On a 96-well plate, divide it into 4 groups, with three replicate wells in each group.

[0060] A1: Add 80 μL of L-DOPA solution, 80 μL of buffer solution, and 80 μL of enzyme solution to each well.

[0061] A2: Add 80 μL of L-DOPA solution and 160 μL of buffer solution to each well.

[0062] A3: Add 80 μL of polypeptide sample solution, 80 μL of L-DOPA solution, and 80 μL of enzyme solution to each well.

[0063] A4: Add 80 μL of polypeptide sample solution, 80 μL of L-DOPA solution, and 80 μL of buffer solution to each well.

[0064] For each group, first add other solutions and then add the enzyme solution. After reacting at 37 °C for 20 min, measure the absorbance value at 475 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The calculation formula for the inhibition rate is as follows: Inhibition rate (%) = [((A1 - A2) - (A3 - A4)) / (A1 - A2)] * 100%.

[0065] II. Determination of the scavenging of DPPH free radicals by CTLEW and its analogues Preparation of DPPH: Weigh 0.008 g of DPPH precisely, completely dissolve it with absolute ethanol, and make up the volume to 100 mL to obtain a solution with a concentration of 0.08 mg / mL. Store it in the dark at -4 °C for use.

[0066] Preparation of BHT: Accurately weigh 1 mg of BHT and prepare test solutions with different concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.625 μg / mL) using 70% ethanol. Preparation of polypeptide sample solution: Accurately weigh 1 mg of the sample and prepare test solutions with concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625 μg / mL by serial dilution method using 70% ethanol.

[0067] Accurately transfer 100 μL of sample solutions with concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625 μg / mL into 96-well plates, add 100 μL of DPPH solution (0.08 mg / mL) respectively, and label them as A1. Each group is measured in parallel three times. Accurately transfer 100 μL of sample solutions into 96-well plates, add 100 μL of absolute ethanol respectively, and label them as A2. Measure in parallel three times. Accurately transfer 100 μL of DPPH solution (0.08 mg / mL) and add 100 μL of 70% ethanol, label it as A0, and measure in parallel three times. React in the dark for 30 min, measure the absorbance at 517 nm, and the inhibition rate = [1 - (A1 - A2) / A0] × 100%. Then calculate the IC 50 value.

[0068] III. Cytotoxicity Assay of CTLEW and Its Analogs on Normal Cells In this experiment, the classical MTS method was used to determine the cytotoxicity of all synthesized bioactive small peptides on human normal lung epithelial cells (BEAS-2B). After cell passage, 10 μL of cell suspension was pipetted onto a cell counting plate, and the cells were counted under a microscope. The cells were seeded into 96-well plates at a density of 4000 cells per well, with 100 μL of cell suspension per well. After incubation for 24 h, the target compounds and positive control drugs were fully dissolved in DMSO and prepared into samples with a concentration of 40 μmol / L, added to the wells and incubated for another 24 h. Finally, MTS solution was added and incubated for 4 h, and the OD value was measured using a microplate reader. The experimental results were expressed as cell viability.

[0069] IV. Experimental Results and Analysis 1. CTLEW and Its Analogs Inhibit Tyrosinase Activity The results of in vitro tyrosinase inhibition activity screening showed that, compared with the positive control kojic acid (164.90±0.85 μg / mL), CTLEW (83.32±0.92 μg / mL) among these compounds showed very strong tyrosinase inhibition activity, CTLVW (143.33±1.66 μg / mL) showed moderate inhibition activity, and other compounds showed weak inhibition activity, indicating that the pentapeptide compound CTLEW of the present invention has special anti-tyrosinase activity. Amino acids with higher molecular docking scores such as phenylalanine (Phe, F) and arginine (Arg, R) were added, and the target compounds all showed good anti-tyrosinase activity, which was basically consistent with the molecular docking results.

[0070] In addition, during the experiment, it was found that CFLWW, CWLIW, and CILFW precipitated. The absolute value of the binding energy was between 4.23 - 5.25, with a relatively high score but poor actual activity. The absolute value of the binding energy of CTLEW and CLEW was less than 3.33, but CTLEW had better actual pharmacodynamic effects. It was speculated that this was related to the water solubility of the compounds. The Log P value (the lower the Log P value, the better the hydrophilicity) of the target compounds was calculated using software. The results showed that the Log P values of CWLIW and CILFW were greater than 0.69, CFLWW was as high as 1.02, and CTLEW was -3.21, the same as the positive control kojic acid. It can be seen that good water solubility may enhance the activity of the compounds in inhibiting tyrosinase.

[0071] The IC 50 values of CTLEW and its analogues for inhibiting tyrosinase are as shown in Table 2 - 3 below: Table 2 - 3 IC 50 values Table 2-3 IC 50 values for tyrosinase inhibition by CTLEW and its analogues 2. Results of CTLEW and its analogues in scavenging DPPH free radicals Free radicals are by-products of normal metabolism in the body and play an important role in life metabolism. The excessive accumulation of free radicals will cause oxidation and deformation of proteins or nucleic acids, and then trigger various diseases. DPPH free radicals are relatively stable and convenient for experimental operations. Currently, DPPH is commonly used in laboratories to evaluate the antioxidant activity of compounds.

[0072] The experimental results are shown in Table 2 - 4. The IC 50The value ranges from 54.52 ± 0.21 μg / mL to 134.70 ± 2.40 μg / mL, and is comparable to the activity of the positive control BHT (103.95 ± 0.01 μg / mL). The IC 50 of CLW is 54.52 ± 0.21 μg / mL, with the highest antioxidant activity. Followed by CLEW (64.47 ± 0.14 μg / mL), CTLW (64.86 ± 0.59 μg / mL) and CTLVW (80.20 ± 0.99 μg / mL). The present invention finds that most of the sequences with good antioxidant activity contain valine, leucine, tryptophan and phenylalanine. It can be seen from the results that the addition of hydrophobic amino acids and aromatic hydrocarbon amino acids can improve the antioxidant activity of walnut small molecule peptides. BHT is the widely used antioxidant 2,6-Di-tert-butyl-4-methylphenol.

[0073] Table 2-4 IC 50 values Table 1-5 IC 50 values for DPPH radical scavenging by CTLEW and its derivatives 3. Cytotoxicity results of CTLEW and its analogues on normal cells To evaluate the safety of CTLEW and its analogues of the present invention to humans, the present invention adopted the MTS method to test the toxicity of 15 synthetic small molecule peptides on human normal lung epithelial cells (BEAS-2B). The experimental results are presented in the form of survival rate, as Figure 4 shown. The survival rates of human normal lung epithelial cells (BEAS-2B) are all above 80%, indicating that all the synthetic active small molecule polypeptides (CTLEW and its analogues) have no inhibitory effect on human normal lung epithelial cells (BEAS-2B) at 40 μmol / L, and the normal cytotoxicity is extremely low. It can be known that walnut small molecule peptides and their analogues (CTLEW and its analogues) have low cytotoxicity and high safety.

[0074] 4. Determination of the inhibition kinetics of CTLEW on tyrosinase The enzyme kinetics was used to determine the inhibitory activity of the compound on tyrosinase by keeping the concentration of tyrosinase constant and changing the concentration of the substrate (L-DOPA). In this experiment, the concentration of the enzyme solution was fixed at 125 U / mL, and a series of solutions of L-DOPA with concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μg / mL were prepared. The solution concentrations of CTLEW were 100, 80, 60, and 0 μg / mL. 80 μL of the polypeptide sample solution, 80 μL of the L-DOPA solution, and 80 μL of the enzyme solution were added to each group, with three replicates. The reaction was carried out at 37 °C for 20 min, and the absorbance value was measured at 475 nm using an enzyme-linked immunosorbent assay reader. A double-reciprocal plot was made using Oring 8.0, and the concentration of the compound was plotted twice against the slope and the intercept on the vertical axis of the double-reciprocal plot and the straight line in the double-reciprocal plot using Lineweaver-Brunk. According to the inhibition type of the compound on tyrosinase, the inhibition constant K i or K is . Among them, K i is the binding constant between the inhibitor and tyrosinase, and K is is the binding constant between the inhibitor and the tyrosinase-substrate complex.

[0075] The enzyme inhibition kinetics of CTLEW on tyrosinase was studied using the Lineweaver-Burk double-reciprocal plot. As Figure 5 -A shows, with the reciprocal of the substrate concentration as the X-axis and the reciprocal of the reaction rate as the Y-axis for plotting, different concentrations of CTLEW show different straight lines in the graph, and these straight lines finally intersect the X-axis. As the concentration of the compound CTLEW increases, the value of K m remains unchanged, and the value of V max gradually decreases, proving that the compound CTLEW is a non-competitive tyrosinase inhibitor. Then, by plotting the reciprocal of V max obtained from the straight lines of different concentrations against the inhibitory concentration ( Figure 5 -B), the binding constant K i was calculated to be 0.045 μg / mL. The obtained binding constant is relatively small, indicating that CTLEW binds strongly to tyrosinase.

[0076] 5. Study on the anti-browning effect of CTLEW on fresh-cut potatoes Three groups of solutions were prepared: water (control blank control group), kojic acid (positive control group, 0.5 mM), and CTLEW (target compound group, 0.5 mM). Fresh potatoes of the same variety, size, and color purchased from the market were sliced, washed with distilled water, and then the surface water was drained with absorbent paper. The potato slices were immersed in the above three groups of solutions for 10 min and observed at room temperature. The apple slices in each group were photographed every day, and the L*, a*, and b* values of each group were measured with a color difference meter, and △E was calculated as a parameter for judging anti-browning. Each group was repeated three times.

[0077] △E = 〔(L t * - L initial *) 2 + (a t * - ainitial *) 2 + (b t * - b initial *) 2 〕 0.5 The results were as Figure 6 shown. As the storage time increased, the L* values of the CTLEW group, kojic acid control group, and blank control group (distilled water) gradually decreased, and the ΔE values gradually increased, indicating that the fresh-cut potato slices underwent browning during this process. By Figure 6 observing the change trend of the L* values of the three groups, the L* value of the blank control group decreased very significantly, indicating that the blank group had no anti-browning ability. The decreasing trends of the L* values of the CTLEW and kojic acid groups were slower than that of the blank group. Among them, CTLEW was better than kojic acid, indicating that both the CTLEW group and the kojic acid positive control group had significant anti-browning ability for fresh-cut potato slices, and the anti-browning effect of CTLEW was better than that of kojic acid. From Figure 6 the total color difference change of the ΔE value in Figure 6 -A and the surface change photos of the potato slices in

[0078] -C on the 6th day, the same results could also be obtained. In this experiment, fresh apples purchased from the market were used as the research object to investigate the anti-browning ability of CTLEW on fresh apple slices. Three groups of solutions were set up in this experiment, namely water (blank control group), kojic acid (positive control group, 0.5 mM), and CTLEW (target compound group, 0.5 mM). After washing the fresh apple slices of the same variety, size, and color purchased, they were sliced, and the surface water of the apple slices was gently wiped with absorbent paper and then immersed in the above three groups of solutions simultaneously for 10 min. After taking them out, they were put into sample bags and placed in a -4°C refrigerator. The apple slices in each group were photographed every day, and the L*, a*, and b* values of each group were measured with a color difference meter, and △E was calculated as a parameter for judging anti-browning. Each group was repeated three times.

[0079] △E = 〔(L t *-L initial *) 2 + (a t *- ainitial *) 2 + (b t *-b initial *) 2 〕 0.5 The results are as Figure 7 shown. At the beginning of the experiment, the brightness and L* values of the three groups were almost exactly the same. As the storage time increased, it was found that the L* values of each group began to gradually decrease, and the photos of the apple slices (C) visibly darkened, indicating that browning reactions occurred in the fresh-cut apple slices during storage. From Figure 7 -A and Figure 7 -B, it can be seen that in the blank control group (distilled water), the L* value decreased from 73.01 to 54.84 within 8 days, and the ΔE value increased from 0 to 19.41. In the kojic acid control group, the L* value decreased from 74.09 to 60.77, and the ΔE value increased from 0 to 14.13. In the CTLEW treatment group, the L* value decreased from 76.6 to 66.29, and the ΔE value increased from 0 to 10.09. Based on the changes in the above L* values and ΔE values, it can be seen that the trend of brightness reduction in the CTLEW group and the kojic acid control group is much slower than that in the blank control group, indicating that CTLEW and kojic acid have an anti-browning effect on fresh-cut apple slices. Comparing the CTLEW group and the kojic acid control group, the brightness change of CTLEW is slower than that of kojic acid, and it can be considered that the anti-browning effect of CTLEW is better than that of kojic acid. In addition, the same conclusion can also be drawn from the appearance presented in the photos ( Figure 7 -C)).

[0080] Enzyme kinetics confirmed that CTLEW is a non-competitive inhibitor and has a strong binding ability to tyrosinase. The anti-browning experiments on fresh-cut fruits and vegetables showed that CTLEW exhibited a stronger anti-browning effect than kojic acid on both potato slices and apple slices. CTLEW is a lead compound of tyrosinase inhibitor with antioxidant function and is expected to be applied in the fields of medicine, food, cosmetics, etc.

[0081] Based on the above experimental results, the CTLEW and its analogs of the present invention can form a composition with a carrier and be applied in the fields of medicine, food, cosmetics, etc. The carrier includes but is not limited to: deionized water, phosphate buffer (pH 6.5), food-grade ethanol (concentration ≤ 30%), glycerol, sodium alginate solution (1 - 2% w / v), β-cyclodextrin, corn starch, lactose, microcrystalline cellulose, food-grade silicon dioxide, chitosan (1 - 3% acetic acid solution), sodium carboxymethyl cellulose (CMC-Na), sunflower oil-based emulsion, polyvinyl alcohol (PVA), gelatin, ascorbic acid (antioxidant), Tween 80 (emulsifier), potassium sorbate (preservative). Preferably, the composition has a dosage form of solution, powder or coating.

[0082] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily achieved.

Claims

1. A walnut pentapeptide compound, characterized in that, It has the following chemical structure: 。 2. A method for preparing the pentapeptide compound according to claim 1, characterized in that, It includes: a) Targeting tyrosinase, using Autodock 4.3 software for molecular docking to design compounds with the structure of general formula A; b) Using 2-CTC resin as a carrier and Oxyma / DIC as a condensing agent to synthesize the target compound by solid-phase synthesis method.

3. Use of the pentapeptide compound described in claim 1 or prepared by the preparation method of claim 2 in the preparation of a food preservative.

4. The application according to claim 3, characterized in that, The preservative is used to inhibit enzymatic browning of fruits and vegetables.

5. The application according to claim 3, characterized in that, The fruits and vegetables include potatoes and apples.

6. Use of the pentapeptide compound described in claim 1 or prepared by the preparation method of claim 2 in the preparation of a tyrosinase inhibitor.

7. A composition, characterized in that, It contains the pentapeptide compound described in claim 1 or prepared by the preparation method of claim 2, and a pharmaceutically or foodologically acceptable carrier.

8. The composition according to claim 7, wherein Its dosage form is solution, powder or coating.

9. An analogue of a walnut pentapeptide compound, characterized in that, Any one of the following chemical structures: 。 10. Use of the analogue of the walnut pentapeptide compound as described in claim 9 in the preparation of an antioxidant.

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