Red rice bran-derived small molecule peptide and application thereof

Abstract

The application discloses a small molecule peptide derived from red rice bran, and the amino acid sequence of the small molecule peptide is Leu-Cys-Trp (LCW). Through in vitro tyrosinase activity and intracellular tyrosinase activity experiments of the small molecule peptide LCW, the results show that LCW is combined with amino acid residues of tyrosinase through hydrogen bond, electrostatic interaction force and hydrophobic force, and is combined with active centers Cu400 and Cu401 of tyrosinase through van der Waals force to inhibit tyrosinase activity, and LCW has copper ion chelating capacity. Experiments prove that LCW has good tyrosinase inhibitory activity, can inhibit the synthesis of melanin, and the small molecule peptide has high safety, good water solubility, simple preparation, and is suitable for industrialized production and market popularization and application.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of red rice bran source small molecule peptide and its application in inhibiting melanin, belong to the field of biotechnology. BACKGROUND

[0002] In the process of melanin generation, tyrosinase is a key rate-limiting enzyme, which can catalyze L-tyrosinase to generate L-dopa, and then generate dopaquinone, and then generate melanin through a series of reactions. However, excessive melanin production caused by pigmentation diseases such as chloasma, seborrheic keratosis, freckles, etc. Some chemical agents on the market such as ascorbic acid, arbutin, kojic acid and hydroquinone, the side effects of these reagents also limit their long-term use in different industries. In recent years, skin whitening research on active peptides has attracted much attention. Bioactive peptides are composed of amino acids, have mild action conditions, stable properties, etc. Especially food-derived active peptides obtained by different enzyme hydrolysis can inhibit tyrosinase activity. Feng Yanxia et al. found that peptide segment Tyr-Arg-Ser-Arg-Lys-Ser-Ser-Trp-Pro (YRSRKSSWP) and Akihito Ochiai et al. found Leu-Gln-Pro-Ser-His-Tyr (L-Q-P-S-H-Y) both have whitening effect. However, there are few reports on tyrosinase inhibitors from Yunnan red rice bran protein, so it is of great significance to carry out related research. SUMMARY

[0003] The present application provides a small molecule peptide obtained from red rice bran, whose amino acid sequence is Leu-Cys-Trp (LCW). The small molecule peptide has good tyrosinase inhibitory activity on both in vitro activity and cell level, and can inhibit the synthesis of melanin. The present application realizes the full utilization of byproduct rice bran resources.

[0004] The application grinds and screens red rice bran, and obtains protein powder by alkali dissolution and acid precipitation after defatting the rice bran powder, and the protein powder is hydrolyzed by alkaline protease, trypsin, neutral protease and papain respectively, the tyrosinase inhibitory activity of the hydrolysate is detected, the results show that the activity of the alkaline protease hydrolysate is higher, the structure of the alkaline protease hydrolysate is identified by LC-MS / MS, 449 peptide segments are identified, 20 peptide segments which are not reported, have high water solubility and high safety are screened out, the 20 peptide segments are docked with tyrosinase (2Y9X), and the peptide segment LCW which has lower binding energy with tyrosinase is screened out, the in vitro tyrosinase activity of the small molecule peptide LCW and the intracellular tyrosinase activity experiment of the small molecule peptide LCW are carried out, the results show that the amino acid residues of LCW and tyrosinase are combined through hydrogen bond, electrostatic interaction force and hydrophobic force, and are combined with the active centers Cu400 and Cu401 of tyrosinase through van der Waals force to inhibit the activity of tyrosinase, and have copper ion chelating ability, the experiment proves that LCW has good tyrosinase inhibitory activity and can inhibit the synthesis of melanin.

[0005] The advantages and technical effects of the application are as follows:

[0006] The small molecule peptide LCW in the application is derived from red rice bran, has high safety and good water solubility, has good tyrosinase inhibitory activity, can inhibit the synthesis of melanin, the enzyme catalytic reaction kinetics shows that the peptide segment LCW has reversible mixed type inhibition effect on tyrosinase, the application provides a new way for melanin inhibition, and the peptide is simple to prepare, suitable for industrial production and market promotion and application. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 The in vitro tyrosinase inhibitory activity results of the products after the protein extracted from red rice bran is hydrolyzed by four different proteases;

[0008] Figure 2 The copper ion chelating ability results of the products after the protein extracted from red rice bran is hydrolyzed by four different proteases;

[0009] Figure 3 The 3D visualization diagram of the docking of the peptide segment LCW and tyrosinase (2Y9X);

[0010] Figure 4 The 2D visualization diagram of the docking of the peptide segment LCW and tyrosinase (2Y9X);

[0011] Figure 5 The in vitro activity evaluation experiment results of the peptide segment LCW on tyrosinase, wherein the A diagram is the tyrosinase inhibitory activity result, and the B diagram is the copper ion chelating ability result;

[0012] Figure 6Results of the effect of the peptide segment LCW on the viability of melanoma cells

[0013] Figure 7 Results of the effect of the peptide segment LCW on the intracellular tyrosinase activity

[0014] Figure 8 Results of the effect of the peptide segment LCW on the intracellular melanin production inhibition

[0015] Figure 9 Effect diagram of the peptide segment LCW on the reduction of melanin production

[0016] Figure 10 Graph of the relationship between the tyrosinase reaction rate and the tyrosinase concentration

[0017] Figure 11 Lineweaver-Burk double-reciprocal graph of the peptide segment LCW and the enzyme

[0018] Figure 12 Graph of the relationship between the slope and the concentration of the peptide segment LCW

[0019] Figure 13 Graph of the relationship between the intercept and the concentration of the peptide segment LCW DETAILED DESCRIPTION

[0020] The technical solutions of the present application are further described below through examples, but the content of the present application is not limited thereto. The methods in the present examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. are all obtained from commercial channels or prepared according to conventional methods unless otherwise specified.

[0021] Example 1: Obtaining of the small molecule peptide LCW

[0022] 1. Red rice bran was ground through a 40-mesh sieve, and the rice bran was mixed with petroleum ether at a mass-volume ratio of g:mL 1:3 for defatting. After stirring at room temperature for 2 h, the mixture was centrifuged at 4000 rpm for 15 min, and the precipitate was collected and dried in a fume hood. The dried rice bran powder was added to a NaOH solution (0.001 mol / L) at pH 11 at a mass-volume ratio of g:mL 1:15, and stirred at room temperature for 4 h. After standing for half an hour, the supernatant was collected, and the pH of the supernatant was adjusted to 4.7 with 1 mol / L HCl. The mixture was centrifuged at 4000 rpm for 15 min, and the precipitate was collected, washed with water, and adjusted to neutral with a 1 mol / L NaOH solution. The precipitate was again centrifuged to obtain a precipitate (the whole process was repeated 3 times), which was vacuum freeze-dried to obtain a protein powder, which was stored at -20℃.

[0023] 2. Add protein powder to ultrapure water to prepare a solution with a mass volume (g: mL) concentration of 5%, then add alkaline protease (RRBP-A, pH 8.5, 50°C), trypsin (RRBP-P, pH 8.5, 37°C), neutral enzyme (RRBP-N, pH 7.0, 50°C), and papain (RRBP-M, pH 7.0, 50°C) respectively for 4 hours, with an enzyme addition amount of 2500 U / g; terminate the hydrolysis process after boiling in water for 10 minutes, centrifuge the enzyme hydrolysate at 4000 rpm for 15 minutes, and collect the supernatant; freeze-dry the supernatant to obtain the hydrolysate, which is subjected to in vitro tyrosinase inhibition activity evaluation;

[0024] 3. In vitro tyrosinase inhibition activity experiment

[0025] Take L-DOPA as the substrate of tyrosinase, mix 80 μL of the hydrolysate of step 2 with different concentrations (1, 5, 10, 15, 20 mg / mL) and 40 μL of tyrosinase solution (200 U / mL) dissolved in phosphate buffer (pH 6.8), incubate in the dark at room temperature for 10 minutes, then add 160 μL of L-DOPA solution (2 mmol / L) dissolved in phosphate buffer (pH 6.8), measure the absorbance value at 475 nm after 15 minutes of reaction, and calculate the tyrosinase inhibition rate according to the following formula; at the same time, take kojic acid as a positive control;

[0026] Tyrosinase inhibition rate (%) = [1- (A3-A2) / (A1-A0)] x 100%;

[0027] In the formula: A1 is the absorbance without hydrolysate, A0 is the absorbance without hydrolysate and tyrosinase, A3 is the absorbance of the hydrolysate, and A2 is the absorbance without tyrosinase.

[0028] The results are shown in Figure 1 As can be seen from the figure, the tyrosinase inhibition effect of the four different enzyme hydrolysates (RRBP-A, RRBP-P, RRBP-N, RRBP-M) obtained by enzyme hydrolysis of red rice bran protein is positively correlated with the concentration; when the concentration is 20 mg / mL, the tyrosinase inhibition activity of RRBP-A hydrolysate is the highest (58.92 ± 3.19%), followed by RRBP-P (50 ± 1.44%), RRBP-N (46.95 ± 4.79%), and RRBP-M (39.05 ± 1.6%), but their inhibition effects on tyrosinase are all lower than that of kojic acid; the results show that the content of tyrosinase inhibitory peptides in the hydrolysate RRBP-A is significantly higher than that in other hydrolysates.

[0029] 4, 10 μL of the hydrolysate solution with different concentrations (4, 6, 8, 10, 12 mg / mL) was mixed with 280 μL of 50 mmol / L sodium acetate buffer, then 10 μL of 1 mg / mL CuSO4·5H2O solution (pH 6.0, 50 mmol / L sodium acetate buffer) and 6 μL of 4 mmol / L catechol violet solution (pH 6.0, 50 mmol / L sodium acetate buffer) were added in turn, mixed and incubated for 6 minutes, then the absorbance value was measured at 632 nm wavelength using a microplate reader, the copper ion complexing capacity was calculated according to the following formula, and a blank control without adding the hydrolysate and a positive control with EDTA were set;

[0030] Copper ion complexing capacity (%) = (A1-A0) / A1x100%; in the formula: A1 is the absorbance of the blank control, and A0 is the absorbance of the hydrolysate.

[0031] The results are shown in Figure 2 From the figure, compared with the other three hydrolysates, the copper chelating activity of the hydrolysate RRBP-A was the best, especially at a concentration of 12 mg / mL, the chelating activity of RRBP-A was 11.53%, 10.87% and 8.59% higher than that of RRBP-N, RRBP-M and RRBP-P respectively (P < 0.05). Of course, compared with all the hydrolysates, EDTA showed the strongest chelating copper activity, which also supported the tyrosinase inhibitory effect of RRBP-A.

[0032] 5、LC-MS / MS was used to identify the structure of the hydrolysate RRBP-A. A total of 449 peptide segments were identified. The desalted hydrolysate RRBP-A (3 μL) was dissolved in 0.1% formic acid solution, analyzed using a C18 analysis column (Acclaim PepMap, 75 μm × 25 cm) and an EASY-nanoLC 1200 system (Thermo Fisher Scientific, Massachusetts, USA), and the gradient process of liquid separation was as follows: 0-53.6 min B liquid (80% acetonitrile + 0.1% FA) linear gradient range 4%-50%, 53.6-54.4 min B liquid gradient range 50%-95%, 54.4-6 min B liquid gradient range 95%. The flow rate was 300 nL / min. The sample eluent was analyzed by mass spectrometer in positive ion mode, the column temperature was 40°C, the electrospray voltage was 2 kV, and the mass spectrometer was operated in data-dependent acquisition (DDA) mode and automatically switched between MS and MS / MS nodes. The full scan mass spectrum (m / z: 350-1500) was collected in an Orbitrap with a resolution of 120000. The normalized automatic gain control (AGC target value was 200%, maximum injection time was 50 ms, and the database was Oryza_sativa_subsp_japonica (version 2022, 43673 entries) downloaded from unipor. When searching the PEAKS database, the fragment ion mass tolerance was 0.02 Da, and the parent ion mass tolerance was ±10 ppm. Methionine oxidation and deamination of asparagine and glutamine were designated as variable modifications. Peptide segments with -10 lgP≥20 were screened, at least containing one unique peptide segment.

[0033] To screen small molecule peptides that have not been reported, further analysis selected peptide segments with molecular weights between 300-1200 Da, and then used Peptide ranker to screen peptide segments with scores greater than 0.85. Then the toxicity and water solubility of the peptide segments were predicted by ToxinPred program (http: / / crdd.osdd.net / raghava / toxinpred / ) and SwissADME (http: / / www.swissadme.ch / ), respectively. Finally, 20 peptide segments with good water solubility and high safety were obtained.

[0034] The 20 peptide segments were subjected to molecular docking with tyrosinase (2Y9X), and the peptide segments with lower binding energy were screened. The process of molecular docking was carried out by using Autodock Vina (version 1.5.7) for molecular docking analysis. The tyrosinase in mushroom (PDB ID: 2Y9X) was downloaded from the RCSB protein database (http: / / www.rcsb.org / pdb / ) as the X-ray structure of the receptor protein, and its structure was modified using Pymol, retaining the A chain containing 391 amino acid residues and the copper ions Cu400 and Cu401; before docking, the water molecules in the tyrosinase structure were removed and hydrogen was added using the Autodock tool, the three-dimensional structure of the screened polypeptide was constructed by Pymol, and the binding parameters were set as center_x = -7.951, center_y = -25.584, center_z = -26.924, and all dimensions were 60 Å. The three-dimensional and two-dimensional structure visualization of the molecular docking results was carried out using Discovery Studio4.5.

[0035] The docking results showed that the binding energy of the peptide segment LCW with tyrosinase was -7.1 Kcal / moL, which had good binding with tyrosinase (2Y9X), and theoretically the inhibitory peptide had the ability to tightly bind with the active center of tyrosinase.

[0036] The 3D and 2D visualization results of the peptide segment LCW docked with tyrosinase (2y9x) are shown in Figure 3 、 4 The results showed that the peptide segment LCW had two hydrogen bond interactions with the ASN260 and MET280 residues, one electrostatic interaction with the GLU256 residue, and six hydrophobic interactions with the VAL248, VAL283, ALA286 and HIS263 residues. The active site of tyrosinase is composed of three regions: solvent open region, hydrophobic tunnel region and substrate binding pocket region, among which the binding site of the solvent open region is the hydrophilic amino acid residues of Glu189 and Arg268 residues, and the hydrophobic tunnel region is composed of Val248, Phe264, Val283 and Pro284 residues. In addition, the binding pocket is composed of two copper ions (Cu400 and Cu401) and six highly conserved histidine residues, among which Cu400 is combined with His61, His85 and His94 residues, and Cu401 is combined with His259, His263 and His296 residues, which indicates that the peptide segment LCW has strong binding ability with tyrosinase.

[0037] 6、The peptide segment LCW was synthesized by Nanjing Jiepeibio Technology Co., Ltd. using solid phase synthesis method, and the purity of the synthesized peptide segment was >95%, which was stored at -80℃ and used for subsequent test experiments.

[0038] Example 2: Experiment on the in vitro activity of peptide LCW against tyrosinase and its copper ion complexing ability.

[0039] 1. The in vitro activity test of peptide LCW against tyrosinase is the same as step 3 in Example 1, except that the concentration of peptide LCW solution is 0.0625-1 mg / mL.

[0040] See results Figure 5 A. The LCW peptide exhibits a strong positive correlation with the inhibitory activity against tyrosinase. Furthermore, the IC50 of the LCW peptide inhibiting tyrosinase is [not specified in the original text]. 50 The value was 153.5 ± 4.95 μg / mL, which was higher than the IC50 of kojic acid (23.5 ± 0.71 μg / mL). 50 value.

[0041] 2. The method for detecting copper ion chelating ability is the same as step 4 in Example 1, except that the concentration of the peptide LCW solution is 0.25, 0.5, 1, 2, and 4 mg / mL. The results will be... Figure 5 B, the LCW peptide also showed good activity in copper ion chelation, with an IC50 value of [missing information]. 50 The concentration was 1245.3 ± 83.1 μg / mL, higher than the IC50 of EDTA. 50 Value: 867.67 ± 47.75 μg / mL;

[0042] The above results indicate that the LCW peptide exhibits strong activity in both tyrosinase inhibition and copper ion complexation.

[0043] Example 3: Experiment on the inhibition of melanin production by peptide LCW

[0044] 1. Effects of LCW peptides on cell viability

[0045] The cell viability of B16-F10 cells was determined by the CCK-8 assay.

[0046] Mouse melanoma cells B16-F10 were seeded in DMEM medium containing 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin and cultured at 37°C with 5% CO2 saturated humidity until the cells reached the logarithmic growth phase. The cells were then digested with trypsin and separated into 5 × 10⁶ cells. 3 Cells were seeded at 37 °C / mL into 96-well plates and cultured for 24 hours. The blank control group only had its culture medium changed. Different concentrations of peptide LCW (2, 1, 0.5, 0.25, 0.125, 0.0625 mg / mL) were added to the sample groups and treated for 48 hours. Then, 10% (v / v) CCK-8 solution was added to each well and incubated for another 4 hours. The absorbance was measured at 450 nm and the cell viability was calculated.

[0047] Cell viability (%) = [A1-A0] / A0 × 100%, where: A1 is the absorbance of the sample and A2 is the absorbance of the blank control group;

[0048] The results are as follows Figure 6 As shown, within the concentration range of 0.0625-2 mg / mL, LCW peptide had no significant effect on the viability of B16-F10 cells, with cell viability remaining above 92.25%, and there was no significant difference between different concentrations of LCW (P > 0.05).

[0049] 2. Effects of LCW peptides on intracellular tyrosinase activity

[0050] The concentration is 5 × 10 3 B16-F10 cells / mL were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. Then, the cells were treated with 100 μL of DMEM medium (blank control) or DMEM medium containing different concentrations of LCW peptides for 48 hours. After washing three times with pre-cooled PBS, 50 μL of a solution containing 1% (v / v) Triton X-100 was added to all wells and incubated at -80°C for 60 minutes. After incubation at 37°C until the cells were completely thawed, 50 μL of L-DOPA (4 mmol / L) was added to each well and the cells were incubated at 37°C for 1 hour. Finally, the absorbance was measured at 475 nm and the intracellular tyrosinase activity was calculated. A positive control group of kojic acid was also set up.

[0051] Cellular tyrosinase activity (%) = A1 / A0 × 100%, where: A1 is the absorbance of the LCW containing peptide, and A0 is the absorbance of the blank control;

[0052] The results are as follows Figure 7 As shown, the addition of peptide LCW significantly inhibited tyrosinase (P<0.05). Intracellular tyrosinase activity in the LCW-treated groups decreased by 10.01% (0.25 mg / mL), 23.66% (0.5 mg / mL), and 29.72% (1.0 mg / mL), respectively. Simultaneously, the positive control kojic acid (0.25 mg / mL) reduced intracellular tyrosinase activity by 37.21%. Peptide LCW possesses the ability to inhibit intracellular tyrosinase activity.

[0053] 3. Effects of LCW peptide on intracellular melanin production

[0054] B16-F10 cells were treated with peptide segment LCW (0.25, 0.5, 1 mg / mL), kojic acid (200 μg / mL), DEME medium (blank) for 48 hours, then the cells were digested with 0.25% trypsin solution, and collected into 1.5 mL sterile EP tubes, centrifuged at 10000 rpm for 10 minutes, the cell precipitate was mixed with 200 μL 1 mol / L NaOH (containing 10% DMSO), incubated at 80°C water bath for 30 minutes, then 200 μL ultrapure water was added to each well, the absorbance was measured at 450 nm wavelength, and the melanin content (%) = A1 / A0 x 100% was calculated, wherein: A1 is the absorbance of the peptide segment LCW, A0 is the absorbance of the blank;

[0055] The results are shown in Figure 8 There is a significant negative correlation between melanin content and LCW concentration, and the degree of significant decrease in melanin content is 29.35% (0.25 mg / mL), 36.55% (0.50 mg / mL) and 51.27% (1.00 mg / mL), respectively. In addition, there is no significant difference between the kojic acid 250 μg / mL group and the LCW 1.00 mg / mL group (P>0.05).

[0056] Figure 9 The results in Table 1 also demonstrate that LCW can significantly reduce the production of melanin, and the color becomes lighter with the increase of LCW concentration. These results show that the peptide segment LCW has a strong melanin inhibitory effect.

[0057] Example 4: Inhibition mechanism of peptide segment LCW on tyrosinase

[0058] 1. The concentration of substrate L-DOPA was set to 0.4 mg / mL, then different concentrations of peptide segment LCW solution (0, 0.05, 0.1 mg / mL) were added, and the reaction was carried out in the dark for 10 minutes, then different concentrations of tyrosinase solution (50, 100, 150, 200, 250 U / mL) were added, the absorbance change of the reaction system was measured, and finally the curve of enzyme reaction rate (ΔA / min) vs. added tyrosinase (TYR) concentration was plotted, and the inhibition mode of peptide segment LCW on tyrosinase was determined according to the curve characteristics: if a group of straight lines intersecting at the origin is obtained, then the inhibition mode of peptide segment LCW on TYR is reversible inhibition; if a group of parallel straight lines is obtained, then it is irreversible inhibition.

[0059] The results are shown in Figure 10As shown in Figure 4, the tyrosinase activity decreased linearly with different concentrations of peptide LCW solution, and all lines were concentrated on the origin, indicating that LCW inhibited the hydroxylation of tyrosine or the catalytic oxidation of L-DOPA by inhibiting the activity of tyrosinase, rather than by reducing the number of tyrosinase. The results showed that the inhibition of peptide LCW on the activity of tyrosinase was a reversible inhibition.

[0060] 2. The peptide LCW solution (0, 0.0625, 0.125, 0.25 mg / mL) was mixed with different concentrations (0.2, 0.4, 0.8, 1.2, 1.6 mg / mL, prepared with PBS) of L-DOPA solution for 10 minutes, then tyrosinase solution (200 U / mL, 40 μL) was added, and the reaction was carried out at 37°C for 30 minutes. The absorbance was measured at 450 nm using a 96-well. The reciprocal of the substrate concentration (1 / [S]) was used as the abscissa, and the reciprocal of the initial rate of enzymatic reaction (1 / V) was used as the ordinate to draw the curve; finally, the type of tyrosinase inhibition was determined.

[0061] The results are shown in Figure 5. Figure 11-13 Figure 11 The Lineweaver-Burk double-reciprocal plot of peptide LCW concentration of 0, 0.125, 0.25, 0.5 mg / mL was analyzed, and in the graph of 1 / V vs. 1 / [S], four lines with different slopes intersected in the second quadrant. It can be concluded that peptide LCW belongs to the mixed type of competition-non-competition, indicating that it can not only bind to free tyrosinase, but also to enzyme-substrate complex, resulting in a decrease in the rate of enzymatic reaction.

[0062] From the graph of slope (K m / V max ) vs. peptide concentration Figure 12 The equilibrium constant (K i ) of peptide LCW binding to E can be obtained from the graph of intercept (1 / V max ) vs. peptide concentration Figure 13 ES (K is ) can be obtained, where K i and K is values are 0.42 and 2.55 mmol / L, respectively. The results show that peptide LCW competes with the substrate for binding to the active center of tyrosinase, thereby inhibiting its activity.​

Claims

1. Use of a small molecule peptide in the manufacture of a melanin inhibitor, characterized in that: Derived from red rice bran, with the amino acid sequence Leu-Cys-Trp.

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