CN109608520B - Pearl-derived functional peptide and application thereof - Google Patents

Abstract

The invention discloses a pearl-derived functional peptide and application thereof. The amino acid sequence of the functional peptide is methionine-isoleucine-leucine-cysteine-serine-leucine (MIILLCSLL). The functional peptide has good functions of eliminating free radicals in organisms, resisting oxidation and inhibiting melanin cytochrome synthesis activity, so the functional peptide can be used for preparing free radical scavengers, antioxidants and cosmetic raw materials with whitening functions.

Description

Pearl-derived functional peptide and application thereof

Technical Field

The invention belongs to the field of marine organism functional peptides, and particularly relates to a novel pearl-derived functional peptide and application thereof.

Background

Since ancient times, pearls are good products for whitening and beautifying, and the record of the compendium of materia medica says that pearls are painted to make people moist and good in color. In modern cosmetics industry, pearls, pearl shells and zymolytes thereof become important components of whitening and freckle removing products and are recorded by the catalogs of cosmetic raw materials of the national food and drug administration.

The pearl protein is converted into functional peptide, which is an effective means for improving the efficacy of the pearl protein. Modern biological, pharmacological and medical researches find that skin color is closely related to the content, size, type and distribution of melanin in skin, and tyrosinase promotes the hydroxylation of tyrosine and the oxidation of dopa, which are necessary conditions for the formation of melanin in cells. The activities of oxidation resistance and free radical resistance become one of the main indexes for measuring the whitening effect, and are widely adopted in the leading countries of the European beautifying cosmetic industry. Meanwhile, a large amount of evidences show that the whitening and freckle removing functions of the pearls are closely related to the oxidation resistance of the pearls, and the pearl proteins are converted into functional peptides with special amino acid sequences, so that the oxidation resistance of the pearls can be obviously improved. The principle of the method is that a large number of amino acid sequences with antioxidant function are inlaid in untreated pearl protein, but the protein molecules are large and complex, and are difficult to directly absorb, and the contact between an antioxidant functional domain and an action target is limited, so that the full play of the whitening effect is restricted. The hydrolysis can convert macromolecules into easily-utilized oligopeptide, and fully expose the antioxidant functional domain, so that the antioxidant and pigment generation inhibiting activities of the pearl protein hydrolysate are obviously higher than those of untreated raw materials. Japanese scholars firstly find that acid hydrolyzed pearl protein has stronger antioxidant and tyrosinase activity inhibition activities, and obtain cosmetic application patents. Through improvement in various countries, biological enzyme is used as a catalyst to replace acid and alkali which have strong pollution and are difficult to control, and pearl protein hydrolysate with various varieties is prepared.

Pearl shell sclerostin zymolyte has been widely used, such as the pearl extract recorded in the catalogue of names of used cosmetic raw materials in China. However, few reports about specific types and structures of antioxidant peptides in zymolyte exist at present, the optimization of activities of high-activity pearl cosmetic raw materials and other novel pearl functional peptides is limited, the establishment of an accurate quality control technology is also restricted, and the product level is difficult to promote.

Disclosure of Invention

The first purpose of the invention is to provide a pearl-derived functional peptide with good functions of scavenging free radicals in organisms, resisting oxidation and inhibiting melanin cytochrome synthesis.

The functional peptide contains 9 amino acid residues, has an amino acid sequence of methionine-isoleucine-leucine-cysteine-serine-leucine (MIILLCSLL), and is a peptide with a novel structure.

The functional peptide of the invention takes the mixture of bromelain and keratinase as a catalyst to catalyze the hydrolysis reaction of pearl protein, and the effective components of the hydrolysate which can remove free radicals in organisms, resist oxidation and inhibit the synthesis activity of melanin cytochrome are prepared. After being separated and purified from the hydrolysate by ultrafiltration and chromatography, the hydrolysate is analyzed by matrix assisted time of flight mass spectrometry (MALDI-TOF-MS), and the molecular weight of the hydrolysate is identified to be 1018.604Da, and the amino acid sequence is methionine-isoleucine-leucine-cysteine-serine-leucine (MIILLCSLL). A search of the SWISS-PROT database at NCBI revealed no significant homologous sequences, and the results indicated that this functional peptide is a novel structural peptide derived from pearls.

The second purpose of the invention is to provide the application of the functional peptide in preparing a free radical scavenger or an antioxidant.

A radical scavenger or antioxidant comprising the above functional peptide as an active ingredient.

The third purpose of the invention is to provide the application of the functional peptide in the preparation of the inhibitor with the melanin cytochrome synthesis inhibiting activity.

A melanin cytochrome synthesis inhibitor comprising the above functional peptide as an active ingredient.

The fourth object of the present invention is to provide the use of the above-mentioned functional peptide for the preparation of a cosmetic raw material, such as a cosmetic raw material having a melanin cytochrome synthesis inhibitory activity.

A cosmetic raw material characterized by comprising the above functional peptide as an active substance.

The invention develops and develops aiming at the problems in the prior art, 1 antioxidant peptide with novel structure and good activity is prepared and identified by tracking and separating the active ingredients for inhibiting the generation of antioxidant and anti-free radical and melanin of human pigment cells in the pearl zymolyte, can be used as a high-quality antioxidant and anti-free radical and pigment cell melanin synthesis inhibitor and can also be used as a quality control index of the pearl zymolyte, and has obvious significance for the optimization of the pearl proteolysis process and the development of a novel product quality control technology.

The functional peptide has good functions of eliminating free radicals in organisms and inhibiting synthesis of human melanocytes pigment, so the functional peptide can be used for preparing cosmetic raw materials.

Description of the drawings:

FIG. 1 is a PPH-I-3ODS HPLC chromatogram;

FIG. 2 is a MALDI-TOF-TOF mass spectrum of the target functional peptide (MIILLCSLL).

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: preparation of enzymolysis protein of pearl

1. Raw materials and reagents

The pearl powder in the examples is provided by QIFUZHU processing Co Ltd in Guangzhou city, and is sieved with 200 mesh sieve, packaged in bags, sealed, and stored at-20 deg.C.

Bromelain (enzyme activity 40 ten thousand U/g) purchased from Pombo bioengineering, Inc. of south-Guangxi; keratinase (enzyme activity 20 ten thousand U/g) available from Jettier biotech, Inc.; DEAE-52 cellulose cation exchange resin was purchased from Whatman, and Sephadex G-15 gel resin was purchased from GE. Other reagents such as ethanol, formaldehyde, isopropanol, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate and the like are all made in China and analyzed. The test water is ultrapure water.

2. Preparation of pearl protein

Weighing about 100g of pearl powder, adding 100mL of water and 50mL of ethanol, and uniformly stirring. 3M hydrochloric acid was added slowly until no bubbles were produced. And (4) carrying out suction filtration on the reactant, collecting precipitates on filter paper, airing to obtain 5.6g of crude pearl protein, and carrying out freeze preservation.

3. Preparation of enzymolysis protein of pearl

Taking 5g of crude pearl protein, adding 50ml of bromelain/keratinase solution (the enzyme-substrate ratio is 4000U/g), wherein the weight ratio of bromelain in the solution is as follows: keratinase ═ 5: 1 (w/w). Adjusting pH to 6.5 with 1mol/L HCl, hydrolyzing at 50 deg.C for 5h, deactivating enzyme in boiling water for 10min after reaction, centrifuging at 6500r/min for 10min, removing insoluble impurities, centrifuging at 15000r/min for 10min, collecting supernatant, filtering with 0.2 μm filter membrane to obtain filtrate, Margarita zymoprotein solution, and lyophilizing to obtain zymolyte 3.8g, which is Margarita zymolyte.

Example 2 separation of novel functional peptides from enzymatic hydrolysate of Margarita

1. Dissolving 3g of pearl hydrolyzed protein obtained in the enzymolysis in the embodiment 1 in 100mL of water to prepare a sample solution, filtering the sample solution by using an ultrafiltration membrane with the molecular weight cutoff of 3kDa, and respectively freeze-drying the cut-off part and the filtrate to obtain two samples: PPH-I (molecular weight)>3kDa), PPH-II (molecular weight)<3 kDa). The DPPH-free radical scavenging ability of PPH-I and II (detailed methods see example 3), and EC of PPH-I, respectively, were measured₅₀EC of PPH-II ═ 4.5mg/mL₅₀The more active component PPH-I was selected accordingly to continue isolation at 40.5 mg/mL.

2. Separating PPH-I by using a G15 gel column to obtain 4 component fragments PPH-I-1-4, respectively freeze-drying, and detecting EC of DPPH free radical scavenging activity₅₀The values were 7.53mg/ml, 7.33mg/ml, 5.88mg/ml, 10.461mg/ml, respectively.

The G15 gel column separation is carried out under the following conditions: dissolving 1G of PPH-I sample in 5mL of deionized water to prepare a chromatographic sample, adding a Sephadex G-15 gel filtration column (2.6 multiplied by 100cm) balanced by pure water in advance, eluting the column by the deionized water at the flow rate of 1mL/min, sequentially collecting 4 chromatographic peak fragments (PPH-I-1-4) according to signals of a UV (280nm) detector, and respectively freeze-drying.

3. Collecting the most active fragment PPH-I-3 in the sample prepared in the previous step, separating with HPLC (ODS column) (chromatogram is shown in figure 1), collecting the chromatographic peak fraction at 11.5min, lyophilizing to obtain target functional peptide PPH-I-3-11, and testing DPPH scavenging activity of the target functional peptide, its EC₅₀The value was 0.82mg/mL, showing good radical scavenging ability.

The HPLC separation is carried out by using Agilent1200HPLC high performance liquid chromatography as a chromatograph, using Zorbax and SB C-18 columns (column size: 4.6mm × 250mm, 5 μm particle size, Agilent) and detecting wavelength of 280 nm. Solvent system: a, 0.1% aqueous trifluoroacetic acid, B, acetonitrile (Merck, pure by chromatography). Flow rate: 1.0 mL/min; elution procedure: 0-5 min, 5% B; 5-30min, 5% B-55% B. PPH-I-3(0.6g) was dissolved in 2ml of a 0.1% aqueous solution of trifluoroacetic acid (TFA) to prepare a sample solution. The sample amount of each separation is 50 μ L, and a chromatographic peak fragment (target functional peptide) with the retention time of 11.5min is collected. Co-injecting, eluting for 40 times, mixing target peak fragments, and lyophilizing to obtain 295.3mg of PPH-I-3-11 powder.

4. Structural identification

The structure of PPH-I-3-11 was identified using matrix assisted-time of flight mass spectrometry (MALDI-TOF-MS). MALDI-tof (ultraflexreeme) mass spectrometers use a positive ion delay extraction reflection mode to achieve the highest resolution and mass accuracy. CHCA was used as the matrix. During the test, 0.5uL of PPH-I-3-11 sample solution is spotted on a MALDI stainless steel target plate, after the solvent is volatilized, 0.5uLCHCA solution (0.5g/L, the solvent is 0.1% trifluoroacetic acid and 50% acetonitrile water solution) is spotted on the sample solution, and the sample is naturally volatilized at room temperature to obtain an analysis sample. The sample was ionized with a 337nm laser reflected from a nitrogen laser, accelerating the ions at 20kv voltage under optimized parameters. The LIFT cell voltage for the MS/MS experiment was set at 19kv to reach a final acceleration voltage (reflected voltage) of 29 kv. The precursor ions were selected using TIS (timing ion selector). After adjusting the laser intensity above the threshold for generating molecular ions for each peptide, 1000 shots were taken to collect MS/MS data.

The mass spectral data and fragment resolution of PPH-I-3-11 are shown in FIG. 2. Molecular ion Peak [ M +1⁺]At 1018.604 Da. In MascotFragment information was retrieved from the MS/MS database and no meaningful results were obtained. Further, analysis of the fragment ions by de novo sequencing using RapideNovo software confirmed that the amino acid sequence of the functional peptide (PPH-I-3-11) was: methionine-isoleucine-leucine-cysteine-serine-leucine (MIILLCSLL, with the specific sequence shown in SEQ ID No. 1). Met-Ile-Ile-Leu-Leu-Cys-Ser-Leu-Leu. The result of the search of SWISS-PROT database in NCBI without finding homologous sequence of PPH-I-3-11 functional peptide can indicate that the purified PPH-I-3-11 functional peptide is a new structural peptide generated from pearl protein.

Example 3 measurement of DPPH radical scavenging action.

The experiment employed a DPPH experiment to evaluate the free radical scavenging ability of the samples. The ability of a pearl protein zymolyte to scavenge DPPH free radical activity is determined according to the literature (Hsu, K.C.; Lu G.H.; Jao, C.L., effective properties of peptides prepended from root tissues with enzymes with orientation (Bacillus subtilis), Food research International 2009,42, (5-6), 647-652.). Dissolving a corresponding amount of sample in water according to a preset initial concentration to obtain a sample solution, adding 100 mu l of the sample solution into a first line of a 96-well plate, performing concentration gradient dilution by using ultrapure water, and respectively adding 100 mu l of the sample solution with the concentration of 2x10^{- 4}Three parallel experiments were performed for each concentration in a mol/L DPPH ethanol solution, and the blank experiment was performed by replacing the sample solution with absolute ethanol. The mixture was left to react at 37 ℃ for 30min in the dark. The absorbance was measured at 517 nm. The DPPH radical clearance was calculated according to the following formula:

in the formula: s is the absorbance of the sample after the reaction of adding DPPH; SB is the absorbance of the absolute ethyl alcohol added into the sample; c is the absorbance of absolute ethyl alcohol plus DPPH; absorbance of blank control with CB

The result is used for drawing a dose-effect curve, and the EC of DPPH scavenging activity can be calculated₅₀Value, i.e. halfThe clearance dose is counted.

Examples 1 and 2 EC for DPPH scavenging of the major samples₅₀See table 1 for values.

TABLE 1, examples 1 and 2 separation of ECs resulting in DPPH scavenging of the major samples₅₀Value of

Sample (I)

PPH

PPH-I

PPH-II

PPH-I-1

PPH-I-2

PPH-I-3

PPH-I-4

PPH-I-3-11

EC50(mg/mL)

30.5±9.2

4.5±1.1

40.5±3.2

7.53±0.53

7.33±0.13

5.88±2.5

10.46±2.3

0.82±0.25

EXAMPLE 4 determination of Pearl hydrolyzed protein and PPH-I-3-11 iron ion reducing Capacity (FRAP)

FRAP is an important measure of antioxidant function. The FRAP value of the sample is determined by the method of the present experimental reference (Eric A. Decker, andBarbara Welch, Role of transfer as a lipid oxidation catalyst in musclefood. J. age. food chem.,1990,38(3), pp 674-677).

Taking 10mmol/L TPTZ stock solution (dissolved with 40mmol/L hydrochloric acid), 20mmol/L Fe Cl₃The solution and 0.3mol/L acetate buffer (pH3.6) are mixed in a volume ratio of 1: 1: 10, mixing and standing at 37 ℃ for 1 hour to prepare a fresh FRAP working solution.

To 100. mu.L of each sample at a set concentration (0.1 and 1.0mg/mL), 300. mu.L of distilled water and 3mL of a freshly prepared FRAP working solution were added, mixed well, reacted at 37 ℃ for 30 minutes, and then the absorbance was measured at a wavelength of 593nm, using distilled water as a blank. Standard curves were plotted against 0-600. mu.M Trolox standard. The reducing power (FRAP value) of the sample is expressed in micromolar concentration of Trolox produced (μ M TE).

The results of the reducing power of the novel functional peptide PPH-I-3-11 (methionine-isoleucine-leucine-cysteine-serine-leucine; MIILLCSLL) of the pearl protein PPH positive control sodium sulfite are shown in Table 2. As can be seen from Table 2, the untreated pearl hydrolyzed protein has a certain reducing power, and the FRAP value reaches 160.58 μ M TE under the dosage of 1 mg/L. The reducing power of the novel functional peptide PPH-I-3-11 reaches 709.8 +/-2.8 mu M TE, and is close to the level of inorganic reagent sodium sulfite, which indicates that the novel functional peptide has good reducing activity.

TABLE 2 reduction force determination experiment of hydrolyzed pearl protein and novel functional peptide PPH-I-3-11

^*PPH is hydrolyzed protein of Margarita, PPH-I-3-11 is purified functional peptide, Na₂SO₃Is a positive control.

Example 5 melanocyte pigment production inhibitory Activity of functional peptide PPH-I-3-11

Human Epidermal Melanocytes (HEM) were provided by southern medical university. Melanocyte medium, pancreatin/EDTA digest, pancreatin stop solution, polylysine were purchased from Langeritaceae Biotech, Inc.

Cell culture, cytotoxicity assay, cell viability, melanin assay were performed according to methods in literature (Cho, M.; Ryu, M.; Jeong, Y.; Chung, Y. -H.; Kim, D. -E.; Cho, H. -S.; Kang, S.; Han, J. -S.; Chang, M. -Y.; Lee, C. -K., Cardamonin Suppresses melangenes by inhibition of Wnt/β -catenning, biochemical and biological research communications 2009,390(3), 500-505.).

After the HEM cells are recovered, the number of the cells is 1 × 10⁴/cm²The cells were inoculated into a culture flask at the density of (1) and the melanocyte medium was added. After 24 hours of culture for adherence, melanocyte medium containing the functional peptide PPH-I-3-11 (methionine-isoleucine-leucine-cysteine-serine-leucine; MIILLCSLL) was added and repeated 3 times per concentration. After further 2 days of culture, the cells were digested with pancreatin and collected into a centrifuge tube. After centrifugation, the cells were resuspended and transferred to an EP tube. The precipitate was collected by centrifugation again, dissolved with 1M NaOH solution and placed in an incubator at 37 ℃. After the melanin granules dissolved, their absorbance at 490nm was measured. The results of the experiment are shown in table 3.

TABLE 3 Effect of PPH-I-3-11 on melanin content in HEM cells

Test specimen	Final concentration (mg/mL)	Melanin content (%)	Cell survival rate
				Control group
	0	100±5.3	100±11.2
				Positive control (arbutin)	1.5	68.3±12.5	86.3±9.5
PPH-I-3-11	1.0	96.6±8.8	91.6±5.8
				PPH-I-3-11	2.0	82.3±15.5	96.9±3.6
PPH-I-3-11	4.0	63.3±6.2	93.4±15.2
				PPH-I-3-11	8.0	41.1±18.8	91.4±7.2
PPH-I-3-11	16.0	40.5±2.8	82.2±5.2

As can be seen from the experimental results, the functional peptide PPH-I-3-11 has an inhibitory effect on the generation of melanin in HEM cells and shows obvious dose dependence. When the content of the functional peptide PPH-I-3-11 in the culture medium is increased from 1.0mg/mL to 16.0mg/mL, the content of melanin is reduced from 96.6 percent of blank control to 40.5 percent. Meanwhile, compared with the positive control arbutin, the compound shows lower cytotoxicity, and the cell survival rate is over 90 percent except for 16mg/mL groups.

Sequence listing

<110> Nanhai ocean institute of Chinese academy of sciences

<120> functional peptide derived from pearl and use thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Met Ile Ile Leu Leu Cys Ser Leu Leu

1 5

Claims (9)

1. A functional peptide having the amino acid sequence: methionine-isoleucine-leucine-cysteine-serine-leucine.

2. Use of the functional peptide of claim 1 for the preparation of a free radical scavenger.

3. A radical scavenger comprising the functional peptide according to claim 1 as an active ingredient.

4. Use of the functional peptide of claim 1 for the preparation of an antioxidant.

5. An antioxidant comprising the functional peptide according to claim 1 as an active ingredient.

6. Use of the functional peptide according to claim 1 for the preparation of a melanin cytochrome synthesis inhibitor.

7. A melanocyte pigment synthesis inhibitor comprising the functional peptide according to claim 1 as an active ingredient.

8. Use of the functional peptide according to claim 1 for preparing a cosmetic raw material.

9. A cosmetic raw material comprising the functional peptide according to claim 1.

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