Tyrosinase activity inhibitory peptide from dictyophora indusiata sporocarp and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a biological small molecule active peptide, in particular to a tyrosinase activity inhibitory peptide derived from dictyophora indusiata sporocarp and application of the tyrosinase activity inhibitory peptide. The amino acid composition of the activity inhibitory peptide DIP-9 is Tyr-Phe-Pro-His-Cys-Lys-Trp-Tyr-Met, and the amino acid composition of the activity inhibitory peptide DIP-9 is Tyr-Phe-Pro-His-Cys-Lys-Trp The relative molecular mass is 1275 Da. The tyrosinase inhibitory activity of the polypeptide obtained by extracting and purifying bamboo fungus sporocarp is verified through a cell model, finally, primary screening finds that one polypeptide DIP-9 has better tyrosinase inhibitory activity, the extraction method is further researched and perfected, and the amino acid sequence of the polypeptide is identified. The DIP-9 can be used as a tyrosinase inhibitor in cosmetics with a whitening effect, and is expected to be developed and utilized as cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a biological small molecule active peptide, in particular to a tyrosinase activity inhibitory peptide derived from bamboo fungus fruiting body and application thereof. Background Art

[0002] With economic development and the improvement of quality of life, Asian women have an increasing demand for whitening. Currently, most of the skin care products with whitening effects are achieved by adding chemical synthetic substances with whitening effects. Such as hydroquinone, arbutin, kojic acid and BHT (2,6-di-tert-butyl-4-methylphenol), but it is found during use that a series of side effects, such as cytotoxicity, accompany the rapid whitening effect. At present, the safety of cosmetics is increasingly valued, and natural whitening substances from plants are becoming more and more popular due to their lower side effects. Melanin formation is a complex process, in which tyrosinase is extremely important as a key enzyme in melanin synthesis. Tyrosinase is a copper-containing multifunctional oxidase that plays a key role in the biosynthesis of melanin. Excessive synthesis of melanin can lead to pigmentation and the formation of skin spots. Existing tyrosinase inhibitors include hydroquinone, kojic acid and its derivatives, but these inhibitors have defects such as skin irritation, low stability or poor safety for long-term use. Therefore, the development of tyrosinase inhibitors with natural sources, stable efficacy and no side effects has important application value.

[0003] Peptide is a compound formed by α-amino acids linked together by peptide bonds. It is also an intermediate product of protein hydrolysis and a multifunctional compound derived from protein. In general, its nutritional and physiological functions are superior to those of protein (macromolecule protein) and amino acids. Compared with macromolecules such as polysaccharides, peptides have a simple structure and can show better physiological activity at lower doses. Compared with small molecules such as compounds, peptides can be efficiently synthesized artificially.

[0004] As a medicinal and edible fungus, bamboo fungus has rich biologically active substances, including polysaccharides, peptides, flavonoids and volatile substances. In summary, if the peptides in bamboo fungus can be used well, it should have broad market application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a tyrosinase activity inhibitory peptide derived from bamboo fungus fruiting body in response to the problems existing in the prior art, and to use the peptide to prepare a tyrosinase activity inhibitor, such as for preparing cosmetics with whitening effect, which can inhibit tyrosinase activity, thereby reducing melanin formation and having a good skin whitening effect.

[0006] In order to achieve the purpose of the present invention, the specific technical scheme of the present invention is as follows:

[0007] A bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9, the amino acid composition of the activity inhibitory peptide DIP-9 is Tyr-Phe-Pro-His-Cys-Lys-Trp-Tyr-Met, as shown in SEQ ID No. 1, and the relative molecular mass is 1275Da.

[0008] Furthermore, the above-mentioned bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 is derived from the bamboo fungus fruiting body.

[0009] The present invention also protects the use of the above-mentioned tyrosinase activity inhibitory peptide DIP-9 in cosmetics with whitening effects.

[0010] Preferably, the cosmetic is a preparation prepared by using an effective amount of tyrosinase activity inhibitory peptide DIP-9 as an active ingredient and adding medically acceptable excipients or auxiliary ingredients. In terms of material-liquid ratio, the effective amount of tyrosinase activity inhibitory peptide DIP-9 is 1-1.5%, that is, 100 mL of emulsion or other forms of cosmetics contain 1-1.5 g of DIP-9.

[0011] Preferably, the cosmetics can be facial masks, lotions, toners, etc., which are preparations prepared with an effective amount of tyrosinase activity inhibitory peptide DIP-9 as an active ingredient and medically acceptable excipients or auxiliary ingredients. The effective amount of tyrosinase activity inhibitory peptide DIP-9 is 1-1.5 mg / day, that is, the recommended dosage of cosmetics, and the daily use of tyrosinase activity inhibitory peptide DIP-9 is 10-15 mg.

[0012] The invention discloses an application of the tyrosinase activity inhibitory peptide DIP-9 in preparing cosmetics for inhibiting the production of melanin in B16 cells induced by L-dopa.

[0013] The present invention also protects the use of the above-mentioned tyrosinase activity inhibitory peptide DIP-9 in the preparation of a tyrosinase activity inhibitor; the tyrosinase activity inhibitor can be a cosmetic.

[0014] Furthermore, the effective amount of the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 is 1-1.5 wt %.

[0015] The present invention also protects the use of the above-mentioned tyrosinase activity inhibitory peptide DIP-9 in a composition for improving whitening effect.

[0016] The present invention also protects a pharmaceutical composition, which contains the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 as described above.

[0017] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable adjuvant.

[0018] Furthermore, the effective amount of the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 is 1-1.5 wt %.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention extracts and purifies the polypeptide obtained from the fruiting body of bamboo fungus, verifies its tyrosinase inhibitory activity through a cell model, and finally, a polypeptide DIP-9 is found to have good tyrosinase inhibitory activity through preliminary screening. Further research improves the extraction method and identifies the amino acid sequence of the polypeptide. DIP-9 can be used as a tyrosinase inhibitor in cosmetics with whitening effect, and is expected to be developed and utilized as cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the amino acid sequence of DIP-9;

[0022] Figure 2 This is the molecular exclusion chromatography separation diagram, A1-A4 are the four elution peaks with decreasing molecular weight;

[0023] Figure 3 This is the chromatogram of component A2 after separation and purification by RP-HPLC;

[0024] Figure 4 is the circular dichroism spectrum of DIP-9. DETAILED DESCRIPTION

[0025] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0026] Any feature disclosed in this specification (including claims and abstract), unless otherwise stated, may be replaced by other equivalent or alternative features having similar purposes. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

[0027] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0029] In this application, unless otherwise specified, % refers to the mass percentage, i.e. wt%.

[0030] Embodiment 1:

[0031] Isolation, purification and sequence identification of tyrosinase inhibitory peptides from bamboo fungus:

[0032] Weigh 50 g of the dried fruiting body of Dictyophora longifolia (Dictyophora longifolia, produced in Yibin, Sichuan), grind it in a traditional Chinese medicine grinder, and then pass it through a 100-mesh sieve to obtain the dried fruiting body powder of Dictyophora longifolia.

[0033] Prepare phosphate buffered saline (PBS buffer): 8 g sodium chloride (NaCl), 0.2 g potassium chloride (KCl), disodium hydrogen phosphate (Na 2 HPO 4 )1.42g, potassium dihydrogen phosphate (KH 2 PO 4 )0.27g, add about 800mL of deionized water and stir thoroughly to dissolve, then add concentrated hydrochloric acid to adjust the pH to 6.8, and finally make up to 1L.

[0034] Weigh 50 g of bamboo fungus fruiting body powder and place it in a beaker, add the sample according to the bamboo fungus fruiting body powder / PBS buffer (pH 6.8) solid-liquid ratio of 1:20 (m / V), put it into an ultrasonic-microwave synergistic extraction instrument, set the microwave power to 80 w, and set the treatment time to 8 min. After the treatment, centrifuge at 4000 rpm / min for 20 min, absorb the supernatant, which is the bamboo fungus protein solution, and calculate the protein extraction rate.

[0035]

[0036] Wherein, V is the total volume of bamboo fungus protein extract (mL); W is 1 , protein content in the extract (g / 100mL); m, mass of bamboo fungus (g); W 2 , protein content in bamboo fungus (g / mL), W 1 and W 2 The nitrogen content was determined by Kjeldahl method. The bamboo fungus protein powder was obtained by freeze drying in a vacuum freeze dryer.

[0037] Take 10 g of bamboo fungus fruiting body protein dry powder, dissolve it in sterile water, prepare a solution with a concentration of 100 mg / mL, load it on a Superdex 30Increase gel filtration column, elute with sterile water, collect the eluate through an automatic collector, then measure the absorbance of the eluate at 215 nm, collect and freeze-dry a single peak, and obtain the bamboo fungus fruiting body polypeptide complex.

[0038] Take 1g of the bamboo fungus fruiting body polypeptide complex dry powder and dissolve it in sterile water to prepare a solution with a concentration of 10mg / mL, purify it by reverse high performance liquid chromatography, collect a single peak, and obtain the bamboo fungus fruiting body polypeptide (DIP-9). The chromatographic conditions are set as follows: the chromatographic column uses a PE C18 column (150mm×4.6mm), phase A: pure water, phase B: acetonitrile containing 0.1% trifluoroacetic acid; the injection volume is set to 30μL; the column temperature is set to 25±5℃; the flow rate is selected to be 1.0mL / min; the detection wavelength is set to 215nm. Elution conditions: phase A: 0-8min, 99%-97%; 8-12min, 97%-96%; 12-16min, 96%-80%; 16-20min, 80%-99%.

[0039] Ultrasonic-microwave synergistic extraction was used to extract 50 g of Dictyophora fruiting bodies, and the protein extraction rate was determined to be 32.34%.

[0040] The results of size exclusion chromatography separation are as follows Figure 1 As shown, the polypeptide extract can obtain 4 elution peaks according to the molecular weight, which are marked as A1-4 from large to small. By collecting the elution peaks respectively, the tyrosinase inhibitory activity is determined, and the A2 component with the best inhibitory activity is selected as the bamboo fungus polypeptide complex for the next step of purification. After component A2 is separated and purified by RP-HPLC ( Figure 2 ), and three components, namely B1-3, were obtained. The tyrosinase inhibitory activity was determined, and B2 with the best inhibitory activity was selected as the bamboo fungus polypeptide, namely DIP-9.

[0041] After 10g of polypeptide extract was filtered by molecular exclusion chromatography Superdex 30Increase gel filtration, 0.69g of polypeptide complex lyophilized powder was obtained, with a yield of 6.9%. 1g of polypeptide complex lyophilized powder was finally purified by reverse-phase high performance liquid chromatography (RP-HPLC) to obtain 0.15g of DIP-9 lyophilized powder, with a yield of 15%.

[0042] The amino acid composition of DIP-9 was analyzed by liquid chromatography-mass spectrometry (LC-MS-MS). Liquid A is an aqueous solution containing 0.1% formic acid, and liquid B is an acetonitrile solution containing 0.1% formic acid. The chromatographic column is balanced with liquid A. The sample is loaded by an automatic sampler and then separated by gradient separation on the chromatographic column at a flow rate of 0.3 mL / min and a column temperature of 70°C. Mass spectrometry conditions: The sample was analyzed by mass spectrometry using a TripleTOF 5600+ mass spectrometer. Analysis time: 18 min, detection mode: positive ion, primary mass spectrometry scanning range: 600-3500 m / z. The antioxidant peptide consists of 9 amino acids with a relative molecular mass of 1275 Da. Tyr-Phe-Pro-His-Cys-Lys-Trp-Tyr-Met (SEQ ID No. 1).

[0043] Example 2 Secondary structure test of bamboo fungus derived tyrosinase activity inhibitory peptide DIP-9

[0044] The amino acid composition of DIP-9 is simple, and its secondary structure was determined by circular dichroism analysis. DIP-9 was dissolved in water and prepared into a solution with a concentration of 0.25 mg / mL. Circular dichroism analysis was performed at 25°C using a quartz cuvette with a step diameter of 2 mm. Data were collected in the range of 190-260 nm with a 1 nm interval and a 1 nm bandwidth. Figure 3 As shown in the circular dichroism spectrum of DIP-9, a strong negative band was observed near 198nm and a weak positive band was observed near 220nm. These two absorption peaks show that DIP-9 has the characteristics of an irregular secondary structure. Further statistical analysis confirmed that 67.5% of DIP-9 in aqueous solution was in a random coil and 27.3% of DIP-9 was in a β-flip.

[0045] Example 3 Effect of DIP-9, a tyrosinase inhibitory peptide derived from bamboo fungus, on the melanin content of B16 cells

[0046] B16 cells growing in logarithmic phase were taken and cultured at a rate of 3×10 5 The cells were inoculated at a concentration of 100 / mL in a 6-well plate, with 1 mL of culture medium per well. 37°C, 5% CO 2 The cells were cultured in an environment for 24 h. The supernatant was discarded, and 1 mL of complete culture medium containing different concentrations of DIP-9 (12.5, 25, 50, 100 and 200 μg / mL) was added to each well. The control group was set to the same volume of culture medium without DIP-9 sample, and the blank well was not inoculated with cells but only 1 mL of culture medium was added. Each group was set to 3 replicates. The above cells were cultured at 37°C and 5% CO 2 Continue to culture in the environment for 24h, 48h, and 72h, and then rinse with PBS buffer for 3 times. Then add 2mL of 0.2mol / L NaOH solution and place in a water bath at 80℃ for 1h to completely dissolve the cell clumps. Measure the absorbance at 492nm, and the relative content of melanin is calculated as follows:

[0047]

[0048] The effect of DIP-9 on the melanin content of B16 cells is shown in Table 1. Based on the previous determination of DIP-9 on the viability of B16 cells, DIP-9 concentrations of 12.5, 25, 50, 100 and 200 μg / mL were selected for the determination of melanin content. At this set concentration, the activity of B16 cells was not significantly inhibited. The lowest concentration of quinoa saponin set in the experiment had no obvious inhibitory effect on the synthesis of melanin in B16 cells, that is, the safe dose of DIP-9. 25μg / mL of DIP-9 only showed an inhibitory effect when cultured for 72h. With the increase of DIP-9 concentration, the inhibitory effect gradually increased. After 72h of DIP-9 treatment of B16 cells with 200μg / mL, the intracellular melanin content was 68.83%, that is, the inhibition rate of DIP-9 on melanin production reached 31.17%. Therefore, DIP-9 is a potential inhibitor of melanin synthesis.

[0049] Table 1 Effect of DIP-9 on melanin synthesis in B16 cells

[0050]

[0051]

[0052] Note: The values ​​are the mean ± standard error of 3 replicates. Different lowercase letters in the same column indicate a significant difference at the 5% level.

[0053] Example 4 Effect of DIP-9, a Tyrosinase Inhibitory Peptide from Dictyophora Indica on Tyrosinase Activity in B16 Cells

[0054] B16 cells growing in logarithmic phase were taken and cultured at a rate of 3×10 5 The cells were inoculated at a concentration of 1 / mL in a 6-well plate, with 1 mL of culture medium per well. Incubate at 37°C in a 5% CO2 environment for 24 hours. Discard the supernatant, add 1 mL of complete culture medium containing different concentrations of DIP-9 (12.5, 25, 50, 100 and 200 μg / mL) to each well, the control group was set to the same volume of culture medium without DIP-9 sample, and the blank well was not inoculated with cells but only 1 mL of culture medium was added, and 3 replicates were set for each group. The cells treated with DIP-9 continued to be cultured for 24 hours, 48 ​​hours, and 72 hours, the culture medium was aspirated and discarded, and then rinsed with PBS buffer 3 times. Add 50 μL of cell lysis solution 1% Triton X-100, freeze at low temperature for 1 hour, and thaw at room temperature after taking out. Incubate at 37°C and add 10 μL of 1% L-DOPA solution, react for 1 hour. Determine the absorbance at 492 nm, and the relative activity of tyrosinase is calculated as follows:

[0055]

[0056] The effect of DIP-9 on the tyrosinase activity of B16 cells is shown in Table 2. Compared with the control group, DIP-9 has a significant inhibitory effect on the tyrosinase activity of B16 cells. Within the experimental concentration range, after 24 hours of treatment with 12.5 μg / mL of DIP-9, the tyrosinase activity decreased to 93.31%, which was significantly different from the control group (p<0.05). With the increase of DIP-9 concentration and the increase of treatment time, the tyrosinase activity of B16 cells gradually decreased, showing a dose-dependent relationship. Within the experimental setting range, the inhibition rate of quinoa saponins on the tyrosinase activity of B16 cells was 8.85-51.17%.

[0057] Table 2 Effect of DIP-9 on tyrosinase activity of B16 cells

[0058]

[0059]

[0060] Note: The values ​​are the mean ± standard error of 3 replicates. Different lowercase letters in the same column indicate a significant difference at the 5% level.

[0061] Example 5 Synthesis of Tyrosinase Activity Inhibitory Peptide DIP-9

[0062] The present invention separates, purifies and identifies a bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9. In actual production applications, due to the scarcity of bamboo fungus fruiting bodies, the present invention provides a solid phase synthesis method for synthesizing and producing DIP-9, including but not limited to an Fmoc-SPPS method, a BOC-SPPS method, and a fragment condensation connection method, thereby more efficiently obtaining the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9, and providing materials for further utilization of the polypeptide.

[0063] Reagents and raw materials required for solid phase synthesis: 2-CL resin, amino acid (hereinafter represented by Fmoc-aa-oh), dimethylformamide (DMF), dichloromethane (DCM), methanol, N,N'-diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIEA), 1-hydroxybenzotriazole (HOBT), trifluoroacetic acid (TFA), triisopropylsilane (TIS), pure water, EDT, and diethyl ether.

[0064] A method for solid phase synthesis of an antioxidant peptide DIP-9, wherein the amino acid composition of the tyrosinase activity inhibitory peptide DIP-9 is Tyr-Phe-Pro-His-Cys-Lys-Trp-Tyr-Met, comprises the following steps:

[0065] 1. Resin selection and preparation

[0066] Select Rink amide resin to synthesize peptides with amide at the C-terminus. Weigh an appropriate amount of resin (0.1 mmol loading) and swell it with dichloromethane (DCM) for 30 min.

[0067] 2. Removal of Fmoc protecting group

[0068] The Fmoc protecting group was removed using a 20% piperidine / DMF solution. After shaking for 10 min, a new piperidine / DMF solution was replaced and the removal was continued for another 10 min.

[0069] The resin was washed with DMF three times for 1 min each time to remove the residual piperidine and Fmoc groups.

[0070] 3. Coupling of amino acids (synthesis from C-terminus to N-terminus)

[0071] The first amino acid (Met) is coupled:

[0072] Fmoc-Met-OH (0.3 mmol, 3 times molar amount) was dissolved in DMF, HBTU (0.3 mmol) and HOBt (0.3 mmol) were added as activating agents, and DIEA (0.6 mmol) was added to adjust the pH.

[0073] The mixed solution was added to the treated resin and reacted for 30 minutes.

[0074] After the coupling was completed, the resin was washed 3 times with DMF.

[0075] 2nd amino acid (Tyr) coupling: Removal of Fmoc protecting group (step 2).

[0076] Fmoc-Tyr-OH was coupled as described above.

[0077] Repeat step 3 and couple other amino acids in the following order: Trp, Lys, Cys (side chain with protecting group), His (side chain with protecting group), Pro, Phe, Tyr.

[0078] 4.Terminal closure

[0079] After the last amino acid (Tyr) was synthesized, a blocking reaction of the terminal amino group was performed using acetic anhydride / pyridine to prevent the exposure of unreacted amine groups.

[0080] 5. Cleavage and deprotection

[0081] The synthesized resin was transferred into a cleavage solution (TFA / water / TIS 95 / 2.5 / 2.5, v / v) and shaken for 2-3 h.

[0082] The cleaved solution was added dropwise to cold ether for precipitation, and the crude peptide was obtained by centrifugation.

[0083] 6. Purification and identification of crude peptides

[0084] The crude peptide was purified by RP-HPLC to obtain high-purity DIP-9, which was then freeze-dried by a vacuum freeze dryer to obtain a dry peptide powder.

[0085] The above experiments prove that the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 has a significant inhibitory effect on the tyrosinase activity of the L-dopa-induced B16 cell model, and further tests show that it can inhibit the formation of melanin. The obtained active peptide is an excellent tyrosinase activity inhibitor, and DIP-9 can be used in cosmetics as a component with whitening activity.

[0086] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

[0087] This background section is provided to generally present the context of the invention, and the work of the presently named inventors, the work to the extent described in this background section, and aspects of this section that did not constitute prior art at the time of application are neither explicitly nor implicitly admitted to be prior art to the present invention.

Claims

1. A bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9, characterized in that: The amino acid composition of the activity inhibitory peptide DIP-9 is Tyr-Phe-Pro-His-Cys-Lys-Trp-Tyr-Met.

2. The bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 according to claim 1, characterized in that: The relative molecular mass of the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 is 1275Da.

3. The bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 according to claim 1 or 2, characterized in that: The bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 is derived from the bamboo fungus fruiting body.

4. Use of the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 as claimed in claim 1 or 2 in the preparation of a tyrosinase activity inhibitor.

5. The use according to claim 4, characterized in that: The effective amount of the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 is 1-1.5 wt %.

6. Use of the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 as claimed in claim 1 or 2 in the preparation of cosmetics for inhibiting L-dopa-induced B16 cell melanin production.

7. The use according to claim 5, characterized in that: The effective amount of the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 is 1-1.5 wt %.

8. A pharmaceutical composition, characterized in that: The invention comprises the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 as claimed in claim 1 or 2.

9. The pharmaceutical composition according to claim 8, characterized in that: Pharmaceutically acceptable adjuvants are also included.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that: The effective amount of the bamboo fungus-derived tyrosinase activity inhibitory peptide DIP-9 is 1-1.5 wt %.

Images