阅读说明：本技术 一种龙眼核多酚-南瓜籽多肽组合物及其制备方法 (Longan seed polyphenol-pumpkin seed polypeptide composition and preparation method thereof ) 是由 孙培冬 王培宇 孙菡峥 杨成 于 2020-05-25 设计创作，主要内容包括：本发明公开了一种龙眼核多酚-南瓜籽多肽组合物及其制备方法,包括,龙眼核多酚提取物和南瓜籽多肽,其中,所述龙眼核多酚在组合物中的浓度为0～10ug/mL,所述南瓜籽多肽在组合物中的浓度为0.2～1.0mg/mL。本发明中制备的龙眼核多酚与一定浓度的南瓜籽多肽复配可以降低龙眼核多酚毒性,同时,本发明中制备的龙眼核多酚与南瓜籽多肽复配赋予其可以降低细胞内黑色素含量的新功能。(The invention discloses a longan seed polyphenol-pumpkin seed polypeptide composition and a preparation method thereof, wherein the longan seed polyphenol composition comprises a longan seed polyphenol extract and pumpkin seed polypeptide, the concentration of the longan seed polyphenol in the composition is 0-10ug/m L, and the concentration of the pumpkin seed polypeptide in the composition is 0.2-1.0mg/m L.)

1. A longan seed polyphenol-pumpkin seed polypeptide composition is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

the composition comprises a longan seed polyphenol extract, pumpkin seed polypeptide and a solvent, wherein the concentration of the longan seed polyphenol in the composition is 0-10ug/m L, the concentration of the pumpkin seed polypeptide in the composition is 0.2-1.0mg/m L, and the solvent is dimethyl sulfoxide and deionized water.

2. The longan seed polyphenol-pumpkin seed polypeptide composition as claimed in claim 1, wherein the concentration of the longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition is 6-8 ug/m L, and the concentration of the pumpkin seed polypeptide in the longan seed polypeptide composition is 0.4-0.8 mg/m L.

3. The longan seed polyphenol-pumpkin seed polypeptide composition as set forth in claim 1, wherein: the preparation method of the longan seed polyphenol comprises the following steps,

drying the cleaned longan seeds at 45 +/-5 ℃ for 24 hours, crushing the longan seeds and sieving the crushed longan seeds with a 60-mesh sieve to obtain longan seed powder;

adding petroleum ether into longan seed powder, stirring for 3-4 h at the temperature of 40 ℃, removing the petroleum ether, adding ethanol, carrying out ultrasonic treatment for 60-80 min at the temperature of 70 ℃, and filtering to obtain a longan seed polyphenol extracting solution, wherein the longan seed polyphenol content in the extracting solution is 53.68-54.78 mg/g;

concentrating longan seed polyphenol extracting solution under reduced pressure, extracting with ethyl acetate for three times, combining three extracting solutions, concentrating under reduced pressure, and freeze-drying to obtain ethyl acetate phase polyphenol powder, namely the longan seed polyphenol L EP.

4. The longan seed polyphenol-pumpkin seed polypeptide composition as claimed in claim 3, wherein petroleum ether is added to longan seed powder, and the ratio of the raw materials to the liquid is 1:4 in g/m L.

5. The longan seed polyphenol-pumpkin seed polypeptide composition as set forth in claim 3, wherein ethanol is added after removing petroleum ether, wherein the volume fraction of ethanol is 60%, and the ratio of the materials to the liquids is 1: 25 in g/m L.

6. The longan seed polyphenol-pumpkin seed polypeptide composition as set forth in claim 3, wherein: and ultrasonic treatment, wherein the ultrasonic power is 100W, and the frequency is 40 Hz.

7. The longan seed polyphenol-pumpkin seed polypeptide composition as claimed in claim 3, wherein the longan seed polyphenol extracting solution is subjected to vacuum concentration and extraction by ethyl acetate, wherein the volume of the longan seed polyphenol extracting solution is 200-500 m L, the longan seed polyphenol extracting solution is subjected to vacuum concentration to 50m L, and the volume ratio of the ethyl acetate to the longan seed polyphenol extracting solution subjected to vacuum concentration is 4: 1.

8. The longan seed polyphenol-pumpkin seed polypeptide composition as set forth in claim 3, wherein: and (3) freeze-drying, wherein the temperature of a cold trap is-30-50 ℃, the pressure is 10-50 Pa, and the time is 3-5 days.

9. The longan seed polyphenol-pumpkin seed polypeptide composition as set forth in claim 1, wherein: the preparation method of the pumpkin seed polypeptide comprises the following steps,

crushing pumpkin seeds by a high-speed crusher, and sieving by a 60-mesh sieve;

adding petroleum ether at a ratio of 1:4 in g/m L, stirring at 40 deg.C for 4 hr, vacuum filtering, repeating for 3 times, mixing filtrates, and removing residual petroleum ether in fume hood to obtain defatted semen Cucurbitae;

adding degreased pumpkin seeds into deionized water according to a material-liquid ratio of 1:30g/m L, adjusting the pH value to 9.5, reacting for 180min at 35 ℃, centrifuging for 5min under the condition of 1000-10000 r/min, collecting supernatant, adjusting the pH value to 4.3, centrifuging for 10min at 5000r/min, collecting precipitate, washing to neutrality with water, and freeze-drying to obtain pumpkin seed protein;

adding pumpkin seed protein into deionized water according to a material-liquid ratio of 1: 5-1: 40g/m L to form a pumpkin seed protein dispersion, adjusting the temperature and pH of the pumpkin seed protein dispersion to 50 ℃ and 9.0, adding alkaline protease according to an enzyme base ratio of 2.5%, performing enzymolysis for 2 hours, inactivating in boiling water, adjusting the temperature and pH of a reaction solution to 37 ℃ and 7.5, adding trypsin according to an enzyme base ratio of 2.5%, performing enzymolysis for 2 hours, inactivating, and adding 0.5 mol/L NaOH solution during enzymolysis to maintain the pH value of the solution unchanged;

after enzymolysis, cooling the mixed solution to room temperature, adding 1 mol/L HCl to adjust the pH to the isoelectric point of 4.3, centrifuging for 10min at 5000r/min, collecting the supernatant, and freeze-drying to obtain polypeptide;

preparing 10mg/m L solution from pumpkin seed polypeptide, sequentially passing through 0.45-micron and 0.22-micron microporous filter membranes, adjusting the ultrafiltration pressure to be 0.1-0.2 MPa, carrying out ultrafiltration for 5 times at normal temperature, collecting the polypeptide solution passing through a 1000Da ultrafiltration membrane, and freeze-drying to obtain the polypeptide, namely PSP for short.

10. A method for preparing the longan seed polyphenol-pumpkin seed polypeptide composition as set forth in any one of claims 1-9, wherein the longan seed polyphenol-pumpkin seed polypeptide composition comprises the following steps: the preparation method comprises the following steps of,

dissolving longan seed polyphenol L EP powder in dimethyl sulfoxide to prepare L EP high-concentration solution, wherein the concentration is more than or equal to 10ug/m L;

dissolving pumpkin seed polypeptide PSP powder in deionized water to prepare a PSP solution with the concentration of more than or equal to 1.0mg/m L;

and fully mixing the L EP high-concentration solution and the PSP solution to prepare the longan seed polyphenol-pumpkin seed polypeptide composition, wherein the longan seed polyphenol concentration in the composition is 0-10ug/m L, the pumpkin seed polypeptide concentration is 0.2-1.0mg/m L, and the dimethyl sulfoxide and the deionized water in the composition are uniformly distributed.

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to a longan seed polyphenol-pumpkin seed polypeptide composition and a preparation method thereof.

Background

At present, a single-enzyme enzymolysis method is mostly adopted for preparing pumpkin seed polypeptide, performance research is focused on oxidation resistance, but the performance of the pumpkin seed polypeptide is poor, and the bioavailability and the effect on human skin cells cannot be evaluated due to the fact that an evaluation method is a chemical method.

The extraction of polyphenol in longan seeds mostly adopts ethanol extraction, but the polyphenol prepared by the method has low content, has excellent antioxidant performance, and is not exceptional for the polyphenol extracted from longan seeds. However, polyphenols are toxic at higher concentrations, which has a certain adverse effect on the human body and limits the use of the polyphenols.

Therefore, how to increase the polyphenol content in the extract and reduce the toxicity of the extract while ensuring the efficacy of the extract is an urgent technical problem to be solved in the field.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.

Therefore, the invention aims to overcome the defects in the prior art and provide a longan seed polyphenol-pumpkin seed polypeptide composition.

In order to solve the technical problems, the invention provides a longan seed polyphenol-pumpkin seed polypeptide composition which comprises a longan seed polyphenol extract, pumpkin seed polypeptide and a solvent, wherein the concentration of longan seed polyphenol in the composition is 0-10ug/m L, the concentration of pumpkin seed polypeptide in the composition is 0.2-1.0mg/m L, and the solvent is dimethyl sulfoxide and deionized water.

The preferable scheme of the longan seed polyphenol-pumpkin seed polypeptide composition is that the concentration of longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition is 6-8 ug/m L, and the concentration of pumpkin seed polypeptide in the longan seed polyphenol-pumpkin seed polypeptide composition is 0.4-0.8 mg/m L

The longan seed polyphenol-pumpkin seed polypeptide composition is prepared by a preferable scheme, wherein the longan seed polyphenol is prepared by drying cleaned longan seeds at 45 +/-5 ℃ for 24 hours, crushing the longan seeds, sieving the crushed longan seeds with a 60-mesh sieve to obtain longan seed powder, adding petroleum ether into the longan seed powder, stirring at 40 ℃ for 3-4 hours, removing the petroleum ether, adding ethanol, performing ultrasonic treatment at 70 ℃ for 60-80 minutes, filtering to obtain an longan seed polyphenol extracting solution, wherein the longan seed polyphenol content in the extracting solution is 53.68-54.78 mg/g, concentrating the longan seed polyphenol extracting solution under reduced pressure, extracting with ethyl acetate for three times, combining the three extracting solutions, concentrating under reduced pressure, and performing freeze drying to obtain ethyl acetate phase polyphenol powder, namely the longan seed polyphenol L EP.

The preferable scheme of the longan seed polyphenol-pumpkin seed polypeptide composition is that petroleum ether is added into longan seed powder, wherein the ratio of materials to liquids in g/m L is 1: 4.

As a preferable scheme of the longan seed polyphenol-pumpkin seed polypeptide composition, ethanol is added after petroleum ether is removed, wherein the volume fraction of the ethanol is 60%, and the ratio of materials to liquids is 1: 25 in g/m L.

As a preferable embodiment of the longan seed polyphenol-pumpkin seed polypeptide composition, the longan seed polyphenol-pumpkin seed polypeptide composition comprises: and ultrasonic treatment, wherein the ultrasonic power is 100W, and the frequency is 40 Hz.

The longan seed polyphenol-pumpkin seed polypeptide composition is preferably prepared by concentrating a longan seed polyphenol extracting solution under reduced pressure and extracting the concentrated longan seed polyphenol extracting solution with ethyl acetate, wherein the volume of the longan seed polyphenol extracting solution is 200-500 m L, the longan seed polyphenol extracting solution is concentrated under reduced pressure to 50m L, and the volume ratio of the ethyl acetate to the longan seed polyphenol extracting solution subjected to reduced pressure concentration is 4: 1.

As a preferable embodiment of the longan seed polyphenol-pumpkin seed polypeptide composition, the longan seed polyphenol-pumpkin seed polypeptide composition comprises: and (3) freeze-drying, wherein the temperature of a cold trap is-30-50 ℃, the pressure is 10-50 Pa, and the time is 3-5 days.

As a preferable embodiment of the longan seed polyphenol-pumpkin seed polypeptide composition, the longan seed polyphenol-pumpkin seed polypeptide composition comprises: the preparation method of the pumpkin seed polypeptide comprises the steps of crushing pumpkin seeds by a high-speed crusher, and sieving by a 60-mesh sieve;

adding petroleum ether at a ratio of 1:4 in g/m L, stirring for 4h at 40 ℃, performing suction filtration, repeating for 3 times, combining filtrates, removing residual petroleum ether in a fume hood to obtain defatted pumpkin seeds, adding the defatted pumpkin seeds at a ratio of 1:30g/m L to deionized water, adjusting pH to 9.5, reacting for 180min at 35 ℃, centrifuging for 5min at 1000-10000 r/min, collecting supernatant, adjusting pH to 4.3, centrifuging for 10min at 5000r/min, collecting precipitate, washing with water to neutrality, performing freeze drying to obtain pumpkin seed protein, adding the pumpkin seed protein at a ratio of 1: 5-1: 40g/m L to deionized water to form a pumpkin seed protein dispersion, adjusting the pH of the pumpkin seed protein dispersion and 50 ℃, adjusting the pH to 9.0, adding alkaline protease at a ratio of 2.5% of the pumpkin seed protein dispersion and the pH to 50 ℃, performing freeze drying for 2h, adding the polypeptide solution at 365-80 μm to a temperature of 1:5, adjusting the pH of the reaction isoelectric point to 37.5 ℃, inactivating the polypeptide at 5000r/m, adding the polypeptide dispersion and the polypeptide solution to 0.0.0.0 μm, performing freeze drying to obtain a polypeptide solution, and drying for 364 μm to obtain a polypeptide solution, and a polypeptide solution, wherein the polypeptide solution is added to obtain a polypeptide solution, the polypeptide solution is added to obtain a polypeptide solution, the polypeptide solution.

It is still another object of the present invention to overcome the deficiencies of the prior art and to provide a method for preparing a longan seed polyphenol-pumpkin seed polypeptide composition.

In order to solve the technical problems, the invention provides the following technical scheme that a preparation method of the longan seed polyphenol-pumpkin seed polypeptide composition comprises the steps of dissolving longan seed polyphenol L EP powder in dimethyl sulfoxide to prepare L EP high-concentration solution with the concentration being more than or equal to 10ug/m L, dissolving pumpkin seed polypeptide PSP powder in deionized water to prepare PSP solution with the concentration being more than or equal to 1.0mg/m L, and fully mixing the L EP high-concentration solution with the PSP solution to prepare the longan seed polyphenol-pumpkin seed polypeptide composition, wherein the concentration of longan seed polyphenol in the composition reaches 0-10ug/m L, the concentration of pumpkin seed polypeptide in the composition reaches 0.2-1.0mg/m L, and the dimethyl sulfoxide in the composition and the deionized water are uniformly distributed.

The invention has the beneficial effects that:

(1) the invention provides a longan seed polyphenol-pumpkin seed polypeptide composition, wherein an ethyl acetate is adopted to extract a longan seed extract, so that the polyphenol content is obviously improved, and the oxidation resistance of the longan seed extract is also improved; the prepared longan seed polyphenol and pumpkin seed polypeptide are compounded, so that the oxidation resistance can be obviously improved, and a synergistic effect is achieved.

(2) The toxicity of longan seed polyphenol can be reduced by compounding the longan seed polyphenol prepared by the method with pumpkin seed polypeptide with a certain concentration, and meanwhile, the longan seed polyphenol prepared by the method has a new function of reducing the content of melanin in cells by compounding the longan seed polyphenol with the pumpkin seed polypeptide, and the requirements for achieving the effects are that the final concentration of the longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition compounded by the longan seed polyphenol and the pumpkin seed polypeptide is 0-10ug/m L, and the final concentration of the pumpkin seed polypeptide is 0.2-1.0mg/m L.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a comparison graph of cytotoxicity of longan seed polyphenol concentration greater than 10ug/m L in longan seed polyphenol-pumpkin seed polypeptide composition according to an embodiment of the present invention.

FIG. 2 is a comparison graph of cytotoxicity of pumpkin seed polypeptide in the concentration of more than 1.0mg/m L in the longan seed polyphenol-pumpkin seed polypeptide composition in the embodiment of the invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The cytotoxicity assay of the invention:

the cell activity of the samples on human skin fibroblasts was examined by the MTT method HSF cells in logarithmic growth phase were taken at 20 × 10³The density of each well is inoculated in a 96-well plate, each well is 100 mu L (except a blank group), after incubation in an incubator for 24h, culture solution in the wells is removed, DMEM is used as a solution for respectively taking a sample solution, 100 mu L of the sample solution is added into each well for incubation for 24h, 100 mu L of DMEM is used for replacing the sample as a control group, the solution in the wells is removed, PBS is used for washing for 2 times, 100 mu L0.5 mg/m L of MTT solution is added into each well, 100 mu L0.5.5 mg/m L of MTT solution is added into the wells without cells as a blank group, the wells are placed in the incubator for incubation for 4h, 100 mu L of DMSO is added into each well after the incubation is finished, the wells are fully shaken for 5min, the absorbance OD at 490nm is measured by using a microplate reader, and the cell viability is calculated according to the.

Cell viability (OD sample-OD blank)/(OD control-OD blank) × 100% (1)

The invention is characterized by the following: respectively measuring the DPPH, OH and O2 clearance rates of the pumpkin seed polypeptide and the longan seed polyphenol when the pumpkin seed polypeptide and the longan seed polyphenol are compounded so as to investigate the antioxidant performance of the polypeptide.

The whitening activity of the invention is as follows:

the method comprises the steps of taking mouse melanoma cells in a logarithmic growth phase, inoculating the mouse melanoma cells into a 6-well plate at a density of 20 × 103 cells/well, inoculating the mouse melanoma cells into each well at 2m L (except for blank groups) for 24 hours, removing culture solution in each well after incubation in an incubator, respectively preparing sample solutions with a certain concentration by taking DMEM as a solution, adding 2m L sample solution into each well for incubation for 24 hours, replacing the DMEM with 100 mu L samples as a control group, removing the solution in each well, washing the cells for 2 times by PBS (phosphate buffer solution), collecting the cells, adding 1m L10% DMSO (prepared by NaOH), fully shaking, sealing and placing for 30min, detecting an absorbance value at 470nm, and expressing the melanin content by absorbance.

The alkaline protease of the invention: the product is B8360, the enzyme activity is more than or equal to 200000U/g; the trypsin of the invention: beijing Soilebao science and technology Limited, cat # T8150, enzyme activity 250. N.F.U/mg; other raw materials, which are not specifically described, are all commercially available.