CN111449979B - Longan seed polyphenol-pumpkin seed polypeptide composition and preparation method thereof - Google Patents
Longan seed polyphenol-pumpkin seed polypeptide composition and preparation method thereof Download PDFInfo
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- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 abstract description 8
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- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/34—Alcohols
- A61K8/347—Phenols
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
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Abstract
The invention discloses a longan seed polyphenol-pumpkin seed polypeptide composition and a preparation method thereof, wherein the longan seed polyphenol-pumpkin seed polypeptide composition comprises a longan seed polyphenol extract and pumpkin seed polypeptide, the concentration of longan seed polyphenol in the composition is 0-10ug/mL, and the concentration of pumpkin seed polypeptide in the composition is 0.2-1.0 mg/mL. The longan seed polyphenol prepared by the invention can be compounded with pumpkin seed polypeptide with a certain concentration to reduce the toxicity of the longan seed polyphenol, and meanwhile, the compound of the longan seed polyphenol and the pumpkin seed polypeptide has a new function of reducing the content of melanin in cells.
Description
Technical Field
The invention 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 the following technical scheme: the longan seed polyphenol-pumpkin seed polypeptide 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/mL, the concentration of the pumpkin seed polypeptide in the composition is 0.2-1.0mg/mL, and the solvent is dimethyl sulfoxide and deionized water.
As a preferable embodiment of the longan seed polyphenol-pumpkin seed polypeptide composition, the longan seed polyphenol-pumpkin seed polypeptide composition comprises: the concentration of longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition is 6-8 ug/mL, and the concentration of pumpkin seed polypeptide in the longan seed polyphenol-pumpkin seed polypeptide composition is 0.4-0.8 mg/mL
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 longan seed polyphenol comprises the steps of drying cleaned longan seeds for 24 hours at the temperature of 45 +/-5 ℃, 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 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 freeze-drying to obtain ethyl acetate phase polyphenol powder, namely the longan seed polyphenol LEP.
As a preferable embodiment of the longan seed polyphenol-pumpkin seed polypeptide composition, the longan seed polyphenol-pumpkin seed polypeptide composition comprises: adding petroleum ether into longan seed powder, wherein the ratio of material to liquid in g/mL is 1: 4.
As a preferable embodiment of the longan seed polyphenol-pumpkin seed polypeptide composition, the longan seed polyphenol-pumpkin seed polypeptide composition comprises: and adding ethanol after removing the petroleum ether, wherein the volume fraction of the ethanol is 60%, and the feed-liquid ratio is 1 in g/mL: 25.
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.
As a preferable embodiment of the longan seed polyphenol-pumpkin seed polypeptide composition, the longan seed polyphenol-pumpkin seed polypeptide composition comprises: concentrating the longan seed polyphenol extracting solution under reduced pressure, and extracting with ethyl acetate, wherein the volume of the longan seed polyphenol extracting solution is 200-500 mL, concentrating under reduced pressure to 50mL, and the volume ratio of the ethyl acetate to the longan seed polyphenol extracting solution after concentration under reduced pressure 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/mL, stirring at 40 deg.C for 4h, vacuum filtering, repeating for 3 times, mixing filtrates, and removing residual petroleum ether in a fume hood to obtain defatted semen Cucurbitae; adding degreased pumpkin seeds into deionized water according to a material-liquid ratio of 1:30g/mL, 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/mL 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 0.5mol/L NaOH solution during enzymolysis to keep the pH value of the solution unchanged; after enzymolysis, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH to the isoelectric point of 4.3, centrifuging for 10min at 5000r/min, collecting supernatant, and freeze-drying to obtain polypeptide; preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes, adjusting ultrafiltration pressure to be 0.1-0.2 MPa, performing 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.
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: a preparation method of longan seed polyphenol-pumpkin seed polypeptide composition comprises the steps of dissolving longan seed polyphenol LEP powder in dimethyl sulfoxide to prepare a LEP high-concentration solution, wherein the concentration of the LEP high-concentration solution is greater than or equal to 10 ug/mL; dissolving pumpkin seed polypeptide PSP powder in deionized water to prepare a PSP solution, wherein the concentration of the PSP solution is more than or equal to 1.0 mg/mL; and fully mixing the LEP high-concentration solution with the PSP solution to prepare the longan seed polyphenol-pumpkin seed polypeptide composition, wherein the concentration of the longan seed polyphenol in the composition reaches 0-10ug/mL, the concentration of the pumpkin seed polypeptide in the composition reaches 0.2-1.0mg/mL, and dimethyl sulfoxide and deionized water in the composition 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 the longan seed polyphenol can be reduced by compounding the longan seed polyphenol prepared by the invention and the pumpkin seed polypeptide with a certain concentration, and meanwhile, the longan seed polyphenol prepared by the invention and the pumpkin seed polypeptide are compounded to have a new function of reducing the melanin content in cells, so that the requirements of the effects are as follows: 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 0-10 ug/mL; the final concentration of the pumpkin seed polypeptide is 0.2-1.0 mg/mL.
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/mL in longan seed polyphenol-pumpkin seed polypeptide composition in an embodiment of the present invention.
FIG. 2 is a comparison graph of cytotoxicity of semen Cucurbitae polypeptide in the longan seed polyphenol-semen Cucurbitae polypeptide composition of the present invention at a concentration of greater than 1.0 mg/mL.
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 MTT method is adopted to examine the cell activity of the sample on human skin fibroblasts. HSF cells were collected at 20X 10 in logarithmic growth phase3Density of cells/holeThe cells were plated in 96-well plates at 100. mu.L per well (excluding blank), and after 24h incubation in an incubator, the culture medium was removed from the wells. DMEM is used as a solution, the sample solution is added into each well by 100 mu L of the sample solution, and the sample is incubated for 24 hours, and 100 mu L of DMEM is used as a control group instead of the sample. The wells were removed from solution, washed 2 times with PBS, and 100. mu.L of 0.5mg/mL MTT solution was added to each well, and 100. mu.L of 0.5mg/mL MTT solution was added to the wells without cells as a blank, which was placed in an incubator for 4 h. After the incubation, 100. mu.L DMSO was added to each well, the mixture was shaken for 5min, and then the absorbance OD at 490nm was measured using a microplate reader, and the cell viability was calculated according to equation 1.
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:
measuring the melanin content in the melanocyte, and investigating the whitening activity after compounding. The melanoma cells of the mice in the logarithmic growth phase were inoculated at a density of 20X 103 cells/well in 6-well plates at 2mL per well (excluding the blank), and after incubation in an incubator for 24h, the culture medium in the wells was removed. DMEM is used as a solution to prepare sample solutions with certain concentrations respectively, 2mL of the sample solutions are added into each well to incubate for 24h, and 100 mu L of DMEM is used as a control group to replace the samples. The wells were removed of solution, washed 2 times with PBS and the cells were collected. Adding 1mL 10% DMSO (prepared by NaOH), shaking thoroughly, sealing, standing for 30min, and detecting absorbance at 470nm to obtain melanin content.
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.
Example 1
The embodiment provides a longan seed polyphenol-pumpkin seed polypeptide composition and a preparation method thereof, and the preparation method comprises the following steps:
(1) drying the cleaned longan seeds at 45 +/-5 ℃ for 24 hours, crushing the longan seeds by using a high-speed crusher and sieving the crushed longan seeds by using a 60-mesh sieve; adding petroleum ether according to a material-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, removing the petroleum ether, adding ethanol, carrying out ultrasonic treatment for 60min, wherein the volume fraction of the ethanol is 60%, the material-liquid ratio is 1/25(g/mL), the temperature is 70 ℃, the ultrasonic power is 100W, the frequency is 40Hz, and filtering after ultrasonic treatment to obtain a longan seed polyphenol extracting solution, wherein the longan seed polyphenol content in the extracting solution is 53.68 mg/g;
taking 500mL of longan seed polyphenol extracting solution, concentrating under reduced pressure to 50mL, extracting with 200mL of ethyl acetate, extracting for three times by using a solvent, combining three extracting solutions, concentrating under reduced pressure, and freeze-drying (the temperature of a cold trap is-30-50 ℃, the pressure is 10-50 Pa, and the time is 3-5 days) to obtain ethyl acetate phase polyphenol powder, namely LEP for short.
(2) 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/mL, stirring at 40 deg.C for 4h, vacuum filtering, repeating for 3 times, mixing filtrates, and removing residual petroleum ether in a fume hood to obtain defatted semen Cucurbitae;
adding degreased pumpkin seeds into deionized water according to a material-liquid ratio of 1:30g/mL, adjusting pH to 9.5, reacting for 180min at 35 ℃, centrifuging for 5min under the condition of 10000r/min, collecting supernatant, adjusting pH 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 1:40g/mL 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 2h, then 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 2h, then inactivating, and dropwise adding 0.5mol/L NaOH solution during enzymolysis to keep the pH value of the solution unchanged;
after enzymolysis, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH to the isoelectric point of 4.3, centrifuging for 10min at 5000r/min, collecting supernatant, and freeze-drying to obtain polypeptide;
preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45 μm and 0.22 μm microporous filter membranes, adjusting ultrafiltration pressure to 0.2MPa, ultrafiltering at room temperature for 5 times, collecting polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze drying to obtain polypeptide, PSP for short.
(3) The preparation method of the longan seed polyphenol-pumpkin seed polypeptide composition comprises the following steps:
dissolving longan seed polyphenol LEP powder in dimethyl sulfoxide to prepare a LEP high-concentration solution, wherein the concentration is more than or equal to 10 ug/mL;
dissolving pumpkin seed polypeptide PSP powder in deionized water to prepare a PSP solution, wherein the concentration of the PSP solution is more than or equal to 1.0 mg/mL;
and fully mixing the LEP high-concentration solution with the PSP solution to prepare the longan seed polyphenol-pumpkin seed polypeptide composition, wherein the concentration of the longan seed polyphenol in the composition reaches 0-10ug/mL, the concentration of the pumpkin seed polypeptide in the composition reaches 0.2-1.0mg/mL, and dimethyl sulfoxide and deionized water in the composition are uniformly distributed, and the concentrations are the final concentrations of the longan seed polyphenol and the pumpkin seed polypeptide in the composition.
Cell viability above 90.00% is shown in numerous documents to be non-toxic.
The cytotoxicity of the longan seed polyphenol and the pumpkin seed polypeptide after compounding are shown in table 1.
TABLE 1
The PSP and LEP concentrations in the table are the final concentrations in the composition. As can be seen from Table 1, when the concentration of longan seed polyphenol is in the range of 0-10ug/mL, after the longan seed polyphenol acts on cells for 24 hours, the cell viability is less than 80%, which indicates that the longan seed polyphenol has certain cytotoxicity in the concentration range. However, when the longan seed polyphenol and the pumpkin seed polypeptide are compounded in different ratios, the cell viability of the combination is significantly improved compared to that of the longan seed polyphenol alone. Therefore, the concentration range of the longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition is as follows: 0-10ug/mL, the concentration range of the pumpkin seed polypeptide is as follows: 0.2-1.0mg/mL can effectively improve the cell activity and reduce the cytotoxicity of longan seed polyphenol. Taking 0.2mg/mL pumpkin seed polypeptide and 6ug/mL longan seed polyphenol as examples, the cell viability of the 6ug/mL longan seed polyphenol when acting on cells alone is 68.40%, but when the two exist in the form of a composition, the final concentrations of the two in the composition are still 0.2mg/mL and 6ug/mL respectively, the cell viability reaches 70.41%, compared with the longan seed polyphenol alone, the cell viability of the composition is improved, and the cell toxicity is reduced.
The antioxidant properties of the longan seed polyphenol and the pumpkin seed polypeptide after compounding are shown in table 2. The formulation method of each composition in the table was: preparing LEP high-concentration solution from certain mass of longan seed polyphenol LEP powder by using dimethyl sulfoxide, wherein the concentration of the LEP high-concentration solution is more than or equal to 10 ug/mL; preparing a certain mass of pumpkin seed polypeptide PSP powder into a PSP solution with the concentration of more than or equal to 1.0mg/mL by using deionized water; and fully mixing the LEP high-concentration solution with the PSP solution to prepare the longan seed polyphenol-pumpkin seed polypeptide composition, keeping the concentration of longan seed polyphenol in the composition to be 0-10ug/mL, keeping the concentration of pumpkin seed polypeptide to be 0.2-1.0mg/mL, and uniformly distributing dimethyl sulfoxide and deionized water in the composition.
TABLE 2
The PSP and LEP concentrations in the table are the final concentrations in the composition. As can be seen from Table 2, the radical scavenging rate was 21.24%, 35.01% and 49.91% when the concentration of longan seed polyphenol was 6, 8 and 10ug/mL, respectively. When the concentration of the pumpkin seed polypeptide is 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL respectively, the clearance rate of free radicals is 5.42 percent, 7.21 percent, 10.54 percent, 15.74 percent and 20.83 percent respectively. However, when the longan seed polyphenol and the pumpkin seed polypeptide are compounded in different proportions, the radical scavenging ability of the combination is significantly improved compared with that of the longan seed polyphenol or the pumpkin seed polypeptide alone. Taking 0.2mg/mL pumpkin seed polypeptide and 6ug/mL longan seed polyphenol as examples, the free radical clearance rates are 5.42% and 21.24% respectively when the two are used alone, but when the two exist in the form of a composition, the final concentrations of the two in the composition are still 0.2mg/mL and 6ug/mL respectively, the free radical clearance rate reaches 38.39%, but the sum of the two free radical clearance rates is not far greater, so that the synergistic effect is achieved. As can be seen from table 2, the concentration ranges of longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition are: 0-10ug/mL, the concentration range of the pumpkin seed polypeptide is as follows: 0.2-1.0mg/mL can effectively improve the oxidation resistance and has a certain synergistic effect.
The whitening activity of the longan seed polyphenol and the pumpkin seed polypeptide after compounding is shown in table 3. The formulation method of each composition in the table was: preparing LEP high-concentration solution from certain mass of longan seed polyphenol LEP powder by using dimethyl sulfoxide, wherein the concentration of the LEP high-concentration solution is more than or equal to 10 ug/mL; preparing a certain mass of pumpkin seed polypeptide PSP powder into a PSP solution with the concentration of more than or equal to 1.0mg/mL by using deionized water; and fully mixing the LEP high-concentration solution with the PSP solution to prepare the longan seed polyphenol-pumpkin seed polypeptide composition, keeping the concentration of longan seed polyphenol in the composition to be 0-10ug/mL, keeping the concentration of pumpkin seed polypeptide to be 0.2-1.0mg/mL, and uniformly distributing dimethyl sulfoxide and deionized water in the composition.
TABLE 3
The PSP and LEP concentrations in the table are the final concentrations in the composition. As can be seen from table 3, when the concentrations of longan seed polyphenol were 6, 8, and 10ug/mL, respectively, the melanin contents were 121.83%, 124.21%, and 126.84%, respectively, indicating that longan seed polyphenol did not have the activity of reducing the intracellular melanin content. When the concentrations of the pumpkin seed polypeptide are respectively 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL, the melanin contents are respectively 110.32%, 114.65%, 115.21%, 117.65% and 119.02% compared with the control group, which indicates that the pumpkin seed polypeptide has no activity of reducing the intracellular melanin content. However, when the longan seed polyphenol and the pumpkin seed polypeptide are compounded in different proportions, the melanin content of the composition is lower than 100 percent, which indicates that the composition has the activity of reducing the melanin content in cells. Taking 0.2mg/mL pumpkin seed polypeptide and 6ug/mL longan seed polyphenol as examples, the melanin contents are 110.32% and 121.83% respectively when the pumpkin seed polypeptide and the longan seed polyphenol are used alone, but when the pumpkin seed polypeptide and the longan seed polyphenol exist in a composition, the final concentrations of the pumpkin seed polypeptide and the longan seed polyphenol in the composition are 0.2mg/mL and 6ug/mL respectively, the melanin content reaches 95.36%, and the capacity of reducing the melanin content is incubated. As can be seen from table 3, the concentration ranges of longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition are: 0-10ug/mL, the concentration range of the pumpkin seed polypeptide is as follows: 0.2-1.0mg/mL, can reduce the secretion of melanin of cells, and has whitening effect.
Example 2
The embodiment of the invention provides a method for increasing polyphenol content in longan seed polyphenol, which comprises the following steps:
(1) drying the cleaned longan seeds at 45 +/-5 ℃ for 24 hours, crushing the longan seeds by using a high-speed crusher and sieving the crushed longan seeds by using a 60-mesh sieve; adding petroleum ether according to a material-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, removing the petroleum ether, adding ethanol, carrying out ultrasonic treatment for 60min, wherein the volume fraction of the ethanol is 60%, the material-liquid ratio is 1/25(g/mL), the temperature is 70 ℃, the ultrasonic power is 100W, the frequency is 40Hz, and filtering after ultrasonic treatment to obtain a longan seed polyphenol extracting solution, wherein the longan seed polyphenol content in the extracting solution is 53.68 mg/g.
(2) Taking 500mL of longan seed polyphenol extracting solution, concentrating under reduced pressure to 50mL, sequentially extracting with 200mL of chloroform, ethyl acetate and n-butanol for three times by using each solvent, combining, concentrating under reduced pressure, and freeze-drying to obtain powder.
(3) The measurement of the polyphenol content in the extract is shown in table 4.
TABLE 4
As can be seen from Table 4, the ethyl acetate phase gave the highest polyphenol content of 663.50 mg/g; the crude extract phase is greatly improved by 285.62 mg/g. The polyphenol content of the chloroform phase and the n-butanol phase is far lower than that of the ethyl acetate phase and slightly lower than that of the crude extract phase, namely 254.23mg/g and 262.25mg/g respectively, and the results show that the polyphenol content of the crude extract is obviously improved after the crude extract is extracted by ethyl acetate. Therefore, ethyl acetate is preferred as the extractant in the present invention.
Comparative example 1
(1) Drying the cleaned longan seeds at 45 +/-5 ℃ for 24 hours, crushing the longan seeds by using a high-speed crusher and sieving the crushed longan seeds by using a 60-mesh sieve; adding petroleum ether according to a material-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, removing the petroleum ether, adding ethanol, carrying out ultrasonic treatment for 60min, wherein the volume fraction of the ethanol is 60%, the material-liquid ratio is 1/25(g/mL), the temperature is 70 ℃, the ultrasonic power is 100W, the frequency is 40Hz, and filtering after ultrasonic treatment to obtain a longan seed polyphenol extracting solution, wherein the longan seed polyphenol content in the extracting solution is 53.68 mg/g;
taking 500mL of longan seed polyphenol extracting solution, concentrating under reduced pressure to 50mL, extracting with 200mL of ethyl acetate, extracting for three times by using a solvent, combining three extracting solutions, concentrating under reduced pressure, and freeze-drying (the temperature of a cold trap is-30-50 ℃, the pressure is 10-50 Pa, and the time is 3-5 days) to obtain ethyl acetate phase polyphenol powder, namely LEP for short.
(2) 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/mL, stirring at 40 deg.C for 4h, vacuum filtering, repeating for 3 times, mixing filtrates, and removing residual petroleum ether in a fume hood to obtain defatted semen Cucurbitae;
adding degreased pumpkin seeds into deionized water according to a material-liquid ratio of 1:30g/mL, adjusting pH to 9.5, reacting for 180min at 35 ℃, centrifuging for 5min under the condition of 10000r/min, collecting supernatant, adjusting pH 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 1:40g/mL 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 2h, then 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 2h, then inactivating, and dropwise adding 0.5mol/L NaOH solution during enzymolysis to keep the pH value of the solution unchanged;
after enzymolysis, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH to the isoelectric point of 4.3, centrifuging for 10min at 5000r/min, collecting supernatant, and freeze-drying to obtain polypeptide;
preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45 μm and 0.22 μm microporous filter membranes, adjusting ultrafiltration pressure to 0.2MPa, ultrafiltering at room temperature for 5 times, collecting polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze drying to obtain polypeptide, PSP for short.
(3) Dissolving longan seed polyphenol LEP powder in dimethyl sulfoxide to prepare a LEP high-concentration solution, wherein the concentration is more than or equal to 10 ug/mL;
dissolving pumpkin seed polypeptide PSP powder in deionized water to prepare a PSP solution, wherein the concentration of the PSP solution is more than or equal to 1.0 mg/mL;
and fully mixing the LEP high-concentration solution with the PSP solution to prepare the longan seed polyphenol-pumpkin seed polypeptide composition, wherein the concentration of the longan seed polyphenol in the composition is more than 10ug/mL, the concentration of the pumpkin seed polypeptide reaches 0.2-1.0mg/mL, and dimethyl sulfoxide and deionized water in the composition are uniformly distributed, and the concentrations are the final concentrations of the longan seed polyphenol and the pumpkin seed polypeptide in the composition.
The cytotoxicity of the prepared composition is shown in figure 1. In the figure, 10.1ug/mL LEP is 10.1ug/mL longan seed polyphenol solution. As can be seen from FIG. 1, when the final concentration of longan seed polyphenol in the composition exceeds 10.0ug/mL, the toxicity continues to increase regardless of the final concentration of pumpkin seed polypeptide.
Comparative example 2
On the basis of comparative example 1, the concentration range requirement of longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition is maintained as follows: 0-10ug/mL, the concentration range of the pumpkin seed polypeptide is more than 1.0mg/mL, the concentrations are the final concentrations of the longan seed polyphenol and the pumpkin seed polypeptide in the composition, and other conditions are the same as those in the comparative example 1.
The cytotoxicity of the prepared composition is shown in fig. 2. In the figure, 10ug/mL LEP is the longan seed polyphenol solution of 10 ug/mL. As can be seen in FIG. 2, the cytotoxicity of the composition increased when the final concentration of pumpkin seed polypeptide in the composition exceeded 1.0 mg/mL.
Comparative example 3
The antioxidant activity of the composition prepared on the basis of comparative example 1 is shown in table 5.
TABLE 5
In the table, 10.1ug/mL LEP is 10.1ug/mL longan seed polyphenol solution, and it can be seen from table 5 that, when the final concentration of longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition is greater than 10.0ug/mL, the antioxidant performance of the composition has no synergistic effect. Taking 0.2mg/mL pumpkin seed polypeptide and 10.1ug/mL longan seed polyphenol as examples, the free radical clearance rates are respectively 5.42% and 52.02% when the two are used alone, but when the two exist in the form of a composition, the final concentrations of the two in the composition are still respectively 0.2mg/mL and 10.1ug/mL, the free radical clearance rate reaches 60.36%, which is close to the sum of the two free radical clearance rates, and the synergistic effect is not obvious.
Comparative example 4
The antioxidant activity of the composition prepared on the basis of comparative example 2 is shown in table 6.
TABLE 6
In the table, 10.1ug/mL LEP is 10.1ug/mL longan seed polyphenol solution; the composition is formulated as described above. As can be seen from Table 6, when the final concentration of the pumpkin seed polypeptide in the longan seed polyphenol-pumpkin seed polypeptide composition is more than 10.0mg/mL, the antioxidant performance of the composition is not synergistic. Taking 10.1mg/mL pumpkin seed polypeptide and 6ug/mL longan seed polyphenol as examples, the free radical clearance rates are respectively 22.82% and 21.24% when the two are used alone, but when the two exist in the form of a composition, the final concentrations of the two in the composition are still respectively 10.1mg/mL and 6ug/mL, the free radical clearance rate reaches 45.76%, which is close to the sum of the two free radical clearance rates, and no significant synergistic effect is achieved.
Comparative example 5
The melanin content of the compositions prepared on the basis of comparative example 1 is shown in table 7.
TABLE 7
In the figure, 10.1ug/mL LEP is 10.1ug/mL longan seed polyphenol solution; the composition is formulated as described above. As can be seen from Table 7, when the final concentration of longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition is greater than 10.0ug/mL, the intracellular melanin content is always greater than 100.00% of the control group, which indicates that no whitening activity exists beyond this concentration range. Taking 0.2mg/mL pumpkin seed polypeptide and 10.1ug/mL longan seed polyphenol as examples, the intracellular melanin contents are 110.32% and 126.84% respectively when the pumpkin seed polypeptide and the longan seed polyphenol are used alone, but when the pumpkin seed polypeptide and the longan seed polyphenol exist in a composition form, the final concentrations of the pumpkin seed polypeptide and the longan seed polyphenol in the composition are 0.2mg/mL and 10.1ug/mL respectively, the intracellular melanin contents reach 124.86% and the whitening activity is not realized.
Comparative example 6
The melanin content of the compositions prepared on the basis of comparative example 2 is shown in table 8.
TABLE 8
The whitening activity of the composition is shown in table 8. As can be seen from table 8, when the final concentration of the pumpkin seed polypeptide in the longan seed polyphenol-pumpkin seed polypeptide composition is greater than 10.0mg/mL, the intracellular melanin content is always greater than 100.00% of the control group, which indicates that no whitening activity exists beyond this concentration range. Taking 10.1mg/mL pumpkin seed polypeptide and 6ug/mL longan seed polyphenol as examples, the intracellular melanin contents are 119.92% and 121.83% respectively when the pumpkin seed polypeptide and the longan seed polyphenol are used alone, but when the pumpkin seed polypeptide and the longan seed polyphenol exist in a composition, the final concentrations of the pumpkin seed polypeptide and the longan seed polyphenol in the composition are 10.1mg/mL and 6ug/mL respectively, the intracellular melanin contents reach 125.66% respectively, and the whitening activity is not realized.
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. The longan seed polyphenol prepared by the invention can be compounded with pumpkin seed polypeptide with a certain concentration to reduce the toxicity of the longan seed polyphenol, and meanwhile, the compound of the longan seed polyphenol and the pumpkin seed polypeptide has a new function of reducing the content of melanin in cells. The toxicity of the longan seed polyphenol can be reduced by compounding the longan seed polyphenol prepared by the method with the pumpkin seed polypeptide with a certain concentration, meanwhile, the longan seed polyphenol prepared by the method is endowed with a new function of reducing the content of melanin in cells by compounding the longan seed polyphenol with the pumpkin seed polypeptide, and the longan seed polyphenol and the pumpkin seed polypeptide do not have the function when acting on the cells independently, which is specifically described in example 1 and comparative examples 5 and 6.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (3)
1. A preparation method of a longan seed polyphenol-pumpkin seed polypeptide composition is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
dissolving longan seed polyphenol LEP powder in dimethyl sulfoxide to prepare a LEP high-concentration solution, wherein the concentration is more than or equal to 10 microgram/mL;
dissolving pumpkin seed polypeptide PSP powder in deionized water to prepare a PSP solution, wherein the concentration of the PSP solution is more than or equal to 1.0 mg/mL;
fully mixing the LEP high-concentration solution with the PSP solution to prepare a longan seed polyphenol-pumpkin seed polypeptide composition, wherein the concentration of longan seed polyphenol in the composition is 6-8 mu g/mL, the concentration of pumpkin seed polypeptide reaches 0.4-0.8 mg/mL, and dimethyl sulfoxide and deionized water in the composition are uniformly distributed; wherein,
the preparation method of the longan seed polyphenol comprises the steps of drying cleaned longan seeds for 24 hours at the temperature of 45 +/-5 ℃, crushing the longan seeds and sieving the crushed longan seeds with a 60-mesh sieve to obtain longan seed powder; adding petroleum ether into the mixture according to the ratio of the material to the liquid in g/mL of 1:4, stirring the mixture for 3 to 4 hours at the temperature of 40 ℃, and removing the petroleum ether, wherein the ratio of the material to the liquid in g/mL of 1: 25, adding 60% ethanol by volume, carrying out ultrasonic treatment at the temperature of 70 ℃ for 60-80 min, 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 LEP; wherein the volume of the longan seed polyphenol extracting solution is 200-500 mL, the longan seed polyphenol extracting solution is concentrated to 50mL under reduced pressure, and the volume ratio of ethyl acetate to the longan seed polyphenol extracting solution after the concentration under reduced pressure is 4: 1;
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/mL, stirring at 40 deg.C for 4h, vacuum filtering, repeating for 3 times, mixing filtrates, and removing residual petroleum ether in a fume hood to obtain defatted semen Cucurbitae; adding degreased pumpkin seeds into deionized water according to a material-liquid ratio of 1:30g/mL, 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/mL 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 0.5mol/L NaOH solution during enzymolysis to keep the pH value of the solution unchanged; after enzymolysis, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH to the isoelectric point of 4.3, centrifuging for 10min at 5000r/min, collecting supernatant, and freeze-drying to obtain polypeptide; preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes, adjusting ultrafiltration pressure to be 0.1-0.2 MPa, performing 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.
2. The method for preparing the longan seed polyphenol-pumpkin seed polypeptide composition as set forth in claim 1, wherein the longan seed polyphenol-pumpkin seed polypeptide composition comprises: and ultrasonic treatment, wherein the ultrasonic power is 100W, and the frequency is 40 Hz.
3. The method for preparing the longan seed polyphenol-pumpkin seed polypeptide composition as set forth in claim 1, wherein the longan seed polyphenol-pumpkin seed polypeptide composition comprises: and (3) freeze-drying, wherein the temperature of a cold trap is-30 to-50 ℃, the pressure is 10 to 50Pa, and the time is 3 to 5 days.
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