CN111449979A - 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 PDF

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CN111449979A
CN111449979A CN202010449006.2A CN202010449006A CN111449979A CN 111449979 A CN111449979 A CN 111449979A CN 202010449006 A CN202010449006 A CN 202010449006A CN 111449979 A CN111449979 A CN 111449979A
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polyphenol
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pumpkin
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CN111449979B (en
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孙培冬
王培宇
孙菡峥
杨成
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Jiangnan University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
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    • A61K8/33Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64Proteins; Peptides; Derivatives or degradation products thereof
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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    • A61Q19/08Anti-ageing preparations
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2800/59Mixtures
    • A61K2800/592Mixtures of compounds complementing their respective functions
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/84Products 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 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.

Description

Longan seed polyphenol-pumpkin seed polypeptide composition and preparation method thereof
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.
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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.
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 cleaned longan seeds at 45 +/-5 ℃ for 24 hours, crushing the longan seeds by using a high-speed crusher, sieving the crushed longan seeds with a 60-mesh sieve, adding petroleum ether according to the material-liquid ratio of 1:4(g/m L), stirring the mixture for 4 hours at 40 ℃, removing the petroleum ether, adding ethanol, performing ultrasonic treatment for 60 minutes, wherein the volume fraction of the ethanol is 60 percent, the material-liquid ratio is 1/25(g/m L), the temperature is 70 ℃, the ultrasonic power is 100W, the frequency is 40Hz, and filtering the mixture after the ultrasonic treatment is performed to obtain longan seed polyphenol extracting solution, wherein the content of longan seed polyphenol in the extracting solution is 53.68 mg/g;
taking longan seed polyphenol extracting solution 500m L, concentrating under reduced pressure to 50m L, extracting with ethyl acetate of L m, extracting with a solvent for three times, combining extracting solutions of the three times, concentrating under reduced pressure, and freeze-drying (cold trap temperature is-30-50 ℃, pressure is 10-50 Pa, and time is 3-5 days) to obtain ethyl acetate phase polyphenol powder, which is referred to as L EP 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/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 10000r/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 1:40g/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 2h, 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, and then inactivating, wherein a 0.5 mol/L NaOH solution is dropwise added during the 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 semen Cucurbitae polypeptide, sequentially passing through 0.45 μm and 0.22 μm microporous filter membranes, regulating ultrafiltration pressure to 0.2MPa, ultrafiltering at room temperature for 5 times, collecting the polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze drying to obtain polypeptide PSP.
(3) The preparation method of the longan seed polyphenol-pumpkin seed polypeptide composition comprises the following steps:
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 concentration of the longan seed polyphenol in the composition reaches 0-10ug/m L, the concentration of the pumpkin seed polypeptide reaches 0.2-1.0mg/m L, and the dimethyl sulfoxide and the deionized water in the composition are uniformly distributed, wherein 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
Figure BDA0002506988080000061
The concentration of longan seed polyphenol in the combination is 0-10ug/m L, and the concentration of longan seed polypeptide in the range of 0.2-1.0mg/m L is effective in increasing cell viability and decreasing cell cytotoxicity of longan seed polyphenol alone, the concentration of longan seed polyphenol in the combination of 0.2-1.0mg/m 5663 is 68.40% when longan seed polyphenol alone is used, but the cell viability of the combination is 68.40% when longan seed polyphenol in the form of 0.2mg/m L is used with longan seed polypeptide in the range of 6ug/m L, while the cell viability of the combination of longan seed in the form of 0.2mg/m L% is increased with the cell viability of the combination of 68.40% when longan seed polypeptide alone is used with the cell viability of 3526.26% when longan seed polypeptide alone is used with the cell viability of L% when longan seed polypeptide in the form of 0.2mg/m L is used with the cell viability of longan seed polypeptide in the form of L% when longan seed polypeptide alone is used.
The preparation method of the longan seed polyphenol and the antioxidant performance of the longan seed polyphenol compounded with the pumpkin seed polypeptide are shown in table 2, the preparation method of each composition in the table comprises the steps of preparing L EP high-concentration solution of the longan seed polyphenol L EP powder with dimethyl sulfoxide, the concentration of which is greater than or equal to 10ug/m L, preparing PSP solution of the pumpkin seed polypeptide PSP powder with deionized water, the concentration of which is greater 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 the longan seed polyphenol in the composition is kept to be 0-10ug/m L, the concentration of the pumpkin seed polypeptide is kept to be 0.2-1.0mg/m L, and the dimethyl sulfoxide and the deionized water in the composition are uniformly distributed.
TABLE 2
Figure BDA0002506988080000071
In table 2, it can be seen that when the concentrations of longan seed polyphenol are 6, 8, 10ug/m L, the radical scavenging rates are 21.24%, 35.01%, 49.91%, respectively, when the concentrations of pumpkin seed polypeptide are 0.2, 0.4, 0.6, 0.8, 1.0mg/m L, respectively, the radical scavenging rates are 5.42%, 7.21%, 10.54%, 15.74%, 20.83%, respectively, but when the concentrations of longan seed polyphenol and pumpkin seed polypeptide are compounded in different ratios, the radical scavenging rates of the combination are significantly improved compared to the radical scavenging rates of longan seed polyphenol alone or pumpkin seed polypeptide, for example, 0.2mg/m L pumpkin seed polypeptide and 6ug/m L, when used alone, the radical scavenging rates are 5.42%, 21.24%, but when both are present in the form of the composition, the synergistic effect of the combination is still greater than the synergistic effect of the concentrations of longan seed polypeptide in the ranges of 0.38-352 mg/m L%, thus the synergistic effect of the combination is not as seen by the synergistic effect of longan seed polypeptide in the ranges of 0.2-352, 352-358, and 358-8 mg/m, respectively, thus the synergistic effect of the combination is not greater than the synergistic effect of longan seed polypeptide in the synergistic effect of the combination of longan seed polypeptide in the combination.
The preparation method of the longan seed polyphenol and the whitening activity of the compound of the longan seed polyphenol and the pumpkin seed polypeptide is shown in table 3, the preparation method of each composition in the table comprises the steps of preparing L EP high-concentration solution of the longan seed polyphenol L EP powder with dimethyl sulfoxide, the concentration of which is greater than or equal to 10ug/m L, preparing PSP solution of the pumpkin seed polypeptide PSP powder with deionized water, the concentration of which is greater 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 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 dimethyl sulfoxide and the deionized water in the composition are uniformly distributed.
TABLE 3
Figure BDA0002506988080000081
In table PSP, L EP concentrations are final concentrations in the composition, it can be seen from table 3 that when the concentrations of longan seed polyphenol are 6, 8, 10ug/m L, respectively, the melanin contents are 121.83%, 124.21%, 126.84% respectively compared to the control group, indicating that longan seed polyphenol does not have the activity of reducing the intracellular melanin content, when the concentrations of pumpkin seed polypeptide are 0.2, 0.4, 0.6, 0.8, 1.0mg/m L, respectively, the melanin contents are 110.32%, 114.65%, 115.21%, 117.65%, 119.02% respectively, indicating that pumpkin seed polypeptide does not have the activity of reducing the intracellular melanin content, but when longan seed polyphenol and pumpkin seed polypeptide are compounded at different ratios, the melanin content of the composition is less than 100%, indicating that the composition has the activity of reducing the intracellular melanin contents, when longan seed polypeptide and pumpkin seed polypeptide are compounded at different ratios, the melanin contents of 0.2mg/m L and 396 ug/m are examples, when longan seed polyphenol alone is used as a core polyphenol, the melanin contents are 110.32%, the melanin contents are still reduced in the composition, when longan seed polypeptide concentration ranges from 0.2mg/m 388 to 638, and the melanin contents of the longan seed polypeptide are still capable of being incubated at ranges of 0.26%, and 0.26% to L, respectively, and the melanin contents of the longan seed polypeptide are still reduced in the range of 0.8, and the melanin contents of the composition when longan seed polypeptide is found from 0.8, and the longan seed polypeptide is still capable of the range of 0.26% to 638.
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 cleaned longan seeds at 45 +/-5 ℃ for 24h, crushing the longan seeds by using a high-speed crusher, sieving the crushed longan seeds with a 60-mesh sieve, adding petroleum ether according to a material-liquid ratio of 1:4(g/m L), stirring the mixture for 4h at 40 ℃, removing the petroleum ether, adding ethanol, performing ultrasonic treatment for 60min, wherein the volume fraction of the ethanol is 60%, the material-liquid ratio is 1/25(g/m L), the temperature is 70 ℃, the ultrasonic power is 100W, the frequency is 40Hz, and filtering the mixture after the ultrasonic treatment is performed to obtain a longan seed polyphenol extracting solution, wherein the longan seed polyphenol content in the extracting solution is 53.68 mg/g.
(2) Concentrating longan seed polyphenol extractive solution 500m L under reduced pressure to 50m L, sequentially extracting with chloroform, ethyl acetate and n-butanol of 200m L for three times, mixing, 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
Figure BDA0002506988080000091
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 cleaned longan seeds at 45 +/-5 ℃ for 24 hours, crushing the longan seeds by using a high-speed crusher, sieving the crushed longan seeds with a 60-mesh sieve, adding petroleum ether according to the material-liquid ratio of 1:4(g/m L), stirring the mixture for 4 hours at 40 ℃, removing the petroleum ether, adding ethanol, performing ultrasonic treatment for 60 minutes, wherein the volume fraction of the ethanol is 60 percent, the material-liquid ratio is 1/25(g/m L), the temperature is 70 ℃, the ultrasonic power is 100W, the frequency is 40Hz, and filtering the mixture after the ultrasonic treatment is performed to obtain longan seed polyphenol extracting solution, wherein the content of longan seed polyphenol in the extracting solution is 53.68 mg/g;
taking longan seed polyphenol extracting solution 500m L, concentrating under reduced pressure to 50m L, extracting with ethyl acetate of L m, extracting with a solvent for three times, combining extracting solutions of the three times, concentrating under reduced pressure, and freeze-drying (cold trap temperature is-30-50 ℃, pressure is 10-50 Pa, and time is 3-5 days) to obtain ethyl acetate phase polyphenol powder, which is referred to as L EP 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/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 10000r/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 1:40g/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 2h, 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, and then inactivating, wherein a 0.5 mol/L NaOH solution is dropwise added during the 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 semen Cucurbitae polypeptide, sequentially passing through 0.45 μm and 0.22 μm microporous filter membranes, regulating ultrafiltration pressure to 0.2MPa, ultrafiltering at room temperature for 5 times, collecting the polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze drying to obtain polypeptide PSP.
(3) 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 concentration of the longan seed polyphenol in the composition is more than 10ug/m L, the concentration of the pumpkin seed polypeptide reaches 0.2-1.0mg/m L, 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/m L L EP is longan seed polyphenol solution of 10.1ug/m L, and as can be seen from figure 1, when the final concentration of longan seed polyphenol in the composition exceeds 10.0ug/m L, the toxicity is continuously improved no matter the final concentration of pumpkin seed polypeptide.
Comparative example 2
On the basis of comparative example 1, the concentration range of longan seed polyphenol in the longan seed polyphenol-pumpkin seed polypeptide composition is maintained to be 0-10ug/m L, the concentration range of pumpkin seed polypeptide is more than 1.0mg/m L, the concentrations are the final concentrations of longan seed polyphenol and pumpkin seed polypeptide in the composition, and other conditions are the same as those in comparative example 1.
The cytotoxicity of the prepared composition is shown in figure 2, wherein 10ug/m L L EP in the figure is longan seed polyphenol solution of 10ug/m L, and as can be seen from figure 2, when the final concentration of the pumpkin seed polypeptide in the composition exceeds 1.0mg/m L, the cytotoxicity of the composition is improved.
Comparative example 3
The antioxidant activity of the composition prepared on the basis of comparative example 1 is shown in table 5.
TABLE 5
Figure BDA0002506988080000111
In the table, 10.1ug/m L L EP is the longan seed polyphenol solution of 10.1ug/m L, 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/m L, the antioxidant performance of the composition has no synergistic effect, taking 0.2mg/m L pumpkin seed polypeptide and 10.1ug/m L longan seed polyphenol as examples, the radical scavenging rates are 5.42% and 52.02% respectively when used alone, but when both exist in the form of the composition, the final concentrations of both in the composition are still 0.2mg/m L and 10.1ug/m L respectively, the radical scavenging rate reaches 60.36%, which is close to the sum of the radical scavenging rates of both, and has no significant synergistic effect.
Comparative example 4
The antioxidant activity of the composition prepared on the basis of comparative example 2 is shown in table 6.
TABLE 6
Figure BDA0002506988080000112
Figure BDA0002506988080000121
In the table, 10.1ug/m L L EP is longan seed polyphenol solution of 10.1ug/m L, and the composition preparation method is the method described in the table 6, it can be seen that when the final concentration of the pumpkin seed polypeptide in the longan seed polyphenol-pumpkin seed polypeptide composition is greater than 10.0mg/m L, the antioxidant performance of the composition has no synergistic effect, taking 10.1mg/m L pumpkin seed polypeptide and 6ug/m L longan seed polyphenol as examples, the free radical scavenging rates are respectively 22.82% and 21.24% when used alone, but when the two exist in the form of the composition, the final concentrations of the two in the composition are still 10.1mg/m L and 6ug/m L respectively, the free radical scavenging rate reaches 45.76%, which is close to the sum of the two free radical scavenging rates, and has no significant synergistic effect.
Comparative example 5
The melanin content of the compositions prepared on the basis of comparative example 1 is shown in table 7.
TABLE 7
Figure BDA0002506988080000122
In the figure, 10.1ug/m L L EP is longan seed polyphenol solution of 10.1ug/m L, and the composition preparation method is the method 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/m L, the intracellular melanin content is always greater than 100.00% of the control group, which indicates that the whitening activity is not achieved beyond the concentration range, taking 0.2mg/m L pumpkin seed polypeptide and 10.1ug/m L longan seed polyphenol as examples, the intracellular melanin content is 110.32% and 126.84% respectively when the two are used alone, but when the two are in the form of the composition, the final concentrations of the two in the composition are still 0.2mg/m L and 10.1ug/m L respectively, the intracellular melanin content reaches 124.86% and the whitening activity is not achieved.
Comparative example 6
The melanin content of the compositions prepared on the basis of comparative example 2 is shown in table 8.
TABLE 8
Figure BDA0002506988080000123
Figure BDA0002506988080000131
The whitening activity of the composition is shown in table 8, it can be seen from table 8 that when the final concentration of the pumpkin seed polypeptide in the longan seed polyphenol-pumpkin seed polypeptide composition is greater than 10.0mg/m L, the intracellular melanin content is always greater than 100.00% of the control group, which indicates that the whitening activity is not achieved beyond the concentration range, in the case of 10.1mg/m L pumpkin seed polypeptide and 6ug/m L longan seed polyphenol, the intracellular melanin content is 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 are in the form of the composition, the final concentrations of the pumpkin seed polypeptide and the longan seed polypeptide in the composition are still 10.1mg/m L and 6ug/m L respectively, and the intracellular melanin content reaches 125.66% and the whitening activity is not achieved.
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.
Longan seed polyphenol and pumpkin seed polypeptide are compounded to reduce toxicity, and antioxidant performance and whitening activity of the longan seed polyphenol and the pumpkin seed polypeptide are tested. Provides a theoretical basis for compounding longan seed polyphenol and pumpkin seed polypeptide in cosmetics as an antioxidant whitening formula.
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 (10)

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.
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