CN114569483A - Ginseng polypeptide extract, preparation method thereof and anti-ultraviolet whitening essence - Google Patents
Ginseng polypeptide extract, preparation method thereof and anti-ultraviolet whitening essence Download PDFInfo
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- CN114569483A CN114569483A CN202210164871.1A CN202210164871A CN114569483A CN 114569483 A CN114569483 A CN 114569483A CN 202210164871 A CN202210164871 A CN 202210164871A CN 114569483 A CN114569483 A CN 114569483A
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
-
- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
The invention provides a ginseng polypeptide extract, a preparation method thereof and an anti-ultraviolet whitening essence, and belongs to the technical field of cosmetics. S1, preprocessing ginseng; s2, extracting with a solvent to obtain a ginseng extracting solution; s3, preparing polar ionic liquid; s4, first purification of the ginseng polypeptide; s5, carrying out secondary purification by using a chromatographic column to obtain the ginseng polypeptide extract. The high-purity ginseng polypeptide compound obtained by the invention has a good ultraviolet radiation resistant effect, has the effects of fading color spots and inhibiting melanin generation, and thus has a whitening and anti-radiation effect, and the prepared ultraviolet radiation resistant whitening essence has good anti-radiation and whitening effects.
Description
Technical Field
The invention relates to the technical field of cosmetics, and particularly relates to a ginseng polypeptide extract, a preparation method thereof and an anti-ultraviolet whitening essence.
Background
The reasons for the pigmentation and the color spots are complex, such as genetic factors, endocrine dyscrasia, environmental pollution, ultraviolet irradiation and the like, and the pigmentation and the color spots caused by any reason are all caused by the pigmentation, so the main approach for whitening the skin and removing the spots is to inhibit the generation of the melanin. There are various methods for inhibiting the production of melanin, such as inhibiting the activity of tyrosinase, scavenging active oxygen or free radicals, reducing ultraviolet irradiation, preventing the occurrence of oxidation reaction, etc., which are effective methods for reducing the formation of melanin.
People can reduce or eliminate color spots by adding various whitening and freckle-removing substances into cosmetics, thereby achieving the effects of whitening and removing freckles.
At present, the commonly used whitening and freckle-removing components on the market comprise arbutin and derivatives thereof, hydroquinone derivatives, kojic acid and derivatives thereof, fruit acids, vitamin C and derivatives thereof, plant extract components with whitening and freckle-removing effects and the like. Wherein, although the arbutin and the derivatives thereof, the hydroquinone derivatives, the kojic acid and the derivatives thereof, the fruit acids and other components have good effects, the cytotoxicity and the irritation are relatively large, and the arbutin and the derivatives thereof are easy to interfere the normal physiological metabolism function of the skin after long-term use, and cause some adverse side effects, such as reduction of the photoprotection of the skin, easy rebound, skin allergy, contact dermatitis and the like. Although the other types of whitening and freckle-removing components have high safety, the effects are slow, and the market application rate is not high.
The dried root and rhizome of Panax ginseng, a plant of Araliaceae, Panax ginseng, as a traditional famous and precious medicinal material, has a long history of application in the medical history of China. Modern medicine shows that ginseng has many functions of regulating central nervous system, increasing myocardial contraction force, slowing down heart rate, increasing cardiac output and coronary blood flow, enhancing sexual function, promoting gonad function and development, enhancing immunity and improving hematopoiesis. Meanwhile, ginseng also has the effects of resisting radiation, viruses, tumors, shock and the like.
In more than thirty years, with the progress of research methods, the extraction, separation and structural identification of chemical components of ginseng have been greatly advanced. Various chemical components such as saponin, volatile components, protein, polypeptide, polysaccharide, amino acid and the like are separated from the ginseng.
The research on ginseng protein and polypeptide starts in the early century, mainly focuses on the extraction method of ginseng polypeptide, and the activity of the polypeptide is not reported. A protein with the molecular weight of 26KD is separated from ginseng such as Tzi Bun Ng and the like, and the in vitro activity shows that the protein has the functions of resisting lipolysis, resisting erythrocyte accumulation, resisting virus, ribonuclease activity, arginase activity and the like. 14 peptides with insulin-like action were isolated in 1980 by Ando et al, Japan, and one 11 and one 12 peptides were isolated in 1990 by Wenjinger et al, the university of Jilin, but no further study was made on the activity. Zhang jin et al, Jilin university, proposed a new process for extracting total ginseng polypeptides, total ginseng polysaccharides and total ginseng saponins from ginseng by controlling ethanol with different concentrations and separating the total ginseng polysaccharides, total ginseng polypeptides and total ginseng saponins at a certain pH value. Tenglirong, etc. separates out pure products according to the hydrophobicity and heat resistance of ginseng polypeptide. In the 90 s, with the wide application of modern biotechnology in the field of traditional Chinese medicine, the research on ginseng protein and polypeptide has been increasing. More and more enterprises have the extraction technology of the ginseng active polypeptide and apply the ginseng active polypeptide to high-end whitening skin care products.
Disclosure of Invention
The invention aims to provide a ginseng polypeptide extract, a preparation method thereof and an anti-ultraviolet whitening essence, so that a high-purity ginseng polypeptide compound is obtained, the compound has a good anti-ultraviolet radiation effect, and has the effects of fading color spots and inhibiting melanin generation, so that the whitening and anti-radiation effects are achieved, and the prepared anti-ultraviolet whitening essence has good anti-radiation and whitening effects.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a ginseng polypeptide extract, which comprises the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving to obtain Ginseng radix powder;
s2, solvent extraction: soaking the ginseng powder prepared in the step S1 in an aqueous solution containing isopropanol, acetone and acetic acid, heating, stirring, extracting, filtering, collecting an extracting solution, and concentrating at low temperature under reduced pressure to obtain a ginseng extracting solution;
s3, preparing polar ionic liquid: introducing CO into the ionic liquid2Gas to obtain polar ionic liquid;
s4, first purification of the ginseng polypeptide: adding the ginseng extract obtained in the step S2 into the polar ionic liquid obtained in the step S3, performing microwave stirring extraction, separating an organic layer, heating and oscillating, collecting a water layer, and performing freeze drying to obtain a ginseng polypeptide water extract;
s5, purifying the chromatographic column for the second time: purifying the ginseng polypeptide water extract obtained in the step S4 by adopting a reversed-phase ODS C18 chromatographic column to obtain the ginseng polypeptide extract.
As a further improvement of the invention, the mesh number of the sieving in the step S1 is 150-200 meshes.
As a further improvement of the invention, the content of the isopropanol in the aqueous solution containing the isopropanol, the acetone and the acetic acid in the step S2 is 5-7 wt%; the acetone content is 2-5 wt%; the acetic acid content is 10-25 wt%; the soaking time is 10-20 min; heating to 70-90 deg.C, and extracting for 2-4 h; the low-temperature reduced pressure concentration is carried out at 40-50 deg.C and 0.01-0.1MPa until the density of extractive solution is 2-3g/cm3。
As a further improvement of the present invention, in step S3, the ionic liquid is at least one selected from the group consisting of 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hexafluoroantimonate, 1-hexyl-3-methylimidazole tetrafluoroborate, 1-hexyl-3-methylimidazole hexafluoroantimonate, and 1-hexyl-3-methylimidazole hexafluoroantimonate; the CO is2The aeration speed of the gas is 2-5mL/min, and the aeration time is 30-60 min.
In a further improvement of the present invention, in step S4, the volume ratio of the ginseng extract to the polar ionic liquid is 1: (2-3); the microwave power is 500-1000W, and the extraction time is 1-2 h; the heating is carried out to 50-60 ℃.
As a further improvement of the present invention, the specific operation of step S5 is as follows: dissolving the ginseng polypeptide water extract with 0.1-0.5 wt% trifluoroacetic acid and 2-4 wt% acetonitrile solution, filtering with 0.22 μm filter membrane, loading, using 0.1-0.5 wt% trifluoroacetic acid and 2-4 wt% acetonitrile aqueous solution to balance chromatography column, then using 0.1-0.5 wt% trifluoroacetic acid and 15 wt% acetonitrile aqueous solution to rinse for 10-30min, using 0.1-0.5 wt% trifluoroacetic acid and 10 wt% acetonitrile aqueous solution to rinse for 20-30min, then using 0.1-0.5 wt% trifluoroacetic acid containing acetonitrile aqueous solution to rinse for 30-50min, the elution speed is 0.5-1.5mL/min, the column specification is 15-20mm in diameter and 50-70cm in length.
The invention further protects the ginseng polypeptide extract prepared by the preparation method.
The invention further protects the application of the ginseng polypeptide extract in the field of cosmetics.
The invention further provides an anti-ultraviolet whitening essence which contains 2-5 wt% of the ginseng polypeptide extract.
As a further improvement of the invention, the health-care food is prepared from the following raw materials in percentage by mass: the ginseng polypeptide extract of claim 7, 2-5%, PEG-100 stearate 0.5-1%, butylene glycol 2-5%, glycerin 3-6%, carbomer 0.5-2%, squalane 2-3%, xanthan gum 0.1-0.5%, nicotinamide 0.5-1%, essence 0.1-0.5%, pearl powder 1-2%, phenoxyethanol 0.1-0.4%, and the balance of deionized water.
The invention has the following beneficial effects: firstly, adding ginseng powder into an aqueous solution containing isopropanol, acetone and acetic acid for solvent extraction, effectively extracting acid soluble protein, alcohol soluble protein, water soluble protein and the like, simultaneously extracting other active substances such as saponin, triterpene and the like, and entering the next purification step;
according to the invention, carbon dioxide is introduced into water-insoluble ionic liquid, so that the polarity of the ionic liquid is improved, other active substances with poor polarity in extract liquid are left in an organic layer, and the ginseng polypeptide compound with high polarity enters an ionic liquid layer, so that polypeptide and other impurities are separated, the extracted high-polarity ionic liquid is heated and returned to common ionic liquid, and after water is added for oscillation, the polar ginseng polypeptide compound dissolved in the ionic liquid is extracted to a water layer, so that not only is the first purification of the ginseng polypeptide compound realized, but also the ionic liquid can be reused;
further, after the ginseng polypeptide compound with high polarity and good hydrophobicity is further separated by the reversed-phase C18 chromatographic column, the high-purity ginseng polypeptide compound is obtained, the compound has a good ultraviolet radiation resistant effect, color spots are lightened, melanin generation is inhibited, and therefore the whitening and anti-radiation effects are achieved, and the prepared ultraviolet-resistant whitening essence has good anti-radiation and whitening effects.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph comparing the effect of aqueous ginseng polypeptide extracts on the proliferation of NIH-3T3 cells in test example 1;
FIG. 2 is a graph comparing the effect of aqueous ginseng polypeptide extract on UVB-induced NIH-3T3 cell damage in test example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a preparation method of a ginseng polypeptide extract, which comprises the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving with 150 mesh sieve to obtain Ginseng radix powder;
s2, solvent extraction: adding 10g of the ginseng powder obtained in step S1 into 100mL of an aqueous solution containing isopropanol, acetone and acetic acid (the content of isopropanol is 5 wt%; and the content of C is less than one hundred)The ketone content was 2 wt%; 10 wt%) of acetic acid, heating to 70 deg.C, stirring and extracting for 2 hr, vacuum filtering, collecting extractive solution, concentrating at 40 deg.C under 0.01MPa to obtain extractive solution with density of 2g/cm3To obtain ginseng extract;
s3, preparing polar ionic liquid: introducing CO into 200mL of ionic liquid 1-butyl-3-methylimidazolium hexafluoroantimonate2Gas, CO2The aeration speed of the gas is 5mL/min, and the aeration time is 30min, so as to obtain the polar ionic liquid;
s4, first purification of the ginseng polypeptide: adding 10g of the Ginseng radix extract obtained in step S2 into 20g of the polar ionic liquid obtained in step S3, extracting with 500W microwave under stirring for 1h, separating the organic layer, heating to 50 deg.C, oscillating, collecting the water layer, and freeze drying to obtain Ginseng radix polypeptide water extract;
s5, purifying the chromatographic column for the second time: purifying the ginseng polypeptide water extract prepared in the step S4 by adopting a reversed-phase ODS C18 chromatographic column to obtain a ginseng polypeptide extract;
the method comprises the following specific steps: dissolving the ginseng polypeptide water extract by using 0.1 wt% trifluoroacetic acid and 2 wt% acetonitrile solution, filtering by using a 0.22 mu m filter membrane, after loading, balancing a chromatographic column by using 0.1 wt% trifluoroacetic acid and 2 wt% acetonitrile aqueous solution, then rinsing for 10min by using 0.1 wt% trifluoroacetic acid and 15 wt% acetonitrile aqueous solution, rinsing for 20min by using 0.1 wt% trifluoroacetic acid and 10 wt% acetonitrile aqueous solution, and rinsing for 30min by using 0.1 wt% trifluoroacetic acid acetonitrile aqueous solution, wherein the elution speed is 0.5mL/min, the specification of the column is 15mm in diameter, and the length is 50 cm.
Example 2
The embodiment provides a preparation method of a ginseng polypeptide extract, which comprises the following steps:
s1, pretreatment of ginseng: cleaning ginseng, slicing, drying, crushing, and sieving with a 200-mesh sieve of 150 meshes to obtain ginseng powder;
s2, solvent extraction: soaking 10g of the powder obtained in step S1 in 100mL of an aqueous solution containing isopropanol, acetone, and acetic acid (isopropanol content of 7 wt%; acetone content of 5 wt%; acetic acid content of 25 wt%) for 20min, heating to 90 deg.C, and stirringExtracting for 4 hr, vacuum filtering, collecting extractive solution, concentrating at 50 deg.C and 0.1MPa until the density of the extractive solution is 3g/cm3To obtain ginseng extract;
s3, preparing polar ionic liquid: introducing CO into 200mL of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate2Gas, CO2The aeration speed of the gas is 2mL/min, and the aeration time is 60min, so as to obtain the polar ionic liquid;
s4, first purification of the ginseng polypeptide: adding 10g of the Ginseng radix extract obtained in step S2 into 30g of the polar ionic liquid obtained in step S3, extracting under stirring with 1000W of microwave for 2h, separating the organic layer, heating to 60 deg.C, oscillating, collecting the water layer, and freeze drying to obtain Ginseng radix polypeptide water extract;
s5, purifying the chromatographic column for the second time: purifying the ginseng polypeptide water extract prepared in the step S4 by adopting a reversed-phase ODS C18 chromatographic column to obtain a ginseng polypeptide extract;
the method specifically comprises the following steps: dissolving the ginseng polypeptide water extract by using an acetonitrile solution containing 0.5 wt% of trifluoroacetic acid and 4 wt%, filtering by using a 0.22 mu m filter membrane, loading, then using an aqueous solution containing 0.5 wt% of trifluoroacetic acid and 4 wt% of acetonitrile to balance a chromatographic column, then using an acetonitrile aqueous solution containing 0.5 wt% of trifluoroacetic acid and 15 wt% of acetonitrile to elute for 30min, using an acetonitrile aqueous solution containing 0.5 wt% of trifluoroacetic acid and 10 wt% of acetonitrile to elute for 30min, and then using an acetonitrile solution containing 0.5 wt% of trifluoroacetic acid to elute for 50min, wherein the elution speed is 1.5mL/min, the column specification is 20mm in diameter, and the length is 70 cm.
Example 3
The embodiment provides a preparation method of a ginseng polypeptide extract, which comprises the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving with 200 mesh sieve to obtain Ginseng radix powder;
s2, solvent extraction: soaking 10g of the powder of Ginseng radix obtained in step S1 in 100mL of aqueous solution containing isopropanol, acetone, and acetic acid (isopropanol content is 6 wt%, acetone content is 3.5 wt%, and acetic acid content is 22 wt%) for 15min, heating to 80 deg.C, stirring and extracting for 3 hr, vacuum filtering, collecting extractive solution, concentrating at 45 deg.C under 0.05MPa to obtain extractive solution with density of 2.5g/cm3To obtain ginseng extract;
s3, preparing polar ionic liquid: introducing CO into 200mL of ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate2Gas, CO2The aeration speed of the gas is 3.5mL/min, and the aeration time is 45min, so as to obtain the polar ionic liquid;
s4, first purification of the ginseng polypeptide: adding 10g of the Ginseng radix extract obtained in step S2 into 25g of the polar ionic liquid obtained in step S3, extracting with 750W microwave under stirring for 1.5h, separating organic layer, heating to 55 deg.C, oscillating, collecting water layer, and freeze drying to obtain Ginseng radix polypeptide water extract;
s5, purifying the chromatographic column for the second time: purifying the ginseng polypeptide water extract prepared in the step S4 by adopting a reversed-phase ODS C18 chromatographic column to obtain a ginseng polypeptide extract;
the method specifically comprises the following steps: dissolving the ginseng polypeptide water extract by using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile, filtering by using a 0.22 mu m filter membrane, after loading, using an equilibrium chromatographic column containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile in water, then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 15 wt% of acetonitrile in water to elute for 20min, using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 10 wt% of acetonitrile in water to elute for 25min, and then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid to elute for 40min, wherein the elution speed is 1mL/min, the column specification is 17mm in diameter, and the length is 60 cm.
Comparative example 1
Compared with example 3, no isopropanol was added in step S2, and other conditions were not changed.
The method comprises the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving with 200 mesh sieve to obtain Ginseng radix powder;
s2, solvent extraction: soaking 10g of the Ginseng radix powder obtained in step S1 in 100mL of aqueous solution containing acetone and acetic acid (acetone content is 3.5 wt%, acetic acid content is 28 wt%) for 15min, heating to 80 deg.C, stirring and extracting for 3h, vacuum filtering, collecting extractive solution, concentrating at 45 deg.C under 0.05MPa to obtain extractive solution with density of 2.5g/cm3To obtain ginseng extract;
s3, preparing polar ionic liquid: introducing CO into 200mL of ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate2Gas, CO2The aeration speed of the gas is 3.5mL/min, and the aeration time is 45min, so as to obtain the polar ionic liquid;
s4, first purification of the ginseng polypeptide: adding 10g of the Ginseng radix extract obtained in step S2 into 25g of the polar ionic liquid obtained in step S3, extracting with 750W microwave under stirring for 1.5h, separating organic layer, heating to 55 deg.C, oscillating, collecting water layer, and freeze drying to obtain Ginseng radix polypeptide water extract;
s5, second purification of the chromatographic column: purifying the ginseng polypeptide water extract prepared in the step S4 by adopting a reversed-phase ODS C18 chromatographic column to obtain a ginseng polypeptide extract;
the method comprises the following specific steps: dissolving the ginseng polypeptide water extract by using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile, filtering by using a 0.22 mu m filter membrane, after loading, using an equilibrium chromatographic column containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile in water, then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 15 wt% of acetonitrile in water to elute for 20min, using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 10 wt% of acetonitrile in water to elute for 25min, and then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid to elute for 40min, wherein the elution speed is 1mL/min, the column specification is 17mm in diameter, and the length is 60 cm.
Comparative example 2
In comparison with example 3, acetic acid was not added in step S2, and other conditions were not changed.
The method comprises the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving with 200 mesh sieve to obtain Ginseng radix powder;
s2, solvent extraction: soaking 10g of the powder of Ginseng radix obtained in step S1 in 100mL of aqueous solution containing isopropanol and acetone (isopropanol content 28 wt%; acetone content 3.5 wt%) for 15min, heating to 80 deg.C, stirring and extracting for 3h, vacuum filtering, collecting extractive solution, concentrating at 45 deg.C under 0.05MPa to obtain extractive solution with density of 2.5g/cm3To obtain ginseng extract;
s3, preparing polar ionic liquid: to 200mL of ionic liquid 1-hexyl-3-methylimidazolium tetraIntroducing CO into fluoborate2Gas, CO2The aeration speed of the gas is 3.5mL/min, and the aeration time is 45min, so as to obtain the polar ionic liquid;
s4, first purification of the ginseng polypeptide: adding 10g of the Ginseng radix extract obtained in step S2 into 25g of the polar ionic liquid obtained in step S3, extracting with 750W microwave under stirring for 1.5h, separating organic layer, heating to 55 deg.C, oscillating, collecting water layer, and freeze drying to obtain Ginseng radix polypeptide water extract;
s5, purifying the chromatographic column for the second time: purifying the ginseng polypeptide water extract prepared in the step S4 by adopting a reversed-phase ODS C18 chromatographic column to obtain a ginseng polypeptide extract;
the method specifically comprises the following steps: dissolving the ginseng polypeptide water extract by using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile, filtering by using a 0.22 mu m filter membrane, after loading, using an equilibrium chromatographic column containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile in water, then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 15 wt% of acetonitrile in water to elute for 20min, using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 10 wt% of acetonitrile in water to elute for 25min, and then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid to elute for 40min, wherein the elution speed is 1mL/min, the column specification is 17mm in diameter, and the length is 60 cm.
Comparative example 3
Step S3 was not performed, and other conditions were not changed, as compared with example 3.
The method comprises the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving with 200 mesh sieve to obtain Ginseng radix powder;
s2, solvent extraction: soaking 10g of the powder of Ginseng radix obtained in step S1 in 100mL of aqueous solution containing isopropanol, acetone, and acetic acid (isopropanol content is 6 wt%, acetone content is 3.5 wt%, and acetic acid content is 22 wt%) for 15min, heating to 80 deg.C, stirring and extracting for 3 hr, vacuum filtering, collecting extractive solution, concentrating at 45 deg.C under 0.05MPa to obtain extractive solution with density of 2.5g/cm3To obtain ginseng extract;
s3, first purification of the ginseng polypeptide: adding 10g of the ginseng extract prepared in the step S2 into 25g of ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate, performing microwave stirring extraction for 1.5h at 750W, separating an organic layer, heating to 55 ℃, oscillating, collecting a water layer, and performing freeze drying to obtain a ginseng polypeptide water extract;
s4, purifying the chromatographic column for the second time: purifying the ginseng polypeptide water extract prepared in the step S3 by adopting a reversed-phase ODS C18 chromatographic column to obtain a ginseng polypeptide extract;
the method specifically comprises the following steps: dissolving the ginseng polypeptide water extract by using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile, filtering by using a 0.22 mu m filter membrane, after loading, using an equilibrium chromatographic column containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile in water, then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 15 wt% of acetonitrile in water to elute for 20min, using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 10 wt% of acetonitrile in water to elute for 25min, and then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid to elute for 40min, wherein the elution speed is 1mL/min, the column specification is 17mm in diameter, and the length is 60 cm.
Comparative example 4
Steps S3 and S4 were not performed, and other conditions were not changed, as compared with example 3.
The method comprises the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving with 200 mesh sieve to obtain Ginseng radix powder;
s2, solvent extraction: adding 10g of the ginseng powder prepared in the step S1 into 100mL of an aqueous solution containing isopropanol, acetone and acetic acid (the content of the isopropanol is 6 wt%, the content of the acetone is 3.5 wt% and the content of the acetic acid is 22 wt%), soaking for 15min, heating to 80 ℃, stirring and extracting for 3h, performing suction filtration, collecting an extracting solution, and performing freeze drying to obtain a ginseng extract;
s3, performing secondary purification on the chromatographic column: purifying the ginseng extract obtained in step S2 by using a reversed-phase ODS C18 chromatographic column to obtain a ginseng polypeptide extract;
the method specifically comprises the following steps: dissolving the ginseng polypeptide water extract by using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile, filtering by using a 0.22 mu m filter membrane, after loading, using an equilibrium chromatographic column containing 0.3 wt% of trifluoroacetic acid and 3 wt% of acetonitrile in water, then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 15 wt% of acetonitrile in water to elute for 20min, using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid and 10 wt% of acetonitrile in water to elute for 25min, and then using an acetonitrile solution containing 0.3 wt% of trifluoroacetic acid to elute for 40min, wherein the elution speed is 1mL/min, the column specification is 17mm in diameter, and the length is 60 cm.
Comparative example 5
Step S5 was not performed, and other conditions were not changed, as compared with example 3.
The method comprises the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving with 200 mesh sieve to obtain Ginseng radix powder;
s2, solvent extraction: soaking 10g of the Ginseng radix powder obtained in step S1 in 100mL of aqueous solution containing isopropanol, acetone and acetic acid (isopropanol content 6 wt%, acetone content 3.5 wt% and acetic acid content 22 wt%) for 15min, heating to 80 deg.C, stirring and extracting for 3h, suction filtering, collecting extractive solution, concentrating at 45 deg.C and 0.05MPa to obtain extractive solution with density of 2.5g/cm3To obtain ginseng extract;
s3, preparing polar ionic liquid: introducing CO into 200mL of ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate2Gas, CO2The aeration speed of the gas is 3.5mL/min, and the aeration time is 45min, so as to obtain the polar ionic liquid;
s4, first purification of the ginseng polypeptide: adding 10g of the Ginseng radix extract obtained in step S2 into 25g of the polar ionic liquid obtained in step S3, extracting with 750W microwave under stirring for 1.5h, separating organic layer, heating to 55 deg.C, shaking, collecting water layer, and freeze drying to obtain Ginseng radix polypeptide water extract.
Test example 1
1. Effect of Ginseng polypeptide Water extracts prepared in examples 1-3 and comparative examples 1-5 on proliferation of NIH-3T3 cells
Culturing NIH-3T3 cells in DMEM complete medium containing 10% FBS and 1% double antibody, digesting cells with pancreatin (containing EDTA), counting, spreading cells in 96-well plate, and culturing at 1 × 104Each group of 4 multiple wells. 135 μ L of cell suspension was added to each well, 4 replicates per group. After 24h, 15 μ L of the ginseng polypeptide water extract medicines prepared in examples 1-3 and comparative examples 1-5 are added into each hole at a concentration of 0.1mg/mL respectively, and deionized water is used as a pairAfter incubation for 24h, 20. mu.L (5mg/ml) of MTT solution was added to each well, incubation was continued for 4h, then the triplicate lysate was added, 100. mu.L per well, and OD was detected by microplate reader overnight at 37 ℃570. The results are shown in FIG. 1. In the figure, P represents the ratio of P to the control group<0.05。
2. Effect of the Water extract of Ginseng radix Polypeptides prepared in examples 1-3 and comparative examples 1-5 on UVB-induced NIH-3T3 cell injury
Culturing NIH-3T3 cells in DMEM complete medium containing 10% FBS and 1% double antibody, digesting cells with pancreatin (containing EDTA), counting, spreading cells in 96-well plate, and culturing at 1 × 104Each group of 4 multiple wells. 135 μ L of cell suspension was added to each well, 4 replicates per group. After 24h, carrying out ultraviolet radiation treatment, wherein the experimental UVB wavelength is 311nm, and the radiation intensity is 13mW/cm2The distance between the cells and the irradiation light source is 11cm, and the irradiation dose is set to be 4000mJ/cm2Immediately after irradiation, 15 μ L of the ginseng polypeptide water extract drug prepared in examples 1-3 and comparative examples 1-5 was added at 0.1mg/mL, 15 μ L of deionized water was used as a blank and no UVB irradiation was performed, 15 μ L of deionized water was used as a UVB model and UVB irradiation was performed, incubation was continued for 24h, 20 μ L (5mg/mL) of MTT solution was added to each well, incubation was continued for 4h, then 100 μ L of triple lysate was added to each well, and after overnight at 37 ℃, OD was detected by a microplate reader570. The results are shown in FIG. 2. In the figure, Δ represents P compared with the blank group<0.05; denotes P compared to the UVB model group<0.05。
As can be seen from FIG. 1, the ginseng polypeptide aqueous extracts prepared in examples 1 to 3 had little effect on NIH-3T3 cell proliferation. The extract of ginseng polypeptide obtained in step S2 of comparative examples 1 and 2 is still mainly polypeptides without adding isopropanol or acetic acid, and has little effect on cell proliferation, while the extract prepared in comparative examples 3-5 contains more other substances, which have a certain effect on cell proliferation but have little effect.
As can be seen from FIG. 2, the ginseng polypeptide water extracts prepared in examples 1-3 have obvious protective effect on UVB-induced NIH-3T3 cell damage, and the ginseng polypeptide water extracts prepared by the method have good anti-ultraviolet radiation effect.
The protective effect of the comparative examples 1 and 2 is reduced, isopropanol or acetic acid is not added in the step S2, the content of the prolamin or the acid soluble protein in the obtained ginseng polypeptide water extract is reduced, the radiation resistant effect is reduced, and the existence of the prolamin and the acid soluble protein in the ginseng polypeptide water extract has a synergistic effect.
Comparative example 3, which did not perform step S3, showed a significant decrease in protection against UVB-induced NIH-3T3 cell damage, because no CO was passed through the ionic liquid2And then, the ionic liquid has poor polarity, and cannot extract the polypeptide substances efficiently, so that the content of active peptides in the prepared ginseng polypeptide water extract is obviously reduced, and the ultraviolet radiation resistant effect is obviously reduced. According to the invention, carbon dioxide is introduced into the water-insoluble ionic liquid, so that the polarity of the ionic liquid is improved, other active substances with poor polarity in the extraction liquid are left in the organic layer, the ginseng polypeptide compound with high polarity enters the ionic liquid layer, so that polypeptide and other impurities are separated, the extracted high-polarity ionic liquid is heated and returned to the common ionic liquid, and after water is added and oscillation is carried out, the polar ginseng polypeptide compound dissolved in the ionic liquid is extracted to the water layer, so that not only is the first purification of the ginseng polypeptide compound realized, but also the ionic liquid can be reused.
Comparative example 4 without performing steps S3 and S4, the protective effect of UVB induced NIH-3T3 cell damage was significantly reduced, and pure reversed-phase ODS C18 column separation could not efficiently obtain a ginseng polypeptide extract with high polypeptide activity.
Comparative example 5 does not carry out step S5, the protective effect is reduced, and after the ginseng polypeptide complex with high polarity and good hydrophobicity is further separated by the reversed phase C18 chromatographic column, the ginseng polypeptide complex with high purity is eluted, so that the ginseng polypeptide complex with high purity is obtained, has a good ultraviolet radiation resistance effect, has the effects of fading color spots and inhibiting melanin generation, can have the effects of whitening and resisting radiation, and the prepared ultraviolet radiation resistant whitening essence has good radiation resistance and whitening effects.
Example 4
The embodiment provides an ultraviolet-resistant whitening essence which is prepared from the following raw materials in percentage by mass: 3.5% of the ginseng polypeptide extract prepared in example 1, 0.7% of PEG-100 stearate, 3.5% of butanediol, 5% of glycerol, 1% of carbomer, 2.5% of squalane, 0.35% of xanthan gum, 0.7% of nicotinamide, 0.35% of essence, 1.5% of pearl powder, 0.2% of phenoxyethanol and the balance of deionized water.
Example 5
Compared with example 4, the ginseng polypeptide extract prepared in example 1 was replaced by the ginseng polypeptide extract prepared in example 2, and other conditions were not changed.
Example 6
Compared with example 4, the ginseng polypeptide extract prepared in example 1 is replaced by the ginseng polypeptide extract prepared in example 3, and other conditions are not changed.
Comparative example 6
Compared with example 4, the ginseng polypeptide extract prepared in example 1 is replaced by the ginseng polypeptide extract prepared in comparative example 1, and other conditions are not changed.
Comparative example 7
Compared with example 4, the ginseng polypeptide extract prepared in example 1 is replaced by the ginseng polypeptide extract prepared in comparative example 2, and other conditions are not changed.
Comparative example 8
Compared with example 4, the ginseng polypeptide extract prepared in example 1 is replaced by the ginseng polypeptide extract prepared in comparative example 3, and other conditions are not changed.
Comparative example 9
Compared with example 4, the ginseng polypeptide extract prepared in example 1 is replaced by the ginseng polypeptide extract prepared in comparative example 4, and other conditions are not changed.
Comparative example 10
Compared with example 4, the ginseng polypeptide extract prepared in example 1 is replaced by the ginseng polypeptide extract prepared in comparative example 5, and other conditions are not changed.
Test example 2 whitening efficacy test
1. And (3) testing a sample: ultraviolet-resistant whitening essence liquids prepared in examples 4 to 6 and comparative examples 6 to 10;
2. blank control group: during the test, the test site was replaced with an equal amount of deionized water without any cosmetic.
3. Experimental population
The subjects were 30 persons in total, male 0 persons, female 30 persons, and the age ranged from 18 to 36 years. The exclusion conditions of the volunteers meet the inclusion and exclusion standards of the diagnosis standard and the treatment principle of the cosmetic contact dermatitis.
4. Test method
The left and right arms of the subject were selected as the test areas. The MPA980 multifunctional skin tester is used for measuring the skin melanin and the skin brightness of the experimental part before the cosmetic is applied to the experimental part. The test product is applied to a test area (the same area) cleaned by clean water at 8 am and 10 pm every day, and the test subject cannot smear any other cosmetics except the tested sample on the test part during the test. Before and after the application of the cosmetics, the subject cleaned the applied part, stood still for 15min, measured the value of the applied part with a test instrument, and analyzed the change law of skin melanin and skin brightness L value, the results are shown in table 1 and table 2.
5. Change in melanin content
The change of the skin melanin content is reflected in the test period, and the change rule of the skin melanin content in the experimental area along with time. The larger the value, the higher the skin melanin content, and vice versa.
Difference in melanin content (Δ T)% (melanin content (T) after use of the product)n) Melanin content (T) before use of the product0)]Melanin content (T) before use0)×100％。
TABLE 1
6. Changes in skin brightness
The change of the skin brightness is reflected in the change rule of the skin brightness of the experimental area along with time in the testing period. The larger the value, the better the skin brightness, and conversely, the worse the skin brightness.
Luminance difference (Δ T)% (luminance value after use of product (T)%)n) Using the pre-product brightness value (T)0)]Brightness value before use of product (T)0)×100％
TABLE 2
| Group of | Luminance difference (%) |
| Example 4 | 4.2 |
| Example 5 | 4.5 |
| Example 6 | 5.0 |
| Comparative example 6 | 3.2 |
| Comparative example 7 | 3.0 |
| Comparative example 8 | 2.6 |
| Comparative example 9 | 2.0 |
| Comparative example 10 | 3.1 |
| Blank group | -1.2 |
As can be seen from the above table, the ultraviolet resistant whitening essence prepared in examples 4 to 6 has a strong inhibitory effect on the melanin content of the skin, and simultaneously can improve the skin brightness, and compared with the ultraviolet resistant whitening essence prepared in example 6, the effect is more prominent.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (10)
1. A preparation method of a ginseng polypeptide extract is characterized by comprising the following steps:
s1, pretreatment of ginseng: cleaning Ginseng radix, slicing, drying, pulverizing, and sieving to obtain Ginseng radix powder;
s2, solvent extraction: adding the ginseng powder prepared in the step S1 into an aqueous solution containing isopropanol, acetone and acetic acid for soaking, heating, stirring, extracting, filtering, collecting an extracting solution, and concentrating at low temperature under reduced pressure to obtain a ginseng extracting solution;
s3, preparing polar ionic liquid: introducing CO into the ionic liquid2Gas to obtain polar ionic liquid;
s4, first purification of the ginseng polypeptide: adding the ginseng extract obtained in the step S2 into the polar ionic liquid obtained in the step S3, performing microwave stirring extraction, separating an organic layer, heating and oscillating, collecting a water layer, and performing freeze drying to obtain a ginseng polypeptide water extract;
s5, purifying the chromatographic column for the second time: purifying the ginseng polypeptide water extract obtained in the step S4 by adopting a reversed-phase ODS C18 chromatographic column to obtain the ginseng polypeptide extract.
2. The method as claimed in claim 1, wherein the sieving of step S1 is performed in a 200-mesh sieve.
3. The method according to claim 1, wherein the isopropanol content in the aqueous solution containing isopropanol, acetone and acetic acid in step S2 is 5-7 wt%; the content of acetone is 2-5 wt%; the acetic acid content is 10-25 wt%; the soaking time is 10-20 min; heating to 70-90 deg.C, and extracting for 2-4 h; concentrating at low temperature and reduced pressure at 40-50 deg.C and 0.01-0.1MPa to obtain extractive solution with density of 2-3g/cm3。
4. The method according to claim 1, wherein the ionic liquid in step S3 is at least one selected from the group consisting of 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hexafluoroantimonate, 1-hexyl-3-methylimidazole tetrafluoroborate, 1-hexyl-3-methylimidazole hexafluorophosphate, 1-hexyl-3-methylimidazole hexafluoroantimonate; the CO is2The aeration speed of the gas is 2-5mL/min, and the aeration time is 30-60 min.
5. The method according to claim 1, wherein the volume ratio of the ginseng extract to the polar ionic liquid in step S4 is 1: (2-3); the microwave power is 500-1000W, and the extraction time is 1-2 h; the heating is carried out to 50-60 ℃.
6. The method according to claim 1, wherein the step S5 is specifically performed as follows: dissolving the ginseng polypeptide water extract with 0.1-0.5 wt% trifluoroacetic acid and 2-4 wt% acetonitrile solution, filtering with 0.22 μm filter membrane, loading, using 0.1-0.5 wt% trifluoroacetic acid and 2-4 wt% acetonitrile aqueous solution to balance chromatography column, then using 0.1-0.5 wt% trifluoroacetic acid and 15 wt% acetonitrile aqueous solution to rinse for 10-30min, using 0.1-0.5 wt% trifluoroacetic acid and 10 wt% acetonitrile aqueous solution to rinse for 20-30min, then using 0.1-0.5 wt% trifluoroacetic acid containing acetonitrile aqueous solution to rinse for 30-50min, the elution speed is 0.5-1.5mL/min, the column specification is 15-20mm in diameter and 50-70cm in length.
7. A ginseng polypeptide extract prepared by the method of any one of claims 1-6.
8. Use of the ginseng polypeptide extract according to claim 7 in the cosmetic field.
9. An anti-ultraviolet whitening essence comprising 2-5 wt% of the ginseng polypeptide extract according to claim 7.
10. The ultraviolet-resistant whitening essence according to claim 9, which is prepared from the following raw materials in percentage by mass: the ginseng polypeptide extract of claim 7, 2-5%, PEG-100 stearate 0.5-1%, butylene glycol 2-5%, glycerin 3-6%, carbomer 0.5-2%, squalane 2-3%, xanthan gum 0.1-0.5%, nicotinamide 0.5-1%, essence 0.1-0.5%, pearl powder 1-2%, phenoxyethanol 0.1-0.4%, and the balance of deionized water.
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