CN114621313A - Euglena protein extract and application thereof in cosmetics - Google Patents
Euglena protein extract and application thereof in cosmetics Download PDFInfo
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- CN114621313A CN114621313A CN202011450703.6A CN202011450703A CN114621313A CN 114621313 A CN114621313 A CN 114621313A CN 202011450703 A CN202011450703 A CN 202011450703A CN 114621313 A CN114621313 A CN 114621313A
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- euglena
- gymnocyanin
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- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 claims description 4
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- 230000000694 effects Effects 0.000 abstract description 19
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
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- 239000011718 vitamin C Substances 0.000 description 1
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- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
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- 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
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/303—Extraction; Separation; Purification by precipitation by salting out
-
- 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
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
-
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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- Engineering & Computer Science (AREA)
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- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
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Abstract
The application relates to the field of cosmetics, in particular to a gymnema algae protein extract and application thereof in cosmetics. The euglena gracilis protein extracting solution obtained by the method has a remarkable repairing function aiming at cells, and has the effects of delaying senescence, improving cell activity and being low in toxic and side effects.
Description
Technical Field
The invention relates to the field of cosmetics, in particular to a gymnema algae protein extract and application thereof in cosmetics.
Background
In recent years, marine algae such as kelp, nemacystus and undaria and microalgae such as gold algae have been increasingly popular in the japanese market. In the fine algae market, the market growth of euglena is remarkable, and the market scale is about 100 hundred million yen;
euglena, a wind delivery company specially operating fine algae products in Japan, develops sales routes through the grouping of sales companies or an M & A mode, and develops a cosmetic market by developing cooperation with more than 50 mechanisms such as Japan universities, pharmaceutical enterprises, Wutian drugs and the like to succeed; the turnover of the brand 'Euglena Farm green juice' under the flag occupies the proportion of the total commercial value of the direct sale of the food of the company, and the proportion is increased by 3 times. In addition to fucoidan and microalgae mentioned above, DHA-rich algae and Nannochloropsis (Nannochloropsis) called "Chlorella marinum" are also receiving great market attention.
In recent years, a product named as "euglena" appears in the microalgae market and is developed and applied as a health food. The algae is Euglena gracilis (Euglena gracilis), and the kinds of bioactive substances in Euglena gracilis cells are rich, such as beta-carotene, vitamin C, vitamin E, beta-1, 3-glucan, PUFA and the like, and can be used as the source of single cell protein. For example: beta-1, 3-glucan has been known to act as an immune response modifier and has been known to enhance immunity in humans. The research also finds that the substance has the effects of resisting cancers, resisting bacterial infection, activating macrophages, inducing cytokine secretion, promoting hematopoiesis, resisting radiation, treating burn, promoting wound healing, reducing blood fat, resisting oxidation and the like, and the derivatives (such as sulfation) of the substance even have the effect of resisting HIV virus;
under Euglenophyta (Euglenophyta), only one Euglenophyta, Euglenophyceae in the prefecture and Euglenophyta, four families (Euglenophyta, Vaginaceae, Bombaginaceae and Euglenophyta), about 40 or more than 1000 species are included, most species are freshwater species, and a large amount of the Euglenophyta generally exist in calm inland water. Euglena species under Euglena, such as Euglena gracills, have a long history as a biological study object, and are one of the best studied species at present, listed as model species in experimental biology. For example: the euglena GRAS dry powder containing paramylon passes through the GRAS certification of FDA in the united states, and also passes through the new resource food certification of euglena in 2013 in 5 months.
However, research on euglena gracilis in cosmetics is not very much, and the product categories are not very rich. Based on the vigorous demand of consumers for nontoxic and harmless pure natural skin care products and cosmetics, the market urgently needs to use euglena extract such as: cosmetics containing euglena protein as the main ingredient are available.
Disclosure of Invention
The invention aims to solve the problems and provides an active protein extract of euglena gracilis and application thereof in preparing cosmetics.
The specific technical scheme for solving the technical problems is as follows:
according to a first aspect, the present invention provides a method for extracting euglena protein, comprising the steps of:
1. a preparation method of euglena protein comprises the following steps:
1) cell disruption: weighing 0.5g of euglena dry powder, adding PBS, mixing, and ultrasonically crushing for 15 min;
2) cell extract: centrifuging at 12000rpm at 4 deg.C for 20min, and transferring the supernatant into a centrifuge tube;
3) protein salting out: slowly adding ammonium sulfate solid into the cell extract, continuously stirring for dissolving until the concentration of ammonium sulfate reaches 60%, continuously stirring for 1 hour at room temperature, and then placing into a 4-degree refrigerator for standing overnight;
4) protein purification: and centrifuging the solution with the separated protein at 4000rpm for 20min, discarding the supernatant, and keeping the precipitate for later use. Dissolving the protein precipitate with a proper amount of PBS solution, adding into a 100D dialysis bag, and dialyzing in deionized water until no ammonium sulfate exists in the solution;
5) sterilization: filtering the dialyzed and purified protein by using a 0.22 mu m filter head, and collecting filtrate to obtain sterile gymnocyanin extract.
According to another aspect, the present invention relates to the use of the aforementioned gymnocyanine protein for the preparation of a cell damage repairing agent.
According to another aspect, the present invention relates to the use of the euglena protein described above for the preparation of a skin care or cosmetic product.
According to another aspect, the invention relates to the use of the gymnocyanine protein described above for the preparation of a repairing agent for oxidative damage of cells.
In some embodiments, the cells include, but are not limited to, immortalized keratinocytes, fibroblasts, macrophages;
according to another aspect, the present invention relates to a euglena cell culture solution comprising: 0.02g/LKH2PO40.6g/L peptone, 0.025g/L MgSO47H2O, 0.4g/L Yeast extract paste, 0.4g/L sodium acetate, 0.5. mu.g/L vitamin B12, 0.04g/L Potassium citrate, 0.4mg/L vitamin B1。
Drawings
FIG. 1: HaCaT cell-CCK 8 cell activity detection effect graph
FIG. 2: HDF cell-CCK 8 cell activity detection effect graph
FIG. 3: HDF cell-ROS detection effect graph
FIG. 4: HDF-antioxidation experiment effect chart
Examples
The euglena gracilis protein extract is also called 848 purified protein
Example 1: culture of euglena gracilis
Algae breeding: euglena gracilis (purchased from fresh water algae seed bank of Chinese academy of sciences, number FACHB-848)
Basic culture conditions: illumination 3000lux (lux), temperature 25 ± 2 ℃, medium HUT (final medium concentration formula, pH 6.4, below):
| numbering | Components | Dosage of | Concentration of mother liquor |
| 1 | KH2PO4 | 1mL | 20g/L |
| 2 | Peptone | 0.6g/L | - |
| 3 | MgSO4·7H2O | 1mL | 25g/L |
| 4 | Yeast extract | 0.4g/L | - |
| 5 | Sodium acetate (NaCI) | 0.4g/L | - |
| 6 | Vitamin B12 | 1mL | 0.5mg/L |
| 7 | Potassium citrate | 1mL | 40g/L |
| 8 | Vitamin B1 | 1mL | 0.4g/L |
The culture mode is as follows: and (5) mixotrophic culture. Taking a proper amount of pure euglena gracilis culture solution, inoculating the euglena gracilis culture solution into 500mL of sterilized culture solution under the aseptic condition, standing and culturing, and shaking the solution three times every day to uniformly disperse the euglena gracilis culture solution. Wherein the sterilized culture medium comprises 0.02g/L KH2PO40.6g/L peptone, 0.025g/L MgSO4·7H2O, 0.4g/L yeast extract, 0.4g/L sodium acetate, 0.5 mu g/L vitamin B12, 0.04g/L potassium citrate, 0.4mg/L vitamin B1.
The preparation process comprises the following steps: weighing solid powder with different components according to the formula of the HUT culture solution, dissolving the solid powder in deionized water, respectively preparing mother liquor with 1000 times of concentration, and standing at normal temperature for later use. Adding a proper amount of deionized water into a volumetric flask with the volume of 1L, simultaneously adding 1mL of mother liquor with each component, fixing the volume to 1L, shaking up, introducing the self-made culture solution into a reagent bottle, and sterilizing at 121 ℃ for 20min to obtain the sterilized culture solution.
Example 2: process for extracting euglena protein
1. Cell disruption: weighing 0.5g of euglena dry powder, adding 30mL of PBS, uniformly mixing, and carrying out ultrasonic crushing for 15min (2s on, 4s off, power 60%);
2. cell extract: centrifuging at 12000rpm at 4 deg.C for 20min, and transferring the supernatant into a centrifuge tube;
3. protein salting out: slowly adding ammonium sulfate solid into the cell extract, continuously stirring for dissolving until the concentration of ammonium sulfate reaches 60%, continuously stirring for 1 hour at room temperature, and then placing into a 4-degree refrigerator for standing overnight;
4. protein purification: and centrifuging the solution with the separated protein at 4000rpm for 20min, discarding the supernatant, and keeping the precipitate for later use. Dissolving the protein precipitate with appropriate amount of PBS solution, adding into 100D dialysis bag, and dialyzing in deionized water until no ammonium sulfate is contained in the solution.
5. And (3) degerming: filtering the dialyzed and purified protein by using a 0.22 mu m filter head, and collecting filtrate to obtain sterile gymnocyanin extract.
Example 3: cell damage repair experiment
1. Experimental Material
HaCaT immortalized keratinocytes (obtained from a Zhonghui organism, from the cell bank of the Chinese academy of sciences)
2. Apparatus, reagents and consumables related thereto
2.1 apparatus
A clean room, a biological safety cabinet, a desk centrifuge, a countess cell counter, a CO2 incubator, a pipette gun, a-80 ℃ refrigerator, a 4 ℃ refrigerator, an inverted microscope and an electric heating constant-temperature water tank.
2.2 reagents
DMEM high-glucose medium, FBS (serum), PBS, double antibody (penicillin 10000U/ml, streptomycin 10,000 mu g/ml), 0.25% Trypsin-EDTA pancreatin, DMSO, and CCK8 kit.
2.3 consumable
T-75 culture flask, 10ml pipet, 15ml centrifuge tube, 50ml centrifuge tube, 96 well plate.
2.4 Positive reference and inducer
Positive reference: 0.1% plant soothing agent Comthing SGS (Jia leaves, production lot PS00231IL09229) inducer: (100-;
3 experimental operating procedure
HaCaT cell-barrier repair model construction
1) Laying a 96-well plate: taking out T-75 culture bottle, discarding used culture medium, adding 3mL pancreatin, standing in 37 deg.C incubator for 2min, adding 3mL culture medium to stop digestion, gently blowing down HaCaT cell at bottom of culture dish, collecting culture medium to centrifuge tube, placing centrifuge tube in centrifuge and balancing, centrifuging at 1000rpm for 3min, discarding supernatant, addingAdding 5ml DMEM culture medium, re-suspending the cells, taking out 180ul, adding 20ul trypan blue, counting the number of the cells by using a countess cell counter, finally adding the culture medium containing the cells into a 96-well plate, wherein each well has 100ul cells, and the final density of the cells is 4-5 multiplied by 104Per well. And after the plate paving is finished, putting the plate into a cell culture box for culturing for 24 h.
2) And (3) induction construction of a model: when the cells were cultured to about 80% confluency, the medium was discarded. The samples are added respectively, and the samples are added in groups as follows: experimental groups: the test object is euglena protein extract, SLS and culture medium, and the negative control group comprises: SLS + medium, positive control group: plant soothing agent SGS + SLS + medium, blank control: and (4) a culture medium. After the administration, the 96-well plate was placed in an incubator (37 ℃, 5% CO2, 95% RH) and incubated for 24 h.
3)CCK8 cell viability assay: after 24 hours of incubation, removing the culture medium, adding 100ul of culture medium and 10ul of CCK8 detection solution into each hole, putting into an incubator, and incubating for 1-4 hours; after being taken out, the cells are detected by using an enzyme-labeling instrument with the wavelength of 450 nm.
The experimental results (as in fig. 1) show that: compared with a blank group, the HaCaT cell activity of the negative control group is obviously reduced under the action of an inducer SLS; in the experimental group, the gymnema algae protein extract can obviously improve the cell damage caused by SLS induction. In conclusion, the gymnocyanin extract has a good repairing function on HaCaT cell barrier damage caused by SLS induction.
Example 4: HDF-Oxidation resistance model experiment
1. Experimental Material
HDF human dermal fibroblasts. (purchased from North Nay, primary extraction)
2. Apparatus, reagents and consumables related thereto
2.1 apparatus
Clean room, biosafety cabinet, bench centrifuge, countess cell counter, CO2An incubator, a pipette gun, a-80 ℃ refrigerator, a 4 ℃ refrigerator, a program cooling box, a liquid nitrogen tank, an inverted microscope, an electric heating constant temperature water tank and a fluorescence quantitative microplate reader.
2.2 reagents
DMEM low-sugar medium, FBS (serum), PBS, double antibody (penicillin 10000U/ml, streptomycin 10,000 mu g/m1), 0.25% Trypsin-EDTA pancreatin, DMSO, CCK8 kit and active oxygen detection kit.
2.3 consumable
T-75 culture flask, 10ml pipet, 15ml centrifuge tube, 50ml centrifuge tube, 96 well plate.
2.4 Positive reference and inducer
Positive reference: 0.5% of VC magnesium phosphate (alatin, cat # S160999); an inducer: 154uM H2O2
3 experimental operating procedure
RAW264.7 (purchased from North Nay, ATCC) cell-anti-inflammatory model
3.1 CCK8 detection
1) Laying a 96-well plate: taking out the T75 culture bottle, discarding the used culture medium, adding 3mL pancreatin (Shanghai source culture, catalog number S312JV), standing for 1min in an incubator at 37 ℃, adding 3mL culture medium to stop digestion, gently blowing down the cells (RAW264.7 cells) at the bottom of the culture dish, collecting the culture medium to a centrifuge tube, placing the centrifuge tube in a centrifuge and balancing, centrifuging for 3min at 1000rpm, discarding the supernatant, adding 5mL DMEM culture medium, taking out 180ul after cell resuspension, adding 20ul trypan blue, counting the number of cells by using a countess cell counter, finally adding the culture medium containing cells to a 96-well plate, each well being 100ul, and the final density of the cells being (0.8-1) multiplied by 103Per well. And after the plate paving is finished, putting the plate into a cell culture box for culturing for 24 hours.
2) And (3) inducing and constructing a model: when the cells were cultured to about 80% confluency, the medium was discarded. The samples are added respectively, and the samples are added in groups as follows: experimental group-test substance Euglena protein extract + H2O2+ Medium, negative control group-H2O2+ culture medium, positive control group-Vc phosphate magnesium + H2O2+ medium, blank control-medium. After the administration, the 96-well plate was placed in an incubator (37 ℃, 5% CO2, 95% RH) and incubated for 24 h.
3)CCK8 cell viability assay: after 24 hours of incubation, the medium was discarded every timeAdding 100ul culture medium and 10ul CCK8 detection solution (Solebao, Cat. CA1210) into the well, placing in an incubator, and incubating for 1-4 h; after being taken out, the cell viability is detected by using an enzyme-labeling instrument with the wavelength of 450 nm.
4)ROS detection: after 24 hours of incubation, the medium was discarded. The ROS concentration in the cells was detected by a fluorescent quantitative microplate reader according to the kit instructions.
According to the results of further experiments (as shown in FIG. 2), the negative group was at H compared to the blank group2O2The cell activity is obviously reduced under the induction of (3); the positive control group VC magnesium phosphate has no obvious repairing effect; compared with the negative group, the cell activity of the experimental group is obviously improved under the action of the extracting solution.
According to further experimental results (as shown in fig. 3), compared with the blank group, the content of ROS in the negative group is obviously increased, and the success of constructing the HDF cell antioxidant model is proved; in the experimental group, the ROS content in HDF cells was significantly down-regulated due to the action of the gymnocyanin extract. Therefore, the euglena gracilis extracting solution obtained by the application can effectively reduce the intracellular ROS content. Euglena gracilis extract for H2O2The induced HDF cell oxidative damage has a certain repair function.
Example 5: RAW 264.7-anti-inflammatory model experiment
1. Experimental Material
RAW264.7 (purchased from North Nay, ATCC)
2. Apparatus, reagents and consumables related thereto
2.1 apparatus
A clean room, a biological safety cabinet, a desk centrifuge, a countess cell counter, a CO2 incubator, a pipette gun, a-80 ℃ refrigerator, a 4 ℃ refrigerator, a program cooling box, a liquid nitrogen tank, an inverted microscope, an electric heating constant temperature water tank and a fluorescence quantitative microplate reader.
2.2 reagents
DMEM high-glucose medium, FBS (serum), PBS, double antibody (penicillin 10000U/ml, streptomycin 10,000 mu g/ml), 0.25% Trypsin-EDTA pancreatin, DMSO, CCK8 kit and mouse tumor necrosis factor alpha (TNF-alpha) enzyme-linked immunosorbent assay kit.
2.3 consumable
T-75 culture flask, 10ml pipet, 15ml centrifuge tube, 50ml centrifuge tube, 96 well plate.
2.4 Positive reference and inducer
Positive reference: 100ug/ml dexamethasone (Beijing Zhongke quality testing Biotechnology Co., Ltd., cat # 8582); an inducer: 1ug/ml LPS (SIGMA, cat # L2880)
3 experimental operating procedure
3.1 CCK8 detection
1) Laying a 96-well plate: taking out a T75 culture bottle, discarding the used culture medium, adding 3mL of pancreatin (Shanghai source culture, product number S312JV), standing in an incubator at 37 ℃ for 1min, adding 3mL of culture medium to stop digestion, gently blowing down cells (RAW264.7 cells) at the bottom of the culture dish, collecting the culture medium into a centrifuge tube, placing the centrifuge tube into a centrifuge and balancing, centrifuging at 1000rpm for 3min, discarding the supernatant, adding 5mL of DMEM culture medium, taking out 180ul after resuspending the cells, adding 20ul of trypan blue, counting the number of the cells by using a countess cell counter, finally adding the culture medium containing the cells into a 96-well plate, 100ul per well, and finally adding the cells to a 96-well plate with a final density of (0.8-1) multiplied by 103Per well. And after the plate paving is finished, putting the plate into a cell culture box for culturing for 24 h.
2) And (3) induction construction of a model: when the cells were cultured to about 60% confluency, the medium was discarded. The samples were added separately, in groups as follows: experiment group-test object euglena gracilis extract + LPS + culture medium, negative control group-LPS + culture medium, positive control group-dexamethasone + LPS + culture medium, blank control group-culture medium. After the administration, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO)295% RH) for 24 h.
3) TNF-alpha detection: after 24 hours of incubation, the medium was removed from the 96-well plate. According to the kit instruction, the concentration of TNF-alpha in the cell supernatant is detected by a fluorescence quantitative enzyme-labeling instrument.
According to the results of the experiment (as shown in fig. 4), the concentration of TNF-alpha protein in the cell supernatant of the negative group was significantly increased under the induction of LPS compared to the blank group; compared with the negative group, the concentration of the TNF-alpha protein of the experimental group is obviously reduced under the action of the extracting solution.
Therefore, the euglena gracilis extracting solution obtained by the application can effectively reduce the expression of TNF-alpha protein in cells. The euglena gracilis extract has a certain inhibiting function on RAW264.7 cell inflammation caused by LPS induction.
In conclusion, the euglena gracilis extracting solution obtained by the invention has a remarkable repairing function aiming at cells, has the effects of delaying senescence and improving cell activity, and is more suitable for being used as a skin care product or a cosmetic component due to the fact that the extracting solution belongs to natural components and the toxic and side effects are greatly reduced compared with chemical reagents with the same or similar functions, and unexpected technical effects are achieved.
Claims (6)
1. A preparation method of a gymnocyanin extract comprises the following steps:
1) cell disruption: weighing 0.5g of euglena dry powder, adding PBS, mixing, and ultrasonically crushing for 15 min;
2) cell extract: centrifuging at 12000rpm at 4 deg.C for 20min, and transferring the supernatant into a centrifuge tube;
3) protein salting out: slowly adding ammonium sulfate solid into the cell extract, continuously stirring for dissolving until the concentration of ammonium sulfate reaches 60%, continuously stirring for 1 hour at room temperature, and then placing into a 4-degree refrigerator for standing overnight;
4) protein purification: centrifuging the solution with separated protein at 4000rpm for 20min, discarding the supernatant, and keeping the precipitate for later use; dissolving the protein precipitate with a proper amount of PBS solution, adding into a 100D dialysis bag, and dialyzing in deionized water until no ammonium sulfate exists in the solution;
5) and (3) degerming: filtering the dialyzed and purified protein by using a 0.22 mu m filter head, and collecting filtrate to obtain sterile gymnocyanin extract.
2. Use of a gymnocyanin extract obtained in claim 1 for the preparation of a cell damage repairing agent.
3. Use of a gymnocyanin extract obtained according to claim 1 for the preparation of a skin-care or cosmetic product.
4. Use of a gymnocyanin extract obtained in claim 1 for preparing an oxidative damage repairing agent.
5. Use according to claim 2, wherein the cells are selected from the group consisting of immortalised keratinocytes, fibroblasts, macrophages.
6. A euglena cell culture fluid comprising: 0.02g/L KH2PO4, 0.6g/L peptone, 0.025g/L MgSO4.7H2O, 0.4g/L yeast extract, 0.4g/L sodium acetate, 0.5. mu.g/L vitamin B12, 0.04g/L potassium citrate, 0.4mg/L vitamin B1.
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| CN114195884A (en) * | 2021-10-27 | 2022-03-18 | 禾美生物科技(浙江)有限公司 | Recombinant human collagen and preparation method thereof |
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