CN117186263A - Acidic polysaccharide, preparation method and application thereof in cosmetic field - Google Patents
Acidic polysaccharide, preparation method and application thereof in cosmetic field Download PDFInfo
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- 150000004805 acidic polysaccharides Chemical class 0.000 title claims abstract description 42
- 229920001284 acidic polysaccharide Polymers 0.000 title claims abstract description 41
- 239000002537 cosmetic Substances 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 37
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 37
- 239000005017 polysaccharide Substances 0.000 claims abstract description 37
- 150000004804 polysaccharides Chemical class 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000012153 distilled water Substances 0.000 claims abstract description 11
- 239000002244 precipitate Substances 0.000 claims abstract description 8
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- 238000002791 soaking Methods 0.000 claims abstract description 5
- 238000005406 washing Methods 0.000 claims abstract description 5
- 238000011068 loading method Methods 0.000 claims abstract description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 241000218993 Begonia Species 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 238000000605 extraction Methods 0.000 claims description 7
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000005349 anion exchange Methods 0.000 claims description 2
- 238000012869 ethanol precipitation Methods 0.000 claims description 2
- 102000008186 Collagen Human genes 0.000 abstract description 6
- 108010035532 Collagen Proteins 0.000 abstract description 6
- 229920001436 collagen Polymers 0.000 abstract description 6
- 238000001556 precipitation Methods 0.000 abstract description 5
- 239000012535 impurity Substances 0.000 abstract description 3
- 241001528553 Malus asiatica Species 0.000 abstract description 2
- 238000005571 anion exchange chromatography Methods 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract 2
- 239000002253 acid Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 230000002500 effect on skin Effects 0.000 description 11
- 210000002950 fibroblast Anatomy 0.000 description 10
- 238000012360 testing method Methods 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 230000002378 acidificating effect Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 102100034343 Integrase Human genes 0.000 description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 230000007794 irritation Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 231100000430 skin reaction Toxicity 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 206010040914 Skin reaction Diseases 0.000 description 2
- 206010050637 Skin tightness Diseases 0.000 description 2
- MZVQCMJNVPIDEA-UHFFFAOYSA-N [CH2]CN(CC)CC Chemical group [CH2]CN(CC)CC MZVQCMJNVPIDEA-UHFFFAOYSA-N 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000005360 mashing Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 230000035483 skin reaction Effects 0.000 description 2
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 2
- 238000003809 water extraction Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 241000218999 Begoniaceae Species 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102000001187 Collagen Type III Human genes 0.000 description 1
- 108010069502 Collagen Type III Proteins 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010040880 Skin irritation Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- UBXYXCRCOKCZIT-UHFFFAOYSA-N biphenyl-3-ol Chemical group OC1=CC=CC(C=2C=CC=CC=2)=C1 UBXYXCRCOKCZIT-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 description 1
- OAABHEHWRQAHEJ-UHFFFAOYSA-N butan-1-ol;chloroform Chemical compound ClC(Cl)Cl.CCCCO OAABHEHWRQAHEJ-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 210000000245 forearm Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000000774 hypoallergenic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007373 indentation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000874 microwave-assisted extraction Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000037393 skin firmness Effects 0.000 description 1
- 230000036556 skin irritation Effects 0.000 description 1
- 231100000475 skin irritation Toxicity 0.000 description 1
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Landscapes
- Cosmetics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an acidic polysaccharide, a preparation method and application thereof in the field of cosmetics, wherein the acidic polysaccharide is prepared by the following steps: soaking dried flowers of Malus asiatica with ethanol solution to remove impurities, extracting with alkaline water, concentrating, adding absolute ethanol into the concentrated solution, standing for alcohol precipitation, centrifuging, collecting precipitate, washing, and drying to obtain crude polysaccharide, deproteinizing by Sevag method to obtain refined polysaccharide; loading the refined polysaccharide on DEAE-52 anion exchange chromatography column, eluting with distilled water to remove impurities, eluting with 0.5mol/L NaCl solution, collecting NaCl solution eluate, dialyzing, and drying. The acidic polysaccharide can promote the expression of collagen in skin, and has the prospect of developing cosmetics for improving the compactness, smoothness and tender feel of skin.
Description
Technical Field
The invention belongs to the field of chemistry, relates to polysaccharide and application thereof, and in particular relates to acidic polysaccharide, a preparation method and application thereof in the field of cosmetics.
Background
Polysaccharides are an important form of sugar that naturally occurs, including structural, storage and bioactive polysaccharides. The bioactive polysaccharide has various important biological activities, so that the bioactive polysaccharide is widely applied in the fields of biological medicine, cosmetics and the like.
Acidic polysaccharides are an important biologically active polysaccharide. The acidic polysaccharides are mainly derived from plants, animals and microorganisms. The acidic polysaccharide is mainly extracted and separated from roots, stems, leaves and fruits of plants. From a chemical structure analysis, the acidic polysaccharide chain may contain neutral monosaccharides or basic monosaccharides in addition to acidic groups (mainly uronic acid, sulfate). As for the acidic groups and the proportion of the acidic groups in the acidic polysaccharide or the acidity thereof are not clearly specified at present, the polysaccharides containing the acidic groups are generally called acidic polysaccharides in the literature.
Because the acidic polysaccharide has wide sources and wide activity, the research of extracting, separating and purifying the acidic polysaccharide, activity and the like has become the research popular direction in the polysaccharide field.
The extraction method of the acidic polysaccharide comprises water extraction, alkaline water extraction, ultrasonic assisted extraction, microwave assisted extraction, enzyme assisted extraction, etc., and the crude polysaccharide is usually obtained by the extraction methods, and is usually separated and purified by precipitation method and column chromatography. The precipitation method comprises a fractional alcohol precipitation method, a quaternary ammonium salt precipitation method and the like. Common column chromatography packing materials include ion exchange type Diethylaminoethyl (DEAE) -dextran, DEAE-cellulose and DEAE-agarose, and gel type packing materials such as Sephadex (Sephadex G), sepharose (Sepharose) and the like. The aim of purifying the acidic polysaccharide is often not achieved by adopting a single separation and purification method, and the acidic polysaccharide is separated by adopting a plurality of methods in combination in the current research.
The acid polysaccharide has various biological activities, and researchers prove that the acid polysaccharide has activities of resisting oxidation, resisting inflammation, regulating immunity, reducing blood fat and the like through in vivo and in vitro experiments and research on the action mechanism of the acid polysaccharide, but the relationship between the molecular structure and the biological activity of the acid polysaccharide is still undefined due to the complexity and the irregularity of the structure of the acid polysaccharide, so that the deep research of the acid polysaccharide is seriously hindered.
The begonia is perennial evergreen herb of begoniaceae, the leaves are many, the leaves are emerald green and bright, the leaf is also very beautiful, and when flowers are bloomed, the flowers are many, clustered flowers are bright and beautiful, the flowering period is super-long, the begonia can bloom in four seasons, and the begonia is a relatively common garden flower plant, the flowering period of which is the same as the name of the begonia, can really bloom all the year round, and can bloom in winter as long as the temperature is proper.
The folk skin care method has the use of mashing the flowers of the begonia, uniformly mixing the mashed flowers with glycerin and applying the mashed flowers to face for skin care, but the description of what components in the flowers of the begonia play the role of skin care is not clear, and the improvement effect on skin is not specifically described. To explain these doubts, the invention was developed for malus asiatica resources.
Disclosure of Invention
The invention aims to provide an acidic polysaccharide, a preparation method and application thereof in the field of cosmetics.
The aim of the invention is achieved by the following technical scheme:
an acidic polysaccharide prepared by the steps of:
weighing dried flowers of crushed begonia, soaking in an ethanol solution with the volume fraction of 80%, filtering, volatilizing ethanol from filter residues, heating for leaching with alkali water, filtering, concentrating the filtrate under reduced pressure, adding absolute ethanol into the concentrated solution to ensure that the volume fraction of the ethanol reaches 80%, standing for ethanol precipitation, centrifuging, collecting the precipitate, washing with absolute ethanol, acetone and diethyl ether in sequence, and vacuum drying to obtain crude polysaccharide;
dissolving the crude polysaccharide with a proper amount of distilled water, deproteinizing by a Sevag method to obtain refined polysaccharide;
dissolving refined polysaccharide with appropriate amount of distilled water, loading onto DEAE-52 anion exchange chromatographic column, eluting with distilled water, and detecting with phenol-sulfuric acid method until no sugar is detected; eluting with 0.5mol/L NaCl solution, and detecting by phenol-sulfuric acid method until no sugar comes out; collecting NaCl solution eluent, dialyzing, and freeze-drying to obtain acidic polysaccharide.
Preferably, the 80% ethanol solution is soaked for 24 hours.
Preferably, the temperature of the aqueous alkaline extraction is 80-85 ℃.
Preferably, the pH control ph=8.0 is measured every 15min during the aqueous alkaline heated extraction.
The use of the above acidic polysaccharides for the preparation of cosmetics for improving skin firmness, smoothness and tenderness.
The beneficial technical effects are as follows:
although flowers of begonia in four seasons have reports of beauty maintenance (glycerin is used for uniformly applying on the face after mashing), the reports do not clearly state what components are active components, nor state what improvement effect is provided on the skin. The invention separates and purifies the flowers of the begonia into an acidic polysaccharide, the polysaccharide can promote the expression of collagen in skin, and the collagen is the most important component for determining the skin tightness, smoothness and tenderness, so the polysaccharide has the prospect of developing cosmetics for improving the skin tightness, smoothness and tenderness.
Drawings
Fig. 1 is an SEM image of acidic polysaccharides, showing the cluster morphology of the particles.
FIG. 2 shows the expression levels of COl a1, COl a1 genes in mouse dermal fibroblasts.
Detailed Description
Example 1: acid polysaccharide preparation and acid group content determination
Weighing 2kg of crushed dried flowers of begonia, soaking for 24 hours by using an ethanol solution with the volume fraction of 80 percent (removing small molecular impurities and fat-soluble substances), filtering, volatilizing ethanol from filter residues, heating and leaching the filter residues by alkali water for 4 hours according to the feed liquid ratio of 1:10 (controlling the temperature to 80-85 ℃ and controlling the pH to be 8.0 once every 15min, controlling the pH to be sodium hydroxide), filtering, repeatedly extracting the filter residues once, combining the two filtrates, concentrating under reduced pressure (55 ℃), adding absolute ethanol into the concentrated solution to ensure that the volume fraction of the ethanol reaches 80 percent, standing and precipitating the concentrated solution for 24 hours at the temperature of 4 ℃, centrifugally separating (5000 rpm and 15 min), collecting the precipitate, washing the precipitate by using absolute ethanol, acetone and diethyl ether in sequence, and carrying out vacuum drying at the temperature of 50 ℃ to obtain crude polysaccharide.
Dissolving crude polysaccharide with appropriate amount of distilled water, deproteinizing by Sevag method, adding chloroform-n-butanol mixed solution (4:1, V/V) with volume of 1/4 of polysaccharide solution, shaking vigorously for 30min, standing for layering, and removing the lower organic solvent and protein precipitate in the middle layer. Repeatedly treating the upper polysaccharide solution with Sevag reagent for 3-5 times until no obvious intermediate layer protein precipitate exists, centrifuging at 10000rpm for 10 minutes, collecting supernatant, adding absolute ethanol to make the ethanol volume fraction reach 80%, standing at 4 ℃ for 24 hours, centrifuging (5000 rpm, 15 minutes), collecting precipitate, washing sequentially with absolute ethanol, acetone and diethyl ether, and vacuum drying at 50 ℃ to obtain deproteinized crude polysaccharide. Dissolving deproteinized crude polysaccharide with distilled water, and dialyzing in dialysis bag for 48 hr to remove monosaccharide, organic reagent and organic salt. And after the dialysis is finished, freeze-drying to obtain the refined polysaccharide.
Dissolving refined polysaccharide with appropriate amount of distilled water, loading onto DEAE-52 anion exchange chromatography column, eluting with distilled water (flow rate: 1.5 mL/min), and detecting with phenol-sulfuric acid method until no sugar is detected; then eluted with 0.5mol/L NaCl solution (flow rate: 1.5 mL/min), and the phenol-sulfuric acid method was examined until no sugar was coming out. Collecting NaCl solution eluent, putting into a dialysis bag for dialysis for 48 hours, and freeze-drying after the dialysis is finished to obtain the acidic polysaccharide.
The content of uronic acid (acid group) in the acidic polysaccharide is determined by using glucose as a standard substance and using galacturonic acid as a standard substance by using a phenol-sulfuric acid method and a m-hydroxybiphenyl method. The sugar content in the acidic polysaccharide is (92.6+/-1.9)%, and the uronic acid content in the acidic polysaccharide is (33.7+/-1.4)%, which is a typical acidic polysaccharide. Fig. 1 is an SEM image of the acidic polysaccharide, showing the cluster morphology of the particles.
Example 2: effects on skin collagen content
The effect of the acidic polysaccharide prepared in example 1 on the expression level of collagen in mouse dermal fibroblasts was measured.
Mouse dermal fibroblasts were purchased from wuhanshang biotechnology limited.
Mouse dermal fibroblasts complete medium was purchased from Shanghai pure Biotechnology Co.
MultiScribe Reverse Transcriptase from Semer Feier technology (China).
SYBR Green Master Mix is available from Shanghai-Mei bioengineering Co.
PCR amplification primers were purchased from Guangzhou Ruibo Biotechnology Inc., and the sequences were as follows:
COl1a1 upstream primer (5 '-3'): GCTCCTCTTAGGGGCCACT;
COl1a1 downstream primer (5 '-3'): CCACGTCTCACCATTGGGG;
COl3a1 upstream primer (5 '-3'): CTGTAACATGGAAACTGGGGAAA;
COl3a1 downstream primer (5 '-3'): CCATAGCTGAACTGAAAACCACC;
beta-actin upstream primer (5 '-3'): GGCTGTATTCCCCTCCATCG;
beta-actin downstream primer (5 '-3'): CCAGTTGGTAACAATGCCATGT.
Experimental operation:
the mice dermal fibroblasts with good growth state are resuspended by the complete culture medium of the mice dermal fibroblasts and inoculated into 6-hole culture plates, 1mL of each hole is placed at 37 ℃ and 5% CO 2 The culture was conditioned overnight, and 1mL of the complete medium of mouse dermal fibroblasts containing the acidic polysaccharide of example 1 was added the next day to give a final acidic polysaccharide concentration of 25, 50. Mu.g/mL, and the complete medium of mouse dermal fibroblasts without acidic polysaccharide was added as a control to continue the culture. After 48h, the cells were collected, total RNA was extracted by Trizol method, the purity and concentration of RNA were determined, and RNA was reverse transcribed into cDNA by strictly following the instructions of reverse transcriptase kit (MultiScribe Reverse Transcriptase). The cDNA, primer and SYBR Green Master Mix were mixed well, amplified using real-time fluorescent quantitative PCR and the fluorescence intensity was detected, the amplification procedure was as follows: cycling for 1 time at 95 ℃ for 5 min; 95 ℃ for 20s; cycling for 45 times at 60 ℃ for 30s and 72 ℃ for 20s; and finally extending at 72 ℃ for 2min. Beta-actin was used as reference gene, 2 -ΔΔCt The relative expression amounts of the genes COl a1 (type I collagen) and COl a 3a1 (type III collagen) were calculated.
The results are shown in Table 1 and FIG. 2, the acidic polysaccharide can effectively improve the expression level of COl a1 and COl a1 genes in the mouse dermal fibroblasts, and promote the synthesis of collagen by the mouse dermal fibroblasts.
TABLE 1 relative expression levels of genes COl a1, COl a1
| Group of | COl1a1 | COl3a1 |
| Control | 1.00±0.07 | 1.00±0.09 |
| Acidic polysaccharide-25. Mu.g/mL | 1.83±0.07 | 1.59±0.12 |
| Acidic polysaccharide-50. Mu.g/mL | 2.77±0.08 | 2.45±0.11 |
Example 3: skin irritation test
In view of the potential for some irritation to the skin, the patch test commonly used in the cosmetic arts was used to test whether the acidic polysaccharide of example 1 would cause irritation and allergy to human skin.
30 healthy volunteers were selected as subjects with reference to cosmetic safety Specification (2015 edition), 15 men and 15 women. After bathing, 0.025g of the test substance (acidic polysaccharide) was placed in a plaque test chamber, and the plaque test with the test substance was applied to the back or forearm side of the subject with hypoallergenic tape, and applied to the skin with gentle palm pressure for 24 hours. Skin reactions were observed according to the criteria of table 2 for 30min (after the disappearance of the indentations), 24h and 48h, respectively, after removal of the subject plaque tester, and the observations were recorded and evaluated with reference to table 2.
TABLE 2 skin response grading Standard Table for skin closed Patch test
The test results showed that 30 healthy volunteers were negative in skin reactions 30min, 24h and 48h after removal of the subject spot tester, and no irritation and allergy to human skin was observed.
Claims (5)
1. An acidic polysaccharide, characterized by being prepared by the steps of:
weighing dried flowers of crushed begonia, soaking in an ethanol solution with the volume fraction of 80%, filtering, volatilizing ethanol from filter residues, heating for leaching with alkali water, filtering, concentrating the filtrate under reduced pressure, adding absolute ethanol into the concentrated solution to ensure that the volume fraction of the ethanol reaches 80%, standing for ethanol precipitation, centrifuging, collecting the precipitate, washing with absolute ethanol, acetone and diethyl ether in sequence, and vacuum drying to obtain crude polysaccharide;
dissolving the crude polysaccharide with a proper amount of distilled water, deproteinizing by a Sevag method to obtain refined polysaccharide;
dissolving refined polysaccharide with appropriate amount of distilled water, loading onto DEAE-52 anion exchange chromatographic column, eluting with distilled water, and detecting with phenol-sulfuric acid method until no sugar is detected; eluting with 0.5mol/L NaCl solution, and detecting by phenol-sulfuric acid method until no sugar comes out; collecting NaCl solution eluent, dialyzing, and freeze-drying to obtain acidic polysaccharide.
2. The acidic polysaccharide according to claim 1, wherein: soaking in 80% ethanol solution for 24 hr.
3. The acidic polysaccharide according to claim 1, wherein: the extraction temperature of the alkaline water is 80-85 ℃.
4. An acidic polysaccharide according to claim 3, characterized in that: pH control ph=8.0 was measured every 15min during the aqueous alkaline heat extraction.
5. Use of an acidic polysaccharide according to any one of claims 1 to 4 for the preparation of a cosmetic for improving the firmness, smoothness and tenderness of the skin.
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| CN202311303229.8A CN117186263A (en) | 2023-10-10 | 2023-10-10 | Acidic polysaccharide, preparation method and application thereof in cosmetic field |
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