CN110452313B - Refined plant polysaccharide, preparation method and application in preparing cosmetics - Google Patents

Refined plant polysaccharide, preparation method and application in preparing cosmetics Download PDF

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CN110452313B
CN110452313B CN201910845102.6A CN201910845102A CN110452313B CN 110452313 B CN110452313 B CN 110452313B CN 201910845102 A CN201910845102 A CN 201910845102A CN 110452313 B CN110452313 B CN 110452313B
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polysaccharide
refined plant
plant polysaccharide
petasites japonicus
total polysaccharide
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洪其昌
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Shanghai sunfeng Chemical Technology Co.,Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • 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
    • A61K2800/70—Biological properties of the composition as a whole
    • A61K2800/72—Hypo-allergenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying

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Abstract

The invention discloses a refined plant polysaccharide, a preparation method and application in the aspect of preparing cosmetics, wherein the refined plant polysaccharide is prepared by the following steps: firstly, water extraction and alcohol precipitation are carried out to obtain the total polysaccharide of the petasites japonicus, then the total polysaccharide of the petasites japonicus is taken and dissolved by deionized water, the obtained sample is loaded on a DEAE-Sepharose Fast Flow chromatographic column as a loading solution, sodium chloride solutions with the concentrations of 0 and 0.2mol/L are sequentially used for elution, 0.2mol/L sodium chloride solution eluent is collected, the concentration is carried out, dialysis is carried out by a dialysis bag to remove salt, and freeze drying is carried out. The refined plant polysaccharide provided by the invention can promote the proliferation and migration of human skin fibroblasts, has the effect of promoting skin repair, has no obvious stimulation to human skin, and has the value of preparing cosmetics for promoting skin repair.

Description

Refined plant polysaccharide, preparation method and application in preparing cosmetics
Technical Field
The invention belongs to the field of cosmetics, relates to application of natural polysaccharide in cosmetics, and particularly relates to refined plant polysaccharide, a preparation method and application in preparing cosmetics.
Background
Beauty is the nature of a person, but with age, the ability of a person to repair skin and the elasticity of skin decreases.
The skin repair ability of a human is mainly determined by the proliferation rate and migration ability of human skin fibroblasts, and after the human ages, the decrease of the proliferation rate and migration ability of the skin fibroblasts is a direct cause of the decrease of the skin repair ability.
Human skin elasticity is primarily related to collagen and elastin content. Collagen and elastin respectively constitute collagen fiber and elastic fiber, and collagen fiber mainly supports the volume, keeps moisture, lets skin plump, and elastic fiber mainly makes skin can stretch and kick-back, and the elasticity of skin is decided to the two jointly.
Therefore, in order to improve the skin repair ability and skin elasticity of human, it is necessary to find a component which can improve the proliferation rate and migration ability of skin fibroblasts and the synthesis of collagen and elastin.
The polysaccharide from natural plants is widely applied to the field of cosmetics, such as whitening, freckle removing, moisturizing and the like, because the polysaccharide has good compatibility with human skin, is mild, has no stimulation and has sufficient sources.
The petasites japonicus is a very common perennial herb, but the utilization degree is extremely low, so that the great resource waste is caused.
The patent application with the application number of 2019108449488 claims a butterbur total polysaccharide and application of the butterbur total polysaccharide in promoting skin repair and improving skin elasticity so as to prepare cosmetics, further researches on two refined polysaccharides separated from the butterbur total polysaccharide and tests the skin repair promoting activity of the two refined polysaccharides.
Disclosure of Invention
The invention aims at providing a refined plant polysaccharide and a second purpose of providing the application of the refined plant polysaccharide in preparing cosmetics.
The technical scheme for realizing the purpose of the invention is as follows:
a refined plant polysaccharide is prepared by the following steps:
(1) preparing total polysaccharide: crushing dry whole herb of the petasites japonicus, adding absolute ethyl alcohol, carrying out hot reflux extraction and degreasing, filtering, adding distilled water into filter residues, carrying out heating extraction, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain an extract, adding an appropriate amount of distilled water, stirring to fully dissolve, carrying out suction filtration, adding 4 times of volume of absolute ethyl alcohol into the filtrate, uniformly stirring, carrying out alcohol precipitation, centrifuging, discarding supernatant, washing with acetone and absolute ethyl alcohol in sequence, dissolving with an appropriate amount of distilled water, and freeze-drying to obtain the total polysaccharide of the petasites japonicus;
(2) refining total polysaccharide: dissolving Petasites japonicus total polysaccharide with deionized water, loading onto DEAE-Sepharose Fastflow chromatographic column as sample solution, sequentially eluting with 0 and 0.2mol/L sodium chloride solution, collecting 0.2mol/L sodium chloride solution eluate, concentrating, dialyzing with dialysis bag to remove salt, and freeze drying.
Preferably, in the degreasing step in the step (1), anhydrous ethanol is added according to the feed-liquid ratio of 1:5 for hot reflux extraction for 3h for degreasing.
Preferably, in the step (1) of extracting the distilled water, the filter residue is added with the distilled water according to the material-liquid ratio of 1:10, and the mixture is heated and extracted at 85 ℃ for 12 hours.
Preferably, the alcohol precipitation time in the step (1) is 12 h.
Preferably, the elution flow rate in step (2) is 1.5 mL/min.
The refined plant polysaccharide is used for promoting skin repair.
The refined plant polysaccharide is used for preparing cosmetics for promoting skin repair.
The beneficial effects are that:
the refined plant polysaccharide provided by the invention can promote the proliferation and migration of human skin fibroblasts, has the effect of promoting skin repair, has no obvious stimulation to human skin, and has the value of preparing cosmetics for promoting skin repair.
Drawings
FIG. 1 is an SEM image of a purified plant polysaccharide A, in which B is a partially enlarged view of A and has high uniformity;
FIG. 2 is an SEM image of refined plant polysaccharide B, in which B is a partial enlarged view of A and has high uniformity;
FIG. 3 shows the results of the cell scratch test.
Detailed Description
First, experiment method
1. Preparation of refined plant polysaccharides A and B
(1) Preparing total polysaccharide: crushing dry whole herb of the petasites japonicus, adding absolute ethyl alcohol according to a material-liquid ratio of 1:5(kg: L), carrying out hot reflux extraction for 3h for degreasing, repeatedly degreasing for 1 time, filtering, adding distilled water into filter residues according to a material-liquid ratio of 1:10(kg: L), heating and extracting at 85 ℃ for 12h, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain an extract, adding distilled water according to a material-liquid ratio of 1:5(kg: L), stirring to fully dissolve, carrying out suction filtration, adding 4 times of volume of absolute ethyl alcohol into the filtrate, stirring uniformly, carrying out alcohol precipitation for 12h, centrifuging, discarding supernatant, washing with acetone and absolute ethyl alcohol in sequence, dissolving with a proper amount of distilled water, and freeze-drying to obtain the total polysaccharide of the petasites japonicus.
(2) Refining total polysaccharide: dissolving 300mg of Petasites japonicus total polysaccharide with 15mL of deionized water, loading the solution as a loading solution onto a DEAE-Sepharose fast flow chromatographic column with the specification of 5cm × 25cm (namely, the inner diameter × the height of a column bed), sequentially eluting with 300mL, 400mL and 400mL of sodium chloride solutions with the concentrations of 0, 0.2 and 0.5mol/L respectively at the elution flow rate of 1.5mL/min, respectively collecting the eluates of the 0.2 and 0.5mol/L of sodium chloride solutions, concentrating, dialyzing and desalting with a dialysis bag (the molecular weight cutoff is 3500, the flattening width is 34mm, and the diameter is 22mm), respectively freezing and drying to obtain refined plant polysaccharides A and B, and scanning with SEM to observe the uniformity.
2. Human skin fibroblast cell culture and passage
Human skin fibroblast HFF-1 cells were cultured in DMEM containing 15% fetal bovine serum and 1% double antibody at 37 deg.C and 5% CO2Culturing in incubator, changing culture solution every 3d, digesting with 0.25% trypsin when cell growth fusion reaches about 85%, and subculturing at 1:3 ratio. Cells in the logarithmic growth phase were taken for the experiment.
3. Skin repair promoting Activity test
3.1 promotion of human skin fibroblast proliferation (MTT test)
Collecting cells in logarithmic growth phase, digesting, and preparing into 1 × 10 concentration DMEM medium containing 15% fetal calf serum and 1% double antibody5The cell suspension was inoculated in a 96-well plate at 150. mu.L/well, 37 ℃ and 5% CO2After culturing in an incubator for 24h, changing the culture medium to DMEM culture medium containing 15 mu g/mL refined plant polysaccharide A or B, 15% fetal calf serum and 1% double antibody for continuous culture, after continuous culture for 24h and 48h, removing the culture medium, washing with PBS, adding 150 mu L DMEM culture medium containing 15% fetal calf serum and 1% double antibody and 20 mu LMTT solution (5mg/mL), 37 ℃ and 5% CO2Culturing for 4h, removing supernatant, adding DMSO150 μ L into each well, and shaking for 10min to dissolve the crystals. Meanwhile, a control group is arranged, compared with the refined plant polysaccharide group, only refined plant polysaccharide is not added in the control group, the rest is completely the same, and each group has 5 multiple holes. The absorbance of each well was measured at 490nm, and the proliferation promoting rate of the purified plant polysaccharide A, B was calculated according to the calculation formula shown below.
Proliferation promoting rate (%) - (absorbance value of refined plant polysaccharide-absorbance value of control group) ÷ absorbance value of control group × 100%
3.2 promotion of human skin fibroblast migration (cell scratch test)
Collecting cells in logarithmic growth phase, digesting, and preparing into 1 × 10 concentration DMEM medium containing 15% fetal calf serum and 1% double antibody5Cell suspension/mLThe solution was inoculated into 6-well plates 2 mL/well at 37 ℃ with 5% CO2After 24 hours of culture in the incubator, the tip of a 1mL pipette is gently used to make straight scratches at the center of each culture well, PBS is used for washing, 2mL DMEM medium containing 15 mug/mL of refined plant polysaccharide A or B, 15% fetal bovine serum and 1% double antibody is added into the refined plant polysaccharide group, 2mL DMEM medium containing 15% fetal bovine serum and 1% double antibody is added into the control group, each 2 multiple wells are formed, the culture is continued for 24 hours, observation and photographing are carried out under an inverted microscope, the scratch distance is measured by ImageJ software, and the migration promotion rate of the refined plant polysaccharide group is calculated according to the calculation formula shown below.
Mobility (%) as [% ] (scratch distance of control group-scratch distance of purified plant polysaccharide group) [% ]. control group scratch distance. times.100%
4. Skin irritation test
According to technical Specification for safety of cosmetics (2015 edition), 30 test persons are selected according to the selection standard of a test subject, qualified patch materials are selected, 0.025g of refined plant polysaccharide A or B is put into a patch tester, and no substance is added into a control hole. The spot tester with the tested object is pasted on the back of a volunteer by using a non-irritating adhesive tape, the spot tester is lightly pressed by hands and palms to be uniformly pasted on the skin, and the spot tester is continuously kept on the skin for 24 hours. The interval is 30min after the plaque remover is removed, and the skin reaction is observed after the indentation disappears. The observation was done 24 and 48h after the patch test. And judging the result, and reacting at the 0 stage: no reaction occurs at the tested part; stage 1 reaction: pale erythema on the skin; 2, stage reaction: erythema, infiltration, or pimples of the skin; 3, stage reaction: edematous erythema, papules, etc. of the skin; 4, stage reaction: the skin appeared noticeably red, swollen, with papules or bullae.
5. Statistical treatment
Using SPSS17.0 analysis, data are presented as mean ± standard deviation, homogeneity of variance is tested using One-way ANOVA with One-way analysis of variance, and analyzed with Student's-ttest, P <0.05 indicating that the difference is significant.
Third, experimental results
1. SEM result of refined plant polysaccharide A, B
SEM images of refined plant polysaccharide A, B as shown in fig. 1 and 2, refined plant polysaccharide A, B has different surface characteristics, but all have higher homogeneity.
2. Refined plant polysaccharide A, B for promoting skin repairing activity
The results of the MTT test are shown in table 1, and compared with the control group, the absorbance values of the refined plant polysaccharide A, B group were significantly increased, and the time-dependent proliferation promoting rate of human skin fibroblasts was observed.
TABLE 1 Absorbance values for each set at different time points
Control group Refined plant polysaccharide A group (proliferation promoting rate) Refined plant polysaccharide B group (proliferation promoting rate)
24h 0.41±0.08 0.65±0.12(58.5％) 0.53±0.09(29.3％)
48h 0.60±0.09 1.07±0.15(78.3％) 0.92±0.13(53.3％)
As shown in fig. 3, the scratch distance of the refined plant polysaccharide A, B group was significantly reduced compared to the control group, and the mobility promoting rate of the refined plant polysaccharide A, B was shown in table 2.
TABLE 2 mobility promotion of purified plant polysaccharide A, B
Multiple holes 1 Multiple holes 2
Refining plant polysaccharide A 100％ 100％
Refined plant polysaccharide B 59.1％ 48.0％
MTT test results and cell scratch test results show that the refined plant polysaccharide A, B can promote the proliferation and migration of human skin fibroblasts, and has the effect of promoting skin repair.
2. Test results of polysaccharide Patch
The patch test results are shown in table 2, and it can be seen that the refined plant polysaccharide A, B is mild to human skin, and can be used for preparing cosmetics to improve the efficacy of the cosmetics in promoting skin repair.
TABLE 2 Patch test results
Figure BDA0002194999380000041
Figure BDA0002194999380000051
The above examples show that the refined plant polysaccharide of the invention can promote the proliferation and migration of human skin fibroblasts, has the effect of promoting skin repair, has no obvious stimulation to human skin, and has value for preparing cosmetics for promoting skin repair.

Claims (1)

1. Use of a refined plant polysaccharide for the preparation of a cosmetic for promoting skin repair, the refined plant polysaccharide being prepared by the steps of:
(1) preparing total polysaccharide: crushing dry whole herb of the petasites japonicus, adding absolute ethyl alcohol according to a material-liquid ratio of 1kg to 5L, carrying out hot reflux extraction for 3h for degreasing, repeatedly carrying out degreasing for 1 time, filtering, adding distilled water into filter residues according to a material-liquid ratio of 1kg to 10L, heating and extracting at 85 ℃ for 12h, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain an extract, adding distilled water according to a material-liquid ratio of 1kg to 5L, stirring for full dissolution, carrying out suction filtration, adding 4 times of volume of absolute ethyl alcohol into the filtrate, uniformly stirring, carrying out alcohol precipitation for 12h, centrifuging, discarding supernatant, washing with acetone and absolute ethyl alcohol in sequence, dissolving with an appropriate amount of distilled water, and freeze-drying to obtain the total polysaccharide of the petasites japonicus;
(2) refining total polysaccharide: dissolving 300mg of Petasites japonicus total polysaccharide with 15mL of deionized water, loading the solution as a loading solution onto a DEAE-Sepharose Fast Flow chromatographic column, sequentially eluting with 300mL and 400mL of sodium chloride solutions with the concentrations of 0 and 0.2mol/L respectively at the elution Flow rate of 1.5mL/min, collecting 0.2mol/L sodium chloride solution eluent, concentrating, dialyzing with a dialysis bag to remove salt, and freeze-drying to obtain the Petasites japonicus total polysaccharide; wherein, the specification of the DEAE-Sepharose Fast Flow chromatographic column is 5cm of inner diameter and 25cm of column bed height.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103479611A (en) * 2012-06-12 2014-01-01 中国科学院上海药物研究所 Application of petasiphenol compound in preparation of tyrosinase inhibitor
CN105477104A (en) * 2016-01-22 2016-04-13 韩冰 Hand sanitizer for personnel in clinical laboratory
CN105998528A (en) * 2016-06-29 2016-10-12 何勤 Dental care tooth brushing powder

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104861080A (en) * 2015-05-06 2015-08-26 广东药学院 Polysaccharide in guava and preparation method and application thereof
CN107997985A (en) * 2017-12-07 2018-05-08 吉林农业大学 Application of the deerskin polysaccharide in moisture-keeping cosmetics are prepared
CN109771449A (en) * 2019-02-24 2019-05-21 温州大学 A kind of Hijiki polysaccharide and its application with promotion wound healing effect

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103479611A (en) * 2012-06-12 2014-01-01 中国科学院上海药物研究所 Application of petasiphenol compound in preparation of tyrosinase inhibitor
CN105477104A (en) * 2016-01-22 2016-04-13 韩冰 Hand sanitizer for personnel in clinical laboratory
CN105998528A (en) * 2016-06-29 2016-10-12 何勤 Dental care tooth brushing powder

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