CN111471119B - Plant polysaccharide and application thereof in preparation of cosmetics for removing freckles and whitening - Google Patents
Plant polysaccharide and application thereof in preparation of cosmetics for removing freckles and whitening Download PDFInfo
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- CN111471119B CN111471119B CN202010479418.0A CN202010479418A CN111471119B CN 111471119 B CN111471119 B CN 111471119B CN 202010479418 A CN202010479418 A CN 202010479418A CN 111471119 B CN111471119 B CN 111471119B
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- 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
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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
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- 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
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- 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
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- 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
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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Abstract
The invention discloses a plant polysaccharide and application thereof in preparing cosmetics for removing freckles and whitening. The plant polysaccharide has the activity of inhibiting the expression of melanin synthesis related protein, can inhibit the activity of tyrosinase, further inhibit the deposition of melanin in cells, and has the effects of removing freckles and whitening, so that the plant polysaccharide has the prospect of being developed into freckles removing and whitening cosmetics, such as freckles removing and whitening facial cleanser, facial cream or facial mask.
Description
Technical Field
The invention belongs to the field of cosmetics, relates to freckle removing and whitening application of plant polysaccharide, and particularly relates to plant polysaccharide and application of plant polysaccharide in preparation of freckle removing and whitening cosmetics.
Background
Melanin is a biological pigment of protein derivatives, which is synthesized by cells themselves and is insoluble in water and soluble in alkaline solution. Melanin can absorb ultraviolet rays and protect skin from skin damage caused by excessive irradiation of ultraviolet rays. However, excessive synthesis of melanin can lead to darkening of skin tone and even the development of cosmetic problems such as freckles.
There are many enzymes involved in melanin synthesis, the most critical one of which is tyrosinase. Under the action of tyrosinase, melanocytes continuously transport melanin granules to cutin. After the human body is irradiated by sunlight ultraviolet rays, tyrosinase is stimulated and activated by the ultraviolet rays, the process of synthesizing melanin by melanocytes is accelerated, and the skin becomes dark.
At present, many scientific researchers still focus on molecular mechanism research in the aspect of melanin synthesis and screening and research and development work of whitening drugs so as to solve the problem of pigmentation of various skins and realize the effects of removing freckles and whitening.
The Chinese violet is a perennial herb, and researches prove that the Chinese violet extract has the effect of resisting skin aging. At present, the research of the viola yedoensis makino polysaccharide on removing melanin and removing freckles and whitening does not exist.
Disclosure of Invention
The invention aims to provide a plant polysaccharide and application thereof in preparing cosmetics for removing freckles and whitening.
The above purpose of the invention is realized by the following technical scheme:
a plant polysaccharide is prepared by the following steps: soaking pulverized herba Violae in petroleum ether for defatting, collecting residue, extracting with water under heating, filtering the extractive solution, concentrating the filtrate, adding 4 times of anhydrous ethanol at 4 deg.C overnight to precipitate polysaccharide, centrifuging, collecting precipitate, dissolving in hot water, precipitating protein with trichloroacetic acid at 4 deg.C overnight, centrifuging to obtain supernatant, concentrating, and drying to obtain plant polysaccharide.
Further, the temperature of extraction by heating with water was 85 ℃.
Further, the ratio of extracted materials to liquid is 1:20, kg/L.
Further, the extraction time was 3 h.
Further, the filtrate was concentrated to 1/10 in its original volume.
The plant polysaccharide is used for removing speckle and whitening skin.
The application of the plant polysaccharide in preparing spot-removing whitening cosmetics.
Further, the cosmetic may be a facial cleanser.
Further, the cosmetic may be a cream.
Further, the cosmetic may be a mask.
Has the advantages that:
the plant polysaccharide has the activity of inhibiting the expression of melanin synthesis related protein, can inhibit the activity of tyrosinase, further inhibit the deposition of melanin in cells, and has the effects of removing freckles and whitening, so that the plant polysaccharide has the prospect of being developed into freckles removing and whitening cosmetics, such as freckles removing and whitening facial cleanser, facial cream or facial mask.
Drawings
FIG. 1 shows the melanin content in the melanoma cells of each group; compared with a control group, the content of melanin in melanoma cells of the polysaccharide group is obviously reduced;
FIG. 2 shows the expression levels of melanin synthesis-related proteins in groups of melanoma cells; compared with a control group, the expression levels of melanin synthesis related proteins TYR and MITF in the melanoma cells of the polysaccharide group are obviously reduced;
FIG. 3 is a graph showing tyrosinase activity in various groups of melanoma cells; tyrosinase activity was significantly reduced in melanoma cells of the polysaccharide group compared to the control group.
Detailed Description
The following examples are given to illustrate the essence of the present invention, but not to limit the scope of the present invention.
Example 1: preparation of polysaccharides
The viola yedoensis makino polysaccharide is extracted according to a conventional polysaccharide extraction process, and specifically comprises the following steps:
soaking pulverized herba Violae whole plant in petroleum ether for 2 days for degreasing, collecting residue, extracting with water at 85 deg.C under heating at a ratio of 1:20(kg/L) for 3 hr, filtering the extractive solution, concentrating the filtrate to 1/10, precipitating polysaccharide with 4 times of anhydrous ethanol at 4 deg.C overnight, centrifuging, retaining the precipitate, redissolving in hot water, precipitating protein with trichloroacetic acid at 4 deg.C overnight, centrifuging, collecting supernatant, concentrating, and drying to obtain herba Violae polysaccharide.
Example 2: activity assay
First, experimental reagent
Mouse melanoma B16-F10 cells were cryopreserved in a laboratory and used after resuscitation.
Fetal bovine serum FBS and DMEM media were purchased from Gibco, usa.
Viola yedoensis Makino polysaccharide was prepared according to the method of example 1.
The various chemical reagents are conventional.
Second, Experimental methods
1. Cell culture
Mouse melanoma B16-F10 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% double antibody at 37 deg.C with 5% CO2Culturing in a constant temperature incubator, digesting and passing after the cells grow over the bottom of the culture flask.
2. Effect on the Melanin content in melanoma cells
2.1 selection of action concentration
The MTT method is adopted to screen out the concentration which has no obvious proliferation inhibition effect on mouse melanoma B16-F10 cells, and the method comprises the following steps:
taking mouse melanoma B16-F10 cells in logarithmic growth phase, digesting, and preparing a cell suspension by using a DMEM complete medium containing 10% fetal calf serum and 1% double antibody, wherein the cell concentration is 2 multiplied by 105Perml, inoculated in a 96-well plate at 100. mu.L per well, 5% CO at 37 ℃2Culturing in a constant temperature incubator. After adaptive culture for 12h, the drug group is changed to DMEM complete medium containing the viola yedoensis makino polysaccharide with different concentrations for culture, and cells cultured without adding drugs are set as a control group and culture solution without cells is set as a blank group. And after continuing culturing for 48h, adding 20 mu L of MTT solution with the concentration of 5mg/mL into each hole, incubating for 4h, removing the supernatant, adding 150 mu L of DMSO into each hole, oscillating to fully dissolve crystals, measuring the OD value of each hole at the wavelength of 490nm, and calculating the proliferation inhibition rate of different drugs on cells according to a formula. 3 replicates per concentration.
Proliferation inhibition (%) was [ 1- (OD drug-OD blank)/(OD control-OD blank) ] × 100%
The result shows that 200 mu g/mL of the viola yedoensis polysaccharide has no obvious proliferation inhibition effect on mouse melanoma B16-F10 cells, and the inhibition rate is (4.69 +/-0.25)%, which is less than 5%. Therefore, a final concentration of 200. mu.g/mL was chosen for subsequent experiments.
2.2 Effect on melanin content
The influence of the drug test on the melanin content in the mouse melanoma B16-F10 cells, which has no obvious proliferation inhibition concentration on the mouse melanoma B16-F10 cells, is tested by the following steps:
taking mouse melanoma B16-F10 cells in logarithmic growth phase, digesting, and preparing a cell suspension by using a DMEM complete medium containing 10% fetal calf serum and 1% double antibody, wherein the cell concentration is 2 multiplied by 105Perml, inoculated in 6-well plates, 2mL per well, 5% CO at 37 ℃2Culturing in a constant temperature incubator. After adaptive culture for 12h, the drug group is replaced by DMEM complete medium containing 200 mug/mL of Chinese violet polysaccharide for culture, and cells cultured without drugs are set as a control group. Continuously culturing for 48h, discarding the culture solution, washing with PBS, digesting the cells, adding NaOH containing 1mol/L, uniformly blowing, carrying out water bath at 80 ℃ for 30min, adding deionized water to enable the concentration of NaOH to be 0.2mol/L, uniformly blowing, transferring into a 96-well plate, measuring the OD value of each well at 475nm wavelength, and calculating the melanin content of the drug group according to a formula with the melanin content of a control group as 100 percent: melanin content (%) ═ OD drug group/OD control group × 100%. 3 replicates per concentration.
3. Effect on expression of protein involved in melanin synthesis in melanoma cells
Taking mouse melanoma B16-F10 cells in logarithmic growth phase, digesting, and preparing a cell suspension by using a DMEM complete medium containing 10% fetal calf serum and 1% double antibody, wherein the cell concentration is 2 multiplied by 105Perml, inoculated in 6-well plates, 2mL per well, 5% CO at 37 ℃2Culturing in a constant temperature incubator. After adaptive culture for 12h, the drug group is replaced by DMEM complete medium containing 200 mug/mL of Chinese violet polysaccharide for culture, and cells cultured without drugs are set as a control group. Continuing culturing for 48h, discarding the culture solution, washing with PBS, digesting the cells, lysing on ice, determining the protein concentration with BCA kit, performing SDS-PAGE electrophoresis on 50 μ g of protein, transferring to PVDF membrane, sealing with 5% skimmed milk powder, adding MITF, TYR and beta-actin primary antibody, incubating at 4 deg.C overnight, washing with TBS-T washing solution, adding secondary antibody, incubating at room temperature for 1h, developing, exposing, and changing color by ECL methodThe chemiluminescence imaging system takes a picture.
4. Effect on tyrosinase Activity in melanoma cells
The effect of the drug test on the tyrosinase activity in the mouse melanoma B16-F10 cells, which has no obvious proliferation inhibition concentration on the mouse melanoma B16-F10 cells, is as follows:
taking mouse melanoma B16-F10 cells in logarithmic growth phase, digesting, and preparing a cell suspension by using a DMEM complete medium containing 10% fetal calf serum and 1% double antibody, wherein the cell concentration is 2 multiplied by 105Perml, inoculated in a 96-well plate at 100. mu.L per well, 5% CO at 37 ℃2Culturing in a constant temperature incubator. After adaptive culture for 12h, the drug group is replaced by DMEM complete medium containing 200 mug/mL of Chinese violet polysaccharide for culture, and cells cultured without drugs are set as a control group. After further culturing for 48h, removing the culture solution, washing with PBS, adding 100 μ L of 0.1% Triton-X-100 (prepared with PBS having pH of 6.8) solution into each well, shaking for 5min to completely lyse the cells, adding 50 μ L of 0.1% L-DOPA (prepared with PBS having pH of 6.8) solution into each well, reacting at 37 deg.C for 20min, measuring OD at 475nm of each well, and calculating the tyrosinase activity of the drug group according to the formula with the tyrosinase activity of the control group as 100%: tyrosinase activity (%) ═ OD drug group/OD control group × 100%. 3 replicates per concentration.
5. Data processing
Data were processed using SPSS17.0 statistical software and experimental results are presented as mean ± SD. Group comparisons using the t-test, p <0.05 indicated significant differences.
Third, experimental results
1. Effect on the Melanin content in melanoma cells
As shown in table 1 and fig. 1, the content of melanin in melanoma cells of the polysaccharide group was significantly reduced compared to the control group, with statistical differences.
TABLE 1 melanin content in groups of melanoma cells
| Group of | Content of |
| Control group | |
| 100％ | |
| Polysaccharide group | (51.5±2.6)％ |
2. Effect on expression of protein involved in melanin synthesis in melanoma cells
As shown in FIG. 2, the expression levels of melanin synthesis-related proteins TYR and MITF were significantly decreased in the melanoma cells of the polysaccharose group, as compared to the control group. It is known to those skilled in the art that the expression and activity of the TYR protein in cells directly determines the melanin content, whereas the TYR protein is regulated by the key transcription factor MITF, both of which are two important related proteins for the synthesis of melanin in melanoma cells.
3. Effect on tyrosinase Activity in melanoma cells
As shown in table 2 and fig. 3, the tyrosinase activity was significantly decreased in the melanoma cells of the polysaccharide group compared to the control group, with statistical differences.
TABLE 2 tyrosinase activity in groups of melanoma cells
| Group of | Tyrosinase |
| Control group | |
| 100％ | |
| Polysaccharide group | (59.1±3.3)％ |
The above results show that the plant polysaccharide provided by the invention has the activity of inhibiting the expression of melanin synthesis-related protein, can inhibit the activity of tyrosinase, further inhibit the deposition of melanin in cells, and has the effects of removing freckles and whitening, so that the plant polysaccharide has the prospect of being developed into freckles-removing and whitening cosmetics, such as freckles-removing and whitening facial cleanser, facial cream or facial mask.
The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.
Claims (4)
1. The application of the plant polysaccharide in preparing the freckle-removing and whitening cosmetics comprises the following steps: soaking pulverized herba Violae whole plant in petroleum ether for 2 days to defat, collecting residue, extracting with water at 85 deg.C under heating at a ratio of 1kg to 20L for 3 hr, filtering the extractive solution, concentrating the filtrate to 1/10, precipitating polysaccharide with 4 times of anhydrous ethanol at 4 deg.C overnight, centrifuging, retaining precipitate, re-dissolving in hot water, precipitating protein with trichloroacetic acid at 4 deg.C overnight, centrifuging, collecting supernatant, concentrating, and drying.
2. Use according to claim 1, characterized in that: the cosmetic is facial cleanser.
3. Use according to claim 1, characterized in that: the cosmetic is a cream.
4. Use according to claim 1, characterized in that: the cosmetic is facial mask.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1883619A (en) * | 2006-05-29 | 2006-12-27 | 广东中科药物研究有限公司 | Pharmaceutical composition of viola yedoensis and method for preparing same |
| CN110192996A (en) * | 2019-06-27 | 2019-09-03 | 福建师范大学 | A kind of viola mandshurica moisturizer and preparation method thereof and the moisturizing moisturizing sleep mask containing the moisturizer |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1883619A (en) * | 2006-05-29 | 2006-12-27 | 广东中科药物研究有限公司 | Pharmaceutical composition of viola yedoensis and method for preparing same |
| CN110192996A (en) * | 2019-06-27 | 2019-09-03 | 福建师范大学 | A kind of viola mandshurica moisturizer and preparation method thereof and the moisturizing moisturizing sleep mask containing the moisturizer |
Non-Patent Citations (4)
| Title |
|---|
| Effects of Aloe and Violae herba Extract on the Anti-oxidant,Anti-inflammatory, Anti-wrinkle and Whitening;Chang-Hun Kim et al;《The Journal of Korean Oriental Medical Ophthalmology &Otolaryngology & Dermatology》;20101231;第23卷(第1期);第23-43页 * |
| 中草药对于痤疮病原菌与黑色素生成的影响;曾文楷;《静宜大学应用化学研究所学位论文》;20101231;摘要 * |
| 天然活性成分及奈米化技术与纺织之应用;林千惠等;《第五届两岸纺织科技研讨会论文集》;20130516;第107-115页 * |
| 紫花地丁多糖提取工艺优化;袁会成等;《中兽医医药杂志》;20191231;第38卷(第04期);第61-63页 * |
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