CN110317279B - Herba Centellae polysaccharide, application of herba Centellae polysaccharide as plant source antiseptic, and application of herba Centellae polysaccharide in cosmetics - Google Patents
Herba Centellae polysaccharide, application of herba Centellae polysaccharide as plant source antiseptic, and application of herba Centellae polysaccharide in cosmetics Download PDFInfo
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- CN110317279B CN110317279B CN201910762107.2A CN201910762107A CN110317279B CN 110317279 B CN110317279 B CN 110317279B CN 201910762107 A CN201910762107 A CN 201910762107A CN 110317279 B CN110317279 B CN 110317279B
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- 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
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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Abstract
The invention discloses a centella asiatica polysaccharide, application of the centella asiatica polysaccharide as a plant-derived preservative and application of the centella asiatica polysaccharide in cosmetics. The centella asiatica polysaccharide is prepared by the following method: weighing herba Centellae, sequentially using petroleum ether and 95% ethanol for reflux degreasing, filtering, evaporating solvent from filter residue, heating and extracting with distilled water, filtering, concentrating the filtrate to 1/5 volume, filtering again, adding 4 times volume of anhydrous ethanol, stirring uniformly, standing, precipitating with ethanol, filtering, washing precipitate with anhydrous ethanol, acetone and ether sequentially, redissolving with distilled water, repeatedly precipitating with ethanol for 1 time, removing protein by Sevag method, dialyzing, and freeze drying. The experimental result shows that the asiatic pennywort herb polysaccharide provided by the invention has excellent inhibitory activity on bacteria and fungi, is high in safety to skin, does not cause skin allergy, can be used as a plant-derived preservative, can be further used for preserving cosmetics, and has the advantages of being natural, effective, mild and non-irritant.
Description
Technical Field
The invention belongs to the field of cosmetics, and relates to application of centella asiatica polysaccharide in cosmetics, in particular to centella asiatica polysaccharide, application of the centella asiatica polysaccharide as a plant-derived preservative and application of the centella asiatica polysaccharide in cosmetics.
Background
The research finds that natural preservative substances can be extracted from various plants in the nature, and have obvious inhibiting effect on bacteria and/or fungi. These natural preservative substances include alkaloids, flavones, coumarins, saponins, etc.
Since natural preservative substances are generally less harmful and safer to the skin, the development of natural preservative plants from plants for use as preservatives and their addition to cosmetics has been a hot spot in the development of cosmetic preservatives.
Centella asiatica is a dry whole plant of centella asiatica of Umbelliferae and contains various triterpenoid active ingredients including asiaticoside, madecassoside, asiatic acid, madecassic acid and the like, and clinical and experimental studies show that the centella asiatica can effectively promote the healing of skin wounds and has an inhibiting effect on pathological scars formed by excessive healing. Modern pharmacological research shows that centella asiatica can promote cell growth, accelerate wound healing and repair skin injury, and has the effects of resisting oxidation, resisting ulcer, diminishing inflammation, protecting nerve cells and the like. Therefore, small molecular compounds such as asiaticoside, madecassoside, asiatic acid and madecassic acid in centella asiatica are widely used in cosmetic products.
Centella asiatica polysaccharide is a macromolecular compound in centella asiatica, and the report that the centella asiatica polysaccharide has antibacterial and antifungal activities is not found yet.
Disclosure of Invention
The invention aims at providing the asiatic pennywort herb polysaccharide, secondly aims at providing the use of the asiatic pennywort herb polysaccharide as a plant-derived preservative, and thirdly aims at providing the application of the asiatic pennywort herb polysaccharide in cosmetics.
The above purpose of the invention is realized by the following technical scheme:
a centella asiatica polysaccharide is prepared by the following steps: weighing the centella asiatica whole plant, sequentially using petroleum ether with a distillation range of 60-90 ℃ and 95% ethanol for reflux degreasing, filtering, volatilizing a solvent from filter residues, heating and extracting by using distilled water, filtering, concentrating a filtrate to 1/5 volumes, filtering again, adding absolute ethanol with a volume 4 times that of the filtrate, uniformly stirring, standing, precipitating with ethanol, performing suction filtration, sequentially washing and precipitating by using absolute ethanol, acetone and diethyl ether, redissolving by using distilled water, repeatedly precipitating with ethanol for 1 time, performing suction filtration, sequentially washing and precipitating by using absolute ethanol, acetone and diethyl ether, removing protein by a Sevag method, dialyzing, and freeze-drying.
Preferably, the feed-liquid ratio of the degreasing step is 1; 5.
preferably, the two solvents are separately degreased and refluxed for 3 h.
Preferably, the feed-liquid ratio of the water extraction step is 1; 10.
preferably, the water is extracted 2 times for 2h each time.
Preferably, dialysis is performed using a dialysis bag with a cut-off of 5000 u.
Preferably, dialysis is performed for 24 h.
Use of the above centella asiatica polysaccharide as a plant-derived preservative.
The application of the centella asiatica polysaccharide as a cosmetic preservative.
The technical effects are as follows:
the invention provides asiatic pennywort herb polysaccharide, and experimental results show that the asiatic pennywort herb polysaccharide provided by the invention has excellent inhibitory activity on bacteria and fungi, is high in safety on skin, does not cause skin allergy, can be used as a plant-derived preservative and further can be used for preserving cosmetics, and has the advantages of being natural, effective, mild and non-irritant.
Drawings
FIG. 1 is an SEM image of polysaccharide prepared in example 1, and it can be seen from the SEM image that the polysaccharide has low regularity, a large number of random cross structures among molecules and more branched chains.
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: extraction and preservative property test of centella asiatica polysaccharide
First, experimental material
Centella asiatica is purchased in the Bozhou Chinese medicinal material market, cleaned, air-dried, crushed and sieved by a 60-mesh sieve for later use.
The test bacteria include bacteria and fungi, wherein: the bacteria include Escherichia coli, Bacillus proteus, Pseudomonas aeruginosa, streptococcus, Staphylococcus aureus, and Bacillus subtilis, and the fungi include Aspergillus niger and yeast. The bacteria adopts beef extract peptone culture medium, the aspergillus niger adopts potato glucose agar culture medium, and the yeast adopts yeast extract peptone glucose agar culture medium.
Second, Experimental methods
1. Preparation and qualitative detection of centella asiatica polysaccharide
Weighing herba Centellae whole plant, sequentially performing reflux degreasing for 3h by using 60-90 ℃ distillation range petroleum ether and 95% ethanol (volume percentage content) according to a material-liquid ratio of 1:5 (1kg whole plant uses 5L solvent), filtering, volatilizing the solvent from filter residue, heating and extracting 2 times by using distilled water at 85 ℃ according to a material-liquid ratio of 1:10, each time for 2h, combining extract, filtering, concentrating the filtrate to 1/5 volume, filtering again, adding 4 times of volume of absolute ethanol, stirring uniformly, standing, precipitating with ethanol for 12h, performing suction filtration, sequentially washing and precipitating with absolute ethanol, acetone and diethyl ether for 2 times, re-dissolving with distilled water, repeatedly precipitating with ethanol for 1 time, performing suction filtration, sequentially washing and precipitating with absolute ethanol, acetone and diethyl ether for 2 times, performing Sevag method protein removal, dialyzing for 24h by using a dialysis bag with the molecular weight of 5000u, and freeze-drying.
Molish reaction: dissolving 1g of the polysaccharide prepared by the method in 20mL of distilled water to prepare a polysaccharide solution, adding 1mL of alpha-naphthol into 1mL of the polysaccharide solution in a test tube, adding 1mL of concentrated sulfuric acid, and obtaining a red-purple ring with a positive Molish reaction.
Phenol-sulfuric acid reaction: 1mL of the polysaccharide solution is put into a test tube, 1mL of phenol is added, 5mL of concentrated sulfuric acid is added, a yellow-brown compound appears, and the reaction of phenol-sulfuric acid is positive.
FIG. 1 is an SEM image of the above polysaccharide, which shows low regularity, numerous random cross-links between molecules, and many branched chains.
2. Inhibition of bacteria and fungi by centella asiatica polysaccharides
2.1 preparation of polysaccharide solutions and suspensions
The centella asiatica polysaccharide prepared in example 1 is weighed, dissolved by distilled water to prepare solutions with different concentrations, and then diluted to other concentrations. The test bacteria are placed in a slant culture medium, the bacteria are activated for 24h at 37 ℃, the mould is activated for 48h at 28 ℃, and the yeast is activated for 24h at 30 ℃. Selecting bacterial lawn, and preparing into 1 × 10 pieces in sterile water6～1×108CFU/mL of bacterial suspension.
2.2 qualitative detection of bacteriostatic action
Slowly pouring the sterilized culture medium into a sterile culture dish under the aseptic condition, cooling and solidifying, and respectively and uniformly coating 0.2mL of test bacterial suspension on a solid plate. Placing a circular filter paper sheet with the diameter of 12mm and made of sterilized filter paper into a 10mg/mL polysaccharide solution for soaking for 1h, drying and then gently placing the circular filter paper sheet on the surface of a culture medium, placing the culture medium inoculated with bacteria at 37 ℃ for inverted culture for 24h, placing the culture medium inoculated with aspergillus niger at 28 ℃ for inverted culture for 48h, and placing the culture medium inoculated with saccharomycetes at 30 ℃ for inverted culture for 24 h. The product without the inhibition zone is negative, the product with the inhibition zone is positive, and the diameter of the inhibition zone is measured by a cross method. 3 portions were worked up in parallel. At the same time, 1 part of blank control containing no polysaccharide is provided.
The results are shown in Table 1.
TABLE 110 mg/mL Centella polysaccharide zone diameter of inhibition
| Bacterial species name | Diameter of bacteriostatic circle (mm) |
| Escherichia coli | 18.5±1.4 |
| Pseudomonas aeruginosa | 19.2±1.6 |
| Proteobacteria | 20.8±1.4 |
| Streptococcus sp | 19.6±1.2 |
| Staphylococcus aureus | 18.1±1.3 |
| Bacillus subtilis | 18.8±1.5 |
| Aspergillus niger | 25.5±1.4 |
| Yeast | 24.8±1.6 |
| Blank control | 12 (remark: filter)Paper diameter, no zone of inhibition) |
The detection result shows that the asiatic pennywort herb polysaccharide has obvious inhibition effect on bacteria and fungi.
2.3 determination of Minimum Inhibitory Concentration (MIC)
Preparing herba Centellae polysaccharide into 0.5, 0.25, 0.125, 0.0625, and 0.0313mg/mL solutions with distilled water, transferring 1mL solution into different culture media respectively, mixing polysaccharide solution and culture medium, solidifying, and cooling. Respectively and uniformly coating 0.2mL of test bacteria suspension on a solid plate, placing a culture medium inoculated with bacteria at 37 ℃ for inverted culture for 24h, placing a culture medium inoculated with aspergillus niger at 28 ℃ for inverted culture for 48h, and placing a culture medium inoculated with microzyme at 30 ℃ for inverted culture for 24 h. "-" represents the growth of a sterile colony, "+" represents the growth of a colony, and the lowest concentration of no colony is the Minimum Inhibitory Concentration (MIC).
The results are shown in Table 2.
TABLE 2 Minimum Inhibitory Concentration (MIC) assay results
The detection result shows that the Minimum Inhibitory Concentration (MIC) of the asiatic pennywort herb polysaccharide to bacteria is 0.25mg/mL, the Minimum Inhibitory Concentration (MIC) to fungi is 0.125mg/mL, and the asiatic pennywort herb polysaccharide has obvious inhibitory action to both bacteria and fungi.
Example 2: allergy test (Patch test)
The test substance was the centella asiatica polysaccharide prepared in example 1.
30 healthy volunteers, 15 male and 15 female, were selected as subjects according to the technical Specification for cosmetic safety (2015 edition). After bathing, 0.025g of the test object is placed in a small chamber of a spot tester, the spot tester with the test object is attached to the back or the curved side of the forearm of the subject by using a hypoallergenic adhesive tape, and the test object is lightly pressed by a palm to be uniformly attached to the skin for 24 hours.
Skin reactions were observed according to the criteria of table 3 30min (after disappearance of the indentation), 24h and 48h after removal of the test article plaque test, respectively, and the observation results were recorded and evaluated with reference to table 3.
TABLE 3 skin response grading Standard for skin Enclosed Patch test
As a result, 30 healthy volunteers were negative, and the rate of negativity was 100%.
The experimental result shows that the asiatic pennywort herb polysaccharide provided by the invention has excellent inhibitory activity on bacteria and fungi, is high in safety to skin, does not cause skin allergy, can be used as a plant-derived preservative, can be further used for preserving cosmetics, and has the advantages of being natural, effective, mild and non-irritant.
Claims (1)
1. Use of centella asiatica polysaccharides for the preparation of a plant-derived preservative inhibiting bacteria such as escherichia coli, proteus vulgaris, pseudomonas aeruginosa, streptococcus, staphylococcus aureus and bacillus subtilis, and fungi such as aspergillus niger and yeast, the centella asiatica polysaccharides being prepared by the following method: weighing the whole centella asiatica, respectively refluxing and degreasing for 3h by using petroleum ether with a distillation range of 60-90 ℃ and ethanol with a volume percentage content of 95% in sequence, filtering, evaporating solvent from filter residues, heating and extracting for 2 times by using distilled water at 85 ℃ after the filter residues volatilize, 2h each time, combining extracting solutions, filtering, concentrating filtrate to 1/5 volume, filtering again, adding absolute ethyl alcohol with 4 times of volume, uniformly stirring, standing and precipitating with ethanol for 12h, performing suction filtration, sequentially washing and precipitating with absolute ethyl alcohol, acetone and ether for 2 times, re-dissolving with distilled water, repeatedly precipitating with ethanol for 1 time, performing suction filtration, sequentially washing and precipitating with absolute ethyl alcohol, acetone and ether for 2 times, removing protein by a Sevag method, dialyzing for 24h by using a dialysis bag with a molecular weight cutoff of 5000u, and performing freeze drying to obtain the centella asiatica.
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| CN101396384B (en) * | 2007-09-28 | 2012-11-21 | 天津天士力现代中药资源有限公司 | Asiatic centella extract and preparation methode thereof |
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| "Effects of Centella asiatica extract on antioxidant status andliver metabolome of rotenone‐treated rats using GC–MS";Thidarat Intararuchikul 等;《Biomedical Chromatography》;20190228;第33卷(第2期);第1697-1700页 * |
| "积雪草中降血糖多糖的研究";王雪松 等;《中国药学杂志》;20051130;第40卷(第22期);第e4395页 * |
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