CN117327207A - Tea branch citrus peel cell wall polysaccharide and preparation method and application thereof - Google Patents
Tea branch citrus peel cell wall polysaccharide and preparation method and application thereof Download PDFInfo
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- CN117327207A CN117327207A CN202311282452.9A CN202311282452A CN117327207A CN 117327207 A CN117327207 A CN 117327207A CN 202311282452 A CN202311282452 A CN 202311282452A CN 117327207 A CN117327207 A CN 117327207A
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- cell wall
- citrus
- peel
- wall polysaccharide
- tea branch
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 100
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 100
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- 241000207199 Citrus Species 0.000 title claims abstract description 56
- 235000020971 citrus fruits Nutrition 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 241001122767 Theaceae Species 0.000 claims abstract description 57
- 241001672694 Citrus reticulata Species 0.000 claims abstract description 36
- 210000004927 skin cell Anatomy 0.000 claims abstract description 28
- 210000003491 skin Anatomy 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 23
- 229920002472 Starch Polymers 0.000 claims abstract description 20
- 235000019698 starch Nutrition 0.000 claims abstract description 20
- 239000008107 starch Substances 0.000 claims abstract description 20
- 238000001035 drying Methods 0.000 claims abstract description 19
- 239000003814 drug Substances 0.000 claims abstract description 15
- 239000000463 material Substances 0.000 claims abstract description 14
- 238000005238 degreasing Methods 0.000 claims abstract description 13
- 239000003960 organic solvent Substances 0.000 claims abstract description 10
- 239000000843 powder Substances 0.000 claims abstract description 10
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- 238000005303 weighing Methods 0.000 claims abstract description 5
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- 238000000034 method Methods 0.000 claims description 24
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- 239000000017 hydrogel Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 15
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
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- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 2
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
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- 244000183685 Citrus aurantium Species 0.000 description 1
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010051246 Photodermatosis Diseases 0.000 description 1
- 241001093501 Rutaceae Species 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
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- HEILIGJNYTWOHU-UHFFFAOYSA-N ethanol 2-hydroxybenzoic acid Chemical compound CCO.OC(=O)C1=CC=CC=C1O HEILIGJNYTWOHU-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
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- 229930003944 flavone Natural products 0.000 description 1
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
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- 239000001814 pectin Substances 0.000 description 1
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- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 230000008845 photoaging Effects 0.000 description 1
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- 239000011573 trace mineral Substances 0.000 description 1
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- 238000010361 transduction Methods 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
Classifications
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/75—Rutaceae (Rue family)
- A61K36/752—Citrus, e.g. lime, orange or lemon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
- 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
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Birds (AREA)
- Sustainable Development (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application provides a tea branch citrus peel cell wall polysaccharide, a preparation method and application thereof. The preparation method of the citrus reticulata skin cell wall polysaccharide comprises the following steps: weighing tea branch orange peel, drying, pulverizing, and sieving; reflux-extracting tea branch and citrus peel powder in an organic solvent, rinsing filter residues, degreasing, and removing starch; rinsing and drying filter residues after starch removal to obtain a tea branch citrus peel cell wall material; extracting the tea branch orange peel cell wall material with an extraction solution, regulating the pH of the extraction solution to be neutral, concentrating, dialyzing, and freeze-drying to obtain tea branch orange peel cell wall polysaccharide. The tea branch citrus peel cell wall polysaccharide is prepared by a preparation method of the tea branch citrus peel cell wall polysaccharide. The application of the citrus reticulata skin cell wall polysaccharide is the application of the citrus reticulata skin cell wall polysaccharide in preparing skin care products or medicines for resisting oxidization and promoting skin injury repair.
Description
Technical Field
The application relates to the field of cell wall polysaccharide preparation, in particular to a tea branch citrus peel cell wall polysaccharide, a preparation method and application thereof.
Background
Chachiensis is a cultivar of Citrus aurantium of Rutaceae, citrus. The tea branch citrus peel can be divided into tea branch citrus embryo peel, citrus green peel (green peel), reddish peel (two red peel) and red peel (red peel) according to the harvesting period and the maturity of the tea branch citrus peel, and also comprises dried mature peel after aging, namely the pericarpium citri reticulatae. The pericarpium citri reticulatae is a main source of the pericarpium citri reticulatae serving as a medicinal material in the Guangdongdao, and is also a Chinese medicine variety for both medicine and food approved by the national ministry of health. The tea branch orange peel contains various active ingredients, and the research and development of volatile oil, flavone and trace elements are more at present. However, plant cell wall structures which have been considered inactive in plants in recent years have been increasingly paid attention to by researchers as research is conducted. As one of the main differences between plant cells and animal cells, cell walls exert various functions not only in the processes of cell metabolism, substance transport between cells, signal induction and transduction, cell fate determination, responses to pathogens and stress, and the like, but also in these processes, influence cell differentiation, defense, and plant growth and development. However, cell wall polysaccharides, which are the main chemical components of cell walls, are also attracting more and more attention.
Cell wall polysaccharides can be broadly divided into pectin, hemicellulose and cellulose. These polysaccharides together constitute the major structure of the cell wall, ensuring plant integrity, supporting and regulating life processes. Cell wall polysaccharides are also a major component of dietary fiber, playing a non-negligible role in our diet today, and are also considered a promising biomedical and food industry feedstock. In recent years, attention has been paid to the fact that they have biological activities such as antioxidation, antibacterial, anticoagulation and antidiabetic. In addition, it has adhesion, biodegradability, non-toxicity and various physicochemical properties.
The skin is the largest organ of the human body and mainly protects the body from potential damages such as microorganisms, radiation, mechanical damage and the like, so the skin is extremely vulnerable to damage. Rapid healing is required after skin injury to reestablish the protective barrier, and wound healing remains an important clinical problem. However, in modern medicine, drugs capable of stimulating wound healing are still limited. The cell wall has the functions of cell differentiation, defense and metabolism in the growth and development process of plants, so that the basis for further searching for new natural compounds with activities of antioxidation, skin repair, wound healing and the like is formed. In fact, it has been reported in the literature that plant polysaccharides can accelerate healing, regulate inflammatory phases, scavenge free radicals and stimulate proliferation of dermal fibroblasts and keratinocytes. However, to date, no related research report has been made on the extraction of polysaccharides with antioxidant and wound healing activity from the cell wall of citrus reticulata.
Disclosure of Invention
The embodiment of the application provides a tea branch citrus peel cell wall polysaccharide, a preparation method and application thereof, so as to solve the problems in the related art, and the technical scheme is as follows:
in a first aspect, an embodiment of the present application provides a method for preparing a citrus reticulata skin cell wall polysaccharide, including:
weighing tea branch orange peel, drying, pulverizing, and sieving;
reflux-extracting tea branch and citrus peel powder in an organic solvent, rinsing filter residues, degreasing, and removing starch;
rinsing and drying filter residues after starch removal to obtain a tea branch citrus peel cell wall material;
extracting the tea branch orange peel cell wall material with an extraction solution, regulating the pH of the extraction solution to be neutral, concentrating, dialyzing, and freeze-drying to obtain tea branch orange peel cell wall polysaccharide.
In one embodiment, the tea branch orange peel is weighed, dried, crushed and screened by a 40-60 mesh screen; wherein the drying temperature is 50-60 ℃ and the drying time is 12-24 hours.
In one embodiment, the organic solvent is ethanol, the concentration of the ethanol is 80-95%, and the feed-liquid ratio is 1:15-25;
reflux extraction temperature is 60-90 ℃;
degreasing for 12-15 hours at the temperature of 4-10 ℃, wherein the degreasing solvent is chloroform and methanol according to the mass ratio of 2:1, the ratio of the feed solution to the liquid is 1:10-20 parts of a base;
treating with dimethyl sulfoxide to remove starch, wherein the concentration of dimethyl sulfoxide is 80-95%, the treatment time is 8-10 hours, and the feed-liquid ratio is 1:10-20.
in one embodiment, the powder of citrus reticulata peel is extracted in an organic solvent under reflux for 2-5 times, each for 1-3 hours, and the residue is rinsed 3-5 times and then defatted, followed by treatment with dimethyl sulfoxide to remove starch;
and rinsing the filter residue after removing the starch for 3-5 times.
In one embodiment, the solvent used for rinsing the filter residue in degreasing after rinsing the filter residue and rinsing the filter residue in rinsing the filter residue after removing starch is acetone or ethanol with the concentration of more than 80%;
the extraction solution is sodium carbonate solution containing sodium borohydride, wherein the sodium carbonate is 0.05mol/L and the sodium borohydride is 0.02mol/L.
In one embodiment, the extraction is performed with an extraction solution at a temperature of 20-30deg.C for a time of 2-4 hours with a feed to liquid ratio of 1:15-25;
dialyzing with 6-8kDa dialysis bag;
the drying is vacuum drying or freeze drying.
In one embodiment, the source of the tea branch citrus peel is tea branch citrus peel, green peel, red peel, or dried orange peel.
In a second aspect, an embodiment of the present application provides a citrus reticulata skin cell wall polysaccharide, which is prepared by the method for preparing any one of the citrus reticulata skin cell wall polysaccharides.
In a third aspect, embodiments of the present application provide an application of a citrus reticulata skin cell wall polysaccharide, where the application is an application of the citrus reticulata skin cell wall polysaccharide in preparing a skin care product or a medicine for resisting oxidation and promoting repair of skin injury.
In one embodiment, the skin care product comprises a lotion, or essence, or hydrogel, or essence, or cream, or emulsion, or mask; the concentration of the tea branch citrus peel cell wall polysaccharide in the skin care product is 2-15%;
the dosage forms of the medicine comprise solution, aerosol, spray, ointment or film.
The advantages or beneficial effects in the technical scheme at least comprise:
the preparation method of the tea branch citrus peel cell wall polysaccharide provided by the invention is innovative and optimized for extracting the plant peel cell wall polysaccharide, and compared with the traditional preparation method of the peel cell wall polysaccharide, the tea branch citrus peel cell wall polysaccharide can provide a polysaccharide raw material with better antioxidant and wound healing activities for the market more pertinently. The invention greatly promotes the comprehensive development and utilization of the tea branch citrus peel and widens the application prospect of the tea branch citrus peel in the aspects of skin care products and medicines.
The preparation method of the tea branch citrus peel cell wall polysaccharide provided by the invention is simple and quick, and experimental researches show that the cell wall polysaccharide has the effects of resisting oxidization and healing wounds, so that the tea branch citrus peel cell wall polysaccharide can be applied to biomedical auxiliary materials for resisting oxidization of skin and healing wounds.
The tea branch citrus peel cell wall polysaccharide prepared by the method has the sugar content of 60-85%, the protein content of 2.0-8.5%, the total phenol content of 0.3-1.5% and the uronic acid content of 40-62% through detection. The antioxidant has good capability of scavenging the hydroxyl free radical in an in-vitro antioxidant activity test, so that the antioxidant can be applied to skin care products or medicine raw materials with antioxidant effect; meanwhile, in the proliferation and migration of human keratinocytes and the wound healing test, the tea branch citrus peel cell wall polysaccharide is found to be capable of remarkably promoting the proliferation and migration of human keratinocytes, accelerating the wound healing and epithelialization processes, and is suitable for preparing skin care products or medicines for promoting the skin to update and repair, especially wound healing.
The foregoing summary is for the purpose of the specification only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present application will become apparent by reference to the drawings and the following detailed description.
Drawings
In the drawings, the same reference numerals refer to the same or similar parts or elements throughout the several views unless otherwise specified. The figures are not necessarily drawn to scale. It is appreciated that these drawings depict only some embodiments according to the disclosure and are not therefore to be considered limiting of its scope.
FIG. 1 is a graph of the hydroxyl radical scavenging ability of a cell wall polysaccharide.
FIG. 2 is a graph showing the effect of CCK8 on HaCaT cell proliferation of cell wall polysaccharide.
Fig. 3 is a microscopic image of HaCaT cell wound healing with cell wall polysaccharide treatment in a cell scoring experiment.
Fig. 4 is a graph comparing wound healing rates of cell wall polysaccharide in scratch experiments with those of control groups.
Fig. 5 is a graph comparing the extent of wound healing of cell wall polysaccharides in a subject wound healing assay with a control group.
Detailed Description
Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present application. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.
In a first aspect, an embodiment of the present application provides a method for preparing a citrus reticulata skin cell wall polysaccharide, including:
step 101, weighing tea branch orange peel, drying, crushing and screening.
In one embodiment, the source of the citrus unshiu peel is citrus unshiu embryo peel, or green peel, or red peel, or two red peel, or dried orange peel over years.
In one embodiment, the tea branch orange peel is weighed, dried, crushed and screened by a 40-60 mesh screen; wherein the drying temperature is 50-60 ℃ and the drying time is 12-24 hours.
And 102, reflux-extracting tea branch orange peel powder in an organic solvent, rinsing filter residues, degreasing, and then removing starch.
In one embodiment, the organic solvent is ethanol, the ethanol concentration is 80-95%, and the feed-to-liquid ratio is 1:15-25;
in one embodiment, the temperature of the reflux extraction is 60-90 ℃.
In one embodiment, degreasing is carried out for 12-15 hours at the temperature of 4-10 ℃, and the degreasing solvent is chloroform and methanol according to the mass ratio of 2:1, the ratio of the feed solution to the liquid is 1:10-20.
in one embodiment, the starch is removed by treatment with dimethyl sulfoxide, the concentration of dimethyl sulfoxide being 80-95%, preferably 90%, the treatment time being 8-10 hours, the ratio of feed to liquid being 1:10-20.
in one embodiment, the powder of citrus reticulata peel is extracted in an organic solvent under reflux for 2-5 times, each for 1-3 hours, and the residue is rinsed 3-5 times and then defatted, followed by treatment with dimethyl sulfoxide to remove starch;
in one embodiment, the filter residue after removal of the starch is rinsed 3-5 times.
And 103, rinsing and drying filter residues after starch removal to obtain the tea branch citrus peel cell wall material.
In one embodiment, the solvent used for rinsing the filter residue in the degreasing after rinsing the filter residue in step 102 and the solvent used for rinsing the filter residue in the rinsing the filter residue after removing starch in step 103 are acetone or ethanol with a concentration of 80% or more.
And 104, extracting the citrus reticulata skin cell wall material with an extraction solution, regulating the pH of the extraction solution to be neutral, concentrating, dialyzing, and freeze-drying to obtain citrus reticulata skin cell wall polysaccharide.
In one embodiment, the extraction solution is a sodium carbonate solution containing sodium borohydride, wherein sodium carbonate is 0.05mol/L and sodium borohydride is 0.02mol/L.
In one embodiment, the extraction is performed with an extraction solution at a temperature of 20-30deg.C for a time of 2-4 hours with a feed to liquid ratio of 1:15-25.
in one embodiment, the drying is vacuum drying or freeze drying, preferably freeze drying.
In a second aspect, embodiments of the present application provide a citrus reticulata skin cell wall polysaccharide prepared by a method for preparing the citrus reticulata skin cell wall polysaccharide.
Through detection, the polysaccharide on the cell wall of the tea branch citrus peel prepared by the application has the sugar content of 60-85%, the protein content of 2.0-8.5%, the total phenol content of 0.3-1.5% and the uronic acid content of 40-62%.
The citrus reticulata skin cell wall polysaccharide has good capability of removing hydroxyl free radicals; meanwhile, the composition has obvious promotion effect on proliferation, migration and wound healing of human keratinocytes.
In a third aspect, embodiments of the present application provide an application of a citrus reticulata skin cell wall polysaccharide, where the application is an application of the citrus reticulata skin cell wall polysaccharide in preparing a skin care product or a medicine for resisting oxidation and promoting repair of skin injury.
In one embodiment, the skin care product comprises a lotion, or a serum, or a hydrogel, or a serum, or a cream, or an emulsion, or a mask; the concentration of the tea branch citrus peel cell wall polysaccharide in the skin care product is 2-15%;
the dosage forms of the medicine comprise solution, aerosol, spray, ointment or film.
Taking hydrogel as an example, the hydrogel is used as an external preparation for skin wound healing, the hydrogel comprises the tea branch citrus peel cell wall polysaccharide obtained by adopting the method, and the preparation method of the hydrogel is as follows:
and (3) weighing the citrus reticulata skin cell wall polysaccharide, dissolving the citrus reticulata skin cell wall polysaccharide in a sterilizing solvent to prepare a polysaccharide aqueous solution, adding a hydrophilic polymer, uniformly mixing, helping to dissolve on a magnetic stirrer, adding a calcium chloride or ferric trichloride solution after the solution is completely dissolved, continuously stirring for reaction, simultaneously adjusting the pH value of the solution to 7.4, and finally, until the hydrogel is converted from a flowing state to a non-flowing state, thus obtaining the hydrogel.
Preferably, the concentration of the citrus reticulata skin cell wall polysaccharide in the hydrogel is 2-15%.
Preferably, the sterilizing solvent includes, but is not limited to, pure water or PBS.
Preferably, the hydrophilic polymer includes, but is not limited to, one or more polymers selected from sodium alginate, hyaluronic acid, chitosan and derivatives thereof, cellulose and derivatives thereof.
Preferably, the concentration of the calcium chloride or ferric chloride solution is 0.5-3%.
The preparation method of the polysaccharide hydrogel applied to wound healing is simple, quick, convenient and environment-friendly.
Example 1: preparation of tea branch citrus peel cell wall polysaccharide
Drying dried ripe tea branch orange peel at 50deg.C, pulverizing, and sieving with 40 mesh sieve. Taking 100g of tea branch orange peel powder and 95% ethanol according to a feed liquid ratio of 1:20, and reflux-extracting at 80deg.C for 2 hr each for 3 times. After cooling, filtration, the residue was rinsed 3 times with 80% ethanol, and then washed with chloroform/methanol (2:1) solution in a feed ratio of 1:10, and pre-treated at 4 ℃ for 15 hours. The obtained residue is treated with a solution of dimethyl sulfoxide with the ratio of 90% to 1:10 for 8 hours. Rinsing the residue with acetone for 3 times, and lyophilizing to obtain the tea branch mandarin orange peel cell wall material.
Taking the obtained tea branch citrus peel cell wall material, and using 0.05mol/L sodium carbonate (containing 0.02mol/L sodium borohydride) solution according to a feed liquid ratio of 1: extracting at 25deg.C for 2 hr, regulating pH to neutrality, concentrating, dialyzing with 6-8kDa dialysis bag, and lyophilizing to obtain polysaccharide of pericarpium Citri Reticulatae Chachiensis cell wall.
The polysaccharide on the cell wall of the tea branch citrus peel prepared by the method has the sugar content of 62.8%, the protein content of 3.5%, the total phenol content of 0.4% and the uronic acid content of 46.7% respectively measured by a phenol sulfuric acid method, a BCA protein method, a gallic acid colorimetric method and a gas chromatography method.
Comparative example 1
The present comparative example provides a citrus peel cell wall polysaccharide, which aims to facilitate comparison of the results of the cell wall polysaccharide prepared by the conventional extraction method with the citrus peel cell wall polysaccharide of the present invention in the following experiments. The preparation method refers to the extraction method of citrus polysaccharide disclosed in Chinese patent document CN 107033256A.
The preparation method comprises the following steps: cleaning mandarin orange peel, shearing, drying in a blast constant temperature dryer at 50deg.C for 12 hr, pulverizing, and sieving with 40 mesh sieve to obtain mandarin orange peel powder. Then taking orange peel powder and mixing according to a feed liquid ratio of 1:15 is fully mixed with petroleum ether, degreased and decolored at 40 ℃, then the petroleum ether is discarded, volatilized, and soaked in 20 times distilled water for 10 minutes. Finally extracting for 10 minutes at the ultrasonic condition of 60 ℃ and 220W and 20KHZ to obtain the citrus peel cell wall polysaccharide.
Example 2: experiment for removing hydroxyl free radicals of polysaccharide on cell wall of citrus reticulata skin
Polysaccharide samples in example 1 and comparative example 1 were each prepared as a series of sample solutions with a concentration gradient (0.1,0.2,0.4,0.8,1.2and 1.6mg/mL), while preparing a control (Vc) solution with the same concentration gradient. 1ml of a solution to be tested under each concentration is taken, 1ml of ferrous sulfate solution (9 mmol/L), 1ml of salicylic acid-ethanol solution (9 mmol/L) and 1ml of hydrogen peroxide solution (9 mmol/L) are sequentially added, the mixture is uniformly mixed, the mixture is incubated at 37 ℃ for 30 minutes, and then centrifuged at 4000r/min for 10 minutes, the supernatant is taken to measure absorbance at 510nm, and deionized water is used as a blank control. Hydroxyl radical Scavenging (SR) was calculated from the formula. SR (%) = (A0-a)/a0×100. Wherein: a0 is the absorbance of the blank, and a is the absorbance of the sample.
The results are shown in fig. 1, and indicate that the citrus peel cell wall polysaccharide prepared in example 1 has better hydroxyl radical scavenging ability in the experimental concentration range than the citrus peel cell wall polysaccharide prepared in the traditional method, and the scavenging rate is proportional to the polysaccharide concentration.
Example 3: effect of Chamaecyparis tanguticum wall polysaccharide on skin keratinocyte proliferation
Human immortalized keratinocytes (HaCaT cells) are immortalized cell lines spontaneously transformed from adult epidermal cells, which participate in the formation of a physical barrier to the skin, protect the skin, and are widely used in the study of skin injury repair, photoaging and a range of skin diseases.
The effect of the tea branch citrus peel cell wall polysaccharide of example 1 on keratinocyte proliferation was examined in this experiment using the CCK8 method. HaCaT cells were first grown in 4X 10 cells 3 The density of cells/well was seeded in 96-well plates, and 100. Mu.L of cell suspension (4X 10) was added to each well 4 cells/mL), after 24h incubation, the supernatant was discarded, 100 μl of polysaccharide samples (25, 50, 100, 200 μg/mL) from example 1 and comparative example 1, respectively, at different concentrations, 3 wells per concentration, after 24h incubation, 10 μl of CCK8 solution per well was added, mixed well, and incubated for 2h. Absorbance was measured at 450nm using a microplate reader.
The experimental results are shown in fig. 2, and compared with the citrus peel cell wall polysaccharide in comparative example 1, the citrus peel cell wall polysaccharide in example 1 has a stronger promoting effect on keratinocyte proliferation in the detection concentration range, and the cell proliferation activity is firstly increased and then decreased with the increase of the concentration. Wherein, the tea branch citrus peel cell wall polysaccharide has the most obvious proliferation promoting effect compared with a blank control group at the optimal proliferation concentration of 100 mug/mL, and the cell proliferation activity reaches 160.9 percent.
Example 4: wound healing Activity of Chazhi citrus peel cell wall polysaccharide
The cell scarification test is an experimental method for determining the migration movement and repair capacity of cells, and is used for researching the mechanism of skin wound healing by establishing an in-vitro wound healing model. The experiment uses the method to obtain HaCaT cells at a ratio of 4×10 5 The density of cells/well was seeded in 12-well plates, 1ml of cell suspension was added to each well, and after 24h incubation, 10. Mu.mThe L gun head was scored vertically on each well, the medium was discarded, the PBS was used for 3 washes, the experimental group was added with serum-free medium containing 100. Mu.g/mL tea branch orange peel cell wall polysaccharide, and the control group was added with blank medium. Cell growth was determined by microscopic photography, scratch areas at 0, 24, 48h were compared and the rate of healing of the scratch areas was calculated using ImageJ software.
The results of the cell scarification experiments are shown in fig. 3 and 4, and the wound healing rate of the citrus peel cell wall polysaccharide of example 1 was higher at 24 and 48 hours after administration and higher by 13% than that of the control group at 48 hours, compared with the citrus peel cell wall polysaccharide. Compared with the traditional citrus peel cell wall polysaccharide, the citrus peel cell wall polysaccharide has more remarkable effect of promoting keratinocyte proliferation and migration, and also has better skin repair and wound healing activities.
Example 5: preparation of tea branch citrus peel cell wall polysaccharide hydrogel and evaluation of wound healing efficacy
The sample polysaccharide 50mg in example 1 and comparative example 1 was weighed and dissolved in 1mL of sterilized PBS (pH 7.4) to prepare aqueous polysaccharide solutions, 4% aqueous sodium alginate solution was added to mix well, the dissolution was assisted on a magnetic stirrer, and after complete dissolution, 2% calcium chloride solution was added to continue stirring reaction until the hydrogel was changed from a flowing state to a non-flowing state, to prepare hydrogels.
Group of subjects: the skin of the selected hand or arm is wounded, and the size of the wound is close to 30 persons, and the age is between 20 and 30 years. The animals were randomized into 3 groups of 10 persons each, two of which were experimental groups and one of which was control group. The test group was applied with the same amount of the hydrogel preparation described above to the wound area every day (once a day and a night), the control group was applied with the same amount of the hydrogel preparation containing no polysaccharide sample to the wound area, and the wound healing condition of each group of subjects was compared after one week of the test (no other drugs or agents for promoting wound healing were administered to each group during the test).
The test results are shown in fig. 5, and the results show that the test subjects have better skin wound healing degree compared with the control subjects, and meanwhile, the hydrogel containing the citrus reticulata skin cell wall polysaccharide has better skin wound healing effect compared with the citrus reticulata skin cell wall polysaccharide hydrogel.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
The foregoing is merely specific embodiments of the present application, but the scope of the present application is not limited thereto, and any person skilled in the art can easily think of various changes or substitutions within the technical scope of the present application, and these should be covered in the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (10)
1. A method for preparing a citrus reticulata skin cell wall polysaccharide, which is characterized by comprising the following steps:
weighing tea branch orange peel, drying, pulverizing, and sieving;
reflux-extracting tea branch and citrus peel powder in an organic solvent, rinsing filter residues, degreasing, and removing starch;
rinsing and drying filter residues after starch removal to obtain a tea branch citrus peel cell wall material;
extracting the tea branch orange peel cell wall material with an extraction solution, regulating the pH of the extraction solution to be neutral, concentrating, dialyzing, and freeze-drying to obtain tea branch orange peel cell wall polysaccharide.
2. The method for preparing the citrus unshiu peel cell wall polysaccharide according to claim 1, wherein the citrus unshiu peel is weighed, dried, crushed and screened by a 40-60 mesh screen; wherein the drying temperature is 50-60 ℃ and the drying time is 12-24 hours.
3. The method for preparing the citrus reticulata skin cell wall polysaccharide according to claim 1, wherein the organic solvent is ethanol, the concentration of the ethanol is 80-95%, and the feed-liquid ratio is 1:15-25;
reflux extraction temperature is 60-90 ℃;
degreasing for 12-15 hours at the temperature of 4-10 ℃, wherein the degreasing solvent is chloroform and methanol according to the mass ratio of 2:1, the ratio of the feed solution to the liquid is 1:10-20 parts of a base;
treating with dimethyl sulfoxide to remove starch, wherein the concentration of dimethyl sulfoxide is 80-95%, the treatment time is 8-10 hours, and the feed-liquid ratio is 1:10-20.
4. the method for preparing the citrus reticulata skin cell wall polysaccharide according to claim 3, wherein citrus reticulata skin powder is extracted by reflux in an organic solvent for 2 to 5 times, each time for 1 to 3 hours, and the residue is rinsed for 3 to 5 times and then defatted, followed by treatment with dimethyl sulfoxide to remove starch;
and rinsing the filter residue after removing the starch for 3-5 times.
5. The method for preparing the tea branch citrus peel cell wall polysaccharide according to claim 1, wherein the solvent used for rinsing the filter residue in degreasing after rinsing the filter residue and for rinsing the filter residue in rinsing the filter residue after removing starch is acetone or ethanol with the concentration of more than 80%;
the extraction solution is sodium carbonate solution containing sodium borohydride, wherein the sodium carbonate is 0.05mol/L and the sodium borohydride is 0.02mol/L.
6. The method for preparing the citrus reticulata skin cell wall polysaccharide according to claim 1, wherein the extraction is carried out by using an extraction solution at 20-30 ℃ for 2-4 hours, and the feed-liquid ratio is 1:15-25;
dialyzing with 6-8kDa dialysis bag;
the drying is vacuum drying or freeze drying.
7. The method for preparing a cell wall polysaccharide of citrus unshiu peel according to any one of claims 1 to 6, wherein said citrus unshiu peel source is citrus unshiu peel, green peel, red peel, or dried orange peel for years.
8. A tea branch citrus peel cell wall polysaccharide, characterized in that it is prepared by the preparation method of the tea branch citrus peel cell wall polysaccharide according to any one of claims 1 to 7.
9. The application of the citrus reticulata skin cell wall polysaccharide is characterized in that the application is the application of the citrus reticulata skin cell wall polysaccharide in the preparation of skin care products or medicines for resisting oxidization and promoting skin injury repair according to claim 8.
10. The use of the citrus reticulata skin cell wall polysaccharide according to claim 9, wherein the skin care product comprises a lotion, or a serum, or a hydrogel, or a serum, or a cream, or an emulsion, or a mask; the concentration of the tea branch citrus peel cell wall polysaccharide in the skin care product is 2-15%;
the dosage forms of the medicine comprise solution, aerosol, spray, ointment or film.
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