CN114404444B - Polysaccharide composition for promoting expression of aquaporin, and preparation method and application thereof - Google Patents
Polysaccharide composition for promoting expression of aquaporin, and preparation method and application thereof Download PDFInfo
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- CN114404444B CN114404444B CN202210097933.1A CN202210097933A CN114404444B CN 114404444 B CN114404444 B CN 114404444B CN 202210097933 A CN202210097933 A CN 202210097933A CN 114404444 B CN114404444 B CN 114404444B
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Classifications
-
- 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
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
-
- 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
- A61P17/18—Antioxidants, e.g. antiradicals
-
- 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
-
- 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
- A61K2800/82—Preparation or application process involves sonication or ultrasonication
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dermatology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Birds (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of medicines and cosmetics, and discloses a polysaccharide composition for promoting aquaporin expression, and a preparation method and application thereof. The polysaccharide composition comprises: the polysaccharide composition can improve water transmission and/or wetting agent transmission in skin, prevent and/or treat diseases caused by skin barrier injury, prevent and/or treat drying syndrome, repair mucous membrane, relieve skin dryness and aging, and can be used for preparing medicines and daily chemicals.
Description
Technical Field
The invention belongs to the technical field of medicines and cosmetics, and particularly relates to a polysaccharide composition for promoting aquaporin expression, and a preparation method and application thereof.
Background
Aquaporin (AQP), also known as Aquaporin, is a protein located on the cell membrane (an integral membrane protein) that forms "tunnels" in the cell membrane that control the ingress and egress of water into and out of the cell, just like a "cellular pump". AQPs primarily promote transmembrane diffusion of water and various small solutes, involved in cell migration and many physiological processes. Functionally differentiated human AQPs are involved in a variety of non-infectious diseases including cancer, renal insufficiency, neurological disorders, epilepsy, dermatological disorders, metabolic syndrome, and even cardiac disorders. Cells regulate their shape and volume by utilizing the most abundant molecule in the body, water, and control dynamic balance. Thus, AQPs may play a critical role in controlling infectious disease cell volume and homeostasis. The role of AQPs in non-infectious diseases such as different tumors, cerebral edema, ischemic stroke, obesity, kidney and skin diseases, cataracts, etc. has been widely studied. AQPs in the skin are involved in skin hydration, cell proliferation and differentiation, migration, immunity and wound healing, AQP3 in the skin is involved in proliferation and migration of epidermal cells, AQP3 is the most abundant and most studied aquaporin in the skin, and its downregulation and/or mislocalization is associated with psoriasis; AQP5 is involved in water secretion in both the outer and basolateral membranes of sweat glands, AQP5 can regulate keratinocyte proliferation and differentiation. AQP5 overexpression induced HaCaT cell proliferation and dedifferentiation, but did not affect HaCaT cell apoptosis, suggesting that AQP5 mediates the balance of epidermal keratinocyte proliferation and differentiation, possibly playing an important role in maintaining keratinocyte potential.
Known actives that promote aquaporin production are relatively few, some of which are traditional Chinese medicine compositions: for example, patent documents CN202011426245.2 and CN202011426243.3 disclose a traditional Chinese medicine composition capable of promoting expression of aquaporins AQP3 and AQP9 in mask liquid, which contains okra extract, gentian extract, sargassum muticum extract and matsutake extract, CN 202011426243.3), and other substances with single effects: for example, patent document CN202110043486.7 discloses that dandelion water extract can promote aquaporin expression, patent document CN201080064285.5 discloses that long bean extract can promote aquaporin 3 expression, and patent document CN201710075675.6 discloses that coix seed extract and cactus stem extract can promote aquaporin 3 expression, wherein substances and compositions capable of simultaneously regulating and controlling various aquaporins are fewer. Therefore, an excellent substance for promoting aquaporin production is desired.
Disclosure of Invention
The object of the first aspect of the present invention is to provide a polysaccharide composition promoting the expression of aquaporins.
The object of the second aspect of the present invention is to provide a method for preparing the polysaccharide composition for promoting expression of aquaporin according to the first aspect.
The object of a third aspect of the present invention is to provide the use of the polysaccharide composition of the first aspect for promoting aquaporin expression in the preparation of a product.
The object of the fourth aspect of the invention is to provide a product.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention there is provided a polysaccharide composition for promoting expression of aquaporin, the polysaccharide composition comprising: dictyophora indusiata polysaccharide, dendrobium polysaccharide and ophiopogon polysaccharide.
Preferably, the polysaccharide composition comprises the following components in parts by mass: 2-8 parts of bamboo fungus polysaccharide, 1-6 parts of dendrobium polysaccharide and 1-6 parts of ophiopogon polysaccharide; the composition further comprises the following components in parts by mass: 4-8 parts of bamboo fungus polysaccharide, 1-4 parts of dendrobium polysaccharide and 1-4 parts of ophiopogon polysaccharide; the composition further comprises the following components in parts by mass: 4-6 parts of bamboo fungus polysaccharide, 2-4 parts of dendrobium polysaccharide and 2-4 parts of ophiopogon polysaccharide.
Preferably, the dendrobium is dendrobium candidum.
Preferably, the aquaporin is at least one of aquaporin 3 and aquaporin 5.
In a second aspect of the present invention, there is provided a method for preparing the polysaccharide composition for promoting expression of aquaporin according to the first aspect of the present invention, wherein the Dictyophora indusiata polysaccharide, the Dendrobium nobile polysaccharide and the ophiopogon polysaccharide are mixed to obtain the polysaccharide composition.
Preferably, the Dictyophora Indusiata polysaccharide, the Dendrobium nobile polysaccharide and the radix Ophiopogonis polysaccharide can be obtained by market purchase, or can be obtained by extracting Dictyophora Indusiata, dendrobium nobile and radix Ophiopogonis respectively.
Preferably, the extraction mode is at least one of acid extraction, alkali extraction, enzyme extraction, ultrafiltration, ultrasonic extraction, subcritical extraction and microwave extraction; further subcritical extraction.
Preferably, the preparation method of the bamboo fungus polysaccharide comprises the following steps: 1) Mixing Dictyophora indusiata with water, homogenizing to obtain homogenate; 2) Subcritical extraction is carried out on the homogenate, solid-liquid separation is carried out, the obtained filtrate is concentrated to a relative density of 1.20-1.40, and the bamboo fungus polysaccharide is obtained through alcohol precipitation.
Preferably, before the Dictyophora indusiata and water are mixed in the step 1), the Dictyophora indusiata is crushed into fine powder with 60-100 meshes.
Preferably, the mass ratio of the bamboo fungus to the water in the step 1) is 1 (8-20).
Preferably, the homogenization of step 1) is preceded by a soaking step.
Preferably, the soaking time is 10-30 min.
Preferably, the homogenization in step 1) is carried out by a colloid mill for 5 to 10 minutes.
Preferably, the subcritical extraction in step 2) is carried out at a temperature of 110 to 150 ℃.
Preferably, the subcritical extraction in step 2) is carried out at a pressure of 2 to 4Mpa.
Preferably, the subcritical extraction in step 2) takes 0.5 to 2 hours.
Preferably, the conditions of concentration in step 2) are 60 to 70 ℃.
Preferably, the method of alcohol precipitation in the step 2) is to add ethanol so that the final concentration of the ethanol is 78-82%.
Preferably, the alcohol precipitation in step 2) further comprises the following steps: mixing, standing for 6-12 h, removing ethanol and water, and drying the obtained polysaccharide precipitate.
Preferably, the preparation method of the dendrobium polysaccharide comprises the following steps: 1) Mixing herba Dendrobii with water, homogenizing to obtain homogenate; 2) Subcritical extraction is carried out on the homogenate, solid-liquid separation is carried out, the obtained filtrate is concentrated to a relative density of 1.20-1.40, and the dendrobium polysaccharide is obtained through alcohol precipitation.
Preferably, the dendrobium nobile in the step 1) is crushed into fine powder of 60-100 meshes before being mixed with water.
Preferably, in the step 1), the mass ratio of the dendrobium to the water is 1 (8-20).
Preferably, the homogenization of step 1) is preceded by a soaking step.
Preferably, the soaking time is 10-30 min.
Preferably, the homogenization in step 1) is carried out by a colloid mill for 5 to 10 minutes.
Preferably, the subcritical extraction in step 2) is carried out at a temperature of 110 to 150 ℃.
Preferably, the subcritical extraction in step 2) is carried out at a pressure of 2 to 4Mpa.
Preferably, the subcritical extraction in step 2) takes 0.5 to 2 hours.
Preferably, the conditions of concentration in step 2) are 60 to 70 ℃.
Preferably, the method of alcohol precipitation in the step 2) is to add ethanol so that the final concentration of the ethanol is 78-82%.
Preferably, the alcohol precipitation in step 2) further comprises the following steps: mixing, standing for 6-12 h, removing ethanol and water, and drying the obtained polysaccharide precipitate.
Preferably, the preparation method of the ophiopogon japonicus polysaccharide comprises the following steps: 1) Mixing radix Ophiopogonis with water, homogenizing to obtain homogenate; 2) Subcritical extraction is carried out on the homogenate, solid-liquid separation is carried out, the obtained filtrate is concentrated to a relative density of 1.20-1.40, and the ophiopogon polysaccharide is obtained through alcohol precipitation.
Preferably, the radix Ophiopogonis in step 1) is pulverized into 60-100 mesh fine powder before mixing with water.
Preferably, in the step 1), the mass ratio of the dwarf lilyturf tuber to the water is 1 (8-20).
Preferably, the homogenization of step 1) is preceded by a soaking step.
Preferably, the soaking time is 10-30 min.
Preferably, the homogenization in step 1) is carried out by a colloid mill for 5 to 10 minutes.
Preferably, the subcritical extraction in step 2) is carried out at a temperature of 110 to 150 ℃.
Preferably, the subcritical extraction in step 2) is carried out at a pressure of 2 to 4Mpa.
Preferably, the subcritical extraction in step 2) takes 0.5 to 2 hours.
Preferably, the conditions of concentration in step 2) are 60 to 70 ℃.
Preferably, the method of alcohol precipitation in the step 2) is to add ethanol so that the final concentration of the ethanol is 78-82%.
Preferably, the alcohol precipitation in step 2) further comprises the following steps: mixing, standing for 6-12 h, removing ethanol and water, and drying the obtained polysaccharide precipitate.
In a third aspect of the invention there is provided the use of a polysaccharide composition according to the first aspect of the invention for promoting expression of aquaporin in the preparation of a product.
Preferably, the product has any one of functions (a) to (f):
(a) Promoting aquaporin expression;
(b) Improving water transport and/or humectant transport in the skin;
(c) Preventing and/or treating diseases caused by damage to the skin barrier;
(d) Preventing and/or treating dry syndrome;
(e) Repairing mucous membrane;
(f) Relieving skin dryness and aging.
Preferably, the aquaporin is at least one of aquaporin 3 and aquaporin 5.
Preferably, the wetting agent is glycerol.
Preferably, the skin barrier damage-causing diseases include eczema, atopic dermatitis, psoriasis, ichthyosis and solar dermatitis.
Preferably, the mucous membranes include oral mucous membranes and gastrointestinal mucous membranes.
Preferably, the products include daily chemicals and pharmaceuticals.
Preferably, the pharmaceutical product is in the form of an oral solution, an oral suspension, a capsule, a tablet, a powder or a granule.
Preferably, the pharmaceutical product is suitable for oral administration.
Preferably, the daily chemical product is in the form of an aqueous solution, emulsion, ointment, cream, foam, lotion, bead, gel or spray.
Preferably, the daily chemical product is suitable for topical application.
Preferably, the product further comprises acceptable auxiliary materials in medicines or daily chemicals.
In a fourth aspect of the invention there is provided a product comprising a polysaccharide composition according to the first aspect of the invention which promotes expression of aquaporin.
Preferably, the product has any one of functions (a) to (f):
(a) Promoting aquaporin expression;
(b) Improving water transport and/or humectant transport in the skin;
(c) Preventing and/or treating diseases caused by damage to the skin barrier;
(d) Preventing and/or treating dry syndrome;
(e) Repairing mucous membrane;
(f) Relieving skin dryness and aging.
Preferably, the aquaporin is at least one of aquaporin 3 and aquaporin 5.
Preferably, the wetting agent is glycerol.
Preferably, the skin barrier damage-causing diseases include eczema, atopic dermatitis, psoriasis, ichthyosis and solar dermatitis.
Preferably, the mucous membranes include oral mucous membranes and gastrointestinal mucous membranes.
Preferably, the products include daily chemicals and pharmaceuticals.
Preferably, the pharmaceutical product is in the form of an oral solution, an oral suspension, a capsule, a tablet, a powder or a granule.
Preferably, the pharmaceutical product is suitable for oral administration.
Preferably, the daily chemical product is in the form of an aqueous solution, emulsion, ointment, cream, foam, lotion, bead, gel or spray.
Preferably, the daily chemical product is suitable for topical application.
Preferably, the product further comprises acceptable auxiliary materials in medicines or daily chemicals.
A toner comprising a polysaccharide composition that promotes aquaporin expression according to the first aspect of the invention and a humectant.
Preferably, the humectant is glycerin.
The beneficial effects of the invention are as follows:
the present invention provides a polysaccharide composition comprising: the polysaccharide composition can improve water transmission and/or wetting agent transmission in skin, prevent and/or treat diseases caused by skin barrier injury, prevent and/or treat drying syndrome, repair mucous membrane, relieve skin dryness and aging, and can be used for preparing medicines and daily chemicals.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are those obtained commercially, and the water may be deionized water, purified water or distilled water, unless otherwise specified.
The preparation method of the ophiopogon root polysaccharide, the dendrobium candidum polysaccharide and the bamboo fungus polysaccharide in the following embodiments comprises the following steps:
the preparation method of the ophiopogon japonicus polysaccharide comprises the following steps: pulverizing radix Ophiopogonis into 80 mesh fine powder, and collecting fine powder; the feed liquid ratio is 1:10 Adding deionized water, soaking for 30min, and treating with colloid mill for 8min to obtain homogenate; putting the homogenate into a subcritical extraction kettle, setting the extraction temperature to 120 ℃, setting the pressure to 3Mpa, extracting the fluid with water for 1h, and collecting filtrate; concentrating the filtrate at 65deg.C to relative density of 1.30, adding ethanol to make ethanol concentration in the solution 80%, stirring, standing for 8 hr, removing ethanol and water, and drying polysaccharide precipitate to obtain radix Ophiopogonis polysaccharide.
The preparation method of the dendrobium candidum polysaccharide comprises the following steps: pulverizing dried herba Dendrobii into 80 mesh fine powder, and collecting fine powder; the feed liquid ratio is 1:10 Adding deionized water, soaking for 30min, and treating with colloid mill for 8min to obtain homogenate; putting the homogenate into a subcritical extraction kettle, setting the extraction temperature to 120 ℃, setting the pressure to 3Mpa, extracting the fluid with water for 1h, and collecting filtrate; concentrating the filtrate at 65deg.C to relative density of 1.30, adding ethanol to reach ethanol concentration of 80%, stirring, standing for 8 hr, removing ethanol and water, and drying polysaccharide precipitate to obtain Dendrobium candidum polysaccharide.
The preparation method of the bamboo fungus polysaccharide comprises the following steps: pulverizing dried Dictyophora Indusiata into 80 mesh fine powder, and collecting the fine powder; the feed liquid ratio is 1:10 Adding deionized water, soaking for 30min, and treating with colloid mill for 8min to obtain homogenate; putting the homogenate into a subcritical extraction kettle, setting the extraction temperature to 120 ℃, setting the pressure to 3Mpa, extracting the fluid with water for 1h, and collecting filtrate; concentrating the filtrate at 65deg.C to relative density of 1.30, adding ethanol to make ethanol concentration in the solution 80%, stirring, standing for 8 hr, removing ethanol and water, and drying polysaccharide precipitate to obtain Dictyophora Indusiata polysaccharide.
Example 1 polysaccharide composition for promoting expression of aquaporin
1. A polysaccharide composition for promoting expression of aquaporin, the polysaccharide composition comprising the following components in parts by mass: 4 parts of bamboo fungus polysaccharide, 4 parts of dendrobium candidum polysaccharide and 2 parts of ophiopogon polysaccharide.
2. The preparation method of the polysaccharide composition for promoting the expression of the aquaporin comprises the step of mixing the Dictyophora Indusiata polysaccharide, the Dendrobium officinale polysaccharide and the ophiopogon japonicus polysaccharide.
Example 2 polysaccharide composition for promoting expression of aquaporin
1. A polysaccharide composition for promoting expression of aquaporin, the polysaccharide composition comprising the following components in parts by mass: 4 parts of bamboo fungus polysaccharide, 2 parts of dendrobium candidum polysaccharide and 4 parts of ophiopogon polysaccharide.
2. The preparation method of the polysaccharide composition for promoting the expression of the aquaporin comprises the step of mixing the Dictyophora Indusiata polysaccharide, the Dendrobium officinale polysaccharide and the ophiopogon japonicus polysaccharide.
Example 3 polysaccharide composition for promoting expression of aquaporin
1. A polysaccharide composition for promoting expression of aquaporin, the polysaccharide composition comprising the following components in parts by mass: 6 parts of bamboo fungus polysaccharide, 2 parts of dendrobium candidum polysaccharide and 2 parts of ophiopogon root polysaccharide.
2. The preparation method of the polysaccharide composition for promoting the expression of the aquaporin comprises the step of mixing the Dictyophora Indusiata polysaccharide, the Dendrobium officinale polysaccharide and the ophiopogon japonicus polysaccharide.
Comparative example 1A substance for promoting expression of aquaporin
A substance for promoting expression of aquaporin, comprising the following components in parts by mass: 10 parts of bamboo fungus polysaccharide.
Comparative example 2A substance for promoting expression of aquaporin
A substance for promoting expression of aquaporin, comprising the following components in parts by mass: 10 parts of dendrobium candidum polysaccharide.
Comparative example 3A substance for promoting expression of aquaporin
A substance for promoting expression of aquaporin, comprising the following components in parts by mass: 10 parts of ophiopogon polysaccharide.
Comparative example 4A polysaccharide composition for promoting expression of aquaporin
1. A polysaccharide composition for promoting expression of aquaporin, the polysaccharide composition comprising the following components in parts by mass: 7 parts of bamboo fungus polysaccharide and 3 parts of dendrobium candidum polysaccharide.
2. The preparation method of the polysaccharide composition for promoting the expression of the aquaporin comprises the step of mixing the Dictyophora indusiata polysaccharide and the Dendrobium officinale polysaccharide.
Comparative example 5A polysaccharide composition for promoting expression of aquaporin
1. A polysaccharide composition for promoting expression of aquaporin, the polysaccharide composition comprising the following components in parts by mass: 7 parts of bamboo fungus polysaccharide and 3 parts of ophiopogon polysaccharide.
2. The preparation method of the polysaccharide composition for promoting the expression of aquaporin comprises the step of mixing the Dictyophora Indusiata polysaccharide and the ophiopogon japonicus polysaccharide.
Comparative example 6A polysaccharide composition for promoting expression of aquaporin
1. A polysaccharide composition for promoting expression of aquaporin, the polysaccharide composition comprising the following components in parts by mass: 5 parts of dendrobium candidum polysaccharide and 5 parts of ophiopogon root polysaccharide.
2. The preparation method of the polysaccharide composition for promoting the expression of the aquaporin comprises the step of mixing dendrobium candidum polysaccharide and ophiopogon root polysaccharide.
Application example A toner
1. A toner comprising: polysaccharide composition of example 3 0.5g, glycerol 9.5g and deionized water 90g.
2. The preparation method of the toner comprises the following steps: mixing the polysaccharide composition of example 3, glycerol and deionized water, stirring to obtain feed liquid, and homogenizing the feed liquid at 3500r/min for 15min to obtain toner.
Effect examples
1. Effect of polysaccharide compositions/substances promoting aquaporin expression on the expression of AQP3 in skin keratinocytes HaCaT
(1) HaCaT cell model
Inoculating human immortalized epidermal cells (HaCaT cell) into DMEM medium containing 10% fetal bovine serum and 1% diabody at 37deg.C and 5% CO 2 Is cultured under the condition of (2). The cultured HaCaT cells were isolated by digestion with 0.25% insulin-EDTA at 5×10 in 96-well plates 3 Cell/well (cell/well) concentration. Adding a complete medium containing the polysaccharide composition/substance for promoting the expression of aquaporin of examples 1 to 3 and comparative examples 1 to 6 into the experimental group, and then adding the complete medium so that the final concentration of the polysaccharide composition/substance for promoting the expression of aquaporin in the medium is 500 mug/mL, respectively; full culture medium is added into the blank group in the whole courseAnd (5) nourishing. 48 After h, cells were collected for qPCR detection. Significance analysis was performed on the data obtained from the experiments. The test was performed in triplicate.
(2) qPCR detection of AQP3 expression at mRNA level
1) Total RNA extraction (gun head and centrifuge tube were both sterilized by humid heat, without RNase)
Taking a homogenizing tube, adding 1mL of Trizol Reagent, and pre-cooling on ice. The collected sample was taken and added to the homogenization tube. The homogenizer is fully ground. The supernatant was centrifuged at 12000rpm for 10min. Adding 250 mu L of chloroform, inverting the centrifuge tube for 15s, fully and uniformly mixing, and standing for 3min. Centrifuge at 12000rpm for 10min at 4 ℃. The supernatant was transferred to a new centrifuge tube, 0.8 volumes of isopropanol was added and mixed upside down. The mixture was left at-20℃for 15min. Centrifuging at 12000rpm for 10min at 4deg.C, and collecting white precipitate at the bottom of the tube to obtain RNA. The liquid was removed by suction, and the precipitate was washed by adding 1.5mL of 75% ethanol. Centrifuge at 12000rpm for 5min at 4 ℃. The liquid is sucked and removed, and the centrifuge tube is placed on an ultra clean bench for blowing for 3min. RNA was dissolved by adding 15. Mu.L of RNase-free water. Incubate at 55℃for 5min. RNA concentration and purity were measured using Nanodrop 2000: after the instrument blank is zeroed, 2.5 mu L of RNA solution to be detected is taken on a detection base, a sample arm is put down, and the detection of the absorbance value is started by using software on a computer. RNA at too high a concentration was diluted in a proper ratio to a final concentration of 200 ng/. Mu.L.
2) Reverse transcription (gun head and PCR are both sterilized by moist heat, no RNase)
A PCR tube was taken and a solution containing 2. Mu.g of RNA was added. mu.L of oligo (dT) 18 was added. The solution was made up to 12. Mu.L with deionized water without ribonuclease. The temperature is kept at 65 ℃ for 5min on a PCR instrument, and the mixture is rapidly cooled on ice. mu.L of 5 Xreaction Buffer, 2. Mu.L of 10mM dNTP mix, 1. Mu. L RiboLock RNAase inhibitor (20U/. Mu.L) and 1. Mu.LRevertaI M-MuLV reverse transcriptase (200U/. Mu.L) were added in this order and mixed by aspiration with a gun. Preserving the temperature on a PCR instrument at 42 ℃ for 60min, and preserving the temperature at 70 ℃ for 5min after the completion of the inactivation of the reverse transcriptase.
3) Quantitative PCR
(1) Taking 0.2mL PCR tube, preparing the following reaction system, and preparing 3 tubes for each reverse transcription product:
2× qPCR Mix12.5μL；
7.5. Mu.M Gene primer (AQP 3-F, AQP 3-R) 2.0. Mu.L;
2.5. Mu.L of the reverse transcription product;
ddH 2 O8.0μL；
wherein AQP3-F is: GGGGAGATGCTCCACATCC (SEQ ID NO. 1), AQP3-R is: AAAGGCCAGGTTGATGGTGAG (SEQ ID NO. 2).
(2) PCR amplification system:
pre-denaturation at 95℃for 10min;
cycling (40 times) at 95deg.C, 15 s-60deg.C, 60s;
the melting curve is 60 ℃ to 95 ℃, and the temperature is raised by 0.3 ℃ every 15 s.
4) Result processing
ΔΔct method:
a=ct (target gene, sample to be tested) -CT (internal standard gene, sample to be tested);
b=ct (gene of interest, control sample) -CT (internal standard gene, control sample);
K=A-B；
fold expression = 2-K.
The results are shown in Table 1: the polysaccharide compositions/substances of examples 1 to 3 and comparative examples 1 to 6 all promote the expression level of AQP3 in HaCaT cells, but the enhancement effect of the polysaccharide compositions of examples 1 to 3 on the expression level of AQP3 in HaCaT cells is significantly higher than that of the polysaccharide compositions/substances of comparative examples 1 to 6, which shows that the enhancement effect of the polysaccharide compositions of examples 1 to 3 on the expression level of AQP3 in HaCaT cells is superior to that of the single action effect of the bamboo fungus polysaccharide, the dendrobium candidum polysaccharide and the ophiopogon polysaccharide of comparative examples 1 to 3 and the effect of the combination of any two of the bamboo fungus polysaccharide, the dendrobium candidum polysaccharide and the ophiopogon polysaccharide of comparative examples 4 to 6, namely, the bamboo fungus polysaccharide, the dendrobium candidum polysaccharide and the ophiopogon polysaccharide of the polysaccharide composition of examples 1 to 3 have a synergistic effect on the promotion of the expression level of AQP3 in HaCaT cells.
TABLE 1 relative expression values of AQP3 at mRNA level
Note that: each group is relative to the blankThe group was subjected to one-way analysis of variance and P values (P < 0.05, P < 0.01, P < 0.001, P < 0.0001) were calculated for significance variance analysis. Examples 1 to 3 were subjected to one-way analysis of variance with respect to comparative example 4, and P value was calculated △ P＜0.05， △△ P＜0.01， △△△ P < 0.001) for significance differential analysis.
2. Effect of polysaccharide compositions/substances promoting aquaporin expression on expression of AQP5 in mandibular gland cell HSG
(1) Human submandibular gland cell (HSG) cell model
Human submandibular gland cells (HSG) were cultured with 10% fetal bovine serum in DMEM medium at 37deg.C, 5% CO 2 Culturing in a cell culture box, starting passage when the cells grow to 90% fusion, enabling the cells to grow in an adherent manner, and performing passage 1 time every 2-3 days, wherein the cells are in a logarithmic growth phase and are used for experiments. The cultured HSG cells were isolated by digestion with 0.25% insulin-EDTA at 5X 10 in 96-well plates 3 Cell/well (cell/well) concentration. Adding a complete medium containing the polysaccharide compositions/substances for promoting aquaporin expression of examples 1 to 3 and comparative examples 1 to 6, and then adding the complete medium so that the final concentration of the polysaccharide composition/substance for promoting aquaporin expression in the medium is 500. Mu.g/mL, respectively; the blank group is added into the complete culture medium for culture in the whole course. 48 After h, cells were collected for qPCR detection. Significance analysis was performed on the data obtained from the experiments. The test was performed in triplicate.
Steps (2) and (3) are identical to step 1, except that the gene primers are different: AQP5-F is CGGGCTTTCTTCTACGTGG (SEQ ID NO. 3) and AQP5-R is GCTGGAAGGTCAGAATCAGCTC (SEQ ID NO. 4).
The results are shown in Table 2: the polysaccharide compositions/substances of examples 1 to 3 and comparative examples 1 to 6 all promote the expression level of AQP5 in HSG cells, but the polysaccharide compositions of examples 1 to 3 have significantly higher effect on enhancing the expression level of AQP5 in HSG cells than the polysaccharide compositions/substances of comparative examples 1 to 6, indicating that the polysaccharide compositions of examples 1 to 3 have better effect on enhancing the expression level of AQP5 in HSG cells than the single effect of Dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon japonicus polysaccharide of comparative examples 1 to 3 and the effect of the combination of any two of Dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon japonicus polysaccharide of comparative examples 4 to 6, i.e. the effect of Dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon japonicus polysaccharide of the polysaccharide compositions of examples 1 to 3 on promoting the expression level of AQP5 in HSG cells.
TABLE 2 relative expression values of AQP5 at mRNA level
Note that: each group was subjected to one-way analysis of variance with respect to the blank group, and P values (< 0.05, < 0.01, < 0.001, < 0.0001,) were calculated for the significance variance analysis. Examples 1 to 3 were subjected to one-way analysis of variance with respect to comparative example 4, and P value was calculated △ P＜0.05， △△ P＜0.01， △△△ P < 0.001) for significance differential analysis.
3. Effect experiment of toner in application example
The human body test of the moisture content of the inside of the arm was performed by 25 persons selected from volunteer raters, the arm was divided into 3 areas, respectively, a blank area, a matrix group (the same as the toner preparation method in application example, except that the polysaccharide composition of example 3 was not contained, the content of the polysaccharide composition was supplemented by deionized water) and an active substance test group (toner group), the test area was cleaned before the test, the skin moisture content before the test was measured, the skin moisture content (%) was measured after the sample was applied (100 μl each time) (the blank area was not subjected to any treatment) for 4 hours, and the statistical analysis was performed on the test results.
The results are shown in Table 3: the toner of the application examples can be used to effectively increase the moisture content in the skin. The following is indicated: the polysaccharide composition of example 3 can significantly improve the skin moisture content control effect (increase skin moisture content) of the product, and has a moisturizing effect.
TABLE 3 moisture content of 4h skin
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
SEQUENCE LISTING
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Claims (12)
1. A polysaccharide composition, which consists of the following components in parts by mass: 2-8 parts of bamboo fungus polysaccharide, 1-6 parts of dendrobium polysaccharide and 1-6 parts of ophiopogon polysaccharide;
the preparation method of the bamboo fungus polysaccharide comprises the following steps: 1) Mixing Dictyophora indusiata with water, homogenizing to obtain homogenate; 2) Carrying out subcritical extraction and solid-liquid separation on the homogenate, concentrating the obtained filtrate until the relative density is 1.20-1.40, and carrying out alcohol precipitation to obtain bamboo fungus polysaccharide, wherein the subcritical extraction temperature is 110-150 ℃, the subcritical extraction pressure is 2-4 Mpa, and the subcritical extraction time is 0.5-2 h;
the preparation method of the dendrobium polysaccharide comprises the following steps: 1) Mixing herba Dendrobii with water, homogenizing to obtain homogenate; 2) Carrying out subcritical extraction and solid-liquid separation on the homogenate, concentrating the obtained filtrate until the relative density is 1.20-1.40, and carrying out alcohol precipitation to obtain dendrobium polysaccharide, wherein the subcritical extraction temperature is 110-150 ℃, the subcritical extraction pressure is 2-4 Mpa, and the subcritical extraction time is 0.5-2 h;
the preparation method of the ophiopogon japonicus polysaccharide comprises the following steps: 1) Mixing radix Ophiopogonis with water, homogenizing to obtain homogenate; 2) Carrying out subcritical extraction and solid-liquid separation on the homogenate, concentrating the obtained filtrate until the relative density is 1.20-1.40, and carrying out alcohol precipitation to obtain ophiopogon japonicus polysaccharide, wherein the subcritical extraction temperature is 110-150 ℃, the subcritical extraction pressure is 2-4 Mpa, and the subcritical extraction time is 0.5-2 h.
2. The polysaccharide composition according to claim 1, wherein:
the polysaccharide composition comprises the following components in parts by mass: 4-8 parts of bamboo fungus polysaccharide, 1-4 parts of dendrobium polysaccharide and 1-4 parts of ophiopogon polysaccharide.
3. The polysaccharide composition according to claim 2, characterized in that:
the polysaccharide composition comprises the following components in parts by mass: 4-6 parts of bamboo fungus polysaccharide, 2-4 parts of dendrobium polysaccharide and 2-4 parts of ophiopogon polysaccharide.
4. A method for preparing the polysaccharide composition according to any one of claims 1 to 3, which is obtained by mixing bamboo fungus polysaccharide, dendrobium polysaccharide and ophiopogon polysaccharide.
5. Use of the polysaccharide composition of any one of claims 1 to 3 in the preparation of a product;
the product has any one of functions (a) - (f):
(a) Promoting aquaporin expression;
(b) Improving water transport and/or humectant transport in the skin;
(c) Preventing and/or treating diseases caused by damage to the skin barrier;
(d) Preventing and/or treating dry syndrome;
(e) Repairing mucous membrane;
(f) Relieving skin dryness and aging;
the product is daily chemical products or medicines.
6. The use according to claim 5, characterized in that:
the aquaporin is at least one of aquaporin 3 and aquaporin 5.
7. The use according to claim 5, characterized in that:
the wetting agent is glycerol; and/or
Diseases caused by damage to the skin barrier include eczema, atopic dermatitis, psoriasis, ichthyosis and solar dermatitis.
8. The use according to claim 7, characterized in that:
the daily chemical product is in the form of water aqua, emulsion, ointment, cream, foam, lotion, bead, gel or spray; and/or
The product also comprises acceptable auxiliary materials in medicines or daily chemicals.
9. A product comprising the polysaccharide composition of any one of claims 1-3;
the product is daily chemical products or medicines.
10. The product according to claim 9, characterized in that:
the product has any one of functions (a) - (f):
(a) Promoting aquaporin expression;
(b) Improving water transport and/or humectant transport in the skin;
(c) Preventing and/or treating diseases caused by damage to the skin barrier;
(d) Preventing and/or treating dry syndrome;
(e) Repairing mucous membrane;
(f) Relieving skin dryness and aging.
11. The product according to claim 10, wherein:
the aquaporin is at least one of aquaporin 3 and aquaporin 5; and/or
The wetting agent is glycerol; and/or
Diseases caused by damage to the skin barrier include eczema, atopic dermatitis, psoriasis, ichthyosis and solar dermatitis.
12. The product according to claim 11, wherein:
the daily chemical product is in the form of water aqua, emulsion, ointment, cream, foam, lotion, bead, gel or spray; and/or
The product also comprises acceptable auxiliary materials in medicines or daily chemicals.
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| CN113440538A (en) * | 2020-03-25 | 2021-09-28 | 无限极（中国）有限公司 | Polysaccharide composition with effect of relieving atopic dermatitis and skin external medical auxiliary preparation |
| CN111707836A (en) * | 2020-07-08 | 2020-09-25 | 无限极（中国）有限公司 | Protein chip for skin moisture detection, preparation method, application and detection method thereof |
| CN113667031A (en) * | 2021-08-26 | 2021-11-19 | 无限极（中国）有限公司 | Preparation method and application of micromolecular dendrobium officinale polysaccharide |
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