CN114404444A - Polysaccharide composition for promoting aquaporin expression and preparation method and application thereof - Google Patents
Polysaccharide composition for promoting aquaporin expression and preparation method and application thereof Download PDFInfo
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- CN114404444A CN114404444A CN202210097933.1A CN202210097933A CN114404444A CN 114404444 A CN114404444 A CN 114404444A CN 202210097933 A CN202210097933 A CN 202210097933A CN 114404444 A CN114404444 A CN 114404444A
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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
Landscapes
- 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 damage, prevent and/or treat dryness syndrome, repair mucosa, relieve dry skin and aging, and can be used for preparing health care products, medicines and daily chemical products.
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 cell membranes (integral membrane protein) that forms "tunnels" in the cell membranes that control the flow of water into and out of the cells, just like "water pumps for cells". AQPs primarily promote the diffusion of water and various small solutes across membranes, and are involved in cellular 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, skin disorders, metabolic syndrome, and even heart diseases. Cells regulate their shape and volume by utilizing the most abundant molecule in the body, water, and control homeostasis. Thus, AQPs may play a crucial 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, cataract, etc. has been widely studied. AQPs in skin are associated with skin hydration, cell proliferation and differentiation, migration, immunity and wound healing, AQP3 in skin is involved in proliferation and migration of epidermal cells, AQP3 is the most abundant and studied aquaporin in skin, whose down regulation and/or localization errors are associated with psoriasis; AQP5 is involved in water secretion in both the outer membrane of sweat glands and basolateral membranes, and AQP5 regulates keratinocyte proliferation and differentiation. The over-expression of AQP5 can induce the proliferation and de-differentiation of HaCaT cells, but does not affect the apoptosis of HaCaT cells, and suggests that AQP5 mediates the balance of proliferation and differentiation of epidermal keratinocytes and may play an important role in maintaining the potential of keratinocytes.
The known active substances for promoting the generation of aquaporin are few, and some of the active substances are traditional Chinese medicine compositions: for example, patent documents CN202011426245.2 and CN202011426243.3 disclose a Chinese medicinal composition for promoting the expression of aquaporins AQP3 and AQP9 in facial mask solution, which contains okra extract, gentian root extract, sargassum muticum extract and agaricus extract, CN202011426243.3), and others are effective substances alone: for example, patent document CN202110043486.7 discloses that the dandelion water extract can promote the expression of aquaporin, patent document CN201080064285.5 discloses that the carob bean extract can promote the expression of aquaporin 3, and patent document CN201710075675.6 discloses that the coix seed extract and the twisted cactus stem extract can promote the expression of aquaporin 3, wherein the substances and compositions capable of simultaneously regulating and controlling various aquaporins are few. Therefore, an excellent aquaporin production-promoting substance is desired.
Disclosure of Invention
The first aspect of the present invention aims to provide a polysaccharide composition for promoting the expression of aquaporins.
The second aspect of the present invention is directed to a method for preparing the aquaporin expression-promoting polysaccharide composition of the first aspect.
The third aspect of the present invention aims to provide the use of the aquaporin expression promoting polysaccharide composition of the first aspect in the preparation of a product.
The fourth aspect of the invention aims to provide a product.
In order to achieve the 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 aquaporin expression, the polysaccharide composition comprising: bamboo fungus 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 dendrobe polysaccharide and 1-6 parts of ophiopogon polysaccharide; the paint further comprises the following components in parts by mass: 4-8 parts of bamboo fungus polysaccharide, 1-4 parts of dendrobe polysaccharide and 1-4 parts of ophiopogon polysaccharide; the paint further comprises the following components in parts by mass: 4-6 parts of bamboo fungus polysaccharide, 2-4 parts of dendrobe polysaccharide and 2-4 parts of ophiopogon polysaccharide.
Preferably, the dendrobium is dendrobium officinale.
Preferably, the aquaporin is at least one of aquaporin 3 and aquaporin 5.
In a second aspect of the present invention, a preparation method of the polysaccharide composition for promoting aquaporin expression in the first aspect of the present invention is provided, wherein the polysaccharide composition is obtained by mixing dictyophora indusiata polysaccharide, dendrobe polysaccharide and ophiopogon root polysaccharide.
Preferably, the dictyophora indusiata polysaccharide, the dendrobium polysaccharide and the ophiopogon polysaccharide can be obtained in the market, or can be obtained by respectively extracting dictyophora indusiata, dendrobium and ophiopogon root as raw materials.
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, and homogenizing to obtain homogenate; 2) and performing subcritical extraction on the homogenate, performing solid-liquid separation, concentrating the obtained filtrate until the relative density is 1.20-1.40, and performing alcohol precipitation to obtain the dictyophora phalloidea polysaccharide.
Preferably, the bamboo fungus is crushed into fine powder of 60-100 meshes before the bamboo fungus is mixed with water in the step 1).
Preferably, the mass ratio of the bamboo fungus to the water in the step 1) is 1 (8-20).
Preferably, the step 1) of homogenizing further comprises a soaking step.
Preferably, the soaking time is 10-30 min.
Preferably, the homogenization method in the step 1) is to use a colloid mill for treatment for 5-10 min.
Preferably, the temperature of the subcritical extraction in the step 2) is 110-150 ℃.
Preferably, the pressure of the subcritical extraction in the step 2) is 2-4 Mpa.
Preferably, the subcritical extraction time in the step 2) is 0.5-2 h.
Preferably, the concentration condition in the step 2) is 60-70 ℃.
Preferably, the alcohol precipitation method in the step 2) is to add ethanol to make the final concentration of the ethanol be 78-82%.
Preferably, the alcohol precipitation in the step 2) further comprises the following steps: and (3) uniformly 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) and performing subcritical extraction on the homogenate, performing solid-liquid separation, concentrating the obtained filtrate until the relative density is 1.20-1.40, and performing alcohol precipitation to obtain the dendrobium polysaccharide.
Preferably, the dendrobium nobile in the step 1) is crushed into fine powder of 60-100 meshes before being mixed with water.
Preferably, the mass ratio of the dendrobium to the water in the step 1) is 1 (8-20).
Preferably, the step 1) of homogenizing further comprises a soaking step.
Preferably, the soaking time is 10-30 min.
Preferably, the homogenization method in the step 1) is to use a colloid mill for treatment for 5-10 min.
Preferably, the temperature of the subcritical extraction in the step 2) is 110-150 ℃.
Preferably, the pressure of the subcritical extraction in the step 2) is 2-4 Mpa.
Preferably, the subcritical extraction time in the step 2) is 0.5-2 h.
Preferably, the concentration condition in the step 2) is 60-70 ℃.
Preferably, the alcohol precipitation method in the step 2) is to add ethanol to make the final concentration of the ethanol be 78-82%.
Preferably, the alcohol precipitation in the step 2) further comprises the following steps: and (3) uniformly mixing, standing for 6-12 h, removing ethanol and water, and drying the obtained polysaccharide precipitate.
Preferably, the preparation method of the ophiopogonpolysaccharide comprises the following steps: 1) mixing radix Ophiopogonis with water, and homogenizing to obtain homogenate; 2) and performing subcritical extraction on the homogenate, performing solid-liquid separation, concentrating the obtained filtrate until the relative density is 1.20-1.40, and performing alcohol precipitation to obtain the ophiopogonpolysaccharide.
Preferably, the radix ophiopogonis is crushed into fine powder of 60-100 meshes before being mixed with water in the step 1).
Preferably, the mass ratio of the radix ophiopogonis to the water in the step 1) is 1 (8-20).
Preferably, the step 1) of homogenizing further comprises a soaking step.
Preferably, the soaking time is 10-30 min.
Preferably, the homogenization method in the step 1) is to use a colloid mill for treatment for 5-10 min.
Preferably, the temperature of the subcritical extraction in the step 2) is 110-150 ℃.
Preferably, the pressure of the subcritical extraction in the step 2) is 2-4 Mpa.
Preferably, the subcritical extraction time in the step 2) is 0.5-2 h.
Preferably, the concentration condition in the step 2) is 60-70 ℃.
Preferably, the alcohol precipitation method in the step 2) is to add ethanol to make the final concentration of the ethanol be 78-82%.
Preferably, the alcohol precipitation in the step 2) further comprises the following steps: and (3) uniformly 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 aquaporin expression promoting polysaccharide composition of the first aspect of the invention in the manufacture of a product.
Preferably, the product has any one of the 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 skin barrier damage;
(d) prevention and/or treatment of sjogren's syndrome;
(e) repairing mucosa;
(f) relieving xerosis cutis and aging.
Preferably, the aquaporin is at least one of aquaporin 3 and aquaporin 5.
Preferably, the humectant is glycerin.
Preferably, the diseases caused by skin barrier damage include eczema, atopic dermatitis, psoriasis, ichthyosis and solar dermatitis.
Preferably, the mucosa comprises the oral mucosa and the gastrointestinal mucosa.
Preferably, the products include health products, daily chemical products and medicines.
Preferably, the pharmaceutical and/or nutraceutical product is in the form of an oral solution, oral suspension, capsule, tablet, powder or granules.
Preferably, the pharmaceutical and/or nutraceutical is suitable for oral administration.
Preferably, the daily chemical product is in the form of a water agent, an emulsion, an ointment, a cream, a foam, a lotion, a roll-on, a gel or a spray.
Preferably, the daily chemical article is suitable for topical application.
Preferably, the product also comprises acceptable auxiliary materials in medicines, health products or daily chemical products.
In a fourth aspect of the invention, there is provided a product comprising a aquaporin expression promoting polysaccharide composition of the first aspect of the invention.
Preferably, the product has any one of the 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 skin barrier damage;
(d) prevention and/or treatment of sjogren's syndrome;
(e) repairing mucosa;
(f) relieving xerosis cutis and aging.
Preferably, the aquaporin is at least one of aquaporin 3 and aquaporin 5.
Preferably, the humectant is glycerin.
Preferably, the diseases caused by skin barrier damage include eczema, atopic dermatitis, psoriasis, ichthyosis and solar dermatitis.
Preferably, the mucosa comprises the oral mucosa and the gastrointestinal mucosa.
Preferably, the products include health products, daily chemical products and medicines.
Preferably, the pharmaceutical and/or nutraceutical product is in the form of an oral solution, oral suspension, capsule, tablet, powder or granules.
Preferably, the pharmaceutical and/or nutraceutical is suitable for oral administration.
Preferably, the daily chemical product is in the form of a water agent, an emulsion, an ointment, a cream, a foam, a lotion, a roll-on, a gel or a spray.
Preferably, the daily chemical article is suitable for topical application.
Preferably, the product also comprises acceptable auxiliary materials in medicines, health products or daily chemical products.
A toner comprising the aquaporin expression-promoting polysaccharide composition of the first aspect of the invention and a humectant.
Preferably, the humectant is glycerin.
The invention has the beneficial effects that:
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 damage, prevent and/or treat dryness syndrome, repair mucosa, relieve dry skin and aging, and can be used for preparing health care products, medicines and daily chemical products.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents, and the like used in this example are commercially available reagents and materials, and the water may be deionized water, purified water, or distilled water, unless otherwise specified.
The preparation methods of ophiopogonpolysaccharide, dendrobium officinale polysaccharide and bamboo fungus polysaccharide in the following embodiments are as follows:
the preparation method of the ophiopogonpolysaccharide comprises the following steps: pulverizing the dried radix Ophiopogonis into 80 mesh fine powder, and collecting the fine powder; according to the material-liquid ratio of 1: 10 (mass ratio), adding deionized water, soaking for 30min, and processing for 8min by a colloid mill to obtain homogenate; putting the homogenate into a subcritical extraction kettle, setting the extraction temperature at 120 ℃, the pressure at 3Mpa and the fluid as water, extracting for 1h, and collecting the filtrate; concentrating the filtrate at 65 deg.C to relative density of 1.30, adding ethanol to make ethanol concentration in the solution be 80%, stirring, standing for 8 hr, removing ethanol and water, and drying the polysaccharide precipitate to obtain ophiopogonpolysaccharide.
The preparation method of the dendrobium officinale polysaccharide comprises the following steps: pulverizing dry herba Dendrobii into 80 mesh fine powder, and collecting fine powder; according to the material-liquid ratio of 1: 10 (mass ratio), adding deionized water, soaking for 30min, and processing for 8min by a colloid mill to obtain homogenate; putting the homogenate into a subcritical extraction kettle, setting the extraction temperature at 120 ℃, the pressure at 3Mpa and the fluid as water, extracting for 1h, and collecting the filtrate; concentrating the filtrate at 65 deg.C to relative density of 1.30, adding ethanol to make ethanol concentration in the solution be 80%, stirring, standing for 8 hr, removing ethanol and water, and drying the polysaccharide precipitate to obtain Dendrobium officinale polysaccharide.
The preparation method of the bamboo fungus polysaccharide comprises the following steps: pulverizing dried Dictyophora Indusiata product into 80 mesh fine powder, and collecting fine powder; according to the material-liquid ratio of 1: 10 (mass ratio), adding deionized water, soaking for 30min, and processing for 8min by a colloid mill to obtain homogenate; putting the homogenate into a subcritical extraction kettle, setting the extraction temperature at 120 ℃, the pressure at 3Mpa and the fluid as water, extracting for 1h, and collecting the filtrate; concentrating the filtrate at 65 deg.C to relative density of 1.30, adding ethanol to make ethanol concentration in the solution be 80%, stirring, standing for 8 hr, removing ethanol and water, and drying the polysaccharide precipitate to obtain Dictyophora indusiata polysaccharide.
Example 1A polysaccharide composition for promoting aquaporin expression
1. A polysaccharide composition for promoting the expression of aquaporin comprises the following components in parts by mass: 4 parts of bamboo fungus polysaccharide, 4 parts of dendrobium officinale polysaccharide and 2 parts of ophiopogon japonicus polysaccharide.
2. The preparation method of the polysaccharide composition for promoting aquaporin expression is to mix dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon japonicus polysaccharide to obtain the polysaccharide composition.
Example 2A polysaccharide composition for promoting aquaporin expression
1. A polysaccharide composition for promoting the expression of aquaporin comprises the following components in parts by mass: 4 parts of bamboo fungus polysaccharide, 2 parts of dendrobium officinale polysaccharide and 4 parts of ophiopogon japonicus polysaccharide.
2. The preparation method of the polysaccharide composition for promoting aquaporin expression is to mix dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon japonicus polysaccharide to obtain the polysaccharide composition.
Example 3A polysaccharide composition for promoting aquaporin expression
1. A polysaccharide composition for promoting the expression of aquaporin comprises the following components in parts by mass: 6 parts of bamboo fungus polysaccharide, 2 parts of dendrobium officinale polysaccharide and 2 parts of ophiopogon polysaccharide.
2. The preparation method of the polysaccharide composition for promoting aquaporin expression is to mix dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon japonicus polysaccharide to obtain the polysaccharide composition.
Comparative example 1 an agent for promoting aquaporin expression
A substance for promoting the expression of aquaporin comprises the following components in parts by mass: 10 parts of bamboo fungus polysaccharide.
Comparative example 2 an agent for promoting aquaporin expression
A substance for promoting the expression of aquaporin comprises the following components in parts by mass: 10 parts of dendrobium officinale polysaccharide.
Comparative example 3 an agent for promoting aquaporin expression
A substance for promoting the expression of aquaporin comprises the following components in parts by mass: 10 parts of ophiopogonpolysaccharide.
Comparative example 4A polysaccharide composition for promoting aquaporin expression
1. A polysaccharide composition for promoting the expression of aquaporin comprises the following components in parts by mass: 7 parts of bamboo fungus polysaccharide and 3 parts of dendrobium officinale polysaccharide.
2. The preparation method of the polysaccharide composition for promoting aquaporin expression is to mix bamboo fungus polysaccharide and dendrobium officinale polysaccharide to obtain the polysaccharide composition.
Comparative example 5A polysaccharide composition for promoting aquaporin expression
1. A polysaccharide composition for promoting the expression of aquaporin comprises the following components in parts by mass: 7 parts of bamboo fungus polysaccharide and 3 parts of ophiopogon root polysaccharide.
2. The preparation method of the polysaccharide composition for promoting aquaporin expression is to mix the dictyophora indusiata polysaccharide and the ophiopogon japonicus polysaccharide to obtain the polysaccharide composition.
Comparative example 6A polysaccharide composition for promoting aquaporin expression
1. A polysaccharide composition for promoting the expression of aquaporin comprises the following components in parts by mass: 5 parts of dendrobium officinale polysaccharide and 5 parts of ophiopogon japonicus polysaccharide.
2. The preparation method of the polysaccharide composition for promoting aquaporin expression is obtained by mixing dendrobium officinale polysaccharide and ophiopogon japonicus polysaccharide.
Application embodiment of toner
1. A toner comprising: 0.5g of the polysaccharide composition of example 3, 9.5g of glycerol and 90g of deionized water.
2. The preparation method of the toner comprises the following steps: mixing the polysaccharide composition obtained in the example 3, glycerol and deionized water, uniformly stirring to obtain a feed liquid, and homogenizing the feed liquid at 3500r/min for 15min to obtain the toner.
Effects of the embodiment
1. Effect of aquaporin-expressing polysaccharide compositions/substances on the expression of AQP3 in HaCaT skin keratinocytes
(1) HaCaT cell model
Inoculating human immortalized epidermal cell (HaCaT cell) into 1% double antibody DMEM medium containing 10% fetal calf blood sodium, and culturing at 37 deg.C with 5% CO2Culturing under the conditions of (1). Cultured HaCaT cells were detached by digestion with 0.25% insulin-EDTA and plated at 5X 10 in 96-well plates3Cell/well (cells/well) concentration. The experimental group added complete culture medium containing the aquaporin expression promoting polysaccharide compositions/substances of examples 1-3 and comparative examples 1-6, and then added complete culture medium, so that the final concentration of the aquaporin expression promoting polysaccharide compositions/substances in the culture medium is 500 mug/mL respectively; the blank group was cultured by adding complete medium throughout the whole course. After 48h, cells were harvested for qPCR detection. And (4) carrying out significance analysis on the data obtained by the experiment. Three replicates were tested.
(2) qPCR detection of AQP3 expression at mRNA level
1) Total RNA extraction (gun head and centrifuge tube are sterilized by moist heat, no RNase)
A homogenate tube was taken, 1mL of Trizol Reagent was added, and the mixture was placed on ice for precooling. The collected sample was taken and added to the homogenization tube. And fully grinding by using a homogenizer. The supernatant was centrifuged at 12000rpm for 10 min. Adding 250 μ L of chloroform, inverting the centrifuge tube for 15s, mixing well, and standing for 3 min. Centrifuge at 12000rpm for 10min at 4 ℃. The supernatant was transferred to a new centrifuge tube, 0.8 times the volume of isopropanol was added, and the mixture was mixed by inversion. Standing at-20 deg.C for 15 min. Centrifuging at 12000rpm at 4 deg.C for 10min to obtain white precipitate as RNA. The liquid was removed by suction and 1.5mL of 75% ethanol was added to wash the precipitate. Centrifuge at 12000rpm for 5min at 4 ℃. The liquid was aspirated off and the centrifuge tube was placed on a clean bench and blown for 3 min. Add 15. mu.L of RNase free water to dissolve the RNA. Incubate at 55 ℃ for 5 min. RNA concentration and purity were determined using Nanodrop 2000: after the blank of the instrument is zeroed, 2.5 mu L of RNA solution to be detected is placed on a detection base, a sample arm is put down, and light absorption value detection is started by using software on a computer. The RNA with the excessive concentration is diluted with an appropriate proportion to make the final concentration be 200 ng/. mu.L.
2) Reverse transcription (gun head and PCR are both sterilized by moist heat, no RNA enzyme)
A PCR tube was taken and a solution containing 2. mu.g of RNA was added. Add 1. mu.L oligo (dT) 18. Make up to 12 μ L with nuclease-free deionized water. Preserving the temperature for 5min at 65 ℃ on a PCR instrument, and quickly cooling on ice. mu.L of 5 × Reaction Buffer, 2. mu.L of 10mM dNTP Mix, 1. mu.L of RiboLockRNAase inhibitor (20U/. mu.L) and 1. mu.L of LReverAi M-MuLV reverse transcriptase (200U/. mu.L) were added in this order and mixed by pipetting. Keeping the temperature of the PCR sample at 42 ℃ for 60min, and keeping the temperature at 70 ℃ for 5min after the completion of the reaction to inactivate the reverse transcriptase.
3) Quantitative PCR
Taking 0.2mL PCR tube, preparing the following reaction system, preparing 3 tubes for each reverse transcription product:
wherein AQP3-F is: GGGGAGATGCTCCACATCC (SEQ ID NO.1), AQP3-R is: AAAGGCCAGGTTGATGGTGAG (SEQ ID NO. 2).
(ii) PCR amplification System:
pre-denaturation at 95 deg.C for 10 min;
cycles (40 times) 95 ℃, 15s → 60 ℃, 60 s;
the melting curve was 60 ℃ → 95 ℃ and the temperature was raised 0.3 ℃ every 15 seconds.
4) Result processing
Δ Δ CT method:
CT (target gene, sample to be tested) -CT (internal standard gene, sample to be tested);
CT (target gene, control sample) -CT (internal standard gene, control sample);
K＝A-B；
expression fold 2-K.
The results are shown in table 1: the polysaccharide compositions/substances in examples 1 to 3 and comparative examples 1 to 6 both promote the expression level of AQP3 in HaCaT cells, but the enhancing effect of the polysaccharide compositions in examples 1 to 3 on AQP3 expression in HaCaT cells is significantly higher than that of the polysaccharide compositions/substances in comparative examples 1 to 6, which indicates that the enhancing effect of the polysaccharide compositions in examples 1 to 3 on AQP3 expression in HaCaT cells is better than that of the polysaccharide compositions in comparative examples 1 to 3 on the single action effect of dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon polysaccharide and on the combination effect of any two of dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon polysaccharide in comparative examples 4 to 6, namely that the dictyophora indusiata polysaccharide, the dendrobium officinale polysaccharide and the ophiopogon polysaccharide in the polysaccharide compositions in examples 1 to 3 have a synergistic effect on promoting expression of AQP3 in HaCaT cells.
TABLE 1 relative expression values of AQP3 at the mRNA level
Note: the groups were analyzed for single factor variance relative to the blank group and significant difference analysis was performed by calculating the P values (. P. < 0.05,. P. < 0.01,. P. < 0.001,. P. < 0.0001). Examples 1-3 Single factor ANOVA with respect to comparative example 4, P value was calculated (△P＜0.05，△△P＜0.01，△△△P < 0.001) were analyzed for significant differences.
2. Effect of polysaccharide composition/substance promoting aquaporin expression on AQP5 expression in HSG of mandibular gland cells
(1) Human Submaxillary Gland (HSG) cell model
Human submaxillary gland cells (HSG) were cultured in DMEM medium containing 10% fetal bovine serum at 37 deg.C with 5% CO2Culturing in a cell culture box, starting passage when the cells grow to be 90% fused, enabling the cells to grow in an adherent manner, carrying out passage for 1 time every 2-3 days, and carrying out experiment when the cells are in a logarithmic growth phase. Cultured HSG cells were isolated by digestion with 0.25% insulin-EDTA and plated at 5X 10 in 96-well plates3Cell/well (cells/well) concentration. The aquaporin expression is promoted by adding the promoter containing the components of examples 1 to 3 and comparative examples 1 to 6The complete medium of the polysaccharide composition/substance of (a), and then adding the complete medium so that the final concentration of the polysaccharide composition/substance promoting aquaporin expression in the medium is 500 μ g/mL, respectively; the blank group was cultured by adding complete medium throughout the whole course. After 48h, cells were harvested for qPCR detection. And (4) carrying out significance analysis on the data obtained by the experiment. Three replicates were tested.
The steps (2) and (3) are the same as those in step 1, and differ only in 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 1-3 and comparative examples 1-6 all promote the expression level of AQP5 in HSG cells, but the enhancing effect of the polysaccharide compositions of examples 1-3 on AQP5 expression in HSG cells is significantly higher than that of the polysaccharide compositions/substances of comparative examples 1-6, which indicates that the enhancing effect of the polysaccharide compositions of examples 1-3 on AQP5 expression in HSG cells is better than that of the polysaccharide compositions/substances of comparative examples 1-3 on the single action effect of dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon root polysaccharide and on the combination effect of any two of dictyophora indusiata polysaccharide, dendrobium officinale polysaccharide and ophiopogon root polysaccharide in comparative examples 4-6, namely, the dictyophora indusiata polysaccharide, the dendrobium officinale polysaccharide and the ophiopogon root polysaccharide in the polysaccharide compositions of examples 1-3 have a synergistic effect on promoting expression of AQP5 in HSG cells.
TABLE 2 relative expression values of AQP5 at the mRNA level
Note: the groups were analyzed for single factor variance relative to the blank group and significant difference analysis was performed by calculating the P values (. P. < 0.05,. P. < 0.01,. P. < 0.001,. P. < 0.0001). Examples 1-3 Single factor ANOVA with respect to comparative example 4, P value was calculated (△P＜0.05，△△P＜0.01，△△△P < 0.001) were analyzed for significant differences.
3. Effect test of toner in application example
25 human volunteer evaluators were selected to conduct a moisture content human test on the inner side of the arm, the arm was divided into 3 zones, blank zones, a substrate group (same as the preparation method of the toner in the application example except that the polysaccharide composition of example 3 was not contained and the content of the polysaccharide composition was replenished with deionized water) and an active test group (toner group), the test zones were cleaned before the test, then the skin moisture content before the test was measured, then samples (100 μ L each time) (blank zones were not treated at all) were applied for 4h and then the skin moisture content (%) was measured, and the test results were statistically analyzed.
The results are shown in Table 3: the toner in the application embodiment can effectively increase the water content in the skin. Shows that: the polysaccharide composition of example 3 can significantly improve the skin moisture content regulation effect (increase the skin moisture content) of the product, and has a moisturizing effect.
TABLE 34 h moisture content of skin
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
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Claims (10)
1. A polysaccharide composition comprising: bamboo fungus polysaccharide, dendrobium polysaccharide and ophiopogon polysaccharide.
2. The polysaccharide composition of claim 1, wherein:
the polysaccharide composition comprises the following components in parts by mass: 2-8 parts of bamboo fungus polysaccharide, 1-6 parts of dendrobe polysaccharide and 1-6 parts of ophiopogon polysaccharide.
3. The polysaccharide composition of claim 2, wherein:
the polysaccharide composition comprises the following components in parts by mass: 4-8 parts of bamboo fungus polysaccharide, 1-4 parts of dendrobe polysaccharide and 1-4 parts of ophiopogon polysaccharide.
4. The polysaccharide composition of claim 3, wherein:
the polysaccharide composition comprises the following components in parts by mass: 4-6 parts of bamboo fungus polysaccharide, 2-4 parts of dendrobe polysaccharide and 2-4 parts of ophiopogon polysaccharide.
5. The method for preparing the polysaccharide composition of any one of claims 1 to 4, wherein the polysaccharide composition is obtained by mixing dictyophora indusiata polysaccharide, dendrobium polysaccharide and ophiopogon polysaccharide.
6. The method of claim 5, wherein:
the bamboo fungus polysaccharide, the dendrobium polysaccharide and the ophiopogon polysaccharide are respectively extracted from bamboo fungus, dendrobium and ophiopogon root as raw materials;
preferably, the extraction method is at least one of acid extraction, alkali extraction, enzyme extraction, ultrafiltration, ultrasonic extraction, subcritical extraction and microwave extraction.
7. The method of claim 6, wherein:
the preparation method of the bamboo fungus polysaccharide comprises the following steps: 1) mixing Dictyophora Indusiata with water, and homogenizing to obtain homogenate; 2) performing subcritical extraction on the homogenate, performing solid-liquid separation, concentrating the obtained filtrate until the relative density is 1.20-1.40, and performing alcohol precipitation to obtain dictyophora indusiata polysaccharide;
preferably, the preparation method of the dendrobium polysaccharide comprises the following steps: 1) mixing herba Dendrobii with water, homogenizing to obtain homogenate; 2) performing subcritical extraction on the homogenate, performing solid-liquid separation, concentrating the obtained filtrate until the relative density is 1.20-1.40, and performing alcohol precipitation to obtain dendrobium polysaccharide;
preferably, the preparation method of the ophiopogonpolysaccharide comprises the following steps: 1) mixing radix Ophiopogonis with water, and homogenizing to obtain homogenate; 2) and performing subcritical extraction on the homogenate, performing solid-liquid separation, concentrating the obtained filtrate until the relative density is 1.20-1.40, and performing alcohol precipitation to obtain the ophiopogonpolysaccharide.
8. Use of a polysaccharide composition as claimed in any one of claims 1 to 4 in the manufacture of a product;
preferably, the product has any one of the 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 skin barrier damage;
(d) prevention and/or treatment of sjogren's syndrome;
(e) repairing mucosa;
(f) relieving xerosis cutis and aging.
9. Use according to claim 8, characterized in that:
the aquaporin is at least one of aquaporin 3 and aquaporin 5.
10. A product comprising the polysaccharide composition of any one of claims 1 to 4.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115998874A (en) * | 2022-12-07 | 2023-04-25 | 西南交通大学 | Use of aquaporin 3 and/or aquaporin 5 as depression drug targets |
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