CN112190593B

CN112190593B - Polysaccharide composition for inhibiting TRPV1 pathway and preparation method and application thereof

Info

Publication number: CN112190593B
Application number: CN202011096212.6A
Authority: CN
Inventors: 杨凯业; 李莉楠; 刘光荣; 杜志云; 彭奕; 林丽
Original assignee: Infinitus China Co Ltd
Current assignee: Infinitus China Co Ltd
Priority date: 2020-10-14
Filing date: 2020-10-14
Publication date: 2021-11-30
Anticipated expiration: 2040-10-14
Also published as: CN112190593A

Abstract

The invention relates to the technical field of medicines and cosmetics, and discloses a polysaccharide composition for inhibiting a TRPV1 pathway, and a preparation method and application thereof. The polysaccharide composition comprises saussurea involucrate polysaccharide, tremella polysaccharide, dendrobe polysaccharide and salvia polysaccharide; the concentration of the saussurea involucrate polysaccharide is not higher than 0.4mg/mL, the concentration of the dendrobium polysaccharide is not higher than 0.35mg/mL, the concentration of the tremella polysaccharide is not higher than 0.2mg/mL, and the concentration of the salvia miltiorrhiza polysaccharide is not higher than 0.2 mg/mL. The polysaccharide composition can effectively inhibit the expression of TRPV1, thereby pre-protecting and repairing the skin nerve barrier, and can be used for preventing and treating skin allergy and preparing related cosmetics and medicaments.

Description

Polysaccharide composition for inhibiting TRPV1 pathway and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines and cosmetics, in particular to a polysaccharide composition for inhibiting a TRPV1 pathway, and a preparation method and application thereof.

Background

With the rapid development of social economy, the living standard of residents is continuously improved, and factors such as environmental pollution and living pressure cause a series of skin problems, such as sensitive skin. According to incomplete statistics, the population of sensitive skin is increasing globally, with 38% for men and 61% for women. Environmental pollution and overuse of cosmetics cause the body's resistance to external stimuli to be gradually weakened, and skin allergy, irritation and the like are more frequent. The common symptoms of skin allergy mainly comprise redness and itching, and symptoms such as blisters, red and swollen dry crumbs, exudation and liquefaction and the like can be accompanied, the specific degree of the symptoms varies from person to person, and symptoms such as numbness, tight chest and swelling can appear in severe cases. Except for pollution allergens, substances such as essence, spice, preservative, hair dye, surfactant and the like in various cosmetics are common skin allergens, most of the substances belong to chemical addition components, so healthy and green plant extracts become the main anti-allergy stream of the cosmetics, and the cosmetics with anti-allergy, anti-inflammation, mildness and effectiveness are increasingly valued by people. While healthy, green and anti-allergic effective cosmetics are derived from materials, so that the plant extracts with stable anti-allergic, anti-irritant and soothing functions tend to be favored by scholars and markets.

Skin allergy is often accompanied by the development of immune and allergic mechanisms. The inflammatory reaction is caused by the fact that the skin stratum corneum is reduced in thickness and the barrier function of the skin is damaged, so that the permeability of the skin to water-soluble irritants in the external environment is increased, external antigens easily enter skin tissues, and the inflammatory reaction is mediated through keratinocytes, Langerhans cells and lymphocytes. Thus, it is shown that the impaired barrier function of the skin is not merely a manifestation of skin inflammation, but more importantly, the fuse that causes skin inflammation to occur. The skin is the largest organ of the human body and is an effective barrier against external environmental pollution. Wherein the skin is composed of 4 layers of barriers of different components, respectively: firstly, microbial barrier: small molecule substances such as Free Fatty Acids (FFAs) and phenol-soluble regulatory Proteins (PSMs) produced by microbial population on the skin surface, and activated immune cells to release anti-inflammatory factors, and effectively resist infection of microorganisms and pathogens; a physical barrier: the outermost skin consists of a physical barrier composed of a Stratum Corneum (SC) composed of anucleated keratinocytes, and a Tight Junction (TJ) in the stratum granulosum; its function is to resist the percutaneous permeation of harmful media and the loss of excess water and salt; (iii) chemical barrier: consists of a lipid lamella composed of hydrophobic mediators between keratinocytes, namely ceramides, free fatty acids and cholesterol, and of antimicrobial peptides (AMPs) produced by keratinocytes. The functions of the skin care product are to keep the oil balance of skin tissues and inhibit the invasion of pathogens and allergens; fourthly, nerve barrier: the immune barrier, which is composed of body fluids and cells in the adaptive immune system, includes a variety of inflammatory factors and lymphocytes, such as Dendritic Cells (DCs), antigen presenting cells, APC and NK cells, and the like. Once the barrier function of the skin is broken, diseases of the skin, such as erythema, darkening, skin aging, skin allergy, skin inflammation and the like, can be caused. Therefore, protecting the skin barrier from damage and reducing the irritation of allergens to the skin is a new area of cosmetic research at present.

Studies indicate that the sensory nerve dysfunction is one of the pathogenesis mechanisms of sensitive skin, and the capsaicin receptor TRPV1(transient receptor potential vanilloid 1) on the surface of skin nerve fibers and keratinocyte membranes is activated by pain, pruritus, heat and chemical stimulation to cause the increase of calcium ion influx, so that apoptosis is caused, and finally the skin barrier function is damaged. Meanwhile, the repair of the damaged skin barrier can be delayed due to the activation of TRPV1, and the repair of the damaged barrier can be effectively promoted by the administration of an exogenous TRPV1 antagonist, so that the allergic skin disease is reduced. Because the pathogenesis of sensitive skin is complex, the prior dermatology clinic mainly adopts physical, medicine and anti-allergic cosmetics for treatment, but the effect is not obvious, and the cosmetics have various types and can aggravate allergy when being improperly used. Therefore, the development of natural cosmetics directed to capsaicin receptor TRPV1 is important for alleviating sensitive skin caused by capsaicin receptor TRPV 1.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a polysaccharide composition capable of significantly inhibiting TRPV1 expression and a preparation method thereof, so that the polysaccharide composition can prevent or repair damage of nerve barrier by inhibiting TRPV1 expression, and can be used in preparation of anti-allergic cosmetics or drugs.

A polysaccharide composition capable of remarkably inhibiting expression of TRPV1 comprises saussurea involucrate polysaccharide, tremella polysaccharide, dendrobe polysaccharide and Salvia miltiorrhiza polysaccharide.

The concentration of the saussurea involucrate polysaccharide is not higher than 0.4mg/mL, the concentration of the dendrobium polysaccharide is not higher than 0.35mg/mL, the concentration of the tremella polysaccharide is not higher than 0.2mg/mL, and the concentration of the salvia miltiorrhiza polysaccharide is not higher than 0.2 mg/mL.

Tianshan saussurea involucrata as medicinal plant is recorded in Ben Cao gang mu Shi Yi (A supplement to the compendium of materia Medica) written by Zhao Zhi Ming Dynasty, which has been used for a long time in folk. Herba Saussureae Involueratae has effects of dispelling cold and removing dampness, promoting blood circulation and dredging channels, strengthening tendons and supporting yang, resisting inflammation, relieving pain, and contracting uterus, and can be used for treating rheumarthritis, female lower abdomen psychroalgia, amenorrhea, retained afterbirth, measles without adequate eruption, cough due to lung cold, and sexual impotence. It contains abundant proteins, amino acids, flavonoids, alkaloids, phenylpropanoids, polysaccharides, volatile oil, etc., and has effects in regulating pH, accelerating skin metabolism, reducing wrinkle, and keeping skin luster and plump. And has good therapeutic effect on facial freckle and chloasma hepaticum.

Tremella contains abundant natural colloid, and has effects of moistening skin and removing facial chloasma and freckle after long-term administration due to its yin nourishing effect. The tremella polysaccharide is a plant source hyaluronic acid substance, and is a main active ingredient in fruiting bodies and cell fermentation spores of tremella fuciformis of Basidiomycetes. The tremella polysaccharide is a mixture of multiple polysaccharides. Mainly comprises five polysaccharides, namely acidic heteropolysaccharide, neutral heteropolysaccharide, muramyl polysaccharide, exopolysaccharide and acidic oligosaccharide. The main chain mannan is an active center, and the 2, 4 and 6 positions of the main chain are connected with side chains consisting of residues such as glucose, xylose, fucose, galactose and the like. The molecular weight of the tremella polysaccharide reaches 130-175 ten thousand. The tremella polysaccharide has special biological activity, such as enhancing organism immunity, and simultaneously has multiple physiological functions of moisturizing property and stability, resisting aging, resisting tumor, reducing blood sugar, reducing blood fat, resisting radiation and the like.

The dendrobium has the effects of treating injury, removing arthralgia, descending qi, tonifying internal organs, resisting fatigue, reducing weight, strengthening yin, thickening intestines and stomach after long-term use, reducing weight and prolonging life. Modern pharmacological research shows that the dendrobium has various functional activities of enhancing immunity, resisting oxidation, resisting tumors, protecting the liver, resisting fatigue and the like, and the dendrobium polysaccharide is used as an important functional active substance of the dendrobium, has wide pharmacological action and mainly has the effects of enhancing immunity, resisting oxidation, resisting tumors, reducing blood sugar and the like.

Salvia miltiorrhiza is dried root and rhizome of Labiatae (Labiatae perennial herb Salvia miltiorrhiza Bunge, which contains 4 kinds of chemical and medicinal components, i.e. liposoluble diterpene quinones, water-soluble phenolic acids, volatile oils and polysaccharides, Salvia miltiorrhiza Bunge polysaccharide is a component which can not be ignored except liposoluble diterpene components and water-soluble phenolic acids in Salvia miltiorrhiza Bunge, and the Salvia miltiorrhiza Bunge polysaccharide is composed of galactose, arabinose, mannose, glucose, ribose, xylose and rhamnose in a molar ratio of 58.8: 15.3: 4.2: 12.7: 8.5: 1.0: 2.8, and has the effects of delaying aging, resisting virus, resisting cancer, enhancing immunity and reducing blood lipid.

The invention aims to fully exert the efficacy advantages of polysaccharide and provide a safe and effective polysaccharide composition with synergistic effect, which can pre-protect or repair the skin nerve barrier.

Preferably, the mass ratio of the saussurea involucrate polysaccharide, the tremella polysaccharide, the dendrobium polysaccharide and the salvia miltiorrhiza polysaccharide (0.01-10) is as follows: (0.01-10): (0.01-10): (0.01-10), more preferably (1-10): (1-10): (1-10): (1-10).

In a specific embodiment of the invention, the mass ratio of the saussurea involucrate polysaccharide, the tremella polysaccharide, the dendrobe polysaccharide and the salvia miltiorrhiza polysaccharide is 1:1:1:10, 1:1:10:1, 1:10:1:1, 10:1:1:1 or 1:1:1: 1.

Compared with each monosaccharide and other polysaccharide compositions, the polysaccharide composition can effectively inhibit the expression of TRPV1, thereby pre-protecting and repairing skin nerve barriers, and can be used for preventing and treating skin allergy and other problems. Therefore, the invention provides application of the polysaccharide composition in preparing a TRPV1 inhibitor, application of the polysaccharide composition in preparing cosmetics or medicines for preventing or repairing skin nerve barrier damage, and application of the polysaccharide composition in preparing anti-allergic cosmetics. The cosmetic can be in various dosage forms such as aqua, gel, face cleaning cream, face cream, emulsion, etc., and specific ingredients or auxiliary materials are used according to the optional materials of various dosage forms in the field of cosmetics.

Simultaneously, the invention also provides a preparation method of the polysaccharide composition, which comprises the steps of decoloring saussurea involucrate, tremella, dendrobium and salvia miltiorrhiza, extracting each polysaccharide by water extraction and alcohol precipitation methods, and mixing each polysaccharide in proportion to obtain the polysaccharide composition.

Preferably, the preparation method comprises the steps of decolorizing saussurea involucrate, tremella fuciformis, dendrobium nobile and salvia miltiorrhiza respectively, then decocting the materials in water for one time or more than two times respectively, combining the filtrates obtained by decocting the materials in water for one time, concentrating, adding ethanol for precipitating, filtering and collecting precipitates to obtain polysaccharides, and finally mixing the polysaccharides in proportion to obtain the polysaccharide composition.

Wherein the decoloring is performed by adopting ethanol. Preferably, the volume ratio of the ethanol to each raw material drug is 1: 1-10: 1, decoloring for 4-10 hours; more preferably, the volume ratio is 3: 1-5: 1, decoloring for 5-7 hours; in the specific implementation process, the volume ratio is 4: 1, decoloring time is 6 hours;

in the specific implementation mode of the invention, the traditional Chinese medicine is decocted in water twice, and the first decoction is prepared according to the ratio of materials to liquid of 1:5-1: 30, adding water, boiling and extracting for 1-4 hours, decocting the filter residue for the second time and above with water, adding 5-20 times of water into the filter residue, boiling and extracting for 1-8 hours; more preferably, the first water decoction is prepared according to the ratio of materials to liquids of 1: 12-1: 18 adding water, boiling and extracting for 1.5-2.5 h, decocting the filter residue for the second time or more with water, adding 8-12 times of water into the filter residue, boiling and extracting for 1.5-3 h; in the specific implementation process, water is added into the first water decoction according to the material-liquid ratio of 1:15, boiling extraction is carried out for 2 hours, and 10 times of water is added into filter residues obtained by the second and above water decoction, boiling extraction is carried out for 2 hours;

preferably, the concentration is carried out at 40-90 ℃ until the concentration is proper in viscosity(solid content 30% -50%)(ii) a The ethanol precipitation is the ethanol precipitation by adopting 1-8 times of absolute ethanol, and preferably the ethanol precipitation by adopting 4 times of absolute ethanol.

In a specific embodiment of the invention, the polysaccharide is extracted, dried to a moisture of less than 6%, pulverized and mixed.

According to the technical scheme, the saussurea involucrate polysaccharide, the tremella polysaccharide, the dendrobe polysaccharide and the salvia miltiorrhiza polysaccharide are used as active ingredients to form the polysaccharide composition with the function of inhibiting the TRPV1 channel in a synergistic manner, the polysaccharide composition can effectively inhibit the expression of the TRPV1 channel, so that the skin nerve barrier is pre-protected and repaired, and the polysaccharide composition can be used for preventing and treating skin allergy and preparing related cosmetics and medicines.

Drawings

FIG. 1 shows the effect of saussurea involucrate polysaccharide, tremella polysaccharide, dendrobe polysaccharide, salvia polysaccharide and 4-t tert-cyclohexanol of different concentrations on BV2 cell viability;

FIG. 2 is a graph showing the pre-protective effect of the mono-and polysaccharides compositions of comparative examples 1-13 and examples 1-5 on the nerve barrier of capsaicin injury; denotes p < 0.001 compared to model group;

FIG. 3 is a graph showing the repair of the nerve barrier of capsaicin damage by the mono-and polysaccharide compositions of comparative examples 1-13 and examples 1-5; denotes p < 0.001 compared to the model group.

Detailed Description

The invention discloses a polysaccharide composition for inhibiting a TRPV1 pathway, and a preparation method and application thereof, and can be realized by appropriately improving process parameters by taking the contents into consideration by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the polysaccharide compositions and methods of making and using the same of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the polysaccharide compositions and methods of making and using the same described herein, as well as other variations and combinations of the polysaccharide compositions and methods of making and using the same, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In a specific embodiment, the present invention provides a method of preparing the polysaccharide composition:

taking a saussurea involucrate medicinal material, crushing into 16-mesh crushed powder, adding absolute ethyl alcohol with a material-liquid ratio (g/mL) of 1:5-1:10, soaking for 6-24h, removing the ethyl alcohol, collecting medicinal material filter residue, adding water with a material-liquid ratio of 1:5-1: 30, boiling and extracting for 1-2 h, filtering through 300-mesh filter cloth, and collecting filtrate for later use; adding water according to the material-liquid ratio of 1:5-1: 20, boiling and extracting for 1-2 h, filtering through 300-mesh filter cloth, and collecting filtrate; mixing the filtrates, and concentrating at 55-70 deg.C until the solid content is 25-40%; adding absolute ethyl alcohol in an amount which is 4 times that of the concentrated solution for alcohol precipitation, filtering and collecting polysaccharide, placing the polysaccharide in an oven at 45-75 ℃ for drying until the water content is less than 8%, and crushing the polysaccharide through a 100-mesh sieve to obtain the saussurea involucrate polysaccharide.

Taking tremella, crushing to 40-mesh crushed powder, adding absolute ethyl alcohol with a material-liquid ratio (g/mL) of 1:8-1:12, soaking for 24-36h, removing the ethyl alcohol, collecting medicinal material filter residue, adding water with a material-liquid ratio of 1: 25-1: 45, boiling and extracting for 1.5-3.5 h, filtering through 300-mesh filter cloth, and collecting filtrate for later use; adding water according to the material-liquid ratio of 1: 10-1: 20, boiling and extracting for 1-2 h, filtering through 300-mesh filter cloth, and collecting filtrate; mixing the filtrates, centrifuging at 5000r/min for 10min, and concentrating the filtrate at 55-70 deg.C until the solid content is 30-40%; adding 3-4 times of anhydrous ethanol according to the amount of the concentrated solution for alcohol precipitation, filtering and collecting polysaccharide, placing in a drying oven at 45-75 ℃ for drying until the water content is less than 8%, and crushing and sieving by a 100-mesh sieve to obtain the tremella polysaccharide.

Taking a dendrobium medicinal material, crushing the dendrobium medicinal material into 16-mesh crushed powder, adding absolute ethyl alcohol with a material-liquid ratio (g/mL) of 1:8-1:10, soaking for 12-24h, removing the ethyl alcohol, collecting medicinal material filter residue, adding water according to the material-liquid ratio of 1: 12-1: 25, boiling and extracting for 1.5-2.5 h, filtering through 300-mesh filter cloth, and collecting filtrate for later use; adding water according to the material-liquid ratio of 1: 10-1: 15, boiling and extracting for 1-2 h, filtering through 300-mesh filter cloth, and collecting filtrate; mixing the filtrates, and concentrating at 60-70 deg.C until the solid content is 25-35%; adding anhydrous ethanol in an amount which is 4 times that of the concentrated solution for alcohol precipitation, filtering and collecting polysaccharide, placing the polysaccharide in a drying oven at 45-75 ℃ for drying until the water content is less than 8%, and crushing the polysaccharide through 100 meshes to obtain the dendrobium polysaccharide.

Crushing a salvia miltiorrhiza medicinal material into 16-mesh crushed powder, adding absolute ethyl alcohol with a material-liquid ratio (g/mL) of 1:5-1:8, soaking for 6-12h, removing the ethyl alcohol, collecting medicinal material filter residue, adding water according to the material-liquid ratio of 1:5-1: 15, boiling and extracting for 1-2 h, filtering through 300-mesh filter cloth, and collecting filtrate for later use; adding water according to the material-liquid ratio of 1:5-1:8, boiling and extracting for 1-2 h, filtering through 300-mesh filter cloth, and collecting filtrate; mixing the filtrates, and concentrating at 55-65 deg.C until the solid content is 25-35%; adding anhydrous ethanol in an amount which is 4 times that of the concentrated solution for alcohol precipitation, filtering and collecting polysaccharide, placing the polysaccharide in an oven at 45-75 ℃ for drying until the water content is less than 8%, and crushing and sieving the polysaccharide by a 100-mesh sieve to obtain the salvia miltiorrhiza polysaccharide.

More preferably, the preparation method is as follows:

taking the saussurea involucrate, crushing into 16-mesh crushed powder, adding absolute ethyl alcohol with a material-liquid ratio (g/mL) of 1:10, soaking for 12h, removing the ethyl alcohol, collecting the filter residue of the medicinal material, adding water with a material-liquid ratio of 1:10, boiling and extracting for 1.5h, filtering through 300-mesh filter cloth, and collecting the filtrate for later use; adding water according to the material-liquid ratio of 1:8, boiling and extracting for 1h, filtering through 300-mesh filter cloth, and collecting filtrate; mixing the filtrates, and concentrating at 60 deg.C to solid content of 30%; adding anhydrous ethanol 4 times the amount of the concentrated solution, precipitating with ethanol, filtering, collecting polysaccharide, drying in a 65 deg.C oven until the water content is less than 8%, pulverizing, and sieving with 100 mesh sieve to obtain Tianshan herba Saussureae Involueratae polysaccharide.

Taking tremella, crushing to 40-mesh crushed powder, adding absolute ethyl alcohol with a material-to-liquid ratio (g/mL) of 1:12, soaking for 24h, removing the ethyl alcohol, collecting medicinal material filter residue, adding water with a material-to-liquid ratio of 1:35, boiling and extracting for 2h, filtering through 300-mesh filter cloth, and collecting filtrate for later use; adding water according to the material-liquid ratio of 1:15, boiling and extracting for 1h, filtering through 300-mesh filter cloth, and collecting filtrate; mixing filtrates, centrifuging at 5000r/min for 10min, and concentrating the filtrate at 60 deg.C to solid content of 30%; adding anhydrous ethanol 4 times the amount of the concentrated solution, precipitating with ethanol, filtering, collecting polysaccharide, drying in a 65 deg.C oven until the water content is less than 8%, pulverizing, and sieving with 100 mesh sieve to obtain Tremella polysaccharide.

Taking a dendrobium medicinal material, crushing to 16-mesh powder, adding absolute ethyl alcohol with a material-liquid ratio (g/mL) of 1:8, soaking for 12h, removing the ethyl alcohol, collecting medicinal material filter residue, adding water according to the material-liquid ratio of 1:25, boiling and extracting for 1.5h, filtering through 300-mesh filter cloth, and collecting filtrate for later use; adding water according to the material-liquid ratio of 1:10, boiling and extracting for 1h, filtering through 300-mesh filter cloth, and collecting filtrate; mixing the filtrates, and concentrating at 60 deg.C to solid content of 30%; adding anhydrous ethanol 4 times the amount of the concentrated solution, precipitating with ethanol, filtering, collecting polysaccharide, drying in a 65 deg.C oven until the water content is less than 8%, pulverizing, and sieving with 100 mesh sieve to obtain herba Dendrobii polysaccharide.

Pulverizing Saviae Miltiorrhizae radix into 16 mesh powder, soaking in anhydrous ethanol with a material-to-liquid ratio (g/mL) of 1:5 for 6h, removing ethanol, collecting residue, adding water with a material-to-liquid ratio of 1:10, boiling for 2h, filtering with 300 mesh filter cloth, and collecting filtrate; adding water according to the material-liquid ratio of 1:8, boiling and extracting for 1h, filtering through 300-mesh filter cloth, and collecting filtrate; mixing the filtrates, and concentrating at 60 deg.C to solid content of 25%; adding anhydrous ethanol 4 times the amount of the concentrated solution, precipitating with ethanol, filtering, collecting polysaccharide, drying in oven at 55 deg.C until the water content is less than 8%, pulverizing, and sieving with 100 mesh sieve to obtain Saviae Miltiorrhizae radix polysaccharide.

In the comparative experiments of the present invention, the concentration of each polysaccharide in the polysaccharide composition of the present invention was at least 0.0077 mg/mL; preferably 0.0077 to 0.077mg/mL, and more preferably 0.025mg/mL, 0.0334mg/mL or 0.05 mg/mL. In the contrast test, except the difference of each group, the other test conditions are kept consistent so as to ensure the comparability.

In the present experiments, the present invention uses mouse glial cell aventurine cytology validation; mouse glial cells are isolated from brain tissue; microglia, a type of glial cell, is a macrophage in the brain and spinal cord, and is the first line of immune defense in the Central Nervous System (CNS) and also the most important line. Microglia account for approximately 20% of the glial cells in the brain, which constantly clear damaged nerves, plaques and infectious material in the central nervous system. Countless clinical and neuropathological studies surface-activated microglia play a significant role in the pathogenesis of neurodegenerative diseases, such as parkinson's disease, multiple sclerosis and alzheimer's disease; but excessive activation or uncontrolled microglia can cause neurotoxicity. They are important sources of proinflammatory factors and oxidative stress, such as Tumor Necrosis Factor (TNF), nitric oxide, interleukins, and other neurotoxic substances. The primary function of glial cells: 1. glia occur during the transition from early brain development to the mature stage; 2. glia can act as macrophages in the brain when programmed cells die, or the central nervous system is damaged or pathologically damaged during brain development, 3. glia express antigens when class II histocompatibility complex expresses CD-4 positive T cells, can undergo Fe-mediated macrophage or macrophage interactions, and share antigens with hematopoietic cells and macrophage tissues.

The polysaccharide composition for inhibiting TRPV1 pathway, the preparation method and the application thereof provided by the invention are further described below.

Examples 1 to 5: polysaccharide compositions of the invention

Example 1

Dissolving the saussurea involucrate polysaccharide, the tremella polysaccharide, the dendrobium polysaccharide and the salvia miltiorrhiza polysaccharide powder in distilled pure water by 1g respectively, and fully stirring and dissolving to obtain the aqueous solution containing the saussurea involucrate polysaccharide, the tremella polysaccharide and the salvia miltiorrhiza polysaccharide. Wherein the final concentrations of herba Saussureae Involueratae polysaccharide, Tremella polysaccharide, herba Dendrobii polysaccharide, and Saviae Miltiorrhizae radix polysaccharide are respectively 0.25 mg/mL.

Example 2

Dissolving the saussurea involucrate polysaccharide, the tremella polysaccharide and the dendrobium polysaccharide in 0.4g and the salvia polysaccharide in 4g respectively in distilled pure water, and fully stirring and dissolving to obtain the aqueous solution containing the saussurea involucrate polysaccharide, the tremella polysaccharide and the salvia polysaccharide. The final concentrations of herba Saussureae Involueratae polysaccharide, Tremella polysaccharide, herba Dendrobii polysaccharide, and Saviae Miltiorrhizae radix polysaccharide are respectively 0.0077mg/mL, and 0.077 mg/mL.

Example 3

Dissolving the saussurea involucrate polysaccharide, the tremella polysaccharide and the salvia miltiorrhiza polysaccharide powder in 0.4g and the dendrobe polysaccharide in 4g respectively in distilled pure water, and fully stirring and dissolving to obtain the aqueous solution containing the saussurea involucrate polysaccharide, the tremella polysaccharide and the salvia miltiorrhiza polysaccharide. The final concentrations of herba Saussureae Involueratae polysaccharide, Tremella polysaccharide, herba Dendrobii polysaccharide, and Saviae Miltiorrhizae radix polysaccharide are respectively 0.0077mg/mL, 0.077mg/mL, and 0.0077 mg/mL.

Example 4

Dissolving the saussurea involucrate polysaccharide, the dendrobium polysaccharide and the salvia miltiorrhiza polysaccharide powder in 0.4g and the tremella polysaccharide in 4g respectively in distilled pure water, and fully stirring and dissolving to obtain the aqueous solution containing the saussurea involucrate polysaccharide, the tremella polysaccharide and the salvia miltiorrhiza polysaccharide. The final concentrations of herba Saussureae Involueratae polysaccharide, Tremella polysaccharide, herba Dendrobii polysaccharide, and Saviae Miltiorrhizae radix polysaccharide are respectively 0.0077mg/mL, 0.077mg/mL, 0.0077mg/mL, and 0.0077 mg/mL.

Example 5

Dissolving the above Tremella polysaccharide, herba Dendrobii polysaccharide, and Saviae Miltiorrhizae radix polysaccharide powder 0.4g respectively, and herba Saussureae Involueratae polysaccharide 4g in distilled pure water, stirring thoroughly to obtain aqueous solution containing herba Saussureae Involueratae polysaccharide, Tremella polysaccharide, and Saviae Miltiorrhizae radix polysaccharide. Wherein the concentrations of Tremella polysaccharide, herba Dendrobii polysaccharide and Saviae Miltiorrhizae radix polysaccharide are respectively 0.0077mg/mL, 0.0077mg/mL and 0.0077mg/mL, and herba Saussureae Involueratae polysaccharide is 0.077 mg/mL.

Comparative examples 1 to 13: control polysaccharide and compositions thereof

Comparative example 1

Dissolving the above herba Saussureae Involueratae polysaccharide powder 10mg in distilled pure water, and stirring to dissolve completely to obtain water solution containing herba Saussureae Involueratae polysaccharide. Wherein the concentration of saussurea involucrate polysaccharide is 0.1 mg/mL.

Comparative example 2

Dissolving 10mg of the above Tremella polysaccharide powder in distilled pure water, and stirring to dissolve completely to obtain water solution containing Tremella polysaccharide. Wherein the concentration of the tremella polysaccharide is 0.1 mg/mL.

Comparative example 3

Dissolving 10mg of the above herba Dendrobii polysaccharide powder in distilled pure water, and stirring to dissolve completely to obtain water solution containing herba Dendrobii polysaccharide. Wherein the concentration of the dendrobium polysaccharide is 0.1 mg/mL.

Comparative example 4

Dissolving 10mg of the above Saviae Miltiorrhizae radix polysaccharide powder in distilled pure water, and stirring to dissolve completely to obtain water solution containing Saviae Miltiorrhizae radix polysaccharide. Wherein the concentration of Saviae Miltiorrhizae radix polysaccharide is 0.1 mg/mL.

Comparative example 5

Dissolving 5mg of the saussurea involucrate polysaccharide and the tremella polysaccharide powder in distilled pure water respectively, and fully stirring and dissolving to obtain the aqueous solution containing the saussurea involucrate polysaccharide and the tremella polysaccharide. Wherein the concentration of the polysaccharide of saussurea involucrate and the concentration of the polysaccharide of tremella are respectively 0.05 mg/mL.

Comparative example 6

Dissolving 5mg of the saussurea involucrate polysaccharide and the dendrobium polysaccharide powder in distilled pure water respectively, and fully stirring and dissolving to obtain the aqueous solution containing the saussurea involucrate polysaccharide and the dendrobium polysaccharide. Wherein the concentration of the polysaccharide of herba Saussureae Involueratae and the concentration of the polysaccharide of herba Dendrobii are respectively 0.05 mg/mL.

Comparative example 7

Dissolving 5mg of the saussurea involucrate polysaccharide and the salvia miltiorrhiza polysaccharide powder in distilled pure water respectively, and fully stirring and dissolving to obtain the aqueous solution containing the saussurea involucrate polysaccharide and the salvia miltiorrhiza polysaccharide. Wherein the concentration of saussurea involucrate polysaccharide and salvia miltiorrhiza polysaccharide is 0.05mg/mL respectively.

Comparative example 8

Dissolving the above Tremella polysaccharide and herba Dendrobii polysaccharide powder 5mg each in distilled pure water, and stirring to dissolve completely to obtain water solution containing Tremella polysaccharide and herba Dendrobii polysaccharide. Wherein the concentration of the polysaccharide of saussurea involucrate and the concentration of the polysaccharide of tremella are respectively 0.05 mg/mL.

Comparative example 9

Dissolving the above Tremella polysaccharide and Saviae Miltiorrhizae radix polysaccharide powder 5mg each in distilled pure water, and stirring to dissolve completely to obtain water solution containing Tremella polysaccharide and Saviae Miltiorrhizae radix polysaccharide. Wherein the concentration of tremella polysaccharide and Salvia miltiorrhiza polysaccharide is 0.05mg/mL respectively.

Comparative example 10

Dissolving the above herba Dendrobii polysaccharide and Saviae Miltiorrhizae radix polysaccharide powder 5mg each in distilled pure water, and stirring to dissolve completely to obtain water solution containing herba Dendrobii polysaccharide and Saviae Miltiorrhizae radix polysaccharide. Wherein the concentration of the dendrobe polysaccharide and the salvia miltiorrhiza polysaccharide is 0.05mg/mL respectively.

Comparative example 11

Dissolving the saussurea involucrate polysaccharide, the tremella polysaccharide and the dendrobium polysaccharide powder in distilled pure water by 3.34mg respectively, and fully stirring and dissolving to obtain the aqueous solution containing the saussurea involucrate polysaccharide, the tremella polysaccharide and the dendrobium polysaccharide. Wherein the concentrations of the saussurea involucrate polysaccharide, the tremella polysaccharide and the dendrobium polysaccharide are respectively 0.0334 mg/mL.

Comparative example 12

Dissolving the above herba Saussureae Involueratae polysaccharide, Tremella polysaccharide, and Saviae Miltiorrhizae radix polysaccharide powder in distilled water (3.34 mg each), and stirring to obtain water solution containing herba Saussureae Involueratae polysaccharide, Tremella polysaccharide, and Saviae Miltiorrhizae radix polysaccharide. Wherein the concentrations of the saussurea involucrate polysaccharide, the tremella polysaccharide and the salvia polysaccharide are respectively 0.0334 mg/mL.

Comparative example 13

Dissolving the above Tremella polysaccharide, herba Dendrobii polysaccharide, and Saviae Miltiorrhizae radix polysaccharide powder in distilled pure water (3.34 mg each), stirring thoroughly to obtain aqueous solution containing herba Saussureae Involueratae polysaccharide, Tremella polysaccharide, and Saviae Miltiorrhizae radix polysaccharide. Wherein the concentrations of the tremella polysaccharide, the dendrobe polysaccharide and the salvia miltiorrhiza polysaccharide are respectively 0.0334 mg/mL.

Example 6: evaluation of cytotoxicity in MTT colorimetric assay

Materials and methods

BV 2: mouse glial cells;

in a cow supplemented with 10% of cowBV2 cells were cultured in DMEM medium with 10. mu.l/ml streptomycin/penicillin. The culture was maintained at 37 ℃ with 5% CO₂The culture medium was changed every 2 to 3 days in the incubator (2).

BV2 cells were then plated on 96-well plates and after 24h of culture, the cells were treated for 24h by adding the drug and the positive control, 4-tert-butylcyclohexanol, to the 96-well plates according to the drug concentrations in Table 1. Each group was replicated three times.

TABLE 1

Categories	Medicine	Concentration of
			Ts	Saussurea involucrata polysaccharide	0.1～1.0mg/mL
T	Tremella polysaccharide	0.1～1.0mg/mL
			D	Dendrobium polysaccharide	0.1～1.0mg/mL
S	Salvia miltiorrhiza polysaccharide	0.1～1.0mg/mL
			B	4-tert-butylcyclohexanol	60～480uM

The results are shown in figure 1, different concentrations of saussurea involucrate polysaccharide, dendrobe polysaccharide, white fungus polysaccharide and red sage polysaccharide have influence on BV2 cell viability, low concentration saussurea involucrate polysaccharide, dendrobe polysaccharide and white fungus polysaccharide, and red sage polysaccharide has no toxicity to cells, while high concentration saussurea involucrate polysaccharide, dendrobe polysaccharide, white fungus polysaccharide and red sage polysaccharide have reduced cell activity and have toxic effect on cells. In the experiment, the cell viability is generally considered to be no toxicity when being not less than 20% compared with the control group, so that relative to BV2 cells, the critical toxicity concentration of the saussurea involucrate polysaccharide cells is 0.4mg/mL, the critical toxicity concentration of the dendrobium polysaccharide cells is 0.35mg/mL, the critical toxicity concentration of the tremella polysaccharide cells is 0.2mg/mL, and the critical toxicity concentration of the salvia miltiorrhiza polysaccharide cells is 0.2mg/mL, so that the safe concentration of the saussurea involucrate polysaccharide, the dendrobium polysaccharide, the tremella polysaccharide and the salvia miltiorrhiza polysaccharide acting on BV2 cells is 0-0.4mg/mL, 0-0.350mg/mL, 0-0.20mg/mL and 0-0.20 mg/mL.

Example 7: intracellular TRPV1 assay on BV2 cells

Materials and methods

BV 2: mouse glial cells

BV2 cells were cultured in DMEM medium with 10% fetal bovine serum and 10. mu.l/ml streptomycin/penicillin. The culture was maintained at 37 ℃ with 5% CO₂The culture medium was changed every 2 to 3 days in the incubator (2).

Polysaccharide injury repair experiment: BV2 cells 5 x 10 per well⁴Inoculating the cells on a 6-well plate, culturing for 24h, adding 10uM Capsaicin 24h into each well, taking one group as a blank control, treating the cells for 24h according to the drug concentration in the table 2 after 30min, taking 240uM 4-t-butylcyclohexanol as a positive control, acting for 24h in the same way, collecting the cells, quantitatively testing the expression of TRPV1 by using an Elisa kit of TRPV1, specifically marking TRPV1 antibody by live cell immunofluorescence, and further qualitatively determining the expression of TRPV1 in the cells by FITC staining.

Polysaccharide pre-protection experiment: will be provided withBV2 cells 5 x 10 per well⁴Inoculating the cells on a 6-well plate, culturing for 24h, treating the cells for 24h according to the drug concentration shown in the table 2, using 240uM 4-t tert-butylcyclohexanol as a positive control, adding 10uM Capsaicin 24h into each well, using one group as a blank control, removing Capsaicin after 30min, cleaning for 3 times by pbs, incubating for 24h in an incubator at 37 ℃, collecting the cells, and quantitatively testing the expression quantity of TRPV1 by using an Elisa kit of TRPV 1. The expression condition of TRPV1 in cells is further qualitatively determined by specific labeling of TRPV1 antibody through immunofluorescence of living cells and FITC staining.

TABLE 2

The results of the pre-protection test in fig. 2 show that the expression of TRPV1 in each group is significantly reduced compared to the model group, and the polysaccharide compositions of the present invention are overall better, wherein the effect of example 1 in regulating the expression level of TRPV1 is most significant compared to the lowest level of TRPV1 in each other treatment group, and is close to the effect of the positive control group.

Meanwhile, in the repair test shown in fig. 3, FITC staining is carried out by using a TRPV1 antibody specifically labeled by living cell immunofluorescence, compared with a model group, the specific expression of TRPV1 of each group is obviously reduced, and each polysaccharide composition of the invention is better in whole, wherein the expression level of TRPV1 regulated and controlled in example 1 is the lowest and most obvious compared with other treatment groups, and the effect is better than that of a positive control group.

The results of the above two experiments demonstrate that there is an accelerating relationship between the polysaccharides, which is particularly evident in the polysaccharide composition of example 1, on the premise that the total concentration of each group of polysaccharides is substantially the same, indicating that the polysaccharide composition of the present invention is capable of achieving the intended purpose both in terms of pre-protection and in terms of repair.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A polysaccharide composition capable of inhibiting TRPV1 expression is characterized by comprising saussurea involucrate polysaccharide, tremella polysaccharide, dendrobe polysaccharide and salvia miltiorrhiza polysaccharide, wherein the mass ratio of the saussurea involucrate polysaccharide to the tremella polysaccharide to the dendrobe polysaccharide to the salvia miltiorrhiza polysaccharide is (1-10): (1-10): (1-10): (1-10).

2. The polysaccharide composition of claim 1, wherein: the concentration of the saussurea involucrate polysaccharide is not higher than 0.4mg/mL, the concentration of the dendrobium polysaccharide is not higher than 0.35mg/mL, the concentration of the tremella polysaccharide is not higher than 0.2mg/mL, and the concentration of the salvia miltiorrhiza polysaccharide is not higher than 0.2 mg/mL.

3. Use of a polysaccharide composition as claimed in claim 1 or 2 for the preparation of a TRPV1 inhibitor.

4. Use of the polysaccharide composition according to claim 1 or 2 for the preparation of a cosmetic or pharmaceutical product for the prevention or repair of damage to the cutaneous nerve barrier.

5. Use of the polysaccharide composition according to claim 1 or 2 for the preparation of an anti-allergic cosmetic.

6. The process for preparing the polysaccharide composition as claimed in claim 1 or 2, wherein the polysaccharide composition is obtained by decolorizing saussurea involucrate, tremella, dendrobium and salvia miltiorrhiza, extracting the polysaccharides by water extraction and alcohol precipitation, and mixing the polysaccharides in proportion.

7. The preparation method of claim 6, wherein the polysaccharide composition is prepared by decolorizing saussurea involucrate, Tremella, herba Dendrobii and Saviae Miltiorrhizae radix respectively, decocting with water for one or more times, mixing the decoctions, concentrating, precipitating with ethanol, filtering, collecting precipitate, and mixing the polysaccharides at a certain ratio.

8. The method according to claim 6 or 7, wherein the decolorization is performed using ethanol.