CN115010822B - Armillarisin mycelium polysaccharide and application thereof - Google Patents
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- A—HUMAN NECESSITIES
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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Abstract
The invention discloses a armillary mycelium polysaccharide and application thereof, wherein the monosaccharide composition and the molar ratio of the armillary mycelium polysaccharide are mannose, galacturonic acid and glucose, and arabinose=9.83:14.52:17.45:8.63; the molecular weight of the armillariella mycelium polysaccharide is 2.95X10 4 Da. Animal experiments show that the armillariella mycelium polysaccharide PAT-W can obviously reduce the inflammatory factor level of ulcer tissues, improve the content of epidermal growth factors, reduce inflammatory cell infiltration, promote ulcer surface healing and regulate the unbalanced oral flora caused by ulcers. The bright mycelium polysaccharide PAT-W can be used as a novel natural active substance for treating canker sore, and can be widely applied to the fields of medicine and oral care.
Description
Technical Field
The invention belongs to the technical field of biological medicine preparation, and particularly relates to armillariella mycelium polysaccharide and application thereof.
Background
Oral ulcers are the most frequent in oral mucosal diseases and have many causes, for example, high mental stress, immune disorders, endocrine disorders, nutrient deficiency, trauma, oral dysbacteriosis, and the like. Although the Chinese herbal medicine has certain self-healing property, once the Chinese herbal medicine is ill, the patient can feel hard to burn and pain at the affected part, the serious patient can affect eating and speaking, the disease is hard to radically cure, the Chinese herbal medicine has repeatability, and great inconvenience is brought to work and life of people. The treatment of the oral ulcer is mainly symptomatic treatment, and the immunity of the organism is regulated, antibiotics, antibacterial drugs and the like are used by inhibiting the immune function, relieving local inflammatory reaction, but the repeated use of the antibacterial drugs and the antibiotics is easy to cause the organism to generate drug resistance and other side reactions. Or local medicines such as lozenge, medicinal film, ointment and the like are applied to achieve the aim of relieving pain, but the substance basis and efficacy relation of the active ingredients are urgent to be clear; and the medicinal components with pungent smell such as medicinal films, ointments and the like can produce certain side effects such as retching and acid regurgitation after entering the body after entering the mouth. In recent years, natural bioactive substances which are efficient and free of side reactions have attracted attention. Researches prove that natural active polysaccharides such as bletilla striata, aloe polysaccharide and the like have the function of treating canker sore.
Armillariella tabiscens (Scop. EX. Fr) Sing, a unique precious medicinal fungus in China, contains natural active ingredients such as armillarisin A and armillarisin B, and has been widely used for treating acute and chronic hepatitis, appendicitis, otitis media and cholecystitis. The polysaccharide is used as an important natural active substance generated in the growth process of armillariella, has pharmacological effects of regulating immunity, resisting bacteria, reducing blood sugar and lipid, improving intestinal microecological disorder and the like, and has a few reports on the effect of treating canker sore. The applicant proves that the Bright fungus polysaccharide PAT-W has the remarkable function of treating canker sore by evaluating indexes such as oral mucosa tissue inflammatory factor level, epidermal growth factor release amount, oral flora polymorphism regulation and the like.
Disclosure of Invention
The invention aims to provide armillariella mycelium polysaccharide and application thereof. The invention uses polyamide method to remove protein and pigment impurity, ultrafiltration membrane separation method and AKTADEAE-FF ion chromatography technique to prepare armillarisin PAT-W with specific chemical composition, which has remarkable curative effect on oral ulcer. The armillarisin mycelium polysaccharide can be widely applied to the medicine and daily chemical industries, such as the research and development of dental ulcer spray and nursing toothpaste containing the armillarisin mycelium polysaccharide, and has important development prospect and market application value.
The armillarisin mycelium polysaccharide has the monosaccharide composition and molar ratio of mannose, galacturonic acid, glucose and arabinose=9.83:14.52:17.45:8.63, and the molecular weight is 2.95 multiplied by 10 4 Da。
The preparation method of the armillariella mycelium polysaccharide comprises the following steps:
step 1: inoculating 4-6g armilliella tabecten (Scop. EX. Fr) strain (supplied by Safebrile Biometrics Co., ltd.) to 1000mL glucose-potato-MgSO 4 Culturing at 28deg.C for 15 days to obtain artificial liquid;
step 2: crushing the armillariella mycelium (2000 g) obtained in the step 1, adding distilled water according to the ratio of 1g to 10mL, leaching for 3 hours at 90 ℃, repeating leaching for 3 times, and combining the extracting solutions;
step 3: concentrating the extract obtained in the step 2 to 1/250 of the original volume, adding 95% ethanol with the volume being 4 times that of the extract, standing at 4 ℃ for 12h, centrifuging at 4500rpm for 10-15min, and collecting precipitate;
step 4: dissolving the precipitate obtained in the step 3 in distilled water, and decoloring and deproteinizing by a polyamide method to obtain crude polysaccharide of armillariella mycelia;
step 5: preparing the crude polysaccharide obtained in the step 4 into 100mg/mL solution, and passing through ultrafiltration membrane with molecular weight cut-off of 1×10 4 Da and 1X 10 5 Da, concentrating the trapped fluid of the molecular weight components between the Da and the Da, and freeze-drying; preparing 5mL polysaccharide solution of 100mg/mL obtained after ultrafiltration and freeze-drying into DEAE-FF anion exchange chromatographic column, eluting with double distilled water and 0.5moL/mL sodium chloride at equal degree, collecting by a branch pipe, dialyzing to remove sodium chloride, and freeze-drying. Agilent HPLC-ELSD liquid chromatography is used to detect the eluent composition as homogeneous composition (PAT-W) by using double distilled water as mobile phase.
In step 1, the glucose-potato-MgSO 4 The culture medium is obtained by the following method: 300g potato is peeled and chopped, boiled for 15min, filtered, then mixed with 20g glucose and 5g MgSO 4 Mixing, adding water to 1000mL, sterilizing at 121deg.C for 20min to obtain glucose-potato-MgSO 4 A culture medium;
in the step 4, the conditions for decoloring and deproteinizing by a polyamide method are as follows: 80g of polyamide with 100-120 meshes and 300mL of precipitate solution are uniformly mixed in a 500mL shaking flask, and the shaking table is vibrated at 200rpm for 30min at room temperature to fully adsorb proteins and pigments, then liquid is filtered, filtrate is collected, concentrated and freeze-dried to obtain crude polysaccharide.
The armillarisin mycelium polysaccharide can obviously reduce the level of inflammatory factors, improve the content of epidermal growth factors, reduce inflammatory cell infiltration and adjust the richness and uniformity of oral flora, so that the armillarisin mycelium polysaccharide can be used for treating canker sores.
The armillarisin mycelium polysaccharide of the invention has no smell, is white powder, and is colorless and transparent. The polysaccharide solution overcomes the defects of retching, acid regurgitation and the like caused by entering the organism after entering the medicine with pungent smell such as other medicine films, ointments and the like.
The application of the armillariella mycelium polysaccharide is used for preparing a medicinal preparation with the function of treating canker sore. The mycelium polysaccharide of the armillariella has the effects of obviously relieving the inflammatory injury of the ulcer surface of the oral mucosa, improving the release of the epidermal growth factor, promoting the healing of the ulcer surface of the oral mucosa, regulating the dysbacteriosis of the oral mucosa and the like.
The invention takes a rat with canker sore induced by combination of mechanical wound and staphylococcus aureus as a study object, and verifies the effect of the mycelium polysaccharide of the armillariella for treating canker sore through animal experiments.
The armillarisin mycelium polysaccharide has remarkable improvement effect on the oral dysbacteriosis of rats with canker sore.
Drawings
FIG. 1 is a liquid chromatogram of standard monosaccharide composition (x-solvent peak, man-mannose, rha-rhamnose, glcA-glucuronic acid, galA-galacturonic acid, glc-glucose, gal-galactose, ara-arabinose, fuc-galactose).
FIG. 2 is a liquid chromatogram of the polysaccharide monosaccharide composition of the armillarisin mycelia (Man-mannose, galA-galacturonic acid, glc-glucose, ara-arabinose).
FIG. 3 is a relative molecular mass detection chromatogram of the armillariella mycelium polysaccharide PAT-W.
FIG. 4 is a graph showing the effect of armillar mycelium polysaccharide on mucosal tissue pathology in rats with canker sores
FIG. 5 is a graph showing the effect of armillar mycelium polysaccharide on alpha diversity of oral flora in rats with canker sores
FIG. 6 is the effect of armillar mycelium polysaccharide on the beta diversity of oral flora in rats with canker sores.
Detailed Description
The preparation of the present invention, reducing inflammatory factors of ulcerated tissues, promoting the release of epidermal growth factor and wound healing, and regulating polymorphism of oral flora will be described by way of specific examples, which are intended to illustrate the present invention and not to limit the scope of the present invention.
Example 1: preparation of armillariella mycelium and polysaccharide PAT-W
Step 1:300g potato is peeled and chopped, boiled for 15min, filtered, 20g glucose and 5g MgSO 4 Adding water to 1000mL, sterilizing at 121deg.C for 20min to obtain glucose-potato-MgSO 4 A culture medium; inoculating 4-6g of Armillariella tabects (Scop. EX. Fr) strain of Sing (supplied by Safebrile Biometrics Co., ltd.) into 1000mL glucose-potato-MgSO 4 Culturing at 28deg.C for 15 days to obtain artificial liquid;
step 2: pulverizing the armillariella mycelium (2000 g) obtained in the step 1, adding distilled water according to the liquid-to-material ratio of 1:10 (g/mL), leaching for 3 hours at 90 ℃, repeatedly leaching for 3 times, and combining the extracting solutions;
step 3: concentrating the extract obtained in the step 2 to 1/250 of the original volume, adding 95% ethanol with the volume being 4 times that of the extract, standing at 4 ℃ for 12h, centrifuging at 4500rpm for 10-15min, and collecting precipitate;
step 4: dissolving the precipitate obtained in the step 3 with distilled water, and decolorizing and deproteinizing by adopting a polyamide method to prepare the crude polysaccharide of the armillariella mycelium. The conditions are as follows: 80g of polyamide with 100-120 meshes and 300mL of precipitate solution are uniformly mixed in a 500mL shaking flask, and the shaking table is vibrated at 200rpm for 30min at room temperature to fully adsorb proteins and pigments, then liquid is filtered, filtrate is collected, concentrated and freeze-dried to obtain crude polysaccharide.
Step 5: preparing the crude polysaccharide obtained in the step 4 into 100mg/mL solution, and passing through ultrafiltration membrane with molecular weight cut-off of 1×10 4 Da and 1X 10 5 Da, and concentrating and freeze-drying the trapped liquid component with the molecular weight in between. Preparing 1mL of solution with concentration of 100mg/mL of the polysaccharide after ultrafiltration and freeze-drying, passing through a DEAE-FF anion exchange column, eluting with double distilled water and sodium chloride, 5 mL/tube, and obtaining the total sugar content of 97.88% by a phenol sulfuric acid method, wherein the flow rate is 1mL/min, and the dialysis and freeze-drying are recorded as PAT-W.
Example 2: monosaccharide composition and molecular weight of armillariella mycelium polysaccharide PAT-W
Monosaccharide composition analysis of PAT-W samples were treated by acid hydrolysis-pre-column PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization method and analyzed by high performance liquid chromatography. 5mg PAT-W was weighed and dissolved in 5mL 2mol/L trifluoroacetic acid, the tube was sealed with nitrogen and allowed to acid-hydrolyze thoroughly by oil bath at 110℃for 8 h. The acid component is spun out by a rotary evaporator, a proper amount of deionized water is added, the rotary evaporation is repeated until the pH of the solution shows neutral, and 1mL of distilled water is added for standby.
Step 1: 50. Mu.L of 0.5mol/LPMP methanol solution and 50. Mu.L of 0.3mol/L NaOH solution are added to the standard monosaccharide and the acidolyzed PAT-W solution, the mixture is reacted in a water bath at 70 ℃ for 30min to carry out PMP pre-column derivatization, and then 50. Mu.L of 0.3mol/L HCl is used for neutralization. The obtained product is detected by high performance liquid chromatography, and a DAD detector is selected. The HPLC column temperature was 30℃and the chromatographic column was Zorbox Eclipse XDB-C18 (4.6 mm. Times.250 mm,5 μm) and the wavelength was 245 nm. The mobile phase A is acetonitrile, and the mobile phase B is 0.05mol/L phosphate buffer solution. Time gradient elution is carried out for 0-60min, and the initial setting is that a mobile phase A: mobile phase b=17%: 83% final elution to mobile phase A: mobile phase b=20%: 80%, the sample injection amount was 10. Mu.L.
Step 2: molecular weight of PAT-W was detected using deionized water elution, agilent high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). 1mL of a solution of 2mg/mL PAT-W and dextran standards (T5, T12, T41, T100, T200) was prepared, a TSK Gel G6000 PWXL chromatographic column was used, and the carrier gas was N 2 The gas flow rate was 2.5L/min, and the sample injection amount was 10. Mu.L. The molecular weight was measured to be 2.98X10 by plotting the logarithm of the relative molecular weight of the standard (LgMw) and the retention time (Rt) 4 Da。
Example 3: therapeutic effect of armillariella mycelium polysaccharide PAT-W on oral ulcer
The prepared armillarisin mycelium polysaccharide PAT-W is used as a raw material, and the efficacy experiment evaluation of the armillarisin mycelium polysaccharide for treating canker sores is carried out by utilizing a canker sore rat model.
1. Preparation of rat model with canker sore
Selecting 48 SPF-class SD female rats (produced by Experimental animal center of university of Anhui medical science, production license SCXK (Anhui) 2017-001), weighing 250+ -10 g, and mixing with ratsThe machine is divided into a normal group, an ulcer model group and 400mg/kg vitamin B 2 Positive drug group, 100mg/kg PAT-W low dose group, 200mg/kg medium dose group, 400mg/kg high dose group, 8 each. All groups of rats except the normal group were modeled as canker sores.
The method comprises the following steps: the medical sterilizing cotton swab is dipped with 0.6mg/mL of phenol solution and burned for 30s at the left mucous membrane of the oral cavity. After 2 hours, the submucosal tissue was burned and injected with 0.05mL 2X 10 3 CFU/mL of Staphylococcus aureus was used to simulate post-traumatic oral bacterial infection.
Rats in the blank group were treated with physiological saline. After 24 hours, the formation of canker sore in the rat was observed. And the model group, the positive medicine group and rats of the PAT-W low-medium-high dose treatment group are used for successfully establishing an oral ulcer model except a normal group, the cheek mucous membrane of the rats presents an elliptical or circular ulcer surface, and the surface of the cheek mucous membrane is provided with an off-white pseudomembrane with the diameter of 4.0mm-5.0mm. The administration method of the positive medicine group is that a medical disinfection cotton swab dips 0.3mL of 400mg/kg vitamin B after 1d administration after molding 2 (Vitamin B 2 ) The solution is applied on the ulcer wound for 60s, and the residual solution is administrated by means of gastric lavage. PAT-W administration was performed in the same manner as in the positive group in the low, medium and high dose groups. Normal and model control groups were given equal amounts of physiological saline, each group was given 1 time per day for 7 days, and changes in body weight and ulcer surface were recorded daily. After 7d anesthesia, the rats are sacrificed, the ulcerated tissues are taken, and the inflammatory factors and the epidermal growth factor content, H, are measured&E, analyzing pathological changes by dyeing; oral mucus was collected and analyzed for relative abundance of oral flora. Experimental data differential significance analysis was performed using GraphPad Prism 8.3 software, oral flora abundance was analyzed based on high throughput sequencing of 16 srrrna.
2. Experimental results
(1) As can be seen from Table 1, the body weight of the ulcerated rats was significantly reduced in 7d due to oral erosion in the model group compared to the normal group. Physiological saline and VitaminB were administered separately from day 1 2 And armillariella mycelium polysaccharide dressing and gastric lavage treatment. The results showed that the weight of the model group still continuously decreased; and Vitamin B 2 Group rats showed some reduction in body weight but appeared from day fiveRecovering again; and the high-dose armillarisin group starts to rise from the weight of the ulcerous rats on the 3 rd day, so that the armillarisin mycelium polysaccharide can obviously improve the emaciation symptoms of the ulcerous rats.
Table 1 change in rat body weight (g) (n=8,
)
| group of | |
Day 1 | Day 3 | |
Day 7 |
| Blank group | 251.16±0.99 | 251.92±1.32 | 253.17±1.30 | 253.83±1.33 | 254.50±1.39 |
| Model group | 251.08±3.67 | 248.92±4.17 | 245.92±3.53 | 244.67±2.53 | 246.00±2.82 |
| Positive medicine group | 253.33±2.52 | 252.42±2.29 | 249.75±2.13 | 248.25±4.65 | 250.17±4.26 |
| Low dose group | 247.00±5.08 | 244.92±4.62 | 242.33±4.30 | 242.33±4.90 | 244.25±4.60 |
| Medium dose group | 250.50±2.45 | 248.08±2.97 | 245.83±1.80 | 247.75±1.73 | 249.08±1.90 |
| High dose group | 252.00±2.51 | 250.42±2.01 | 248.25±3.09 | 250.67±2.43 | 251.42±2.54 |
(2) As can be seen from Table 2, rats in the model group, the positive drug group and the armillarisin low, medium and high dose group all appeared canker sore surfaces of substantially the same size after molding 1d, except for the normal group. From 1d onPre-physiological saline, vitamin B 2 And armillariella mycelium polysaccharide is subjected to gastric lavage treatment after being applied, and the result shows that the ulcer surface of the model group has certain self-healing property but slow healing degree; compared with the model group, the positive medicine group and the armillarisin low-medium-high dose group significantly promote the healing of ulcer surface (P<0.05 or P<0.01 A) is provided; with the same concentration of Vitamin B 2 In contrast, after high doses of leucin treatment of ulcerous rats, the degree of healing was superior to that of Vitamin B 2 。
TABLE 2 variation of ulcer wound area in rats (mm) 2 )(n＝8，
)
| Group of | Day 1 | Day 3 | |
Day 7 |
| Blank group | - | - | - | - |
| Model group | 4.65±0.53 | 4.09±0.12 | 3.49±0.48 | 2.85±0.65 |
| Positive medicine group | 4.68±0.13 | 3.52±0.36 | 2.07±0.16** | 0.91±0.36** |
| Low dose group | 4.63±0.81 | 3.99±0.87 | 3.62±0.41 | 2.14±0.14 |
| Medium dose group | 4.67±0.12 | 3.63±0.29* | 2.52±0.15* | 1.28±0.31* |
| High dose group | 4.60±0.23 | 3.15±0.14* | 1.94±0.51** | 0.87±0.25** |
Note that: -no ulcer surface appears; p <0.05, significant, P <0.01, very significant compared to model group.
(3) The contents of tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and interleukin 1 beta (IL-1 beta) are important detection indexes of the inflammation of the ulcer tissue, and when the content of the inflammatory factor is in a stable content, the ulcer mucosa tissue can promote the healing of the ulcer surface without being damaged by the inflammation. The results are shown in Table 3: TNF-alpha, I in ulcerated tissue of rats in model group compared to blank groupThe indexes such as L-6, IL-1 beta and the like are obviously increased (P<0.01 A) is provided; compared with the model group, vitamin B 2 And a significant decrease in TNF-alpha, IL-6, and IL-1β of low, medium and high dose group leucins polysaccharide (P)<0.05、P<0.01 or P<0.001 A) is provided; and with the same concentration of Vitamin B 2 In contrast, after high dose of leucin treatment, the decrease in inflammatory factor was not significant, but the value was lower than that of Vitamin B 2 。
Epidermal Growth Factor (EGF) can promote cell proliferation and is an important index for detecting epithelial cell regeneration. The results are shown in Table 3: compared to the blank group, the model group showed significantly decreased EGF (P<0.01 A) is provided; compared with the model group, EGF of the positive medicine group and the armillarisin low-medium-high dosage group are obviously increased (P)<0.05 A) is provided; with the same concentration of Vitamin B 2 In contrast, the degree of decline was not statistically different after treatment of ulcerous rats with high doses of leucin, but was numerically better than that of Vitamin B 2 。
The results show that: the oral ulcer of the rat caused by mechanical wound combined bacterial infection has obvious abnormality of the inflammatory factor content, and has obvious improvement effect on the inflammatory factor level of the oral ulcer when the armillarisin mycelium polysaccharide is given. The armillarisin mycelium polysaccharide promotes the secretion of epidermal growth factor, and promotes the proliferation of cells so as to promote the healing of ulcer surfaces. More importantly, the index detection result shows that the armillariella mycelium polysaccharide is superior to the positive control medicine with the same concentration.
Table 3 the content of inflammatory factors and epidermal growth factors (pg/mg) of the ulcerated tissue of the rat (n=8,
)
note that: compared with the blank group, #p <0.01, very significant; # # # P <0.001, extremely significant. P <0.05, significant compared to model group; * P <0.01, very significant; * P <0.001, extremely significant.
As shown in fig. 4, the oral mucosa tissue of the rat in the normal group is complete, the epithelial cells are clear in structure and ordered in arrangement, and no inflammatory cells infiltrate; compared with the normal group, the mucous membrane tissue of the model group has obvious inflammatory cell infiltration, and the whole epidermis tissue is broken and accompanied with erythrocyte extravasation; compared with the model group, the rat mucous membrane epithelial tissue is basically completely recovered in the treatment group, especially in the high-dose armillarisin, and no obvious inflammatory cell infiltration exists.
As shown in fig. 5 and 6, microbial α and β diversity analysis patterns obtained by high throughput sequencing. It can be found that the abundance of oral bacteria in the model group is extremely significantly reduced compared to the normal group; compared with the model group, the flora abundance of the positive medicine group and the armillarisin low-medium dose group is improved; the flora enrichment of the high-dose group of the armillarisin is obviously regulated, the flora abundance and uniformity are promoted, the dysbacteriosis is improved, and the regulating effect is better than that of a positive medicine group.
Claims (2)
1. An application of armillariella mycelium polysaccharide, which is characterized in that:
the armillarisin mycelium polysaccharide is used for preparing a medicinal preparation with the function of treating canker sore;
the monosaccharide composition and the molar ratio of the armillariella mycelium polysaccharide are mannose, galacturonic acid, glucose, arabinose=9.83, 14.52, 17.45 and 8.63; the molecular weight of the armillariella mycelium polysaccharide is 2.95X10 4 Da。
2. Use according to claim 1, characterized in that:
the pharmaceutical preparation can obviously reduce the level of inflammatory factors, improve the content of epidermal growth factors, reduce inflammatory cell infiltration and regulate the richness and uniformity of oral flora.
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