Application of exopolysaccharide from Falmioporus occidentalis

Abstract

The present invention relates to the field of drug design technology, and specifically relates to an application of an extracellular polysaccharide of F. cyrtonema, which can be used to prepare a skin wound healing agent, especially a skin scald healing agent; specifically: the extracellular polysaccharide of F. cyrtonema can effectively inhibit the inflammatory response of the wound, promote the production of mouse cytokines, accelerate tissue repair and regeneration, and has a good effect on the healing of mouse scald skin. The present invention provides a new effective ingredient for the preparation of wound healing agents, and also provides a new direction for the application of the extracellular polysaccharide of F. cyrtonema.

Description

Technical Field

[0001] The invention relates to the technical field of medicaments, and in particular to an application of extracellular polysaccharide of Familiar occidentalis. Background Art

[0002] Fungal polysaccharides are polysaccharide secondary metabolites produced by fungal mycelium during the fermentation process. They are widely present in mycelium, fruiting bodies and fermentation broth. They are a type of active substance that can control cell differentiation, regulate cell growth and aging. At present, related research mainly focuses on the separation and extraction of fungal polysaccharides, content analysis and detection, material structure analysis, and pharmacological activity evaluation. Studies have shown that when active polysaccharides are used as biological effect regulators, they have multiple biological activities such as antioxidant, anti-inflammatory, immunomodulatory, anti-tumor, antiviral, and lipid-lowering.

[0003] Fomes fomentarius is a fungus of the order Polyporales, belonging to the class Basidiomycetes, order Polyporales, family Polyporaceae, and genus Fomes. According to the Compendium of Materia Medica, studies have found that the ethanol extract of its fruiting body has a strong inhibitory effect on mouse S-180 tumors, and Fomes fomentarius has antioxidant, anti-inflammatory, and anti-diabetic activities, but no article has reported whether the polysaccharide of Fomes fomentarius has good activity against mouse scalded skin. Summary of the invention

[0004] The inventors conducted experiments on the healing ability of the extracellular polysaccharide of F. oxysporum on the wounds of scalded mice and found that the extracellular polysaccharide of F. oxysporum can be used to prepare a skin wound healing agent, especially a skin scald healing agent; specifically, the extracellular polysaccharide of F. oxysporum can effectively inhibit the inflammatory response of the wound, promote the production of mouse cytokines, accelerate tissue repair and regeneration, and has a good effect on the healing of scalded skin in mice.

[0005] The exopolysaccharide of Faecalibacterium oxysporum is prepared by the following method:

[0006] After deep fermentation of the fruiting body of the woody porphyra, the mycelium and the fermentation liquid were obtained by filtering with gauze respectively; after the fermentation liquid was concentrated by vacuum decompression, 95% ethanol was added to the concentrated fermentation liquid (3:1), and the fermentation liquid was placed in a refrigerator at 4°C overnight to obtain a precipitate, which was the crude polysaccharide;

[0007] The crude polysaccharide was washed with distilled water for three times, and then deproteinized and decolorized with 30% H2O2. The decolorized and deproteinized polysaccharide solution was dialyzed with a retention rate of 8000-14,000Da. The dialysis time was: 24 hours with tap water, 24 hours with distilled water, and freeze-dried to obtain the primary purified extracellular polysaccharide of the wood hoof fungus. FFEP was eluted in series with two DEAE-52 chromatography columns, and the primary purified polysaccharide was further purified. A 1.0 mg / mL polysaccharide solution was accurately prepared and evenly added to the DEAE-52 chromatography column (1.6 cm×50 cm, filling height 25 cm). The sample was eluted with double distilled water at a flow rate of 0.5 mL / min. After the eluate was concentrated and freeze-dried, a light yellow powder was obtained, which was the pure polysaccharide.

[0008] The invention provides a new effective ingredient for the preparation of wound healing agents and also provides a new direction for the application of the extracellular polysaccharide of the phyllostachys occidentalis. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The skin sections of the wound surface of mice in different drug-administered groups are shown;

[0010] In the figure: A. blank control group, B. high-dose group, C. positive control group, D. healthy mice without burns; ×40 (the numbers after the letters represent days).

[0011] Figure 2 The changes of TGF-β content in the wound surface of mice in different drug-treated groups.

[0012] Figure 3 The figure shows the changes of IL-6 content in the wound surface of mice in different drug-administered groups.

[0013] Figure 4 The changes of MMP-1 content in the wound surface of mice in each group receiving different medications. DETAILED DESCRIPTION

[0014] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0015] Example

[0016] Extracellular polysaccharide of Falmioporus oxyphylla: provided by the Natural Products Research Laboratory, College of Biological Sciences and Engineering, Northern Minzu University. Method: The polysaccharide product was obtained by deep fermentation, filtration, alcohol precipitation, decolorization, deproteinization, dialysis and alcohol precipitation of the fungus Falmioporus oxyphylla.

[0017] Experimental animals

[0018] 52 Kunming mice weighing about 25±2g were provided by the Animal Room of the College of Biological Sciences and Engineering of Northern Minzu University, with the qualification certificate number: Ningyishidongzhun No. 01. The living environment of mice: temperature 20±2℃, relative humidity 40±5%, normal light and darkness.

[0019] Reagent configuration

[0020] (1) Depilatory agent: Accurately weigh 2.00 g of washing powder, 6.0000 g of sodium sulfide, and 14.0000 g of starch, and mix them into a paste according to the proportions for later use.

[0021] (2) Physiological saline: Weigh 0.9000 g of sodium chloride, accurately measure 100 ml of distilled water, add the weighed sodium chloride into the distilled water, and stir to fully dissolve it to obtain 0.9% physiological saline.

[0022] (3) 1.0% extracellular polysaccharide ointment of Fa. oxyphylla (low dose): Accurately weigh 0.5000 g of extracellular polysaccharide of Fa. oxyphylla, measure 1.0 ml of dimethyl sulfoxide, and 50.00 g of vaseline, mix them according to the proportion, stir evenly and set aside.

[0023] (4) 5.0% extracellular polysaccharide ointment of Fa. oxyphylla (high dose): Accurately weigh 2.5000 g of extracellular polysaccharide of Fa. oxyphylla, measure 1.0 ml of dimethyl sulfoxide, and 50.00 g of vaseline, mix them according to the proportion, stir evenly and set aside.

[0024] Animal modeling of mice

[0025] Hair removal on the back of mice:

[0026] After one week of adaptive feeding of the experimental mice, the mice were anesthetized in a closed beaker filled with ether gas for about 1 minute. After the mice were anesthetized, they were taken out of the beaker and placed on the operating table. Scissors were used to cut out a 3.0cm×3.0cm exposed area. The hair on the back of the mice was cut as short as possible, but the skin should not be damaged. The depilatory agent was evenly applied to the north part. After about 3 minutes, it was wiped clean with absorbent cotton dipped in warm water.

[0027] Start modeling:

[0028] Mice were anesthetized with ether for about 1 min, and the anesthetized mice were fixed on the dissecting tray. Cotton wool (or iron rod with a diameter of 1 cm) was soaked in 90℃ hot water for 2 min. The iron rod was taken out and quickly pressed against the back of the anesthetized mice. The scalding area was 2.0 cm 2 , lasting 15 seconds. It can be observed that the scald area is clearly identifiable, white, the epidermis is wrinkled, and the subcutaneous tissue is obviously edematous after 30 minutes, indicating that the deep II degree scald model is successfully established.

[0029] Experimental mice grouping:

[0030] 52 mice were randomly divided into 4 groups, 13 mice in each group, including 5% FFEP-1 group, 1% FFEP-1 group, negative control group (0.9% saline or allowed to recover naturally), and positive control group (polymyxin B ointment). The negative control group was not given any drugs and allowed to recover naturally.

[0031] feed:

[0032] All mice were fed normally, and the burned parts were smeared with medicine twice a day and exposed to air, about 0.1 ml each time.

[0033] Observation on the appearance and pathological histology of scalded skin

[0034] The skin of the wound surface is about 1.5×1.5cm 2 The skin tissue was sliced ​​with paraffin, stained with HE, and observed under a 40x optical microscope.

[0035] Paraffin sectioning steps:

[0036] (1) Material collection: The materials must be fresh and complete. In this experiment, 3 mice were randomly selected from each group and placed in a large beaker filled with ether at 0, 4, 8, 12, and 15 days after the mouse model was established. The beaker was sealed for about 5 minutes. After the mice died, they were taken out and the wounds and the skin of the depilated parts of the mice were carefully cut with dissecting scissors to remove fat.

[0037] (2) Fixation: The treated skin was immediately placed in 4% paraformaldehyde, sealed, and stored in a refrigerator at 4°C overnight.

[0038] (3) Washing and dehydration: Remove the tissue from the fixative paraformaldehyde, rinse off the fixative with running water, cut off the excess, and trim into small pieces suitable for slicing. The trimmed tissue is placed in the following concentrations of alcohol for dehydration: 50% alcohol (2h) - 70% alcohol (2h) - 80% alcohol (1.5h) - 90% alcohol (1.5h) - 95% alcohol (1h) - 100% alcohol (0.5h)

[0039] (4) Transparency: The dehydrated tissue was immersed in a 1:1 solution of anhydrous alcohol and xylene for 1 h, then immersed in xylene i for 30 min, and finally immersed in xylene ii (30 min).

[0040] (5) Wax immersion: Mix soft wax liquid and xylene in a ratio of 1:1, immerse the skin tissue in the wax liquid for 2.0 h, then immerse it in the soft wax liquid for 2.0 h, and finally immerse it in the hard wax liquid for 2.0 h. Try to keep the entire process in a 60°C oven. If the wax liquid solidifies, it will seriously affect the slicing process. Insufficient or excessive wax immersion time will lead to slicing failure.

[0041] (6) Embedding: Place the wax-impregnated tissue blocks into the wax liquid. After solidification, remove the wax blocks and use a blade to gently cut off the excess blank wax blocks. Trim the wax blocks into cubes and finally fix them for 5 μm sectioning.

[0042] (7) Slicing and pasting: Pick up the slices with a brush and spread them flatly on a glass slide coated with chrome alum gelatin adhesive sheet, and bake them on a slide spreader until the adhesive sheet is dry.

[0043] (8) Dewaxing: The mounted sections were immersed in xylene i for 30 min, xylene ii for 30 min, and a mixture of xylene and anhydrous ethanol (1:1) for 1 h.

[0044] (9) HE staining: After the tissue sections are drained, they are stained with nuclear stain (hematoxylin stain) for 5 min, rinsed under running water for about 5 min, then stained with pulp stain (eosin stain) for 2 min, rinsed with a color enhancer, and the residual water on the slide was absorbed with filter paper.

[0045] (10) Dehydration: HE-stained sections were placed in different ethanol solutions for dehydration;

[0046] (11) After staining, the sections were sealed with neutral gum and examined under a microscope at 40x magnification after drying overnight.

[0047] Treatment of burnt skin surface of mice and determination of related indexes

[0048] Mice were randomly divided into a low-dose group (1% FFEP), a high-dose group (5% FFEP), a negative control group, and a positive control group (polymyxin ointment). The scalded surface of the mouse skin was evenly smeared with equal amounts of 1% and 5% FFEP ointment and polymyxin ointment, respectively. The negative control group was not treated, and the latter was treated with 0.9% saline twice a day at a fixed time (morning and evening). The scab formation and scabbing, healing of skin wounds, etc. of mice in each group were observed and recorded daily. On the 4th, 8th, 12th, and 18th days after scalding, 3 mice in each group were killed, and the wound skin was taken for measurement. The wound area of ​​each mouse at different time points was analyzed, and the healing days and scabbing days were counted. The formula is as follows:

[0049] Wound healing rate = (initial scald area - wound area at each time point) / initial scald area × 100%

[0050] The remaining damaged skin was taken and weighed, and pre-cooled saline was added at 1 ml / 100 mg, and the mixture was manually homogenized to make a 50% homogenate. The mixture was centrifuged at 10,000 r / min for 15 min at 4°C, and the supernatant was taken. The contents of mouse interleukin-6 (IL-6), mouse transforming growth factor β (TGF-β), and mouse matrix metalloproteinase (MMP-1) were determined according to the instructions of the enzyme-linked immunosorbent assay kit.

[0051] Statistical analysis

[0052] At least three mice were treated each time, and all data were processed by GraphPad Prism 5 statistical software and expressed as mean ± standard deviation. The differences between groups were analyzed by t test. Image J image processing and analysis software was used to analyze the wound area of ​​each mouse at different time points.

[0053] Results and Analysis

[0054] Appearance and pathological histological observation of skin burns

[0055] Soak a 1cm diameter iron rod in 90℃ hot water for 2 minutes, take it out and quickly place it close to the back of Kunming mice, keep the scald area uniform, and continue the treatment for 15 seconds for each mouse. The skin at the scald site is obviously red and swollen, and subcutaneous blood vessels are visible; after 30 minutes, the subcutaneous tissue of the mouse is obviously edematous, the blisters are relatively flat and thin, the wound surface is light red or red and white, the reticular embolism of blood vessels is obviously seen, and the bottom of the skin can be seen. The typical symptoms of deep second-degree scald appear, indicating that the modeling is successful.

[0056] After modeling, mice were randomly divided into four groups: negative control group B, 1.0% FFEP low-dose group C, 5.0% FFEP high-dose group D, and positive control group E. They were free to eat every day, and the medicine was applied twice a day at a fixed time. The dosage was controlled and applied evenly each time. We can clearly see that the scalded tissue of mice in the negative control group without any treatment healed the slowest at 4d, 8d, 12d, and 15d of medication, and the wound had obvious suppuration at 8d; different amounts of polysaccharides have a certain repair ability on scalded skin, and show a certain dose-effect relationship. Compared with the negative control group, the suppuration phenomenon of mice in the low-dose group was reduced, and the scab time was fast; mice in the high-dose group had no yellow liquid outflow, and at the same treatment time, the wound could obviously see obvious healing effect, which was close to the positive control polymyxin; after 15d of treatment, the scalds in the negative control group and the low-dose group were not completely healed, and the high-dose group and the positive control group almost completely shed scabs, and new hair could be clearly seen growing on the scalded part, completely covering the wound. This indicates that the extracellular polysaccharide of Falmioporus occidentalis has a certain healing ability on the scalded tissue of mice, and presents a certain dose-effect relationship. The effect of the high-dose group of 5.0% FFEP is close to that of the positive control group.

[0057] Paraffin sections of the burned skin were taken for microscopic observation. Figure 1 It is obvious that compared with the blank control group, the high-dose group had less inflammatory cell infiltration in the skin of scalded mice, which indicates that the polysaccharide of F. fusca can effectively inhibit inflammation. After 15 days of medication, the skin of the mice in the high-dose group regenerated well. Compared with the normal mice, the skin tissue was clearly identifiable, and many new microvessels were visible (indicated by the white arrows); compared with the blank group, the thickness of the skin epidermis was obvious, close to the epidermis of normal mice.

[0058] Determination of skin wound healing rate

[0059] Table 1 Effect of FFEP on the healing rate of burn wounds in mice

[0060]

[0061]

Note

[0062] By measuring the wound healing area of ​​the scalded tissue at different times, it can be seen from the table above that when the wound was 4 days old, there was no significant difference in the low-dose group compared with the negative control group, while the high-dose group and the positive group had a certain significance (p<0.05); after 4 days of wound treatment, the healing rate of the skin of the mice in the drug-treated group increased significantly (p<0.01), and the healing rate was close to that of the polymyxin positive control group; after 15 days of treatment, the wounds of the mice in the high-dose group and the positive control group were basically scabbed, and elastic and ruddy regenerated skin had been formed, while the wounds of the mice in the negative group had not yet completely healed. This shows that the extracellular polysaccharide of the woody porphyra can significantly promote the scabbing and regeneration of scalded tissue in mice, and effectively shorten the wound healing time.

[0063] Determination of cytokines in skin wounds

[0064] Mouse transforming growth factor TGF-β is an important growth factor that regulates wound regeneration. Low levels of expression after skin trauma will affect skin regeneration and remodeling. Figure 2 It can be clearly seen that after 4 days of drug administration, the TGF-β content of the drug-treated group was significantly higher than that of normal mice, while the negative control group without any treatment decreased, and the inflammatory response worsened; with the extension of the drug administration time, when it was 12 days, the high-dose group showed a very significant increase compared with the normal mice, which can indicate that the extracellular polysaccharide of the wood-tied porphyra has a certain promoting effect on the expression of TGF-β in the body of scalded mice. The self-repair of the mice in the negative control group also showed a significant increase in TGF-β; by 15 days, the skin of the mice in the drug-treated group was basically completely detached, and the expression of TGF-β in the body tended to be normal mice.

[0065] Mouse interleukin IL-6 is also an important growth factor that regulates skin wound regeneration. Low levels of expression after skin trauma will affect skin regeneration and remodeling. Figure 3 It is obvious that after 4 days of drug administration, the IL-6 content of the drug-treated group was significantly higher than that of normal mice, while the IL-6 content of the negative control group without any treatment decreased due to the aggravated inflammatory response and yellow liquid infiltration; as the drug administration time prolonged, when it was 4-12 days, compared with normal mice, the interleukin of the low-dose group and high-dose mice increased significantly, which may be that the extracellular polysaccharide has a certain promoting effect on the expression of IL-6 in the scalded mice, which helps the healing of the mouse wound. In the self-repair of the mice in the negative control group, IL-6 also showed an increasing trend; by 15 days, the skin of the mice in the drug-treated group was basically completely scabbed, and the expression of IL-6 in the body tended to be normal mice.

[0066] Mouse matrix metalloproteinase MMP-1 is a collagenase, and its content is closely related to the healing of skin wounds. The synthesis and degradation of collagen are inseparable from collagenase. After skin wounds, collagenase will be stimulated to produce and help skin tissue regeneration and remodeling. Figure 4 It is obvious that after 4 days of drug administration, the MMP-1 content of the drug-treated groups (low-dose group, high-dose group, and positive control group) increased compared with normal mice, thereby accelerating the decomposition and regulation of collagen in the wound skin, while the MMP-1 content of the negative control group without any treatment decreased due to the aggravated inflammatory response; with the extension of the drug-treated time, when 4-12 days, compared with normal mice, the matrix metalloproteinases of the low-dose group and high-dose group mice increased significantly, which may be due to the treatment of wounds by extracellular polysaccharides, which has a certain stimulating effect on the expression of MMP-1 in the wounds of scalded mice and helps to decompose collagen in the wounds of mice. The mice in the negative control group did not use drugs, and the body needed a long period of self-repair, and MMP-1 also showed a slow increase trend; by 15 days, the skin of the drug-treated group mice and the positive control group mice was basically completely scabbed, and the expression of MMP-1 in the body tended to be normal mice.

[0067] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. The application of the exopolysaccharide of Phyllostachys occidentalis is characterized by: Can be used to prepare skin burn healing agent; The exopolysaccharide of Faecalibacterium oxysporum is prepared by the following method: After deep fermentation of the fruiting body of the woody porphyra, the mycelium and the fermentation liquid are obtained by filtering with gauze respectively; after the fermentation liquid is concentrated by vacuum decompression, 95% ethanol is added to the concentrated fermentation liquid, the volume ratio of the ethanol to the fermentation liquid is 3:1, and the fermentation liquid is placed in a refrigerator at 4°C overnight to obtain a precipitate, which is crude polysaccharide; The crude polysaccharide is washed three times with distilled water, deproteinized and decolorized with 30% H2O2, and then the decolorized and deproteinized polysaccharide solution is dialyzed with a retention capacity of 8000-14,000Da, and the dialysis time is: 24 hours with tap water, 24 hours with distilled water, and freeze-dried to obtain the primary purified extracellular polysaccharide of the fungus; the primary purified polysaccharide is further purified by eluting with two DEAE-52 chromatography columns in series: 1.0 mg / mL polysaccharide solution is accurately prepared and evenly added to the DEAE-52 chromatography column, and the sample is eluted with double distilled water at a flow rate of 0.5 mL / min. After the eluate is concentrated and freeze-dried, a light yellow powder is obtained, which is the pure polysaccharide.

2. The use according to claim 1, characterized in that: The extracellular polysaccharides of Falmioporus occidentalis can effectively inhibit the inflammatory response of the wound, promote the production of cytokines in mice, and accelerate tissue repair and regeneration.

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