Preparation method of snail mucus polysaccharide and chronic wound repair medicine

Abstract

The invention belongs to the technical field of medical chronic wound dressing. The preparation method of the snail mucus polysaccharide is characterized by comprising the following steps: (1) treating snail mucus at high temperature to denature and inactivate protein; (2) filtering the treated snail mucus through four layers of medical gauze to remove impurities; (3) carrying out reduced pressure suction filtration on the filtrate obtained in the step (2); (4) filtering the filtrate obtained in the step (3) by using a water system filter membrane; and (5) freeze-drying to obtain the snail mucus polysaccharide freeze-dried powder. The chronic wound repair medicine comprises the snail mucus polysaccharide freeze-dried powder and an auxiliary agent, the mass ratio of the snail mucus polysaccharide freeze-dried powder to the auxiliary agent is 1: 9. The invention is used for solving the technical problems of high cost and to-be-verified clinical effect of the existing chronic wound repair medicine.

Description

Technical Field

[0001] The invention relates to the technical field of medical chronic wound dressings, and in particular to a method for preparing snail mucus polysaccharide, and application of the snail mucus polysaccharide obtained by the preparation method in preparing chronic wound repair drugs. Background Art

[0002] About 1% of the world's population suffers from wounds, and about 5% of medical expenses are spent on wound repair. Chronic wounds have complex pathogenesis, are lingering, and are prone to recurrence, which not only causes heavy economic and mental pressure on patients, but also brings a heavy burden to the medical system. With the aging of the population and changes in dietary structure, the incidence of arteriosclerosis, diabetes, etc. has increased significantly, and the incidence of chronic wounds related to this has increased accordingly.

[0003] Chronic wounds refer to wounds that cannot go through the normal and orderly repair process in time, cannot proceed according to the normal healing stages, fail to heal for a long time and are in a state of continuous inflammation, and cannot reach a complete anatomical and functional healthy state. Clinically, they are usually classified according to etiology, that is, according to the main cause of the wound. Chronic wounds can be divided into diabetic wounds, wounds caused by various types of trauma (burns, surgery, etc.), pressure sores, vascular skin ulcers of the lower limbs, radiation skin ulcers, infected ulcers, cancerous ulcers, iatrogenic ulcers, chronic ulcers caused by other reasons, etc.

[0004] Wound healing is a complex process. The body repairs and regenerates wounds that are difficult to heal to achieve functional and anatomical integrity. Its formation mechanism is relatively complex and involves many aspects, including local tissue hypoxia, inflammatory factors, and growth factor deficiency.

[0005] With the in-depth study of chronic wounds, there are a variety of treatment methods for the etiology and pathogenesis of chronic wounds in clinical practice, all of which have achieved certain therapeutic effects. However, in specific clinical applications, it is still necessary to select appropriate treatment methods based on the actual situation of the patient.

[0006] Debridement is the most basic means of treating chronic wounds. No matter what treatment plan is adopted, the wound must be kept fresh. Negative pressure wound therapy, hyperbaric oxygen, new silver ion dressings, and photobiomodulation play an auxiliary role in the treatment of chronic wounds, but the treatment cycle is relatively long. At present, some new technologies, such as platelet-rich plasma therapy and stem cell therapy, are in the clinical research stage, with high treatment costs, and a large number of clinical trials are still needed to prove their efficacy. Summary of the invention

[0007] The first object of the present invention is to provide a method for preparing snail mucus polysaccharide, so as to solve the technical problems that the existing chronic wound repair drugs are high in cost and the clinical effects need to be verified.

[0008] In order to solve the above technical problems, the present invention adopts the following technical scheme, a method for preparing snail mucus polysaccharide, characterized in that the preparation method is prepared by separating protein from snail mucus, comprising the following steps: (1) Treat snail mucus at high temperatures to denature and inactivate proteins; (2) The processed snail mucus is filtered through four layers of medical gauze to remove impurities; (3) The filtrate obtained in step (2) is filtered under reduced pressure; (4) The filtrate obtained in step (3) is filtered using a water filter membrane; (5) Freeze-drying to obtain freeze-dried powder.

[0009] The technical solution of the present invention is further improved, and the snail mucus polysaccharide includes: 53.9-54.01wt% of snail mucus total sugar, 11.37-11.41wt% of protein. According to literature reports, the remaining substances may be a variety of functional small molecules, such as allantoin, glycolic acid, vitamins, minerals, etc.

[0010] The technical solution of the present invention is further improved, and the total sugar of snail mucus includes the following components in weight percentage: 92.64% glucose, 4.83% arabinose, 0.86% rhamnose, 0.56% galactose, 0.36% fucose, 0.22% xylose, and 0.53% glucuronic acid. Snail mucus polysaccharide is obtained by separating snail mucus, has clear components, and is safe and effective.

[0011] In order to solve the technical problem of the source of snail mucus, the present invention adopts the following technical solution, wherein the snail mucus is derived from the Chinese white jade snail. The snail mucus polysaccharide used in the present invention is obtained by processing the mucus of the Chinese white jade snail. The breeding cost of the Chinese white jade snail is low, and it has become an industrialized breeding, and the source of raw materials is simple.

[0012] In order to solve the technical problem of high temperature treatment of snail mucus, the present invention adopts the following technical solution, wherein the high temperature in step (1) is maintained at 105-120°C.

[0013] In order to solve the technical problem of how to achieve vacuum filtration, the present invention adopts the following technical solution, wherein the filter paper used in the vacuum filtration in step (3) is an analytical quantitative filter paper with a pore size of 10 μm.

[0014] In order to solve the technical problem of water filter membrane parameters, the present invention adopts the following technical solution: the pore size of the water filter membrane used for filtration in step (4) is 0.45 μm.

[0015] The second object of the present invention is to provide a chronic wound repairing drug, comprising the above-mentioned snail mucus polysaccharide and an adjuvant; the mass ratio of the snail mucus polysaccharide to the adjuvant is 1:9.

[0016] In order to solve the above technical problems, the present invention adopts the following technical scheme, wherein the auxiliary agent is vaseline.

[0017] The application of traditional Chinese medicine in chronic wounds is also a current research focus, among which snails have attracted widespread attention for their medical and health value. Li Shizhen of the Ming Dynasty described the morphology and medicinal value of snails in detail in the Compendium of Materia Medica: "It can treat umbilical wind in children, promote urination, eliminate throat numbness, stop nosebleeds, relieve deafness, treat various toxic hemorrhoids and fistulas, and control centipede, scorpion and scorpion poisons." Modern Chinese medicine also recognizes that snails are salty and cold in nature, and enter the bladder, stomach, and large intestine meridians, and have the effects of clearing away heat and detoxification, calming the nerves, and reducing swelling.

[0018] Snail mucus is a protective liquid secreted by snails during crawling. Literature records that snail mucus is a mixture of 90-99.7% water and a small amount of protein, polysaccharide, etc., and contains a variety of functional small molecules, such as allantoin, glycolic acid, vitamins, minerals, etc. A large number of in vitro and in vivo studies have shown that snail mucus has a variety of pharmacological effects that promote human health, including promoting cell proliferation and migration, promoting angiogenesis, antibacterial, antioxidant, anticancer and other activities, most of which are related to skin repair and wound healing. Application of snail mucus polysaccharide in the preparation of chronic wound repair drugs. The chronic wound repair drug of the present invention can reduce tissue inflammation, improve tissue blood supply and effectively promote wound healing. In the evaluation of animal models, snail mucus polysaccharide can effectively reduce inflammation, improve blood vessels, and promote wound healing, and its effect is significantly increased compared with snail mucus. The preparation method is simple, the cost is low, and it can be mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a monosaccharide composition analysis diagram of snail mucus in Example 1 of the present invention; Figure 2 is a graph of fibroblast activity at different concentrations of the present invention; Figure 3 This is a diagram showing the effects of the snail mucus group, the experimental group (the present invention) and the control group on the wound healing speed of rats; Figure 4 It is a diagram showing the effects of the snail mucus group, the experimental group (the present invention) and the control group on the density of tissue capillaries; Figure 5 It is a diagram showing the effects of the snail mucus group, the experimental group (the present invention) and the control group on capillary density; Figure 6 It is a diagram showing the anti-inflammatory effects of the snail mucus group, the experimental group (the present invention) and the control group on TNF-α and IL-6. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] Snail mucus is safe and widely used in the field of beauty and skin care. There is no report on its effect on animal cells, so it has broad prospects for clinical application. In addition, the active ingredients of snail mucus have not yet been purified. How to create a repair drug with high safety, no allergic reaction, and multiple healing effects is one of the important research topics in this field.

[0022] Example 1 Preparation of snail mucus polysaccharide: 1. Snail Mucus Extract: Select fresh white jade snails weighing more than 20 grams, wash the snails with clean water and place them in a clean environment. Place the snails on a flat plate at a temperature below 28°C, and use a vibration motor to drive the plate up and down to promote the snail to secrete mucus. Use a container to collect the mucus secreted by the snail's foot gland and store the extracted mucus in an environment of 0-5°C.

[0023] 2. Preparation of snail mucus polysaccharide: (1) Preparation of snail mucus polysaccharide 1.5 L of snail mucus protein was denatured and inactivated, then filtered through four layers of medical gauze to remove impurities such as dust, feed, and broken shells. It was then vacuum filtered through an analytical quantitative filter paper with a pore size of 10 μm. The filtrate was then filtered using a water filter membrane with a pore size of 0.45 μm and freeze-dried to obtain a total of 20 g of freeze-dried powder with a yield of about 13%.

[0024] (2) Determination of total sugar content and protein content Glucose was used as the standard and prepared into a stock solution with a concentration of 1 mg / mL using ultrapure water, which was then diluted step by step into standard working solutions with concentrations of 0.8, 0.4, 0.2, 0.1, and 0.05 mg / mL. Snail mucus samples were prepared into a solution with a concentration of 1 mg / mL using ultrapure water. 100 μL of the above prepared standard solution and sample solution were taken, and 100 μL of 5% phenol solution was added respectively. After reacting at room temperature for 30 min, 1 mL of concentrated sulfuric acid was added, mixed, and 100 μL was aspirated into a 96-well plate. The absorbance was measured at a wavelength of 490 nm using an ELISA reader.

[0025] The protein content in the sample was determined using the BCA kit. According to the instructions of the kit, standard bovine serum albumin (BSA) was dissolved in phosphate buffer (1×PBS) at pH=7.2 to form a 25 mg / mL stock solution. An appropriate amount of the stock solution was further diluted to 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL standard solutions, and the sample to be tested was made into a 1 mg / mL solution. 20 μL of the standard solution and sample solution were taken in a 96-well plate, and the working solution A and B in the kit were mixed at a ratio of 50:1, and 200 μL of the mixed working solution was added. After incubation at 37°C for 30 min, the absorbance was measured at a wavelength of 540 nm using an enzyme reader.

[0026] (3) Analysis of monosaccharide composition of snail mucus Prepare 2 M trifluoroacetic acid (TFA) by adding 2.28 mL of TFA to a volumetric flask and dilute to 10 mL with ultrapure water.

[0027] Prepare a mixed standard solution: take 3 mg of each of fucose, rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid and glucuronic acid and add 3 mL of ultrapure water to make a 1 mg / mL sample solution. Dilute it stepwise to 100, 80, 60, 40, 20, 10, 5, and 2.5 ppm and filter it with a 0.22 μm water filter.

[0028] Weigh 6 mg of snail mucus sample and dissolve it in 0.6 mL of ultrapure water to prepare a 10 mg / mL sample solution. Take a brown bottle, draw 500 μL of 5 mg / mL sample solution and 2 mL of 2M TFA, shake well; cover the bottle cap tightly, wrap the bottle mouth with plastic wrap to prevent acid volatilization, and react in a 100℃ oven for 12 h. After returning to room temperature, take 500 μL of the reaction solution and adjust it to neutral with 1M NaOH; add water to make up to 5 mL; filter with a 0.22 μm water filter and analyze.

[0029] High performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) was used for analysis, and different monosaccharides were quantitatively analyzed by external standard method. The chromatographic column used was DionexCarboPac TM PA10 (4 mm×250 mm). Mobile phase A was 15 mM sodium hydroxide, and mobile phase B was 15 mM sodium hydroxide and 150 mM sodium acetate. The single injection volume was 25 μL, and the sample was eluted at a flow rate of 1 mL / min at 30°C. Figure 1 This is a monosaccharide composition analysis diagram of snail mucus in Example 1. Figure 1 As shown, the polysaccharides in this snail mucus have glucose as the main monosaccharide and contain a small amount of arabinose.

[0030] Example 2 Snail Mucus Polysaccharide Cell Proliferation Experiment (1) Cell proliferation assay The cells were divided into three groups: snail mucus group, experimental group (snail mucus polysaccharide) and control group. About 1000-2000 cells (100ul) were added to each well, and 4-6 duplicate wells were set for each group. A blank group was also set up. PBS buffer was added to the outermost wells to reduce the effect of evaporation. Then the cells were placed at 37℃ 5%CO 2 The cells were cultured in a cell culture incubator for 12-24 h. Drug addition and incubation time after drug addition: Observe that the cells adhere well to the wall, aspirate the culture medium of each well, add culture medium containing different concentrations of drugs to the experimental group, and add culture medium without drugs to the blank group, and then incubate the 96-well plate at 37°C and 5% CO 2 Incubate cells in a culture incubator.

[0031] Add 10ul of CCK-8 solution to each well, and be careful not to generate bubbles during the addition process. Immerse the tip of the pipette into the culture medium and add slowly. Incubation time after adding CCK-8: Place the culture plate with CCK-8 added in a 37℃ 5% CO 2 Incubate in the incubator for 1-4 hours. Measure OD value: Take out the 96-well plate and use a microplate reader to measure the OD value of each well at a wavelength of 450nm. Select 0h, 12h, 24h, 48h, and 72h to record the values, analyze and process the data, and draw a proliferation curve.

[0032] (2) Results analysis like Figure 2 As shown in Table 1: Snail mucus can promote cell proliferation, among which snail mucus polysaccharide (experimental group) has the best proliferation promoting effect compared with ordinary mucus.

[0033] Table 1 Fibroblast proliferation effects under different environments Example 3 Animal Experimental Study on the Effectiveness of Snail Mucus Polysaccharide (1) Animal experiment on snail mucus polysaccharide The rats were divided into three groups: snail mucus group, experimental group (snail mucus polysaccharide) and control group. The hair on the back surface of the rats was shaved with an electric shaver. After 12 hours of adaptive culture, the rats were anesthetized with 50 mg / kg sodium pentobarbital. After the rats were anesthetized, a skin sampler was used to cut a circular area with a diameter of 20 mm on the left and right sides of the midline of the back of the rats, causing symmetrical and equal-sized full-thickness skin wounds.

[0034] Snail mucus, snail mucus polysaccharide and an equal amount of vaseline were mixed to prepare vaseline medicine. The treatment was carried out once a day and the burn wound was photographed and observed, and the wound area was measured and recorded for 8 days. A ruler was placed near the wound to set a measuring scale, and the wound size was measured and recorded using Image J software.

[0035] (2) Analysis of wound healing results like Figure 3 As shown, the healing speed of snail mucus polysaccharide (experimental group) was significantly faster than that of the snail mucus group and the vaseline control group.

[0036] (3) Pathological analysis After collecting the wound tissue samples from the back of the rats, they were immediately fixed in a 10% formalin solution. After fixation, the tissues were embedded in paraffin, and the paraffin blocks were cut into 4 μm thick tissue slices for later use.

[0037] Conventional dewaxing: Gently rinse three times with 1xPBS solution, each time for 5 minutes. Place the tissue sections in a freshly prepared PBS solution containing 3% H 2 0 2 The reaction time was about 10 min, and then the slices were removed and washed with ddH 2 Rinse gently with 0 water for 5 min, and then rinse gently 3 times with 1x PBS solution, each time for 5 min.

[0038] The sections were then processed according to the following procedure: 100% alcohol I for 5 min, 100% alcohol II for 5 min, 95% alcohol I for 5 min, 95% alcohol II for 5 min, 85% alcohol for 3 min, 75% alcohol for 2 min, and distilled water rinse for 5 min.

[0039] Then, HE staining was performed according to the following process: hematoxylin staining for 5 minutes, water washing, hydrochloric acid alcohol differentiation treatment for 15 seconds, water washing (microscope examination to confirm the staining), water washing for 15 minutes, distilled water washing for 2 minutes, 0.5% eosin staining for 1 minute, 75% alcohol treatment for 2 minutes, 85% alcohol treatment for 3 minutes, 95% alcohol I treatment for 5 minutes, 95% alcohol II treatment for 5 minutes, 100% alcohol I treatment for 5 minutes, 100% alcohol II treatment for 5 minutes, xylene I treatment for 10 minutes, and xylene II treatment for 5 minutes. HE staining was completed.

[0040] Observe the specimens under a microscope and record the minimum distance of the scar on the skin surface, such as Figure 4As shown, the snail mucus polysaccharide (experimental group) had a smaller average minimum distance and faster wound healing speed than the snail mucus group and the vaseline control group.

[0041] Observe the specimen under a microscope and record the density of tissue capillaries, such as Figure 5 As shown, the healing speed of snail mucus polysaccharide (experimental group) and the capillary density were higher in the snail mucus group compared with the vaseline control group.

[0042] Example 4 Anti-inflammatory effect of snail mucus polysaccharide (1) Anti-inflammatory experiment of snail mucus polysaccharide Take the wound and surrounding tissues of the rat, rinse the tissues with pre-cooled PBS (0.01M, pH=7.4), remove residual blood, weigh and mince the tissues. Add the minced tissues and PBS into a glass homogenizer and grind them thoroughly on ice. In order to further lyse tissue cells, the homogenate can be repeatedly frozen and thawed or ultrasonically disrupted. Finally, centrifuge the homogenate at 2-8℃, 5000×g for 5-10 minutes and take the supernatant.

[0043] Set up standard wells, blank wells, and sample wells respectively. Add 100 μL of diluted standard to the standard wells, 100 μL of standard & sample diluent to the blank wells, and 100 μL of the sample to be tested to the remaining wells, cover the ELISA plate, and incubate at 37°C for 90 minutes. Shake off the liquid in the wells, add 100 μL of biotinylated antibody working solution to each well, cover the ELISA plate, and incubate at 37°C for 1 hour.

[0044] Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Add 100μL of enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37℃ for 30 minutes. Add 90μL of substrate solution to each well, cover the ELISA plate with a film, and incubate at 37℃ in the dark for about 15 minutes. Add 50μL of stop solution to each well to stop the reaction. Immediately measure the OD value of each well at a wavelength of 450 nm using an ELISA reader.

[0045] (2) Results analysis like Figure 6 As shown, the snail mucus polysaccharide is the experimental group. The levels of TNF-α and IL-6 in the experimental group were significantly lower than those in the control group, indicating that mucus polysaccharide has an anti-inflammatory effect.

Claims

1. A method for preparing snail mucus polysaccharide, characterized in that: The preparation method is prepared by separating protein from snail mucus, comprising the following steps: (1) Treat snail mucus at high temperatures to denature and inactivate proteins; (2) The processed snail mucus is filtered through four layers of medical gauze to remove impurities; (3) The filtrate obtained in step (2) is filtered under reduced pressure; (4) The filtrate obtained in step (3) is filtered using a water filter membrane; (5) Freeze-drying to obtain snail mucus polysaccharide freeze-dried powder.

2. The method for preparing snail mucus polysaccharide according to claim 1 is characterized in that: The snail mucus polysaccharide freeze-dried powder contains 53.9-54.01 wt % of total snail mucus sugar and 11.37-11.41 wt % of protein.

3. The snail mucus polysaccharide according to claim 2, characterized in that: The total sugar of the snail mucus includes the following components in weight percentage: 92.64% glucose, 4.83% arabinose, 0.86% rhamnose, 0.56% galactose, 0.36% fucose, 0.22% xylose, and 0.53% glucuronic acid.

4. The method for preparing snail mucus polysaccharide according to claim 1, characterized in that: The snail mucus is derived from the Chinese white jade snail.

5. The method for preparing snail mucus polysaccharide according to claim 1, characterized in that: The high temperature in step (1) is maintained at 105-120°C.

6. The method for preparing snail mucus polysaccharide according to claim 1, characterized in that: The filter paper used in the reduced pressure filtration in step (3) is an analytical quantitative filter paper with a pore size of 10 μm.

7. The method for preparing snail mucus polysaccharide according to claim 1, characterized in that: The pore size of the water filter membrane used for filtration in step (4) is 0.45 μm.

8. Chronic wound repair medicine, characterized by: The invention comprises the snail mucus polysaccharide obtained by the snail mucus polysaccharide preparation method according to any one of claims 1 to 7, and an adjuvant; The mass ratio of the total sugar in the snail mucus to the auxiliary agent is 1:

9.

9. The chronic wound repairing drug according to claim 8, characterized in that: The auxiliary agent is vaseline.

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