A composition for treating stubborn skin infection, sore and inflammation, and a preparation method and application thereof

Abstract

The application discloses a composition for treating stubborn skin infection, ulcer and inflammation and a preparation method and application thereof, and belongs to the technical field of functional compositions. The composition is prepared from components containing baicalin 1-5 parts, antibacterial alkaloids 0.5-3 parts, asiaticoside 2-6 parts, salvianolic acid B 3-8 parts, gallic acid 1-4 parts, white plant polysaccharide 5-10 parts, konjac glucomannan 3-10 parts, chitosan 3-5 parts, glycerol 8-20 parts, genipin 0.5-2 parts, calcium chloride 1-3 parts, laurazepam 1-3 parts and water 40-80 parts. The antibacterial alkaloids include berberine or phellodendrine. The application scientifically matches the above components based on deep understanding of the stubborn skin pathological mechanism, and the composition exhibits a significant synergistic effect in in-vitro antibacterial, antibiofilm and anti-inflammatory experiments. The preparation process is simple, the cost is low, and the application is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional compositions, and particularly relates to a composition for treating stubborn skin infections, ulcers and inflammations, and a preparation method and application thereof. BACKGROUND

[0002] Stubborn skin infections (such as folliculitis and furuncles caused by drug-resistant bacteria), chronic ulcers (such as diabetic foot ulcers and pressure ulcers), and severe skin inflammations (such as severe eczema and dermatitis) are difficult to treat clinically. Their common pathological features include: severe damage to the wound barrier function, formation of bacterial biofilm, persistent excessive inflammatory response, and low tissue repair and regeneration capacity. The current mainstream treatment programs such as antibiotics (such as mupirocin ointment) and corticosteroid drugs often have poor efficacy, drug resistance, high recurrence rate, and significant side effects when facing biofilm-related infections and chronic inflammation.

[0003] Natural active ingredients such as baicalin, obacunine, and berberine have been proven to have anti-inflammatory, antibacterial, antioxidant, and healing-promoting effects. High molecular materials such as white pith polysaccharide and konjac glucomannan are often used as pharmaceutical excipients. However, the existing technology is mostly the application of single components or simple physical mixtures, and cannot solve the compatibility, stability, and synergistic delivery problems of multiple components. For example, a simple mixed ointment cannot penetrate the biofilm barrier and the drug is released too quickly, which cannot maintain a long-acting effective concentration at the wound surface.

[0004] Therefore, it is of great clinical significance and market value to develop a new drug composition that can have multi-target synergistic effect, effectively penetrate and remove biofilm, regulate the inflammatory microenvironment, and continuously promote tissue regeneration. SUMMARY

[0005] In order to overcome the defects of the existing technology that the biofilm-related infection treatment is difficult and the multiple pathological links of chronic wounds are difficult to be considered, the present application provides a composition for treating stubborn skin infections, ulcers and inflammations, and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] In one aspect, the present application provides a composition for treating stubborn skin infections, ulcers and inflammations, which is prepared from the following components by mass fraction: baicalin 1-5 parts, antibacterial alkaloids 0.5-3 parts, asiaticoside 2-6 parts, salvianolic acid B 3-8 parts, gallic acid 1-4 parts, white pith polysaccharide 5-10 parts, konjac glucomannan 3-10 parts, chitosan 3-5 parts, glycerol 8-20 parts, genipin 0.5-2 parts, calcium chloride 1-3 parts, laurocapram 1-3 parts, and water 40-80 parts.

[0008] The antibacterial alkaloid includes berberine or phellodendrine.

[0009] Optionally, the composition for treating stubborn skin infection, sore and inflammation is prepared from components comprising: 3 parts of baicalin, 2 parts of antibacterial alkaloid, 4 parts of asiaticoside, 5 parts of salvianolic acid B, 3 parts of gallic acid, 7 parts of bletilla striata polysaccharide, 5 parts of konjac glucomannan, 4 parts of chitosan, 12 parts of glycerol, 1 part of genipin, 2 parts of calcium chloride, 2 parts of laurazepam, and 50 parts of water.

[0010] The antibacterial alkaloid includes berberine or phellodendrine.

[0011] Optionally, the degree of deacetylation of the chitosan is greater than or equal to 85%, and the molecular weight of the chitosan is 5000-20000 Da.

[0012] In a second aspect, the application provides a preparation method of the composition for treating stubborn skin infection, sore and inflammation, comprising the following steps:

[0013] (1) mixing bletilla striata polysaccharide and konjac glucomannan with water preheated to 55-60°C, stirring and heating to 80-85°C, and then keeping warm and standing, and cooling to obtain phase A;

[0014] (2) mixing chitosan with acidified water, and stirring until completely dissolved to obtain phase B;

[0015] (3) mixing baicalin, antibacterial alkaloid, asiaticoside, salvianolic acid B and gallic acid with glycerol, and homogenizing to obtain phase C;

[0016] (4) mixing laurazepam with glycerol to obtain phase D;

[0017] (5) dissolving genipin and calcium chloride in water respectively to obtain a genipin solution and a calcium chloride solution;

[0018] (6) adding phase B into phase A, stirring uniformly, then adding phase C, continuing to stir for 10-15 min, then adding the genipin solution, reacting for 10-20 min, and then adding the calcium chloride solution to obtain a light yellow transparent gel;

[0019] (7) adding phase D into the light yellow transparent gel, and adding the remaining water, stirring uniformly, and vacuum defoaming to obtain the composition for treating stubborn skin infection, sore and inflammation.

[0020] Optionally, in step (1), the stirring speed is 200-300 r / min.

[0021] The keeping warm and standing time is 25-35 min.

[0022] The cooling is to 25-40°C.

[0023] Optionally, in step (1), the stirring speed is independently selected from any value or a range between any two values of 200 r / min, 210 r / min, 220 r / min, 230 r / min, 240 r / min, 250 r / min, 260 r / min, 270 r / min, 280 r / min, 290 r / min, 300 r / min.

[0024] Optionally, in step (1), the incubation time is independently selected from any value or a range between any two values of 25 min, 28 min, 30 min, 32 min, 35 min.

[0025] Optionally, in step (1), the cooling is independently selected from any value or a range between any two values of cooling to 25°C, 30°C, 35°C, 40°C.

[0026] Optionally, in step (2), the pH of the acidified water is 4-5;

[0027] The acidification is performed using glacial acetic acid;

[0028] The concentration of the glacial acetic acid is 1-3 wt%;

[0029] The stirring speed is 300-500 r / min.

[0030] Optionally, in step (2), the pH of the acidified water is independently selected from any value or a range between any two values of 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.

[0031] Optionally, in step (2), the concentration of the glacial acetic acid is independently selected from any value or a range between any two values of 1 wt%, 2 wt%, 3 wt%.

[0032] Optionally, in step (2), the stirring speed is independently selected from any value or a range between any two values of 300 r / min, 350 r / min, 400 r / min, 450 r / min, 500 r / min.

[0033] Optionally, in step (3), the antibacterial alkaloid comprises berberine or phellodendrine;

[0034] The homogenization speed is 3000-4000 r / min.

[0035] The homogenization time is 5-10 min.

[0036] Optionally, in step (3), the rotation speed of the homogenization is independently selected from any value or a range between any two values of 3000 r / min, 3200 r / min, 3400 r / min, 3500 r / min, 3600 r / min, 3800 r / min, 4000 r / min.

[0037] Optionally, in step (3), the time of the homogenization is independently selected from any value or a range between any two values of 5 min, 6 min, 7 min, 8 min, 9 min, 10 min.

[0038] Optionally, the ratio of the amount of the glycerol in step (3) to the amount of the glycerol in step (4) is 1-3:1.

[0039] Optionally, in step (6), the rotation speed of the stirring is 50-100 r / min.

[0040] Optionally, in step (6), the rotation speed of the stirring is independently selected from any value or a range between any two values of 50 r / min, 60 r / min, 70 r / min, 80 r / min, 90 r / min, 100 r / min.

[0041] Optionally, in step (7), the rotation speed of the stirring is 300-500 r / min.

[0042] The pressure of the vacuum defoaming is -0.1 to -0.05 Mpa.

[0043] The time of the vacuum defoaming is 10-15 min.

[0044] Optionally, in step (7), the rotation speed of the stirring is independently selected from any value or a range between any two values of 300 r / min, 350 r / min, 400 r / min, 450 r / min, 500 r / min.

[0045] Optionally, in step (7), the pressure of the vacuum defoaming is independently selected from any value or a range between any two values of -0.1 Mpa, -0.09 Mpa, -0.08 Mpa, -0.07 Mpa, -0.06 Mpa, -0.05 Mpa.

[0046] Optionally, in step (7), the time of the vacuum defoaming is independently selected from any value or a range between any two values of 10 min, 11 min, 12 min, 13 min, 14 min, 15 min.

[0047] In a third aspect, the application provides the use of the above-mentioned composition in the preparation of a medicament for treating intractable skin infections, ulcers and inflammation.

[0048] Compared with the prior art, the application has the following advantages:

[0049] (1) The composition for treating intractable skin infections, ulcers and inflammation provided by the application combines berberine / baicalein (destroying cell membranes / structures) with gallic acid (inhibiting enzyme activity) to have a synergistic antibacterial and antibiofilm effect; combining baicalin (immunomodulation) with salvianolic acid B (antioxidation, improving microcirculation) and asperosaponin (stimulating collagen) achieves seamless connection from inhibiting excessive and continuous inflammatory response to actively promoting repair; using white yam polysaccharide (natural adhesive, having film-forming and moisturizing properties), konjac glucomannan (forming a heat irreversible gel when encountering alkali) and low molecular weight chitosan (antibacterial, promoting healing, and bioadhesion) as a composite matrix, using genipin and calcium chloride to crosslink the above-mentioned natural polysaccharide matrix, a stable hydrogel is formed in situ on the skin wound, the gel can closely adhere to irregular wounds, provide a persistent moist environment, and at the same time, through crosslinking of the polysaccharide chain, the release rate of the active ingredients is regulated, long-acting sustained release is achieved, the action time is prolonged, and the curative effect is improved; the added laurocapram is a penetration enhancer that can effectively overcome the barrier dysfunction of the skin and the obstruction of the stratum corneum, ensuring that both hydrophobic active ingredients (such as baicalin and berberine) and hydrophilic ingredients can be efficiently absorbed through the skin and reach the deep part of the lesion.

[0050] (2) The preparation method of the composition for treating intractable skin infections, ulcers and inflammation provided by the application adopts a unique step-by-step in-situ crosslinking preparation process, first crosslinking chitosan and the like with genipin (reacting with amino groups), then introducing calcium chloride (ionically crosslinking konjac glucomannan and the like), and finally forming a dense gel structure. This step-by-step process precisely controls the gel formation process, ensuring that the active ingredients (especially hydrophobic ingredients) are uniformly dispersed and encapsulated in the gel structure, guaranteeing the sustained-release characteristics and stability of the final product. Moreover, the preparation process of the application is simple, low in cost, and suitable for industrial production. DETAILED DESCRIPTION

[0051] The application will be further described below in combination with specific examples. The following description is only a few examples of the application and does not limit the application in any form. Although the preferred embodiments are disclosed as follows, the application is not limited thereto, and any skilled person in the art can make some changes or modifications to the disclosed technical content without departing from the scope of the technical solution of the application, which are equivalent to equivalent embodiments and belong to the scope of the technical solution.

[0052] Unless otherwise specified, the raw materials in the examples of the application are purchased through commercial channels and used directly without any special treatment.

[0053] Unless otherwise specified, the analytical methods in the examples all use the conventional settings of the instruments or devices and the conventional analytical methods.

[0054] Scutellarein in the following examples and comparative examples was purchased from Wuhan Kangqiong Biomedicine Technology Co., Ltd. (the active ingredient content was 90%); Phellodendrine, asiaticoside, genipin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (the purity was all: HPLC≥98%); Berberine, konjac glucomannan were purchased from Jiangsu Cailei Biotechnology Co., Ltd. (the purity was all 99%); Salvianolic acid B was purchased from Nanjing Bencao Yikang Biotechnology Co., Ltd. (CAS No.: 121521-90-2, purity≥98%); Gallic acid was purchased from Hubei Xinrunde Chemical Co., Ltd. (CAS No.: 149-91-7, purity 99%); Bletilla striata polysaccharide was purchased from Xi'an Green Bio-technology Co., Ltd. (purity 98%); Chitosan was purchased from Wuhan Jiyesheng Chemical Co., Ltd. (the molecular weight was 5000 Da); Glycerol was purchased from Jiangxi Yikang Natural Spice Oil Refining Factory (purity 99%); Calcium chloride was purchased from Puyang Kaisi Chemical Co., Ltd. (purity 99%); Laurazepine was purchased from Hubei Boishi Chemical Co., Ltd. (purity 99%); Sophoridine was purchased from Xi'an Bai Nian Kang Biotechnology Co., Ltd.; Paeonol was purchased from Xi'an Miao Gu Biotechnology Co., Ltd.

[0055] Example 1

[0056] A preparation method of a composition for treating intractable skin infections, ulcers and inflammation, the steps are as follows:

[0057] (1) Take the following mass parts of raw materials: 1 part of scutellarein, 0.5 parts of phellodendrine, 2 parts of asiaticoside, 3 parts of salvianolic acid B, 1 part of gallic acid, 5 parts of Bletilla striata polysaccharide, 3 parts of konjac glucomannan, 3 parts of chitosan, 8 parts of glycerol, 0.5 parts of genipin, 1 part of calcium chloride, 1 part of laurazepine, and 71 parts of water;

[0058] (2) Mix Bletilla striata polysaccharide, konjac glucomannan with 25 parts of water preheated to 55℃, stir at 200 r / min and warm to 80℃, keep warm for 25 min, cool to 25℃, get phase A;

[0059] (3) Mix chitosan with 10 parts of acidified water (pH 4, the acidification is carried out with 1wt% glacial acetic acid), stir at 300 r / min until completely dissolved, get phase B;

[0060] (4) Mix scutellarein, phellodendrine, asiaticoside, salvianolic acid B, gallic acid with 6 parts of glycerol, homogenize at 3000 r / min for 5 min, get phase C;

[0061] (5) mix the laurocapram with the remaining 2 parts of glycerol to obtain phase D;

[0062] (6) dissolve the genipin in 5 parts of water and dissolve the calcium chloride in 5 parts of water to obtain a genipin solution and a calcium chloride solution;

[0063] (7) add phase B to phase A and stir uniformly at 50 r / min, then add phase C, continue to stir at 50 r / min for 10 min, then add the genipin solution, react for 10 min, and then quickly add the calcium chloride solution to obtain a light yellow transparent gel;

[0064] (8) add phase D to the light yellow transparent gel and supplement the remaining 26 parts of water, stir uniformly at 300 r / min, vacuum degassing (-0.1 Mpa, 10 min), and then distribute to obtain the composition for treating stubborn skin infections, ulcers and inflammation.

[0065] Example 2

[0066] A preparation method of a composition for treating stubborn skin infections, ulcers and inflammation, comprising the following steps:

[0067] (1) weigh the following mass parts of raw materials: baicalin 3 parts, berberine 2 parts, asiaticoside 4 parts, salvianolic acid B 5 parts, gallic acid 3 parts, white plant polysaccharide 7 parts, konjac glucomannan 5 parts, chitosan 4 parts, glycerol 12 parts, genipin 1 part, calcium chloride 2 parts, laurocapram 2 parts, and water 50 parts;

[0068] (2) mix the white plant polysaccharide, konjac glucomannan and 25 parts of water preheated to 57℃, stir at 250 r / min and heat to 82℃, keep warm for 30 min, cool to 30℃, to obtain phase A;

[0069] (3) mix the chitosan with 10 parts of acidified water (pH 4.5, the acidification is performed by using 2 wt% glacial acetic acid), stir at 400 r / min until completely dissolved to obtain phase B;

[0070] (4) mix baicalin, berberine, asiaticoside, salvianolic acid B and gallic acid with 9 parts of glycerol, homogenize at 3500 r / min for 8 min to obtain phase C;

[0071] (5) mix the laurocapram with the remaining 3 parts of glycerol to obtain phase D;

[0072] (6) dissolve the genipin in 5 parts of water and dissolve the calcium chloride in 5 parts of water to obtain a genipin solution and a calcium chloride solution;

[0073] (7) add B phase into A phase, stir uniformly under 80 r / min, then add C phase, continue to stir under 80 r / min for 12 min, then add genipin solution, react for 15 min, and then quickly add calcium chloride solution to obtain a light yellow transparent gel;

[0074] (8) add D phase into the light yellow transparent gel, and supplement with the remaining 5 parts of water, stir uniformly under 400 r / min, vacuum degassing (-0.08 Mpa, 13 min), and then distribute to obtain the composition for treating stubborn skin infection, sore and inflammation.

[0075] Example 3

[0076] A preparation method of a composition for treating stubborn skin infection, sore and inflammation, comprising the following steps:

[0077] (1) weigh the following raw materials: baicalin 5 parts, obakunine 3 parts, asiaticoside 6 parts, salvianolic acid B 6 parts, gallic acid 4 parts, white plant polysaccharide 5 parts, konjac glucomannan 3 parts, chitosan 3 parts, glycerol 15 parts, genipin 1 part, calcium chloride 1 part, laurazepam 3 parts, and water 45 parts;

[0078] (2) mix white plant polysaccharide, konjac glucomannan and 20 parts of water preheated to 60℃, stir under 300 r / min and heat to 85℃, keep warm for 35 min, cool to 40℃ to obtain A phase;

[0079] (3) mix chitosan and 8 parts of acidified water (pH 5, the acidification is performed by using 3 wt% glacial acetic acid) to obtain B phase, and stir under 500 r / min until completely dissolved;

[0080] (4) mix baicalin, obakunine, asiaticoside, salvianolic acid B and gallic acid with 10 parts of glycerol, homogenize under 4000 r / min for 10 min to obtain C phase;

[0081] (5) mix laurazepam with the remaining 5 parts of glycerol to obtain D phase;

[0082] (6) dissolve genipin with 5 parts of water and dissolve calcium chloride with 5 parts of water to obtain genipin solution and calcium chloride solution;

[0083] (7) add B phase into A phase, stir uniformly under 100 r / min, then add C phase, continue to stir under 100 r / min for 15 min, then add genipin solution, react for 20 min, and then quickly add calcium chloride solution to obtain a light yellow transparent gel;

[0084] (8) Add D phase into the light yellow transparent gel, and supplement the remaining 7 parts of water, stir evenly at 500 r / min, vacuum degassing (-0.05 Mpa, 15 min), and then perform dispensing to obtain the composition for treating stubborn skin infection, sore and inflammation.

[0085] Comparative Example 1

[0086] A preparation method of a composition for treating stubborn skin infection, sore and inflammation, comprising the following steps:

[0087] Compared with Example 2, the difference is that baicalin and gallic acid are omitted:

[0088] (1) Take the following mass parts of raw materials: berberine 2 parts, asiaticoside 4 parts, salvianolic acid B 5 parts, white yam polysaccharide 7 parts, konjac glucomannan 5 parts, chitosan 4 parts, glycerol 12 parts, genipin 1 part, calcium chloride 2 parts, laurazepam 2 parts, and water 50 parts;

[0089] (2) Mix white yam polysaccharide, konjac glucomannan and 25 parts of water preheated to 57℃, stir at 250 r / min and heat to 82℃, and then keep warm for 30 min, and cool to 30℃ to obtain phase A;

[0090] (3) Mix chitosan with 10 parts of acidified water (pH 4.5, the acidification is performed by using 2wt% glacial acetic acid) and stir at 400 r / min until completely dissolved to obtain phase B;

[0091] (4) Mix berberine, asiaticoside and salvianolic acid B with 9 parts of glycerol, and homogenize at 3500 r / min for 8 min to obtain phase C;

[0092] (5) Mix laurazepam with the remaining 3 parts of glycerol to obtain phase D;

[0093] (6) Dissolve genipin with 5 parts of water and dissolve calcium chloride with 5 parts of water to obtain a genipin solution and a calcium chloride solution;

[0094] (7) Add phase B into phase A and stir evenly at 80 r / min, then add phase C, continue to stir at 80 r / min for 12 min, then add the genipin solution and react for 15 min, and then quickly add the calcium chloride solution to obtain a light yellow transparent gel;

[0095] (8) Add phase D into the light yellow transparent gel, and supplement the remaining 5 parts of water, stir evenly at 400 r / min, vacuum degassing (-0.08 Mpa, 13 min), and then perform dispensing to obtain the composition for treating stubborn skin infection, sore and inflammation.

[0096] Comparative Example 2

[0097] A preparation method of a composition for treating stubborn skin infection, sore and inflammation, comprising the following steps:

[0098] Compared with Example 2, the difference is that baicalin is replaced by matrine, and gallic acid is replaced by paeonol:

[0099] (1) Take the following mass parts of raw materials: matrine 3 parts, berberine 2 parts, asiaticoside 4 parts, salvianolic acid B 5 parts, paeonol 3 parts, white yam polysaccharide 7 parts, konjac glucomannan 5 parts, chitosan 4 parts, glycerol 12 parts, genipin 1 part, calcium chloride 2 parts, laurazepam 2 parts, and water 50 parts;

[0100] (2) Mix white yam polysaccharide, konjac glucomannan and 25 parts of water preheated to 57°C, stir at 250 r / min and heat to 82°C, and keep still for 30 min, and then cool to 30°C to obtain phase A;

[0101] (3) Mix chitosan with 10 parts of acidified water (pH 4.5, the acidification is carried out by using 2wt% glacial acetic acid) and stir at 400 r / min until completely dissolved to obtain phase B;

[0102] (4) Mix matrine, berberine, asiaticoside, salvianolic acid B and paeonol with 9 parts of glycerol, and homogenize at 3500 r / min for 8 min to obtain phase C;

[0103] (5) Mix laurazepam with the remaining 3 parts of glycerol to obtain phase D;

[0104] (6) Dissolve genipin with 5 parts of water and dissolve calcium chloride with 5 parts of water to obtain a genipin solution and a calcium chloride solution;

[0105] (7) Add phase B to phase A and stir uniformly at 80 r / min, then add phase C, continue to stir at 80 r / min for 12 min, then add the genipin solution and react for 15 min, and then quickly add the calcium chloride solution to obtain a light yellow transparent gel;

[0106] (8) Add phase D to the light yellow transparent gel and supplement the remaining 5 parts of water, and stir uniformly at 400 r / min, vacuum defoam (-0.08 Mpa, 13 min), and then perform vacuum defoaming to obtain the composition for treating stubborn skin infection, sore and inflammation.

[0107] Comparative Example 3

[0108] A preparation method of a composition for treating stubborn skin infection, sore and inflammation, comprising the following steps:

[0109] Compared with Example 2, the difference is that the preparation process is only a simple physical mixing:

[0110] (1) Take the following mass parts of raw materials: baicalin 3 parts, berberine 2 parts, asiaticoside 4 parts, salvianolic acid B 5 parts, gallic acid 3 parts, white yam polysaccharide 7 parts, konjac glucomannan 5 parts, chitosan 4 parts, glycerol 12 parts, genipin 1 part, calcium chloride 2 parts, loperamide 2 parts, and water 50 parts;

[0111] (2) Mix the above raw materials, stir uniformly at 400 r / min, vacuum degassing (-0.08 Mpa, 13 min), and then sub-packaging to obtain the composition for treating stubborn skin infections, ulcers and inflammation.

[0112] Experimental Example 1

[0113] In vitro release rate study:

[0114] Franz diffusion cell method was used, with 0.9% phosphate buffer (PBS, pH 7.4) as the release medium, and the temperature was 37±0.5℃. The gel product obtained in Example 2 of the present application and the product obtained in Comparative Example 3 were placed in the supply pool. At the predetermined time points (1, 2, 4, 6, 8, 12, 24 h), samples were taken, and the concentration of salvianolic acid B was determined by HPLC method, and the cumulative release rate (%) was calculated. The results are shown in Table 1.

[0115] Table 1 Cumulative release rate of salvianolic acid B (%)

[0116] Time (h) 1 2 4 6 8 12 24 Example 2 12.3±1.5 21.1±2.1 33.4±1.8 42.7±2.3 52.6±1.9 63.2±1.3 75.8±2.5 Comparative Example 3 45.6±3.2 68.9±2.8 82.1±2.5 87.5±1.9 89.2±1.7 90.5±1.5 91.8±1.2

[0117] As can be seen from Table 1, the salvianolic acid B in the product of Comparative Example 3 is rapidly released by more than 85% within 8 hours, and then enters a release plateau. The gel product of Example 3 of the present application shows a steady slow-release trend within 24 hours, and the cumulative release rate is 75.8% at 24 hours. The results show that, compared with the simple physical mixing process, the step-by-step in-situ crosslinking preparation process used in the present application successfully constructs a slow-release system, which can significantly prolong the action time of the drug.

[0118] Experimental Example 2

[0119] In vitro antibacterial experiment:

[0120] The products in Examples 1-3 and the products in Comparative Examples 1-3 were evaluated for in vitro antibacterial activity against drug-resistant bacteria by agar diffusion method.

[0121] 1. Experimental strains:

[0122] Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300, purchased from Bao Sai Plasmid Strain Resource Co., Ltd.);

[0123] Pseudomonas aeruginosa (ATCC 27853, purchased from Qingdao Haibo Biotechnology Co., Ltd.).

[0124] 2. Experimental medium:

[0125] Mueller-Hinton (MH) agar medium.

[0126] 3. Experimental grouping and treatment:

[0127] Example 1: sterile water dilution 10 times;

[0128] Example 2: sterile water dilution 10 times;

[0129] Example 3: sterile water dilution 10 times;

[0130] Comparative Example 1: sterile water dilution 10 times;

[0131] Comparative Example 2: sterile water dilution 10 times;

[0132] Comparative Example 3: sterile water dilution 10 times;

[0133] Negative control group: sterile normal saline;

[0134] Positive control group: mupirocin ointment, sterile water dilution 10 times.

[0135] 4. Experimental method:

[0136] The prepared MRSA bacterial suspension (concentration 1 x 10 8 CFU / mL) was uniformly coated on the MH agar plate by coating method; a sterile puncher was used to punch a 6 mm diameter hole on the agar, and a sterile needle was used to pick out the agar in the hole; a micro pipettor was used to accurately add equal amount (50 μL) of the above-mentioned product liquid to the center of each group of holes; the plate was placed vertically in a 4°C refrigerator for pre-diffusion for 3 h, and then transferred to a 37°C constant temperature incubator for culture for 24 h; the diameter of each bacteriostatic circle (including the hole diameter) was measured with a vernier caliper, and the average value was taken twice with cross intersection, with an accuracy of 0.1 mm. Three repeated holes were set in each group.

[0137] The experimental results are shown in Table 2.

[0138] Table 2 Diameter of bacteriostatic circle of each group on different bacteria (n = 3, mm)

[0139] Group Methicillin-resistant Staphylococcus aureus Pseudomonas aeruginosa Example 1 17.2±0.5 14.9±0.7 Example 2 18.8±0.7 15.8±0.6 Example 3 17.6±0.9 15.2±0.2 Comparative Example 1 11.8±0.4 9.1±0.3 Comparative Example 2 10.2±0.8 8.5±0.5 Comparative Example 3 12.3±0.6 9.8±0.2 Negative control group 6.0 ± 0.0 (only hole diameter) 6.0±0.0 Positive control group 20.5±0.9 6.0 ± 0.0 (only hole diameter)

[0140] As shown in Table 2, the negative control group had no inhibition zone, indicating that the experimental system was reliable. Compared with Comparative Examples 1-3, the inhibition zones of Examples 1-3 of the present application were significantly larger, and the effect of Example 2 was optimal. Compared with the first-line drug mupirocin ointment, the products of Examples 1-3 of the present application not only had excellent activity comparable to mupirocin against methicillin-resistant Staphylococcus aureus, but also could effectively resist Pseudomonas aeruginosa, which was completely ineffective for mupirocin, thus exhibiting a broader antibacterial spectrum. The results directly indicated that the specific component combination and preparation process of the present application produced a significant synergistic antibacterial effect between the components.

[0141] Experimental Example 3

[0142] Evaluation of Anti-inflammatory Effect on LPS-induced Macrophage Inflammation Model:

[0143] 1. Experimental Method:

[0144] A mouse macrophage (RAW264.7) inflammation model was established by stimulating with lipopolysaccharide (LPS, 1 μg / mL). After different drug interventions, the cells were cultured for 24 h. The concentrations of key inflammatory factors, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the cell supernatant were detected by ELISA.

[0145] 2. Experimental Groups:

[0146] Example 2: LPS stimulation + Example 2 product;

[0147] Comparative Example 1: LPS stimulation + Comparative Example 1 product;

[0148] Comparative Example 2: LPS stimulation + Comparative Example 2 product;

[0149] Comparative Example 3: LPS stimulation + Comparative Example 3 product;

[0150] Blank control group: normal cell culture without LPS stimulation and any drug;

[0151] Model control group: cells were stimulated with LPS, but no therapeutic drug was added.

[0152] The experimental results are shown in Tables 3 and 4.

[0153] Table 3: Effect on LPS-induced release of inflammatory factors from RAW264.7 cells

[0154] Group TNF-α concentration (pg / mL) IL-6 concentration (pg / mL) Blank control group 85.5 65.3 Model control group 1250.8 980.5 Example 2 275.4 270.8 Comparative Example 1 740.2 625.8 Comparative Example 2 810.5 670.2 Comparative Example 3 562.3 505.4

[0155] Based on the above data, the inhibition rate (%) of each group relative to the model control group was calculated:

[0156] Inhibition rate (%) = [1- (inflammatory factor concentration measured in each treatment group-inflammatory factor concentration measured in blank control group) / (inflammatory factor concentration measured in model control group-inflammatory factor concentration measured in blank control group)] x 100%.

[0157] Table 4 Inhibition rate (%) of different products on inflammatory factor release

[0158] Group TNF-α inhibition rate IL-6 inhibition rate Example 2 83.7 77.5 Comparative Example 1 43.8 38.8 Comparative Example 2 37.8 33.9 Comparative Example 3 59.1 51.9

[0159] As can be seen from Table 3, the concentrations of TNF-a and IL-6 in the model control group were extremely significantly higher than those in the blank control group, indicating that the LPS inflammation model was successfully established. As can be seen from Table 4, compared with Comparative Examples 1-3, the product of Example 2 of the present application had the lowest inflammatory factor concentration, and its inhibition rate was also significantly higher. This shows that, through specific component compounding and preparation process, the present application produces a significant synergistic anti-inflammatory effect between active ingredients, rather than just a simple additive effect of mixing. The present application can effectively inhibit the release of key inflammatory factors by macrophages, which provides a solid celluar basis for its in vivo relief of excessive inflammatory reaction at wound surface.

[0160] Experimental Example 4

[0161] Cell scratch experiment for evaluating the healing-promoting effect:

[0162] 1. Experimental principle

[0163] Simulated wound surface: A cell-free "scratch area" is made on the cell-covered culture plate to simulate a skin wound.

[0164] Intervention treatment: Add culture medium containing different drugs.

[0165] Observation of repair: Cells will gradually migrate and proliferate to the scratch area until closure. By measuring the change in scratch width at different time points, the "healing rate" can be quantitatively calculated to determine the repair-promoting ability of the drug.

[0166] 2. Experimental materials and reagents

[0167] Cell line: Mouse fibroblast cells (L929).

[0168] Culture medium: DMEM high-sugar culture medium, supplemented with 10% fetal bovine serum (FBS) and 1% double-antibiotic.

[0169] Experimental apparatus: 6-well plate, 200 μL sterile gun head, microscope equipped with digital camera.

[0170] 3. Experimental grouping

[0171] Negative control group: Complete culture medium containing 0.1% DMSO (solvent control).

[0172] Positive control group: complete medium containing 10 ng / mL epidermal growth factor (EGF).

[0173] Example 2 and Comparative Examples 1-3 groups: each group was extracted with complete medium (100 mg / mL), and the supernatant was obtained by centrifugation, filtered to remove bacteria, and used (i.e., the obtained drug-containing medium).

[0174] 4. Experimental procedure

[0175] Cell plating: the cells were inoculated in a 6-well plate and cultured to 100% confluence.

[0176] Making a scratch: a sterile gun tip was used to smoothly draw a straight line scratch perpendicular to the bottom of the plate. Ensure that all scratches are of uniform width.

[0177] Washing: gently washed 3 times with PBS to remove floating cell debris.

[0178] Drug treatment: the negative control, positive control, Example 2 and Comparative Examples 1-3 drug-containing media were added, respectively.

[0179] Observation and photography: the plate was placed in a 37°C, 5% CO2 incubator for culture. At 0h, 12h, 24h time points, photographs were taken at the same position under the microscope to record the scratch situation.

[0180] Image analysis: Image J and other image analysis software were used to measure the width of the scratch at each time point (multiple points can be measured and averaged).

[0181] 5. Data processing and result analysis

[0182] Calculate the scratch healing rate:

[0183] Healing rate (%) = [(W0-W t ) / W0] x 100%

[0184] W0: average width of the scratch at 0h

[0185] W t : average width of the scratch at a certain time point (12h, 24h)

[0186] Statistical analysis: all experiments were repeated 3 times.

[0187] The results are shown in Table 5.

[0188] Table 5 Promoting effect of different treatments on the scratch healing rate of fibroblasts

[0189] Group 12 h healing rate (%) 24 h healing rate (%) Negative control group 25.3±3.5 55.1±4.2 Positive control group 45.8±4.1 85.6±3.8 Example 2 65.5±5.2 95.8±2.5 Comparative Example 1 42.1±3.4 72.3±1.8 Comparative Example 2 38.7±5.7 67.9±4.5 Comparative Example 3 46.3±1.5 78.2±2.6

[0190] As shown in Table 5, the negative control group cells have certain migration ability by themselves, and the healing rate is about 55.1% in 24 hours. The positive control group (EGF) shows a significant healing-promoting effect, and the healing rate reaches 85.6% in 24 hours, which proves that the experimental system is effective. Compared with Comparative Examples 1-3, the composition group of Example 2 of the present application shows the strongest healing-promoting ability, and the healing rates (65.5% and 95.8%) at the time points of 12 hours and 24 hours are significantly higher than those of other groups. This indicates that the composition of the present application can significantly promote the migration and proliferation of fibroblasts, thereby accelerating the closure of the "wound surface". This provides cytological evidence for the in vivo efficacy of the composition in promoting granulation tissue generation and epithelial repair.

[0191] The above is only a few embodiments of the present application, and does not limit the present application in any form. Although the preferred embodiments are disclosed above, the present application is not limited thereto. Any person skilled in the art can make some changes or modifications to the above disclosed technical contents without departing from the scope of the technical solutions of the present application, and such changes or modifications are equivalent to equivalent embodiments, and are within the scope of the technical solutions.

Claims

1. A composition for treating intractable skin infections, sores, and inflammation, characterized in that, It is prepared from the following components in parts by weight: baicalin 1-5 parts, antibacterial alkaloids 0.5-3 parts, asiaticoside 2-6 parts, salvianolic acid B 3-8 parts, gallic acid 1-4 parts, Bletilla striata polysaccharide 5-10 parts, konjac glucomannan 3-10 parts, chitosan 3-5 parts, glycerol 8-20 parts, genipin 0.5-2 parts, calcium chloride 1-3 parts, laurocapram 1-3 parts, and water 40-80 parts; The antibacterial alkaloids include berberine or phellodendron alkaloid; The method for preparing the composition for treating intractable skin infections, sores, and inflammation includes the following steps: (1) Mix Bletilla striata polysaccharide, konjac glucomannan and water preheated to 55~60℃, stir and heat to 80~85℃, keep warm and stand, cool to obtain phase A; (2) Mix chitosan with acidified water and stir until completely dissolved to obtain phase B; (3) Baicalin, antibacterial alkaloids, asiaticoside, salvianolic acid B, gallic acid and glycerol were mixed and homogenized to obtain phase C; (4) Mix laurocapram with glycerol to obtain phase D; (5) Dissolve genipin and calcium chloride in water to obtain genipin solution and calcium chloride solution respectively; (6) Add phase B to phase A and stir evenly. Then add phase C and continue stirring for 10-15 min. Then add genipin solution and react for 10-20 min. Then add calcium chloride solution to obtain a light yellow transparent gel. (7) Add phase D to the pale yellow transparent gel, add the remaining water, stir evenly, and perform vacuum degassing to obtain the composition for treating stubborn skin infections, sores and inflammation.

2. The composition for treating intractable skin infections, sores, and inflammation according to claim 1, characterized in that, The degree of deacetylation of the chitosan is ≥85%, and the molecular weight of the chitosan is 5000~20000 Da.

3. A method for preparing a composition for treating intractable skin infections, sores, and inflammation as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Mix Bletilla striata polysaccharide, konjac glucomannan and water preheated to 55~60℃, stir and heat to 80~85℃, keep warm and stand, cool to obtain phase A; (2) Mix chitosan with acidified water and stir until completely dissolved to obtain phase B; (3) Baicalin, antibacterial alkaloids, asiaticoside, salvianolic acid B, gallic acid and glycerol were mixed and homogenized to obtain phase C; (4) Mix laurocapram with glycerol to obtain phase D; (5) Dissolve genipin and calcium chloride in water to obtain genipin solution and calcium chloride solution respectively; (6) Add phase B to phase A and stir evenly. Then add phase C and continue stirring for 10-15 min. Then add genipin solution and react for 10-20 min. Then add calcium chloride solution to obtain a light yellow transparent gel. (7) Add phase D to the pale yellow transparent gel, add the remaining water, stir evenly, and perform vacuum degassing to obtain the composition for treating stubborn skin infections, sores and inflammation.

4. A method for preparing a composition for treating intractable skin infections, sores, and inflammation according to claim 3, characterized in that, In step (1), the stirring speed is 200~300 r / min; The heat preservation and static setting time is 25~35 minutes; The cooling process involves cooling the temperature to 25-40°C.

5. A method for preparing a composition for treating intractable skin infections, sores, and inflammation according to claim 3, characterized in that, In step (2), the pH of the acidified water is 4-5; The acidification was carried out using glacial acetic acid; The concentration of the glacial acetic acid is 1-3 wt%; The stirring speed is 300~500 r / min.

6. A method for preparing a composition for treating intractable skin infections, sores, and inflammation according to claim 3, characterized in that, In step (3), the antibacterial alkaloids include berberine or phellodendron chinense; The rotational speed of the homogenizer is 3000~4000 r / min; The homogenization time is 5-10 minutes.

7. A method for preparing a composition for treating intractable skin infections, sores, and inflammation according to claim 3, characterized in that, The ratio of the amount of glycerol used in step (3) to the amount of glycerol used in step (4) is 1~3:

1.

8. A method for preparing a composition for treating intractable skin infections, sores, and inflammation according to claim 3, characterized in that, In step (6), the stirring speed is 50~100 r / min.

9. A method for preparing a composition for treating intractable skin infections, sores, and inflammation according to claim 3, characterized in that, In step (7), the stirring speed is 300~500 r / min; The vacuum degassing pressure is -0.1 to -0.05 MPa; The vacuum degassing time is 10-15 minutes.

10. The use of the composition according to claim 1 or 2, or the composition prepared by the preparation method according to any one of claims 3 to 9, in the preparation of a medicament for treating intractable skin infections, sores and inflammations caused by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa.