Traditional Chinese medicine extract gel for promoting skin wound healing and resisting infection and composition

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to traditional Chinese medicine extract gel for promoting skin wound healing and resisting infection and a composition. The traditional Chinese medicine extract gel is prepared from a traditional Chinese medicine extract and a gel matrix according to a certain mass ratio, wherein the traditional Chinese medicine extract is a water extract of rhizoma bletillae and radix notoginseng. The traditional Chinese medicine extract gel can be independently used or further combined with a phage composition consisting of a phage composition such as a pseudomonas aeruginosa phage, a staphylococcus aureus phage, an escherichia coli phage and an enterococcus faecalis phage for use, and is used for developing products for promoting skin wound healing and resisting infection. The method can also be used for developing antibacterial products aiming at mixed bacteria such as pseudomonas aeruginosa, staphylococcus aureus, escherichia coli and enterococcus faecalis, and replacing antibiotics to be used for preventing and treating diseases caused by the mixed bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a traditional Chinese medicine extract gel and a composition for promoting wound healing and resisting infection. Background Art

[0002] Skin trauma refers to the severance or defect of skin tissue caused by external force, which destroys the normal structure of body parts and causes a series of symptoms, such as redness, swelling, ulcers, etc. In severe cases, exudate will seep out. Traditional Chinese medicine has unique advantages in treating trauma. It usually uses Chinese herbal medicines that have the effects of dissipating blood stasis, reducing swelling and promoting tissue regeneration, astringing and stopping bleeding, relieving pain and healing sores, and clearing away heat and detoxifying for treatment. Bletilla striata and Panax notoginseng are representative medicines for treating trauma. Bletilla striata is the dried tuber of the orchid plant Bletilla striata, which has the effects of astringing and stopping bleeding, reducing swelling and promoting tissue regeneration. Panax notoginseng is the dried root of the Araliaceae plant Panax notoginseng, which has the effects of removing blood stasis and stopping bleeding, promoting blood circulation and relieving pain, and detoxifying and reducing swelling. The combination of the two can promote the healing of the old and the new, reduce inflammation and relieve pain, and promote wound healing.

[0003] Skin trauma infection refers to a disease in which various microorganisms (including bacteria, fungi, etc.) invade the body from the damaged skin and colonize and multiply to a certain number, causing local or even systemic inflammatory reactions in the wound. Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter and Enterococcus are common pathogens that cause clinical trauma infection, which can cause inflammation and suppuration of the wound surface, mainly manifested as redness, swelling, pain, increased secretions, and odor in the wound. At present, the drugs used in modern medicine to treat skin trauma and trauma infection are mainly antibiotics, which can play a bactericidal and anti-inflammatory role, control trauma infection, and promote wound healing. Although antibiotic treatment has a certain effect, it has large side effects and is prone to drug resistance. In addition, antibiotics mainly play an antibacterial role and have limited promoting effect on wound healing. Therefore, it is urgent to develop safer and more effective alternative drugs. Bacteriophages are host-specific bacterial viruses that can use hosts to reproduce and co-evolve with hosts, and are not easy to develop resistance. The phage composition is a form of phage administration, which has the characteristics of a wide host spectrum, strong infection ability, and a wide range of temperature and acid-base tolerance, and can be used to develop antibacterial products that simultaneously inhibit mixed infections of multiple bacteria. Therefore, the present invention uses the Bletilla striata-Sanqi drug pair in combination with the phage composition, which has the effects of promoting skin wound healing and anti-infection, and has the potential to be developed into a new drug for treating skin wounds. Summary of the invention

[0004] In view of the lack of safe and effective drugs for treating skin wound infections in the prior art, the present invention provides a Chinese medicine extract gel and composition that has good efficacy and high safety and promotes wound healing and anti-infection.

[0005] In a first aspect, the present invention provides a Chinese herbal extract gel for promoting skin wound healing and anti-infection, comprising a Chinese herbal extract and a gel matrix, wherein the mass ratio of the Chinese herbal extract to the gel matrix is ​​1 to 4:2, and the Chinese herbal extract is an aqueous extract of Bletilla striata and Panax notoginseng.

[0006] In some embodiments, the Chinese medicine extract is prepared as follows: Bletilla striata and Panax notoginseng are mixed in a mass ratio of 1:0.8-1.2, 9-11 times the mass of water are added, and reflux extraction is performed for 3 times, each time for 0.5-1.5 hours, the extracts are combined, and then concentrated and freeze-dried.

[0007] In some embodiments, the gel matrix comprises the following components in parts by weight: 15-20 parts by weight of gelatin, 6-8 parts by weight of citric acid, 650-720 parts by weight of glycerol, and 400-450 parts by weight of purified water.

[0008] In a second aspect, the present invention provides a composition comprising: i) a first preparation: the aforementioned Chinese medicine extract gel; ii) a second preparation: a phage composition whose active ingredients are composed of Pseudomonas aeruginosa phage, Staphylococcus aureus phage, Escherichia coli phage and Enterococcus faecalis phage.

[0009] In some embodiments, the Pseudomonas aeruginosa phage is Pseudomonas aeruginosa phage BD815, with the deposit number being CGMCC No.45873.

[0010] In some embodiments, the Staphylococcus aureus phage is Staphylococcus aureus phage SaBD224, with the deposit number being CGMCC No.46185.

[0011] In some embodiments, the Escherichia coliphage is Escherichia coliphage EcBD91, with the deposit number being CGMCC No.46183.

[0012] In some embodiments, the Enterococcus faecalis phage is Enterococcus faecalisphage EfBD915, with the deposit number being CGMCC No.46184.

[0013] In some embodiments, in the phage composition, the ratio of the number of live Pseudomonas aeruginosa phage, Staphylococcus aureus phage, Escherichia coli phage and Enterococcus faecalis phage is 1:1:1:1.

[0014] In some embodiments, the contents of Pseudomonas aeruginosa phage, Staphylococcus aureus phage, Escherichia coli phage and Enterococcus faecalis phage are not less than 10 14 pfu / mL.

[0015] In some embodiments, the second preparation is a phage combination or a liquid dispersion preparation thereof after expansion, wherein Pseudomonas aeruginosa phage, Staphylococcus aureus phage, Escherichia coli phage and Enterococcus faecalis phage are respectively inoculated into a liquid culture medium containing the corresponding host bacteria, and cultured at 37°C with shaking, and when the bacterial liquid becomes clear, a phage suspension is obtained by filtering with a filter membrane; the phage suspension is centrifuged to discard the supernatant, and the precipitated phage is resuspended with sterile PBS to obtain PBS suspensions of each phage; and the PBS suspensions of each phage are mixed to obtain a second preparation with the phage combination as the active ingredient.

[0016] In some embodiments, the active ingredient content in the second formulation is 10 14 pfu / mL.

[0017] In a third aspect, the present invention provides a use of the aforementioned Chinese herbal extract gel or composition in the preparation of a product for promoting skin wound healing and anti-infection.

[0018] In a fourth aspect, the present invention provides an application of the aforementioned Chinese medicine extract gel or composition in the preparation of a product for preventing and treating wound infection caused by one or a combination of more than two of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Enterococcus faecalis, for example, for treating mixed-strain infection-type wounds or mixed-strain infection-type skin diseases.

[0019] The beneficial effects of the present invention are:

[0020] The Chinese medicine extract gel provided by the present invention contains water extracts of Bletilla striata and Panax notoginseng. By compounding these two Chinese medicine components, the old can be regenerated and the new can be created, wound healing can be promoted, and a synergistic effect can be exerted in curing mixed strain infection-type wounds. The gel has great application value and drug development potential.

[0021] The combined composition provided by the present invention, by combining the traditional Chinese medicine extract gel with the phage composition, can significantly improve skin wound infection caused by mixed bacteria consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Enterococcus faecalis, effectively promote wound healing, and the treatment process is safe, has no toxic side effects, and will not produce drug resistance, providing a new therapeutic drug for the treatment of mixed bacterial infection-type wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The plaque and transmission electron microscopy morphology of bacteriophage SaBD224, A: plaque morphology, scale bar is 1 cm; B: transmission electron microscopy morphology, scale bar is 50 nm;

[0023] Figure 2 The plaque and transmission electron microscopy morphology of bacteriophage EcBD91, A: plaque morphology, scale bar is 1 cm; B: transmission electron microscopy morphology, scale bar is 50 nm;

[0024] Figure 3 The plaque and transmission electron microscopy morphology of bacteriophage EfBD915, A: plaque morphology, scale bar is 1 cm; B: transmission electron microscopy morphology, scale bar is 50 nm;

[0025] Figure 4 This is the complete genome map of bacteriophage SaBD224;

[0026] Figure 5 is the phylogenetic tree of bacteriophage SaBD224;

[0027] Figure 6 This is the complete genome map of bacteriophage EcBD91;

[0028] Figure 7 is the phylogenetic tree of bacteriophage EcBD91;

[0029] Figure 8 This is the complete genome map of bacteriophage EfBD915;

[0030] Fig. 9 is the phylogenetic tree of bacteriophage EfBD915;

[0031] Fig.10 Schematic diagram of the optimal infection multiplicity experiment for bacteriophage SaBD224;

[0032] Fig.11 Schematic diagram of the one-step growth curve experiment of bacteriophage SaBD224;

[0033] Fig.12 Schematic diagram of pH stability experiment of bacteriophage SaBD224;

[0034] Fig.13 Schematic diagram of the thermal stability experiment of bacteriophage SaBD224;

[0035] Fig.14 Schematic diagram of the optimal infection multiplicity experiment for bacteriophage EcBD91;

[0036] Fig.15 Schematic diagram of the one-step growth curve experiment of bacteriophage EcBD91;

[0037] Fig.16 Schematic diagram of pH stability experiment of bacteriophage EcBD91;

[0038] Fig.17Schematic diagram of the thermal stability experiment of bacteriophage EcBD91;

[0039] Fig.18 This is a schematic diagram of the optimal infection multiplicity experiment for bacteriophage EfBD915;

[0040] Fig.19 This is a schematic diagram of the one-step growth curve experiment of bacteriophage EfBD915;

[0041] Fig. 20 Schematic diagram of pH stability experiment of bacteriophage EfBD915;

[0042] Fig.21 Schematic diagram of the thermal stability experiment of bacteriophage EfBD915;

[0043] Fig. 22 Changes of wounds in mice within two weeks of drug administration;

[0044] Fig.23 The change value of wound area within two weeks of drug administration to mice;

[0045] Fig.24 Comparison of the expression of VEGF, a gene related to wound healing, in each group;

[0046] Fig.25 Comparison of the expression of wound healing-related gene bFGF in each group;

[0047] Fig.26 Comparison of the expression of wound healing-related gene TGF-β in each group;

[0048] Fig. 27 Comparison of the antibacterial effect of PBS suspension of phage combination (experimental group 1) with the model group and positive drug group;

[0049] Fig.28 These are scans of HE-stained sections of skin tissue at the wound site of mice. A: blank control group; B: model group; C: Chinese medicine positive control group; D: Western medicine positive control group; E: experimental group 1; F: experimental group 5; G: experimental group 6; H: experimental group 7. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] In the following examples, Pseudomonas aeruginosa phage BD815 is a phage for efficiently lysing Pseudomonas aeruginosa disclosed in Chinese invention patent application No. 202410424765.1, and the sources of other raw materials are shown in Table 1.

[0052] Table 1 Source of raw materials

[0053]

[0054]

[0055] Example 1 Screening and purification of bacteriophage

[0056] Preparation of LB liquid culture medium: weigh 10 g of tryptone, 5 g of yeast extract powder, and 10 g of NaCl and dissolve them in 800 mL of pure water. Stir until all dissolved, adjust the pH to 7.2-7.4 with 10% NaOH, and then make up to 1000 mL. Sterilize at high temperature and high pressure for later use.

[0057] Preparation of LB agar medium: Add 15 g agar to LB liquid medium and heat until the agar is completely dissolved, adjust the pH to 7.2-7.4 with 10% NaOH, and then make up to 1000 mL, sterilize at high temperature and high pressure for later use.

[0058] Preparation of LB semi-solid medium: Add 5 g agar to LB liquid medium and heat until the agar is completely dissolved, adjust the pH to 7.2-7.4 with 10% NaOH, and then make up to 1000 mL, sterilize at high temperature and high pressure for later use.

[0059] Preparation of GAM liquid medium: weigh 10g of peptone, 3g of soy peptone, 5g of yeast extract, 2.2g of beef extract, 13.5g of serum digestion powder, 1.2g of beef liver extract powder, 3g of glucose, 2.5g of potassium dihydrogen phosphate, 3g of NaCl, 5g of soluble starch, 0.3g of L-cysteine ​​and 0.3g of sodium thioglycolate were dissolved in 800mL of pure water, the pH was adjusted to 7.2-7.4 with 10% NaOH, and the volume was made up to 1000mL. Sterilize at high temperature and high pressure for use.

[0060] Preparation of GAM agar medium: Add 15 g agar to GAM liquid medium and heat until the agar is completely dissolved, adjust the pH to 7.2-7.4 with 10% NaOH, and then make up to 1000 mL, sterilize at high temperature and high pressure for later use.

[0061] Preparation of GAM semi-solid medium: Add 5 g agar to GAM liquid medium and heat until the agar is completely dissolved, adjust the pH to 7.2-7.4 with 10% NaOH, and then make up to 1000 mL, sterilize at high temperature and high pressure for later use.

[0062] Sewage from the natural environment of Fangshan District, Beijing was used as a sample for screening phages. Staphylococcus aureus (ATCC 6538) and Escherichia coli (CMCC (B) 44102) were cultured in LB liquid medium, and Enterococcus faecalis (CGMCC 1.2135) was cultured in GAM liquid medium. The three strains of bacteria were taken out from -80℃ cryopreservation tubes and inoculated into their respective liquid culture media at a ratio of 2%. They were cultured in an oscillating incubator at 37℃ and 120r / min for 12h, and the bacteria were subcultured at a ratio of 2%. The subculture operation was repeated for 3 rounds to obtain the three activated bacteria. After the three activated bacteria were cultured to the logarithmic growth phase, 5mL of the sewage sample was added to 100mL of the three bacterial liquids in the logarithmic growth phase, and cultured in an oscillating incubator at 37℃ and 150r / min for 24h. The suspension was filtered with a 0.22μm microporous filter membrane to obtain three enriched phage suspensions. The three phage suspensions were diluted to a certain multiple and cultured for 12 hours using the double-layer plate method to obtain a single plaque. The plaque was transferred to 1 mL of the three bacterial suspensions in the logarithmic growth phase using a sterile needle. After 120 r / min shaking culture at 37°C for 12 hours, the phage suspension was filtered using a 0.22 μm microporous filter membrane to obtain a purified phage suspension after one round. The above operation was repeated 3 times to obtain three purified phage suspensions, named Staphylococcus aureus phage SaBD224, Escherichia coli phage EcBD91, and Enterococcus faecalis phage EfBD915, all of which were deposited in the China General Microbiological Culture Collection Management Center, with the deposit numbers CGMCC No. 46185, CGMCC No. 46183, and CGMCC No. 46184, respectively.

[0063] Example 2 Phage Morphology Observation

[0064] The three phage strains were observed for plaque morphology using the double-layer plate method. 10 μL of each of the three phage suspensions was dropped onto the copper mesh, and after it was allowed to naturally precipitate for 10 minutes, it was blotted from the side with dry filter paper. After leaving it in the air for about 1 minute, 1% osmium acid was added to the copper mesh for fixation, and then stained with 2% phosphotungstic acid for 2 minutes. Then, the excess dye was carefully blotted from the side with dry filter paper, and after leaving it in the air for 30 minutes away from light, it was observed using a transmission electron microscope. The results showed that the diameter of the SaBD224 plaque was about 0.1 mm. Figure 1 The microscopic image shows that the head length of SaBD224 is 74.9±2.6nm, the width is 76.9±1.7nm, and the tail length is 111.8±5.9nm. The tail has a base plate and a retractable tail sheath, so SaBD224 is a phage of the family Myocaudaphaga in the order Caudata. The plaque diameter of EcBD91 is about 0.1 to 0.25cm. Figure 2The microscopic image shows that the head length of EcBD91 is 60.6±2.5nm, the width is 54.2±2.5nm, and the tail length is 119.4±5.1nm. The tail has a base plate and a retractable tail sheath, so EcBD91 is a phage of the family Myocaudaphaga in the order Caudata. The plaque diameter of EfBD915 is about 1.0 to 1.5mm, see Figure 3 The microscopic image showed that the head of EfBD915 was 96.7±2.2nm long, 34.7±1.6nm wide, and the tail was 147.6±3.5nm long. The tail obviously lacked a base plate, tail filaments, and a retractable tail sheath, so EfBD915 was a phage of the family Caudovirales and the family Caudoviridae.

[0065] Example 3 Whole genome sequencing and identification of bacteriophage

[0066] Illumina NextSeq500 was used to sequence the DNA of the samples. The genome of SaBD224 is double-stranded circular DNA, with a complete genome size of 136342 bp and a GC content of 29.88%. The genome of EcBD91 is double-stranded circular DNA, with a complete genome size of 53487 bp and a GC content of 44.15%. The genome of EfBD915 is double-stranded circular DNA, with a complete genome size of 35615 bp and a GC content of 38.12%. Prokka (v.1.14.6) was used for gene prediction, and Circos software was used for genome visualization to obtain a full genome map, see Figure 4 , Figure 6 , Figure 8 The top ten genome sequences with the highest similarity to the three phage genomes in NCBI BLAST were downloaded, and the phylogenetic tree was constructed using MEGA10.0. The phylogenetic analysis was constructed using the ClustalW and Neighbor-Joining methods, see Figure 5 , Figure 7 , Fig. 9 .

[0067] Example 4 Biological characteristics of bacteriophage

[0068] 1. Biological characteristics of Staphylococcus aureus phage SaBD224

[0069] The titer of SaBD224 was determined by double-layer plate method and was 2.5×10 4PFU / mL. Mixed culture with host bacteria at a multiplicity of infection (MOI) of 0.0001, 0.001, 0.01, 0.1, 1, 10. Cultured at 37°C with shaking at 150r / min for 12h, centrifuged at 12000r / min for 2min, filtered with a 0.22μm microporous filter, and counted plaques in the supernatant using a double-layer plate method to determine the titer. The optimal multiplicity of infection was determined to be 0.01. Fig.10 .

[0070] SaBD224 and host bacteria were incubated at 37°C, 150r / min for 10 minutes under the optimal infection multiplicity, and then centrifuged at 12000r / min for 10 minutes. The supernatant was discarded, and the precipitate was resuspended with LB liquid culture medium. The resuspension was continued to be placed in a shaking incubator and cultured at 37°C, 150r / min. 500μL of the suspension was taken at 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, and 90 minutes, and filtered with a 0.22μm microporous membrane. The plaque count was performed using the double-layer plate method, and a one-step growth curve was drawn based on the titer of SaBD224. The results showed that the incubation period of SaBD224 was 0-10 minutes, the outbreak period was 10-70 minutes, and it entered the plateau period after 70 minutes. Fig.11 .

[0071] The titer was 6×10 4 The SaBD224 suspension with PFU / mL was divided into 12 tubes, and the pH of the liquid in the 12 tubes was adjusted to 1-12. The SaBD224 suspensions with different pH were placed at 37°C for 2 hours, and the titer of SaBD224 was determined by the double-layer plate method. The results showed that SaBD224 was completely inactivated under pH ≤ 2 and pH ≥ 11, and pH = 6-9 was the optimal condition for maintaining activity. Therefore, SaBD224 has good tolerance to acid and alkali. Fig.12 .

[0072] The titer was 4.5×10 4 The SaBD224 suspension with a PFU / mL was divided into 6 portions and incubated in a water bath at 30°C, 40°C, 50°C, 60°C, 70°C and 80°C for 1 hour. The titer of SaBD224 was determined by the double-layer plate method. The results showed that SaBD224 had good biological activity at 30-40°C, but completely lost its biological activity at temperatures above 60°C. Fig.13 .

[0073] 2. Biological characteristics of Escherichia coli phage EcBD91

[0074] The titer of EcBD91 was determined by double-layer plate method and was 10 4PFU / mL. Mixed culture with host bacteria at MOI = 0.1, 1, 10, 100, 1000, 10000. Cultured at 37°C with shaking at 150r / min for 12h. Centrifuged at 12000r / min for 2min, filtered the supernatant with a 0.22μm microporous filter and counted the plaques by double-layer plate method. The titer of EcBD91 at each MOI was determined. The optimal MOI was 10. Fig.14 .

[0075] After mixing according to the optimal MOI ratio, incubate at 37°C at 150r / min for 10min, centrifuge at 12000r / min for 10min, discard the supernatant, resuspend the precipitate with LB liquid culture medium, and continue to place the resuspension in a shaking incubator at 37°C and 150r / min. Take 500μL of the suspension at 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, and 110min, filter with a 0.22μm microporous filter membrane, count the plaques with the double-layer plate method, and draw a one-step growth curve based on the titer of EcBD91. The results showed that 0-5min was the incubation period of EcBD91, 5-90min was the outbreak period, and it entered the plateau period after 90min. Fig.15 .

[0076] The titer is 10 7 The EcBD91 suspension with a PFU / mL was divided into 12 tubes, and the pH of the 12 tubes was adjusted to 1-12. The EcBD91 suspensions with different pH were placed at 37°C for 2 hours, and the titer of EcBD91 was determined by the double-layer plate method. The results showed that EcBD91 had good activity under pH = 5-10. Fig.16 .

[0077] The titer is 10 9 The EcBD91 suspension of PFU / mL was divided into 6 parts and incubated in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃ for 1h. After incubation, the titer of EcBD91 was determined by double-layer plate method. The results showed that the optimal temperature for maintaining the activity of EcBD91 was 30-60℃. Fig.17 .

[0078] 3. Biological characteristics of Enterococcus faecalis phage EfBD915

[0079] The titer of EfBD915 was determined by double-layer plate method and was 10 4PFU / mL, and mixed with host bacteria according to the optimal infection multiplicity MOI = 0.01, 0.1, 1, 10, 100, 1000. Under 37℃ condition, shake culture at 150r / min for 12h, centrifuge at 12000r / min for 2min, filter the supernatant with 0.22μm microporous filter membrane, count plaques by double-layer plate method, and measure the titer of EfBD915 at each MOI to determine the optimal infection multiplicity of 10, see Fig.18 .

[0080] EfBD915 and host bacteria were incubated at 37°C, 150r / min for 10 minutes under the optimal infection multiplicity, and then centrifuged at 12000r / min for 10 minutes. The supernatant was discarded, and the precipitate was resuspended with LB liquid culture medium. The resuspended liquid was placed in a shaking incubator and cultured at 37°C, 150r / min. 500μL of the suspension was taken at 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, and 90 minutes, and filtered with a 0.22μm microporous membrane. The plaque count was performed using the double-layer plate method, and a one-step growth curve was drawn based on the titer of SaBD224. The results showed that 0-10 minutes was the incubation period of EfBD915, 10-80 minutes was the outbreak period, and it entered the plateau period after 80 minutes. Fig.19 .

[0081] The titer is 10 4 The EfBD915 suspension with a PFU / mL was divided into 12 tubes, and the pH of the 12 tubes was adjusted to 1-12. The EfBD915 suspensions with different pH were placed at 37°C for 2 hours, and the titer of EfBD915 was determined by the double-layer plate method. The results showed that EfBD915 could maintain good activity under the condition of pH ≥ 6 and had strong alkaline resistance. Fig. 20 .

[0082] The titer is 10 4 The EfBD915 suspension of PFU / mL was divided into 6 parts and incubated in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃ for 1h. After incubation, the titer of EfBD915 was determined by double-layer plate method. The results showed that EfBD915 had good biological activity at 30-40℃. Fig.21 .

[0083] Example 5 Preparation of Composition

[0084] 1. Preparation of Bletilla striata-Panax notoginseng extract gel

[0085] The powders of Bletilla striata and Panax notoginseng were mixed in a ratio of 1:1, and 10 times the amount of water was added for reflux extraction for 3 times, each time for 1 hour. After the extracts were combined, the liquid was concentrated by rotary evaporation and vacuum freeze-dried. 17.5g of gelatin and 7g of citric acid were weighed, 690g of glycerol and 420mL of pure water were added, heated and stirred until completely dissolved, and a gel matrix for administration was prepared. Three doses of Bletilla striata-Panax notoginseng extract gels of high, medium and low doses were prepared according to the ratio of crude drug amount to gel matrix of 2:1 (120g:60g), 1:1 (60g:60g), and 1:2 (30g:60g).

[0086] 2. Preparation of PBS suspension of phage combination

[0087] According to the method of Example 1, Pseudomonas aeruginosa (CGMCC 1.10612), Staphylococcus aureus (ATCC6538), Escherichia coli (CMCC (B) 44102) and Enterococcus faecalis (CGMCC 1.2135) were cultured to the logarithmic growth phase, respectively, and mixed with Pseudomonas aeruginosa phage BD815, Staphylococcus aureus phage SaBD224, Escherichia coli phage EcBD91, and Enterococcus faecalis phage EfBD915 according to the optimal infection multiplicity ratio, and cultured at 37°C, 120r / min for 12h, and when the bacterial solution becomes clear, 0.22μm filter membrane is filtered to obtain a phage suspension. The phage suspension is centrifuged at 30000r / min for 2h, the supernatant is discarded, and the precipitated phage is resuspended with sterile PBS to obtain a single phage PBS suspension, and the phage titer is determined by the double-layer plate method, and the titer is greater than 10 14 pfu / mL. The phage suspensions of BD815, SaBD224, EcBD91 and EfBD915 were mixed in a ratio of 1:1:1:1, and the titer of the mixed phage was greater than 10 14 pfu / mL.

[0088] Example 6 Experimental study on treating wound infection in mice

[0089] ICR male mice were used as experimental animals. After the mice were anesthetized with sodium pentobarbital, a 1 cm 2 Pseudomonas aeruginosa (CGMCC 1.10612) 、 Staphylococcus aureus (ATCC 6538), Escherichia coli (CMCC(B)44102) and Enterococcus faecalis (CGMCC 1.2135) were cultured to the logarithmic growth phase, centrifuged and resuspended in sterile PBS to adjust the concentration of the bacterial solution to 10 9 CFU / mL and mixed at a ratio of 1:1:1:1. Take 100 μL of the mixed bacterial solution and drop it on the wound. After the water evaporates, bandage the wound with sterile gauze and tape. The model is successfully established after 24 hours.

[0090] The blank control group, model group, Chinese medicine positive control group (Jingwanhong), western medicine positive control group (levofloxacin), experimental group 1 (phage combination PBS suspension), experimental group 2 (low dose of Bletilla striata-Sanqi extract gel), experimental group 3 (medium dose of Bletilla striata-Sanqi extract gel), experimental group 4 (high dose of Bletilla striata-Sanqi extract gel), experimental group 5 (low dose of Bletilla striata-Sanqi extract gel + phage combination PBS suspension), experimental group 6 (medium dose of Bletilla striata-Sanqi extract gel + phage combination PBS suspension), experimental group 7 (high dose of Bletilla striata-Sanqi extract gel + phage combination PBS suspension) were set up, with 6 ICR mice in each group. After the model was established, the dressing was changed every day within 2 weeks, and the wound area and mouse weight were recorded by taking photos. In each group containing phage combination PBS suspension, 10 μL of phage combination PBS suspension was first added to the wound, and Bletilla striata-Sanqi extract gel was applied to the wound after the liquid dried. The remaining groups first dripped 10 μL of sterile PBS on the wound, and then applied the drug. The model group dripped PBS and applied the gel matrix. Two weeks after administration, the mice were killed by cervical dislocation, and the skin near the wound was cut and placed in 4% tissue fixative. After embedding with wax blocks, the sections and HE staining were performed, and the scans were taken. The specific values ​​of the wound area were determined using Image J. After the skin near the wound was crushed, RNA was extracted using a universal RNA extraction kit, and the mRNA expression levels of vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF) and transforming growth factor-β (TGF-β) were determined by real-time fluorescence quantitative PCR.

[0091] The wound healing was good when Bletilla striata-Sanqi extract gel was used alone, and the effect was better when used in combination with phage combination PBS suspension. Fig. 22 The wound area change value of Bletilla striata-Sanqi extract gel alone was significantly higher than that of the model group and the positive drug group. The wound healing effect was better when it was combined with phage combination PBS suspension. Fig.23 The Bletilla striata-Panax notoginseng extract gel used alone can significantly promote the expression of wound healing-related genes VEGF, bFGF and TGF-β, and there is no significant difference between the combined use of phage combination PBS suspension and the use alone. Figure 24 to Figure 26 The synergistic antibacterial effect of phage combination PBS suspension was significantly better than that of the model group and positive drug group. Fig. 27 HE staining results showed that the skin structure of the model group was abnormal, the epidermal spinous layer was thickened, and no skin appendages were seen. The skin was mainly composed of proliferating inflammatory cells and fibroblasts, and there were hemorrhagic foci under the skin. The degree of skin damage in the Jingwanhong group and the levofloxacin group was significantly improved compared with the model group. The skin condition of the low-, medium-, and high-dose combination groups was more significantly improved, especially in the medium- and high-dose groups, where the skin appendages increased, the epidermal spinous layer was significantly thinned, and there was almost no inflammatory cell infiltration. Fig.28 .

[0092] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A Chinese herbal medicine extract gel for promoting skin wound healing and anti-infection, characterized in that: The invention is composed of a Chinese medicine extract and a gel matrix, wherein the mass ratio of the Chinese medicine extract to the gel matrix is ​​1 to 4:2, and the Chinese medicine extract is a water extract of Bletilla striata and Panax notoginseng.

2. The Chinese medicine extract gel according to claim 1, characterized in that: The Chinese medicine extract is prepared by the following method: Bletilla striata and Panax notoginseng are mixed in a mass ratio of 1:0.8-1.2, 9-11 times the mass of water are added, and reflux extraction is performed for 3 times, each time for 0.5-1.5 hours, the extracts are combined, and then concentrated and freeze-dried.

3. The Chinese herbal medicine extract gel according to claim 1, characterized in that: The gel matrix comprises the following components in parts by weight: 15 to 20 parts by weight of gelatin, 6 to 8 parts by weight of citric acid, 650 to 720 parts by weight of glycerol and 400 to 450 parts by weight of pure water.

4. A composition, characterized in that The composition comprises: i) a first preparation: the Chinese medicine extract gel according to any one of claims 1 to 3; ii) a second preparation: a phage composition whose active ingredients are Pseudomonas aeruginosa phage, Staphylococcus aureus phage, Escherichia coli phage and Enterococcus faecalis phage.

5. The composition according to claim 4, characterized in that The Pseudomonas aeruginosa phage is Pseudomonas aeruginosa phage BD815, with a deposit number of CGMCC No.45873; and / or, the Staphylococcus aureus phage is Staphylococcus aureus phage SaBD224, with a deposit number of CGMCC No.46185; and / or, the Escherichia coli phage is EcBD91, with a deposit number of CGMCC No.46183; and / or, the Enterococcus faecalis phage is EfBD915, with a deposit number of CGMCC No.46184.

6. The composition according to claim 4 or 5, characterized in that In the phage composition, the ratio of the number of live Pseudomonas aeruginosa phage, Staphylococcus aureus phage, Escherichia coli phage and Enterococcus faecalis phage is 1:1:1:

1.

7. The composition according to claim 4 or 5, characterized in that The contents of Pseudomonas aeruginosa phage, Staphylococcus aureus phage, Escherichia coli phage and Enterococcus faecalis phage are not less than 10 14 pfu / mL.

8. Use of the Chinese herbal extract gel according to any one of claims 1 to 3, or the composition according to any one of claims 4 to 7 in the preparation of products for promoting skin wound healing and anti-infection.

9. Use of the Chinese herbal extract gel according to any one of claims 1 to 3, or the composition according to any one of claims 4 to 7, in the preparation of a product for preventing and treating skin wound infections caused by one or a combination of two or more of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli or Enterococcus faecalis.

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