Boa oil compound for prevention and adjuvant treatment of diabetic foot and preparation method of boa oil compound

Abstract

The invention provides a boa oil compound for prevention and adjuvant therapy of diabetic foot and a preparation method of the boa oil compound. The boa oil compound comprises the following components in parts by weight: 5-15 parts of boa oil enzymolysis extract, 4-6 parts of centella triterpenoids and the like. The python oil enzymolysis extract is hydrolyzed step by step through trypsin and lipase, the recombinant human epidermal growth factor microspheres are of a double-layer coating structure, and the transdermal absorption enhancer is azone, oleic acid, menthol and the like in a specific proportion. The preparation method comprises the steps of enzymolysis and extraction of boa oil, compounding of active components, construction of a sustained-release system, preparation of a final product and the like, wherein each step comprises specific condition control, enzymolysis temperature, pH, stirring rate and time, ultrasonic emulsification parameters, coating operation, homogenization conditions and the like. The components of the compound have a synergistic effect, so that the compound can effectively prevent and assist in treating diabetic foot, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of python oil compounds, and in particular to a python oil compound for preventing and assisting in the treatment of diabetic foot and a preparation method thereof. Background Art

[0002] As one of the serious complications of diabetes, the incidence of diabetic foot is increasing year by year worldwide, which has a great negative impact on the physical and mental health and quality of life of patients. The pathogenesis of diabetic foot is extremely complex, involving the interaction of multiple factors such as neuropathy, vascular disease, immune dysfunction and infection, which leads to the obstruction of wound healing process, difficulty in treatment, long cycle, and easy recurrence, which seriously affects the quality of life of patients and may even lead to serious consequences such as amputation.

[0003] At present, there are various clinical treatments for diabetic foot, but there are still many problems that need to be solved. Traditional drug treatments often face many difficulties. For example, it is difficult to maintain local drug concentrations at effective treatment levels, resulting in poor efficacy; at the same time, systemic medication may cause a series of adverse reactions, increasing patients' pain and medical risks. In terms of topical medications, due to the natural barrier effect of the skin's stratum corneum, many drugs have poor permeability and are difficult to effectively penetrate the skin to reach the lesion site, thus failing to fully exert their therapeutic effects. In addition, some existing drug preparations have obvious deficiencies in encapsulation rate and drug release control, resulting in unstable drug efficacy and failure to provide continuous and effective support for wound repair.

[0004] Moreover, the microenvironment of diabetic foot wounds is complex and changeable, often accompanied by inflammatory reactions, bacterial infections and other problems, and existing treatment methods often lack comprehensive consideration and effective response measures for these complex factors. Some treatment products may also cause greater irritation to the skin, causing allergies and other adverse reactions, further affecting patient compliance and treatment effects.

[0005] Python oil, as a natural oil with great potential, has gradually attracted attention in the fields of scientific research and medicine in recent years. It is rich in a variety of unsaturated fatty acids, vitamins and bioactive ingredients. These unsaturated fatty acids have good skin permeability, can quickly nourish the skin and promote skin metabolism; the vitamins contained have antioxidant effects, can effectively reduce inflammatory responses, and protect cells from free radical damage. Previous studies have shown that python oil has been used in traditional medicine to treat skin trauma, burns, etc., showing the effect of promoting wound healing and relieving pain. However, in the field of diabetic foot prevention and treatment, python oil has not yet been fully developed and applied. Summary of the invention

[0006] In view of this, the present invention provides a python oil complex for preventing and adjuvantly treating diabetic foot and a preparation method thereof to solve the above problems.

[0007] The technical solution of the present invention is realized as follows: A python oil complex for preventing and adjuvantly treating diabetic foot: includes the following components by weight: 5-15 parts of python oil enzymolysis extract, 4-6 parts of asiaticoside triterpenoids, 2-5 parts of silybin-chitosan nanoconjugate, 0.1-0.5 parts of recombinant human epidermal growth factor microspheres, 0.3-0.9 parts of transdermal absorption promoter, 0.1-0.5 parts of silver-loaded mesoporous silica antibacterial agent, 0.2-1 part of aloe polysaccharide, 0.3-0.8 parts of quercetin-β-cyclodextrin inclusion complex; the python oil enzymolysis extract is obtained by stepwise hydrolysis of python adipose tissue with trypsin and lipase; the asiaticoside triterpenoids are selected from asiaticoside or madecassic acid, the recombinant human epidermal growth factor microspheres adopt a double-layer coating structure, the inner layer is a chitosan polyelectrolyte composite membrane, and the outer layer is a methyl methacrylate-methyl acrylate copolymer film, and the transdermal absorption promoter is azone, oleic acid and menthol with a mass ratio of (4-6):(1-3):(0.3-0.7).

[0008] Further, a python oil complex for preventing and adjuvantly treating diabetic foot includes the following components by weight: 10 parts of python oil enzymolysis extract, 5 parts of asiaticoside triterpenoids, 3 parts of silybin-chitosan nanoconjugate, 0.3 parts of recombinant human epidermal growth factor microspheres, 0.6 parts of transdermal absorption promoter, 0.3 parts of silver-loaded mesoporous silica antibacterial agent, 0.8 part of aloe polysaccharide, 0.5 parts of quercetin-β-cyclodextrin inclusion complex.

[0009] Further, the silybin-chitosan nanoconjugate is prepared by the following method: reacting silybin with succinic anhydride in a molar ratio of 1:1-2 in dimethylformamide under nitrogen protection at 40-60 °C for 3-5 hours to obtain succinylated silybin, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of 1:1.0-2.2:0.5-0.9, stirring in the dark at 20-30 °C for 25-35 minutes to generate an active solution, slowly dropping a 1-3% w / v carboxymethyl chitosan solution into the active solution, with a molar ratio of 1:0.3-0.6, stirring in the dark at 35-40 °C for 20-40 hours, maintaining the pH at 6.0±0.2, and obtaining nanoparticles with a particle size of 80-150 nm through ultrafiltration purification.

[0010] Further, the silver-loaded mesoporous silica antibacterial agent is prepared by impregnating mesoporous silica with a pore diameter of 2 - 4 nm in a 0.1 - 0.3 M silver nitrate solution, ultrasonically treating it at 50 - 70 °C for 1 - 1.5 hours, and then calcining it at 300 - 500 °C for 1 - 3 h to obtain an antibacterial agent with a silver loading of 5 - 8%.

[0011] Further, the quercetin-β-cyclodextrin inclusion complex is specifically prepared by dissolving β-cyclodextrin in deionized water at 50 - 70 °C, stirring until completely dissolved to obtain a β-cyclodextrin solution with a concentration of 8 - 13% w / v, slowly adding quercetin to a 40 - 50% w / v ethanol solution at a molar ratio of 1:1.2 - 1.5, stirring evenly and then dropping it into the β-cyclodextrin solution, and carrying out ultrasonic-assisted stirring at 40 - 60 °C for 5 - 7 hours, with the rate controlled at 500 - 700 rpm, the ultrasonic power at 200 - 400 W, and the ultrasonic frequency at 20 - 60 kHz.

[0012] Further, a preparation method of a python oil complex for preventing and adjuvantly treating diabetic foot includes the following steps:

[0013] S1. Enzymatic extraction of python oil: The fresh python adipose tissue is frozen rapidly by liquid nitrogen and then pulverized to 200 - 300 mesh, and is subjected to stepwise enzymatic hydrolysis with trypsin and lipase in sequence. The enzyme activity dosage of trypsin is 3000 - 4000 U / g adipose tissue, and the enzyme activity dosage of lipase is 1500 - 2000 U / g oil phase. The upper oil phase is collected by centrifugation;

[0014] S2. Composite of active components: The asiaticoside triterpenoids and silybin-chitosan nanoconjugates are ultrasonically emulsified at 40 - 50 °C for 30 - 60 minutes;

[0015] S3. Construction of a sustained-release system: The microspheres are immersed in 0.3 - 0.7% chitosan and oscillated at 20 - 30 °C for 20 - 40 minutes to form a 5 - 10 μm thick polyelectrolyte membrane as the inner layer, and then a 10 - 15 μm thin film of an ethanol solution of 5 - 8% w / v methyl methacrylate-methyl acrylate copolymer is formed by fluidized bed spraying as the outer layer, with an encapsulation efficiency ≥95% to form double-layer coated microspheres;

[0016] S4. Preparation of the final product: The products obtained in steps S1 - S3, aloe polysaccharide, and a transdermal absorption promoter are homogenously mixed under the protection of an inert gas, sterilized by a 0.22 μm filter membrane, and then subpackaged.

[0017] Further, the trypsin enzymatic hydrolysis conditions in step S1 are: pH 7.5 ± 0.2, 45 °C ± 1 °C, stirring rate 180 - 220 rpm, enzymatic hydrolysis for 2 - 4 hours, and the lipase enzymatic hydrolysis conditions are: pH 6.0 ± 0.1, 50 °C ± 0.5 °C, stirring rate 120 - 180 rpm, enzymatic hydrolysis for 4 - 5 hours.

[0018] Furthermore, the phacoemulsification parameters described in step S2 are as follows: the frequency is 25 - 30 kHz, the power is 300 - 600 W, and the pulsed working time is 2 s / interval 1 s.

[0019] Furthermore, in the homogenization process described in step S4, high-pressure microfluidization is adopted, the operating pressure is 1200 - 1500 bar, and it circulates 3 - 5 times, controlling the product particle size D90 < 500 nm.

[0020] Furthermore, the application of the python oil complex for preventing and adjuvant treating diabetic foot in the preparation of external medications, and the external medications are ointments, creams or gels.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) Synergy of anti-inflammation and repair: The high content of free fatty acids in the enzymolysis extract of python oil has excellent anti-inflammatory properties, which can effectively reduce the inflammatory reaction of diabetic foot wounds; the triterpenoids of Centella asiatica further enhance the anti-inflammatory effect and at the same time promote the repair and regeneration of wound tissues; silybin-chitosan nanoconjugates not only have antioxidant and anti-inflammatory effects, but also, due to their nanoscale particle size advantage, can better penetrate into wound tissues and play a synergistic repair effect. Multiple components cooperate with each other, starting from two key aspects of inflammation control and tissue repair, and comprehensively improving the wound condition.

[0023] (2) Concurrent antibacterial and wound healing promotion: The silver-loaded mesoporous silica antibacterial agent can effectively inhibit the growth and reproduction of bacteria on the wound surface and reduce the risk of infection; while the recombinant human epidermal growth factor microspheres achieve slow release through a double-layer coating structure, continuously providing growth factors that promote cell proliferation and migration for the wound surface and accelerating wound healing. The concurrent antibacterial and wound healing promotion effects provide a good repair environment for diabetic foot wounds and contribute to improving the treatment effect.

[0024] (3) High-efficiency drug slow release and permeability: The double-layer coating structure of the recombinant human epidermal growth factor microspheres can precisely control the drug release rate, enabling it to maintain a stable blood drug concentration for a long time, avoiding the problems of too high or too low local concentration caused by the rapid release of traditional drugs, and improving the bioavailability and treatment effect of the drug. The transdermal absorption promoter (a specific proportion combination of azone, oleic acid and menthol) can effectively reduce the skin barrier effect and increase the ability of the drug to penetrate through the skin, enabling the active ingredients to reach the wound site more smoothly and play a therapeutic role. At the same time, the reasonable design of the particle sizes of components such as silybin-chitosan nanoconjugates and quercetin-β-cyclodextrin inclusion complexes helps to improve the skin permeability of the drug. Specific embodiments

[0025] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0026] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0027] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can all be obtained from commercial channels.

[0028] Example 1

[0029] A python oil complex for preventing and adjuvantly treating diabetic foot: It comprises the following components by weight parts: 5 - 15 parts of enzymatically hydrolyzed extract of python oil, 4 parts of asiatic triterpenoids, 2 parts of silybin-chitosan nanoconjugate, 0.1 part of recombinant human epidermal growth factor microspheres, 0.3 part of transdermal absorption promoter, 0.1 part of silver-loaded mesoporous silica antibacterial agent, 0.2 part of aloe polysaccharide, 0.3 part of quercetin-β-cyclodextrin inclusion complex;

[0030] The asiatic triterpenoids are selected from asiaticoside;

[0031] The silybin-chitosan nanoconjugate is prepared by the following method: Silybin and succinic anhydride are reacted in dimethylformamide at a molar ratio of 1:1 under nitrogen protection at 40°C for 3 hours to obtain succinylated silybin. EDC and NHS are added at a molar ratio of 1:1.0:0.5, and stirred in the dark at 20°C for 25 minutes to generate an active solution. A 1% w / v carboxymethyl chitosan solution is slowly added dropwise to the active solution at a molar ratio of 1:0.3, and stirred in the dark at 35°C for 20 hours while maintaining pH 5.8. After ultrafiltration purification, nanoparticles with a particle size of 80 nm are obtained;

[0032] The transdermal absorption promoter is azone, oleic acid and menthol with a mass ratio of 4:1:0.3;

[0033] The silver-loaded mesoporous silica antibacterial agent is obtained by impregnating mesoporous silica with a pore size of 2 nm in a 0.1 M silver nitrate solution, ultrasonically treating it at 50°C for 1 hour and then calcining it at 300°C for 1 h to obtain an antibacterial agent with a silver loading of 5%;

[0034] The quercetin-β-cyclodextrin inclusion complex is specifically prepared by dissolving β-cyclodextrin in deionized water at 60°C and stirring until completely dissolved to prepare an 8% w / v β-cyclodextrin solution. Quercetin is slowly added to a 40% w / v ethanol solution at a molar ratio of 1:1.2, stirred evenly and then added dropwise to the β-cyclodextrin solution, and ultrasonically assisted and stirred at 40°C for 5 hours, with the rate controlled at 500 rpm, the ultrasonic power at 200 W, and the ultrasonic frequency at 20 kHz.

[0035] Example 2

[0036] A python oil complex for preventing and adjuvant treating diabetic foot: comprising the following components by weight parts: 15 parts of enzymatically hydrolyzed extract of python oil, 6 parts of asiatic acid triterpenoids, 5 parts of silybin-chitosan nanoconjugate, 0.5 part of recombinant human epidermal growth factor microspheres, 0.9 part of transdermal absorption enhancer, 0.5 part of silver-loaded mesoporous silica antibacterial agent, 1 part of aloe polysaccharide, 0.8 part of quercetin-β-cyclodextrin inclusion complex;

[0037] The asiatic acid triterpenoids are selected from asiatic acid;

[0038] The silybin-chitosan nanoconjugate is prepared by the following method: reacting silybin with succinic anhydride at a molar ratio of 1:2 in dimethylformamide under nitrogen protection at 60 °C for 5 hours to obtain succinylated silybin, adding EDC and NHS at a molar ratio of 1:2.2:0.9, stirring in the dark at 30 °C for 35 minutes to generate an active solution, slowly dropping a 3% w / v carboxymethyl chitosan solution into the active solution at a molar ratio of 1:0.6, stirring in the dark at 40 °C for 40 hours, maintaining pH 6.2, and obtaining nanoparticles with a particle size of 150 nm through ultrafiltration purification;

[0039] The transdermal absorption enhancer is azone, oleic acid and menthol with a mass ratio of 6:3:0.7;

[0040] The silver-loaded mesoporous silica antibacterial agent is obtained by impregnating mesoporous silica with a pore diameter of 4 nm in a 0.3 M silver nitrate solution, performing ultrasonic treatment at 70 °C for 1.5 hours and then calcining at 500 °C for 3 h to obtain an antibacterial agent with a silver loading of 8%;

[0041] The quercetin-β-cyclodextrin inclusion complex is specifically prepared by dissolving β-cyclodextrin in deionized water at 60 °C, stirring until completely dissolved to prepare a β-cyclodextrin solution with a concentration of 13% w / v, slowly adding quercetin at a molar ratio of 1:1.5 to a 50% w / v ethanol solution, stirring evenly and then dropping it into the β-cyclodextrin solution, performing ultrasonic-assisted stirring at 60 °C for 7 hours, controlling the rate at 700 rpm, the ultrasonic power at 400 W, and the ultrasonic frequency at 60 kHz.

[0042] Example 3

[0043] A python oil complex for preventing and adjuvant treating diabetic foot: comprising the following components by weight parts: 10 parts of enzymatically hydrolyzed extract of python oil, 5 parts of asiaticoside triterpenoids, 3 parts of silybin-chitosan nanoconjugate, 0.3 part of recombinant human epidermal growth factor microspheres, 0.6 part of transdermal absorption enhancer, 0.3 part of silver-loaded mesoporous silica antibacterial agent, 0.8 part of aloe polysaccharide, 0.5 part of quercetin-β-cyclodextrin inclusion complex;

[0044] The asiatic acid triterpenoids are selected from asiaticoside;

[0045] The silybin-chitosan nano-conjugate is prepared by the following method: Silybin and succinic anhydride are reacted in dimethylformamide at a molar ratio of 1:2 under nitrogen protection at 50 °C for 4 hours to obtain succinylated silybin. EDC and NHS are added at a molar ratio of 1:1.5:0.7, and stirred in the dark at 25 °C for 30 minutes to generate an active solution. A 2% w / v carboxymethyl chitosan solution is slowly added dropwise to the activated solution at a molar ratio of 1:0.5, and stirred in the dark at 38 °C for 30 hours while maintaining pH 6.0. Nanoparticles with a particle size of 120 nm are obtained through ultrafiltration purification;

[0046] The transdermal absorption enhancer is azone, oleic acid and menthol with a mass ratio of 5:2:0.5;

[0047] The silver-loaded mesoporous silica antibacterial agent is prepared by impregnating mesoporous silica with a pore diameter of 3 nm in a 0.2 M silver nitrate solution, ultrasonicating at 60 °C for 1.3 hours, and then calcining at 400 °C for 2 h to obtain an antibacterial agent with a silver loading of 7%;

[0048] The quercetin-β-cyclodextrin inclusion complex is specifically prepared by dissolving β-cyclodextrin in deionized water at 60 °C and stirring until completely dissolved to obtain a 10% w / v β-cyclodextrin solution. Quercetin is slowly added to a 45% w / v ethanol solution at a molar ratio of 1:1.3, stirred evenly, and then added dropwise to the β-cyclodextrin solution. Ultrasonic-assisted stirring is carried out at 50 °C for 6 hours, with the rate controlled at 600 rpm, the ultrasonic power at 300 W, and the ultrasonic frequency at 40 kHz;

[0049] The above Examples 1-3 adopt the following preparation method:

[0050] S1. Enzymatic hydrolysis extraction of python oil: Fresh python adipose tissue is frozen quickly in liquid nitrogen and then crushed to 250 meshes. Sequential enzymatic hydrolysis is carried out using trypsin and lipase. The enzyme activity dosage of trypsin is 3500 U / g adipose tissue, and the enzyme activity dosage of lipase is 1800 U / g oil phase. The upper oil phase is collected by centrifugation. The enzymatic hydrolysis conditions of trypsin are: pH 7.5, 45 °C, stirring rate 200 rpm, enzymatic hydrolysis for 3 hours. The enzymatic hydrolysis conditions of lipase are: pH 6.0, 50 °C, stirring rate 150 rpm, enzymatic hydrolysis for 4 hours;

[0051] S2. Composite of active components: The asiatic acid triterpenoids and the silybin-chitosan nano-conjugate are ultrasonically emulsified at 45 °C for 50 minutes. The ultrasonic emulsification parameters are: frequency 28 kHz, power 500 W, pulse working 2 s / intermittent 1 s;

[0052] S3. Sustained-release system construction: Immerse the microspheres in 0.5% chitosan and oscillate at 25°C for 30 minutes to form an 8-μm-thick polyelectrolyte membrane as the inner layer. Then, use fluidized bed spraying to form a 12-μm-thick film as the outer layer with an ethanol solution of 7% w / v methacrylic acid-methyl methacrylate copolymer. The encapsulation efficiency is ≥95% to form double-coated microspheres;

[0053] S4. Final product formulation: Homogeneously mix the products obtained in steps S1 - S3, aloe polysaccharide, and transdermal absorption promoter under the protection of inert gas. The homogenization process uses high-pressure microfluidization with an operating pressure of 1300 bar and circulates 4 times. Control the product particle size D90 < 500 nm, and then sterilize through a 0.22-μm filter membrane and subpackage.

[0054] Comparative Example 1

[0055] The difference between this comparative example and Example 3 is that python oil was not enzymatically extracted;

[0056] Formulation: The same as Example 3;

[0057] Preparation method:

[0058] S1: Extract python oil using the traditional hot melt method and heat at 120°C for 3 h;

[0059] S2 - S4 are the same as Example 3.

[0060] Comparative Example 2

[0061] The difference between this comparative example and Example 3 is that the recombinant human epidermal growth factor microspheres were directly added without encapsulation.

[0062] Formulation: The same as Example 3

[0063] Preparation method:

[0064] S1 and S2 are the same as Example 3

[0065] S3 Final product formulation: Homogeneously mix the products obtained in steps S1 - S2, recombinant human epidermal growth factor microspheres, aloe polysaccharide, and transdermal absorption promoter under the protection of inert gas. The homogenization process uses high-pressure microfluidization with an operating pressure of 1300 bar and circulates 4 times. Control the product particle size D90 < 500 nm, and then sterilize through a 0.22-μm filter membrane and subpackage

[0066] Comparative Example 3

[0067] The difference between this comparative example and Example 3 is that a single transdermal absorption promoter was used,

[0068] Formulation: Only azone was used as the transdermal absorption promoter, and other components are the same as Example 3;

[0069] Comparative Example 4

[0070] The difference between this comparative example and Example 3 is that silybin-chitosan nanoconjugate was not used.

[0071] I. Performance Test

[0072] 1. Extraction efficiency and active ingredient content of python oil

[0073] Test method:

[0074] (1) Free fatty acid content: Determined by acid-base titration (using KOH as the titrant and phenolphthalein as the indicator), and calculate the proportion of free fatty acids in the total oil phase;

[0075] (2) Retention rate of active ingredients: Detect the content of active ingredients (squalene, tocopherol) in python oil by HPLC, and compare the retention rates of enzymatic hydrolysis extraction and traditional hot melting method.

[0076] (3) Test results:

[0077] Table 1:

[0078]

[0079]

[0080] As can be seen from the above results, the enzymatic hydrolysis extraction process has been preliminarily optimized with a high retention rate of active ingredients. By increasing the enzyme activity dosage and reaction time, the free fatty acid content and the retention rate of active ingredients are further improved, and the retention of active ingredients under the optimal enzymatic hydrolysis conditions of Example 3 is the best. For the traditional hot melting method extraction, the active ingredients are damaged at high temperature, and the free fatty acid content is significantly reduced.

[0081] 2. Sustained-release performance

[0082] Test method:

[0083] (1) Disperse the microspheres in PBS buffer (pH 7.4, 37 °C), and regularly take samples to determine the rhEGF concentration (ELISA method).

[0084] (2) Calculate the cumulative release rate curve to evaluate the influence of the encapsulation structure and materials on the release rate.

[0085] (3) Test results

[0086] Table 2:

[0087] 24-hour release rate (%) 72-hour cumulative release rate (%) Example 1 38.5±2.1 62.3±3.5 Example 2 35.2±1.8 58.7±3.2 Example 3 32.5±2.8 58.3±3.2 Comparative Example 2 68.7±3.5 92.1±4.1

[0088] As can be seen from the test results, the initial release of the example group is slower and the release is stable. The unencapsulated microspheres result in a rapid release of rhEGF, with a release rate of over 65% at 24 hours, and almost complete release at 72 hours, with a short drug effect.

[0089] 3. Transdermal absorption performance

[0090] Test method: The Franz diffusion cell method was adopted, using excised rat skin as a barrier to measure the transdermal rates (μg / cm 2 / h) and 24-hour cumulative permeation amounts (μg / cm 2 ) of squalene and tocopherol.

[0091] Table 3:

[0092]

[0093]

[0094] The multi-component transdermal penetration enhancer system and double-layer coating microsphere protection strategy of the present invention significantly improved the transdermal absorption performance of squalene and tocopherol, and the cumulative permeation amounts reached 24.8 μg / cm 2 (squalene) and 19.3 μg / cm 2 (tocopherol) respectively, which were significantly better than other groups. In Comparative Example 2 (microspheres not encapsulated), since the active ingredients were directly exposed to the matrix, the transdermal rate might be affected due to aggregation or degradation; in Comparative Example 3 (single azone), due to the lack of protection or synergistic mechanism, the transdermal efficiency was significantly reduced. It shows that the multi-component combination of azone, oleic acid, and menthol used in Example 3 has significant synergistic effects. Azone dissolves the lipids in the skin stratum corneum, reduces the barrier resistance, and significantly improves the permeability of lipophilic components (squalene, tocopherol); oleic acid enhances the diffusion rate of lipophilic components, especially has a significant effect on enhancing the permeability of squalene and tocopherol; menthol regulates the surface tension of the skin and promotes drug penetration, especially for the transdermal absorption of tocopherol.

[0095] II. Wound healing effect

[0096] Test method:

[0097] 1. Animal model: 70 diabetic rats (induced by STZ, blood glucose ≥ 16.7 mmol / L) were selected, and wounds with a diameter of 1.5 cm were created on their backs. They were randomly divided into 7 groups (10 rats in each group).

[0098] 2. Drug administration plan: The corresponding drugs were applied daily for 28 days.

[0099] 3. Detection indexes:

[0100] Wound healing rate: The change in wound area was measured, and the healing rate (%) was calculated.

[0101] Inflammatory factor level: On the 7th day after the operation, wound tissue homogenates were taken, and the concentrations of TNF-α and IL-6 were detected by ELISA method.

[0102] Histopathological scoring: On the 28th day after surgery, wound tissues were taken, and HE staining was used to evaluate the length of newly formed epithelium, the thickness of granulation tissue, and the degree of inflammatory cell infiltration.

[0103] 4. Test results

[0104] Table 4:

[0105]

[0106] Table 5:

[0107]

[0108] From the above results, it can be seen that Example 3 is significantly superior to other groups in terms of wound healing rate, inhibition of inflammatory factors, and tissue repair score, indicating that the synergistic effect of enzymatically hydrolyzed python oil, double-layer coated microspheres, multi-component transdermal promoters, and nano-conjugates significantly improves the wound healing effect of diabetic foot.

[0109] Compared with Comparative Example 1, it shows that the enzymolysis extraction process has a high retention rate of active ingredients and a good healing effect.

[0110] Compared with Comparative Examples 2 and 3, it can effectively control the release of active ingredients and promote the transdermal efficiency.

[0111] Compared with Comparative Example 4, the addition of silybin-chitosan nano-conjugate enhances the anti-inflammatory effect and promotes the arrangement of collagen fibers and the improvement of tissue density.

[0112] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A python oil complex for preventing and adjuvant treating diabetic foot, characterized in that: It comprises the following components by weight parts: 5-15 parts of enzymatically hydrolyzed python oil extract, 4-6 parts of asiaticoside triterpenoids, 2-5 parts of silybin-chitosan nanoconjugate, 0.1-0.5 parts of recombinant human epidermal growth factor microspheres, 0.3-0.9 parts of transdermal absorption enhancer, 0.1-0.5 parts of silver-loaded mesoporous silica antibacterial agent, 0.2-1 part of aloe polysaccharide, 0.3-0.8 parts of quercetin-β-cyclodextrin inclusion complex; the enzymatically hydrolyzed python oil extract is obtained by stepwise hydrolysis of python adipose tissue with trypsin and lipase; the asiaticoside triterpenoids are selected from asiaticoside or madecassic acid, the recombinant human epidermal growth factor microspheres adopt a double-layer coating structure, the inner layer is a chitosan polyelectrolyte composite membrane, and the outer layer is a methyl methacrylate-methyl acrylate copolymer film, and the transdermal absorption enhancer is azone, oleic acid and menthol with a mass ratio of (4-6):(1-3):(0.3-0.7).

2. The python oil complex for preventing and adjuvantly treating diabetic foot according to claim 1, wherein: It comprises the following components by weight parts: 10 parts of enzymatically hydrolyzed python oil extract, 5 parts of asiaticoside triterpenoids, 3 parts of silybin-chitosan nanoconjugate, 0.3 parts of recombinant human epidermal growth factor microspheres, 0.6 parts of transdermal absorption enhancer, 0.3 parts of silver-loaded mesoporous silica antibacterial agent, 0.8 part of aloe polysaccharide, 0.5 parts of quercetin-β-cyclodextrin inclusion complex.

3. The python oil complex for preventing and adjuvant treating diabetic foot according to claim 1, wherein: The silybin-chitosan nanoconjugate is prepared by the following method: reacting silybin with succinic anhydride in a molar ratio of 1:1-2 in dimethylformamide under nitrogen protection at 40-60 °C for 3-5 hours to obtain succinylated silybin, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of 1:1.0-2.2:0.5-0.9, stirring in the dark at 20-30 °C for 25-35 minutes to generate an active solution, slowly dropping a 1-3% w / v carboxymethyl chitosan solution into the active solution, with a molar ratio of 1:0.3-0.6, stirring in the dark at 35-40 °C for 20-40 hours, maintaining the pH at 6.0±0.2, and obtaining nanoparticles with a particle size of 80-150 nm through ultrafiltration purification.

4. The python oil complex for preventing and adjuvantly treating diabetic foot according to claim 1, characterized in that: The silver-loaded mesoporous silica antibacterial agent is obtained by impregnating mesoporous silica with a pore size of 2-4 nm in a 0.1-0.3 M silver nitrate solution, performing ultrasonic treatment at 50-70 °C for 1-1.5 hours, and then calcining at 300-500 °C for 1-3 h to obtain an antibacterial agent with a silver loading of 5-8%.

5. A python oil complex for preventing and adjuvantly treating diabetic foot according to claim 1, characterized in that: The quercetin-β-cyclodextrin inclusion complex is specifically prepared by dissolving β-cyclodextrin in deionized water at 50-70 °C, stirring until completely dissolved to obtain a β-cyclodextrin solution with a concentration of 8-13% w / v, slowly adding quercetin in a molar ratio of 1:1.2-1.5 to a 40-50% w / v ethanol solution, stirring evenly and then dropping it into the β-cyclodextrin solution, performing ultrasonic-assisted stirring at 40-60 °C for 5-7 hours, controlling the rate at 500-700 rpm, the ultrasonic power at 200-400 W, and the ultrasonic frequency at 20-60 kHz.

6. The preparation method of a python oil complex for preventing and adjuvantly treating diabetic foot according to claim 1, characterized in that: It includes the following steps: S1. Enzymatic extraction of python oil: The fresh python adipose tissue is quickly frozen in liquid nitrogen and then crushed to 200 - 300 mesh. Sequentially, trypsin and lipase are used for stepwise enzymatic hydrolysis. The enzyme activity dosage of trypsin is 3000 - 4000 U / g of adipose tissue, and the enzyme activity dosage of lipase is 1500 - 2000 U / g of oil phase. The upper oil phase is collected by centrifugation. S2. Composite of active components: Centella asiatica triterpenoids and silybin-chitosan nanoconjugates are ultrasonically emulsified at 40 - 50 °C for 30 - 60 minutes. S3. Construction of sustained-release system: The microspheres are immersed in 0.3 - 0.7% chitosan and oscillated at 20 - 30 °C for 20 - 40 minutes to form a 5 - 10 μm thick polyelectrolyte membrane as the inner layer. Then, an ethanol solution of 5 - 8% w / v methyl methacrylate-methyl acrylate copolymer is sprayed using a fluidized bed to form a 10 - 15 μm thin film as the outer layer, with an encapsulation efficiency ≥ 95%, forming double-coated microspheres. S4. Preparation of the final product: The products obtained in steps S1 - S3, aloe polysaccharide, and a transdermal absorption promoter are homogenously mixed under the protection of inert gas, sterilized through a 0.22 μm filter membrane, and then sub-packed.

7. The preparation method of a python oil complex for preventing and adjuvantly treating diabetic foot as described in claim 6, characterized in that: The trypsin enzymatic hydrolysis conditions in step S1 are: pH 7.5 ± 0.2, 45 °C ± 1 °C, stirring rate 180 - 220 rpm, enzymatic hydrolysis for 2 - 4 hours. The lipase enzymatic hydrolysis conditions are: pH 6.0 ± 0.1, 50 °C ± 0.5 °C, stirring rate 120 - 180 rpm, enzymatic hydrolysis for 4 - 5 hours.

8. The preparation method of a python oil complex for preventing and adjuvantly treating diabetic foot according to claim 6, characterized in that: The ultrasonic emulsification parameters in step S2 are: frequency 25 - 30 kHz, power 300 - 600 W, pulsed working for 2 s / intermittent for 1 s.

9. The preparation method of a python oil complex for preventing and adjuvantly treating diabetic foot according to claim 6, characterized in that: In step S4, the homogenization process uses high-pressure microfluidization, with an operating pressure of 1200 - 1500 bar, circulating 3 - 5 times, and controlling the product particle size D90 < 500 nm.

10. Use of the python oil complex according to any one of claims 1-5 for preventing and adjuvant treating diabetic foot in the preparation of an external medicine, characterized in that, The topical drug is an ointment, cream, or gel.

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