Application of birch sap thermosensitive in-situ gel in repairing burnt and scalded skin

Abstract

The invention relates to a thermosensitive in-situ gel prepared with birch sap as a main raw material. The birch sap thermosensitive in-situ gel can effectively promote the proliferation of wound fibroblasts and the secretion of anti-inflammatory factors, reduce the level of pro-inflammatory factors, and can be used as a drug delivery carrier in combination with other drugs. When used as a drug delivery carrier in combination with drugs, it has a zero-order drug release characteristic. The birch sap gel provided by the invention is in a liquid state before drug administration, and spontaneously changes into a semi-solid state after contacting with a human wound surface. It has good sustained-release and long-acting therapeutic effects, effectively reduces the number of times of giving and changing dressings for patients with burns and scalds, has good tissue compatibility and biological adhesion, and is suitable for the treatment and repair of burned and scalded skin.

Description

Technical Field

[0001] The invention relates to a temperature-sensitive in-situ gel, which is prepared by taking birch sap as an effective component and a solvent, is suitable for repairing and caring for burned and scalded skin, and belongs to the technical field of medicine. Background Art

[0002] Natural birch sap is rich in oligosaccharides, polysaccharides, amino acids, pentacyclic triterpenoids, fatty acids, proteins and other substances. Pentacyclic triterpenoids such as betulinic acid and betulin can effectively promote the secretion of anti-inflammatory factors IL-4 and IL-10, inhibit the levels of pro-inflammatory factors IL-6 and TNF-α, and reduce the risk of wound inflammation and infection; its rich birch polysaccharides can promote the proliferation of fibroblasts, thereby accelerating the proliferation and repair of wound cells and shortening the healing time of wounds.

[0003] In situ gel is also called instant gel or environmentally sensitive gel. It is a solution with a certain fluidity at room temperature. When the temperature, pH value, and ionic environment of the surrounding environment change, its phase changes to form a semisolid preparation with a certain adhesion. Among them, thermosensitive in situ gel is the most widely studied and relatively mature in situ gel. Thermosensitive gel has a critical phase transition temperature and can undergo phase transition with changes in ambient temperature. It has good adhesion and tissue compatibility. When it is used in transdermal preparations, it can effectively prolong the retention time of the drug at the medication site, achieve long-acting and sustained release of the drug, reduce the number of dosing times, reduce blood drug concentration fluctuations, and greatly improve patient compliance. Summary of the invention

[0004] The invention relates to a temperature-sensitive in-situ gel prepared by taking birch sap as a main raw material, which is suitable for repairing and caring for burned and scalded skin of human body.

[0005] The birch sap thermosensitive in-situ gel has a gelling temperature of 32°C to 34°C. It is in a liquid state before contacting the wound skin. When applied to the wound surface of the human body, it changes into a semi-solid gel state, achieving the sustained-release and long-acting therapeutic effect of the birch sap.

[0006] The birch sap thermosensitive in-situ gel can not only be used to repair burned and scalded skin, but can also be used as a drug delivery carrier in combination with other drugs to treat burned and scalded skin.

[0007] When used as a drug delivery vehicle in combination with other drugs, the birch sap in situ gel has zero-order drug release characteristics.

[0008] The birch sap thermosensitive in-situ gel contains 11.5-35% of temperature-sensitive gel material, 54-88% of birch sap, 0.4-10% of moisturizer and 0.1-1% of preservative, based on 100% of the weight percentage of the gel matrix.

[0009] The temperature-sensitive gel material may be one or a combination of poloxamer 407, poloxamer 188, N-isopropylacrylamide copolymer, and chitosan.

[0010] The moisturizer is one or a combination of glycerin, propylene glycol and sorbitol.

[0011] The preservative is one or a combination of benzoic acid, sodium benzoate, ethylparaben, benzalkonium bromide, methylparaben, ethylparaben, propylparaben, butylparaben, sorbic acid, and potassium sorbate.

[0012] Meanwhile, the preferred formulation can be prepared as follows: based on the weight percentage of the gel matrix being 100%, 72% birch juice, 21.6% poloxamer P407, 3.7% poloxamer P188, 2.5% sorbitol, and 0.2% sodium benzoate.

[0013] The birch sap thermosensitive in-situ gel involved in the present invention can be realized by the following technical approaches.

[0014] (1) Take a prescribed amount of moisturizer and preservative and add them to birch sap, and ultrasonicate them for 30 minutes to completely dissolve them to obtain a birch sap mixture.

[0015] (2) Under normal temperature and magnetic stirring conditions, the prescribed amount of gelling agent is slowly added to the birch sap mixture, and after being stirred evenly, the mixture is refrigerated at 4-10°C for 24 hours to fully swell, thereby obtaining a birch sap thermosensitive gel solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the in vitro release curve of the silver sulfadiazine-birch juice in situ gel in Example 3 of the present invention. DETAILED DESCRIPTION

[0017] The present invention is specifically described by the following examples, which are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention.

[0018] Example 1 Preparation and property investigation of birch sap thermosensitive in-situ gel

[0019] The thermosensitive gel was prepared by the "cold dissolution method".

[0020] Take 2.5g of sorbitol and 0.2g of sodium benzoate and add them to 72g of birch juice. Ultrasonicate for 30min. After complete dissolution, slowly add 21.6g of poloxamer P407 and 3.7g of poloxamer P188 in an ice-water bath, stir evenly, and then refrigerate in a refrigerator at 4-10℃ for 24h to allow it to fully swell, thereby obtaining birch juice thermosensitive in situ gel.

[0021] Determination of in situ gel phase transition temperature (Tg).

[0022] The phase transition temperature of the thermosensitive gel was determined by the "stirring bar method". 20 mL of the prepared birch sap thermosensitive gel was added to a beaker, and a probe thermometer was inserted to measure the temperature of the gel in real time. The gel was placed on a magnetic stirrer for magnetic stirring. The speed was set to 250 r / min and the temperature of the gel was continuously increased at a rate of 1 to 2 °C / min. The time when the gel completely stopped rotating was observed and recorded. The temperature at this time was the phase transition temperature of the in situ gel. Twelve parallel measurement experiments were carried out and the results were averaged.

[0023] The experimental results show that the gelation temperature (Tg) of the birch sap thermosensitive in-situ gel is 32.8°C.

[0024] In situ gel stability study.

[0025] 5 ml of birch sap in situ gel solution was taken and placed in a centrifuge tube. It was centrifuged at 5000 r / min for 45 min and stored at 60°C and -10°C for 48 h. No stratification was observed.

[0026] Determination of gelation time of birch sap thermosensitive in situ gel.

[0027] Take 5 mL of birch sap in-situ gel solution and place it in a 45° inclined test tube. In a constant temperature water bath at 33°C, observe the changes in the sample state and record the time when the gel solution stops flowing. Repeat the measurement 12 times and take the average value of the results.

[0028] The experimental results show that the time required for the birch sap thermosensitive in situ gel to transform from liquid to semi-solid gel is 105±10s, that is, after the birch sap thermosensitive in situ gel is applied to the human epidermal wound, it can quickly complete the transformation from liquid to semi-solid gel within 95s to 115s.

[0029] Example 2 Experiment on the effect of birch sap thermosensitive in situ gel on rats with deep second-degree burns The birch sap thermosensitive in situ gel was prepared by referring to the method described in Example 1.

[0030] 60 male SD rats were randomly divided into 4 groups, 15 in each group, namely normal group, model group, birch sap thermosensitive in situ gel group and positive control erythromycin ointment group. The normal group was only anesthetized, depilated and disinfected; except for the normal group, the rats in the other groups were anesthetized by intraperitoneal injection of 10% chloral hydrate, and then fixed on a wooden board in prone position, and the back hair was removed for disinfection. The back of the rat was immersed in boiling water for 20 seconds, and the scald area was about 10% of the body surface area, causing deep II degree scald wounds.

[0031] The normal group and the model group only applied physiological saline to the hair removal area once a day for 15 consecutive days. The birch sap gel group and the control drug erythromycin ointment group applied the drug to the wound surface with a thickness of about 2 mm, once a day for 15 consecutive days.

[0032] Six rats were randomly selected from each group at 5, 10, and 15 days after scalding. The wounds were photographed and the rats were anesthetized. Blood was collected from the abdominal aorta and centrifuged at 6000 r / min for 30 min. The serum was refrigerated at -15℃ for later use.

[0033] Wound healing time and healing rate were measured.

[0034] The wound photos of rats in each group were taken on days 5, 10, and 15 after scalding, and the wound area was calculated using Image-Pro Plus software.

[0035] Wound healing rate = (original wound area - unhealed wound area) / original wound area × 100%.

[0036] Determination of serum IL-6 and TNF-α levels in rat serum.

[0037] The sera of rats in each group obtained on the 5th, 10th and 15th days after scalding were used to detect the levels of IL-6 and TNF-α by ELISA.

[0038] The experimental results showed that compared with the model group, the wound healing time of rats in the birch sap in situ gel group and the erythromycin ointment group was significantly shortened. The wound healing rates of rats in the birch sap in situ gel group at 5, 10 and 15 days were significantly higher than those in the model group and the positive control erythromycin ointment group.

[0039] After establishing a rat deep II degree burn model, the levels of inflammatory factors IL-6 and TNF-α in the serum of the model group were significantly higher than those in the normal group. After detecting the inflammatory factors in the serum of rats at 5d, 10d, and 15d, the levels of IL-6 and TNF-α in the serum of rats in the model group, positive control group, and birch sap in situ gel group decreased. Among them, the levels of IL-6 and TNF-α in the birch sap in situ gel group and the control drug erythromycin ointment group were significantly lower than those in the model group. The experimental results show that birch sap thermosensitive in situ gel can effectively reduce the level of pro-inflammatory factors in serum in the case of skin burns and scalds, reduce the incidence of wound inflammation and infection, and effectively promote wound healing.

[0040] Table 1 Wound healing time and healing rate of rats in each group

[0041]

[0042] Table 2 IL-6 content in serum of rats in each group

[0043] Group 5d(pg / ml) 10d(pg / ml) 15d(pg / ml) Normal group 119.62±28.75 117.45±16.64 122.72±13.73 Model Group 347.24±25.33 268.51±39.87 210.02±20.19 Birch sap gel in situ 276.35±30.20 153.42±18.69 129.43±11.96 Erythromycin ointment group 291.83±29.92 174.82±34.24 140.82±19.10

[0044] Table 3 TNF-α content in serum of rats in each group

[0045] Group 5d(pg / ml) 10d 15d Normal group 67.96±8.73 72.57±2.05 68.25±9.13 Model Group 242.27±19.12 167.73±18.18 119.03±14.85 Birch sap gel in situ 178.03±11.74 111.721±14.57 73.82±8.77 Erythromycin ointment group 197.92±16.01 132.84±13.92 90.44±14.19

[0046] Example 3 Birch sap thermosensitive in situ gel as a drug delivery vehicle combined with silver sulfadiazine Silver sulfadiazine, a commonly used drug for skin burns, was selected as a model drug.

[0047] The birch sap thermosensitive in situ gel was prepared by referring to the method described in Example 1. After the preparation, 1 g of silver sulfadiazine was added to 100 ml of birch sap in situ gel at room temperature and magnetically stirred for 30 minutes to obtain the birch sap in situ gel containing silver sulfadiazine.

[0048] (1) Determination of in vitro drug release from silver sulfadiazine-birch sap in situ gel.

[0049] 0.5g of sulfadiazine silver-birch juice in-situ gel was added to a beaker and placed in a 37°C constant temperature water bath oscillator for 15 minutes to solidify it. After the in-situ gel completed phase transition, it was placed in a 37°C dissolution cup and 1mL of phosphate buffer (PBS) was added as a release medium (pH=6.8). The rotation speed was 120r / min. 1mL of the release medium was taken at 1h, 2h, 6h, 8h, and 12h, and the volume was fixed to 10mL after filtering with a 0.22μm microporous membrane for HPLC analysis to determine the concentration of sulfadiazine silver. The cumulative release rate of sulfadiazine silver was plotted against time to obtain a drug release curve.

[0050] The experimental results are shown in Figure 1 The results showed that silver sulfadiazine with birch sap thermosensitive in situ gel as the carrier could be released for a long time within 12 hours; the in vitro release behavior of silver sulfadiazine with birch sap in situ gel as the drug delivery carrier was fitted with zero-order kinetics.

[0051] (2) Experiment on the effect of silver sulfadiazine-birch sap in situ gel on rats with deep second-degree burns.

[0052] Silver sulfadiazine-birch juice in situ gel was prepared according to the above method of this example.

[0053] Preparation of blank in situ gel.

[0054] Take 2.5 g of sorbitol and 0.2 g of sodium benzoate and add them to 72 g of purified water. Ultrasonicate for 30 min. After complete dissolution, slowly add 21.6 g of poloxamer P407 and 3.7 g of poloxamer P188 in an ice-water bath, stir evenly, and then place in a refrigerator at 4-10°C for 24 h to allow it to fully swell to obtain a blank in-situ gel.

[0055] Preparation of silver sulfadiazine in situ gel.

[0056] At room temperature, add 1 g of silver sulfadiazine to 100 ml of blank in-situ gel and stir magnetically for 30 minutes to obtain the silver sulfadiazine in-situ gel.

[0057] Sixty male SD rats were selected and randomly divided into 5 groups, with 12 rats in each group, namely, a normal group, a model group, a positive control silver sulfadiazine cream group, a silver sulfadiazine in situ gel group, and a silver sulfadiazine-birch sap in situ gel group. The deep II degree burn model of rats was established by referring to the method described in Example 2.

[0058] After the model was established, the normal group and the model group were smeared with blank in situ gel on the hair removal area, and each drug-treated group was smeared with the drug on the wound surface with a thickness of about 2 mm. After 12 hours, 6 rats were randomly selected from the abdominal aorta to collect blood, and the supernatant was obtained by centrifugation at 6000r / min for 30min. The levels of anti-inflammatory factors IL-4 and IL-10 in the serum were detected by ELISA.

[0059] The experimental results showed that compared with the normal group, the levels of anti-inflammatory factors IL-4 and IL-10 in the serum of rats in each group after burns increased to varying degrees, and the increase level of each drug-treated group was significantly higher than that of the model group. The levels of anti-inflammatory factors in the serum of rats in the sulfadiazine silver-birch sap in situ gel group were significantly increased compared with the commercially available sulfadiazine silver ointment group and the sulfadiazine silver in situ gel group. The experimental results show that the use of birch sap in situ gel as a drug delivery carrier in combination with sulfadiazine silver can effectively increase the level of anti-inflammatory factors in the serum of burns, accelerate wound repair, and reduce the risk of infection.

[0060] Table 4 IL-4 and IL-10 levels in serum of rats in each group

[0061]

[0062] Example 4 Experiment on the effect of birch sap in situ gel on the proliferation of rat fibroblasts Referring to the method described in Example 1, birch sap thermosensitive in situ gel was prepared.

[0063] 36 male SD rats were selected and randomly divided into 3 groups, 12 in each group, respectively, a negative model group, a birch sap thermosensitive in situ gel group and an aloe gel positive control group, and a rat deep II degree burn model was established by referring to the method described in Example 2. The model group only used physiological saline to rub the hair removal area, once a day, for 21 consecutive days. The birch sap gel group and the control drug aloe gel group applied the drug to the wound surface, with a thickness of about 2mm, and administered once a day for 15 consecutive days. Six rats were randomly selected from each group at 7d, 14d, and 21d after the burn, and wound tissues of 1cm×1cm in size were cut from the wound surface for conventional histological staining with hematoxylin-eosin (HE). Five fields of view were randomly selected for each section, and fibroblasts were counted and averaged.

[0064] The experimental results showed that compared with the model group, the fibroblast content in the wound of the rats in the drug group was significantly increased at 7d, 14d and 21d after scalding, and the fibroblast content in the wound of the rats in the birch sap in situ gel group increased most significantly. The experimental results showed that birch sap in situ gel can effectively promote the proliferation of fibroblasts in the wound tissue, thereby effectively accelerating the healing time of the wound.

[0065] Table 5 Comparison of fibroblast content at different times

[0066]

Claims

1. A birch sap thermosensitive in-situ gel, characterized in that: The birch sap temperature-sensitive in-situ gel is composed of birch sap and an in-situ gelling agent; based on the weight percentage of the gel matrix being 100%, it contains 11.5% to 35% of the temperature-sensitive gelling material, 54% to 88% of the birch sap, 0.4% to 10% of the moisturizer, and 0.1% to 1% of the preservative; The birch sap thermosensitive in-situ gel is prepared by a method comprising the following steps: (1) Add the prescribed amount of moisturizer and preservative to birch sap, and perform ultrasonic treatment for 30 minutes to completely dissolve them to obtain a birch sap mixture; (2) Slowly add the prescribed amount of temperature-sensitive gel material to the birch sap mixture under magnetic stirring at room temperature, stir evenly, and refrigerate at 4-10°C for 24 hours to fully swell, thereby obtaining a birch sap temperature-sensitive in-situ gel; The birch sap thermosensitive in-situ gel can be used as a drug delivery carrier and combined with other drugs to treat and repair burned and scalded skin.

2. The birch sap thermosensitive in-situ gel according to claim 1, characterized in that: The temperature-sensitive gel material is one or a combination of poloxamer 407, poloxamer 188, and N-isopropylacrylamide copolymer.

3. The birch sap thermosensitive in-situ gel according to claim 1, characterized in that: The moisturizing agent is one or a combination of glycerol, propylene glycol and sorbitol.

4. The birch sap thermosensitive in-situ gel according to claim 1, characterized in that: The preservative is one or a combination of benzoic acid, sodium benzoate, ethylparaben, benzalkonium bromide, methylparaben, nano silver ion, triclosan, propylparaben, butylparaben, sorbic acid and potassium sorbate.

5. The birch sap thermosensitive in-situ gel according to claim 1, characterized in that: The preferred formulation can be prepared as follows: based on the weight percentage of the gel matrix being 100%, 72% birch juice, 21.6% poloxamer P407, 3.7% poloxamer P188, 2.5% sorbitol, and 0.2% sodium benzoate.

6. The birch sap thermosensitive in-situ gel according to claim 1, characterized in that: The gelling temperature of the thermosensitive gel is 32°C to 34°C.

7. Use of the birch sap thermosensitive in-situ gel according to claim 1 in the preparation of a medicament for repairing skin damaged by burns or scalds.

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