Compound bear gall pain-relieving gel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly discloses a compound bear gall pain-relieving gel as well as a preparation method and application thereof. The compound bear gall pain-relieving gel comprises the following components in percentage by mass: 15-25% of a traditional Chinese medicine composition, 2.5-3.5% of a matrix, 7-13% of a humectant, 1-10% of a penetration enhancer and a proper amount of a pH regulator. The invention discloses a compound bear gall pain-relieving gel as well as a preparation method and application thereof. The compound bear gall pain-relieving gel has a remarkable treatment effect on inflammatory pain and can quickly relieve pain and relieve swelling, the gel dosage form is convenient to smear and can directly reach a focus, and a safe, efficient and convenient new choice is provided for treatment of the inflammatory pain.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a compound bear bile analgesic gel, a preparation method and an application thereof. Background Art

[0002] Inflammatory pain (IP) is a common clinical condition that severely impacts patients' quality of life. Currently, nonsteroidal anti-inflammatory drugs (NSAIDs) are the primary treatment for inflammatory pain. However, long-term use can cause side effects such as gastrointestinal irritation and liver and kidney damage. Traditional Chinese medicine (TCM) topical preparations have become a research hotspot in recent years due to their ability to target lesions and reduce systemic toxicity.

[0003] Bear bile, a traditional and precious Chinese medicine, is composed mainly of bile acids (such as tauroursodeoxycholic acid, TUDCA), amino acids, and bile pigments, which have significant anti-inflammatory, analgesic, sedative, and antispasmodic effects. However, most bear bile preparations currently available on the market are oral or injectable, with a lack of topical preparations for swelling and pain relief. Furthermore, the efficacy of a single ingredient is limited, necessitating the development of a compound topical preparation based on bear bile to achieve synergistic effects.

[0004] However, research on traditional Chinese medicine compound gels mostly focuses on a single efficacy, lacking a multi-target synergistic treatment plan for inflammatory pain. More importantly, traditional topical preparations face major technical bottlenecks in terms of stability, drug loading, and transdermal absorption efficiency: high concentrations of traditional Chinese medicine extracts can easily lead to instability of the gel system, conventional penetration enhancement schemes are difficult to balance penetration efficiency and safety, and preparations are prone to degradation under high temperature and high humidity conditions. In the existing technology, although borneol has been proven to promote drug absorption by interfering with the lipid arrangement of the stratum corneum, and the Chuanxiong-Angelica drug pair (1:1) has also been shown to have a synergistic anti-inflammatory effect, how to scientifically combine these ingredients with bear bile and develop a gel preparation with high drug loading, excellent transdermal permeability and stability remains a key technical problem that needs to be solved in this field. Summary of the Invention

[0005] The present invention aims to provide a compound bear bile analgesic gel, a preparation method and an application thereof. The compound bear bile analgesic gel has a significant therapeutic effect on inflammatory pain, can quickly relieve pain and reduce swelling, and the gel dosage form is easy to apply and can directly reach the lesion, providing a safe, efficient and convenient new option for the treatment of inflammatory pain.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A compound bear bile analgesic gel comprises the following components by mass percentage: 15%-25% of a traditional Chinese medicine composition, 2.5%-3.5% of a matrix, 7%-13% of a moisturizing agent, 1%-10% of a permeation enhancer, and an appropriate amount of a pH regulator.

[0008] Preferably, the traditional Chinese medicine composition comprises bear bile powder, borneol and Chuanxiong-Angelica extract.

[0009] Preferably, the mass ratio of the bear bile powder, borneol and Chuanxiong-Angelica extract is 4:1:8.

[0010] Preferably, the matrix is carbomer; the moisturizing agent is glycerin; the penetration enhancer is propylene glycol; and the pH regulator is triethanolamine.

[0011] The present invention also provides a method for preparing the compound bear bile analgesic gel, comprising the following steps:

[0012] S1. Mix bear bile powder, borneol, and Chuanxiong-Angelica extract in a mass ratio of 4:1:8 to obtain a traditional Chinese medicine composition;

[0013] S2. Mix the matrix and moisturizer, add distilled water and swell for 12 hours to obtain a matrix solution;

[0014] S3, adding a penetration enhancer to the traditional Chinese medicine composition obtained in S1 and stirring evenly to obtain a medicinal solution;

[0015] S4. Mix the matrix solution obtained in S2 with the medicinal solution obtained in S3, add a pH regulator to adjust the pH value to 6.0-7.0, then add enough water, stir evenly, and obtain the compound bear bile analgesic gel.

[0016] Preferably, in S1, the method for preparing the Chuanxiong-Angelica extract comprises the following steps:

[0017] S11, mix the slices of Chuanxiong and Angelica in a mass ratio of 1:1, add 12 times the amount of water and soak for 2 hours;

[0018] S12, decoct and extract twice, each time for 120 min, combine the extracts and filter;

[0019] S13. The filtrate is centrifuged, concentrated, and freeze-dried to obtain the Chuanxiong-Angelica extract.

[0020] Preferably, in S13, the centrifugal speed is 5000 rpm, the centrifugal time is 30 min, the freezing temperature is -60 to -40°C, and the freezing time is 18 to 24 h.

[0021] The present invention also provides an application of the compound bear bile analgesic gel in preparing a medicine for treating inflammatory pain.

[0022] Preferably, the inflammatory pain includes but is not limited to pain or swelling induced by glacial acetic acid, xylene or carrageenan.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] The present invention discloses a compound bear bile analgesic gel, a preparation method, and an application thereof. The process is stable and the product quality is stable and reliable. Relying on the synergistic effect of core ingredients such as bear bile powder, borneol, and Chuanxiong-Angelica extract, the gel prepared through scientific proportioning and advanced preparation technology has excellent transdermal absorption performance. A large amount of animal experimental data shows that the gel can significantly reduce the pain response of animal models of inflammatory pain, effectively alleviate the degree of pain induced by glacial acetic acid, xylene, etc., and at the same time exhibit obvious anti-inflammatory activity and inhibit tissue swelling. The compound bear bile analgesic gel has a significant therapeutic effect on inflammatory pain and provides a reliable drug option for clinical pain treatment.

[0025] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A flow chart showing the preparation process of the compound bear bile analgesic gel provided in Example 1;

[0027] Figure 2 Fourier transform infrared spectroscopy (FT-IR) analysis results of the gelling agents provided in Example 1 and Comparative Example 1;

[0028] Figure 3 The differential scanning calorimetry (DSC) analysis results of the gelling agents provided in Example 1 and Comparative Example 1;

[0029] Figure 4 The wetting angle evaluation results of the gelling agents provided in Example 1 and Comparative Example 1 are as follows: Figure 4 A in the figure is the wetting angle evaluation result of the gel provided in Comparative Example 1. Figure 4 B in the figure is the wetting angle evaluation result of the gel provided in Example 1;

[0030] Figure 5 The effect of the bear bile analgesic gel provided in Example 1 on the writhing reaction of mice induced by glacial acetic acid;

[0031] Figure 6 The results of the IL-1β, IL-6 and PGE2 level detection in tissue homogenate are shown in Figure 2. Figure 6 A in the figure is the result of IL-1β level detection in tissue homogenate. Figure 6 B in the figure is the result of IL-6 level test in tissue homogenate. Figure 6C in the figure is the result of PGE2 level detection in tissue homogenate;

[0032] Figure 7 Electrophoresis of TNF-α and COX-2 protein expression in the inflamed foot of a chronic inflammatory pain mouse model induced by the compound bear bile analgesic gel provided in Example 1 (n=6), wherein A is a blank group; B is a model group; C is a positive drug B group; D is an XBD-L group; E is an XBD-M group; and F is an XBD-H group.

[0033] Figure 8 This is the electrophoresis statistical diagram of the expression of TNF-α and COX-2 proteins in the inflamed foot of the chronic inflammatory pain mouse model induced by the compound bear bile analgesic gel provided in Example 1, wherein: Figure 8 A in the figure is the electrophoresis statistical diagram of COX-2 protein expression, Figure 8 B in the figure is the electrophoresis statistical diagram of TNF-α protein expression. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0035] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0036] Sources of experimental materials: CMC-Na was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; normal saline was purchased from Liaoning Minkang Pharmaceutical Co., Ltd.; heparin sodium injection was purchased from Jiangsu Wanbang Biochemical Pharmaceutical Group Co., Ltd.; PBS was purchased from Israel BioInd Company; interleukin-6 test kit was purchased from ThermoFisher; tumor necrosis factor α kit was purchased from ThermoFisher; interleukin-1β test kit was purchased from ThermoFisher; Hoechst 33258 kit was purchased from Shanghai Biyuntian Pharmaceutical Co., Ltd., China; Annexin V-FITC / PI cell apoptosis kit was purchased from BD Biosciences, USA; analgesic and anti-inflammatory ointment was purchased from Guangdong Hengjian Pharmaceutical Co., Ltd. Diclofenac sodium gel was purchased from Mayinglong Pharmaceutical Group Co., Ltd.; RIPA lysis buffer (strong) was purchased from Servicebio; 50× cocktail protease inhibitor was purchased from Servicebio; PMSF (100 mM) was purchased from Servicebio; phosphorylation protease inhibitor was purchased from Servicebio; BCA protein quantification kit was purchased from Servicebio; 5× SDS-PAGE protein loading buffer (odorless, reduced type) was purchased from Servicebio; paraformaldehyde tissue fixative was purchased from Servicebio; xylene was purchased from Aladdin; 10% chloral hydrate was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0037] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0038] Reference to the embodiments of the present invention Figure 1 The preparation process flow chart of the compound bear bile analgesic gel is shown.

[0039] Example 1

[0040] Example 1 of the present invention provides a compound bear bile analgesic gel, the preparation method of which comprises the following steps:

[0041] S1. Evenly mix 60 g of bear bile powder, 15 g of borneol, and Chuanxiong-Angelica extract to obtain a Chinese medicine composition;

[0042] The preparation method of the Chuanxiong-Angelica extract comprises the following steps:

[0043] S11, mix 300g of Chuanxiong and 300g of Angelica slices, add 12 times the amount of water and soak for 2h;

[0044] S12, decoct and extract twice, each time for 120 min, combine the extracts and filter;

[0045] S13. The filtrate was centrifuged at 5000 rpm for 30 min, concentrated, and freeze-dried at -60°C for 18 h to obtain the Chuanxiong-Angelica extract.

[0046] S2. Mix 27 g of carbomer with 105 g of glycerol, add 500 mL of distilled water, and swell for 12 h to obtain a matrix solution;

[0047] S3, adding 40 mL of propylene glycol to the traditional Chinese medicine composition obtained in S1 and stirring evenly to obtain a medicinal solution;

[0048] S4. Mix the matrix solution obtained in S2 with the medicinal solution obtained in S3, add an appropriate amount of triethanolamine, adjust the pH value to 6.5, add water to a sufficient amount, stir evenly, and obtain 1000 g of compound bear bile analgesic gel.

[0049] Comparative Example 1

[0050] S1. Mix 27 g of carbomer with 105 g of glycerol, add 600 mL of distilled water, and swell for 12 h to obtain a matrix solution.

[0051] S2. Mix the matrix solution obtained in S2 with 40 mL of propylene glycol, add an appropriate amount of triethanolamine, adjust the pH to 7.0, add water to a sufficient amount, and stir evenly to obtain 1000 g of blank gel.

[0052] The effect of the compound bear bile analgesic gel of Example 1 was verified by the following test.

[0053] 1. The appearance of the compound bear bile analgesic gel provided in Example 1 was tested, and the results were as follows: the compound bear bile analgesic gel was brown-yellow in appearance, in a semi-solid state, and was uniform overall without bubbles or lumps. It had good spreadability and the skin surface felt comfortable and smooth after application.

[0054] 2. Determine pH, viscosity, particle size and stability. The test plan is as follows:

[0055] The pH values of three batches of gel were measured using a pHS-3C pH meter. Three samples of gel were added to purified water, sonicated, filtered, and then measured using a pH meter. The results are shown in Table 1.

[0056] Table 1 pH measurement results of compound bear bile analgesic gel

[0057] batch number pH 1 6.45 2 6.21 3 6.57

[0058] As shown in Table 1, the pH values of the three batches of gels ranged from 6.21 to 6.57, which were weakly acidic to neutral.

[0059] According to the Chinese Pharmacopoeia (2020 Edition), the gel was coated on a glass slide and measured. According to the first method of General Rule 0982, the particle size did not exceed 180 μm. The results are shown in Table 2.

[0060] Table 2 Particle size test results of compound bear bile analgesic gel

[0061] batch number Particle size (μm) 1 <180 2 <180 3 <180

[0062] It can be seen from Table 2 that the particle size of the three batches of gels is less than 180 μm, which is in line with the requirements of the 2020 edition of the "Chinese Pharmacopoeia".

[0063] The viscosity of three batches of gel was measured using an NDJ-5S viscometer. Three samples were taken using a No. 4 rotor at 6 rpm. Each sample was measured three times, and the average value was taken. The test results are shown in Table 3.

[0064] Table 3 Viscosity test results of compound bear bile analgesic gel

[0065] batch number Viscosity (Pa.s) RSD (%) 1 65.384 0.15 2 66.275 0.60 3 65.808 1.14

[0066] As shown in Table 3, the viscosity of the three batches of gels is in the range of 65.384 to 66.275 Pa.s, and they have a good application feel.

[0067] To determine the moisturizing rate of the gel, accurately weigh an appropriate amount of the compound bear bile analgesic gel (m0), evenly spread it on a glass slide, and place it in a 25°C oven. After every 24 hours, measure the mass of the gel (m t ), the moisturizing rate H is calculated as follows:

[0068]

[0069] m0 is the initial mass of the sample; m t The quality of the gel was measured for 24 hours. The results are shown in Table 4 below.

[0070] Table 4 Test results of the moisturizing rate of compound bear bile analgesic gel

[0071] batch number Moisturizing rate (%) 1 89.23 2 90.07 3 89.47

[0072] As shown in Table 4, the moisturizing rates of the three batches of gels were all greater than 89%, indicating that the compound bear bile analgesic gel had good moisturizing properties.

[0073] According to the provisions of the "Chinese Pharmacopoeia (2020 Edition)", the test was performed according to the filling inspection method (General Chapter 0942 Method 1). Two 30g and three 60g test samples were taken, respectively, and the weight was determined. The weight of the empty container was also determined to calculate the filling amount and average filling amount of each container. The pharmacopoeia requirements for the minimum filling amount of gels are shown in Table 5 below, and the test results are shown in Table 6 below.

[0074] Table 5 Pharmacopoeia requirements for minimum filling volume of gels

[0075] Labeled packing quantity Average loading volume Quantity per container <20g Not less than the labeled capacity Not less than 93% of the labeled capacity 20g-50g Not less than the labeled capacity Not less than 95% of the labeled capacity >50g Not less than the labeled capacity Not less than 97% of the labeled capacity

[0076] Table 6 Differences in minimum filling volume of compound bear bile analgesic gel

[0077]

[0078]

[0079] It can be seen from Table 6 that the minimum filling weight difference limit of the compound bear bile analgesic gel meets the requirements of the pharmacopoeia.

[0080] To determine the swelling property of the gel, the freeze-dried hydrogel (W0) was immersed in PBS at room temperature. The gel was taken out and weighed at different times (W s ), and the experiment was repeated three times. The swelling ratio (SR) was calculated as follows;

[0081]

[0082] W0 is the initial weight of the freeze-dried hydrogel; W S The results are shown in Table 7.

[0083] Table 7 Swelling rate of compound bear bile analgesic gel in different time periods

[0084] Time (h) Swelling rate (%) 1 1.84±0.19 2 6.61±1.4 4 19.76±0.51 8 29.76±0.51 12 31.87±0.25 24 35.17±0.16

[0085] As shown in Table 7, after immersion for 24 h, the hydrogel basically reached swelling equilibrium, and its swelling ratio was 35.17±0.16%.

[0086] 3. Use Fourier transform infrared spectroscopy (FT-IR) to analyze the binding between the gel matrix and the drug; use differential scanning calorimetry (DSC) to examine the changes in the crystallization of the preparation; and measure the wetting angle to evaluate its hydrophilicity. The experimental plan is as follows:

[0087] Fourier transform infrared spectrometer at 400 and 4000 cm -1 The spectral characteristics of the drug-loaded gel and the blank gel were scanned within the spectral range to reflect the compatibility between the drug and the gel matrix. Figure 2 .

[0088] Depend on Figure 2 It can be seen that the blank gel provided in Comparative Example 1 has a -1 There is a stretching vibration of hydroxyl OH at 2943 cm -1 The stretching vibration of the alkyl CH is at 1702 cm -1 The absorption peak at 1455 cm is the stretching vibration of carboxylic acid C=O; -1 The absorption peak at 1251cm is the angular vibration of methylene; -1 The absorption peak at 799cm is the stretching vibration of carboxylic acid CO; -1 The absorption peak at is the in-plane rocking vibration of methylene. After drug loading, 3446ccm -1 The absorption peak of OH shifts to 3379 cm -1 A red shift occurred, indicating that after drug loading, hydrogen bond coupling occurred between substances, causing the absorption peak to move to a low frequency.

[0089] The melting points of the blank gel provided in Comparative Example 1 and the drug-loaded gel provided in Example 1 were measured using differential scanning calorimetry. An appropriate amount of hydrogel sample was placed in a crucible and the sample was placed in a differential scanning calorimeter for measurement. The temperature program was set to a test temperature range of 0-350°C and a heating rate of 10°C / min. The thermal stability of the blank and drug-loaded gel was evaluated. The results are shown in Figure 2. Figure 3 .

[0090] Depend on Figure 3It can be seen that the blank sample provided in Comparative Example 1 has two sharp endothermic peaks during the heating process. The first endothermic peak at 144.91°C is believed to be due to the destruction of the hydrogen bonds in the matrix molecules, resulting in the neutralization of carboxyl groups in the carbomer. Due to the mutual repulsion of negative charges, the molecular chains diffuse and stretch, and form a viscous expansion state. The viscous gel undergoes an amorphous transformation, resulting in melting behavior. As the DSC heating time continues, the second endothermic peak at 250.43°C is attributed to the decomposition of carbomer. The drug-loaded sample has two small endothermic peaks that superimpose into a broad endothermic peak. The first endothermic peak at 136.24°C is due to the volatilization of traditional Chinese medicine ingredients, and the second endothermic peak at 148.46°C is believed to be due to the amorphous transformation of the gel.

[0091] The blank gel provided in Comparative Example 1 and the drug-loaded gel provided in Example 1 were placed on the observation platform of the ThetaQC optical contact angle meter, with a droplet volume of 4 μL. The droplets were measured after stabilization for 10 seconds, and the angles on both sides of the droplets were recorded to investigate the effect of the addition of drugs on the gel. When the contact angle is less than 90°, this indicates that the gel surface exhibits hydrophilic properties, and the smaller the contact angle, the better the wetting properties of the gel. Conversely, if the contact angle is greater than 90°, the gel surface is hydrophobic, and as the contact angle increases, the wetting properties of the material gradually weaken. When the contact angle reaches 180°, it can be determined to be completely hydrophobic. The results are shown in Tables 8 and Figure 4 .

[0092] Table 8 Gel wetting angle results

[0093] <![CDATA[Wetting angle( ° )]]> Blank gel 87.52±0.99 Drug-loaded gel 77.43±5.93

[0094] From Table 8 and Figure 4 It can be seen that the wetting angles of the blank gel and the drug-loaded gel are both less than 90°, indicating that the surface of the compound bear bile analgesic gel is hydrophilic and has good wettability.

[0095] 4. Stability test:

[0096] (1) Centrifugation test: Take three batches of compound bear bile analgesic gel, weigh appropriate amounts of gel into centrifuge tubes, centrifuge at 1000 r / min for 10 min, and observe the state of the gel before and after centrifugation. The results are shown in Table 9.

[0097] Table 9 Centrifugation test results of compound bear bile analgesic gel

[0098] batch Traits 1 No stratification or flocculation occurs 2 No stratification or flocculation occurs 3 No stratification or flocculation occurs

[0099] As shown in Table 9, after centrifugation, the three batches of compound bear bile analgesic gel showed no stratification or flocculation, indicating that the compound bear bile analgesic gel had good centrifugal stability.

[0100] (2) High temperature test: Weigh about 1 g of three batches of compound bear bile analgesic gel respectively, put them into open syringe bottles, and place them in a constant temperature box at 60°C for 24 hours. After cooling to room temperature, observe the state of the gel, observe the appearance of the gel, and test its spreadability and the content of the index components.

[0101] Table 10 High temperature test results of compound bear bile analgesic gel

[0102] batch Traits pH TUDCA content (%) 1 The whole is uniform, without lumps, and has high viscosity 6.32 98.34 2 The whole is uniform, without lumps, and has high viscosity 6.18 97.21 3 The whole is uniform, without lumps, and has high viscosity 6.05 98.47

[0103] As shown in Table 10, the compound bear bile analgesic gel placed in an open vial will lose water to a certain extent under high temperature conditions, causing the viscosity of the gel to increase, but it has almost no effect on the total content of the indicator component TUDCA, and the pH remains unchanged. The results show that the compound bear bile analgesic gel is not resistant to high temperatures.

[0104] (3) High humidity test: Weigh about 1 g of the three batches of compound bear bile analgesic gel respectively, put them into an open vial, and place them in a humidity of 90±5% for 24 hours. Observe the state of the gel and its appearance and test its spreadability and the content of the index components.

[0105] Table 11 High humidity test results of compound bear bile analgesic gel

[0106] batch Traits pH TUDCA content (%) 1 The whole is uniform, without lumps, and the viscosity is reduced 5.98 97.36 2 The whole is uniform, without lumps, and the viscosity is reduced 6.23 96.59 3 The whole is uniform, without lumps, and the viscosity is reduced 6.42 96.24

[0107] As shown in Table 11, the stability of the three batches of gels was tested in a constant humidity closed environment (92.5%) and at room temperature. The viscosity of the compound bear bile analgesic gel decreased under high humidity, and the pH and content did not change significantly, indicating that it is suitable for storage in a dry place.

[0108] (4) Accelerated test: Weigh about 1 g of the three batches of compound bear bile analgesic gel, put them into a vial, and place them at a temperature of 30 ± 2 ° C and a humidity of 60 ± 5% for 6 months. Samples were taken at the beginning of 0, 1, 2, 3, and 6 months to examine the appearance, pH value, and TUDCA content of the compound bear bile analgesic gel.

[0109] Table 12 Results of accelerated test of compound bear bile analgesic gel

[0110]

[0111] As shown in Table 12, after six months of accelerated testing on the compound bear bile analgesic gel, at room temperature, the properties, pH value and content of the gel remained almost unchanged, indicating that it has good stability.

[0112] 5. Pharmacodynamic study on the compound bear bile analgesic gel of Example 1

[0113] The analgesic and anti-inflammatory effects of the compound bear bile analgesic gel were investigated in the glacial acetic acid-induced writhing pain model in mice, the xylene-induced ear swelling model in mice, and the carrageenan-induced paw swelling model in rats. The experimental scheme is as follows:

[0114] Animal Source: All animals were purchased from the Experimental Animal Center of Yanbian University, Animal License No. SCXK (Ji) 2017-0003. Forty-two SPF-grade male Sprague-Dawley rats (weighing 250–300 g) and 78 Kunming mice (weighing 20–30 g) were housed in SPF-grade conditions at a temperature of 22 ± 4°C and a humidity of 40%–50%. Adaptive feeding was performed for 7 days prior to the experiment.

[0115] Study on analgesic effect: The experimental scheme is as follows: 36 Kunming mice were taken and randomly divided into 6 groups, with 6 mice in each group, namely model group, positive drug A group (analgesic and anti-inflammatory ointment 0.65g / kg), positive drug B (diclofenac sodium gel, 0.05g / kg), XBD-L (0.30g / kg), XBD-M (0.60g / kg) and XBD-H (1.20g / kg). Before administration, use depilatory cream to depilate the mouse abdomen and apply it to the back skin once a day for 7 consecutive days. Wipe off the abdominal drug 1 hour after the last administration. Each mouse was intraperitoneally injected with 0.6% glacial acetic acid at 0.1ml / 10g, start timing, observe the latency of the writhing reaction, and record the number of writhing reactions within 30 minutes. And calculate the analgesic rate. The analgesic rate calculation formula is as follows:

[0116]

[0117] The results are as follows Figure 5 .

[0118] Depend on Figure 5 As shown, the number of writhing times was significantly reduced in mice treated with the positive drug groups A and B, as well as XBD-H, compared to the model group. Furthermore, the XBD-M and XBD-H groups prolonged the latency to writhing compared to the model group. These results demonstrate that bear bile analgesic gel can prolong the latency period of glacial acetic acid-induced pain in mice, improve pain severity, and demonstrate an analgesic effect.

[0119] For the study of anti-inflammatory effects, the experimental protocol is as follows:

[0120] Xylene-induced ear swelling method: 42 Kunming mice were randomly divided into 7 groups, with 6 mice in each group, namely blank control group, model group, positive drug A group (analgesic and anti-inflammatory ointment 0.65g / kg), positive drug B (diclofenac sodium gel, 0.05g / kg), XBD-L (0.30g / kg), XBD-M (0.60g / kg) and XBD-H (1.20g / kg). 1 hour after the last administration, xylene 0.1mL / mouse was applied to both sides of the right ear of each group of mice to induce inflammation. The left ear of the mouse was not applied as a control and was killed 30 minutes later. The two ears of the mouse were cut along the auricle, and circular ear pieces were punched out at the same position on the left and right ears with a 6mm diameter punch. They were weighed separately, and the difference in mass between the two ear pieces was used as the ear swelling degree to calculate the swelling inhibition rate. The calculation formula is as follows:

[0121] Swelling degree (mg) = right ear piece weight - left ear piece weight;

[0122]

[0123] The results are shown in Table 13 below.

[0124] Table 13 Effect of compound bear bile analgesic gel on xylene-induced ear swelling in mice

[0125]

[0126] As shown in Table 13, one hour after modeling, the right ears of mice in the model group swelled after xylene treatment, with the degree of swelling significantly higher than that in the blank control group. Compared with the model group, all doses of XBD significantly reduced the difference in weight between the left and right ear pieces and the swelling rate in a dose-dependent manner.

[0127] Carrageenan-induced paw swelling test: 42 SD rats were randomly divided into 7 groups, with 6 rats in each group, namely blank group, model group, positive drug A group (analgesic and anti-inflammatory ointment 0.33g / kg), positive drug B group (diclofenac sodium gel, 0.03g / kg), XBD-L (0.15g / kg), XBD-M (0.30g / kg), and XBD-H (0.60g / kg). After depilation, the drug was evenly applied to the back skin once a day for 7 days. One hour after the last administration, 0.1mL of 1% carrageenan solution was subcutaneously injected into the left hind paw of rats in all groups except the blank group, with the right paw serving as a reference. The paw thickness of the rats was measured with a vernier caliper 1h, 2h, 3h, 4h, 5h, and 6h after modeling, and the paw swelling of the rats was calculated using the following formula:

[0128] Plantar swelling degree = left plantar thickness - right plantar thickness;

[0129]

[0130] The results are shown in Table 14 below.

[0131] Table 14 Effects of the compound bear bile analgesic gel on paw swelling in rats at different times after inflammation (Mean±SD, n=6)

[0132]

[0133] As shown in Table 14, the thickness of the left hind paw continued to increase in the right hind model group, indicating that inflammation was not controlled. All XBD groups demonstrated anti-inflammatory effects. The XBD-H group demonstrated significant anti-inflammatory effects from 3 to 6 hours after inflammation. Compared with the model group, the positive drug group B demonstrated a significant anti-inflammatory effect. The XBD-M and XBD-H groups showed no significant difference in anti-inflammatory effects compared with the positive drug group B. Starting from the 4th hour, the XBD-M and XBD-H groups demonstrated significant anti-inflammatory effects.

[0134] The levels of IL-1β, IL-6, and PGE2 in rat swollen paw tissue were determined as follows: 6 hours after the last administration, the rats were sacrificed, the left posterior paw tissue was cut, weighed, minced, homogenized, and centrifuged. The supernatant was prepared into a 10% tissue homogenate, and the levels of IL-1β, IL-6, and PGE2 were determined according to the instructions of the kit. The results are shown in Figure 2. Figure 6 .

[0135] IL-1β, IL-6 and PGE2 are key inflammatory mediators. IL-1β can activate endothelial cells and immune cells and promote inflammatory cell infiltration. IL-6 can participate in acute inflammatory response, immune regulation and tissue repair. PGE2 is synthesized by macrophages, fibroblasts, etc. under the catalysis of COX-2. It can induce pain, fever and the release of inflammatory mediators. The content of IL-1β, IL-6 and PGE2 in tissues was determined by ELISA. Figure 6 The results showed that compared with the blank group, the levels of IL-1β, IL-6, and PGE2 in the inflamed foot tissue of the model group rats were significantly increased; compared with the model group, the levels of IL-1β, IL-6, and PGE2 in the tissue of the XBD-H group were significantly reduced, indicating that the bear bile analgesic gel may exert its anti-inflammatory effect by regulating the levels of IL-1β, IL-6, and PGE2.

[0136] The Western Blot method was used to detect tissue protein levels. The experimental scheme was as follows: the left posterior plantar tissues of rats in each group were collected, proteins were extracted, and the protein content was detected using a BCA kit. Next, gel preparation - sample loading - electrophoresis - transfer, the polyvinylidene difluoride (PVDF) membrane was placed in 5% skim milk powder for blocking. The membrane was placed in the primary antibody (TNF-α and COX-2) culture medium and incubated on a shaker at 4°C overnight. The membrane was washed 3 times with TBST, each time for 5 minutes. Then, horseradish peroxidase-conjugated secondary antibody was added and incubated at room temperature for 60 minutes, and then the image was captured with enhanced chemiluminescence (ECL) reagent, and the grayscale value of the target band was analyzed using Image J software. The results are shown in Figure 2. Figure 7 and Figure 8 .

[0137] Depend on Figure 7-Figure 8 The results showed that compared with the blank group, the protein levels of TNF-α and COX-2 in the inflamed foot tissue of the rats in the model group were significantly increased. Compared with the model group, the XBD dose groups and the positive drug group B were able to reduce the protein expression levels of TNF-α and COX-2 in the inflamed foot tissue of the rats to varying degrees. This suggests that the bear bile analgesic gel may exert its anti-inflammatory effect by acting on TNF-α and COX-2 proteins.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A compound bear bile analgesic gel, characterized in that: The invention comprises the following components by mass percentage: 15%-25% of a traditional Chinese medicine composition, 2.5%-3.5% of a matrix, 7%-13% of a moisturizing agent, 1%-10% of a penetration enhancer, and an appropriate amount of a pH regulator.

2. A compound bear bile analgesic gel according to claim 1, characterized in that: The traditional Chinese medicine composition comprises bear bile powder, borneol and chuanxiong-angelica extract.

3. A compound bear bile analgesic gel according to claim 2, characterized in that: The mass ratio of the bear bile powder, borneol and Chuanxiong-Angelica extract is 4:1:

8.

4. The compound bear bile analgesic gel according to claim 1, characterized in that: The matrix is carbomer; the moisturizing agent is glycerin; the penetration enhancer is propylene glycol; and the pH regulator is triethanolamine.

5. A method for preparing the compound bear bile analgesic gel according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Mix bear bile powder, borneol, and Chuanxiong-Angelica extract in a mass ratio of 4:1:8 to obtain a traditional Chinese medicine composition; S2. Mix the matrix and moisturizer, add distilled water and swell for 12 hours to obtain a matrix solution; S3, adding a penetration enhancer to the traditional Chinese medicine composition obtained in S1 and stirring evenly to obtain a medicinal solution; S4. Mix the matrix solution obtained in S2 with the medicinal solution obtained in S3, add a pH regulator to adjust the pH value to 6.0-7.0, then add enough water, stir evenly, and obtain the compound bear bile analgesic gel.

6. The preparation method according to claim 5, characterized in that: In S1, the method for preparing the Chuanxiong-Angelica extract comprises the following steps: S11, mix the slices of Chuanxiong and Angelica in a mass ratio of 1:1, add 12 times the amount of water and soak for 2 hours; S12, decoct and extract twice, each time for 120 min, combine the extracts and filter; S13. The filtrate is centrifuged, concentrated, and freeze-dried to obtain the Chuanxiong-Angelica extract.

7. The preparation method according to claim 6, characterized in that: In S13, the centrifugal speed is 5000 rpm, the centrifugal time is 30 min, the freezing temperature is -60 to -40°C, and the freezing time is 18 to 24 h.

8. Use of the compound bear bile analgesic gel according to any one of claims 1 to 4 in the preparation of a medicament for treating inflammatory pain.

9. The application according to claim 8, characterized in that: The inflammatory pain includes, but is not limited to, pain or swelling induced by glacial acetic acid, xylene, or carrageenan.

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