Propolis gauze for treating osteoarthritis and preparation method and application thereof
By preparing propolis gauze, using the combination of propolis extract and specific solvents, the existing osteoarthritis treatment methods have limited efficacy and major side effects, and provide a simple, effective and safe topical treatment plan.
Patent Information
- Application Number
- CN202510400092.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-01
- Publication Date
- 2025-07-01
AI Technical Summary
Existing treatment methods for osteoarthritis such as oral nonsteroidal anti-inflammatory drugs and articular injection of hyaluronic acid have limited long-term efficacy and may cause side effects, and lack a treatment method with good effect and convenient administration.
Propolis gauze is used to extract the crude propolis extract, ultrasonic treatment and full temperature oscillation extraction are performed using 70% ethanol solution, followed by rotary evaporation, drying and freeze-drying to obtain propolis lyophilized powder. Then, the propolis lyophilized powder was dissolved in an aqueous solution containing DMSO, ethanol and PEG400, and a 1.25-25 mg/mL propolis extract solution was prepared, mixed thoroughly, centrifuged and left to stand, and finally the solution was poured into a medical gauze to prepare a propolis gauze for patching.
When treating osteoarthritis, propolis gauze is simple to use, easy to carry and store, and has good results. It can significantly reduce joint redness and swelling, improve joint structure, and has fewer side effects.
Smart Images
Figure CN120227399A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of medicine, and particularly relates to a propolis gauze for treating osteoarthritis, its preparation method and application. Background Art
[0002] Arthritis generally refers to inflammatory diseases occurring in human joints and their surrounding tissues, and the clinical manifestations are joint redness, swelling, heat, pain, dysfunction and joint deformity. The commonly seen arthritis in clinic mainly includes the following several kinds: ankylosing spondylitis, osteoarthritis, gouty arthritis, etc. Osteoarthritis is a common joint degenerative skeletal disease, and its main characteristics are the slow progressive destruction of articular cartilage and the reactive hyperplasia of bone spurs formed at the attachment of ligaments at the joint edge and subchondral bone, and thus joint pain, stiffness deformity and dysfunction are caused.
[0003] At present, patients with knee osteoarthritis tend to adopt conservative treatments such as oral non-steroidal anti-inflammatory drugs, glucosamine and intra-articular injection of hyaluronic acid to relieve clinical symptoms. However, the long-term efficacy of the above drugs is limited, and side effects such as gastrointestinal discomfort and allergic reactions may be caused. Therefore, it is necessary to find a treatment method with good effect and convenient administration.
[0004] Propolis is an aromatic and sticky solid processed by worker bees of the Italian honeybee by mixing the secretions of the mandibular gland into the secretions such as resins collected from plant buds and barks. Propolis is a highly concentrated plant medicinal ingredient and has rich pharmacological effects, and it plays an important role in aspects such as anti-inflammation and immune regulation. Its main components chrysin, galangin and caffeic acid phenethyl ester have been reported to have the effect of anti-osteoarthritis. Summary of the Invention
[0005] The purpose of the present invention is to prepare a propolis gauze for treating osteoarthritis and its preparation method.
[0006] To achieve the above purpose, the present invention adopts the following technical scheme:
[0007] Weigh the crude propolis extract, add an appropriate volume of 70% ethanol solution, with the solid-liquid ratio of 1:6 - 1:20, and fully dissolve it. Place it in an ultrasonic cleaner for ultrasonic mixing, and then put it into a constant temperature shaking incubator and extract it on a shaker at 37°C for 1 - 3 times, 24 hours each time. After combining the extraction solutions, filter to remove impurities, transfer the filtrate to a rotary evaporator, and rotary evaporate to remove ethanol. Transfer the remaining liquid to a 1000 mL beaker, place it in a drying oven at 55°C and dry it to an extract state, then put it into a refrigerator at -20°C for freezing treatment, and after freezing, place it in a freeze dryer for freeze drying, with the drying time of 24 hours. Collect the propolis freeze-dried powder, and the yield > 60%.
[0008] The extract mainly contains 3 phenolic acid esters and 16 flavonoids and their derivatives, specifically pinocembrin-5-methyl ether, quercetin-3-methyl ether, pinocembrin, kaempferol, apigenin, isorhamnetin, luteolin methyl ether, 2 quercetin dimethyl ethers, galangin-5-methyl ether, isoprenyl caffeate, chrysin, galangin, pinocembrin-3-acetate, caffeic acid phenethyl ester, methoxychrysin, caffeic acid cinnamate, pinocembrin-3-butyrate, and pinocembrin-3-pentenoate.
[0009] The propolis freeze-dried powder prepared by the preparation method of the propolis extract of the present invention is used to prepare a propolis gauze, which is characterized in that it is prepared by the following method: First, prepare an aqueous solution with a solvent containing 2%-5% DMSO, 10%-40% absolute ethanol, and 10%-30% PEG400. Weigh an appropriate amount of propolis freeze-dried powder and dissolve it in the above solvent to prepare a propolis extract solution with a concentration of 1.25-25 mg / mL. Mix well, sonicate for 30 min, then centrifuge at a speed of 4000 rpm for 10 min, and finally let it stand at room temperature for 10 min. Absorb the supernatant and transfer it to a glassware for use. Cut the medical gauze into a 10 cm × 10 cm square and fold it into a 2.5 cm × 2.5 cm small square. Pour 40 mL of the supernatant into a bottle to soak the gauze. The drug loading amount of each piece of medical gauze is 5-100 mg of propolis freeze-dried powder, and cover it tightly. When in use, take out a piece of gauze, unfold it, and apply it to the affected area of arthritis for 1-4 h.
[0010] Application of the propolis gauze of the present invention in the treatment of osteoarthritis.
[0011] The advantages of the present invention are as follows: At present, patients with knee osteoarthritis tend to adopt conservative treatments such as oral non-steroidal anti-inflammatory drugs, glucosamine, and intra-articular injection of hyaluronic acid to relieve clinical symptoms. However, the long-term efficacy of the above drugs is limited, and they may cause side effects such as gastrointestinal discomfort and allergic reactions. The present invention provides for the first time an external propolis gauze for the treatment of osteoarthritis. The propolis gauze is simple to use for the treatment of osteoarthritis, convenient to carry and store, and has good effects, with good novelty, creativity, and practicality. Description of the Drawings
[0012] Figure 1This is the HPLC-MS chromatogram of the ethanol extract of propolis of the present invention. Among them, (1) pinocembrin-5-methyl ether, (2) quercetin-3-methyl ether, (3) pinocembrin, (4) kaempferol, (5) apigenin, (6) isorhamnetin, (7) luteolin methyl ether, (8) quercetin dimethyl ether, (9) galangin-5-methyl ether, (10) quercetin dimethyl ether, (11) caffeic acid isoprenyl ester, (12) chrysin, (13) galangin, (14) pinocembrin-3-acetate, (15) caffeic acid phenethyl ester, (16) methoxychrysin, (17) caffeic acid cinnamate, (18) pinocembrin-3-butyrate, and (19) pinocembrin-3-pentenoate.
[0013] Figure 2 This is a photo of the positive drug patch and propolis gauze for mouse experiments of the present invention.
[0014] Figure 3 The propolis gauze and positive drug patch of the present invention are applied to the surface of the knee joint of a mouse and fixed with medical tape. In the figure: A: blank control group; B: model group (osteoarthritis); C: positive drug control group (loxoprofen sodium patch); D is the low-dose propolis group; E is the medium-dose propolis group; F is the high-dose propolis group.
[0015] Figure 4 This is a picture of the knee joint of a mouse after the propolis gauze of the present invention is applied externally for 4 weeks. In the figure: A: blank control group; B: model group (osteoarthritis); C: positive drug control group (loxoprofen sodium patch); D is the low-dose propolis group; E is the medium-dose propolis group; F is the high-dose propolis group.
[0016] Figure 5 This is the transverse diameter (A) and anteroposterior diameter (B) (measured with a vernier caliper) of the knee joint of a mouse after the propolis gauze of the present invention is applied externally for 4 weeks. Control, blank control group; OA, osteoarthritis model group; OA+LOX, positive drug control group (loxoprofen sodium patch); OA+LEEP, low-dose propolis group; OA+MEEP, medium-dose propolis group; OA+HEEP, high-dose propolis group. Compared with the blank control group in the model group, # P<0.05; compared with the model group in the medicine-applied group, * P<0.05, ** P<0.01.
[0017] Figure 6 This is a HE staining picture of the knee joint of a mouse after the propolis gauze of the present invention is applied externally for 4 weeks. In the figure: A: blank control group; B: model group (osteoarthritis); C: positive drug control group (loxoprofen sodium patch); D is the low-dose propolis group; E is the medium-dose propolis group; F is the high-dose propolis group.
[0018] Figure 7This is a Masson staining picture of the knee joints of mice after 4 weeks of applying propolis gauze externally according to the present invention. In the figure: A: blank control group; B: model group (osteoarthritis); C: positive drug control group (lornoxicam patch); D: low-dose propolis group; E: medium-dose propolis group; F: high-dose propolis group. Detailed implementation manners
[0019] In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific implementation manners, but the present invention is not limited thereto.
[0020] Example 1
[0021] Weigh the crude propolis extract (purchased from Fujian Shenfeng Science and Technology Development Co., Ltd.), add an appropriate volume of 70wt% ethanol solution, and the mass ratio of the crude propolis extract to the 70wt% ethanol solution is 1:6. Make it fully dissolve, place it in an ultrasonic cleaner for ultrasonic mixing, and then put it in a constant temperature shaking incubator and extract it once at 37°C on a shaker for 24 hours each time. Filter to remove impurities, transfer the filtrate to a rotary evaporator, and rotate to evaporate the ethanol. Transfer the remaining liquid to a 1000 mL beaker, place it in a drying oven at 55°C until it becomes an extract paste, then put it in a -20°C refrigerator for freezing treatment, and then place it in a freeze dryer for freeze drying. The drying time is 24 hours. Collect the propolis freeze-dried powder, and the yield > 60%.
[0022] Then prepare a solvent which is an aqueous solution containing 2wt% DMSO, 10wt% absolute ethanol and 10wt% PEG400. Weigh an appropriate amount of propolis freeze-dried powder and dissolve it in the above solvent to prepare a 1.25 mg / mL propolis freeze-dried powder solution. Mix it well, sonicate for 30 minutes, then centrifuge at a speed of 4000 rpm for 10 minutes, and finally let it stand at room temperature for 10 minutes. Absorb the supernatant and transfer it to a glassware for use. Cut the medical gauze into 10 cm × 10 cm squares and fold them into 2.5 cm × 2.5 cm small squares. Stack 10 pieces of gauze and put them into the same medical plastic bottle. Pour 40 mL of the supernatant into the bottle to cover the gauze. The drug loading amount of each piece of medical gauze is about 5 mg of propolis freeze-dried powder, and then cover it tightly. When in use, take out one piece of medical gauze and unfold it, and apply it to the affected area of arthritis for 1 - 4 hours.
[0023] Example 2
[0024] Weigh the crude propolis extract (purchased from Fujian Shenfeng Science and Technology Development Co., Ltd.), add an appropriate volume of 70wt% ethanol solution. The mass ratio of the crude propolis extract to the 70wt% ethanol solution is 1:10. Make it dissolve completely, place it in an ultrasonic cleaner to mix evenly by ultrasonic wave, then put it into a constant temperature shaking incubator and extract it twice at 37°C on a shaker, 24 hours each time. After combining the extracts, filter to remove impurities by suction filtration, transfer the filtrate to a rotary evaporator to evaporate the ethanol. Transfer the remaining liquid to a 1000 mL beaker, place it in a drying oven at 55°C to dry until it becomes an extract paste, then put it into a refrigerator at -20°C for freezing treatment. After freezing, place it in a freeze dryer for freeze-drying, and the drying time is 24 hours. Collect the freeze-dried propolis powder, and the yield is >60%.
[0025] Then prepare a solvent which is an aqueous solution containing 5wt% DMSO, 30wt% absolute ethanol and 20wt% PEG400. Weigh an appropriate amount of freeze-dried propolis powder and dissolve it in the above solvent to prepare a 10mg / mL freeze-dried propolis powder solution. Mix it evenly, ultrasonicate for 30 minutes, then centrifuge at a speed of 4000 rpm for 10 minutes, and finally let it stand at room temperature for 10 minutes. Absorb the supernatant and transfer it to a glassware for standby. Cut the medical gauze into a 10 cm × 10 cm square and fold it into a 2.5 cm × 2.5 cm small square. Stack 10 pieces of gauze and put them into the same medical plastic bottle. Pour 40 mL of the supernatant into the bottle to cover the gauze. The drug loading amount of each piece of medical gauze is about 40 mg of freeze-dried propolis powder, and then cover it tightly. When in use, take out one piece of medical gauze and unfold it, apply it to the affected area of arthritis for 1 - 4 hours.
[0026] Example 3
[0027] Weigh the crude propolis extract (purchased from Fujian Shenfeng Science and Technology Development Co., Ltd.), add an appropriate volume of 70wt% ethanol solution. The mass ratio of the crude propolis extract to the 70wt% ethanol solution is 1:20. Make it dissolve completely, place it in an ultrasonic cleaner to mix evenly by ultrasonic wave, then put it into a constant temperature shaking incubator and extract it three times at 37°C on a shaker, 24 hours each time. After combining the extracts, filter to remove impurities by suction filtration, transfer the filtrate to a rotary evaporator to evaporate the ethanol. Transfer the remaining liquid to a 1000 mL beaker, place it in a drying oven at 55°C to dry until it becomes an extract paste, then put it into a refrigerator at -20°C for freezing treatment. After freezing, place it in a freeze dryer for freeze-drying, and the drying time is 24 hours. Collect the freeze-dried propolis powder, and the yield is >60%.
[0028] Then, the solvent was configured as an aqueous solution containing 5 wt% DMSO, 40 wt% absolute ethanol, and 30 wt% PEG400. An appropriate amount of freeze-dried propolis powder was weighed and dissolved in the above solvent to prepare a 25 mg / mL freeze-dried propolis powder solution. After thorough mixing, it was ultrasonicated for 30 min, then centrifuged at 4000 rpm for 10 min, and finally left standing at room temperature for 10 min. The supernatant was aspirated and transferred to a glassware for later use. Medical gauze was cut into 10 cm × 10 cm squares and folded into 2.5 cm × 2.5 cm small squares. Ten pieces of gauze were stacked and placed in the same medical plastic bottle. 40 mL of the supernatant was poured into the bottle to cover the gauze. The drug loading of each piece of medical gauze was approximately 100 mg of freeze-dried propolis powder, and the bottle was sealed with a cap. When in use, one piece of medical gauze was taken out and unfolded, and applied to the affected area of arthritis for 1 - 4 h.
[0029] Example 4
[0030] Take the freeze-dried propolis powder prepared in Example 2, redissolve it with a 75 wt% ethanol solution, vortex for 2 min, ultrasonicate for 30 min, and then centrifuge at 13000 rpm for 10 min. Take the supernatant as the sample solution and collect it into a sample vial for HPLC-MS analysis, as shown in Figure 1 . It was measured that the extract mainly contained 3 phenolic acid esters and 16 flavonoids and their derivatives, specifically pinosylvin-5-methyl ether, quercetin-3-methyl ether, pinosylvin, kaempferol, apigenin, isorhamnetin, luteolin methyl ether, 2 quercetin dimethyl ethers, galangin-5-methyl ether, isoprenyl caffeate, chrysin, galangin, pinosylvin-3-acetate, caffeic acid phenethyl ester, methoxychrysin, caffeic acid cinnamate, pinosylvin-3-butyrate, and pinosylvin-3-pentenoate.
[0031] Protective effect of propolis gauze of Example 5 on osteoarthritis in mice
[0032] Medical gauze with a drug loading of approximately 20, 40, and 80 mg of freeze-dried propolis powder was taken out from the medical plastic bottle, cut into small pieces of 0.55 cm * 0.7 cm, and labeled as low-dose propolis gauze, medium-dose propolis gauze, and high-dose propolis gauze, as shown in Figure 2 . After 1 week of adaptive feeding of male clean-grade ICR mice, 36 mice were randomly divided into 6 groups, with 6 mice in each group, namely the blank control group, osteoarthritis model group, positive drug control group (loxoprofen sodium patch), low-dose propolis group, medium-dose propolis group, and high-dose propolis group. For mice in other groups except the blank group, sodium iodoacetate (16 mg·kg -1 ) was injected into the left knee joint cavity to induce an osteoarthritis model. Two weeks after modeling, the propolis gauze was applied to the knee joint of the mice, and then the left leg of the mice was fixed with medical white tape once a day for 1 h each time, and the drug was administered continuously for 4 weeks, as shown in Figure 3 .
[0033] The knee joint bone pictures of mice after 4 weeks of external application of propolis gauze are shown in Figure 4 . In the arthritis model group, the knee joint of the mice was significantly red and swollen. After 4 weeks of external application of propolis gauze, the redness and swelling were significantly reduced, and the effects of medium- and high-dose propolis gauze were close to those of the positive drug. The transverse diameter and anteroposterior diameter of the knee joint of mice after 4 weeks of external application of propolis gauze are shown in Figure 5 . Compared with the blank control group, the transverse diameter and anteroposterior diameter of the knee joint of mice in the osteoarthritis model group were significantly increased (P<0.05); compared with the model group, after 4 weeks of external application of propolis gauze, the transverse diameter and anteroposterior diameter of the knee joint of mice were significantly decreased (P<0.05, P<0.01). After the experiment, the left knee joint tissues of the mice were collected, osteoarthritis sections were prepared, and H&E staining and Masson staining were performed, as shown in Figure 6 and Figure 7 . In the blank control group (A in Figure 6 and A in Figure 7 ), the joint staining of the mice was uniform, the surface was smooth, the number of chondrocytes was normal, and a clear tidemark was visible. In the model group (B in Figure 6 and B in Figure 7 ), a decrease in the number of articular chondrocytes and a blurred tidemark were visible. In the drug administration group (C-F in Figure 6 and C-F in Figure 7 ), the articular cartilage damage of the mice was alleviated. For example, in the medium-dose propolis group (E in Figure 6 and E in Figure 7 ), the joint surface was smooth, the cartilage matrix staining was uniform, and the tidemark was relatively clear. The above results indicate that propolis gauze has a good protective effect on the knee joint of osteoarthritis mice.
Claims
1. A method for preparing a propolis extract, characterized in that: Here are the steps: Weigh the crude propolis extract, add an appropriate volume of 70wt% ethanol solution, the mass ratio of the crude propolis extract to the 70wt% ethanol solution is 1:6-1:20, so that it is fully dissolved, place it in an ultrasonic cleaner for ultrasonic mixing, and then place it in a full-temperature shaking incubator at 37℃ for shaking extraction 1-3 times, each time for 24 hours, combine the extracts extracted on the shaking table and filter to remove impurities, transfer the filtrate to a rotary evaporator, rotary evaporate to remove ethanol, transfer the remaining liquid to a 1000mL beaker, place it in a 55℃ oven to dry until it is a paste, and then place it in a -20℃ refrigerator for freezing. After freezing, place it in a freeze dryer for freeze drying, the drying time is 24 hours, and collect propolis freeze-dried powder with a yield of >60%.
2. The method for preparing the propolis extract according to claim 1, characterized in that: The propolis freeze-dried powder mainly contains 3 phenolic acid ester compounds and 16 flavonoids and their derivatives, specifically pine 5-methyl ether, quercetin-3-methyl ether, pine tin, kaempferol, apigenin, isorhamnetin, luteolin methyl ether, 2 quercetin dimethyl ethers, galangin-5-methyl ether, caffeic acid isoprenyl ester, chrysin, galangin, pine tin-3-acetate, caffeic acid phenethyl ester, methoxychrysin, caffeic acid cinnamyl ester, pine tin-3-butyrate and pine tin-3-pentenoate.
3. Propolis gauze is prepared from the propolis freeze-dried powder obtained by the preparation method of the propolis extract according to claim 1 or 2, characterized in that: The method is prepared by the following method: first, a propolis freeze-dried powder solution is prepared, and the solvent used is an aqueous solution containing 2wt%-5wt% DMSO, 10wt%-40wt% anhydrous ethanol and 10wt%-30wt% PEG400; an appropriate amount of propolis freeze-dried powder is weighed and dissolved in the above solvent to prepare a 1.25-25 mg / mL propolis freeze-dried powder solution; the mixture is fully mixed, ultrasonicated for 30 minutes, and then centrifuged at a speed of 4000 rpm for 10 minutes; finally, the mixture is allowed to stand at room temperature for 10 minutes, and the supernatant is transferred to a glass container for standby use; medical gauze is cut into 10cm×10cm squares and folded into 2.5cm×2.5cm small squares; 10 pieces of medical gauze cut into 10cm×10cm squares are placed in the same medical plastic bottle; 40mL of supernatant is poured into the plastic bottle to soak the medical gauze; the drug loading of each piece of medical gauze is 5-100 mg propolis freeze-dried powder, sealed with a lid; when using, take a piece of medical gauze, unfold it, and apply it to the arthritis affected area for 1 to 4 hours.
4. Use of the propolis gauze as claimed in claim 3 in the treatment of osteoarthritis.