Use of a composition of neo-glycoquons and astaxanthin in the preparation of a transdermal medicament for the treatment of osteoarthritis and a composite gel formulation

Abstract

The application belongs to the technical field of biotechnology, and discloses application of a new sialyloligosaccharide and astaxanthin composition in preparation of a transdermal drug for treating osteoarthritis and a composite gel preparation. The composition provided by the application comprises new sialyloligosaccharide, astaxanthin, carbomer and alginate with a mass ratio of (1-10):(1-10):(0.01-0.5):(0.1-1). The composition can realize efficient enrichment of the new sialyloligosaccharide and astaxanthin in the joint cavity through a diffusion pathway of skin penetration, deep tissue diffusion, joint capsule penetration and joint cavity enrichment of the substance, and the new sialyloligosaccharide and astaxanthin have excellent synergistic effects, can efficiently relieve acute inflammatory symptoms of gouty osteoarthritis, block the disease progression and repair the joint tissue pathological changes, and have excellent application prospects in preparation of the transdermal drug for treating osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to the application of a composition of a novel agar oligosaccharide and astaxanthin in the preparation of transdermal drugs for the treatment of osteoarthritis and a composite gel formulation. Background Technology

[0002] Gouty osteoarthritis (GA) is a metabolic inflammatory disease caused by the abnormal deposition of monosodium urate (MSU) crystals in joints and cartilage tissues. MSU crystals, acting as a damage-associated molecular model (DAMP), activate the NLRP3 inflammasome in synovial macrophages, triggering an IL-1β / TNF-α storm cascade, leading to joint redness, swelling, severe pain, and dysfunction. Simultaneously, MSU crystals induce a burst of mitochondrial-derived reactive oxygen species (ROS), forming a vicious cycle of "oxidative damage-inflammatory amplification": ROS not only directly damage the extracellular matrix of chondrocytes but also promote the release of inflammatory factors by activating the NF-κB pathway, accelerating structural damage to the joints.

[0003] Currently, the mainstream medications for gouty osteoarthritis are divided into acute-phase drugs and uric acid-lowering drugs. Among acute-phase drugs, nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac and etoricoxib can quickly relieve inflammatory pain, have a rapid onset of action, and are widely applicable, but they have side effects such as causing gastrointestinal damage and affecting kidney function; selective COX-2 inhibitors have fewer gastrointestinal side effects but may increase cardiovascular risk; colchicine can effectively inhibit inflammation at low doses, and is especially suitable for early use in the acute phase, but it has a narrow therapeutic window, and high doses can easily cause gastrointestinal reactions, and patients with liver or kidney dysfunction need to carefully adjust the dosage; glucocorticoids such as prednisone have strong anti-inflammatory effects and are suitable for patients who have contraindications to or do not respond to other drugs, but long-term use can easily lead to hyperglycemia and osteoporosis, and short-term use also requires rapid dose reduction to prevent rebound. Among uric acid-lowering drugs, allopurinol can effectively inhibit uric acid production, is inexpensive, and is suitable for those with excessive uric acid production, but may cause severe rashes and needs to be started at a low dose; febuxostat has a stronger uric acid-lowering effect and fewer side effects, is suitable for those allergic to allopurinol, has little impact on kidney function, but there is controversy regarding the potential risk of cardiovascular events; benzbromarone can promote uric acid excretion and is suitable for those with reduced uric acid excretion, but may increase the risk of urinary tract stones; the novel PEG-uricase can break down uric acid and is suitable for refractory gout, but the disadvantages are that it requires intravenous injection, may cause allergic reactions, and is more expensive.

[0004] Therefore, there is an urgent need for a drug with low systemic toxicity and side effects that can effectively relieve the inflammation, pain, and joint destruction symptoms of GA. Summary of the Invention

[0005] The primary objective of this invention is to address the limitations of existing drugs for treating gouty osteoarthritis, including limited therapeutic efficacy, high systemic toxicity, and difficulty in maintaining effective concentrations within the joint cavity. This invention provides a composition with specific components for the preparation of transdermal drugs for treating osteoarthritis. Specifically, this composition comprises neoagar oligosaccharides, astaxanthin, carbomer, and alginate in a mass ratio of (1-10):(1-10):(0.01-0.5):(0.1-1). It exhibits excellent transdermal efficacy, achieving targeted delivery within the joint cavity through skin penetration, deep tissue diffusion, joint capsule penetration, and diffusion of substances enriched in the joint cavity. Furthermore, this composition can inhibit the activation of core inflammatory pathways, repair mitochondrial function and rebuild the antioxidant barrier, promote cartilage matrix synthesis, inhibit inflammatory cell aggregation, and inhibit the damage of degrading enzymes to the synovium. It effectively relieves acute inflammatory symptoms of gouty osteoarthritis, blocks disease progression, and repairs pathological changes in joint tissues, demonstrating excellent application prospects in the preparation of transdermal drugs for treating osteoarthritis.

[0006] A second objective of this invention is to provide a composite gel formulation.

[0007] Specifically, the composition provided by the present invention is used in the preparation of a transdermal drug for the treatment of osteoarthritis. The composition comprises neoagar oligosaccharide, astaxanthin, carbomer and alginate in a mass ratio of (1~10):(1~10):(0.01~0.5):(0.1~1).

[0008] Furthermore, the mass ratio of the new oligosaccharide, astaxanthin, carbomer, and alginate is (3~5):(2~3):(0.05~0.1):(0.1~0.2).

[0009] Furthermore, the degree of polymerization of the new agar oligosaccharide is 2 to 4.

[0010] Furthermore, the purity of the all-trans isomer in the astaxanthin is 90%~100%.

[0011] Furthermore, the alginate is sodium alginate and / or potassium alginate.

[0012] Furthermore, the composition is a gel formulation, and the preparation of the composition includes: mixing astaxanthin, neoagar oligosaccharide and solvent to obtain an active mixture; adding the carbomer and alginate to the active mixture, adjusting the pH value to 5.0~7.0, and then performing electrostatic self-assembly to obtain the gel formulation.

[0013] Further, the preparation of the composition includes at least one of the following technical features: (1) the solvent is one or more of propylene glycol, DMSO and water; (2) the mass ratio of astaxanthin to solvent is (1~10):(78~95); (3) the electrostatic self-assembly temperature is 25℃~45℃, the stirring speed is 200rpm~400rpm, and the time is 10min~60min.

[0014] Furthermore, in the topical administration method, the composition has a substance diffusion pathway that includes skin penetration, deep tissue diffusion, joint capsule penetration, and joint cavity enrichment.

[0015] Further, the composition has one or more of the following characteristics: (1) the composition is used as a catalase activator; (2) the composition is used as a mitochondrial protectant and / or mitochondrial repair agent; (3) the composition is used as a Keap1-Nrf2-ARE signaling pathway expression regulator; (4) the composition is used as a TNF-α expression inhibitor; (5) the composition is used as an anti-inflammatory drug; (6) the composition is used as a synovial protectant and / or synovial repair agent; (7) the composition is used as an inflammatory cell infiltration inhibitor, an anti-inflammatory chemotactic regulator and / or an arthritic cell migration inhibitor; (8) the composition is used as a drug for treating gouty osteoarthritis.

[0016] The composite gel formulation provided by the present invention comprises neoagar oligosaccharide, astaxanthin, carbomer and alginate in a mass ratio of (1~10):(1~10):(0.01~0.5):(0.1~1).

[0017] Beneficial effects:

[0018] The composition provided by this invention comprises neo-Agar oligosaccharides, astaxanthin, carbomer, and sodium alginate in a specific mass ratio. Neo-Agar oligosaccharides serve as a transdermal carrier, while carbomer and sodium alginate form a sustained-release gel system. This enables the delivery of neo-Agar oligosaccharides and astaxanthin from the skin to the joint cavity, allowing astaxanthin to successfully penetrate the skin, deep tissues, and joint capsule into the joint cavity, achieving long-term accumulation within the joint cavity to maintain a high concentration while significantly reducing systemic exposure. Furthermore, neo-Agar oligosaccharides and astaxanthin exhibit excellent synergistic effects in relieving inflammation and improving joint tissue morphology—firstly, by regulating the Keap1-Nrf2-ARE signaling pathway. Firstly, it can inhibit the activation of core inflammatory pathways. Secondly, it can rebuild the antioxidant barrier by enhancing the activity of endogenous antioxidant enzyme CAT and protecting or repairing mitochondrial structure and function. Thirdly, it can repair and rebuild damaged joint tissues by promoting cartilage matrix synthesis, slowing the accumulation of inflammatory cells, and inhibiting the destruction of degrading enzymes. Through the synergistic network formed by neo-agar oligosaccharides and astaxanthin, a multi-dimensional pathological intervention mechanism for GA can be constructed to block the progression of the disease while relieving acute symptoms, thereby effectively alleviating the inflammation, pain, and joint destruction symptoms of GA. It has excellent application prospects in the preparation of transdermal drugs for the treatment of osteoarthritis, especially gouty osteoarthritis.

[0019] In some specific embodiments, when the purity of the all-trans isomer in the astaxanthin is preferably 90%~100%, the astaxanthin and neo-agar oligosaccharide have a better synergistic effect, and have a more ideal effect of relieving acute inflammatory symptoms of gouty osteoarthritis, blocking disease progression, and repairing pathological changes in joint tissues. Attached Figure Description

[0020] Figure 1 This is one of the experimental results of detecting the reactive oxygen species level in RAW264.7 cells after incubation with the composite gel formulation provided in the test examples of this invention for 24 hours (all scales are consistent).

[0021] Figure 2 Figure 2 shows the experimental results of detecting the reactive oxygen species level in RAW264.7 cells after incubation with the composite gel formulation provided in the test example of this invention for 24 hours.

[0022] Figure 3 The figure shows the experimental results of detecting the intracellular MDA content of RAW264.7 cells after incubating the composite gel formulation provided in the test example of the present invention for 24 hours.

[0023] Figure 4The figure shows the experimental results of detecting intracellular CAT enzyme activity in RAW264.7 cells after incubation with the composite gel formulation provided in the test example of this invention for 24 hours.

[0024] Figure 5 This is one of the experimental results of detecting the content of mitochondrial peroxides in RAW264.7 cells after incubation with the composite gel formulation provided in the test examples of the present invention for 24 hours;

[0025] Figure 6 Figure 2 shows the experimental results of detecting the content of mitochondrial peroxides in RAW264.7 cells after incubation with the composite gel formulation provided in the test example of this invention for 24 hours.

[0026] Figure 7 Figure 3 shows the experimental results of detecting the content of mitochondrial peroxides in RAW264.7 cells after incubation with the composite gel preparation provided in the test example of this invention for 24 hours.

[0027] Figure 8 The figure shows the experimental results (ΔΔCT value) of the relative expression level of the Keap1 gene in RAW264.7 cells after incubation with the composite gel preparation provided in the test example of the present invention for 24 hours.

[0028] Figure 9 The figure shows the experimental results (ΔΔCT value) of the relative expression level of Nrf2 gene in RAW264.7 cells after incubation with the composite gel preparation provided in the test example of the present invention for 24 h.

[0029] Figure 10 The figure shows the experimental results (ΔΔCT value) of the relative expression level of NQO1 gene in RAW264.7 cells after incubation with the composite gel preparation provided in the test example of the present invention for 24 h.

[0030] Figure 11 The figure shows the experimental results of detecting intracellular TNF-α levels in RAW264.7 cells after incubation with the composite gel formulation provided in the test example of this invention for 24 hours.

[0031] Figure 12 The images show the HE staining results of mouse ankle joint samples after joint application of the composite gel formulation provided in the test examples of this invention to an MSU mouse model (the scale bars between each image and its enlarged image are consistent).

[0032] Figure 13 This is a diagram showing the experimental results of testing the number of infiltrating cells in the ankle joint sample of an MSU mouse model after applying the compound gel formulation provided in the test example of this invention to the joint. Detailed Implementation

[0033] The present invention relates to the application of the composition in the preparation of a transdermal drug for the treatment of osteoarthritis. Specifically, the composition comprises neoazolam oligosaccharide, astaxanthin, carbomer, and alginate, and the mass ratio of the neoazolam oligosaccharide, astaxanthin, carbomer, and alginate is (1~10):(1~10):(0.01~0.5):(0.1~1), such as 1:1:0.01:0.1, 2:3:0.01:0.2, 4:3:0.07:0.175, 5:4:0.1:0.3, 8:5:0.3:1, 10:10:0.5:1, or any value between them.

[0034] In some specific embodiments, the preferred mass ratio of the neo-agar oligosaccharide, astaxanthin, carbomer, and alginate is (3~5):(2~3):(0.05~0.1):(0.1~0.2), such as 3:2:0.05:0.1, 3.5:2:0.06:0.15, 4:3:0.07:0.175, 5:3:0.01:0.2, or any value between them.

[0035] In this invention, the neo-agar oligosaccharide refers to an oligosaccharide with a degree of polymerization of 2 to 20 obtained by hydrolyzing agar polysaccharide. It is composed of neo-agar disaccharide as repeating units and has β-D-galactose residues as reducing ends in its molecular chain structure.

[0036] In some specific embodiments, the degree of polymerization of the neo-astaxanthin oligosaccharide is preferably 2 to 4, such as 2, 3, or 4. In this case, the transdermal delivery system obtained by combining the neo-astaxanthin oligosaccharide, carbomer, and sodium alginate has a superior transdermal effect, enabling the neo-astaxanthin oligosaccharide and astaxanthin to reach and accumulate in the joint cavity more efficiently, thereby exerting their corresponding bioactive effects.

[0037] In this invention, the astaxanthin is a non-vitamin A provitamin A carotenoid, specifically comprising a long carbon chain composed of 11 consecutive conjugated double bonds and a β-ionone ring structure located at both ends of the long carbon chain. Based on the spatial relationship of the groups on all double bonds in the molecular structure that can produce spatial configuration differences, astaxanthin can be further divided into 9-cis isomers, 13-cis isomers, 15-cis isomers and all-trans isomers.

[0038] In some specific embodiments, the purity of the all-trans isomer of astaxanthin is preferably 90% to 100%, such as 90%, 91%, 92.5%, 93%, 94%, 95%, 96%, 97.5%, 99%, 100%, or any value between them. In this case, compared to other isomers, the astaxanthin has a linear structure, which exhibits a more superior synergistic effect with neoazolam oligosaccharides. It can better cross the cell membrane and enter the cell to exert its biological activity, resulting in a more ideal effect of relieving acute inflammatory symptoms of gouty osteoarthritis, blocking disease progression, and repairing pathological changes in joint tissue.

[0039] In this invention, the alginate refers to a natural polysaccharide extracted from brown algae such as seaweed and Sargassum. It is a linear polymer composed of β-D-mannuronic acid and α-L-guluronic acid linked by β-1,4-glycosidic bonds. Specific examples include, but are not limited to, sodium alginate and / or potassium alginate.

[0040] In this invention, the composition is preferably prepared by liquid-phase electrostatic self-assembly to form a gel formulation. More specifically, the preparation of the composition includes: mixing astaxanthin, neoagar oligosaccharide, and a solvent to obtain an active mixture; adding the carbomer and alginate to the active mixture, adjusting the pH to 5.0-7.0, and then performing electrostatic self-assembly to obtain the gel formulation. In some specific embodiments, the pH condition for performing the electrostatic self-assembly is more preferably 5.5-6.5.

[0041] In some specific embodiments, examples of the solvent include, but are not limited to, one or more of propylene glycol, DMSO, and water. In some preferred embodiments, the solvent is preferably an aqueous solution of propylene glycol with a concentration of 5% (v / v) to 15% (v / v).

[0042] In some specific embodiments, the preferred mass ratio of astaxanthin to solvent is (1~10):(78~95), such as 1:78, 1:80, 1:85, 1:90, 1:95, 5:78, 9:94, 10:95 or any value between them.

[0043] In some specific embodiments, the conditions for electrostatic self-assembly specifically include a temperature preferably between 25°C and 45°C, such as 25°C, 26.5°C, 28°C, 30°C, 35°C, 40°C, 45°C, or any value between them, more preferably 30°C to 40°C; a stirring speed preferably between 200 rpm and 400 rpm, such as 200 rpm, 210 rpm, 220 rpm, 230 rpm, 250 rpm, 300 rpm, 320 rpm, 380 rpm, 400 rpm, or any value between them; and a time preferably between 10 min and 60 min, such as 10 min, 12.5 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 55 min, 60 min, or any value between them.

[0044] In this invention, the composition exhibits excellent transdermal delivery, allowing the neoagar oligosaccharide and astaxanthin to pass smoothly through the skin, deep tissues, and joint capsule, thereby accumulating within the joint cavity. In other words, the composition possesses a substance diffusion pathway encompassing skin penetration, deep tissue diffusion, joint capsule penetration, and joint cavity accumulation.

[0045] In this invention, the composition can effectively enhance the activity of the endogenous antioxidant enzyme catalase (CAT) in an inflammatory cell model constructed using monosodium urate (MSU) stimulation and reduce intracellular malondialdehyde levels, demonstrating excellent potential as a catalase activator.

[0046] In this invention, the composition can effectively alleviate the damage to intracellular mitochondrial structure caused by monosodium urate (MSU) stimulation and restore mitochondrial integrity, while inhibiting the production of superoxide, and has excellent potential as a mitochondrial protectant and / or mitochondrial repair agent.

[0047] In this invention, the composition can effectively reverse the imbalance of Keap1-Nrf2-ARE signaling pathway expression caused by monosodium urate (MSU) stimulation, reduce the expression level of keap1 gene, and increase the expression levels of Nrf2 and NQO1 genes, and has excellent potential as a regulator of Keap1-Nrf2-ARE signaling pathway expression.

[0048] In this invention, the composition can effectively inhibit the upregulation of TNF-α expression induced by monosodium urate (MSU) stimulation, thereby reducing the TNF-α expression level, and has excellent potential as a TNF-α expression inhibitor.

[0049] In this invention, the composition can effectively alleviate inflammation caused by monosodium urate (MSU) stimulation by inhibiting the activation of core inflammatory pathways and rebuilding the antioxidant barrier, with ideal inflammation relief effect and excellent potential for application as an anti-inflammatory drug.

[0050] In this invention, the composition can effectively improve the synovial structure damage and cartilage matrix degradation caused by monosodium urate (MSU) stimulation in mouse ankle joint tissues, thereby restoring the integrity of the mouse joint synovial structure and showing excellent potential as a joint synovial protectant and / or joint synovial repair agent.

[0051] In this invention, the composition can effectively improve symptoms of inflammatory cell accumulation in periarticular tissues, reduce the accumulation of inflammatory cells in periarticular tissues and damage to joint tissues, and has excellent potential as an inhibitor of inflammatory cell infiltration, an anti-inflammatory chemotactic regulator and / or an inhibitor of joint inflammatory cell migration.

[0052] In this invention, the composition can repair and reconstruct damaged joint tissues by promoting cartilage matrix synthesis, slowing the accumulation of inflammatory cells and inhibiting degradation enzymes, and has excellent potential as a drug for treating gouty osteoarthritis.

[0053] The present invention also provides a composite gel formulation, which specifically comprises, by mass ratio (1-10):(1-10):(0.01-0.5):(0.1-1), a novel agar oligosaccharide, astaxanthin, carbomer, and alginate. The novel agar oligosaccharide, astaxanthin, carbomer, and alginate are identical or partially identical to those in the above-described composition, and will not be described in detail here.

[0054] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0055] Preparation Example

[0056] This preparation example illustrates a method for preparing a novel agar oligosaccharide, specifically comprising: dissolving agar powder completely in Tris-HCl buffer solution (50 mM, pH=5.8) at a final concentration of 15 g / L; then, adding recombinant β-agarase AgaP4383 at a final concentration of 2 U / mL. [1]The product was added to a solution and enzymatically hydrolyzed at 40°C for 1 hour to obtain the hydrolysate. The hydrolysate was then subjected to alcohol precipitation and freeze-drying to obtain neo-agar oligosaccharide powder. High-performance liquid chromatography (HPLC) analysis showed that the degree of polymerization of the obtained neo-agar oligosaccharide powder was 4–6.

[0057] [1] Liu Xiao, Yang Ziyi, Wu Chaocheng, et al. Isolation, purification and moisturizing activity of neoagartetrasaccharide [J]. Journal of Biology, 2025, 42(01): 55-60.

[0058] Example 1

[0059] This embodiment illustrates a composite gel formulation and its preparation method, specifically including:

[0060] S1. Dissolve astaxanthin (Maclean, catalog number 768722, all-trans isomer purity ≥90%) in 10% propylene glycol aqueous solution (m 虾青素 :m 10%丙二醇水溶液 =1:8) in; according to m 新琼寡糖 :m 虾青素 The ratio of added ingredients is 4:3. Take the new agar oligosaccharide provided in the preparation example and mix it evenly with astaxanthin to obtain an active mixture.

[0061] S2. Carbomer (Maclean, catalog number C886117) and sodium alginate (Maclean, catalog number S817374) were added to the active mixture at final concentrations of 1.0 wt% and 2.5 wt%, respectively. After adjusting the pH to 6.0, electrostatic self-assembly was carried out at 40°C and 300 rpm for 30 min. The pH was then adjusted to 7 to obtain the composite gel formulation.

[0062] Example 2

[0063] This embodiment uses the method provided in Example 1 to prepare a composite gel formulation. The difference is that the mass ratio of neo-agar oligosaccharide and astaxanthin is 1:1, while other conditions remain the same, to obtain the composite gel formulation.

[0064] Example 3

[0065] This embodiment uses the method provided in Example 1 to prepare a composite gel formulation. The difference is that the mass ratio of neo-agar oligosaccharide to astaxanthin is 5:3, while other conditions remain the same, to obtain the composite gel formulation.

[0066] Example 4

[0067] This embodiment uses the method provided in Example 1 to prepare a composite gel formulation. The difference is that an equal mass of new agar oligosaccharide (degree of polymerization of 2-4, Blue Brain Technology (Xiamen) Co., Ltd., catalog number AN24) is added instead of the new agar oligosaccharide in the example, while other conditions remain the same, to obtain the composite gel formulation.

[0068] Example 5

[0069] This embodiment uses the method provided in Example 1 to prepare a composite gel formulation. The difference is that an equal mass of new agar oligosaccharide (degree of polymerization 4, Maclean, catalog number N861204) is added instead of the new agar oligosaccharide in the example, while other conditions remain the same, to obtain the composite gel formulation.

[0070] Comparative Example 1

[0071] The comparative example uses the method provided in Example 1 to prepare a composite gel formulation. The difference is that astaxanthin and neo-agar oligosaccharides are not added to the composite gel formulation. That is, the concentrations of astaxanthin and neo-agar oligosaccharides in the composite gel formulation are both 0. Other conditions are kept the same to obtain the composite gel formulation.

[0072] Comparative Example 2

[0073] The comparative example uses the method provided in Example 1 to prepare the composite gel formulation. The difference is that no astaxanthin is added to the composite gel formulation. That is, the concentration of astaxanthin in the composite gel formulation is 0. Other conditions are kept the same to obtain the composite gel formulation.

[0074] Comparative Example 3

[0075] The comparative example uses the method provided in Example 1 to prepare the composite gel formulation. The difference is that no neo-agar oligosaccharides are added to the composite gel formulation. That is, the concentration of neo-agar oligosaccharides in the composite gel formulation is 0. Other conditions are kept the same to obtain the composite gel formulation.

[0076] Test case

[0077] This embodiment is used to illustrate the relevant performance of the composite gel formulations provided in the above embodiments and comparative examples. The specific tests include:

[0078] 1. In vitro experiments

[0079] RAW264.7 cells (Wuhan Pronosei Life Sciences Co., Ltd., catalog number CL-0190) were cultured according to the instructions until the cell confluence reached 60%~70%, and then randomly divided into 10 groups for the following operations:

[0080] i. MSU model group: Monosodium urate (MSU) was added to the cell culture medium at a final concentration of 100 μg / mL and cultured at 37℃ and 5% CO2 for 30 h;

[0081] ii. Gel intervention group: Monosodium urate (MSU) was added to the cell culture medium at a final concentration of 100 μg / mL and cultured at 37℃ and 5% CO2 for 6 h. Then, the composite gel provided in Comparative Example 1 was added at a concentration of 5% (w / v) and cultured at 37℃ and 5% CO2 for 24 h.

[0082] iii. New agar oligosaccharides (NAOs) intervention group: MSU was added to the cell culture medium at a final concentration of 100 μg / mL and cultured at 37℃ and 5% CO2 for 6 h. Then, the composite gel provided in Comparative Example 2 was added at a concentration of 5% (w / v) and cultured at 37℃ and 5% CO2 for 24 h.

[0083] iv. Astaxanthin (ASTA) intervention group: MSU was added to the cell culture medium at a final concentration of 100 μg / mL and cultured at 37℃ and 5% CO2 for 6 h. Then, the composite gel provided in Comparative Example 3 was added at a concentration of 5% (w / v) and cultured at 37℃ and 5% CO2 for 24 h.

[0084] v. Combined intervention group: MSU was added to the cell culture medium at a final concentration of 100 μg / mL and cultured at 37°C and 5% CO2 for 6 h. Then, the composite gels provided in Examples 1-5 were added at 5% (w / v) and cultured at 37°C and 5% CO2 for 24 h.

[0085] vi. Blank control group: The procedure is basically the same as that of the MSU model group, except that an equal volume of serum-free DMEM medium is used instead of MSU.

[0086] In addition to the conditions mentioned above, the operations, reagents, and conditions used by each group were kept consistent, and the following tests were conducted:

[0087] (1) Reactive oxygen species (ROS) level: After culture, the supernatant was aspirated, and the cells were washed with serum-free DMEM medium. Then, 10 μL of 10 μM DCFH-DA was added, and the cells were incubated at 37℃ in the dark for 30 min. The cells were washed three times with serum-free DMEM medium. The cells were observed under a fluorescence microscope and analyzed using ImageJ 1.8.0 software. The relative fluorescence level of ROS in each group was calculated with the blank control group as a reference. The results are as follows: Figure 1 and 2 As shown.

[0088] Depend on Figure 1 and 2The results show that the ROS level in RAW264.7 macrophages in the MSU model group was significantly higher than that in the blank control group, indicating that MSU induced a severe redox imbalance in the cells, and the model was successfully constructed.

[0089] Compared with Comparative Examples 2 and 3, the treatment of RAW264.7 macrophages with the composite gel formulation provided in Examples 1-5 of this invention can effectively alleviate the redox imbalance induced by MSU, and the ROS level in RAW264.7 macrophages can be restored to near physiological levels.

[0090] (2) Malondialdehyde (MDA) content and catalase (CAT) activity: After culture, the supernatant was aspirated, the cell pellet was collected, and the MDA content and CAT activity were determined using the MDA kit (Solepro, catalog number BC0025) and the CAT kit (Solepro, catalog number BC0200) according to the instructions. The results are as follows: Figure 3 and 4 As shown.

[0091] Depend on Figure 3 and 4 The results show that, compared with the blank control group, the content of lipid peroxidation marker MDA in RAW264.7 macrophages in the MSU model group was significantly increased, and the activity of endogenous antioxidant enzyme CAT was significantly decreased.

[0092] Compared with Comparative Examples 2 and 3, the treatment of RAW264.7 macrophages with the composite gel formulation provided in Examples 1-5 of this invention can effectively reduce the level of intracellular lipid peroxidation marker MDA and enhance the activity of endogenous antioxidant enzyme CAT, thereby improving the antioxidant defense capacity of cells.

[0093] (3) Mitochondrial and superoxide content: After culture, the supernatant was aspirated, and the cell pellet was collected. After culture, the cells were washed three times with serum-free DMEM medium. The nuclei, mitochondria, and mitochondrial superoxide were fluorescently labeled with Hochest 33342, MitoTracker Green, and MitoSOX Red, respectively. After incubation at 37°C in the dark for 25 min, the cells were washed three times with serum-free DMEM medium. The cells were then observed under a fluorescence microscope and analyzed using ImageJ 1.8.0 software. The relative fluorescence levels of superoxide and mitochondria in each group were calculated using the blank control group as a reference. The results are as follows: Figures 5-7 As shown.

[0094] Depend on Figures 5-7The results show that, compared with the blank control group, the mitochondrial content in RAW264.7 macrophages in the MSU model group was significantly reduced, while the superoxide level was increased.

[0095] Compared with Comparative Examples 2 and 3, RAW264.7 macrophages were treated with the composite gel formulations provided in Examples 1-5 of this invention, resulting in increased mitochondrial content and decreased superoxide levels. This indicates that the combined use of neo-agar oligosaccharides and astaxanthin can restore mitochondrial integrity and inhibit superoxide production.

[0096] (4) Expression of inflammation-related genes: After culture, the cell pellet was collected by centrifugation. RNA was extracted from the cell pellet using the FastPure Cell / Tissue Total RNA Extraction Kit V2 (Novazia, catalog number RC112-01) according to the instructions. 1 μg of RNA was amplified using HiScript IV One-Step Ultrasensitive Reverse Transcription Premix (Novazia, catalog number R433-01) according to the instructions to obtain the first-strand cDNA. The first-strand cDNA was detected using a blue universal dye qPCR detection kit according to the instructions. Reverse transcription-polymerase chain reaction was performed on the Roche Digital LightCycler digital PCR system. The measured RNA level (CT value) was standardized according to the GAPDH level. The expression level of the target gene was calculated using the ΔΔCT value. The relative expression level of the target gene in each group was calculated using the blank control group as a reference. The results are as follows: Figures 8-10 As shown.

[0097] Depend on Figures 8-10 The results show that, compared with the blank control group, the expression of Keap1 gene in RAW264.7 macrophages in the MSU model group was significantly increased, while the expression of Nrf2 gene and its downstream effector NQO1 was inhibited.

[0098] Compared with Comparative Examples 2 and 3, the treatment of RAW264.7 macrophages with the composite gel formulation provided in Examples 1-5 of this invention can effectively reverse the imbalance of Keap1-Nrf2-ARE signaling pathway expression caused by MSU stimulation.

[0099] (5) TNF-α level: After culture, the cell pellet was collected by centrifugation, and the TNF-α level was detected using an ELISA kit (Proteintech, catalog number MTA00B-1) according to the instructions. The results are as follows: Figure 11 As shown.

[0100] Depend on Figure 11 The results show that, compared with the blank control group, the level of TNF-α in RAW264.7 macrophages in the MSU model group was significantly increased.

[0101] Compared with Comparative Examples 2 and 3, the composite gel formulation provided in Examples 1-5 of this invention can effectively inhibit the expression of TNF-α when RAW264.7 macrophages are treated.

[0102] 2. In vivo experiments

[0103] Eight-week-old C57BL / 6 mice were used as experimental animals. After one week of acclimatization, they were randomly divided into 10 groups (n=6 per group) and the following procedures were performed:

[0104] i. MSU model group: MSU-containing PBS buffer (1×) was injected into the right ankle joint cavity of mice at a dose of 0.5 mg / mouse (injection volume of 20 μL) (recorded as h0), and no topical administration was performed during the subsequent feeding stage;

[0105] ii. Gel intervention group: MSU-containing PBS buffer (1×, Solarbio, catalog number P1020, pH=7.2, the same below) was injected into the right ankle joint cavity of mice at a dose of 0.5 mg / mouse (injection volume of 20 μL) (recorded as h0). At h2, h5, h8 and h11, 20 mg of the compound gel preparation provided in Comparative Example 1 was applied to the right ankle joint of mice.

[0106] iii. NAOs-only intervention group: MSU-containing PBS buffer (1×, pH=7.2) was injected into the right ankle joint cavity of mice at a dose of 0.5 mg / mouse (injection volume of 20 μL) (recorded as h0). At h2, h5, h8 and h11, 20 mg of the compound gel preparation provided in Comparative Example 2 was applied to the right ankle joint of mice.

[0107] iv. ASTA-only intervention group: MSU-containing PBS buffer (1×, pH=7.2) was injected into the right ankle joint cavity of mice at a dose of 0.5 mg / mouse (injection volume of 20 μL) (recorded as h0). At h2, h5, h8 and h11, 20 mg of the compound gel preparation provided in Comparative Example 3 was applied to the right ankle joint of mice.

[0108] v. Combined intervention group: MSU-containing PBS buffer (1×, pH=7.2) was injected into the right ankle joint cavity of mice at a dose of 0.5 mg / mouse (injection volume of 20 μL) (recorded as h0). At h2, h5, h8 and h11, 20 mg of the compound gel preparation provided in Examples 1-5 was applied to the right ankle joint of mice.

[0109] vi. Blank control group: The procedure was basically the same as that of the MSU model group, except that an equal volume of PBS buffer (1×, pH=7.2) was used instead of PBS buffer containing MSU (1×, pH=7.2).

[0110] In addition to the conditions mentioned above, the operations, reagents, and conditions used by each group were kept consistent, and the following tests were conducted:

[0111] (1) After 24 hours, the ankle joint fluid of each mouse was collected, and the contents of neoazolam oligosaccharide and astaxanthin in the synovial fluid were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The results are shown in Table 1.

[0112] Table 1.

[0113]

[0114] As shown in Table 1, compared with Comparative Examples 1-3, the composite gel formulations provided in Examples 1-5 of this invention, when applied to the ankle joints of mice, showed excellent transdermal efficiency of neo-agar oligosaccharides and astaxanthin.

[0115] (2) Mice were sacrificed 24 hours later, and ankle joints (1cm×1cm×1cm) were collected to obtain ankle joint samples. The ankle joint samples were fixed in 10% paraformaldehyde, embedded in paraffin, sectioned, dewaxed in xylene, hydrated with alcohol, and stained with hematoxylin and eosin (HE). The samples were then photographed and observed under a microscope, and the number of infiltrating cells in the field of view was counted. The results are as follows: Figure 12 and 13 As shown.

[0116] Depend on Figure 12 and 13 The results show that in the blank control group, the structure of the mouse ankle joint tissue was normal, intact, and clear, with smooth articular surfaces and intact cartilage matrix. In contrast, in the MSU model group, the synovial epithelial cells of the mouse ankle joint were discontinuously arranged, the cartilage matrix showed multiple sites of degradation, and a large number of inflammatory cells accumulated in the periarticular tissue.

[0117] Compared with Comparative Examples 1 and 2, when the composite gel formulations provided in Examples 1-5 of this invention were applied to the ankle joints of MSU mouse models, the synovial structure of the mouse joints was significantly improved. The microscopic structure was similar to that of the normal group, the synovium was more continuous and intact, the cartilage matrix was complete, and the symptoms of a large number of inflammatory cells accumulating in the periarticular tissues were effectively improved.

[0118] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. The use of the composition in the preparation of a transdermal drug for the treatment of osteoarthritis, characterized in that, The composition comprises neoagar oligosaccharide, astaxanthin, carbomer, and alginate in a mass ratio of (1~10):(1~10):(0.01~0.5):(0.1~1); wherein the purity of the all-trans isomer of the astaxanthin is 90%~100%. The composition is a gel formulation. The preparation of the composition includes: mixing astaxanthin, neoagar oligosaccharide and solvent to obtain an active mixture; adding the carbomer and alginate to the active mixture, adjusting the pH to 5.0~7.0, and then performing electrostatic self-assembly to obtain a gel formulation.

2. The use of the composition according to claim 1 in the preparation of a transdermal drug for the treatment of osteoarthritis, characterized in that, The degree of polymerization of the new agar oligosaccharide is 2 to 4.

3. The use of the composition according to claim 1 in the preparation of a transdermal drug for the treatment of osteoarthritis, characterized in that, The alginate is sodium alginate and / or potassium alginate.

4. The use of the composition according to claim 1 in the preparation of a transdermal drug for the treatment of osteoarthritis, characterized in that, The preparation of the composition includes at least one of the following technical features: (1) The solvent is one or more of propylene glycol, DMSO and water; (2) The mass ratio of astaxanthin to solvent is (1~10):(78~95); (3) The electrostatic self-assembly temperature is 25℃~45℃, the stirring speed is 200rpm~400rpm, and the time is 10min~60min.

5. The use of the composition according to claim 1 in the preparation of a transdermal drug for the treatment of osteoarthritis, characterized in that, In the topical administration method, the composition has a substance diffusion pathway that includes skin penetration, deep tissue diffusion, joint capsule penetration, and joint cavity enrichment.

6. The use of the composition according to claim 1 in the preparation of a transdermal drug for the treatment of osteoarthritis, characterized in that, The composition has one or more of the following characteristics: (1) The composition is used as a catalase activator; (2) The composition is used as a mitochondrial protectant and / or mitochondrial repair agent; (3) The composition is used as a regulator of the Keap1-Nrf2-ARE signaling pathway; (4) The composition is used as a TNF-α expression inhibitor; (5) The composition is used as an anti-inflammatory drug; (6) The composition is used as a synovial protectant and / or synovial repair agent; (7) The composition is used as an inhibitor of inflammatory cell infiltration, an anti-inflammatory chemotactic regulator and / or an inhibitor of joint inflammatory cell migration; (8) The composition is used as a medicine for treating gouty osteoarthritis.

7. A composite gel formulation, characterized in that, The composite gel formulation comprises neoagar oligosaccharide, astaxanthin, carbomer and alginate in a mass ratio of (1~10):(1~10):(0.01~0.5):(0.1~1), wherein the purity of the all-trans isomer of the astaxanthin is 90%~100%.

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