Composite hydrogel containing saussurea involucrate extract as well as preparation method and application of composite hydrogel

Abstract

According to the composite hydrogel containing the saussurea involucrata extract and the preparation method and application of the composite hydrogel, the composite hydrogel containing the saussurea involucrata extract has the good drug carrying capacity and the good photothermal effect, and the composite hydrogel has the good drug release control capacity under the near-infrared illumination condition; and in combination with the photothermal effect of the black phosphorus nanosheet, the skin permeability of the effective components of the saussurea involucrata extract can be improved under the condition of near-infrared illumination, and the treatment effect of the saussurea involucrata extract on arthritis diseases is enhanced. And the composite hydrogel containing the saussurea involucrate extract does not generate a hemolysis effect under a treatment concentration, and has good biocompatibility and safety.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical hydrogels, and in particular relates to a composite hydrogel containing an extract of Saussurea involucrata and a preparation method and application thereof. Background Art

[0002] Arthritis is a general term for inflammatory diseases that occur in the joints and surrounding tissues of the human body. It can be suffered by children to the elderly. Most of them are accompanied by redness, swelling, fever, pain, stiffness, joint deformation and movement disorders in the joints. Severe cases can cause cramps and disability. The joints are mainly composed of the articular surface, joint capsule, joint cavity, ligament, articular disc and articular lip. Damage to these parts can cause inflammatory reactions, so the clinical manifestations of arthritis are varied and the causes are extremely complex. Arthritis mainly includes the following 4 types: 1. Autoimmune arthritis: including rheumatoid arthritis and ankylosing spondylitis. 2. Osteoarthritis: It is also the most familiar type, caused by degenerative lesions, and is more common in middle-aged and elderly people. 3. Gouty arthritis: It is related to high uric acid and metabolic disorders. 4. Systemic arthritis: Arthritis caused by systemic lupus erythematosus, mixed connective tissue disease, etc. The most common arthritis diseases are rheumatoid arthritis and osteoarthritis. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, and the etiology is not clearly indicated. It is characterized by chronic arthritis, synovial tissue hyperplasia and varying degrees of bone and cartilage erosion, which can cause chronic arthritis destruction and ultimately lead to functional loss. The most prominent feature of rheumatoid arthritis is symmetrical pain and swelling of the hands, wrists, feet and knees (polyarthritis). Osteoarthritis (OA) is a degenerative disease caused by the degeneration of articular chondrocytes and cartilage. The imbalance of the synthesis of articular chondrocytes, extracellular matrix and subchondral bone is a pathological feature of the degenerative process. Articular cartilage is an important component of the joint surface, which can play a role in buffering, promoting the flow of synovial fluid and dispersing mechanical conduction of the lower limbs. The main clinical manifestations are swelling, pain, stiffness and poor mobility around the joints. At present, its risk factors and pathological mechanisms are not yet clear. With the aging of the population and the increasing obesity rate year by year. Although the treatment methods for arthritis are constantly improving clinically, the cure rate of arthritis is still very low.

[0003] Saussurea involucrata (SI) is a plant of the Asteraceae family. It contains chemical components such as flavonoids, lactones, lignins, alkaloids, volatile oils, and polysaccharides. Among them, flavonoids are the main components, mainly rutin. It also contains many phenylpropanoid compounds, mainly phenylpropanoid chlorogenic acid. The main functions of traditional Chinese medicine: warming the kidney and supporting yang, dispelling wind and dampness, and promoting blood circulation. It is used for diseases such as wind-cold dampness arthralgia, rheumatoid arthritis, and lower abdominal cold pain. Modern research shows that Saussurea involucrata also has pharmacological effects such as anti-inflammatory and analgesic, anti-cancer, vasodilation, blood pressure reduction, free radical scavenging, and anti-fatigue.

[0004] Some nano-biomaterials such as hydrogels, liposomes, and microemulsions can be used as transport carriers for arthritis treatment drugs. Among them, hydrogel is a special type of hydrophilic polymer and a unique cross-linked three-dimensional network structure. It can form a polymerization cross-linking system with the target drug through van der Waals forces and other means, thereby extending its retention time, allowing the target drug to be sustained-released at physiological temperature and improving the bioavailability of the drug. At the same time, it has strong material exchange capacity, good biocompatibility, and biodegradability, which can reduce the adverse reactions of drugs, enhance drug efficacy and other application advantages. Therefore, it has good application prospects in drug delivery systems for arthritis treatment. Summary of the invention

[0005] Therefore, based on the above background, the present invention provides a composite hydrogel containing Tianshan Saussurea extract, a preparation method and an application thereof, which constructs a system with good drug loading capacity and biocompatibility by combining a hydrogel body with layered black phosphorus nanosheets. It can load a high amount of Saussurea extract and has good skin permeability. It can have significant anti-arthritis ability through transdermal sustained-release administration.

[0006] The technical solution of the present invention is:

[0007] A composite hydrogel containing Tianshan Saussurea extract, comprising a hydrogel body, black phosphorus nanosheets loaded with Saussurea extract, menthol and borneol;

[0008] The black phosphorus nanosheets are surface-modified by polyethylene glycol.

[0009] Furthermore, the hydrogel is made of the following raw materials: hyaluronic acid, polyvinyl alcohol, and glycerol.

[0010] Furthermore, the average size of the black phosphorus nanosheets is 100-200 nm.

[0011] Based on the same inventive concept, the present invention also provides a method for preparing a composite hydrogel containing an extract of Saussurea tianshanica, which comprises the following steps:

[0012] S1: taking black phosphorus nanosheets and preparing a black phosphorus nanosheet dispersion;

[0013] S2: adding a polyethylene glycol diamine aqueous solution to the black phosphorus nanosheet dispersion to react, thereby preparing a polyethylene glycol-modified black phosphorus nanosheet (BP-PEG) dispersion;

[0014] S3: centrifugally filter the polyethylene glycol-modified black phosphorus nanosheet dispersion prepared in step S2, take the precipitate, take the Saussurea tianshanica extract (SI) according to the amount, dissolve it in ethanol, add the solution to the precipitate, mix well, ultrasonicate, oscillate in the dark, centrifuge, and take the precipitate;

[0015] The ratio of black phosphorus nanosheet precipitate to Saussurea involucrata extract is 1:(5-10);

[0016] The precipitate was resuspended in ultrapure water to obtain a black phosphorus nanosheet (BP-SI) solution loaded with Saussurea involucrata extract;

[0017] S4: Take hyaluronic acid (HA) according to the amount, add a certain amount of ultrapure water, stir evenly, add polyvinyl alcohol (PVA) according to the amount, stir evenly, then slowly add glycerol (GLY), stir evenly, and prepare a hydrogel;

[0018] S5: Pour the black phosphorus nanosheet solution loaded with Saussurea tussock extract prepared in step S3 into the hydrogel in step S4, vortex and mix evenly, then add Saussurea tussock extract to the system, stir evenly, add menthol and borneol with a mass fraction of no more than 1% to the system, stir until mixed evenly, and the composite hydrogel containing Saussurea tussock extract (BP-SI@HPG) can be prepared.

[0019] Furthermore, the mass ratio of hyaluronic acid, polyvinyl alcohol and glycerol in step S4 is 3:3:2.

[0020] The mass ratio of menthol and borneol in step S5 to the hyaluronic acid in step S4 is 2:2:75.

[0021] The preparation of the black phosphorus nanosheet dispersion in step S1 comprises the following steps:

[0022] S1.1 Take black phosphorus powder and place it in a mortar, add an organic solvent, and grind it in the dark; the organic solvent is selected from one of N-methylpyrrolidone (NMP), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0023] The ground black phosphorus powder suspension is transferred to an EP tube to obtain a black phosphorus suspension;

[0024] The obtained black phosphorus suspension was subjected to ultrasonic treatment in the dark;

[0025] The black phosphorus suspension is further subjected to ultrasonic treatment in the dark by using a cell disruptor, so as to split the larger black phosphorus nanosheets into smaller black phosphorus nanosheets through molecular collision and other effects;

[0026] S1.2 After the black phosphorus suspension is allowed to stand, the upper layer of the suspension is taken, and after centrifugation, the upper layer is taken;

[0027] After two more centrifugations, the organic solvent is replaced with an equal volume of ultrapure water to prepare a black phosphorus nanosheet dispersion.

[0028] Furthermore, the preparation of the Saussurea involucrata extract used in step S3 is specifically as follows:

[0029] Take the whole herb of Saussurea tianshanica, add ethanol in an amount of 15-20 times the weight of the whole herb of Saussurea tianshanica, soak for 10-20 days, filter, take the filtrate; and concentrate the filtrate to contain 1g of the original medicinal material per 1mL.

[0030] Furthermore, in step S5, the weight ratio of the black phosphorus nanosheet solution loaded with the Saussurea involucrata extract: the hydrogel: the Saussurea involucrata extract is 2:10:1.

[0031] Based on the same inventive concept, the present invention also provides the use of the composite hydrogel containing the Saussurea involucrata extract in the preparation of drugs for treating arthritis.

[0032] Based on the same inventive concept, the present invention also provides the use of the composite hydrogel containing Tianshan Saussurea extract in the preparation of inflammatory factor inhibitors, characterized in that the inflammatory factors include TNF-α, IL-6 and IL-17.

[0033] The present invention uses hyaluronic acid and polyvinyl alcohol to form a three-dimensional network structure of the hydrogel, which plays a role in increasing adhesion. The added glycerol can increase the moisture retention and plasticity of the hydrogel, so that the hydrogel can have a good moisturizing effect and allow the drug to penetrate the skin better. And an appropriate amount of menthol and borneol are used as skin penetrants. When the two are used together, it is more conducive to increasing the amount of drug penetration in the skin.

[0034] The above technical solution has the following beneficial effects:

[0035] ① The present invention utilizes the principle of electrostatic adsorption to functionalize the surface of PEG and small molecules of Saussurea involucrata extract onto negatively charged black phosphorus nanosheets, and can make the black phosphorus nanosheet composite hydrogel loaded with Saussurea involucrata extract more permeable to skin tissue through near-infrared laser irradiation.

[0036] ② The black phosphorus nanosheet hydrogel prepared by the present invention is applied to the diseased joints. Under near-infrared 808nm irradiation, the skin surface temperature gradually increases. The black phosphorus nanosheet hydrogel can use the photothermal effect to slowly release the effective ingredients of the Saussurea involucrata extract loaded on the black phosphorus nanosheets, thereby improving the utilization rate of the drug and reducing the number of medications. Animal results show that the composite hydrogel containing Saussurea involucrata extract of the present invention has significant inflammatory factor inhibition ability and significant anti-rheumatoid arthritis ability.

[0037] ③ In the gel system of the present invention, hyaluronic acid and polyvinyl alcohol not only form a three-dimensional network structure of the hydrogel, but also make the hydrogel have excellent adhesion. The addition of glycerol increases the moisture retention and plasticity of the system. The addition of menthol and borneol increases the skin permeability of the system drugs, which can better promote the treatment of arthritis and rheumatoid arthritis.

[0038] ④ The composite hydrogel containing the Saussurea involucrata extract of the present invention does not produce a hemolytic effect at a therapeutic concentration and has good biocompatibility and safety.

[0039] ⑤ The composite hydrogel containing snow lotus extract of the present invention has a simple preparation process, is easy to apply, is simple to administer, and can reduce damage to the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 A transmission electron micrograph of black phosphorus nanosheets in an embodiment of the present invention (A) and a transmission electron micrograph of black phosphorus nanosheets loaded with Saussurea involucrata extract (B).

[0041] Figure 2 The drug loading rates of black phosphorus nanosheets and drugs of different masses (A) and the particle size distribution diagram of black phosphorus nanosheets and black phosphorus nanosheets loaded with Saussurea involucrata extract (B) and Zeta potential distribution diagram (C) in the embodiments of the present invention.

[0042] Figure 3 Figure 2 is a temperature change diagram of a blank hydrogel, a black phosphorus nanosheet solution and a composite hydrogel containing Saussurea involucrata extract under near-infrared laser irradiation in an embodiment of the present invention (A), a temperature change diagram of a black phosphorus nanosheet solution and a composite hydrogel containing Saussurea involucrata extract under near-infrared laser irradiation of different concentrations (B) and (C), a temperature change diagram of a blank hydrogel, a black phosphorus nanosheet solution and a composite hydrogel containing Saussurea involucrata extract under near-infrared laser irradiation of different powers (D), (E) and (F), a temperature change diagram of a black phosphorus solution after 5 cycles of near-infrared light switching (G), and a temperature change diagram of a composite hydrogel drug delivery system containing Saussurea involucrata extract after 5 cycles of near-infrared light switching (H).

[0043] Figure 4This is a graph showing the results of a hemolysis experiment of black phosphorus nanosheet composite hydrogels loaded with rutin at different concentrations in an embodiment of the present invention.

[0044] Figure 5 The figures are the therapeutic efficacy of rheumatoid arthritis in different groups in the embodiments of the present invention (A), the rat plantar swelling value (B) and the joint index evaluation (C).

[0045] Figure 6 Figures (A), (B) and (C) show the expression levels of inflammatory factors TNF-α, IL-6 and IL-17 in serum in the examples of the present invention. DETAILED DESCRIPTION

[0046] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0049] Example 1: Preparation of Saussurea involucrata Extract

[0050] Weigh 1000 g of the whole herb of Saussurea tianshanica, add 85% ethanol (the amount added is 16 times the mass of Saussurea tianshanica), soak for 14 days at room temperature, filter, concentrate the filtrate to contain 1 g of the original medicinal material per 1 mL, store in a 4°C refrigerator, and keep sealed.

[0051] Determination of the content of active ingredients in the extract of Saussurea involucrata, which is mainly composed of rutin and chlorogenic acid, was measured by ultraviolet spectrophotometer. The results showed that 1 g of the extract of Saussurea involucrata prepared in this example contained 13.36 mg of rutin and 5.52 mg of chlorogenic acid.

[0052] Example 2: Preparation of black phosphorus nanosheets loaded with Saussurea involucrata extract (BP-SI)

[0053] First, weigh 20 mg of black phosphorus powder in a mortar, add 20 mL of N-methylpyrrolidone (NMP) and grind it at low temperature and away from light. Place it in an ultrasonic cleaner (power: 500w) and ultrasonicate it in a low-temperature water bath away from light for 8 hours. Then replace the cell disruptor and continue to ultrasonicate the black phosphorus suspension at low temperature and away from light for 4 hours. After completion, take out the suspension and let it stand overnight at 4°C. Then centrifuge for 5 minutes (2000rpm, 4°C) to remove the unpeeled large-sized black phosphorus and collect the supernatant. Take the supernatant and place it in an empty centrifuge tube, centrifuge it for 20 minutes (15000rpm, 4°C), add 1 mL of NMP to store the prepared black phosphorus nanosheet suspension in a 4°C refrigerator and seal it for storage.

[0054] Take 2 mL of the prepared black phosphorus nanosuspension, place it in an EP tube, centrifuge it at 12000 rpm, 4°C for 30 min, remove the supernatant, add an equal volume of ultrapure water, and vortex until mixed evenly to obtain a black phosphorus nanosheet dispersion.

[0055] Weigh 10 times the weight of black phosphorus PEG-NH2 (MW: 3400), add it to the black phosphorus nanosheet dispersion, ultrasonicate at 500W for 30min, vortex for 5min, shake at 300rpm, 4℃ on a shaker in the dark for 12h, centrifuge at 12000rpm, 4℃ for 25min, remove the supernatant, and take the precipitate. Take 5 and 10 times the weight of the above black phosphorus nanosheet precipitate (Saussurea involucrata extract, dissolve it with an equal volume of 85% ethanol, add the Saussurea involucrata extract solution to the BP-PEG precipitate, shake at 300rpm, 4℃ on a shaker in the dark for 24h, centrifuge at 12000rpm, 4℃ for 25min, add ultrapure water to the precipitate and resuspend it to obtain the black phosphorus-loaded Saussurea involucrata extract (BP-SI) solution.

[0056] The intermediates and products prepared above were characterized and their properties were analyzed.

[0057] Electron microscopy observations of black phosphorus nanosheets and black phosphorus nanosheets loaded with Saussurea involucrata extract (BP-SI) were performed. Figure 1 shown.

[0058] Results Figure 1 (A) is a transmission electron microscopy image of black phosphorus nanosheets. Black phosphorus is a single-layer or multi-layer sheet structure with a particle size of about 100-200 nm and uniform size. Figure 1 (B) is a transmission electron micrograph of black phosphorus nanosheets loaded with Saussurea involucrata extract. The loaded Saussurea involucrata extract is round-shaped black particles. The particle size of the black phosphorus nanosheets after loading with Saussurea involucrata extract is about 200-300nm. In the figure, black particles can be clearly seen on the black phosphorus nanosheets, indicating that the Saussurea involucrata extract is successfully loaded on the black phosphorus nanosheets.

[0059] In order to compare the effect of black phosphorus and different weight ratios of effective drugs on the overall drug loading rate, the dosage of Tianshan Saussurea extract was 5 and 10 times the weight of black phosphorus nanosheets (drug loading, the results are shown in Figure 2). Figure 2 shown.

[0060] Depend on Figure 2 (A) It can be seen that the different mass ratios of black phosphorus nanosheets and snow lotus extract affect the drug loading rate. When the mass ratio of black phosphorus nanosheets to snow lotus extract is 1:10, the drug loading rate of black phosphorus loaded with snow lotus extract is higher than that of the mass ratio of 1:5, and the drug loading rate can reach 14.72% (calculated with rutin as the main active ingredient) and 18.44% (calculated with chlorogenic acid as the main active ingredient).

[0061] The particle size distribution and potential distribution of black phosphorus nanosheets and black phosphorus nanosheets loaded with Tianshan Saussurea involucrata extract (BP-SI) were analyzed. Figure 2 shown.

[0062] from Figure 2 As shown in (B), the size of BP is 185.00±12.34nm, and the size of BP-SI is 283.37±16.83nm. The black phosphorus nanosheets aggregate after drug loading, resulting in a larger particle size. Figure 2 (C) shows that the Zeta potential of black phosphorus nanosheets is -40.27±3.23mV, and the Zeta potential of BP-SI is -32.27±12.34mV. The absolute value of the Zeta potential of BP-SI is lower than that of BP.

[0063] Example 3: Preparation of BP-SI@HPG

[0064] ① Preparation of blank hydrogel

[0065] Weigh 0.3 g of hyaluronic acid, add 8 mL of ultrapure water, stir thoroughly until mixed, add 0.3 g of polyvinyl alcohol, continue to stir thoroughly until mixed, then slowly add 0.2 g of glycerol, stir until mixed, and prepare an empty hydrogel (i.e., HPG). ② Preparation of composite hydrogel containing Saussurea tianshanica extract (BP-SI@HPG)

[0066] Take the black phosphorus nanosheet solution loaded with Saussurea involucrata extract and slowly pour it into the prepared hydrogel colloid, vortex and mix evenly, then add the Saussurea involucrata extract to the hydrogel colloid, stir evenly, and finally add 1% by mass of menthol and borneol, wherein the mass ratio of menthol to borneol is 1:1, stir until the mixture is evenly mixed, and the composite hydrogel containing Saussurea involucrata extract (ie, BP-SI@HPG) is obtained. The weight ratio of the black phosphorus nanosheet solution loaded with Saussurea involucrata extract: hydrogel: Saussurea involucrata extract is 2:10:1;

[0067] In this step, the amount of menthol and borneol added is controlled to be no more than 1%. Experimental verification shows that if it is greater than 1%, toxic side effects may occur.

[0068] Embodiment 4:

[0069] In order to compare the photothermal effect of black phosphorus nanosheets and black phosphorus nanosheet hydrogels, the temperature changes of HPG, black phosphorus nanosheet solutions and composite hydrogels containing Saussurea involucrata extract under near-infrared laser irradiation, the temperature changes of black phosphorus nanosheets and composite hydrogels containing Saussurea involucrata extract at different concentrations under near-infrared laser irradiation, the influence of near-infrared laser irradiation of different powers on the temperature of HPG, black phosphorus nanosheets and composite hydrogels containing Saussurea involucrata extract, and the temperature changes of black phosphorus nanosheets and composite hydrogels containing Saussurea involucrata extract after 5 cycles of near-infrared light switching were investigated. The results are shown in Figure 2. Figure 3 shown.

[0070] from Figure 3 As can be seen from (A), compared with the blank HPG group, the temperature change of the composite hydrogel of black phosphorus nanosheets and Saussurea involucrata extract was larger. Figure 3 It can be seen from (B) and 3(C) that as the concentration of the solution increases, the photothermal temperature of the composite hydrogel of black phosphorus nanosheets and Saussurea involucrata extract gradually increases after 10 minutes of irradiation. Figure 3 It can be seen from Figures 3(D), 3(E) and 3(F) that as the near-infrared light power increases, the temperature of the composite hydrogel of black phosphorus nanosheets and Saussurea involucrata extract gradually increases after 10 minutes of irradiation. Figure 3 As can be seen from Figures 3(G) and 3(H), in the five cycles of near-infrared laser irradiation switching, the peak temperature of the black phosphorus nanosheets dropped from 40.0°C to 38.7°C, and the peak temperature of the composite hydrogel containing Tianshan Saussurea extract dropped from 43.4°C to 43.0°C after five photothermal cycles. By comparison, the trend of the temperature curve remains basically unchanged, and the photothermal performance of BP is improved after drug loading and combining with hydrogel.

[0071] Embodiment 5:

[0072] In this example, the biosafety of composite hydrogels containing Saussurea tussock extract (BP-SI@HPG) at different concentrations was evaluated.

[0073] In order to investigate the hemolysis rate of BP-SI@HPG at different concentrations, a hemolysis experiment was conducted. Rat blood was taken and placed in an EP tube with sodium heparin, centrifuged at 1500rpm, 4℃ for 10min, the supernatant was removed to obtain mouse red blood cells, and saline was added to wash 2-3 times until the supernatant was clear. The purified red blood cells obtained were made into a 4% red blood cell suspension, and 500uL of 5% red blood cell suspension was added to a 1.5mL centrifuge tube containing 500uL of hydrogel pre-treated with 0.9% saline, and then the centrifuge tube was placed in a 37℃ constant temperature incubator and incubated for 3h. After that, the liquid in the centrifuge tube was centrifuged at 2500rpm, 5min, 4℃, and then 200uL of the supernatant was transferred to a 96-well plate, and its absorbance value at 540nm was measured. The hemolysis rate is calculated according to the formula:

[0074] Hemolysis rate (%) = [(Ap-Ab) / (At-Ab)] × 100%

[0075] Wherein, Ap is the absorbance value of the supernatant, At is the absorbance value of the 0.1% Triton x-100 positive control, and Ab is the absorbance value of the 0.9% saline negative control. 200μg / mL, 100μg / mL, 50μg / mL, and 25μg / mL represent that 1mL of black phosphorus nanosheet hydrogel contains 200μg, 100μg, 50μg, and 25μg of black phosphorus nanosheets, respectively. The results are shown in Figure 4 shown.

[0076] from Figure 4 It can be seen that the hemolysis rate of BP-SI@HPG does not exceed 5% as the concentration continues to increase, and no obvious hemolysis is shown.

[0077] Example 6: Animal Experiment

[0078] Grouping and treatment of experimental animals

[0079] Animal breeding environment

[0080] Barrier level: SPF level; temperature: 23±2℃; relative humidity: 50±10%; environmental noise: ≤60dB; provide sufficient feeding feed and drinking water.

[0081] Experimental animal groups:

[0082] After 7 days of animal experiment in the new environment, the animal model was established. After fasting for 12-16 hours, the rats were divided into 4 modeling groups (n=32) and a blank control group (n=8) according to the random number table method. The modeling groups were divided into RA group, HPG group, BP-SI@HPG group, and BP-SI@HP-G+NIR group. The skin of the right hind foot was disinfected with iodine solution. The rats in the modeling group were injected with 0.1 mL of complete Freund's adjuvant subcutaneously at the plantar of the right foot with a disposable medical 1 mL syringe to induce inflammation; on the 7th day after injection, 0.1 mL of complete Freund's adjuvant was injected intradermally at the plantar of the right hind foot of the rats to strengthen immunity and replicate the RA model. The blank group rats were injected with an equal volume of normal saline in the same way. 14 days after injection, the inflammatory scores of the bilateral hind foot joints of the rats in the modeling group were scored according to the "0-4 grade arthritis scoring method" to evaluate the success of the modeling. After 14 days of modeling, 5 mg / kg of rutin, the main active ingredient in the Tianshan Saussurea extract, was used as the dosage. The drug was applied every other day for 28 days, a total of 42 days. Joints were photographed on days 0, 7, 14, 21, and 28 after grouping and modeling.

[0083] During the treatment period, the joints were photographed with a digital camera on days 0, 7, 14, 21, and 28, and the square ruler was used to record the results. Figure 5 (A) shown.

[0084] Index detection

[0085] ① Plantar swelling value (mm) measurement:

[0086] The degree of toe swelling is an important indicator for measuring adjuvant arthritis. On day 0, the same place on the sole of the rat's foot was marked, and the thickness of the rat's sole was measured with an electronic vernier caliper based on the mark. The thickness of the rat's right sole was measured every 2 days thereafter, and the right sole of the rat was used as a control and as a control between groups. The results are shown in Figure 5 (B) shown.

[0087] ② Arthritis index (AI) evaluation:

[0088] The degree of joint inflammation was evaluated by the arthritis index. Except for the right hind foot at the induction site, each paw was scored according to the degree of lesions in the other three uninjected limbs and whether there was inflammation between the toes, with a score of 0 to 4. The cumulative score was the arthritis index of each rat. The results are shown in Figure 5 (C) shown.

[0089] 0 points: no evidence of erythema and swelling;

[0090] 1 point: erythema and mild swelling limited to the midfoot or ankle joint;

[0091] 2 points: erythema and mild swelling extending from the ankle joint to the midfoot;

[0092] 3 points: erythema and mild swelling spread from the ankle to the joint;

[0093] 4 points: erythema and severe swelling involving the ankles, feet, and toes;

[0094] From Figure 5 As shown, they were divided into five groups: control group, RA group, HPG group, BP-SI@HPG group and BP-SI@HP-G+NIR group. Figure 5 (A) shows that on the 0th day after modeling, the right hind paws of SD rats except the blank group were all in a red and swollen state. After 7, 14, 21 and 28 days of treatment, the swelling degree of the right hind paws of SD rats in the BP-SI@HP-G+NIR group was less than that in the BP-SI@HPG group, the HPG group and the RA group. Figure 5 (B) and Figure 5 (C) shows the changes in the swelling value of the right hind paw of rats and the evaluation of the arthritis index. It can be seen that the comparison before and after medication shows that the swelling value and arthritis evaluation index of the rats' paws have decreased. The paw swelling value of the BP-SI@HP-G+NIR group was 8.83 mm before medication and 5.98 mm after 28 days of treatment. The arthritis evaluation index score was 4 points before medication and 0.83 points after 28 days of treatment. It can be concluded that the BP-SI@HP-G+NIR group has a better therapeutic effect on rheumatoid arthritis.

[0095] Example 7: Detection of the expression of inflammatory factors TNF-α, IL-6, and IL-17 in serum

[0096] Rats were anesthetized with 4% chloral hydrate, 4-5 mL of blood was collected from the lower abdominal aorta using a disposable syringe, centrifuged at 4000 r / min for 10 min, and the supernatant was collected and stored for later use. The concentrations of TNF-α, IL-6, and IL-17 in serum were detected by enzyme-linked immunosorbent assay (ELISA) in strict accordance with the operating instructions of the kit.

[0097] The basic steps are as follows:

[0098] The various reagents were brought to room temperature (18-25°C) to equilibrate for at least 30 minutes.

[0099] Solution preparation: Gradual dilution of standard products, 20 times concentrated washing solution diluted 20 times with distilled water for later use

[0100] Sample addition: Set up blank wells (blank control wells without adding samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50uL of standard samples of different concentrations to the standard wells on the enzyme-labeled plate, and add 50uL of the 5-fold diluted test sample to the test sample wells.

[0101] Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min.

[0102] Washing: Carefully peel off the sealing film, discard the liquid, spin dry, add 350uL of washing solution to each well, let it stand for 30s, discard and spin dry. After each wash, pat dry on filter paper and repeat washing 5 times.

[0103] Except for the blank wells, add 50uL of enzyme-labeled reagent, add the sample to the bottom of the well of the ELISA plate, try not to touch the well wall, and shake gently to mix.

[0104] Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min.

[0105] Washing: Carefully peel off the sealing film, discard the liquid, spin dry, add 350uL of washing solution to each well, let it stand for 30s, discard and spin dry. After each wash, pat dry on filter paper and repeat washing 5 times.

[0106] Color development: First add 50 uL of color developer A to each well, then add 50 uL of color developer B, gently shake to mix, and develop the color at 37℃ in the dark for 10 minutes.

[0107] Stop: Add 50uL of stop solution to each well to stop the reaction.

[0108] Determination: Use the blank well to set the zero value, and measure the absorbance value (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution. Figure 6 .

[0109] from Figure 6 (AC) It can be seen that the expression of inflammatory factors TNF-α, IL-6 and IL-17 increased after modeling, and the expression of inflammatory factors TNF-α, IL-6 and IL-17 can be inhibited after application. Among them, the expression ability of TNF-α, IL-6 and IL-17 in the BP-SI@HP-G+NIR group was significantly higher than that in other groups. Compared with the RA group, the inflammatory factors TNF-α in the BP-SI@HP-G+NIR group decreased from 346.9ng / mL to 265.4ng / mL, IL-6 decreased from 112.2pg / mL to 79.4pg / mL, and IL-17 decreased from 33ng / mL to 20.7ng / mL. It is confirmed that the composite hydrogel containing Tianshan Saussurea extract (BP-SI@HP-G+NIR) can achieve the anti-rheumatoid arthritis effect by inhibiting the expression of TNF-α, IL-6 and IL-17.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite hydrogel containing an extract of Saussurea involucrata, characterized in that: The invention comprises a hydrogel, black phosphorus nanosheets loaded with snow lotus extract, menthol and borneol; The black phosphorus nanosheets are surface-modified by polyethylene glycol.

2. The composite hydrogel containing Saussurea involucrata extract according to claim 1, characterized in that: The hydrogel is made of the following raw materials: hyaluronic acid, polyvinyl alcohol and glycerin.

3. The composite hydrogel containing Saussurea involucrata extract according to claim 1, characterized in that: The average size of the black phosphorus nanosheets is 100-200 nm.

4. The method for preparing a composite hydrogel containing an extract of Saussurea involucrata according to any one of claims 1 to 3, characterized in that: It includes the following steps: S1: taking black phosphorus nanosheets and preparing a black phosphorus nanosheet dispersion; S2: adding a polyethylene glycol diamine aqueous solution to the black phosphorus nanosheet dispersion to react, thereby preparing a polyethylene glycol-modified black phosphorus nanosheet dispersion; S3: centrifugally filter the polyethylene glycol-modified black phosphorus nanosheet dispersion prepared in step S2, take the precipitate, take the Tianshan Saussurea extract according to the amount, dissolve it in ethanol, add the solution to the precipitate, mix well, ultrasonicate, oscillate in the dark, centrifuge, and take the precipitate; The ratio of black phosphorus nanosheet precipitate to Saussurea involucrata extract is 1:(5-10); The precipitate was resuspended in ultrapure water to obtain a black phosphorus nanosheet solution loaded with the Saussurea involucrata extract; S4: Take a certain amount of hyaluronic acid, add a certain amount of ultrapure water, stir evenly, add polyvinyl alcohol according to the amount, stir evenly, then slowly add glycerol, stir evenly, and prepare a hydrogel; S5: Pour the black phosphorus nanosheet solution loaded with Saussurea tianshanica extract prepared in step S3 into the hydrogel in step S4, vortex and mix evenly, then add Saussurea tianshanica extract to the system, stir evenly, add menthol and borneol with a mass fraction of no more than 1% to the system, stir until mixed evenly, and a composite hydrogel containing Saussurea tianshanica extract can be prepared.

5. The method for preparing a composite hydrogel containing Saussurea involucrata extract according to claim 4, characterized in that: The mass ratio of hyaluronic acid, polyvinyl alcohol and glycerol in step S4 is 3:3:

2.

6. The method for preparing a composite hydrogel containing Saussurea involucrata extract according to claim 4, characterized in that: The preparation of the Saussurea involucrata extract used in step S3 is specifically as follows: Take the whole herb of Saussurea tianshanica, add ethanol in an amount of 15-20 times the weight of the whole herb of Saussurea tianshanica, soak for 10-20 days, filter, take the filtrate; and concentrate the filtrate to contain 1g of the original medicinal material per 1mL.

7. The method for preparing a composite hydrogel containing Saussurea involucrata extract according to claim 4, characterized in that: In step S5, the weight ratio of the black phosphorus nanosheet solution loaded with the Saussurea involucrata extract: the hydrogel: the Saussurea involucrata extract is 2:10:

1.

8. Use of the composite hydrogel containing Saussurea involucrata extract according to any one of claims 1 to 7 in the preparation of drugs for treating arthritis.

9. Use of the composite hydrogel containing Saussurea involucrata extract according to any one of claims 1 to 7 in the preparation of inflammatory factor inhibitors, characterized in that: The inflammatory factors include TNF-α, IL-6 and IL-17.

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