Codonopsis pilosula polysaccharide gel containing tripterygium glycosides ethosome as well as preparation method and application of codonopsis pilosula polysaccharide gel

Abstract

The invention discloses radix codonopsis polysaccharide gel containing tripterygium glycosides ethosome in the technical field of medicine and pharmacy. The radix codonopsis polysaccharide gel is prepared from tripterygium glycosides ethosome, a gel matrix, ultrapure water, a humectant, a thickening agent, a preservative and a hydrochloric acid solution. The ratio of the tripterygium glycosides ethosome to the gel matrix to the ultrapure water to the humectant to the thickening agent to the preservative is (0.5-2.5) ml: (150-1200) mg: (6-8) ml: (0.5-2) ml: (50-200) mg: (5-30) mg; the gel matrix is formed by cross-linking carboxymethylated chitosan and derivatized codonopsis pilosula polysaccharide, the mass ratio of the carboxymethylated chitosan to the derivatized codonopsis pilosula polysaccharide is (2: 1)-(4: 1), and the hydrochloric acid solution is used for adjusting the pH value of the codonopsis pilosula polysaccharide gel containing the tripterygium glycosides ethosome to be neutral. The radix codonopsis polysaccharide gel containing the tripterygium glycosides ethosome can be used as a medicine for resisting rheumatoid joints and other autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the technical fields of medicine and pharmacy, and particularly relates to a codonopsis pilosula polysaccharide gel containing triptolide liposomes, and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a highly disabling autoimmune disease characterized by morning stiffness of proximal interphalangeal joints, symmetrical swelling and pain, and limited mobility, with synovitis and cartilage destruction of joints as the main clinical manifestations. As the disease progresses, joint damage will ultimately lead to deformity and disability, seriously affecting the quality of life of patients. Currently, the drugs commonly used for the treatment of RA mainly include non-steroidal anti-inflammatory drugs, glucocorticoid drugs, traditional Chinese herbs such as Tripterygium wilfordii and Paeonia lactiflora. Among them, Tripterygium wilfordii has a significant effect on RA.

[0003] Tripterygium wilfordii Hook.f (TwHF) is the root of a woody vine plant of the genus Tripterygium in the Celastraceae family. Triptolide is an effective compound extracted from TwHF, and its activity is synergistically produced by components such as diterpenoids, triterpenoids, and alkaloids, and has significant anti-inflammatory and immunosuppressive effects, and is widely used in the clinical treatment of RA. However, the terpene and alkaloid components in triptolide have obvious immune toxicity, liver and kidney toxicity, reproductive system toxicity, and adverse reactions in the hematological and digestive systems, which limit its clinical application. Summary of the Invention

[0004] The present invention aims to provide a codonopsis pilosula polysaccharide gel containing triptolide liposomes, in which the gel matrix is formed by chemically modifying traditional Chinese medicine polysaccharides and forming Schiff base bonds through chemical cross-linking to achieve combined administration with triptolide liposomes, so as to solve the problem of large oral side effects of triptolide drugs.

[0005] A codonopsis pilosula polysaccharide gel containing triptolide liposomes in this solution includes triptolide liposomes, a gel matrix, ultrapure water, a humectant, a thickener, a preservative, and a hydrochloric acid solution; the ratio of triptolide liposomes, the gel matrix, ultrapure water, the humectant, the thickener, and the preservative is 0.5 - 2.5 ml: 150 - 1200 mg: 6 - 8 ml: 0.5 - 2 ml: 50 - 200 mg: 5 - 30 mg; the gel matrix is cross-linked by carboxymethylated chitosan and derivatized codonopsis pilosula polysaccharide, and the mass ratio of carboxymethylated chitosan to derivatized codonopsis pilosula polysaccharide is 2:1 - 4:1; the concentration of the hydrochloric acid solution is 0.01 - 0.1 mol / L, and the hydrochloric acid solution is used to adjust the pH of the codonopsis pilosula polysaccharide gel containing triptolide liposomes to neutral.

[0006] Furthermore, the tripterygium glycosides ethosomes include tripterygium glycosides, lecithin, cholesterol, vitamin E, ultrapure water and absolute ethanol; the ratio of tripterygium glycosides, lecithin, cholesterol, vitamin E, ultrapure water and absolute ethanol is 80 - 240 mg: 400 - 1200 mg: 80 - 240 mg: 12 - 56 mg: 11 - 18 ml: 2 - 9 ml.

[0007] Furthermore, the tripterygium glycosides ethosomes are prepared by the injection - ultrasound method or the thin - film dispersion method.

[0008] Furthermore, the lecithin is at least one of egg yolk lecithin, soybean phospholipid, phosphatidylinositol.

[0009] Furthermore, the humectant is at least one of hyaluronic acid, glycerol, propylene glycol, polyethylene glycol.

[0010] Furthermore, the thickener is at least one of sodium hyaluronate, hypromellose, sodium carboxymethylcellulose.

[0011] Furthermore, the preservative is at least one of ethyl paraben, potassium sorbate, benzalkonium bromide.

[0012] Furthermore, the particle size of the tripterygium glycosides ethosomes is 135 - 210 nm, and the encapsulation efficiency of triptolide in the tripterygium glycosides ethosomes is above 80%.

[0013] Furthermore, the codonopsis pilosula polysaccharide is extracted from the codonopsis pilosula of the campanulaceae family by the water extraction - alcohol precipitation method.

[0014] The preparation method of the codonopsis pilosula polysaccharide gel containing tripterygium glycosides ethosomes includes the following steps:

[0015] Step 1, extract the codonopsis pilosula polysaccharide from the codonopsis pilosula of the campanulaceae family and derivatize the codonopsis pilosula polysaccharide to obtain the derivatized codonopsis pilosula polysaccharide rich in aldehyde groups; carboxymethylate chitosan to obtain carboxymethylated chitosan rich in amino groups;

[0016] Step 2, dissolve the derivatized codonopsis pilosula polysaccharide and carboxymethylated chitosan in step 1 in ultrapure water according to the ratio, then add the tripterygium glycosides ethosomes, humectant, thickener, and preservative to the dissolved derivatized codonopsis pilosula polysaccharide solution and mix evenly; then pour it into the carboxymethylated chitosan solution and mix well, and adjust the pH to neutral to obtain the codonopsis pilosula polysaccharide gel containing tripterygium glycosides ethosomes.

[0017] Furthermore, the tripterygium glycosides ethosomes are prepared by the following method:

[0018] Step 1, stir and dissolve tripterygium glycosides, lecithin, cholesterol and vitamin E in absolute ethanol at 25 - 35 °C according to the ratio as the alcohol phase;

[0019] Step 2: At the same time, take ultrapure water as the aqueous phase, place it on a magnetic stirrer, seal it and heat it up to the same temperature as the alcohol phase for standby.

[0020] Step 3: Slowly drop the aqueous phase solution obtained in Step 2 into the alcohol phase solution and stir. After all the dropping is completed, continue stirring for 30 min, perform ultrasonic treatment, and then filter with a 0.22 μm filter membrane to obtain tripterygium glycosides ethosomes.

[0021] The codonopsis pilosula polysaccharide gel containing tripterygium glycosides ethosomes of the present application can be applied in drugs for treating rheumatoid arthritis and other autoimmune diseases, and the codonopsis pilosula polysaccharide participates in drug administration as a gel matrix.

[0022] The present invention has the following beneficial effects:

[0023] 1. The present invention uses ethosomes to encapsulate tripterygium glycosides. Compared with the ethanol aqueous solution of tripterygium glycosides, it has better stability and transdermal absorption. The prepared gel has a certain curative effect on rheumatoid arthritis, and there is no skin allergy phenomenon during use.

[0024] 2. In the gel prepared by the present invention, traditional Chinese medicine polysaccharide is used as the gel matrix. The lone pair of electrons contained in the amino nitrogen atom in the carboxymethylated chitosan structure attacks the positively charged carbon atom on the carbonyl group in the derivatized codonopsis pilosula polysaccharide to complete the nucleophilic addition reaction, forming an intermediate α-hydroxyamine compound, and then further dehydrating to form a Schiff base, which is then administered in combination with tripterygium glycosides to play the role of combining drug and adjuvant.

[0025] 3. The present invention uses gel to co-load ethosomes to encapsulate tripterygium glycosides for drug administration, which has good stability and transdermal absorption, has a certain curative effect on rheumatoid arthritis, and has less irritation and no skin allergy phenomenon during use.

[0026] 4. The tripterygium glycosides ethosome gel prepared by the present invention is a topical transdermal drug delivery preparation. The gel can reduce the irritation to the skin while having a certain sustained release effect, and the gel uses traditional Chinese medicine polysaccharide as the matrix and plays a certain medicinal effect at the same time, further enhancing the medicinal effect of the tripterygium glycosides ethosome gel on rheumatoid arthritis. Description of the Drawings

[0027] Figure 1 It is the particle size distribution diagram of the tripterygium glycosides ethosomes obtained in Example 2 of the present invention;

[0028] Figure 2 It is the diagram showing the change of the cumulative transdermal amount and skin retention amount of triptolide (TP), the active ingredient in TG ethanol aqueous solution, TG-Eths and TG-Eths@COP-Gel, with time;

[0029] Figure 3 The comparative diagram of the ankle joints of rats in each experimental group 21 days after drug administration;

[0030] Figure 4 The comparative diagram of the changes in the foot volume of rats in each group during the treatment process;

[0031] Figure 5 The comparative diagram of the changes in the joint scores of rats in each experimental group before and after drug administration;

[0032] Figure 6 The comparative diagram of the organ indices of rats in each experimental group;

[0033] Figure 7 The pathological sections of the synovial tissue of the ankle joints of rats in each experimental group;

[0034] Figure 8 The change diagram of inflammatory factors in the bodies of rats in each experimental group. Specific implementation manners

[0035] The following is a further detailed description through specific implementation manners:

[0036] The method for extracting codonopsis pilosula polysaccharide from the codonopsis pilosula plant of the campanulaceae family refers to the reference (Gao Di, Luo Cheng, Zhou Kai, et al. Optimization of the extraction process of bletilla striata polysaccharide by response surface methodology [J]. Strait Pharmaceutical Journal, 2024, 36(01): 30-35).

[0037] The method for derivatizing codonopsis pilosula polysaccharide to obtain aldehyde group-rich derivatized codonopsis pilosula polysaccharide refers to the reference (Zheng Qi, Bu Yan, Zhang Yufei, et al. Preparation and exploration of lignin-modified oxidized starch adhesives [J]. Light Textile Industry and Technology, 2021, 50(02): 14-16+22.).

[0038] The method for carboxymethylating chitosan (purchased from Beijing Solarbio Science & Technology Co., Ltd.) to obtain amino group-rich carboxymethyl chitosan refers to the reference (Cheng F, He J M, Yan T S, et al. Antibacterial and hemostatic composite gauze of N,O-carboxymethyl chitosan / oxidized regenerated cellulose [J]. RSC Advances, 2016, 6(97): 94429-94436.).

[0039] Example 1

[0040] Step 1: Weigh 500 mg of egg yolk lecithin, 100 mg of cholesterol, 12 mg of vitamin E, and 120 mg of tripterygium glycosides, dissolve them in 6 mL of absolute ethanol. Under the condition of a 30°C water bath, stir and dissolve them in a closed container to form an alcohol phase. At the same time, take 14 mL of ultrapure water as the water phase and place it on a magnetic stirrer. Heat it in a closed container to the same temperature as the alcohol phase, and slowly drip the water phase into the alcohol phase with a syringe. Stir at 600 r / min for 30 min to obtain crude tripterygium glycosides ethosomes.

[0041] Step 2: Ultrasonic the suspension obtained in Step 1 with a probe-type ultrasonic instrument for 3 min, with an ultrasonic power of 100 W and an intermittent ultrasonic working mode (ultrasonic for 3 s, intermittent for 3 s). Then filter it through a 0.22-μm filter membrane at room temperature to obtain tripterygium glycosides ethosomes.

[0042] Step 3: Dissolve 100 mg of derivatized codonopsis pilosula polysaccharide in 2 mL of ultrapure water, and add 1 mL of tripterygium glycosides ethosomes, 100 mg of sodium hyaluronate, 1 mL of glycerol, and 5 mg of ethylparaben to it, and mix them evenly.

[0043] Step 4: Dissolve 200 mg of carboxymethyl chitosan in 4 mL of ultrapure water. After complete dissolution, mix it evenly with the solution in Step 3. Adjust the pH to neutral with 0.01 mol / L hydrochloric acid solution and add 1 mL of ultrapure water, and stir evenly to obtain a codonopsis pilosula polysaccharide gel containing tripterygium glycosides ethosomes.

[0044] Example 2

[0045] Step 1: Weigh 800 mg of egg yolk lecithin, 240 mg of cholesterol, 12 mg of vitamin E, and 120 mg of tripterygium glycosides, dissolve them in 7.5 mL of absolute ethanol. Under the condition of a 30°C water bath, stir and dissolve them in a closed container to form an alcohol phase. At the same time, take 12.5 mL of ultrapure water as the water phase and place it on a magnetic stirrer. Heat it in a closed container to the same temperature as the alcohol phase, and slowly drip the water phase into the alcohol phase with a syringe. Stir at 800 r / min for 30 min to obtain crude tripterygium glycosides ethosomes.

[0046] Step 2: Ultrasonic the suspension obtained in Step 1 with a probe-type ultrasonic instrument for 3 min, with an ultrasonic power of 150 W and an intermittent ultrasonic working mode (ultrasonic for 3 s, intermittent for 3 s). Then filter it through a 0.22-μm filter membrane at room temperature to obtain tripterygium glycosides ethosomes.

[0047] Step 3: Dissolve 200 mg of derivatized codonopsis pilosula polysaccharide in 2 mL of water, and add 1 mL of tripterygium glycosides ethosomes, 50 mg of hypromellose, 2 mL of hyaluronic acid, and 5 mg of potassium sorbate to it, and mix them evenly.

[0048] Step 4, dissolve 400 mg of carboxymethylated chitosan in 4 mL of water, mix it evenly with the solution in step 3 after it is completely dissolved, add 0.05 mol / L hydrochloric acid solution to adjust the pH to neutral, add 1 ml of ultrapure water, and stir evenly to obtain a Codonopsis pilosula polysaccharide gel containing Tripterygium wilfordii polyglycosides.

[0049] Example 3

[0050] Step 1, weigh 1200 mg soybean lecithin, 180 mg cholesterol, 12 mg vitamin E and 120 mg tripterygium wilfordii polyglycosides and dissolve them in 6 mL anhydrous ethanol, and stir and dissolve them in a closed manner under a 30° C. water bath to form an alcohol phase. At the same time, take 14 mL of ultrapure water as the water phase and place it on a magnetic stirrer, heat it to the same temperature as the alcohol phase in a closed manner, slowly drop the water phase into the alcohol phase with a syringe, and stir at 1200 r / min for 30 min to obtain a crude tripterygium wilfordii polyglycosides alcohol plasmid.

[0051] Step 2, ultrasonicating the suspension obtained in step 1 for 3 minutes using a probe ultrasonic instrument, with an ultrasonic power of 300 W, an indirect ultrasonic working mode, ultrasonicating for 3 seconds, and resting for 3 seconds, and then filtering with a 0.22 μm filter membrane at room temperature to obtain the Tripterygium wilfordii polyglycosides plastid;

[0052] Step 3, dissolving 300 mg of derivatized Codonopsis pilosula polysaccharide in 2 mL of water, and adding 1 mL of Tripterygium wilfordii polyglycosides, 200 mg of sodium carboxymethyl cellulose, 2 mL of propylene glycol and 5 mg of benzalkonium bromide thereto and mixing evenly;

[0053] Step 4, dissolve 600 mg of carboxymethylated chitosan in 3 mL of water, mix it evenly with the solution in step 3 after it is completely dissolved, add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral, add 1 ml of ultrapure water, and stir evenly to obtain a Codonopsis pilosula polysaccharide gel containing Tripterygium wilfordii polyglycosides.

[0054] The above description of the present invention is only a partial embodiment, but the present invention is not limited to the above specific implementation. The above specific implementation is illustrative and not restrictive. All specific expansions of the materials and methods of the present invention, without departing from the scope of protection of the present invention and the claims, are within the scope of protection of the present invention.

[0055] The present invention utilizes ethosomes to encapsulate tripterygium wilfordii polyglycosides, thereby preparing tripterygium wilfordii polyglycoside ethosome gel. Since tripterygium wilfordii polyglycosides have the effects of treating rheumatoid arthritis, and have significant anti-inflammatory and immunosuppressive effects, and Codonopsis pilosula polysaccharide has significant antioxidant, anti-cancer and immune-regulating effects, the Codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycoside ethosomes prepared by the present invention also has the functions of anti-inflammatory, immune-regulating, treating rheumatoid arthritis and the like.

[0056] The particle size, encapsulation efficiency of triptolide ethosomes prepared by the method of the present invention, and the in vitro cumulative transdermal amount of the triptolide ethosome codonopsis pilosula polysaccharide gel prepared are evaluated by the following methods:

[0057] 1. Determination of the particle size of triptolide ethosomes

[0058] Take 200 μL of triptolide ethosomes, dilute it 20 times, and then use a Malvern laser particle size analyzer to measure the particle size and particle size distribution of the ethosomes; the particle size distribution of the triptolide ethosomes obtained in Example 2 of the present invention is as Figure 1 shown. From Figure 1 it can be seen that the particle size of the triptolide ethosomes is 135 - 210 nm, and the particle size distribution is relatively narrow.

[0059] 2. Determination of the encapsulation efficiency of triptolide ethosomes

[0060] Take 2.00 mL of triptolide ethosomes in an ultrafiltration tube, centrifuge at a centrifugal force of 6400×g for 40 min, take the lower layer solution of the ultrafiltration, dilute it to 1 mL with methanol, and detect it by high performance liquid chromatography injection to calculate the free drug m2.

[0061] Another precisely measure 2 mL of triptolide ethosomes, demulsify with methanol and make up the volume to a 5 mL volumetric flask, filter with a 0.22 μm filter membrane, and detect by HPLC to obtain m1. Calculate the drug encapsulation efficiency (Encapsulation efficiency, EE) according to the following formula.

[0062] EE(%) = (m1 - m2) / m1 × 100%

[0063] Table 1 shows the stability observation data of triptolide ethosomes placed at room temperature and 4°C. Through the observation of the storage stability, it shows that the preservation at 4°C is better than that at room temperature, and it can be stably stored for 1 month, and the change rate of the encapsulation efficiency is less than 10%.

[0064] Table 1 Results of stability investigation

[0065]

[0066] 3. In vitro transdermal experiment

[0067] After anesthetizing SD rats, depilate the abdominal skin with depilatory cream. After washing it clean with warm water, peel off the abdominal skin of the rats, and carefully remove the subcutaneous fat and connective tissue with a scraper. Wash it clean with normal saline, lay it flat on filter paper and blot dry, write a label and store it at -20°C. When used, thaw it naturally in normal saline and ensure that the rat skin is intact.

[0068] The Franz diffusion cell method was used for in vitro transdermal experiments. After the naturally thawed rat skin was blotted dry with filter paper, it was cut into an appropriate size and covered on the Franz diffusion cell with the stratum corneum facing up and the dermis facing the receiving chamber, and the dermis should be kept in close contact with the receiving solution. The receiving solution was 30% ethanol physiological saline. Air bubbles were exhausted, and the temperature was maintained at 37 ± 1 °C and the rotation speed was 300 r / min. After assembly, the skin was equilibrated for 30 min, and then TG ethanol aqueous solution (the TG ethanol aqueous solution was obtained by dissolving tripterygium glycosides in absolute ethanol, and the ratio of tripterygium glycosides to absolute ethanol was 120 mg: 7.5 mL), TG-Eths (the tripterygium glycosides ethosomes prepared in Example 2), and TG-Eths@COP-Gel (the codonopsis pilosula polysaccharide gel containing tripterygium glycosides ethosomes prepared in Example 2) with the same amount of tripterygium glycosides were evenly applied on the skin surface. Keeping the rotation speed and temperature, at 2, 4, 6, 8, 10, 12, and 24 h, all the receiving solutions were concentrated to an appropriate concentration, filtered through a 0.22 μm microporous membrane and then injected, and the peak areas of TP (triptolide) at each time period were measured by HPLC method, and the TP content was calculated. The cumulative permeation amount Qn of the drug can be calculated by the following formula.

[0069]

[0070] In the formula: Qn is the cumulative transdermal amount of the drug; V is the total volume of the receiving cell (8 mL); Cn is the mass concentration of the drug in the receiving cell at the nth sampling; Ci is the drug mass concentration detected before the nth sampling; Vi is the sampling volume; A is the effective diffusion area (2.26865 cm 2 ).

[0071] 4. Determination of skin retention amount

[0072] After the in vitro transdermal experiment was completed, the skin retention amount was determined with reference to the literature method (Chen Ji, Zhao Xiaoqian, Ma Yanqiao, et al., Preparation and in vitro transdermal effect of compound zedoary turmeric oil ethosomes gel [J]. Chinese Journal of New Drugs, 2018, 27(07): 830-838). The rat skin was taken out, the administration site was cut off and repeatedly rinsed with physiological saline until the drug on the skin surface was washed clean, soaked with 1.00 mL of methanol, vortexed for 3 min, sonicated in a water bath for 60 min, centrifuged at 12000 r / min for 30 min, then the rat skin was discarded, the supernatant was blown to 0.2 mL with nitrogen, filtered through a 0.22 μm microporous membrane and then injected, and the peak area of TP was measured by HPLC method. The skin retention amount Q of the drug S :

[0073]

[0074] In the formula: V is the volume after concentration of the extraction solution; C is the drug concentration after concentration of the extraction solution; A is the effective diffusion area (2.26865 cm2 )。

[0075] Figure showing the cumulative transdermal amount and skin retention amount of triptolide (TP), the active ingredient in the triptolide ethanol aqueous solution (TG ethanol aqueous solution, i.e., the TG ethanol aqueous solution is obtained by dissolving 120 mg of triptolide in 7.5 mL of absolute ethanol), triptolide ethosomes (TG-Eths), and the Codonopsis pilosula polysaccharide gel containing triptolide ethosomes obtained in Example 2 of the present invention, varying with time Figure 2 )。From Figure 2 It can be seen that the Q24 of TG-Eths is significantly higher than that of the TG ethanol aqueous solution, and the Q24 of TG-Eths@COP-Gel is significantly lower than that of TG-Eths. The application of ethosomes can increase the transdermal permeability of drugs, and the application of the gel dosage form can slow down the release of drugs, having a certain sustained-release effect. Moreover, the skin retention amount of TG-Eths is significantly greater than that of the TG ethanol aqueous solution, and the skin retention amount of TG-Eths@COP-Gel is significantly greater than that of TG-Eths. Compared with the TG ethanol aqueous solution, the skin retention amounts of TG-Eths and TG-Eths@COP-Gel are increased to 1 - 3 times respectively, indicating that TG-Eths@COP-Gel can increase the skin permeation amount of drugs by increasing the retention amount of drugs in the skin.

[0076] 5. Pharmacodynamic evaluation

[0077] Evaluate the pharmacodynamics of a Codonopsis pilosula polysaccharide gel containing triptolide ethosomes. Select SD rats that have successfully established a rheumatoid arthritis model, all of which are male rats aged 5 - 6 weeks. The arthritis index ≥ 4 indicates successful modeling. The joint index scoring criteria are as follows:

[0078] Table 2 Joint index scoring criteria

[0079]

[0080] Set 8 healthy rats as the control group (Control group), and the remaining successfully modeled rats were randomly divided into a model group (Model), a diclofenac positive drug group (DDE), a high-dose group of TG-Eths@COP-Gel (TGG-H), a medium-dose group of TG-Eths@COP-Gel (TGG-M), a low-dose group of TG-Eths@COP-Gel (TGG-L), and a Codonopsis pilosula polysaccharide gel matrix group (COP-Gel, with the same dosage as the high-dose group, without triptolide ethosomes), with 8 rats in each group. The control group and the model group were smeared with normal saline every day and wrapped with adhesive plaster. The other drug administration groups were smeared with the corresponding topical drugs and wrapped with adhesive plaster, once a day. Administration started on the 14th day after the first immunization and lasted for 21 days.

[0081] Using the foot volume and joint index score as the criteria, the efficacy was preliminarily evaluated. During the experiment, the body weights of rats in each group were measured at 0 d (before modeling), 7 d, 14 d (before drug administration), 21 d, 28 d, and 35 d. The volume of the left hind paw of rats was measured by the foot volume drainage method, and the local inflammation changes were reflected by the foot volume. The joint scores were also evaluated according to the redness and swelling degrees of the ankle and toe joints of the front and hind limbs of rats and the affected joint index.

[0082] Meanwhile, the efficacy was further evaluated by calculating the thymus and spleen indices of rats, as well as the pathological analysis of the ankle joint and the changes of in vivo inflammatory factors. After the drug administration was completed, the rats were sacrificed, and the thymus and spleen of rats in each group were weighed to calculate the corresponding organ indices. The left hind ankle joint was dissected and fixed in 4% paraformaldehyde for more than 16 h, decalcified with 10% EDTA, dehydrated, infiltrated with wax, embedded, and sectioned routinely. After HE staining, the pathological changes of the ankle joint synovial tissue were observed under a microscope. Heparin sodium was used for anticoagulation, blood was collected from the orbital cavity, the supernatant was taken after centrifugation at 4500 r / min for 15 min, and stored at -20 °C. The expression levels of IL-1β, IL-4, and TNF-α inflammatory factors in the plasma of rats in each group were detected using an ELISA kit.

[0083] Test results: Figure 3 This is a representative picture of the ankle joint of rats in each experimental group after 21 days of drug administration. After 21 days of treatment, compared with the Control group, the ankle joint swelling in the Model group was obvious, and the joint swelling in the TGG-H, TGG-M, and TGG-L groups was alleviated. Figure 4 This shows the changes in the foot volume of rats in each group during the treatment process. Three days after the primary immunization, the modeled rats were in the acute attack stage, and the foot volume was significantly increased compared with the Control group. From 3 to 7 days, due to the autoimmunity of rats, the foot volume of rats in the Model group decreased. On the 7th day, with the secondary immunization, secondary swelling occurred in some rats, and the foot volume reached the highest value 21 days after modeling. At the end of the treatment, the foot volumes of all rats except the COP-Gel group were significantly lower than that of the Model group (P<0.05, P<0.01). The experimental results indicate that TG-Eths@COP-Gel can significantly alleviate the joint swelling of rats, and the therapeutic effect is concentration-dependent.

[0084] Figure 5 This shows the changes in the joint scores of rats in each experimental group before and after drug administration. It can be seen that the joint scores of each modeled group reached the highest value on the 21st day, which is consistent with the results of the foot volume. At the end of the treatment, the joint scores of each treatment group were significantly different from those of the Model group (P<0.01), and the joint score of the TGG-H group was the lowest. This indicates that the TGG-H group has a good alleviating effect on the joint swelling of CIA rats. Figure 5 ​

[0085] Figure 6 It is for the comparison of the organ indices of rats in each group. The level of the body's immunity is closely related to the development status of the thymus and spleen in the body, and the organ index is a preliminary indicator for measuring the development of the thymus and spleen. Figure 6 A shows the measurement results of the thymus index: The thymus index of the Model group was significantly higher than that of the Control group. The TGG-M group and the TGG-L group had no obvious effect on the decrease of the thymus index, and the other groups could significantly reduce the thymus index. Figure 6 B shows the measurement results of the spleen index: Compared with the Control group, the spleen index of the Model group increased significantly. The treatments of other drug-administered groups could significantly reduce the spleen index of rats. From the test results, the application of TGG-H can improve the immunity of RA rats to a certain extent. Compared with the Control group, **P<0.01; compared with the Model group, #P<0.05, ##P<0.01

[0086] Figure 7 It is the HE staining results of the ankle joint synovium of rats in each group. The joint surface of the rats in the Control group was smooth, covered with a thin layer of synovial cells, and no obvious synovial tissue hyperplasia and inflammatory cell infiltration were seen. Compared with the Control group, the synovium and blood vessels of the rats in the Model group were significantly hyperplastic, and there were large-scale damages, the tissue arrangement was disordered and a large number of inflammatory cell infiltrations were seen. After drug administration, it could be seen that in the DDE group, the TGG-H group, the TGG-M group, and the TGG-L group, the synovium of the joints became thinner to varying degrees, the damages decreased, and the inflammatory cell infiltration decreased with the increase of the administration concentration of TG-Eths@COP-Gel. At the same time, the cell arrangement also tended to be neat. It shows that both DDE and TG-Eths@COP-Gel can improve the synovial hyperplasia and inflammatory infiltration of the joints of CIA rats, and the treatment effect of the TGG-H group is the best. The synovial hyperplasia of the COP-Gel group showed villous shape and extended deep into the joint cavity, and there was a certain cell infiltration in the villi, but the inflammation was also relieved compared with the Model group, indicating that COP-Gel can also relieve the inflammation of CIA rats.

[0087] Figure 8 It is the change level of inflammatory factors in rats. The change of the IL-1β content in the plasma of rats is as Figure 8 shown in A. The levels of pro-inflammatory factors in the DDE group, the TGG-H group, the TGG-M group, and the COP-Gel group were all significantly lower than those in the Model group. The experimental results show that TG-Eths@COP-Gel can relieve the inflammation caused by RA and shows a dose-dependent manner. The change of the IL-4 content in the plasma of rats is as Figure 8As shown in Figure B, compared with the Control group, the content of anti-inflammatory factors in the Model group was significantly decreased, and the IL-4 content in the DDE group and the TGG-H group was significantly increased compared with the Model group. The experimental results showed that TG-Eths@COP-Gel could up-regulate the content of anti-inflammatory factors in CIA rats and play a therapeutic role in RA. The change in the content of TNF-α in rat plasma was as Figure 8 shown in Figure C. There was a significant difference between the Model group and the Control group. The TNF-α content in the TGG-H group and the TGG-M group was significantly decreased compared with the Model group. The experimental results showed that the therapeutic effect of TGG-H was the best, and the level of inflammatory factors tended to be that of the Control group; compared with the Control group, **P<0.01; compared with the Model group, #P<0.05, ##P<0.01.

[0088] It can be seen from the test results that the gel prepared by the present invention has a certain curative effect on rheumatoid arthritis. At the same time, COP-Gel can relieve joint inflammation, indicating that COP-Gel plays a synergistic role in the treatment of RA, suggesting that the concept of "combining medicine and excipient" has been applied.

Claims

1. A codonopsis polysaccharide gel containing tripterygium wilfordii polyglycosides, characterized in that: The invention comprises tripterygium wilfordii polyglycosides, a gel matrix, ultrapure water, a moisturizer, a thickener, a preservative and a hydrochloric acid solution; the ratio of the tripterygium wilfordii polyglycosides, the gel matrix, the ultrapure water, the moisturizer, the thickener and the preservative is 0.5-2.5 ml: 150-1200 mg: 6-8 ml: 0.5-2 ml: 50-200 mg: 5-30 mg; the gel matrix is ​​cross-linked with carboxymethylated chitosan and derivatized codonopsis pilosula polysaccharide, and the mass ratio of the carboxymethylated chitosan to the derivatized codonopsis pilosula polysaccharide is 2:1-4:1; the concentration of the hydrochloric acid solution is 0.01-0.1 mol / L, and the hydrochloric acid solution is used to adjust the pH value of the codonopsis pilosula polysaccharide gel containing the tripterygium wilfordii polyglycosides to neutral.

2. The codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to claim 1, characterized in that: The tripterygium wilfordii polyglycosides plastids include tripterygium wilfordii polyglycosides, lecithin, cholesterol, vitamin E, ultrapure water and anhydrous ethanol; The ratio of tripterygium wilfordii polyglycosides, lecithin, cholesterol, vitamin E, ultrapure water and anhydrous ethanol is 80-240 mg: 400-1200 mg: 80-240 mg: 12-56 mg: 11-18 ml: 2-9 ml.

3. The codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to claim 2, characterized in that: The lecithin is at least one of egg yolk lecithin, soybean lecithin, and phosphatidylinositol.

4. The codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to claim 3, characterized in that: The moisturizing agent is at least one of hyaluronic acid, glycerol, propylene glycol and polyethylene glycol.

5. The codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to claim 4, characterized in that: The thickener is at least one of sodium hyaluronate, hydroxypropyl methylcellulose and sodium carboxymethyl cellulose.

6. The codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to claim 5, characterized in that: The preservative is at least one of ethylparaben, potassium sorbate and benzalkonium bromide.

7. The codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to claim 6, characterized in that: The particle size of the tripterygium wilfordii polyglycoside plastid is 135-210 nm, and the encapsulation rate of triptolide in the tripterygium wilfordii polyglycoside plastid is above 80%.

8. The method for preparing a codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step 1, extracting codonopsis polysaccharide from Codonopsis pilosula of Campanulaceae, and derivatizing the codonopsis polysaccharide to obtain derivatized codonopsis polysaccharide rich in aldehyde groups; and carboxymethylating chitosan to obtain carboxymethylated chitosan rich in amino groups; Step 2, dissolving the derivatized Codonopsis pilosula polysaccharide and carboxymethylated chitosan in the above step 1 in ultrapure water according to the ratio, and then adding tripterygium wilfordii polyglycosides, moisturizer, thickener, and preservative to the dissolved derivatized Codonopsis pilosula polysaccharide solution and mixing evenly; then pouring the carboxymethylated chitosan solution into the solution and mixing evenly, adjusting the pH to neutral, and obtaining a Codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides.

9. The method for preparing a codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to claim 8, characterized in that: The Tripterygium wilfordii polyglycosides plastid is prepared by the following method: Step 1, according to the ratio, stir and dissolve tripterygium wilfordii polyglycosides, lecithin, cholesterol and vitamin E in anhydrous ethanol at 25-35° C. as an alcohol phase; Step 2, simultaneously placing ultrapure water as the water phase on a magnetic stirrer, heating it in a sealed manner to be isothermal with the alcohol phase, and setting aside; Step 3, slowly drop the aqueous solution obtained in step 2 into the alcohol solution and stir, continue stirring for 30 minutes after all the dropwise addition is completed, filter with a 0.22 μm filter membrane after ultrasonic treatment, and obtain the alcohol plastid of Tripterygium wilfordii polyglycosides.

10. Use of the codonopsis pilosula polysaccharide gel containing tripterygium wilfordii polyglycosides according to any one of claims 1 to 7 in the preparation of drugs for treating rheumatoid arthritis and other autoimmune diseases.

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