Application of total alkaloids from wild poppy (Papaver nudicaule L.) in the preparation of drugs for treating rheumatoid arthritis

Abstract

This invention discloses the application of total alkaloids from *Papaver nudicaule* L. in the preparation of a medicament for treating rheumatoid arthritis. This invention also provides a pharmaceutical composition containing total alkaloids from *Papaver nudicaule* L. and its preparation method. Pharmacodynamic experiments show that the ointment containing total alkaloids from *Papaver nudicaule* L. has a significant therapeutic effect on adjuvant-induced arthritis model rats: it can effectively reduce joint swelling, inhibit the levels of inflammatory factors such as IL-6, IL-1β, TNF-α, PGE2, and NO in serum, increase SOD activity and GSH concentration, reduce MDA levels, and improve joint tissue pathological damage. The extraction method of total alkaloids from *Papaver nudicaule* L. described in this invention is simple, the ointment preparation process is mature, the efficacy is comparable to commercially available diclofenac sodium ointment, and it is non-addictive, suitable for long-term topical use by patients with rheumatoid arthritis, and has good clinical application prospects and industrial production value.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine and pharmaceutical technology, specifically relating to the application of total alkaloids from wild poppy (Papaver nudicaule L.) in the preparation of drugs for treating rheumatoid arthritis, especially topical preparations and their preparation methods. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by destructive and symmetrical joint lesions and synovitis. Patients often experience persistent joint pain, swelling, and stiffness, which not only reduces motor function but can also lead to various complications affecting the cardiovascular, pulmonary, and skeletal systems, severely impacting their lives and work. Currently used clinical medications, such as nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and biologics, can control the progression of inflammation to some extent, but long-term use often results in adverse reactions such as gastrointestinal damage, liver and kidney dysfunction, and immunosuppression. Furthermore, some patients experience poor efficacy or drug resistance, highlighting numerous unresolved challenges in clinical treatment. Against this backdrop, naturally derived drugs, with their unique advantages, are gradually becoming a research hotspot in the field of RA treatment. Compared to chemical drugs, the advantages of natural drugs lie in their multi-target, multi-pathway holistic regulatory mechanism: they can not only effectively inhibit the release of inflammatory factors and reduce synovial inflammation, but also regulate the body's immune balance and delay articular cartilage degeneration, achieving a comprehensive treatment goal that addresses both the symptoms and the root cause. Furthermore, natural drugs are derived from nature and have been validated through long-term human use, demonstrating high safety and a relatively low incidence of adverse reactions, making them more suitable for the long-term medication needs of RA patients. Therefore, developing novel RA treatments from natural sources is of great significance.

[0003] Wild poppy (Papaver nudicaule L.) is a perennial herb belonging to the genus Papaver in the family Papaveraceae. Its main active ingredient is total alkaloids. Modern pharmacological studies have confirmed that wild poppy total alkaloids have significant anti-inflammatory, analgesic, and immunomodulatory pharmacological activities, without addictive properties, showing good potential in the treatment of inflammation-related diseases. However, the specific efficacy and mechanism of action of its topical preparations against rheumatoid arthritis are not yet clear, and systematic animal experiments are lacking. Adjuvant arthritis (AA) rat model is a classic animal model that simulates the pathological characteristics of human RA. After modeling, the rats exhibit joint swelling, inflammatory factor disorder, and other manifestations highly consistent with RA patients, making it suitable for screening the efficacy and exploring the mechanisms of anti-RA drugs. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a new use of total alkaloids from wild poppy in the preparation of drugs for treating rheumatoid arthritis, especially the preparation method of topical preparations and the verification of their efficacy.

[0005] The use of total alkaloids of wild poppy (Papaver nudicaule L.) or pharmaceutically acceptable salts thereof in the preparation of medicines for the treatment and / or prevention of rheumatoid arthritis.

[0006] The drug is a topical preparation.

[0007] The topical preparations are ointments, creams, gels, patches, sprays, lotions, or liniments.

[0008] The total alkaloids of the wild poppy were prepared by the following method: the whole wild poppy plant was extracted with an acidic solvent, alkalized, and then extracted with an organic solvent.

[0009] The acidic solvent is an aqueous solution of hydrochloric acid, sulfuric acid, or acetic acid; the organic solvent is chloroform, dichloromethane, ethyl acetate, or a mixture thereof.

[0010] The symptoms of rheumatoid arthritis include joint swelling, elevated inflammatory factors, oxidative stress imbalance, or pathological damage to joint tissues.

[0011] The total alkaloids of the wild poppy exert their effects through one or more of the following mechanisms:

[0012] (a) Reduce joint swelling; (b) Reduce serum levels of IL-6, IL-1β, TNF-α, PGE2 and / or NO; (c) Increase serum SOD activity and / or GSH concentration; (d) Reduce serum MDA levels; (e) Improves joint tissue pathological damage.

[0013] A pharmaceutical composition for treating rheumatoid arthritis, comprising a therapeutically effective amount of total alkaloids from wild poppy and pharmaceutically acceptable excipients.

[0014] The total alkaloids of the wild poppy in the composition have a mass percentage of 0.1%-20%, preferably 0.5%-10%, and more preferably 1%-5%.

[0015] The pharmaceutically acceptable excipients are selected from one or more of the following: matrix, emulsifier, thickener, preservative, antioxidant, and transdermal penetration enhancer.

[0016] The matrix is ​​one or more of the following: petrolatum, lanolin, paraffin, silicone oil, carbomer, sodium alginate, and gelatin.

[0017] The dosage form of the pharmaceutical composition is an ointment.

[0018] The base of the ointment is a mixture of petrolatum and lanolin in a mass ratio of (5-9):(1-5), preferably 7:3.

[0019] A method for preparing the pharmaceutical composition includes the following steps: (a) Extraction of total alkaloids from wild poppy; (b) Heat the matrix to a molten state; (c) Add the total alkaloids of wild poppy obtained in step (a) to the molten matrix and stir to mix well; (d) Cooling, and you get the product.

[0020] The extraction method of total alkaloids from poppy described in step (a) includes: taking the whole poppy plant, crushing it, extracting it by reflux with an alcohol solvent, and concentrating it to obtain an extract; dissolving the extract in acidic water, adjusting the pH to acidic, and extracting it with an organic solvent to remove impurities; adjusting the pH of the remaining acidic water to alkaline with alkali, extracting it with an organic solvent, and concentrating it to obtain the final product.

[0021] The alcohol solvent is ethanol or methanol; the acid solution is hydrochloric acid aqueous solution with pH adjusted to 1.0-3.0; the alkali is ammonia solution with pH adjusted to 9.0-12.0.

[0022] A method for treating rheumatoid arthritis, comprising applying the total alkaloids of the wild poppy or the pharmaceutical composition thereof to the affected area.

[0023] The application frequency is 1-3 times daily, with each application amount being 0.1-0.5 g / cm³. 2 .

[0024] The application of total alkaloids from wild poppy in the preparation of drugs that improve oxidative stress damage associated with rheumatoid arthritis, wherein the improvement is manifested by increasing SOD activity and GSH concentration, and decreasing MDA level. The total alkaloids from wild poppy contain a total alkaloid content of not less than 80%, preferably not less than 85%, and more preferably not less than 88%.

[0025] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: This discovery marks the first time that a topical preparation of total alkaloids from wild poppy has shown significant therapeutic effects on rheumatoid arthritis, filling a gap in this technological field.

[0026] The total alkaloids of wild poppy exert their anti-RA effects through multiple pathways, including inhibiting the release of inflammatory factors (IL-6, IL-1β, TNF-α, PGE2, NO), regulating oxidative stress balance (increasing SOD and GSH, and decreasing MDA), and improving joint tissue pathological damage. This demonstrates the advantages of natural drugs with multiple targets.

[0027] Compared with oral or injection administration, topical preparations can avoid the first-pass effect and systemic adverse reactions, and are suitable for long-term use in RA patients.

[0028] The extraction method is mature, and the preparation process is simple, which is suitable for industrial production. The total alkaloids of Papaver nudicaule are different from traditional poppies and do not have addictive properties, with good safety. Description of the Drawings

[0029] Figure 1 : Standard curve of sinomenine.

[0030] Figure 2 : Comparison photos of the foot morphology of rats in each group.

[0031] Figure 3 : Histogram comparing the toe swelling degrees of rats in each group.

[0032] Figure 4 : Histogram comparing the ankle swelling degrees of rats in each group.

[0033] Figure 5 : HE staining photos of the ankle joint tissues of rats in each group (×200), where A is the blank group, B is the model group, C is the diclofenac group, D is the low-dose TAPN group, E is the medium-dose TAPN group, and F is the high-dose TAPN group. Detailed Implementation Modes

[0034] Example 1 Experimental Materials 36 SPF-grade male SD rats, with a body weight of (180 - 200) g, were provided by the Experimental Animal Center of China Three Gorges University. The animal license number is: SCXK (E) 2022 - 0061; the certificate number is: No. No. 42254221791201120. Feeding conditions: SPF-grade animal house, temperature (22 ± 1) °C, relative humidity (55 ± 15)%, free access to water and food. After one week of adaptive feeding, the experiment was carried out.

[0035] Drugs and Reagents The whole herb of Papaver nudicaule was collected from Kaikukang Town, Tahe County, Daxing'anling Region, China in early July 2024 (G331 - gravelly hillside, 339.12 m). It was identified as Papaver nudicaule L. of the Papaveraceae family by Professor Wang Yubing of the College of Biology and Pharmaceutical Sciences, China Three Gorges University.

[0036] Voltaren ointment (batch number: MR6N, Sino-American Tianjin SmithKline Pharmaceutical); Freund's incomplete adjuvant (batch number: F5506, Sigma-Aldrich); glutathione (GSH) (batch number: A006-1-1), malondialdehyde (MDA) (batch number: A003-1), and SOD (batch number: A001-3) kits were all purchased from Nanjing Jiancheng Bioengineering Institute; rat interleukin-6 (IL-6) ELISA kit (catalog number: 1310602), rat tumor necrosis factor-α (TNF-α) ELISA kit (catalog number: 1317202), and rat interleukin-1β (IL-1β) ELISA kit (catalog number: 1310122) were all purchased from Dakowei Biotechnology Co., Ltd.

[0037] High-speed refrigerated centrifuge (model: FC5515R, Eppendorf GmbH, Germany); microplate reader (model: INFINITE200PR, Tecan GmbH, Switzerland); cryogenic grinder (model: JXFSPRP-CL, Shanghai Jingxin Industrial Development Co., Ltd.).

[0038] Methods—Extraction, content determination and ointment preparation of total alkaloids from wild poppy Extraction of TAPN: Take 10 kg of whole wild poppy plant, crush it, and extract it three times by reflux with 95% ethanol. Concentrate the extract under reduced pressure to obtain an extract. Dissolve and acidify the extract with 2% hydrochloric acid, adjust the pH to 2.0, filter, and extract the acid water three times with chloroform at a 1:1 volume ratio. Adjust the pH of the remaining acid water solution to 11.0 with ammonia water, and then extract it six times with chloroform. Concentrate under reduced pressure to obtain the total alkaloid (TAPN) extract.

[0039] Determination of total alkaloids in TAPN extract: Using sinomenine as a reference, a standard curve for sinomenine was plotted using the bromocresol green acid dye colorimetric method. The content of total alkaloids in TAPN extract was calculated based on the regression equation of the standard curve and the dilution factor.

[0040] Preparation of TAPN ointment: A petrolatum-lanolin (7:3) matrix was used as the base. After melting at 85°C, TAPN extract powder was added, stirred until homogeneous, and then cooled to room temperature to obtain the TAPN ointment. For low, medium, and high doses of TAPN ointment, 0.56, 1.13, and 2.26 g of TAPN extract were added per 100 g of base, respectively.

[0041] Preparation of reagents Freund's complete adjuvant (CFA): Add 6 mg of inactivated Mycobacterium tuberculosis powder to 1 mL of Freund's incomplete adjuvant, and aspirate back and forth with a syringe until complete emulsification is achieved, finally obtaining a Mycobacterium tuberculosis suspension with a concentration of 6 mg / mL.

[0042] Animal modeling, grouping and administration After one week of acclimatization feeding, rats were randomly divided into 6 groups (n=6) according to their body weight. The right hind paw of each rat was routinely disinfected. In the control group, 0.1 mL of 0.9% saline was injected intradermally into the right hind paw of each rat. In the other 5 groups, 0.1 mL of Freund's complete adjuvant was injected intradermally into the right hind paw of each rat (recorded as day 0). On day 7, except for the control group, 0.05 mL of Freund's complete adjuvant was injected into the same location to enhance inflammation. On day 8, rats were randomly divided into 5 groups based on body weight and paw swelling, making a total of 6 groups including the control group: control group, model group, positive control group, and low, medium, and high dose groups of total alkaloids ointment from *Papaver somniferum*. From day 8 to day 32 after model establishment, rats in each group received drug treatment.

[0043] (1) Control group: Apply a base ointment without the drug, covering an area of ​​approximately 0.2 g / cm², twice a day. (2) Model group: No ointment applied. (3) Positive control group: Apply 1.0% diclofenac ointment to an area of ​​approximately 0.2 g / cm². 2 2 times / day (4) Low-dose TAPN ointment group: 0.5% TAPN ointment was applied at a concentration of 0.2 g / cm³. 2 2 times / day (5) Medium-dose group of TAPN ointment: Apply 1.0% TAPN ointment, 0.2 g / cm 2 2 times / day (6) High-dose TAPN ointment group: 2.0% TAPN ointment was applied at a concentration of 0.2 g / cm³. 2 2 times / day.

[0044] detection indicators Foot and ankle swelling On day 0, day 8, and every other day, the diameter of the right hind paw and right hind paw ankle joint of the rats were measured using vernier calipers. Two measurements were taken at the same location on the same toe and ankle each time, and the average value was recorded until the end of the experiment. The difference between the degree of paw swelling and ankle swelling before and after inflammation was used to represent the degree of swelling of the primary lesion in the rats, to observe the effect of the treatment drug on the primary lesion.

[0045] Foot swelling (mm) = thickness after swelling - thickness before swelling; Ankle swelling (mm) = thickness after swelling - thickness before swelling.

[0046] ELISA method for detecting cytokine levels in rat serum Whole blood was collected from the aorta of rats after anesthesia. The blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3000 r / min for 10 min at 4 ℃. Serum was collected and stored at -80 ℃. Serum TNF was measured according to the kit instructions. α, IL 6. IL 1β, PGE2 and NO levels.

[0047] Detection of oxidative stress-related indicators in rat serum According to the kit instructions, the concentrations of SOD, GSH, and MDA in the serum of rats in each group were detected using an ELISA reader.

[0048] Ankle joint histopathological observation After the rats were euthanized by anesthesia, the right hind foot ankle joint tissue was taken and quickly fixed and preserved in 4% paraformaldehyde solution. The hematoxylin-eosin staining (HE staining) method was used for histopathological observation of the ankle joint tissue.

[0049] Data were statistically analyzed using GraphPad Prism software. Data for each group are expressed as mean ± standard deviation. x ± s This indicates that one-way ANOVA was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0050] Determination of alkaloid content in TAPN extract The standard curve of sinomenine is as follows Figure 1 As shown, its regression equation is Y = 5.17X - 0.0088, r 2 =0.9994, linear range 0.04–0.14 mg / mL. After three repeated experiments, the extraction rate of TAPN from the dried whole herb of *Papaver somniferum* was 0.27 ± 0.06%, and the total alkaloid content in the TAPN extract was 88.63 ± 3.6%. Based on this, the alkaloid contents in the prepared low, medium, and high dose TAPN ointments were calculated to be 0.5%, 1%, and 2%, respectively.

[0051] Effect on paw edema in AA rats Effects of TAPN on paw morphology in AA rats, such as Figure 2 As shown.

[0052] The effect of TAPN on paw edema in AA rats, as follows: Figure 3 As shown in Table 1.

[0053] Compared with the control group, the paw swelling of rats in all groups was significantly increased on day 8 after modeling (P<0.001), indicating that the adjuvant arthritis model was successfully established. Compared with the model group, the paw swelling of rats in all groups was significantly increased on day 9 after TAPN administration (P<0.001), indicating that the adjuvant arthritis model was successfully established. Compared with the model group, the paw swelling of rats at medium and high doses of TAPN decreased significantly from day 9 after administration (P<0.05), and the inhibitory effect continued until the end of the experiment, with an effect intensity comparable to that of the positive control drug.

[0054] In conclusion, TAPN can effectively inhibit paw swelling in adjuvant-induced arthritis model rats, and this effect is dose-dependent.

[0055] Table 1. Effect of TAPN ointment on paw swelling in AA rats with arthritis [ x ± s [n=6]

[0056] Note: Compared with the blank group, ### P <0.001; compared with the model group P <0.05、 P <0.01、 P <0.001.

[0057] Note: ### P <0.001 vs. blank group; P <0.05, P <0.01, P <0.001 vs. modelgroup. Effects on ankle swelling in AA rats Compared with the control group, the ankle swelling of rats in all groups was significantly increased on day 8 after modeling (P<0.001), indicating that the adjuvant-induced arthritis model was successfully established. Compared with the model group, the swelling of rat paws at medium and high doses of TAPN decreased significantly starting on day 9 after administration (P<0.05), and showed a very significant inhibitory effect on day 15 (P<0.01). The inhibitory effect continued until the end of the experiment, and the intensity of the effect was comparable to that of the positive control drug.

[0058] In conclusion, TAPN ointment effectively inhibited ankle swelling in an adjuvant-induced arthritis model rat, exhibiting a dose-dependent effect. The results are shown in Table 2. Figure 4 .

[0059] Table 2 Effect of TAPN ointment on ankle thickness in rats with adjuvant-induced arthritis [ x ± s [n=6]

[0060] Note: Comparison with model group p<0.05 p<0.01 p<0.001; compared with the blank control group, the model group, # p<0.05 ## p<0.01 ### p<0.001) Levels of cellular inflammatory factors in the serum of AA rats Compared with the control group, the levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, and inflammatory mediators PGE2 and NO in the serum of rats in the model group were significantly increased (P<0.01), indicating that the adjuvant-induced arthritis model was accompanied by a severe systemic inflammatory response. The medium and high doses of TAPN significantly inhibited the levels of IL-6, IL-1β, TNF-α, and inflammatory mediators PGE2 and NO (P<0.001~P<0.05); the low dose of TAPN significantly reduced the levels of IL-6, IL-1β, and PGE2 (P<0.05), but had a weaker inhibitory effect on TNF-α. The results are shown in Table 3.

[0061] Table 3 Effects of TAPN ointment on inflammatory factors in the serum of AA rats ( x ± s (n=6)

[0062] Note: Compared with the blank group ### P <0.001; compared with the model group P <0.05、 P <0.01、 P <0.001 3.5 Effects on oxidative stress in serum of AA rats Compared with the control group, the serum SOD activity and GSH concentration in the model group rats were significantly decreased (P<0.001~P<0.01), while the MDA level was significantly increased (P<0.001). Compared with the model group, high dose of TAPN significantly increased SOD activity and GSH concentration (P<0.01~P<0.05) and significantly decreased MDA concentration (P<0.01). The results are shown in Table 4.

[0063] Table 4. Effects of TAPN ointment on oxidative stress in serum of rats with arthritis ( x ± s (n=6)

[0064] Note: Compared with the blank group ### P <0.001; compared with the model group P <0.05、 P <0.01、 P <0.001.

[0065] Effects on the histopathology of AA rats Histopathological results showed that, compared with the control group, the model group exhibited significant histopathological damage, with disordered and disrupted structures and extensive infiltration of inflammatory cells, indicating successful model establishment. After drug intervention, the tissue damage in the positive control group and the low, medium, and high dose groups all showed varying degrees of improvement compared to the model group, with reduced inflammatory infiltration and more intact structures. Overall, the improvement showed a dose-dependent trend: the low-dose group showed only mild improvement; the medium-dose group showed more significant improvement; and the high-dose group showed the most significant repair effect on histopathological damage, with tissue structure approaching normal levels, demonstrating superior efficacy compared to the low and medium dose groups, and comparable to or better than the positive control group. In conclusion, TAPN ointment can significantly improve the histopathological damage induced by this model, with the high-dose group showing the best efficacy and a clear dose-response relationship.

Claims

1. Use of total alkaloids of wild poppy (Papaver nudicaule L.) or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment and / or prevention of rheumatoid arthritis.

2. The application according to claim 1, characterized in that, The drug is a topical preparation, which may be an ointment, cream, gel, patch, spray, lotion, or liniment.

3. The application according to claim 1, characterized in that, The total alkaloids of the wild poppy were prepared by the following method: the whole wild poppy plant was extracted with acidic solvent, alkalized, and then extracted with organic solvent. The acidic solvent is an aqueous solution of hydrochloric acid, sulfuric acid, or acetic acid; The alkalization process involves adjusting the pH value to 10-12 using ammonia. The organic solvent is chloroform, dichloromethane, ethyl acetate, or a mixture thereof.

4. The application according to claim 1, characterized in that, The symptoms of rheumatoid arthritis include joint swelling, elevated inflammatory factors, oxidative stress imbalance, or pathological damage to joint tissues; the total alkaloids of the wild poppy exert their effects through one or more of the following mechanisms: (a) Reduce joint swelling; (b) Reduce serum levels of IL-6, IL-1β, TNF-α, PGE2 and / or NO; (c) Increase serum SOD activity and / or GSH concentration; (d) Reduce serum MDA levels; (e) Improves joint tissue pathological damage.

5. A pharmaceutical composition for treating rheumatoid arthritis, characterized in that, It contains therapeutically effective amounts of total poppy alkaloids and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that, The total alkaloids of the wild poppy in the composition have a mass percentage of 0.1% to 20%, preferably 0.5% to 10%, and more preferably 1% to 5%. The pharmaceutically acceptable excipients are selected from one or more of the following: matrix, emulsifier, thickener, preservative, antioxidant, and transdermal penetration enhancer. The matrix is ​​one or more of the following: petrolatum, lanolin, paraffin, silicone oil, carbomer, sodium alginate, and gelatin; The dosage form of the pharmaceutical composition is an ointment; The base of the ointment is a mixture of petrolatum and lanolin in a mass ratio of (5~9):(1~5), preferably 7:

3.

7. A method for preparing the pharmaceutical composition according to any one of claims 5 to 6, characterized in that, Includes the following steps: (a) Extraction of total alkaloids from wild poppy; (b) Heat the matrix to a molten state; (c) Add the total alkaloids of wild poppy obtained in step (a) to the molten matrix and stir to mix well; (d) Cooling, and you get the product.

8. The method according to claim 7, characterized in that, The extraction method of total alkaloids from poppy in step (a) includes: taking the whole poppy plant, crushing it, extracting it by reflux with an alcohol solvent, and concentrating it to obtain an extract; dissolving the extract in acidic water, adjusting the pH to acidic, and extracting it with an organic solvent to remove impurities; adjusting the pH of the remaining acidic water to alkaline with alkali, extracting it with an organic solvent, and concentrating it to obtain the final product. The alcohol solvent is ethanol or methanol; the acidic water is hydrochloric acid aqueous solution with pH adjusted to 1.0–3.0; and the alkali is ammonia water with pH adjusted to 9.0–12.

0.

9. A method for treating rheumatoid arthritis, characterized in that, Apply the total alkaloids of wild poppy or the pharmaceutical composition according to any one of claims 5 to 6 to the affected area.

10. The application of total alkaloids from wild poppy in the preparation of drugs to improve oxidative stress damage associated with rheumatoid arthritis, characterized in that, The improvements are manifested in increased SOD activity and GSH concentration, and decreased MDA levels.

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