Ardisia gigantifolia leaf extract, preparation method thereof and application of ardisia gigantifolia leaf extract in preparation of medicine for preventing and treating gouty arthritis

Abstract

The invention discloses a giantleaf ardisia rhizome leaf extract, a preparation method thereof and application of the giantleaf ardisia rhizome leaf extract in preparation of medicines for preventing and treating gouty arthritis. The method comprises the following steps: extracting leaves of giantleaf ardisia rhizome with ethanol or an ethanol aqueous solution, dissolving an extract in water, and extracting with n-hexane and ethyl acetate in sequence to obtain an ethyl acetate extract, namely the giantleaf ardisia rhizome leaf extract. The treatment potential of the AK leaves on gouty arthritis is systematically evaluated for the first time, and it is found that the ethyl acetate component (EF) of the AK leaves has the remarkable effects of resisting inflammation, relieving synovial cell apoptosis and protecting cartilage. Through network pharmacological analysis, the AK is identified to have a multi-target and multi-channel anti-inflammatory mechanism. The medicinal value of the AK leaves is disclosed, a new normal form is provided for sustainable utilization of wild medicinal plants, and the dual innovative significance of ecological protection and drug development is considered.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine, and particularly relates to a kind of walking horse fetus leaf extract, a preparation method thereof and application thereof in preparing medicines for preventing and treating gouty arthritis. BACKGROUND

[0002] Gouty arthritis (GA) is a pathological state caused by hyperuricemia (HUA) due to disorders of purine metabolism, which leads to the deposition of monosodium urate (MSU) crystals in the joint cavity and connective tissue (Lyu et al., 2022). Clinically, it is usually manifested as severe joint pain, accompanied by redness, burning sensation and limited mobility. In addition, it is closely related to the occurrence of metabolic syndrome, myocardial infarction, diabetes and cardiovascular disease (Abbott et al., 1988; Krishnan et al., 2006; Choi et al., 2007). The pathogenesis of GA is complex. Traditional Chinese medicine (TCM) believes that its onset and progression are influenced by congenital factors, environmental conditions and dietary habits, and it is classified as "Bi syndrome". Western medicine usually regards primary GA as a hereditary disease with strong familial genetic tendency, and secondary GA is mainly related to renal insufficiency, hematological diseases and high-purine diet (Reginato et al., 2012). Statistical data shows that the incidence of GA in China has increased significantly, with the number of patients increasing from 5.86 million to 16.16 million, and showing a trend of youth, becoming an important public health problem (Zhang et al., 2023; Fang et al., 2024).

[0003] Hyperuricemia was once considered the fundamental pathological basis of GA, and reducing blood uric acid levels was the key to treatment (Lin et al., 2015). However, new evidence suggests that only a portion of hyperuricemia patients develop clinical GA (Dalbeth et al., 2018). This difference prompted research to focus on elucidating the role of inflammatory mediators and immune regulatory mechanisms in the pathogenesis of GA. The inflammatory process of GA is often described as a sequential, two-signal mechanism involving different but interdependent stages. In the initial stage, components of the innate immune system, particularly TLR-2, TLR-4, and CD14, interact with monocytes, promoting the uptake of MSU crystals, thereby activating NF-κB, leading to the synthesis of precursor IL-1β and inflammasome components (Liu-Bryan et al., 2005; Bauernfeind et al., 2009; So, 2008). Subsequently, the NLRP3 inflammasome, a cytoplasmic multi-protein complex including the NLRP3 receptor protein, the ASC adaptor protein, and the caspase-1 inflammatory protease, is activated by MSU crystals, leading to the release of IL-1β (Huang et al., 2021; Schroder et al., 2010). IL-1β subsequently triggers a strong inflammatory response, including vasodilation and rapid recruitment of neutrophils to crystal deposition sites (Schett et al., 2015). Neutrophils amplify the inflammatory process by suppressing apoptosis, releasing granules, producing superoxide, releasing cytokines, and, most recently, forming neutrophil extracellular traps (NETs) (Mitroulis et al., 2013).

[0004] The primary goal of GA treatment is to control inflammation. Standard treatments include non-steroidal anti-inflammatory drugs (NSAIDs), colchicine, glucocorticoids, biologics, or combined regimens (Dalbeth et al., 2016; Wilson and Saseen, 2016). However, increasing clinical evidence shows that these treatments have significant limiting adverse reactions involving the gastrointestinal, cardiovascular, hepatic-renal, neurological, and pulmonary systems (Billy et al., 2018; Bindu et al., 2020). Notably, although colchicine remains the primary treatment drug, its long-term use is associated with dose-dependent myotoxicity and neuromuscular sequelae (Borstad et al., 2004; Niel and Scherrmann, 2006; Sun et al., 2019; Zhao et al., 2022). These limitations have prompted increasing interest in developing safer natural alternative therapies. Traditional herbal medicines show great application prospects due to their multi-component, multi-target mechanisms of action and good safety profiles (Song et al., 2019). In addition, preclinical studies have highlighted their strong anti-inflammatory effects and immune-regulatory dual abilities. These pharmacological foundations provide a basis for systematic exploration of plant-derived GA treatment drugs aimed at developing clinically effective drugs with optimized safety.

[0005] Ardisia kteniophylla A. DC. (AK), also known as Matia (Guangdong), Shanzhuyao (Hainan), and Zoumafeng (Guangxi), is mainly distributed in Yunnan, Guangxi, Guangdong, Jiangxi, and Fujian provinces of China. Its roots and rhizomes are widely used in traditional medicine to treat rheumatoid arthritis and bone fracture (Tang, 2007; Xiang and Feng, 2002; Yang, 2007). Although recent studies have highlighted the antioxidant (Yang et al., 2008; Mu et al., 2013) and anti-inflammatory (Dai et al., 2017; Dai et al., 2018) properties of AK, its efficacy in treating GA has not been evaluated. Moreover, to meet the growing demand for medicinal plants, current unsustainable harvesting practices can put this plant at risk of extinction, and researchers are addressing overharvesting through innovative approaches, such as using leaves and branches instead of barks or roots of the species (Zschocke & Van Staden, 2000; Zschocke et al., 2000; Jain et al., 2012). Given the overexploitation and habitat loss issues faced by AK, our study focused on its environmentally friendly leaves, rather than the traditionally used roots. SUMMARY

[0006] The purpose of the present application is to provide an Ardisia kteniophylla leaf extract, a preparation method thereof, and an application thereof in preparing a drug for preventing and treating gouty arthritis.

[0007] In the present application, we established in vivo (monosodium urate (MSU) crystal-induced rat model) and in vitro (inflammatory cell model) systems to comprehensively evaluate the therapeutic effect of AK on gouty arthritis (GA). By integrating metabolomics and network pharmacology methods, we further identified the key bioactive components in the leaf extract of AK and elucidated their potential mechanisms in the treatment of GA. As the first systematic study on the GA-relieving properties of AK leaves, this study highlights the previously unexplored medicinal value of this plant's renewable tissues. Our research results not only propose a promising natural GA treatment candidate drug, but also demonstrate a model for the sustainable use of medicinal plants - bridging conservation and resource development. This work provides an example for the development of non-destructive extraction techniques for wild medicinal plants, ensuring ecological balance while advancing pharmacological discoveries.

[0008] The Ardisia kteniophylla leaf extract of the present application is prepared by the following method: the leaves of Ardisia kteniophylla are extracted with ethanol or aqueous ethanol solution, the extract is dissolved in water, and then extracted with n-hexane and ethyl acetate successively, and the ethyl acetate extract is the Ardisia kteniophylla leaf extract.

[0009] Preferably, the aqueous ethanol solution is an aqueous ethanol solution with a volume fraction of 95%.

[0010] A second object of the present application is to provide the use of the extract of the leaves of Akebia quinata in the preparation of a medicament for treating gouty arthritis.

[0011] Preferably, the medicament for treating gouty arthritis is a medicament having anti-inflammatory, synoviocyte apoptosis-relieving, and / or cartilage-protecting effects.

[0012] Preferably, the medicament is a medicament having the extract of the leaves of Akebia quinata as an active ingredient, and further comprising a pharmaceutically acceptable excipient.

[0013] Preferably, the medicament can be prepared in a dosage form, such as a solid dosage form (tablet, capsule, granule), a liquid dosage form (oral solution), and the like.

[0014] The present application also provides a medicament for treating gouty arthritis, which comprises the extract of the leaves of Akebia quinata as an active ingredient.

[0015] The present application first systematically evaluates the therapeutic potential of the leaves of Akebia quinata for gouty arthritis, and finds that the ethyl acetate fraction (EF) thereof has significant anti-inflammatory, synoviocyte apoptosis-relieving, and cartilage-protecting effects. Through network pharmacology analysis, the anti-inflammatory mechanism of Akebia quinata is identified to have multiple targets and multiple pathways. The research not only reveals the medicinal value of the leaves of Akebia quinata, but also provides a new paradigm for the sustainable use of wild medicinal plants, and has dual innovative significance of ecological protection and drug development. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Effects of alcohol extracts from different tissue parts of Akebia quinata on cell viability and the ability to inhibit NO production in inflammatory cells

[0017] Figure 2 Effects of extracts from different extraction phases of the leaves of Akebia quinata on cell viability and the ability to inhibit NO production in inflammatory cells

[0018] Figure 3 HE-stained sections of the liver, spleen, and kidney of experimental rats

[0019] Figure 4 Knee joint appearance and gait analysis

[0020] Figure 5 Expression levels of inflammatory factors in serum

[0021] Figure 6 Joint tissue sections

[0022] Figure 7 GO functional enrichment analysis and KEGG enrichment analysis of potential target points of Akebia quinata for treating gouty osteoarthritis

[0023] Figure 8Network diagram of “Active ingredients of Ardisia kteniophylla A. DC. - Gouty osteoarthritis - Pathway - Target point” DETAILED DESCRIPTION

[0024] The following examples are further illustrations of the application and are not intended to limit the same.

[0025] Example 1:

[0026] 1. Materials and methods

[0027] 1.1 Chemical reagents and drugs

[0028] MSU (U2875-5G) and colchicine were purchased from Sigma-Aldrich (St. Louis, MO, USA). CCK-8 cell counting kit was purchased from US EVERBRIGHT (Suzhou, China). NO assay kit was purchased from Beyotime (Shanghai, China). ELISA kits for IL-1β, TNF-α and IL-6 were purchased from Elabscience (Wuhan, China). Methanol (chromatographically pure) and acetonitrile (chromatographically pure) were purchased from Merck (Darmstadt, Germany). Formic acid (chromatographically pure) was purchased from Aladdin (Shanghai, China). High-glucose Dulbecco's Modified Eagle Medium (high-glucose DMEM) was purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). Fetal bovine serum (FBS) was purchased from Ponsay Biotech (Wuhan, China).

[0029] 1.2 Plant materials

[0030] Ardisia kteniophylla A. DC. was collected from Guangzhou, Guangdong Province, China, and identified by Dr. Hongfeng Chen, South China Botanical Garden (SCBG, CAS). The voucher specimen was deposited in the Herbarium of South China Botanical Garden, CAS.

[0031] The dried leaves, roots, barks, and xylem of A. kteniophylla were extracted with 95% ethanol for 3 times (60 min each time) to obtain root extract (ER), bark extract (ESB), xylem extract (ESX), and leaf extract (EL), respectively. The leaf extract (LE) was dissolved in water and successively extracted with n-hexane and ethyl acetate at a volume ratio of 1:1 to obtain non-polar fraction (n-hexane extract, HF), moderately polar fraction (ethyl acetate extract, EF), and polar aqueous fraction (AF). The corresponding extracts were concentrated under reduced pressure to obtain the corresponding extracts.

[0032] 1.3 Cell culture and MSU crystal preparation

[0033] Raw264.7 cells were purchased from Wuhan, China. The complete medium was prepared before the experiment and was prepared fresh, and the specific preparation method of the complete medium was as follows: 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin (P / S) were added to high-sugar DMEM. Cultured in a 37°C, 5% CO2 incubator. In vitro experiments, 20 mg MSU powder was sterilized by high pressure and dissolved in 1 mL PBS as a stock solution (Akahoshi et al., 2003; Xiao et al., 2023).

[0034] 1.4 In vitro experiment

[0035] 1.4.1 Cell viability detection

[0036] CCK-8 kit was used to detect cell viability. 100 μL of Raw264.7 cell suspension (1×10 5 cells / mL) was inoculated in a 96-well plate and divided into 3 groups:

[0037] Blank control group: high-sugar DMEM medium without cells;

[0038] Normal control group: high-sugar DMEM medium containing cells;

[0039] Treatment group: high-sugar DMEM medium containing cells and different concentrations of drugs (ER, ESB, ESX, EL; HF, EF, AF).

[0040] After co-culturing for 24 hours, 10 μL of CCK-8 and 90 μL of DMEM were added to each well, and incubation was continued for 1 hour. The OD value was measured at 450 nm using a microplate reader. Cell viability (%) = (treatment group OD - blank group OD) / (normal group OD - blank group OD) x 100%.

[0041] 1.4.2 MSU stimulates NO production in Raw264.7 cells

[0042] 100 μL of cell suspension (1×10 6 cells / mL) was inoculated in a 96-well plate and cultured for 24 hours and divided into 4 groups:

[0043] Normal control group: 100 μL high-sugar DMEM + 1 μL PBS;

[0044] MSU model group: 100 μL high-sugar DMEM + 1.0 μL MSU (final concentration 200 μg / mL);

[0045] Positive control group: 100 μL colchicine (final concentration 130 μg / mL) + 1 μL MSU (final concentration 200 μg / mL);

[0046] Treatment group: 100 μL different concentrations of drugs + 1.0 μL MSU (final concentration 200 μg / mL).

[0047] After 24 h of incubation, the NO level was determined using the NO detection kit according to the instructions.

[0048] 1.5 In vivo experiment

[0049] SD rats (6-8 weeks old, about 250 g) were purchased from Guangzhou Ruige Biological Technology Co., Ltd. and were raised in single cages at a temperature of 22 ± 2 °C and a relative humidity of 55.5 %, with a 12 h light-dark cycle.

[0050] 1.5.1 MSU-induced GA model and drug administration

[0051] Acute GA model: 50 μL of MSU suspension (20 mg / mL) was injected into the medial tibialis tendon from the back of the right hind limb joint at an angle of 45°. Drug administration began 1 h before MSU injection and continued for 3 d after injection. The animals were randomly divided into 6 groups (n = 10):

[0052] Normal control group (NC): 50 μL of sterile normal saline was injected into the joint cavity, and 6 mL of normal saline was administered by gavage.

[0053] Model group (MSU): 50 μL of MSU (20 mg / mL) was injected into the joint cavity, and an equal amount of normal saline was administered by gavage.

[0054] Positive control group (Col): 50 μL of MSU (20 mg / mL) was injected into the joint cavity, and 6 mL of colchicine 1 mg / kg / d was administered by gavage.

[0055] Drug administration group (160, 320, 640 mg / kg): 50 μL of MSU (20 mg / mL) was injected into the joint cavity, and 6 mL of AK leaf EF was administered by gavage at doses of 160, 320, and 640 mg / kg / d (doses were converted based on the maximum crude drug dose of 15 g for humans and the EtOAc yield).

[0056] 1.5.2 Foot swelling determination

[0057] The foot volume was measured by the drainage method before MSU injection, 1 h, 6 h, 24 h, 48 h, and 72 h after injection. Foot swelling (mL) = volume after injection - volume before injection.

[0058] 1.5.3 Gait score

[0059] Blind score, 0-3 grade: 0 = normal; 1 = mild claudication, injected limb hyperflexion; 2 = moderate claudication, affected limb only briefly touches the ground; 3 = severe claudication, three-legged gait.

[0060] 1.5.4 Serum inflammatory factors

[0061] At 1 h after the last administration, 6 rats in each group were anesthetized and blood was collected from the abdominal aorta, centrifuged at 2500 x g for 10 min to obtain serum. TNF-α, IL-1β, and IL-6 were determined by ELISA according to the kit instructions.

[0062] 1.5.5 Histology and immunohistochemistry of cartilage

[0063] The knee joints were fixed with 4% paraformaldehyde for 24 h, decalcified for 14 d, embedded in paraffin, and sectioned at 6 μm for HE, Safranin-O / Fast Green, and Toluidine blue staining.

[0064] Immunohistochemistry: The sections were deparaffinated, hydrated, and antigen-repaired, and endogenous peroxidase was inactivated. The primary antibody was diluted IL-6 (1:200), TNF-α (1:200), and IL-1β (1:200), and incubated at 4 °C overnight. The secondary antibody (dilution ratio 1:1000) was incubated at room temperature, and DAB was used for color development. The sections were counterstained with hematoxylin, dehydrated, and mounted. Photographs were taken under a microscope.

[0065] 1.6 AK chemical composition analysis

[0066] UPLC (ExionLC™ AD) -MS / MS, Agilent SB-C18 column (1.8 μm, 2.1 mm x 100 mm). Mobile phase A: 0.1% formic acid water; B: 0.1% formic acid acetonitrile. Gradient: 0-1 min 5% B, 1-9 min to 95% B, 9-10 min 95% B, 10-11.1 min to 5% B, and equilibration to 14 min. Injection volume 2.0 μL, flow rate 0.3 mL / min, column temperature 40 °C.

[0067] ESI source temperature 550 °C; positive ion 5500 V, negative ion -4500 V; GSI 50 psi, GSII 60 psi, CUR 25 psi; CID high, MRM mode, N2 collision gas medium. MRM ion pairs were monitored according to the retention period, and isotopes, metal adducts (K⁺, Na⁺, NH4⁺), and secondary fragments were removed. The MS / MS spectra were compared with the MWDB database for annotation.

[0068] 1.7 Network pharmacology research

[0069] 1.7.1 Component and disease target screening

[0070] AK components were screened by Swiss ADME (http: / / www.swissadme.ch / ), at least 3 of which met the five rules of drug-likeness (Lipinski, Ghose, Veber, Egan, Muegge) and high gastrointestinal absorption. The active ingredients were introduced into SwissTargetPrediction (http: / / www.swisstargetprediction.ch / ), with the species limit "Homosapiens" and a probability of >0.

[0071] GA disease targets came from GeneCards (https: / / www.genecards.org / ), OMIM (https: / / www.omim.org / ), DisGeNET (https: / / www.disgenet.org / ), and were integrated after de-duplication. The intersection of component-disease target points was obtained using Venny 2.1.0 (https: / / bioinfogp.cnb.csic.es / tools / venny / ).

[0072] 1.7.2 Enrichment analysis

[0073] DAVID (https: / / david.ncifcrf.gov / tools.jsp) was used to do GO, KEGG enrichment on the intersection target points, with identifier "OFFICIAL_GENE_SYMBOL" and species "Homo sapiens". The top 10 (P<0.01) of BP, CC, and MF were taken; the top 20 inflammation-related pathways (P<0.01) were taken, and R package ggplot2 was used for plotting.

[0074] Cytoscape 3.10.2 was used to construct the "Zuomatia active ingredients-gouty osteoarthritis-pathway-target" network, remove isolated nodes, and screen core components and targets according to node size and color.

[0075] 1.8 Statistical analysis

[0076] Data was expressed as mean ± SD, and R 4.0.5 was used for one-way ANOVA and Tukey post-hoc test, with P<0.05 being significant and P<0.01 being extremely significant. AUC and plotting were completed using GraphPad Prism 9.

[0077] 2. Results

[0078] 2.1 Cell viability and analysis of MSU-stimulated NO production in cells

[0079] 2.1.1 AK different tissue ethanol extracts

[0080] As shown in Figure 1 A, the concentrations without significant effect on cell viability were: root extract (ER) and barks extract (ESB) ≤ 1 mg / mL, leaf extract (EL) ≤ 0.5 mg / mL, and stem xylem extract (ESX) ≤ 0.125 mg / mL. For the convenience of activity comparison, two doses were selected for subsequent anti-inflammatory experiments: 62.5 μg / mL and 31.25 μg / mL.

[0081] The anti-inflammatory activity of AK different tissue ethanol extracts was systematically studied by detecting NO production in MSU-stimulated Raw264.7 cells Figure 1 B). Compared with the normal control group (NC), MSU stimulation significantly increased the intracellular NO level (P < 0.05). The results showed that ESX and EL significantly inhibited NO production in a dose-dependent manner. Comparative analysis showed that the anti-inflammatory effect of ESX and EL was significantly better than that of ER and ESB, while there was no significant difference between ESX and EL. Cell viability detection further showed that the maximum non-toxic concentration of ESX and EL was 0.125 mg / mL and 0.5 mg / mL, respectively. In summary, EL not only has strong anti-inflammatory activity, but also has better safety, so EL is preferred for subsequent pharmacological studies, which provides a basis for using AK leaves as a sustainable medicinal resource.

[0082] 2.1.2 Solvent partition components of leaf extract (EL)

[0083] EL was partitioned with n-hexane, ethyl acetate, and water successively, obtaining three components: n-hexane component (HF), ethyl acetate component (EF), and aqueous component (AF). As shown in Figure 2 A-C, the maximum non-toxic concentrations of HF and EF were 62.5 μg / mL, and that of AF was 1.0 mg / mL, which had no significant difference compared with the NC group (P > 0.05). To compare the activity of each component, three concentrations (62.5, 31.25, and 15.625 μg / mL) were selected for in vitro anti-inflammatory experiments.

[0084] As shown in Figure 2D showed that compared with the MSU model group, HF and EF significantly inhibited NO production at all test concentrations, AF also significantly inhibited NO at 62.5 and 31.25 μg / mL, and 15.625 μg / mL had no significant effect. The NO inhibition effect of EF at 62.5 and 31.25 μg / mL had no significant difference from the NC group (P > 0.05), and was equivalent to the dexamethasone (DEX) positive control. In summary, EF had the strongest inhibitory effect on MSU-induced NO production in a dose-dependent manner, so EF was selected for subsequent in vivo experiments.

[0085] 2.2 Therapeutic effect of EF in GA rat model

[0086] A total of 60 rats were used for in vivo experiments. The treatment groups were low dose (160 mg / kg / d), medium dose (320 mg / kg / d), and high dose (640 mg / kg / d) of EF, respectively. No rats died during the treatment period, and no obvious toxic reactions were observed.

[0087] 2.2.1 Toxicity evaluation of EF on liver, spleen, and kidney

[0088] As shown in Figure 3 , the liver, spleen, and kidney tissues of rats treated with EF were observed by HE staining to evaluate potential organ damage. The results showed that:

[0089] Spleen: The red-white pulp boundary was clear, and the structure was not blurred;

[0090] Kidney: The glomerular structure was complete, and there was no cell vacuolar degeneration, flattening, or shedding;

[0091] Liver: The EF treatment group showed no hepatotoxicity, while the colchicine group showed hepatocyte loosening and widened interstitial space, which may be related to its induction of cell cycle arrest.

[0092] In summary, EF showed no toxicity to the liver, spleen, and kidney at therapeutic doses, while colchicine showed hepatotoxicity, suggesting that EF may be a safer alternative drug.

[0093] 2.2.2 Joint swelling and gait function

[0094] Compared with the NC group, the knee joints of rats in the MSU model group were significantly swollen ( Figure 4 A). Macroscopic observation showed that the high-dose EF (640 mg / kg) and colchicine groups had significantly reduced swelling, and the high-dose EF group had no significant difference from the NC group.

[0095] The paw volume was measured by the drainage method ( Figure 4 B). The foot swelling of the MSU group continued to worsen, while the colchicine and EF groups gradually relieved after reaching the peak at 24 hours. AUC analysis ( Figure 4C) shows that the MSU group was significantly higher than the NC group in the swelling of the foot within 72 hours (P < 0.0001), and the medium and high dose EF significantly alleviated the swelling (P < 0.01).

[0096] Gait score Figure 4 D) shows that the model group was significantly abnormal in gait (P < 0.0001). The EF dose groups and the colchicine group significantly improved the gait, and the medium and high dose EF had a very significant effect.

[0097] 2.2.3 Serum inflammatory factor levels

[0098] The levels of TNF-a, IL-1β and IL-6 in the serum of MSU-induced GA group rats were significantly increased (P < 0.0001). The TNF-a (P < 0.01) and IL-1β (P < 0.0001) of each dose group of EF and the colchicine group were significantly reduced. The IL-6 of the medium and high dose EF was significantly inhibited (P < 0.0001), and the low dose was significantly reduced (P < 0.05). In summary, EF can effectively down-regulate the key inflammatory mediators in the serum of GA rats, and it is dose-dependent Figure 5 ).

[0099] 2.2.4 Reduction of cartilage degeneration, synovial cell apoptosis and inflammation

[0100] Cartilage degeneration and synovial cell shedding are the main pathological features of GA. As shown in Figure 6 A, the cartilage of the MSU model group was significantly thinned, the chondrocytes were hypertrophic, vacuolated (indicating apoptosis), and arranged in disorder; the synovial cell apoptosis and shedding were obvious Figure 6 G). EF treatment effectively improved these pathological changes, and the 160 and 320 mg / kg groups were comparable to colchicine, and the 640 mg / kg group was close to normal cartilage morphology; after drug treatment, the synovial apoptosis of each dose group was relieved, and the 640 mg / kg group had a stronger ability to relieve synovial hyperplasia than the positive drug group.

[0101] Alcian blue staining shows that Figure 6 B), the MSU group lost a significant amount of proteoglycan, and the cartilage layer was thinned. EF (320, 640 mg / kg) significantly restored the content and distribution of proteoglycan, close to normal levels.

[0102] Methylthioninium blue staining shows that Figure 6 C), the MSU group had severe loss of chondrocyte apoptosis and matrix. EF (320, 640 mg / kg) significantly improved apoptosis, and the 640 mg / kg group restored the chondrocyte content to the NC group level.

[0103] Immunohistochemical analysis Figure 6DF) showed that IL-1β ( ) was present in chondrocytes of the MSU group. Figure 6 D), IL-6 ( Figure 6 E), TNF-α Figure 6 F) expression was significantly increased (dark brown staining), and IL-1β expression in synovial cells was significantly increased ( Figure 6 H). Treatment with EF (160, 320, 640 mg / kg) significantly reduced the expression of inflammatory factors. The levels of IL-1β and TNF-α in chondrocytes in the medium and high dose groups were comparable to those in the NC group, while IL-6 decreased in a dose-dependent manner. Furthermore, IL-1β expression was significantly reduced in synovial cells in the medium and high dose groups, with the high-dose group showing a treatment effect close to that of the NC group. All EF groups exhibited superior anti-inflammatory effects compared to colchicine.

[0104] 2.3 Network Pharmacological Analysis

[0105] 2.3.1 Construction of Pharmacological Network

[0106] Using the Swiss ADME and SwissTargetPrediction platforms, 217 active ingredients in AK were identified, corresponding to 1101 potential targets. 2213 disease targets related to GA were retrieved from the GeneCards, OMIM, and DisGeNET databases. Cross-analysis revealed 337 common targets, which will serve as key targets for subsequent analysis.

[0107] 2.3.2 GO enrichment and KEGG pathway analysis

[0108] GO enrichment analysis of common targets yielded 822 GO entries, including 580 biological processes (BP), 66 cellular components (CC), and 176 molecular functions (MF). BP mainly involves chromatin remodeling and protein phosphorylation; CC is mainly located in the cell membrane, cytoplasm, and cytosol; MF mainly includes ATP binding and histone kinase activity, etc. Figure 7 A).

[0109] KEGG pathway analysis showed that key pathways in AK treatment of GA include inflammatory signaling pathways such as PI3K-Akt, MAPK, and NF-κB. Figure 7 (B) suggests that it exerts its therapeutic effect by regulating inflammation and immune responses.

[0110] 2.3.3 Drug Composition-Disease-Pathway-Target Network

[0111] Building a network using Cytoscape 3.10.2 Figure 8), which contains 445 nodes and 3289 edges with an average node degree of 11.011. Key targets include PTGS2, KDR, IGF1R, MMP3, SYK, Mapk8, PIK3CG, etc. The core pathways are PI3K-Akt (hsa04151) and MAPK (hsa04010) signaling pathways, indicating that AK treats GA through multi-component, multi-target, and multi-pathway mechanisms.

[0112] 3. Conclusion

[0113] The present application first systematically evaluates the therapeutic potential of AK leaves for gouty arthritis and finds that the ethyl acetate fraction (EF) has significant anti-inflammatory, synoviocyte apoptosis-relieving, and cartilage-protecting effects. Through network pharmacology analysis, the anti-inflammatory mechanism of AK is identified as multi-target and multi-pathway. The present application not only reveals the medicinal value of AK leaves but also provides a new paradigm for the sustainable use of wild medicinal plants, with dual innovative significance of ecological protection and drug development.

Claims

1. A method for preparing an extract of *Eriocaulon buxiflorum* leaf, characterized in that, The leaves of Asparagus cochinchinensis are immersed with ethanol or aqueous ethanol solution, the extract is dissolved in water, and then extracted with n-hexane and ethyl acetate successively, and the ethyl acetate extract is the extract of Asparagus cochinchinensis leaves.

2. The production method according to claim 1, characterized by, The aqueous ethanol solution is an aqueous ethanol solution with a volume fraction of 95%.

3. The extract of Asparagus cochinchinensis leaves prepared by the preparation method according to claim 1 or 2.

4. The use of the extract of Asparagus cochinchinensis leaves according to claim 3 in the preparation of a drug for treating gouty arthritis.

5. Use according to claim 4, characterized in that, The drug for treating gouty arthritis is a drug with anti-inflammatory, synovial cell apoptosis relieving and / or cartilage protecting effects.

6. Use according to claim 4, characterized in that, The drug is a drug with the extract of Asparagus cochinchinensis leaves as an active ingredient and further containing pharmaceutically acceptable adjuvants.

7. Use according to claim 4, characterized in that, The drug is in a solid dosage form or a liquid dosage form.

8. Use according to claim 7, characterized in that, The solid dosage form is a tablet, a capsule or a granule.

9. Use according to claim 7, characterized in that, The liquid dosage form is an oral liquid.

10. A medicament for treating gouty arthritis, characterized by comprising a compound of the formula (I) as an active ingredient. The extract of Asparagus cochinchinensis leaves according to claim 3 is used as an active ingredient.

Images