Ammonium salt of 2,4-dichlorophenoxyacetic acid and its use in the preparation of herbicides

Abstract

This invention belongs to the field of Traditional Chinese Medicine and discloses an extract of the stems of *Ilex chinensis* from Xinjiang, prepared by the following method: dried stems of *Ilex chinensis* from Xinjiang are pulverized, soaked in 75%–95% ethanol (8–10 times their weight by volume) for 0.5–1 hour, and extracted by reflux 1–4 times, 1–3 hours each time. The extract is then filtered, the filtrates are combined, and concentrated to obtain the *Ilex chinensis* stem extract. This *Ilex chinensis* stem extract has anti-inflammatory and analgesic effects with few toxic side effects and can be used to treat rheumatoid arthritis. This invention also discloses the application of the *Ilex chinensis* stem extract in the preparation of drugs for treating rheumatoid arthritis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine and relates to the extract of Xinjiang sand lily stem and its application in the preparation of drugs for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic, inflammatory synovitis, manifested as joint redness, swelling, tenderness, and significantly limited mobility. In Traditional Chinese Medicine (TCM), RA falls under the category of "Bi syndrome," also known as "Li Jie Feng" or "He Xi Feng." Studies show that the disability rate of RA is second only to diseases like malaria. Therefore, finding highly effective and low-toxicity treatments is an urgent problem. In recent years, monomeric compounds derived from natural medicines (such as curcumin) or effective fractions (such as total alkaloids from Tripterygium wilfordii) have been used in the treatment of RA. Therefore, the discovery of new anti-rheumatoid arthritis drugs shows great promise.

[0003] *Ammopiptanthus nanus* (M.Pop.) Cheng f., also known as dwarf sand holly, is a medicinal plant endemic to Xinjiang, found only in Wuqia County, Kizilsu Kyrgyz Autonomous Prefecture. It possesses a variety of resistances, including drought, high temperature, frost, salt and alkali tolerance, and corrosion resistance, making it a very precious relict plant in arid regions. *Ammopiptanthus nanus* is a commonly used medicinal herb among the Kyrgyz people, possessing properties that dispel wind and dampness, relax muscles and tendons, and calm the mind. It is used in folk medicine to treat neurasthenia, boils, and carbuncles, and the entire plant can be used medicinally. Currently, there are no research reports, either domestically or internationally, on the treatment of rheumatoid arthritis with *Ammopiptanthus nanus*. Summary of the Invention

[0004] This invention established an acute inflammation model in rats using carrageenan-induced paw edema and a writhing analgesia model in mice using acetic acid. The anti-inflammatory and analgesic effects of *Ilex chinensis* stem extract were investigated from three perspectives: paw edema inhibition rate, inflammatory factor expression, and the number of writhing episodes in mice. The results showed that *Ilex chinensis* stem extract possesses anti-inflammatory and analgesic effects with few toxic side effects and can be used to treat rheumatoid arthritis. This invention is the first to utilize network pharmacology, combined with literature review, to construct a *Ilex chinensis* component-rheumatoid arthritis target-pathway network, analyzing the mechanism of action of *Ilex chinensis* in treating rheumatoid arthritis. Lipopolysaccharide (LPS) was used to induce pro-inflammatory responses in classical (M1) RAW264.7 macrophages. Western blotting was used to comprehensively and systematically study the mechanism of *Ilex chinensis* in treating rheumatoid arthritis from a holistic perspective of traditional Chinese medicine.

[0005] The purpose of this invention is to provide a stem extract of *Ilex chinensis* from Xinjiang, which is prepared by the following method: pulverizing dried stems of *Ilex chinensis* from Xinjiang, soaking them in 75%–95% ethanol at 8–10 times their weight (g / mL) for 0.5–1 h, extracting by reflux 1–4 times, each time for 1–3 h, filtering, combining the filtrates, and concentrating to obtain the stem extract of *Ilex chinensis* from Xinjiang.

[0006] Alternatively, the Xinjiang Sandalwood stem extract is prepared by the following method: the dried stems of Xinjiang Sandalwood are pulverized, soaked in 75%–95% ethanol at 8–10 times the weight of the medicinal material (g / mL) for 0.5–1 h, heated under reflux for 1–4 times, each time for 1–3 h, filtered, the filtrates are combined, concentrated and dried to obtain the Xinjiang Sandalwood stem extract.

[0007] The reflux extraction temperature is 80–90°C.

[0008] The endpoint of the concentration is: the volume of the concentrated paste no longer decreases.

[0009] The extract of the stem of *Ilex chinensis* from Xinjiang contains resveratrol, 3'-methoxydaidzein, luteolin, sophoridine, gentianin, glycyrrhizin, and white ketone.

[0010] Another object of the present invention is to provide the application of the stem extract of Xinjiang sand lily in the preparation of a drug for treating rheumatoid arthritis.

[0011] Another objective of this invention is to provide the application of the stem extract of *Ilex chinensis* from Xinjiang in the preparation of a drug for treating rheumatoid arthritis with anti-inflammatory and analgesic effects.

[0012] Preferably, the application is the use of Xinjiang Sandalwood stem extract in the preparation of a drug for treating rheumatoid arthritis by reducing at least one of the inflammatory factors TNF-α, IL-6, and IL-1β and reducing inflammatory cell infiltration.

[0013] Preferably, the application is the use of the stem extract of *Ilex chinensis* from Xinjiang in the preparation of a drug for treating rheumatoid arthritis by inhibiting the activation of the NF-κB / COX2 pathway.

[0014] Preferably, the drug is made from the stem extract of *Ilex chinensis* from Xinjiang as the active ingredient, along with a pharmaceutically acceptable carrier or excipient.

[0015] Preferably, the dosage form of the drug is any dosage form that is acceptable in pharmacological terms; specifically, the dosage form of the drug is a hydrogel or an ointment.

[0016] Another object of the present invention is to provide a pharmaceutical composition which is a drug made of the aforementioned *Ilex chinensis* stem extract as the active ingredient and a pharmaceutically acceptable carrier or excipient.

[0017] The dosage form of the drug is hydrogel or ointment.

[0018] Another object of the present invention is to provide the use of the pharmaceutical composition in the preparation of a medicament for treating rheumatoid arthritis.

[0019] Another objective of this invention is to provide a thermosensitive hydrogel, which is prepared by the following method: The dried stems of *Ilex chinensis* from Xinjiang are pulverized and soaked in 75%–95% ethanol (8–10 times their weight by volume) for 0.5–1 h. The mixture is then heated under reflux for extraction 1–4 times, each time for 1–3 h. The extract is filtered, the filtrates are combined, and concentrated to obtain a thick paste-like extract. Tween 80 and sodium benzoate are added to obtain an extract solution. Poloxamer 407 and poloxamer 188 are added to the extract solution, and the mixture is placed at 4°C to obtain a transparent, flowable solution, which is the thermosensitive hydrogel. Specifically, for every 1 g of extract obtained from dried *Ilex chinensis* stems from Xinjiang, 2–3 mL of Tween 80 and 1–2 g of sodium benzoate are added; for every 10 mL of extract solution, 1–2 g of poloxamer 407 and 0.05–0.15 g of poloxamer 188 are added.

[0020] Preferably, for every 1g of the extract obtained from the dried stems of *Ilex chinensis* from Xinjiang, 2.4mL of Tween 80 and 1.5g of sodium benzoate are added; for every 10mL of the extract solution, 1.75g ​​of poloxamer 407 and 0.1g of poloxamer 188 are added. Attached Figure Description

[0021] Figure 1 The results show the chemical composition identification of the stem extract of *Ilex chinensis* from Xinjiang; where A is the HPLC chromatogram of the stem extract of *Ilex chinensis* from Xinjiang; and B is the HPLC chromatogram of the mixed reference solution.

[0022] Figure 2 The graph shows the inhibition rate of toe swelling in rats with an acute inflammatory model at different time points using the stem extract of *Ilex chinensis* from Xinjiang.

[0023] Figure 3 The effect of *Ilex chinensis* stem extract from Xinjiang on serum inflammatory factors in rats with an acute inflammation model; where A: IL-1β, B: TNF-α, and C: IL-6; the model group was compared with the blank control group. #### p<0.0001; Compared with the model group, the dexamethasone group, the high, medium and low dose groups of Xinjiang Sand Wintergreen stem extract, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns indicates no statistical difference.

[0024] Figure 4The image shows the H&E results of the swollen right hind paw of a rat.

[0025] Figure 5 Venn diagram of the intersection target point of Xinjiang Sandalwood and RA.

[0026] Figure 6 PPI map of the core target point at the intersection of Xinjiang Sandalwood and RA.

[0027] Figure 7 Bubble graph of KEGG enrichment analysis for treating RA with Xinjiang ginseng.

[0028] Figure 8 The effect of the stem extract of *Ilex chinensis* from Xinjiang on cell viability was investigated; compared with the control group, ****p<0.0001, *p<0.05.

[0029] Figure 9 The effect of Xinjiang Sandy Winter Ivy stem extract on lipopolysaccharide-induced RAW 264.7 inflammation model was investigated, with the model group compared to the blank control group. #### p<0.0001; Compared with the model group, ****p<0.0001, ***p<0.001, ns indicates no statistical difference.

[0030] Figure 10 Western blot diagram showing the effect of Xinjiang Sandy Winter Ivy stem extract on protein expression in a lipopolysaccharide-induced RAW 264.7 inflammation model.

[0031] Figure 11 This is a sample image of a thermosensitive gel spray made from the stem extract of Xinjiang sand lily. Detailed Implementation

[0032] Example 1

[0033] I. Preparation of stem extract of Xinjiang Sandy Winter Jasmine

[0034] Preparation of Xinjiang Sandy Winter Ivy Stem Extract: Take 100g of dried Xinjiang Sandy Winter Ivy stems, grind them into powder, add 10 times the amount of 95% ethanol (1L) to soak for 1h, heat and reflux to extract 3 times, 90min each time, filter, combine the filtrates, concentrate under reduced pressure, freeze dry to obtain 24.9g of Xinjiang Sandy Winter Ivy Stem Extract in powder form, with a yield of 24.9%.

[0035] Table 1. Yield of extract from the stems of *Ilex chinensis* (Xinjiang)

[0036]

[0037] II. Chemical composition analysis of the stem extract of *Ilex chinensis* from Xinjiang

[0038] 1. Experimental instruments: Shimadzu LC20-ADXR high performance liquid chromatograph from Japan; chromatographic column: Thermo Acclaim120C18, with specifications of 250 mm × 4.6 mm, 5 μm; electronic analytical balance; ultrasonic cleaner, etc.

[0039] 2. Preparation of test solution: Weigh 1 g of the extract from the stem of Ammopiptanthus nanus (M. Pop.) Cheng f. precisely, dissolve it ultrasonically in methanol, make the volume up to 50 mL, filter it through a 0.45 μm microporous filter membrane, and conduct HPLC analysis.

[0040] 3. Preparation of mixed reference substance solution: Take appropriate amounts of resveratrol, 3'-methoxydaidzein, luteolin, maackiain, formononetin, licoflavonol, and wighteone reference substances, mix them, and dissolve them in methanol to obtain the mixed reference substance solution.

[0041] 4. Chromatographic conditions: The injection volume is 20 μL; the mobile phase is water (phase A) containing 0.1% formic acid and methanol (phase B), the flow rate is 1 mL / min, gradient elution is carried out (Table 2), the column oven temperature is 40 °C, and the detection wavelength is 300 nm.

[0042] Table 2. Gradient elution program

[0043]

[0044] 5. Identification of chemical components in Ammopiptanthus nanus (M. Pop.) Cheng f.: By comparing with the chromatographic retention time of the reference substance and the ultraviolet absorption spectrogram, 7 compounds were identified from the extract of the stem of Ammopiptanthus nanus (M. Pop.) Cheng f. For detailed information, see Figure 1 and Table 3. The main components of the extract from the stem of Ammopiptanthus nanus (M. Pop.) Cheng f. are formononetin, licoflavonol, and wighteone, and its active components are mostly flavonoid compounds, providing a material basis for Ammopiptanthus nanus (M. Pop.) Cheng f. to treat rheumatoid arthritis.

[0045] Table 3. Information of reference substances for chemical components in Ammopiptanthus nanus (M. Pop.) Cheng f.

[0046]

[0047] Example 2

[0048] Study on the curative effect of the extract from the stem of Ammopiptanthus nanus (M. Pop.) Cheng f. on the acute inflammation model of rats

[0049] 1. Test substances: The extract from the stem of Ammopiptanthus nanus (M. Pop.) Cheng f. prepared in Example 1 (ANCE), dexamethasone sodium phosphate injection (DXMT, positive drug).

[0050] 2. Experimental animals: SD rats, male, with a body weight of 180 - 220 g, SPF grade, provided by the Animal Center of Nanjing Medical University, animal license number: SYSK (Jiangsu) 202,020 - 0022.

[0051] 3. Instruments and equipment: Gavage needle, Shanghai Bolige Industry & Trade Co., Ltd., specification: 12#; Timer, Jiangsu Kaiji Biotechnology Co., Ltd.

[0052] 4. Experimental Methods

[0053] Grouping: After 2 days of acclimatization, SD rats were randomly divided into 6 groups according to their body weight: blank control group, model group, dexamethasone group (30 mg / kg), and high, medium and low dose groups of Xinjiang Sandy Winter Ivy stem extract (2000, 1000 and 500 mg / kg), with 5 rats in each group.

[0054] Dosage and administration method: Rats in the blank control group and model group were administered 0.5% CMC-Na solution by gavage at a volume of 1 mL / 100 g; rats in the dexamethasone group were injected intraperitoneally with dexamethasone at a dose of 1 mL / 100 g; rats in the high, medium and low dose groups of Xinjiang Sandalwood stem extract were administered 0.5% CMC-Na dispersion solution of Xinjiang Sandalwood stem extract at three different doses by gavage at a volume of 1 mL / 100 g; the model was established 1 hour after administration of the above methods.

[0055] Modeling: One hour after administration, 1% carrageenan solution (0.1 mL / 100 g) was injected into the right hind paw of rats. The thickness of the inflamed paw was measured with calipers at 0, 1, 2, 3, 4, 5 and 6 hours, and the paw swelling rate of rats was calculated. The left paw was not treated in any way.

[0056] Rat paw edema rate (%) = [(post-inflammatory toe thickness - pre-inflammatory toe thickness) / pre-inflammatory toe thickness] × 100

[0057] 5. Experimental Results

[0058] Table 4. Effects of Xinjiang Sandwort Stem Extract on Carrageenan-Induced Paw Edema in Rats

[0059]

[0060] The results are shown in Table 4 and Figure 2 The results showed that the thickness of the toes of rats injected with carrageenan increased significantly after the model group, indicating that the model was successfully established. Both dexamethasone and *Ilex chinensis* stem extract could reduce toe swelling in rats to varying degrees, demonstrating a significant inhibitory effect on toe swelling in the anti-inflammatory experimental model rats. Furthermore, the *Ilex chinensis* stem extract showed a clear dose-dependent effect, with higher concentrations showing better inhibitory effects on toe swelling than lower concentrations.

[0061] Example 3

[0062] Enzyme-linked immunosorbent assay (ELISA) for detecting serum inflammatory markers in rats

[0063] The inflammatory factors IL-1β, TNF-α and IL-6 in the serum of rats with a paw edema model injected with 1% carrageenan solution for 6 hours in Example 2 were measured using an ELISA kit.

[0064] Table 5. Effects of Xinjiang Sand Fern Stem Extract on Serum Cytokines in Rats with Acute Inflammation Model

[0065]

[0066] The results are shown in Table 5. Figure 3 As shown, compared with the blank control group, the levels of inflammatory factors IL-1β, TNF-α, and IL-6 in the serum of rats in the model group were significantly increased. The inflammatory factors in the model group showed significant differences compared with the blank control group, indicating successful model establishment. Compared with the model group, the stem extract of *Ilex chinensis* from Xinjiang reduced the expression of inflammatory factors, and the levels of IL-1β, TNF-α, and IL-6 in rat serum were significantly reduced, showing a trend comparable to that of the positive control drug dexamethasone.

[0067] Example 4

[0068] Effects of Xinjiang Sandy Winter Stem Extract on Histopathological Changes of Swollen Paw in Rats with Acute Inflammation

[0069] The right hind paw of the rat in Example 2 was taken, fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The H&E histopathological sections can be observed under a microscope. Figure 4 It was found that the right hind paw of the control group rats exhibited normal histology. Subcutaneous injection of carrageenan into the right hind paw of rats induced severe edema, characterized by inflammatory cell infiltration and tissue structure destruction. Administration of dexamethasone and extracts from the stems of *Ilex chinensis* significantly inhibited edema in the right hind paw of rats and reduced the degree of inflammatory cell infiltration.

[0070] Example 5

[0071] Study on the effect of Xinjiang Sandy Winter Ivy stem extract on writhing inhibition in a mouse analgesia model

[0072] 1. Test substance: the stem extract of Xinjiang sand lily prepared in Example 1, and aspirin (positive control).

[0073] 2. Experimental animals: ICR mice, male, weighing 18-22g, SPF grade, provided by the Animal Center of Nanjing Medical University, animal license number: IACUC-181201.

[0074] 3. Instruments and equipment: Gavage needle, Shanghai Bolige Industry & Trade Co., Ltd., specification: 8#; Timer, Jiangsu Kaiji Biotechnology Co., Ltd.

[0075] 4. Experimental Methods

[0076] Grouping: After 2 days of acclimatization, ICR mice were randomly divided into 6 groups of 5 mice each according to their body weight. The groups were: blank control group, model group, aspirin group (50 mg / kg), and high, medium and low dose groups of Xinjiang Sandy Winter Ivy stem extract (2000, 1000 and 500 mg / kg, respectively).

[0077] Dosage and administration method: Mice in the blank control group and model group were administered 0.5% CMC-Na solution by gavage; mice in the aspirin group were administered 0.5% CMC-Na dispersion of aspirin by gavage at a dose of 0.1 mL / 10 g; rats in the high, medium, and low dose groups of Xinjiang Sandy Winter Ivy stem extract were administered 0.5% CMC-Na dispersion of Xinjiang Sandy Winter Ivy stem extract by gavage at a dose of 0.1 mL / 10 g; the model was established 1 hour after administration by gavage in the above manner.

[0078] Modeling: One hour after gavage, mice in the model group, aspirin group, and high, medium, and low dose groups of Xinjiang Sandy Wintergreen stem extract were intraperitoneally injected with 0.6% glacial acetic acid solution (0.1 mL / 10 g), while mice in the blank control group were injected with an equal volume of physiological saline. The number of writhing responses in mice within 15 minutes after injection (using a 5-minute latency period) was counted, and the writhing response inhibition rate was calculated.

[0079] Inhibition rate of writhing count (%) = [(Number of writhing counts in the treatment group - Number of writhing counts in the model group) / Number of writhing counts in the model group] × 100

[0080] Among them, the positive drug group was the aspirin group, and the drug administration groups were the high, medium and low dose groups of Xinjiang Sand Wintergreen stem extract.

[0081] 5. Experimental Results

[0082] Table 6. Number of writhing movements in mice after intraperitoneal injection of 0.6% acetic acid solution (%, n=5)

[0083]

[0084] The results, shown in Table 6, indicate that the treatment group exhibited a significant analgesic effect compared to the model group. Specifically, the extract of *Ilex chinensis* stems from Xinjiang reduced the number of writhing movements in the pain model mice. This invention demonstrates that the extract of *Ilex chinensis* stems from Xinjiang has a significant inhibitory effect on acetic acid-induced pain responses in mice, providing a basis for its use in alleviating pain symptoms of rheumatoid arthritis.

[0085] Example 6

[0086] Network pharmacological study of Xinjiang Sandy Winter Ivy stem extract for the treatment of rheumatoid arthritis

[0087] 1. Target prediction of Xinjiang Sand Holly

[0088] A literature search yielded 37 reported compounds of *Ilex chinensis*, and the 2D structures of the active ingredients were downloaded from the PubChem database. Then, the SwissADME database was used for ADME screening to identify compounds with favorable pharmacokinetic properties. Based on the selected components, the SwissTargetPrediction database was used to predict the targets of *Ilex chinensis*, and the protein target information was standardized using the Uniprot protein database, resulting in 453 target compounds of *Ilex chinensis*.

[0089] 2. Screening of targets related to rheumatoid arthritis

[0090] Using "Rheumatoid arthritis" as the keyword and "Homo Sapiens" as the species selection, 1409 targets related to RA were obtained from the GeneCards database.

[0091] 3. Construction of PPI network for intersection target points of *Ilex chinensis* and RA in Xinjiang

[0092] The intersection of 1409 target components from Xinjiang *Ilex chinensis* and 453 RA-related targets was obtained, yielding 126 intersection targets. A Venn diagram of these intersection targets was then plotted. Figure 5 These shared targets were considered potential targets for treating rheumatoid arthritis (RA) with *Ilex chinensis* from Xinjiang. These shared targets were imported into the STRING database platform for Protein-Protein Interaction (PPI) analysis. The results were then imported into Cytoscape 3.9.1 software. The network point size was adjusted based on the Degree value to screen for key targets, many of which were related to anti-inflammatory effects. Figure 6 ).

[0093] 4. Functional and pathway enrichment analysis of the intersection target sites of *Ilex chinensis* and RA.

[0094] 126 overlapping target sites were imported into the DAVID database platform, with "Homo Sapiens" selected as the species, for enrichment analysis. Figure 7 The relevant pathways involved mainly include the PI3K-AKT signaling pathway and the NF-κB signaling pathway. PI3K activation leads to phosphorylation of phosphatidylinositol, which in turn activates the downstream major target AKT. AKT plays multiple important roles in regulating cell growth, differentiation, adhesion, and inflammatory responses. PI3K / AKT activation plays an important role in the expression of iNOS and COX2 in macrophages. NF-κB regulates the production of inflammatory mediators and induces the transcription of pro-inflammatory genes. It also participates in the regulation of cell proliferation and survival, mediating the secretion of cytokines (IL-1β, TNF-α, IL-6) and the expression of COX2.

[0095] Example 7

[0096] Verification of the pharmacodynamic mechanism of the extract from the stems of *Ilex chinensis* in mouse mononuclear macrophage leukemia cells RAW 264.7

[0097] 1. Cytotoxicity test of stem extract of *Ilex chinensis* from Xinjiang

[0098] RAW264.7 cells were loaded at a rate of 6 × 10⁻⁶. 3 The cells were seeded at a density of / well in 96-well plates and cultured in DMEM medium containing 10% Gibco Australian fetal bovine serum (hereinafter referred to as complete medium). When the cells in the control group reached 60% confluence, the medium was replaced with fresh complete medium and cultured for 8 hours. When the cells in the drug-treated groups reached 60% confluence, the medium was replaced with complete medium containing the drug solution (using complete medium prepared from the *Ilex chinensis* stem extract prepared in Example 1 at concentrations of 1, 2, 4, 8, 16, 32, 64, and 128 μg / mL), and cultured for 8 hours. Subsequently, 10 μL of CCK8 solution was added to each well, and the plates were cultured for another 30 minutes. The plates were then shaken for 1 minute, and the absorbance at 450 nm was measured using a full-wavelength microplate reader. The effect of different concentrations of *Ilex chinensis* stem extract on cell viability was evaluated based on the absorbance; the results are shown below. Figure 8 When the concentration of the extract of Xinjiang Sandalwood was 64 μg / mL, the survival rate of RAW264.7 cells was still greater than 80%, proving that the extract of Xinjiang Sandalwood was a low-toxicity traditional Chinese medicine. The next experiment was carried out with a safe concentration (64 μg / mL) of Xinjiang Sandalwood extract.

[0099] 2. The anti-inflammatory activity of the stem extract of *Ilex chinensis* from Xinjiang was tested using the CCK8 assay.

[0100] RAW264.7 cells were loaded at a rate of 6 × 10⁻⁶. 3The cells were seeded at a density of 1 / 2 well in 96-well plates and cultured in DMEM medium containing 10% Gibco Australian fetal bovine serum. In the control group, when cells reached 60% confluence, the medium was replaced with fresh complete medium and cultured for 8 hours. In the model group, when cells reached 60% confluence, the medium was replaced with complete medium containing lipopolysaccharide (LPS) (LPS concentration 1 μg / mL) and cultured for 8 hours. In the drug-treated group, when cells reached 60% confluence, the medium was replaced with complete medium containing both the drug solution and LPS (a complete medium containing both LPS and different concentrations of *Ilex chinensis* stem extract was prepared using complete medium; the LPS concentration was 1 μg / mL, and the *Ilex chinensis* stem extract concentrations were 1, 5, 10, 20, and 40 μg / mL, respectively), and cultured for 8 hours. Subsequently, 10 μL of CCK8 solution was added to each well, and the plates were cultured for another 30 minutes. After shaking for 1 minute, the absorbance at 450 nm was measured using a full-wavelength microplate reader. The anti-inflammatory activity of different concentrations of *Ilex chinensis* stem extract from Xinjiang was evaluated based on absorbance. The results are shown in [Figure 1]. Figure 9 This indicates that the extract of Xinjiang sand lily stem has a good anti-inflammatory effect, and it is dose-dependent.

[0101] 3. The pharmacodynamic mechanism of the extract from the stems of *Ilex chinensis* from Xinjiang was verified using Western blotting.

[0102] RAW264.7 cells were cultured at 20 × 10⁻⁶. 4 Cells were seeded at a density of 1 / 2 well in 6-well plates and cultured in DMEM medium containing 10% Gibco Australian fetal bovine serum. In the control group, when cells reached 60% confluence, the medium was replaced with fresh complete medium and cultured for 8 hours. In the model group, when cells reached 60% confluence, the medium was replaced with complete medium containing lipopolysaccharide (LPS) at a concentration of 1 μg / mL and cultured for 8 hours. In the drug-treated group, when cells reached 60% confluence, the medium was replaced with complete medium containing both the drug solution and LPS (a complete medium containing both LPS and different concentrations of *Ilex chinensis* stem extract was prepared, with LPS concentration of 1 μg / mL and *Ilex chinensis* stem extract concentrations of 10, 20, and 30 μg / mL, respectively), and cultured for 8 hours. Proteins were extracted, and Western blotting was used to detect changes in protein expression in the control, model, and drug-treated groups.

[0103] Based on the network pharmacology results analysis in Example 6, proteins such as p-PI3K, p-AKT, COX2, iNOS, NF-κB, and p-P65 were selected to study their mechanism of action and their protein expression was measured.

[0104] The measurement results are as follows Figure 10As shown, the stem extract of Xinjiang Sandalwood relieves inflammation through the PI3K / AKT / NF-κB signaling pathway. COX2 is the main target of antipyretic, analgesic and anti-inflammatory drugs in clinical practice, which also explains the potential reason why Xinjiang Sandalwood has good anti-inflammatory and analgesic effects.

[0105] Example 8

[0106] Study on formulation of extract from the stems of Xinjiang sandwort

[0107] 1. Preparation of a thermosensitive gel spray made from the stem extract of *Ilex chinensis* from Xinjiang

[0108] A method for preparing a thermosensitive gel spray includes the following steps: Take 1g of dried stems of *Ilex chinensis* from Xinjiang, grind them into powder, add 10 times the weight of the raw material in 80% ethanol (weight unit: g, volume unit: mL) and soak for 0.5h. Extract by reflux at 80℃ for 3h, cool to room temperature, filter the extract, combine the filtrates, and concentrate until the volume of the thick paste no longer decreases, obtaining a thick paste-like *Ilex chinensis* stem extract. Add 2.4mL of Tween 80 and 1.5g of sodium benzoate to obtain a *Ilex chinensis* stem extract solution. Measure 10mL of the *Ilex chinensis* stem extract solution, weigh 1.75g ​​of poloxamer 407 and 0.1g of poloxamer 188, and slowly add them to the *Ilex chinensis* stem extract solution. Refrigerate overnight at 4℃ to allow the poloxamer to fully swell, obtaining a transparent, flowable solution. Fill into spray bottles to obtain the thermosensitive gel spray. Figure 11 .

[0109] In this embodiment, the thermosensitive gel is a liquid at room temperature. At body temperature (36-37°C), the formulation undergoes a phase change to form a gel due to the increased temperature. The viscosity increases and the adhesion is enhanced, which can effectively retain the drug at the administration site, prolong the retention time, and achieve continuous and slow drug release. It is particularly suitable for the treatment of rheumatoid arthritis.

[0110] 2. Preparation of extract ointment from the stems of *Ilex chinensis* (Xinjiang sand lily).

[0111] A solution of Xinjiang sandwort stem extract was prepared into an ointment using a composite oil phase of stearic acid, white petrolatum and lanolin in a mass ratio of 1:3:4, Span 60 as an emulsifier and Tween 80 as an auxiliary emulsifier.

[0112] Take 1g of dried stems of *Ilex chinensis* from Xinjiang, grind them into powder, add 10 times the volume (g, mL) of the raw material in 80% ethanol and soak for 0.5h. Heat under reflux at 80℃ for 3h, cool to room temperature, filter the extract, combine the filtrates, and concentrate until the volume of the thick paste no longer decreases, obtaining a thick paste-like *Ilex chinensis* stem extract. Add 2.4mL of Tween 80 and 1.5g of sodium benzoate to obtain a *Ilex chinensis* stem extract solution. Measure 10mL of the *Ilex chinensis* stem solution (aqueous phase), weigh 0.75g of stearic acid, 2.25g of white petrolatum, and 3g of lanolin. Using stearic acid, white petrolatum, and lanolin as the oil phase, incubate in an 80℃ water bath, add 0.6g of Span 60, and stir thoroughly to dissolve. Slowly pour the aqueous phase into the oil phase while stirring constantly until it becomes a cream. Cool naturally to below 30℃, stop stirring, and let cool to obtain the *Ilex chinensis* stem extract paste.

[0113] The ointment in this embodiment has strong adhesion and can remain on the application site for a long time under body temperature, achieving continuous and slow drug release, which is suitable for the treatment of rheumatoid arthritis.

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. The application of *Ilex chinensis* stem extract from Xinjiang in the preparation of drugs for treating rheumatoid arthritis; characterized in that: The active ingredients of the stem extract of *Ilex chinensis* from Xinjiang are flavonoids, mainly including gentianin, glycyrrhizin, and white ketone. It is prepared by the following method: The dried stems of *Ilex chinensis* from Xinjiang are pulverized, soaked in 75%–95% ethanol (8–10 times their weight by volume) for 0.5–1 hour, and extracted by reflux 1–4 times, 1–3 hours each time. The extract is then filtered, the filtrates are combined, and concentrated to obtain the stem extract of *Ilex chinensis* from Xinjiang. Alternatively, the dried stems of *Ilex chinensis* from Xinjiang are pulverized, soaked in 75%–95% ethanol (8–10 times their weight by volume) for 0.5–1 hour, and extracted by reflux 1–4 times, 1–3 hours each time. The extract is then filtered, the filtrates are combined, concentrated, and dried to obtain the stem extract of *Ilex chinensis* from Xinjiang.

2. Use according to claim 1, characterized in that: The application described is the use of Xinjiang Sand Wintergreen stem extract in the preparation of a drug for treating rheumatoid arthritis by reducing at least one of the inflammatory factors TNF-α, IL-6, and IL-1β and decreasing inflammatory cell infiltration.

3. Use according to claim 1, characterized in that: The application described is the use of the stem extract of Xinjiang Sandalwood in the preparation of a drug for treating rheumatoid arthritis by inhibiting the activation of the NF-κB / COX2 pathway.

4. Use according to claim 1, characterized in that: The drug is made from the stem extract of Xinjiang sand lily as the active ingredient, along with a pharmaceutically acceptable carrier or excipient.

5. Use according to claim 4, characterized in that: The dosage form of the drug is hydrogel or ointment.

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