Medical application of isopentenyl flavonoid compound or aryl naphthalene lignan compound in podophyllum hexandrum

Abstract

The invention discloses a medical application of an isopentenyl flavonoid compound or an aryl naphthalene lignan compound in podophyllum hexandrum. The isopentenyl flavone compound and the aryl naphthalene lignan compound can obviously inhibit proliferation of FLS cells induced by TNF-alpha, and show anti-RA activity in a rat RA model induced by a complete Freund's adjuvant; the isopentenyl flavonoid compound can play an anti-RA effect by inhibiting JAK1, inhibit phosphorylation of proteins such as PLC-gamma, p38 and JNK in FLS cells induced by TNF-alpha, inhibit NF-kappa B intracellular expression, and inhibit a JAK1 downstream signal channel at the cellular level; the aryl naphthalene lignan compound can play an anti-RA effect by inhibiting TNF-alpha, inhibit expression of RIP-1 and TRAF2 / 4 / 5 in FLS cells induced by TNF-alpha, inhibit expression in NF-kB nucleus, and inhibit a TNF-alpha downstream signal channel at the cellular level.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to the medical use of isoprenylated flavonoids or aryl naphthalide lignans in preparing a medicine for treating rheumatoid arthritis (RA). BACKGROUND

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease with erosive arthritis as the main clinical manifestation, characterized by chronic inflammation of the synovium and progressive destruction of the joint, often manifested as joint swelling, pain, and difficulty in flexion and extension [1,2] . It is currently believed that the causes of RA are related to genetics, infection, sex hormone levels, environment, and other factors. The basic pathological changes are chronic synovitis and pannus, which gradually leads to destruction of the joint cartilage and bone, and eventually results in joint deformity and loss of function [3] . RA is most common in people aged 25-50 years. Untreated RA has a high rate of disability, and can also cause extra-articular damage, affecting the heart, lungs, kidneys, and other organs, severely affecting the patient's quality of life. The global incidence of RA is about 0.5-1%, and the direct cost of treatment and indirect economic loss due to loss of labor each year is very large [2] .

[0003] Currently, the drugs used for the treatment of RA mainly include hormone drugs, non-steroidal anti-inflammatory drugs (NSAIDs), and disease-modifying antirheumatic drugs (DMARDs), etc., but all have certain shortcomings. For example, hormone drugs have large toxic and side effects, and can only be used as short-term treatment drugs; NSAIDs can relieve symptoms and take effect quickly, but cannot control disease progression; DMARDs can delay and control disease progression to some extent, but have a slow onset and risks such as bone marrow suppression and abnormal liver function [4] . The low clinical remission rate, frequent drug side effects, and difficult-to-contain bone destruction and complications are still objective treatment difficulties [5] . In recent years, researchers have carried out a lot of work on the anti-RA effects of traditional Chinese medicines, including antibacterial, anti-inflammatory, analgesic, and immunoregulatory aspects. A variety of RA treatment drugs have been developed from traditional anti-rheumatic and anti-inflammatory Chinese medicines, such as Tripterygium glycosides tablets, Qingdai alkaline injection, and total glycosides of white peony root capsules [6-8] . The above drugs can relieve the clinical symptoms of RA patients and control disease progression to some extent. At present, there is an urgent need for good drugs with high-quality evidence-based evidence in clinical practice. It is of great clinical significance to find new anti-RA drugs with high efficiency, low toxicity, and strong selectivity from traditional Chinese medicines or ethnic medicines.

[0004] *Sinopodophyllum hexandrum (Royle) Ying* is a perennial herbaceous plant belonging to the genus *Sinopodophyllum* in the family Berberidaceae. [9] Peach berry, first recorded under the name "Guijiu" in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), contains flavonoids as its main chemical components. [10-12] Lignans [13,14] Small amounts of sterols

[15] and phenolic acid components

[16] It possesses various biological activities, including anti-tumor, antiviral, anti-inflammatory, antibacterial, and insecticidal effects. [17,18] .

[0005] References

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[0007] [2] Chinese Rheumatology Society. 2018 Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis [J]. Chinese Journal of Internal Medicine, 2018, 57(4):242-251.

[0008] [3]McInnes IB, Schett G. The Pathogenesis of Rheumatoid Arthritis[J]. New England Journal of Medicine, 2011, 365(23):2205-2219.

[0009] [4] Geng Yan, Xie Xi, Wang Yu, Jiang Dexun, Zhang Wen, Zhang Zhuoli, Zhao Yan, representing the Chinese Rheumatology Association. Guidelines for the diagnosis and treatment of rheumatoid arthritis [J]. Chinese Journal of Internal Medicine, 2022, 61(1):51-59.

[0010] [5] Zhang Xiaoxiao, Lu Yao, Hou Wei, Zhu Mingjun, Tang Xudong, Xiong Lei, Jiang Quan, Zeng Xianyu, Li Haisong, Zhao Ruihua, Wang Guochen, Zhang Boli. Clinical needs list for the research and development of new Chinese medicine drugs (first batch) [J]. Journal of Traditional Chinese Medicine, 2024, 65(1):17-25.

[0011] [6] Feng Z, Fu L, Wang J, Zhu Y, He X, Zhou L, Zhou X. Efficacy of tripterygium glycosides (TG) in rheumatoid arthritis as a disease-modifying anti-rheumatic drug (DMARD) in combination with conventional DMARDs: A systematic review and meta-analysis of randomized controlled trials [J]. Pharmacological Research, 2022, 184: 106405.

[0012] [7] Duan D, Wang Y, Pan D, Jin X, Yan Y, Song P, Wang L, Xiao J, Wang Z, Wang X. Rheumatoid arthritis drug sinomenine induces apoptosis of cervical tumor cells by targeting thioredoxin reductase in vitro and in vivo [J]. Bioorganic Chemistry, 2022, 122: 105711.

[0013] [8] Qu B, Wang X L, Zheng D C, Mai C T, Liu Z Q, Zhou H, Xie Y. Novel treatment for refractory rheumatoid arthritis with total glucosides of paeony and nobiletin codelivered in a self-nanoemulsifying drug delivery system [J]. Acta Pharmacologica Sinica, 2022, 43: 2094-2108.

[0014] [9] Fu LG. Chinese Plant Red Book - Rare and Endangered Plants (First Edition) [M]. Beijing: Science Press, 1992: 691.

[0015]

[10] Sun Y J, Zhou W, Chen H, Li Z L, Hua H M. Isolation and identification of flavonoids from Sinopodophyllum hexandrum Royle [J]. Journal of Shenyang Pharmaceutical University, 2012, 29(3): 185-189.

[0016]

[11] Sun Y J, Chen H, Xue G M, Chen H, Zhang Y L, Li M, Du K, Wang J M, Feng W S. Two new flavonoid glucosides from the fruits of Sinopodophyllum hexandrum [J]. Natural Product Research, 2021, 35: 2164-2169.

[0017]

[12] Sun Y, Chen H, Wang J, Gao M, Zhao C, Han R, Chen H, Li M, Xue G, Feng W. Sixteen new prenylated flavonoids from the fruit of Sinopodophyllum hexandrum [J]. Molecules, 2019, 24: 3196.

[0018]

[13] Jackson D E, Dewick P M. Aryltetralin lignans from Podophyllum hexandrum and Podophyllum peltatum [J]. Phytochemistry, 1984, 23: 1147-1152.

[0019]

[14] Sun Y J, Li Z L, Chen H, Liu X Q, Zhou W, Hua H M. Three new cytotoxic aryltetralin lignans from Sinopodophyllum emodi [J]. Bioorganic & Medicinal Chemistry Letters, 2011, 21: 3794-3797.

[0020]

[15] Sun Y J, Li Z L, Chen H, Zhou W, Hua H M. Chemical constituents of Sinopodophyllum hexandrum [J]. Chinese Herbal Medicines, 2012, 35: 1607-1609.

[0021]

[16] Sun YJ, Zhou W, Chen H, Li ZL, Hua HM. Studies on phenolic constituents from Radix Daturae Stramonii [J]. Chinese Herbal Medicines, 2012, 43(2): 226-229.

[0022]

[17] Yan ST, Fan H, Li RL, Guo YL, Liu Q, Gao F, Ou L, Chen L, Li M, Wei PF, Zhang L. Research Progress on Chemical Constituents and Pharmacological Effects of Radix Daturae Stramonii [J]. Chinese Wild Plant Resources, 2020, 39(7): 43-50.

[0023]

[18] Anand U, Biswas P, Kumar V, Ray D, Ray P, Loake V I, P., Kandimalla R, Chaudhary A, Singh B, Routhu N K, Chen Z-S, J, Dey A. Podophyllum hexandrum and its active constituents: Novel radioprotectants [J]. Biomedicine & Pharmacotherapy, 2022, 146: 112555-112566. SUMMARY

[0024] The inventors used the TNF-α-induced rat fibroblast-like synoviocyte (FLS cell) proliferation model to screen the in vitro anti-RA activity of different extraction fractions of the Radix Daturae Stramonii root alcohol extract, and the results showed that the dichloromethane extraction fraction of Radix Daturae Stramonii (SHDFr) had an IC 50The value is 8.45 μg / mL, and has in-vitro anti-RA activity. In a CFA-induced RA model of rats, oral SHD Fr (25 mg / kg and 50 mg / kg) can dose-dependently reduce the swelling degree of the feet of the RA rats and joint damage, and inhibit the levels of various inflammatory factors in the blood and synovial tissue. The inventors have isolated and identified isoprenyl flavonoids and aryl naphthalide lignans with anti-RA activity from SHD Fr, which can significantly inhibit the proliferation of FLS cells induced by TNF-α and exhibit anti-RA activity in a CFA-induced RA model of rats; the isoprenyl flavonoids can exert anti-RA effect by inhibiting JAK1, dose-dependently inhibit the phosphorylation of PLC-γ, p38, JNK and other proteins in FLS cells induced by TNF-α, inhibit the nuclear expression of NF-κB, and inhibit the JAK1 downstream signaling pathway at the cellular level; the aryl naphthalide lignans can exert anti-RA effect by inhibiting TNF-α, dose-dependently inhibit the expression of RIP-1 and TRAF2 / 4 / 5 in FLS cells induced by TNF-α, inhibit the nuclear expression of NF-κB, and inhibit the TNF-α downstream signaling pathway at the cellular level.

[0025] The present application aims to provide isoprenyl flavonoids with the structure shown in Formula I:

[0026]

[0027] R1=H, R3=H, R4=H; R1=OH, R3=CH3, R4=H; R1=H, R2=H, R3=H,

[0028] The present application further aims to provide the use of the isoprenyl flavonoids in the preparation of a drug for treating rheumatoid arthritis.

[0029] Preferably, the use is the use of the isoprenyl flavonoids in the preparation of a drug for treating rheumatoid arthritis by inhibiting JAK1.

[0030] The present application further aims to provide aryl naphthalide lignans with the structures shown in Formula II, III, IV and V:

[0031]

[0032] R1=H, 1a R3=H, R4=H; R1=OH, 2a R3=CH3, R4=H; R1=H, R2=H, R3=H, 1a R3=CH3, R4=H; R1=H, R2=H, R3=H,2a = CH3; R 1a = OH, R 2a = H; R 1b = OH, R 2b = CH3; R 1b = H, R 2b = CH3.

[0033] It is another object of the present application to provide the use of the aryl naphthalide lignan compound in the preparation of a medicine for treating rheumatoid arthritis.

[0034] Preferably, the use is the use of the aryl naphthalide lignan compound in the preparation of a medicine for treating rheumatoid arthritis by inhibiting TNF-α. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 In Vitro Anti-RA Activity of Different Extracts of Anemone Raddeana Fr. Schmidt; Wherein, A is the effect of different extracts of Anemone Raddeana Fr. Schmidt on the proliferation of TNF-α induced rat FLS cells; B is the IC 50 value of SHD Fr on the proliferation of TNF-α induced rat FLS; compared with the control group, *** p<0.001; compared with the model group, ## p<0.01, ## p<0.001.

[0036] Figure 2 In Vivo Anti-RA Efficacy of SHD Fr on CFA Induced Rat RA; Wherein, A is the dynamic curve of rat foot thickness at different time points; B is the actual shot of the foot bottom, HE staining of the knee joint, and Safranin O-fast green staining results after the last day of administration.

[0037] Figure 3 Effect of SHD Fr on the Contents of TNF-α (A), IL-6 (B), and IL-1β (B) in the Synovial Tissue of CFA Induced Rat RA Model; Compared with the blank control group, *** p<0.001; compared with the model group, # p<0.05, ## p<0.01, ## p<0.001.

[0038] Figure 4 Effect of SHD Fr on the Contents of TNF-α (A), IL-6 (B), and IL-1β (B) in the Blood of CFA Induced Rat RA Model; Compared with the blank control group, *** p<0.001; compared with the model group, # p<0.05, ## p<0.01, ##p<0.001.

[0039] Figure 5 The results of the study on the anti-TNF-α induced FLS cell proliferation of compounds 1-15; wherein, compared with the blank control group, *** p<0.001; compared with the negative control group, ## p<0.01, ### p<0.001.

[0040] Figure 6 The influence of SHD Fr on TNF-α and JAK1 downstream signal pathways in synovial tissue; wherein, A is the influence of SHD Fr on the expression of proteins related to TNF-α downstream signal pathways; B is the influence of SHD Fr on the expression of proteins related to JAK1 downstream signal pathways; C is the level of NF-κB and NFATc1 in synovial tissue measured by immunofluorescence.

[0041] Figure 7 The influence of compounds 11 and 2 on TNF-α and JAK1 downstream signals in vitro respectively. (A) Western-blot method was used to detect the influence of compound 11 on TNF-α downstream signal pathways; (B) Western-blot method was used to detect the influence of compound 2 on JAK1 downstream signal pathways; (C) Immunofluorescence was used to detect the influence of compounds 11 and 2 on the expression of NF-κB. DETAILED DESCRIPTION

[0042] Example 1

[0043] Extraction and extraction of Anemone raddeana Regel medicinal materials

[0044] Take 20 kg of dried roots of Radix Podophyli, crush and pass through a No. 2 sieve. First, add 95% ethanol according to a solid-liquid ratio of 1:10 kg / L, heat to reflux for 1 h, cool to room temperature, and collect the extract liquid by filtration with gauze. Then, add 95% ethanol to the residue according to a solid-liquid ratio of 1:8 kg / L, heat to reflux for 2 times, 1 h each time, and collect the extract liquid by filtration with gauze. Combine the extract liquids, recover ethanol by rotary evaporation until no alcohol taste and nearly dry, to obtain an ethanol extract (SHE Ext). Mix the SHE Ext in warm water to obtain a dispersoid of extract, repeatedly extract with petroleum ether for 3 times, combine the petroleum ether extract, and concentrate to dryness to obtain a petroleum ether extract part (denoted as SHP Fr, 209 g). Extract the mother liquor with dichloromethane for 3 times, combine the dichloromethane extract, and concentrate to dryness to obtain a dichloromethane extract part (denoted as SHD Fr, 783 g). Continue to extract the mother liquor with water-saturated n-butanol for 3 times, combine the n-butanol extract, and concentrate to dryness to obtain an n-butanol extract part (denoted as SHB Fr, 511 g). Concentrate the remaining mother liquor to dryness to obtain a water layer (denoted as SHW Layer, 325 g).

[0045] In vivo and in vitro anti-RA activity of different extract parts of Radix Podophyli

[0046] To explore the anti-“wind-damp arthralgia” and anti-RA material basis of Radix Podophyli, the inventors used a TNF-α-induced rat synoviocyte fibroblast (FLS cell) proliferation model to screen the in vitro anti-RA activity of the different extract parts (SHP Fr, SHD Fr, SHB Fr) of Radix Podophyli prepared in Example 1.

[0047] Cell culture: Take the cryopreserved tube containing FLS cells out of liquid nitrogen, and perform cell recovery. Seed the cells in DMEM medium containing 10% FBS, 1% streptomycin and penicillin, so that the cells are uniformly spread on the bottom of the culture bottle. Place the culture bottle in a cell culture incubator at 5% CO2, 95% relative humidity and 37°C, and select FLS cells that grow well and are passed to 3-6 generations for experiments.

[0048] Experimental grouping, dosing regimen and establishment of cell model: Collect FLS cells in the logarithmic growth phase, and seed 2×10 5cells / well were plated in 6-well plates, 2 mL per well, and the cells were grown to 70-80% confluence. The cells were divided into blank control group, model group, and different concentrations of drug administration group (SHP Fr, SHD Fr, SHB Fr, 10, 20, 40, 80, 100 μg / mL). The blank control group was replaced with fresh culture medium, and the other groups were replaced with culture medium containing TNF-α (final concentration 20 ng / mL) per well, and cultured for 12 h. The blank control group was replaced with fresh culture medium, the model group was replaced with culture medium containing TNF-α (final concentration 20 ng / mL), and the drug administration group was replaced with culture medium containing the corresponding concentration of the test drug (containing TNF-α final concentration 20 ng / mL), and cultured for 24 h. The MTS cell proliferation kit (MTS Cell Proliferation Colorimetric Assay Kit) was used to detect the optical density D(λ) value of the cells in each group according to the instructions, and the inhibition rate of the drug on the cells was calculated.

[0049] The results are shown in Figure 1 , which show that SHD Fr can inhibit FLS cell proliferation in a concentration-dependent manner, while other extraction sites do not have this activity, and the IC 50 values are all greater than 200 μg / mL Figure 1 A). The IC 50 of SHD Fr on TNF-α-induced FLS cell proliferation is 8.45 μg / mL Figure 1 B). The above results show that the dichloromethane extraction fraction of the root of Anopterus chinesis has strong in vitro anti-RA activity.

[0050] The inventors further evaluated the in vivo anti-RA activity of SHD Fr using a CFA-induced RA model in rats.

[0051] Model establishment: After isoflurane anesthesia of the rats, the right foot of the rats was disinfected, and the normal control group of rats was injected with normal saline (0.1 mL per rat), and the other groups were injected with 0.1 mL CFA on the right foot to induce inflammation and develop into adjuvant arthritis (AIA), and the modeling day was counted as day 0.

[0052] Experimental grouping and drug administration scheme: randomly divided into blank control group, model group, positive control group (Tofacitinib, 15 mg / kg), and different dose administration groups, 10 animals per group, male, 8-10 weeks old. The blank control group was not treated, and the model group and drug group were treated with normal saline by gavage 11 days after modeling, the positive control group of rats was given Tofacitinib (15 mg / kg) by gavage, and the drug administration group of rats was given the corresponding dose concentration of the drug (SHD Fr: 25 and 50 mg / kg) by gavage, and the administration was continued for 10 days. The rats were sacrificed on day 21 and the relevant experiments were performed.

[0053] Measurement of ankle or wrist joint swelling degree of AIA rats: After modeling, the degree of redness and swelling of the ankle joint, metatarsophalangeal joint and interphalangeal joint of the hind limbs of AIA rats was measured every 3 days using a vernier caliper to observe the inhibitory effect of the drug on the ankle or wrist joint swelling degree of AIA rats.

[0054] Analysis of joint tissue of AIA rats by staining method: On day 21, rats were sacrificed by cervical dislocation, and the ankle joint was taken, fixed with 4% paraformaldehyde, decalcified with 10% EDTA, routinely dehydrated, paraffin-embedded, sectioned, HE stained, and ponceau-fuchsin stained, and X-rayed.

[0055] The contents of TNF-α, IL-6 and IL-1β in synovial tissue and blood were determined by ELISA.

[0056] Results are shown in Figures 2-4 , which show that oral administration of SHD Fr (25, 50 mg / kg) can dose-dependently reduce CFA-induced rat foot swelling ( Figure 2 A) Pathological results show that the cells in the normal group are arranged uniformly and neatly, the nucleus is clearly visible, the staining is uniform, and there is no synovial thickening and neovascularization; while in the model group, the cells are arranged in disorder, the synovium is severely proliferated, part of the cell nucleus is necrotic, the staining is uneven, and there are defects and vascularization; compared with the model group, SHD Fr has mild proliferation, mild neovascularization and partial inflammatory cell infiltration ( Figure 2 B). In addition, SHD Fr can significantly reduce the levels of various inflammatory factors TNF-α, IL-6 and IL-1β in synovial tissue and blood ( Figure 3 and Figure 4 ). The above results show that the dichloromethane extract fraction of the root stem of Anemone raddeana (SHD Fr) has strong anti-RA activity in vivo, and is an effective extract fraction of A. raddeana against RA.

[0057] Example 2

[0058] Chemical constituent study of dichloromethane extract fraction of A. raddeana medicinal material (SHD Fr)

[0059] Qualitative analysis of chemical constituents of SHD Fr by high-resolution mass spectrometry

[0060] High-resolution mass spectrometry (AB Sciex TripleTOF TM 5600 +The sample after SHD Fr treatment was determined by UHPLC-QTOF-MS / MS) information-dependent acquisition (IDA) and dynamic background subtraction (DBS) technology, combined with literature data, mass spectrometry database (mzCloud, MoNA and Metlin, etc.) and mass spectrometry data of separated compounds, the fragmentation rules of different types of compounds were analyzed, and 56 relatively high content chemical components in SHD Fr were identified, mainly flavones and lignans, and a small amount of phenolic acid components (see Table 1 for main chemical components).

[0061] Example 3

[0062] Separation and structure identification of part of compounds in SHD Fr

[0063] Fifteen compounds were separated from the dichloromethane active extraction fraction, which were compound 1-compound 15. Among them, compound 1 (dysoverine A), compound 5 (kaempferol), compound 6 (4'-methyl kaempferol), compound 7 (quercetin), and compound 8 (3-O-methyl quercetin) showed no anti-RA activity, and the remaining 10 compounds had significant anti-RA activity. The specific separation process is as follows:

[0064] The dichloromethane extraction fraction (SHD Fr) prepared in Example 1 was subjected to silica gel column chromatography, and dichloromethane and methanol (7:3→5:5, v / v) were used as eluents to divide it into 7 fractions, denoted as Fr1-Fr7;

[0065] Fr3 was subjected to semi-preparative liquid chromatography separation, and the chromatographic column was YMC-Pack ODS-A preparative column, the mobile phase was acetonitrile: water = 65:35, v / v, formic acid was added to adjust the pH to 3.0, the flow rate was 8 mL / min, and the detection wavelength was 254 nm, to obtain compound 2 and compound 3;

[0066] Fr4 was subjected to silica gel column chromatography, and dichloromethane and methanol (48:52, v / v) were used as eluents to divide it into 2 fractions, denoted as Frs41 and Frs42; Frs41 was recrystallized (recrystallization solvent: methanol, recrystallization temperature: room temperature) to obtain compound 4; Frs42 was subjected to semi-preparative liquid chromatography separation, and the chromatographic column was YMC-Pack ODS-A preparative column, the mobile phase was acetonitrile:methanol:water = 25:20:55, v / v / v, formic acid was added to adjust the pH to 3.5, the flow rate was 8 mL / min, and the detection wavelength was 254 nm, to obtain compound 10;

[0067] Fr5 was subjected to HPLC separation on a YMC-Pack ODS-A preparative column with a mobile phase of acetonitrile:methanol:water = 15:22:63, v / v / v, adjusted to pH 3.5 with formic acid, at a flow rate of 8 mL / min, and detection at 254 nm to give compound 14 and compound 15;

[0068] Fr6 was subjected to recrystallization (recrystallization solvent: methanol, recrystallization temperature: room temperature) to give compound 9;

[0069] Fr7 was subjected to MCI column chromatography (filler: CHP-20P 75-150 pm, Trilink Chemicals) with gradient elution using 40% ethanol, 60% ethanol, 80% ethanol, and 100% ethanol, to give four fractions: Frs71, Frs72, Frs73, and Frs74. Frs71 was first subjected to reverse silica gel column chromatography (filler: Chromatorex C18 SMB 100-20 / 45, Catalog No. HU200601; acetonitrile:water = 70:30, v / v, adjusted to pH 3.0 with formic acid), and then subjected to HPLC separation on a YMC-Pack ODS-A preparative column with a mobile phase of acetonitrile:water = 48:52, v / v, adjusted to pH 3.5 with formic acid, at a flow rate of 8 mL / min, and detection at 254 nm to give compound 11. Frs72 was first subjected to reverse silica gel column chromatography (filler: Chromatorex C18 SMB 100-20 / 45, Catalog No. HU200601; acetonitrile:water = 55:45, v / v, adjusted to pH 3.5 with formic acid), and then subjected to HPLC separation on a YMC-Pack ODS-A preparative column with a mobile phase of acetonitrile:methanol:water = 35:20:45, v / v / v, adjusted to pH 3.5 with formic acid, at a flow rate of 8 mL / min, and detection at 254 nm to give compound 13. Frs74 was subjected to HPLC separation on a YMC-Pack ODS-A preparative column with a mobile phase of acetonitrile:methanol:water = 40:25:35, v / v / v, adjusted to pH 3.5 with formic acid, at a flow rate of 8 mL / min, and detection at 254 nm to give compound 12.

[0070] Compound 2: yellow powder, HR-ESI-MS m / z: 355.1167 [M+H] + (cacld for C 20 H 19 O6355.1176); 1H-NMR (400 MHz, DMSO-d6) δ: 12.49 (1H, s, 5-OH), 10.75 (1H, s, 7-OH), 10.05 (1H, s, 4'-OH), 9.30 (1H, s, 3-OH), 7.91 (1H, d, J = 2.3 Hz, H-2'), 7.88 (1H, dd, J = 2.4, 8.52 Hz, H-6'), 6.94 (1H, d, J = 8.5 Hz, H-5'), 6.42 (1H, d, J = 2.1 Hz, H-8), 6.19 (1H, d, J = 2.0 Hz, H-6), 5.30 (1H, t, J = 7.3 Hz, H-2"), 3.28 (2H, d, J = 7.3 Hz, H-1"), 1.71 (3H, s, 4"-CH3), 1.70 (3H, s, 5"-CH3); 13 C-NMR (100 MHz, DMSO-d6) δ: 147.03 (C-2), 135.64 (C-3), 175.87 (C-4), 160.75 (C-5), 98.19 (C-6), 163.87 (C-7), 93.42 (C-8), 156.16 (C-9), 103.05 (C-10), 122.48 (C-l'), 129.15 (C-2'), 127.69 (C-3'), 156.97 (C-4'), 114.91 (C-5'), 127.06 (C-6'), 28.13 (C-l"), 121.66 (C-2"), 131.85 (C-3"), 25.54 (C-4"), 17.73 (C-5"). Compound 2 was identified as isolicoflavonol by comparison with the reported data.

[0071] Compound 3: yellow brown powder, HR-ESI-MS m / z: 385.1272 [M+H] + (cacld for C 21 H 21 O7385.1282); 1H-NMR (400 MHz, DMSO-d6) δ: 12.71 (1H, s, 5-OH), 10.81 (1H, s, 7-OH), 9.75 (1H, s, 4'-OH), 9.12 (1H, s, 3'-OH), 7.45 (1H, d, J = 2.2 Hz, H-6'), 7.37 (1H, dd, J = 2.2 Hz, H-2'), 6.38 (1H, d, J = 2.0 Hz, H-8), 6.19 (1H, d, J = 2.0 Hz, H-6), 5.31 (1H, t, J = 7.4 Hz, H-2"), 3.77 (1H, s, 3"-OCH3), 3.28 (2H, d, J = 7.4 Hz, H-1"), 1.71 (3H, s, 4"-CH3), 1.69 (3H, s, 5"-CH3); 13 C-NMR (100 MHz, DMSO-d6) δ: 146.44 (C-2), 137.67 (C-3), 177.90 (C-4), 161.31 (C-5), 98.54 (C-6), 164.11 (C-7), 93.49 (C-8), 155.74 (C-9), 104.18 (C-10), 122.44 (C-l'), 112.71 (C-2'), 144.76 (C-3'), 156.30 (C-4'), 128.23 (C-5'), 131.88 (C-6'), 28.05 (C-l"), 120.79 (C-2"), 131.88 (C-3"), 25.55 (C-4"), 17.66 (C-5"), 59.59 (3-OCH3). Compound 3 was identified as uralenol-3-methylether by comparison with the reported data.

[0072] Compound 4: yellow powder, HR-ESI-MS m / z: 355.1179 [M+H] + (cacld for C 20 H 19 O6355.1176); 1H-NMR (400 MHz, DMSO-d6) δ: 12.41 (1H, s, 5-OH), 10.69 (1H, s, 7-OH), 10.10 (1H, s, 4'-OH), 9.34 (1H, s, 3-OH), 8.03 (2H, d, J = 8.7 Hz, H-2', 6'), 6.93 (2H, d, J = 8.7 Hz, H-3', 5'), 6.29 (1H, s, H-6), 5.17 (1H, t, J = 7.0 Hz, H-2"), 3.42 (2H, d, J = 6.8 Hz, H-1"), 1.74 (3H, s, 4"-CH3), 1.62 (3H, s, 5"-CH3); 13 C-NMR (100 MHz, DMSO-d6) δ: 146.74 (C-2), 135.55 (C-3), 176.15 (C-4), 161.16 (C-5), 97.80 (C-6), 158.30 (C-7), 105.60 (C-8), 153.48 (C-9), 103.03 (C-10), 122.60 (C-1'), 129.39 (C-2', 6'), 115.48 (C-3', 5'), 159.19 (C-4'), 21.23 (C-1"), 122.02 (C-2"), 130.95 (C-3"), 25.46 (C-4"), 17.83 (C-5"). Compound 4 was identified as 8-prenylkaempferol by comparison with the data reported in the literature.

[0073] Compound 9: white powder, HR-ESI-MS m / z: 415.1380 [M+H] + (cacld for C 22 H 23 O8415.1387); 1H-NMR (400 MHz, CDC13) δ: 7.11 (1H, s, H-8), 6.51 (1H, s, H-5), 6.37 (2H, s, H-2', 6'), 5.99 (1H, d, J = 1.3 Hz, H-13a), 5.97 (1H, d, J = 1.3 Hz, H-13β), 4.77 (1H, d, J = 9.0 Hz, H-1), 4.60 (1H, dd, J = 7.2, 8.6 Hz, H-11a), 4.08 (1H, dd, J = 8.8, 10.0 Hz, H-11β), 4.59 (1H, d, J = 4.4 Hz, H-4), 3.81 (3H, s, 4'-OCH3), 3.76 (6H, s, 3', 5'-OCH3), 2.84 (1H, overlapped, H-3), 2.78 (1H, m, H-2); 13 C-NMR (100 MHz, CDC13) δ: 72.87 (C-1), 40.82 (C-2), 45.33 (C-3), 44.12 (C-4), 109.83 (C-5), 147.74 (C-6), 147.85 (C-7), 106.28 (C-8), 133.12 (C-9), 131.23 (C-10), 71.32 (C-11), 174.36 (C-12), 101.48 (C-13), 135.40 (C-1'), 108.45 (C-2', 6'), 152.64 (C-3', 5'), 137.32 (C-4'), 60.78 (4'-OCH3), 56.32 (3', 5'-OCH3). Compound 9 was identified as podophyllotoxin by comparison with the reported data.

[0074] Compound 10: white powder, HR-ESI-MS m / z: 399.1437 [M+H] + (cacld for C 22 H 23 O7399.1438); 1H-NMR (400 MHz, DMSO-d6) δ: 6.81 (1H, s, H-8), 6.51 (1H, s, H-5), 6.30 (2H, s, H-2', 6'), 5.97 (1H, s, H-13a), 5.95 (1H, s, H-13β), 4.51 (1H, d, J = 5.1 Hz, H-4), 4.41 (1H, t, J = 7.6 Hz, H-11a), 3.95 (1H, dd, J = 8.3, 10.5 Hz, H-11β), 3.02 (1H, dd, J = 5.1, 15.8 Hz, H-1a), 2.74 (1H, dd, J = 11.6, 15.8 Hz, H-1β), 3.64 (6H, s, 3', 5'-OCH3), 3.62 (3H, s, 4'-OCH3), 2.98 (1H, dd, J = 5.2, 13.7 Hz, H-3), 2.62 (1H, m, H-2); 13 C-NMR (100 MHz, DMSO-d6) δ: 32.54 (C-1), 31.98 (C-2), 46.06 (C-3), 42.99 (C-4), 109.92 (C-5), 145.90 (C-6), 146.32 (C-7), 108.52 (C-8), 130.55 (C-9), 129.06 (C-10), 71.54 (C-11), 174.95 (C-12), 100.91 (C-13), 136.28 (C-1'), 108.14 (C-2', 6'), 151.94 (C-3', 5'), 137.01 (C-4'), 55.75 (3', 5'-OCH3). Compound 10 was identified as deoxypodophyllotoxin by comparison with the reported data.

[0075] Compound 11: white powder, HR-ESI-MS m / z: 401.1235 [M+H] + (cacld for C 21 H 21 O8401.1231); 1H-NMR (400 MHz, DMSO-d6) δ: 8.27 (1H, s, 4'-OH), 7.10 (1H, s, H-8), 6.46 (1H, s, H-5), 6.30 (2H, s, H-2', 6'), 5.99 (1H, s, H-13a), 5.96 (1H, s, H-13β), 5.77 (1H, d, J = 7.1 Hz, 1-OH), 4.61 (1H, dd, J = 7.6, 9.4 Hz, H-1), 4.47 (1H, t, J = 7.8 Hz, H-11a), 4.44 (1H, d, J = 4.9 Hz, H-4), 4.08 (1H, dd, J = 8.8, 10.3 Hz, H-11β), 3.63 (6H, s, 3', 5'-OCH3), 3.08 (1H, dd, J = 5.0, 14.2 Hz, H-3), 2.61 (1H, m, H-2); 13 C-NMR (100 MHz, DMSO-d6) δ: 70.66 (C-1), 40.13 (C-2), 44.29 (C-3), 43.23 (C-4), 109.14 (C-5), 146.53 (C-6), 146.43 (C-7), 106.31 (C-8), 131.17 (C-9), 131.01 (C-10), 71.05 (C-11), 174.81 (C-12), 100.99 (C-13), 131.01 (C-1'), 108.83 (C-2', 6'), 147.14 (C-3', 5'), 134.78 (C-4'), 56.14 (3', 5'-OCH3). Compound 11 was identified as 4'-demethylpodophyllotoxin by comparison with the reported data.

[0076] Compound 12: white powder, HR-ESI-MS m / z: 397.1273 [M-H2O+H] + (cacld for C 22 H 21 O7397.1282); 1H-NMR (400 MHz, DMSO-d6) δ: 7.06 (1H, s, H-8), 6.59 (2H, s, H-2', 6'), 6.00 (1H, s, H-5), 5.94 (1H, d, J = 6.2 Hz, 1-OH), 5.90 (1H, s, H-13a), 5.91 (1H, s, H-13β), 4.50 (1H, d, J = 8.2 Hz, H-1), 4.38 (1H, dd, J = 6.4, 16.0 Hz, H-11a), 4.36 (1H, dd, J = 6.1, 16.8 Hz, H-11β), 3.91 (1H, d, J = 7.6 Hz, H-4), 3.68 (3H, s, 4'-OCH3), 3.74 (6H, s, 3', 5'-OCH3), 3.42 (1H, dd, J = 7.5, 9.7 Hz, H-3), 2.51 (1H, m, H-2); 13 C-NMR (100 MHz, DMSO-d6) δ: 67.71 (C-1), 43.25 (C-2), 44.13 (C-3), 43.74 (C-4), 104.94 (C-5), 146.14 (C-6), 146.17 (C-7), 107.68 (C-8), 131.95 (C-9), 135.18 (C-10), 69.72 (C-11), 178.37 (C-12), 101.11 (C-13), 138.85 (C-1'), 106.76 (C-2', 6'), 153.32 (C-3', 5'), 136.55 (C-4'), 60.40 (4'-OCH3), 56.29 (3', 5'-OCH3). Compound 12 was identified as picropodophyllotoxin by comparison with the reported data.

[0077] Compound 13: white powder, HR-ESI-MS m / z: 399.1437 [M+H] + (cacld for C 22 H 23 O7399.1438); 1H-NMR (400 MHz, DMSO-d6) δ: 6.80 (1H, s, H-8), 6.68 (1H, s, H-5), 6.46 (2H, s, H-2', 6'), 5.96 (1H, s, H-13a), 5.95 (1H, s, H-13β), 4.25 (1H, d, J = 3.3 Hz, H-4), 4.44 (1H, dd, J = 7.6, 9.2 Hz, H-11a), 3.93 (1H, dd, J = 2.8, 9.1 Hz, H-11β), 2.71 (1H, dd, J = 6.9, 15.4 Hz, H-la), 2.48 (1H, overlapped, H-1β), 3.71 (6H, s, 3', 5'-OCH3), 3.63 (3H, s, 4'-OCH3), 3.61 (1H, dd, J = 3.4, 9.7 Hz, H-3), 3.07 (1H, m, H-2); 13 C-NMR (100 MHz, DMSO-d6) δ: 32.28 (C-1), 31.40 (C-2), 44.74 (C-3), 44.43 (C-4), 109.15 (C-5), 145.87 (C-6), 146.07 (C-7), 108.86 (C-8), 131.27 (C-9), 128.82 (C-10), 72.78 (C-11), 178.30 (C-12), 100.71 (C-13), 136.05 (C-1'), 105.11 (C-2', 6'), 152.78 (C-3', 5'), 136.05 (C-4'), 55.88 (3', 5'-OCH3). Compound 13 was identified as deoxypicropodophyllotoxin by comparison with the reported data.

[0078] Compound 14: white powder, HR-ESI-MS m / z: 411.1085 [M+H] + (cacld for C 22 H 19 O8411.1074); 1 H-NMR (300 MHz, DMSO-d6) δ: 10.45 (1H, s, 1-OH), 7.62 (1H, s, H-8), 6.86 (1H, s, H-5), 6.54 (2H, s, H-2', 6'), 6.18 (2H, s, H-13), 5.37 (2H, s, H-11), 3.77 (3H, s, 4'-OCH3), 3.73 (6H, s, 3', 5'-OCH3); 13C-NMR (150 MHz, DMSO-d6) δ: 125.12 (C-l), 130.84 (C-2), 122.84 (C-3), 119.59 (C-4), 103.07 (C-5), 149.26 (C-6), 148.86 (C-7), 98.53 (C-8), 131.40 (C-9), 131.34 (C-10), 67.06 (C-l l), 169.82 (C-12), 102.48 (C-13), 145.90 (C-l'), 108.33 (C-2', 6'), 152.91 (C-3', 5'), 137.37 (C-4'), 60.56 (4'-OCH3), 56.42 (3', 5'-OCH3). Compound 14 was identified as dehydropodophyllotoxin by comparison with the reported data.

[0079] Compound 15: white powder, HR-ESI-MS m / z: 413.1239 [M+H] + Calcd for C 22 H 21 O8413.1231); 1 H-NMR (300 MHz, CDCl3) δ: 7.58 (1H, s, H-8), 6.73 (1H, s, H-5), 6.41 (2H, s, H-2', 6'), 6.13 (1H, d, J = 1.2 Hz, H-13a), 6.11 (1H, d, J = 1.2 Hz, H-13β), 4.38 (1H, dd, J = 9.2, 10.4 Hz, H-11a), 4.59 (1H, dd, J = 7.5, 9.2 Hz, H-11β), 4.81 (1H, d, J = 4.3 Hz, H-4), 3.84 (3H, s, 4'-OCH3), 3.77 (6H, s, 3', 5'-OCH3), 3.30 (1H, dd, J = 4.3, 15.5 Hz, H-3), 3.55 (1H, m, H-2); 13C-NMR (150 MHz, CDC13) δ: 192.42 (C-l), 43.49 (C-2), 46.74 (C-3), 44.70 (C-4), 109.67 (C-5), 148.16 (C-6,7), 106.14 (C-8), 128.25 (C-9), 141.55 (C-10), 66.99 (C-l l), 173.03 (C-12), 102.39 (C-13), 132.10 (C-l'), 107.79 (C-2',6'), 153.13 (C-3',5'), 137.83 (C-4'), 60.78 (4'-OCH3), 56.33 (3',5'-OCH3). Compound 15 was identified as podophyllotoxone by comparison with the reported data.

[0080] Compound 2, Compound 3, Compound 4 are isoprenyl flavonoids, the structures are as follows:

[0081]

[0082] Compound 2: R1= H, R3= H, R4= H;

[0083] Compound 3: R1= OH, R3= CH3, R4= H;

[0084] Compound 4: R1= H, R2= H, R3= H,

[0085] Compound 9-Compound 15 are aryl naphthalide lignans, the structures are as follows:

[0086]

[0087] Compound 9: R 1a = OH, R 2a = CH3; Compound 10: R 1a = H, R 2a = CH3; Compound 11: R 1a = OH, R 2a = H.

[0088]

[0089] Compound 12: R 1b = OH, R 2b = CH3; Compound 13: R 1b = H, R 2b = CH3.

[0090] Compound 14: Compound 15:

[0091] Example 4

[0092] Study on anti-RA activity of compounds 2-4 and 9-15

[0093] To determine the pharmacodynamic basis of SHD Fr anti-RA, the isolated compounds were further evaluated for in vitro anti-RA activity using the TNF-a induced FLS cell proliferation model.

[0094] FLS cells were seeded in modified Dulbecco's medium containing 10% fetal bovine serum (FBS; Gibco) and cultured at 37°C to a density of 70%, then transfected with 0.6 μg ViaFect transfection reagent (Promega) and 0.4 μg pGL4.74 (hRluc / TK plasmid) for 24 h. Transfected cells were seeded in 96-well plates at 40,000 cells per well and incubated for 12 h, then stimulated with TNF-a (final concentration of 10 ng / mL per well) and isoprenyl flavonoid compounds (compounds 1-8, final concentration of 1 μM) or aryl naphthalide lignan compounds (compounds 9-15, final concentration of 1 μM) for 6 h. Equal amounts of TNF-a were added as a negative control, and normal FLS cells were used as a blank control. Luciferase assay was performed using Dual-Glo Luciferase Assay System (Promega) and BioTek Synergy4 multimode microplate reader. The percentage of activity inhibition was calculated.

[0095] The results are shown in Table 1, which shows that compounds 2-4 and 9-15 can significantly inhibit the proliferation of FLS cells induced by TNF-a at a concentration of 1 μM. Figure 5

[0096] The in vivo anti-RA activity of the above compounds was evaluated using the rat RA model induced by complete Freund's adjuvant (CFA) according to the method of Example 1 "SHD Fr in vivo anti-RA activity". The above compounds also showed anti-RA activity.

[0097] Example 5

[0098] Study on the target of anti-RA action of active ingredients in SHD Fr

[0099] Prediction of the target of the main active ingredients of SHD Fr based on network pharmacology

[0100] ​Potential targets of isoprenylated flavonoids in SHD Fr for treating RA

[0101] The structure of the isoprenylated flavonoids obtained by mass spectrometric qualitative analysis was introduced into the Swiss Target Prediction (http: / / www.swisstargetprediction.ch / ) database to obtain its target points. After removing duplicates, a total of 173 target points were obtained. With "rheumatoid arthritis, RA" as the keyword, the Genecards (https: / / www.genecards.org / ), DrugBank (https: / / www.drugbank.ca / ), GeneNCBI database (https: / / www.ncbi.nlm.nih.gov / gene), and Therapeutic Target Database (https: / / db.idrblab.nttt) were searched to collect and collate the search results and remove duplicates to obtain the RA target database (referred to as "RA target"), a total of 6051 target points. The active ingredient target points of Anemone raddeana Regel were introduced into VENNY2.1 (https: / / bioinfogp.cnb.csic.es / tools / venny / ) to obtain the drug-disease intersection target points, and after removing duplicates, the potential action target points of Anemone raddeana Regel for treating rheumatoid arthritis were obtained. Mapping them with the above-mentioned potential action target points of the compounds obtained 113 potential target points of isoprenylated flavonoids in Anemone raddeana Regel for treating rheumatoid arthritis. The above-mentioned compound-disease common target points were input into the STRING (https: / / string-db.org / ) database for retrieval, with the protein species set as "Homosapiens" and the minimum interaction threshold set as 0.4. The PPI network of protein interaction was constructed, which was introduced into Cytoscape software to draw the protein interaction network diagram, and the size and color of the network nodes were set. Among them, the size, color and depth of the nodes represent the size of the Degree value, and the results show that the top five target proteins are TP53, AKT1, TNF-α, SRC and JAK1, indicating that these proteins have an important position in the PPI network, and the isoprenylated flavonoids in Anemone raddeana Regel may produce therapeutic effects on RA through these target points.

[0102] Potential targets of aryl naphthalide lignans in SHD Fr for treating RA

[0103] The structures of the qualitatively identified arylnaphthalene lignan compounds were imported into the Swiss Target Prediction (http: / / www.swisstarget prediction.ch / ) database to obtain their targets. After deduplication, a total of 590 targets were obtained. The remaining methods were the same as above; after deduplication, these were the potential targets for the treatment of rheumatoid arthritis by *Taorui*. Mapping these targets with the potential targets of the aforementioned compounds yielded 349 potential targets for the treatment of rheumatoid arthritis by *Taorui* flavonoids. Figure 6 A) The common targets of the above compounds and diseases were entered into the STRING database (https: / / string-db.org / ) for retrieval. The protein type was set to "Homo sapiens," and the minimum interaction threshold was set to 0.4. A protein-protein interaction (PPI) network was constructed and imported into Cytoscape software to draw the protein-protein interaction network diagram. The size and color of the network nodes were set. The size, color, and intensity of the nodes represent the degree value. The results showed that the top five target proteins were TNF-α, GAPDH, TP53, TLR4, and SRC, indicating that these proteins play an important role in the PPI network. The arylnaphthalene lignans in *Prunus persica* may exert a therapeutic effect on RA through these targets.

[0104] Experimental Study on the Inhibitory Effect of SHD Fr on Predicted Targets

[0105] The selected targets (TP53, AKT1, TLR4, TNF-α, JAK1, GAPDH, SRC) were measured.

[0106] Drug preparation: Using DMSO as a solvent, a stock solution of SHD Fr with a concentration of 10 mg / mL was prepared. The SHD Fr stock solution was diluted with DMEM medium containing 10% FBS to obtain drug-containing mediums with different concentrations (final SHD Fr concentrations of 0.1, 0.5, 1, 10, 25, 50, 150, 200, 300 μg / mL).

[0107] Construction of HEK293 cell lines overexpressing TP53, TLR4, TNF-a, GAPDH: HEK293 cells were cultured in DMEM medium containing 10% FBS to reach 70-80% confluency. Using the above protein expression plasmids (TP53: 5'-GUAGAUUACCACUGGAGUCdTdT-3'; TLR4: 5'-CCACCAUCUUCACCAACUAdTdT-3'; TNF-a: 5'-UAAAGCCGAUUUGAGAUUCdTdT-3'; GAPDH: 5'-CUUGAGGCUGUUGUCAUACdTdT-3') and reporter plasmids (e.g. luciferase reporter plasmid), the above plasmids and transfection reagent were mixed in serum-free medium, the mixture contained 2 pg of protein plasmid and 2 pg of reporter plasmid. The transfection mixture was added to the cell culture plate, gently shaken to mix evenly, and then incubated in an incubator at 37°C for 4-6 hours. Then, the medium was replaced with drug-containing medium containing 10% FBS, and the culture was continued for 24-48 hours. The cells were collected and lysed with lysis buffer, and the supernatant was collected. According to the instructions of the luciferase detection kit, luciferase detection reagent was added to the sample. Finally, the luciferase activity was detected using a fluorescence or luminescence detector, and the relative fluorescence units (RLU) were recorded. The background fluorescence of the untransfected control was subtracted from the detection data. The fluorescence units of the sample were normalized to the internal reference plasmid Renilla luciferase, and the IC 50 values were calculated.

[0108] The results are shown in Table 2, which shows that SHD Fr has stronger inhibitory effect on TNF-a and JAK1, with IC 50 values of 1.45 pg / mL and 0.58 pg / mL, respectively, while the IC 50 values for other targets are all greater than 200 pg / mL (Table 2), indicating that SHD Fr exerts anti-RA activity by inhibiting TNF-a and JAK1.

[0109] Experimental study on the inhibitory effect of compounds 2 and 11 on predicted targets

[0110] To further verify the network pharmacology results and determine that Taoerqi exerts anti-RA activity through "double active components-double targets", the inventors selected aryl naphthalide lignan compound 11 and isoprenyl flavonoid compound 2 for TNF-a and JAK1 inhibitory activity determination.

[0111] Drug preparation: using DMSO as solvent, compound 2 stock solution with concentration of 10 mM, compound 11 stock solution with concentration of 10 mM, diluting the stock solution with DMEM medium containing 10% FBS to obtain different concentrations (the final concentrations of compound 2 and compound 11 are 2.5, 5, 10, 20, 50, 100, 150, 200 μM respectively) of drug-containing medium.

[0112] Construction of HEK293 cell lines overexpressing TP53, TLR4, TNF-α, GAPDH: HEK293 cells were cultured in DMEM medium containing 10% FBS to reach 70-80% confluence. Using the above-mentioned protein expression plasmids (TP53: 5'-GUAGAUUACCACUGGAGUCdTdT-3'; TLR4: 5'-CCACCAUCUUCACCAACUAdTdT-3'; TNF-α: 5'-UAAAGCCGAUUUGAGAUUCdTdT-3'; GAPDH: 5'-CUUGAGGCUGUUGUCAUACdTdT-3') and reporter genes, the above-mentioned plasmids and transfection reagents were mixed in serum-free medium, the mixture contained 2 μg of protein plasmid and 2 μg of reporter gene plasmid, the transfection mixture was added to the cell culture plate, shaken gently to make it uniform, and then incubated in an incubator at 37°C for 4-6 hours; then, the medium was replaced with drug-containing medium containing 10% FBS, and the cells were cultured for another 24-48 hours. The cells were collected and lysed with lysis buffer, and the supernatant was collected. According to the instructions of the luciferase detection kit, luciferase detection reagent was added to the sample. Finally, the luciferase activity was detected using a fluorescence or luminescence detector, and the relative fluorescence units (RLU) were recorded. The background fluorescence of the untransfected control was subtracted from the detection data. The fluorescence units of the sample were normalized to the internal reference plasmid Renilla luciferase, and the IC 50 values were calculated.

[0113] The results are shown in Table 2, which show that compound 11 has an IC 50 value of 1.21 μM for inhibiting TNF-α, but has no inhibitory effect on JAK1 (IC 50 > 100 μM); compound 2 has an IC 50 value of 1.08 μM for inhibiting JAK1, but has no inhibitory effect on TNF-α (IC 50 > 100 μM). The above results show that aryl naphthalide lignan compounds in Anoectochilus roxburghii can exert anti-RA effect by inhibiting TNF-α, and isoprenyl flavonoids can exert anti-RA effect by inhibiting JAK1.

[0114] Table 2. IC 50 values of SHD Fr, compound 2 and compound 11 for inhibiting different targets

[0115]

[0116] Effect of SHD Fr on TNF-α and JAK1 downstream signaling pathways

[0117] Activated T cell nuclear factor 1 (NFATc1) is essential for RA disease. TNF-α and JAK1 in synovial tissue can regulate the expression of NFATc1 through different signaling pathways, thereby causing the occurrence and development of RA disease. After TNF-α binds to TNFR1, it causes TRADD to bind to TNFR1, recruits RIP-1 and TRAF2 / 4 / 5, and further activates NF-κB, JNK and p38 signaling, prompting effective activation of NFATc1. On the other hand, the activation of PLC-γ by JAK1 also causes an increase in calcium concentration, increasing the expression of Calcinurin, thereby causing an increase in NFATc1.

[0118] The effect of SHD Fr on TNF-α and JAK1 downstream signaling pathways was evaluated using a rat RA model induced by complete Freund's adjuvant (CFA). The model was established, the experimental groups were divided, and the drug administration scheme was the same as in Example 3.

[0119] The rat synovial tissue was isolated, the synovial tissue was cut into small pieces, and total protein was obtained by adding lysis solution. The total protein content was determined by BCA kit, and the sample protein was denatured. Constant voltage SDS-PAGE electrophoresis was performed, and the protein was transferred to the PVDF membrane by constant current. 5% skim milk powder was blocked, and RIP-1, TRAF2, TRAF4, TRAF5, AP-1, c-fos, p38, JNK, PLC and internal reference, etc. Primary antibody (1:1000) was added, incubated overnight in the refrigerator at 4°C, washed the membrane with TBST three times, incubated with HRP-labeled secondary antibody (1:10000), washed the membrane with TBST, chemiluminescence detection, and density analysis of protein content was performed using Image J image analysis software.

[0120] The synovial tissue of rats was taken out and immediately placed in 4% paraformaldehyde (PFA) fixative solution, fixed at 4°C for 4-6 hours. After fixation, gradient ethanol dehydration was carried out, and then paraffin immersion embedding was carried out, and the tissue was cut into 5 μm thick sections using a microtome, and attached to a glass slide. 5% bovine serum albumin in PBS was blocked at room temperature for 30 minutes to block non-specific binding. The primary antibodies against NFATc1 and NF-κB were added to the slices at an appropriate dilution ratio (1:200), and incubated at 4°C overnight. The slices were washed with PBS 3 times, 5 minutes each time. Secondary antibodies with different fluorescent labels (Alexa Fluor 488 labeled anti-NFATc1 secondary antibody and Alexa Fluor 594 labeled anti-NF-κB secondary antibody, diluted according to the instructions) were added, and incubated at room temperature for 1 hour in the dark. The slices were washed with PBS 3 times, 5 minutes each time. DAPI solution (or other nuclear dyes) was added and incubated in the dark for 5-10 minutes. The slices were sealed with an anti-fluorescence quenching mounting medium, and covered with a cover glass. Observed and photographed under a fluorescence microscope.

[0121] The results of Western-blot and immunofluorescence are shown in Figure 6 , which show that SHD Fr can inhibit the expression of RIP-1 and TRAF2, TRAF4, TRAF5 in the synovial tissue of RA rats, inhibit the nuclear translocation of NF-κB, inhibit JNK and p38 phosphorylation. In addition, SHD Fr can also inhibit the phosphorylation of PLC-γ in the synovial tissue of RA rats, inhibit the expression of Calcinurin, reduce the concentration of calcium ions, and ultimately reduce NFATc1. The above results show that SHD Fr has an effect on the downstream signaling pathways of TNF-α and JAK1, further verifying the target prediction results of network pharmacology.

[0122] Effect of aryl naphthalide lignan compound 11 on TNF-α downstream signaling pathway in FLS cells and effect of prenyl flavonoid compound 2 on JAK1 downstream signaling pathway

[0123] The inventors further investigated the effect of aryl naphthalide lignan compound 11 on the TNF-α downstream signaling pathway, and the effect of prenyl flavonoid compound 2 on the JAK1 downstream signaling pathway.

[0124] TNF-α modeling: the frozen tube containing FLS cells was taken out from liquid nitrogen, and the cell recovery operation was performed. The cells were uniformly spread on the bottom of the culture bottle with DMEM medium containing 10% FBS, 1% streptomycin and penicillin, and were placed in a 37°C cell culture box with 5% CO2 and 95% relative humidity. The FLS cells that grew well and were passed to 3-6 generations were used for experiments. This experiment was divided into blank control group, model group, and different concentrations of test drug group. The blank control group was replaced with fresh culture medium, and the other groups were replaced with culture medium containing TNF-α (final concentration 20 ng / mL) and cultured for 12 h. The blank control group was continuously replaced with fresh culture medium, the model group was replaced with culture medium containing TNF-α (final concentration 20 ng / mL), and the positive control group and the test drug group were replaced with culture medium containing the test drug (the final concentrations of compounds 2 and 11 were 0.5, 1, and 2 μM, respectively, and the final concentration of TNF-α was 20 ng / mL). The culture was continued for 24 h, and was ready for use.

[0125] Immunofluorescence: FLS cells were inoculated in a 24-well plate (cell climbing sheet was placed before inoculation), 500 μL per well, and were divided into blank control group, model group, and test drug group. The blank control group was cultured with culture medium, the model group was cultured with culture medium containing TNF-α (final concentration 500 ng / mL), and the test drug group was cultured with culture medium containing compound 2 or 11 and TNF-α (the final concentration of compound 2 or 11 was 2 μM, and the final concentration of TNF-α was 500 ng / mL). After 24 h of culture, the cell fusion degree reached 80%-90%, and the cells were washed with PBS, fixed with 40 g / L paraformaldehyde-0.1 mol / L phosphate buffer for 30 min, discarded the fixing solution, washed with PBS, permeated with 1% Triton X-100-PBS at room temperature for 20 min, washed with PBS, added an appropriate amount of endogenous peroxidase blocking agent, incubated at room temperature for 15 min, washed with PBS, added an appropriate amount of normal goat serum blocking solution, incubated at 37°C for 15 min, absorbed the liquid with filter paper (do not wash), added NFκB, NFATc1 primary antibody (1:50), incubated in a wet box at 4°C overnight, rewarming for 30 min, washed with PBS, added an appropriate amount of biotin-labeled goat anti-rabbit IgG, incubated at room temperature for 20 min, washed with PBS, added an appropriate amount of horseradish enzyme-labeled streptavidin working solution, incubated at room temperature for 15 min, washed with PBS, added an appropriate amount of freshly prepared DAPI staining solution, and observed under a light microscope.

[0126] The results are shown in Figure 7 , which shows that after TNF-α modeling, the expression of RIP-1 and TRAF2 / 4 / 5 in FLS cells is significantly increased. Pre-administration of compound 11 can dose-dependently inhibit the expression of the above proteins Figure 7 (A), and inhibit the nuclear expression of NF-κB Figure 7C). The above results show that compound 11 can inhibit the downstream signal pathway of TNF-α at the cellular level.

[0127] TNF-α can significantly promote the phosphorylation of PLC-γ, p38, JNK and other proteins in FLS cells. Pre-administration of compound 2 can dose-dependently inhibit the phosphorylation of the above proteins Figure 7 B), inhibit the nuclear expression of NF-κB Figure 7 C). The above results show that compound 2 can inhibit the downstream signal pathway of JAK1 at the cellular level.

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Claims

1. A prenyl flavonoid compound having a structure as shown in Formula I: ###0001### wherein R1= H, R3= H, R4= H; R1= OH, R3= CH3, R4= H; R1= H, R2= H, R3= H, 2. Use of the prenyl flavonoid compound of claim 1 in the preparation of a medicament for treating rheumatoid arthritis.

3. Use according to claim 2, characterized in that: The use is the use of the prenyl flavonoid compound in the preparation of a medicament for treating rheumatoid arthritis by inhibiting JAK1.

4. An aryl naphthalide lignan compound having a structure as shown in Formula II, III, IV, V: ###0002### wherein R 1a = OH, R 2a = CH3; R 1a = H, R 2a = CH3; R 1a = OH, R 2a = H; R 1b = OH, R 2b = CH3; R 1b = H, R 2b = CH3.

5. Use of the aryl naphthalide lignan compound of claim 4 in the preparation of a medicament for treating rheumatoid arthritis.

6. Use according to claim 5, characterized in that: The use is the use of the aryl naphthalide lignan compound in the preparation of a medicament for treating rheumatoid arthritis by inhibiting TNF-α.

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