Use of a class of alkaloid compounds and pharmaceutical compositions thereof for treating inflammation

Abstract

The application belongs to the technical field of medicine, and discloses a class of alkaloid compounds and the use of pharmaceutical compositions thereof in treating inflammation. In vitro cell experiments prove that the components have a significant inhibitory effect on the secretion of important inflammatory factors TNF-alpha and NO of lipopolysaccharide-induced RAW264.7 cells, and have a significant down-regulation effect on the phosphorylation level of key proteins of the NF-kappa B pathway in the inflammatory state. In vivo animal experiments prove that the components have a significant inhibitory effect on the tincture of croton oil-induced mouse ear swelling, and can be used for preventing and / or treating inflammation-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to indole and quinoline alkaloid compounds, pharmaceutical combinations thereof, and their use in preventing and treating inflammation. Pharmacological experiments have confirmed that indole alkaloid components can inhibit the secretion of inflammatory factors by regulating the NF-κB pathway, thereby suppressing inflammation. Animal experiments have also demonstrated that these components can inhibit croton oil-induced ear swelling in mice, demonstrating preventive and therapeutic effects on inflammation and other related diseases. Background Art

[0002] Inflammation is a complex host immune response to defend against a variety of harmful physiological stimuli. It involves activating various immune cells and tissues, which work together to restore normal cellular structure and function. Although self-limited inflammation is physiological and necessary for pathogen clearance, persistent inflammation can be detrimental to the organ in which it originates and to systemic responses in other organs. Numerous studies have shown that inflammation plays a crucial role in the development and progression of many complex diseases, such as rheumatoid arthritis, gout, and atherosclerosis. In clinical practice, anti-inflammatory drugs are the second most commonly used medication, second only to anti-infective drugs. Numerous related drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SAIDs), are commercially available. However, long-term use can cause adverse reactions, limiting their use. Natural products, with their potent activity, novel structures, multi-target effects, and minimal toxicity and side effects, have become a key area of ​​research for novel anti-inflammatory drugs. Small molecules of natural origin have always been an important area of ​​drug development. Flavonoids, alkaloids, and terpenes can reduce inflammation by regulating the NF-κB signaling pathway. [1-6] Indole alkaloids are a class of compounds that use tryptophan or tryptamine as precursors and are composed of a bicyclic structure consisting of a benzene ring fused with a five-membered pyrrole ring. Currently, this type of ingredient has been found in about 70 plant species. Pharmacological activity studies have shown that indole alkaloids have anti-inflammatory, antibacterial, antiviral and anti-tumor properties. [7-9] Activities in other aspects.

[0003] Research has found that indole alkaloids can inhibit the secretion of TNF-α inflammatory factors in RAW 264.7 cells induced by lipopolysaccharide by downregulating the expression of proteins in the NF-κB pathway. Furthermore, these ingredients can reduce croton oil-induced ear swelling in mice.

[0004] References

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[0006] [2]Wu J,Zhao F,Fan K,et al.Dihydromyricetin inhibits inflammation ofFibroblast-Like synoviocytes through Regulation of Nuclear Factor-κBSignaling in Rats with Collagen-Induced Arthritis[J].J.Pharmacol.Exp.Ther,2019,368(2):218-228.

[0007] [3]Lu L,Hu J,Wu Q,et al.Berberine prevents human nucleus pulposuscells from IL-1β-induced extracellular matrixdegradationandapoptosisbyinhibitingtheNF-κBpathway[J].Int.J.Mol.Med,2019,43(4):1679-1686.

[0008] [4]Li X,Wang M,Hong H,et al.Sophocarpine attenuates murine lupusnephritis via inhibiting NLRP3inflammasomeandNF-κBactivation[J].Immunol.Res,2018,66(4):521-527.

[0009] [5] Liu C, Zhao S, Zhu C, et al. Ergosterol ameliorates renal inflammatory responses in mice model of diabetic nephropathy[J]. Biomed. Pharmacother, 2020, 128: 110252.

[0010] [6] Shin JS, Im HT, Lee KT. Saikosaponin B2 Suppresses Inflammatory Responses Through IKK / IκBα / NF-κB Signaling Inactivation in LPS-Induced RAW264.7 Macrophages[J]. Inflammation, 2019, 42(1): 342-353.

[0011] [7] Rosales PF, Bordin GS, Gower AE, et al. Indole alkaloids: 2012 until now, highlighting the new chemical structures and biological activities[J]. Fitoterapia, 2020, 143: 104558.

[0012] [8] Singh TP, Singh OM. Recent progress in biological activities of indole and indole alkaloids[J]. Mini Rev Med Chem, 2018, 18(1): 9-25.

[0013] [9] Jia Y, Wen X, Gong Y, et al. Current scenario of indole derivatives with potential anti-drug-resistant cancer activity[J]. Eur J Med Chem, 2020, 200: 112359. SUMMARY

[0014] The technical problem solved by the present application is to provide indole, quinoline alkaloids or pharmaceutically acceptable salts thereof and their use in the preparation of anti-inflammatory drugs.

[0015] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0016] The first aspect of the technical solution of the present invention is to provide an alkaloid compound or a pharmaceutically acceptable salt thereof, characterized in that it has the following general formula:

[0017]

[0018] R1 is hydrogen, hydroxy, methoxy or ethoxy; R2 is hydrogen, hydroxy, methoxy or ethoxy; R3 and R4 are independently selected from hydrogen, hydroxy, methoxy, ethoxy, glucosyl or apiose (1→6) glucosyl; R5 is carboxyl; R6 is methoxy.

[0019] Preferred compounds include, but are not limited to:

[0020]

[0021] The second aspect of the technical solution of the present invention is to provide a method for preparing the above 12 alkaloid compounds:

[0022] The dried roots and rhizomes of Clematis chinensis were extracted with 5–10 times the volume of 70% ethanol under reflux three times for 2 hours each time, followed by vacuum distillation to obtain a crude extract. An appropriate amount of water was added to uniformly disperse the extract, and the extract was extracted three times with an equal volume of ethyl acetate to the water to obtain an ethyl acetate fraction and an aqueous fraction. The ethyl acetate fraction was separated by chromatography on silica gel and / or Sephadex LH-20 columns, eluting with petroleum ether–ethyl acetate and / or petroleum ether–dichloromethane–methanol solvent systems, respectively, to obtain subfractions. Compounds 1, 5, 6, 8, 9, and 12 were prepared using a high-performance liquid chromatography-reversed-phase silica gel column with a mobile phase of CH₃OH–H₂O. The aqueous fraction was separated by chromatography on a macroporous resin HP-20 and / or MCI resin column, eluting with a C₂H₅OH–H₂O solvent system to obtain subfractions. The subfractions were then separated by reverse-phase silica gel and / or Sephadex LH-20 column chromatography, eluting with CH3OH-H2O as the mobile phase to obtain the components. Preparative separation using HPLC-reverse-phase silica gel column eluting with CH3OH-H2O or CH3CN-H2O solvent system yielded compounds 2, 3, 4, 7, 10, and 11.

[0023] The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which includes at least one of the aforementioned alkaloid compounds or pharmaceutically acceptable salts and a carrier.

[0024] The fourth aspect of the technical solution of the present invention provides the use of the alkaloid compound or pharmaceutically acceptable salt thereof described in the first aspect and the pharmaceutical composition described in the third aspect in anti-inflammatory drugs.

[0025] Beneficial technical effects

[0026] The present invention is the first to isolate and identify 12 alkaloid compounds (1-12) from the roots and rhizomes of Clematis chinensis, and the anti-inflammatory effect of the compounds was evaluated using a lipopolysaccharide-induced RAW 264.7 cell inflammation model. The results showed that compounds 1-12 could significantly inhibit the secretion of the inflammatory factor TNF-α. Compounds 4, 5, 7 and 8 could significantly inhibit the release of the inflammatory factor NO. In addition, compounds 7 and 8 can reduce inflammation by regulating the NF-κB inflammatory pathway. Animal experiments also demonstrated that compounds 7, 8 and 9 can inhibit croton oil-induced mouse ear swelling. Therefore, the alkaloid compounds prepared by the present invention have potential pharmaceutical value for preventing and treating inflammation, and are expected to be developed into anti-inflammatory leads or drugs based on targeted regulation of the NF-κB pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Effects of compounds 7 and 8 on the expression of inflammatory factors such as iNOS, TNF-α and IL-6.

[0028] Figure 2 Effects of compounds 7 and 8 on the expression of NF-κB signaling pathway proteins. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments do not limit the present invention.

[0030] Example 1

[0031] Preparation of compounds 1–12

[0032] The roots and rhizomes of Clematis chinensis (36 kg) were crushed, soaked in 70% ethanol for 2 h, and refluxed for 2 h three times to obtain a 70% ethanol extract. The extract was then concentrated under reduced pressure until no ethanol residue remained. After dispersion with water, the extract was extracted three times with an equal volume of ethyl acetate. The organic phase was concentrated under reduced pressure to obtain an ethyl acetate extract fraction. The remaining water fraction was separated by macroporous resin and eluted with H2O, 50% ethanol, and 95% ethanol, respectively, to obtain a water-eluted fraction, a 50% ethanol-eluted fraction, and a 95% ethanol-eluted fraction. The ethyl acetate fraction was separated by silica gel column chromatography. After 30 times the volume of silica gel column chromatography, the extract was eluted with petroleum ether:ethyl acetate = 50:1, 10:1, 3:1, 95% ethanol, and 55% ethanol to obtain five elution fractions (AE). The C site was separated into 14 components (C-1 to C-14) by silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate (10:1-1:1). C-3 and C-4 were combined and separated by Sephadex LH-20 and eluted with petroleum ether: dichloromethane: methanol = 5:5:1 to obtain four subcomponents C-(3-4)-1 to 4. Component C-(3-4)-4 was purified by preparative HPLC to obtain compound 5. The C-5 site was separated by Sephadex LH-20 and eluted with petroleum ether: dichloromethane: methanol = 5:5:1 to obtain five components (C-5A to C-5E). C-5B was separated by ODS and gradient eluted with methanol and water to obtain five subcomponents (C-5B-1 to C-5). C-5B-2 was separated by preparative HPLC to obtain compounds 6 and 8. The C-6 site was separated by Sephadex LH-20 and eluted with petroleum ether: dichloromethane: methanol = 5:5:1 to obtain five components (C-5A to C-5E). LH-20 separation, eluted with petroleum ether: dichloromethane: methanol = 5:5:1, yielded 10 fractions (C6A-C6J). C6E was separated by preparative HPLC to yield compound 12. The C-10 fraction was separated by Sephadex LH-20, eluted with petroleum ether: dichloromethane: methanol = 5:5:1, yielding 9 fractions (C10-1-9). Subfraction C10-9 was separated by preparative HPLC to yield compound 1. The C-11 fraction was separated by Sephadex LH-20, eluted with petroleum ether: dichloromethane: methanol = 5:5:1, yielding 9 fractions (C11-1-9). Subfraction C11-5 was separated by preparative HPLC to yield compound 9. The 50% ethanol elution fraction of the macroporous resin weighed approximately 520 g, a portion of which was insoluble in water, representing the 50% ethanol-water insoluble fraction. The 50% ethanol-soluble portion was separated by MCI resin and eluted with H2O, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol and 95% ethanol in sequence to obtain 8 elution fractions.10% ethanol elution fraction was separated by Sephadex LH-20 to give 7 fractions (10A-G), fraction 10B was separated by Toyoperal HW-40C to give 16 fractions (10B-1-16), fraction 10C was separated by Toyoperal HW-40C to give 18 fractions (10C-1-18), according to the thin layer chromatography, fractions 10B-6-8 and 10C-6-8 were combined and named 10B+C (1.9 g) and separated by ODS to give 24 sub-fractions (10B+C-1-24), 10B+C-16 was purified by HPLC to give compound 10; fractions 10B-9-13 and 10C-9-12 were combined and named 10B1C1 and separated by ODS to give 24 sub-fractions (10B1C1-1-24), 10B1C1-12 was purified by HPLC to give compound 3; 10B-15 was purified by HPLC to give compound 2; 10D and 10E were dissolved in methanol, compound 4 was directly precipitated; 50% ethanol elution fraction was separated by MCI, eluted with ethanol-water to give 8 fractions (20MCI-95MCI), fraction 20MCI was separated by ODS, eluted with 5%-50% methanol-water to give 29 sub-fractions (20MCI-1-29), 20MCI-10 was purified by HPLC to give compound 11; 40MCI was separated by Sephadex LH-20, eluted with 30% methanol-water and 60% methanol-water to give 16 sub-fractions (40MCI-1-16), 40MCI-14 was purified by HPLC to give compound 7.

[0033] The structures were identified according to the physicochemical properties and spectral data of compounds 1-12.

[0034] Structure identification of compound 1

[0035] yellow amorphous powder; (c 0.05, CH3OH), UV (CH3OH) λ max (log ε) 215 (2.21), 285 (1.85), 308 (1.93); IR (Microscope) v max 3427, 2974, 1698, 1525, 1440, 1339, 1280, 1143 cm -1 ; HRESIMS (m / z 274.1072 [M+H] + , calcd for C 15 H 16 O4N, 274.1079).1 H NMR (400 MHz, CDCl3) δ H :7.81(1H,d,J=3.1Hz,H-2),6.84(1H,s,H-7),7.96(1H,d,J=10.1Hz,H-8 ),5.68(1H,d,J=10.1Hz,H-9),3.84(3H,s,-OCH3),1.48(6H,s,H-11,12); 13 C NMR (100 MHz, CDCl3) δ C :131.4(C-2),108.9(C-3),115.0(C-3a),143.2(C-4),113.8(C-5),136.6(C-6),96.4(C-7 ),131.5(C-7a),122.4(C-8),129.0(C-9),76.4(C-10),27.4(C-11),27.4(C-12),51.2(CO AND 3),165.4(C=O).

[0036] Structural identification of compound 2

[0037] Yellow amorphous powder; (c 0.1,CH3OH),UV(CH3OH)λ max (logε)212(3.96),278(3.45),295(3.43); IR(Microscope)v max 3363,2932,1678,1530,1465,1306,1200,1075cm -1 ;HRESIMS(m / z 382.1142[MH] - ,calcd for C 17 H 20 O9N,382.1138). 1 H NMR (500 MHz, CD3OD) δ H:7.83(1H,s,H-2),7.83(1H,s,H-4),7.05(1H,s,H-7),4.91(1H,d,J=7.4Hz,H-1′),3.53(1H,overlapped,H-2′),3.50(1H,overlapped,H-3′),3.47(1H,over lapped,H-4′),345(1H,overlapped,H-5′),3.91(1H,dd,J=12.0,2.2Hz,H-6′a),3.76(1H,dd,J=12.0,4.7Hz,H-6′b),3.90(3H,s,-OCH3),3.87(3H,s,-OCH3); 13 C NMR (125 MHz, CD3OD) δ C :132.2(C-2),108.5(C-3),120.8(C-3a),110.2(C-4),145.2(C-5),149.2(C-6),96.9(C-7),133. 9(C-7a),104.1(C-1′),75.2(C-2′),78.0(C-3′),71.3(C-4′),78.3(C-5′),62.5(C-6′),51.5(CO AND 3), 57.0 (6-OCH3), 168.0 (C=O).

[0038] Structural identification of compound 3

[0039] White amorphous powder; (c 0.3, CH3OH), UV(CH3OH)λmax(logε)215(3.44),274(2.93),291(2.83); IR(Microscope)v max 3370,2946,1680,1534,1451,1277,1169,HRESIMS(m / z 486.1602[M+H] + ,calcd for C 21 H 28 O 12 N,486.1612). 1 H NMR (500 MHz, CD3OD) δ H7.88 (1H, s, H-2), 7.92 (1H, d, J = 8.8 Hz, H-4), 7.25 (1H, d, J = 2.2 Hz, H-7), 7.02 (1H, dd, J = 8.8, 2.2 Hz, H-5), 4.84 (1H, d, J = 7.4 Hz, H-1'), 3.48 (1H, overlapped, H-2'), 3.47 (1H, overlapped, H-3'), 3.34 (1H, t, J = 9.0 Hz, H-4'), 3.61 (1H, overlapped, H-5'), 4.07 (1H, br. d, J = 9.2 Hz, H-6'a), 3.60 (1H, overlapped, H-6'b), 5.01 (1H, d, J = 2.2 Hz, H-1"), 3.98 (1H, d, J = 2.2 Hz, H-2"), 3.96 (1H, d, J = 9.6 Hz, H-4"a), 3.76 (1H, d, J = 9.6 Hz, H-4"b), 3.64 (2H, ABq, J = 11.4 Hz, H-5"), 3.86 (3H, s, -OCH3); 13 C NMR (125 MHz, CD3OD) δ C : 132.9 (C-2), 108.3 (C-3), 122.8 (C-3a), 122.4 (C-4), 114.4 (C-5), 156.2 (C-6), 100.6 (C-7), 138.6 (C-7a), 103.5 (C-1'), 75.0 (C-2'), 78.1 (C-3'), 71.8 (C-4'), 77.1 (C-5'), 68.8 (C-6'), 111.0 (C-1"), 77.8 (C-2"), 80.5 (C-3"), 75.0 (C-4"), 65.4 (C-5"), 51.4 (CO AND 3), 167.8 (C=O).

[0040] Structural identification data of compound 4

[0041] White amorphous powder, ESI-MS m / z 354.3 [M+H] + ; 1 H NMR (400 MHz, CD3OD) δ H:7.92(1H,d,J=8.7Hz,H-4),7.88(1H,s,H-2),7.21(1H,d,J=2.1Hz,H-7),7.03(1 H,dd,J=8.7,2.1Hz,H-5),4.91(1H,d,J=7.3,H-1′),3.92(1H,br.d,J=12.0Hz,H-6 ′a),3.86(3H,s,-OCH3),3.73(1H,dd,J=12.0,4.9Hz,H-6′b),3.49-3.47(2H,overlapped,H-2′,H-5′),3.43(1H,overlapped,H-4′),3.41(1H,overlapped,H-3′); 13 C NMR (125 MHz, CD3OD) δ C :132.9(C-2),108.2(C-3),122.8(C-3a),122.3(C-4),114.2(C-5),156.1(C-6),100.6(C-7),138.5(C-7 a),167.8(C-8),103.4(C-1′),75.1(C-2′),78.0(C-3′),71.4(C-4′),78.2(C-5′),62.6(C-6′),51.4(CO AND 3).

[0042] Structural identification of compound 5

[0043] White powder, ESI-MS m / z 220.03[M+H] + , 1 H-NMR (400MHz, CDCl3), δ: 8.05 (1H, d, J = 8.7Hz, H-4), 7.82 (1H, s, H-2), 6.93 (1H, dd, J = 8.7, 1.8Hz, H-5), 6.88(1H,d,J=1.8Hz,H-7), 4.38(2H,q,J=7.1Hz,-CH2), 3.86(3H,s,-OCH3), 1.42(3H,t,J=7.1Hz,-CH3); 13 C-NMR (125MHz, CDCl3), δ: 129.8 (C-2), 109.2 (C-3), 122.2 (C-3a), 119.9 (C-4), 111. 7(C-5),157.0(C-6),94.8(C-7),136.8(C-7a),165.3(C-10),59.8(-OCH2-),55.6(CO AND 3),14.6(-CH3).

[0044] Structure identification of compound 6

[0045] White powder, ESI-MS m / z 206.04 [M+H] + , 1 H-NMR (400 MHz, CDC13), δ: 8.04 (1H, d, J = 8.8 Hz, H-4), 7.81 (1H, d, J = 2.9 Hz, H-2), 6.94 (1H, dd, J = 8.8, 2.2 Hz, H-5), 6.89 (1H, d, J = 2.2 Hz, H-7), 3.91 (3H, s, -OCH3), 3.86 (3H, s, 6-OCH3); 13 C-NMR (125 MHz, CDC13), δ: 129.8 (C-2), 108.9 (C-3), 122.2 (C-3a), 119.9 (C-4), 111.8 (C-5), 157.1 (C-6), 94.8 (C-7), 136.8 (C-7a), 165.6 (C-10), 55.6 (6-OCH3), 51.1 (CO AND 3).

[0046] Structure identification of compound 7

[0047] Colorless oil, ESI-MS m / z 192.20 [M+H] + , 1 H-NMR (400 MHz, CD3OD) δ: 7.83 (1H, d, J = 8.6 Hz, H-4), 7.79 (1H, s, H-2), 6.83 (1H, d, J = 2.2 Hz, H-7), 6.74 (1H, dd, J = 8.6, 2.2 Hz, H-5), 3.85 (3H, s, -OCH3); 13 C-NMR (100 MHz, CD3OD); δ: 168.0 (C=O), 155.1 (C-6), 139.2 (C-7a), 132.0 (C-2), 122.4 (C-4), 120.8 (C-3a), 112.7 (C-5), 108.2 (C-3), 98.2 (C-7), 51.3 (CO AND 3).

[0048] Structure identification of compound 8

[0049] Yellow powder, ESI-MS m / z 175.98 [M+H] + , 1H-NMR(400MHz,CD3OD)δ:8.04(1H,dd,J=7.0,2.0Hz,H-4),7.95(1H,s,H-2),7.43(1H,dd,J=7.0,2 .0Hz,H-7),7.21(1H,td,J=7.0,2.0Hz,H-5),7.18(1H,td,J=7.0,2.0Hz,H-6),3.88(3H,s,-OCH3); 13 C-NMR (125MHz, CD3OD), δ: 167.9 (C-8), 133.2 (C-2), 108.2 (C-3), 127.3 (C-3a ),121.9(C-4),122.5(C-5),123.7(C-6),113.0(C-7),138.1(C-7a),51.4(CO AND 3).

[0050] Structural identification data of compound 9

[0051] Pale yellow solid, ESI-MS m / z 146.02[M+H] + , 1 H-NMR (CD3COCD3, 400MHz), δ: 10.03 (1H, s, H-8), 8.22 (1H, dd, J = 7.4, 1.8 Hz, H-4), 8.19 ( 1H, d, J = 2.9 Hz, H -2),7.54(1H,dd,J=7.4,1.8Hz,H-7),7.27(1H,td,J=7.4,1.8Hz,H-5),7.24(1H,td,J=7.4,1.8Hz,H-6); 13 C-NMR (CD3COCD3, 125MHz), δ: 138.1 (C-2), 120.1 (C-3), 125.5 (C-3a), 124. 5(C-4),123.0(C-5),122.2(C-6),113.0(C-7),138.3(C-7a),185.4(C-8).

[0052] Structural identification data of compound 10

[0053] White powder, ESI-MS m / z: 216.4[M+H] + , 1H NMR(400MHz,DMSO-d6)δ:10.88(1H,s,9-NH),7.44(1H,d,J=7.8Hz,H-5),7.32(1H,d,J=7.8Hz,H-8),7.07(1H,t,J=7.5Hz,H-7),6 .98(1H,t,J=7.2Hz,H-6),4.18(2H,dd,J=28.5,15.8Hz,H-1),3.42(1H,overlapped,H-3),3.11(1H,m,H-4a),2.81(1H,m,H-4b); 13 C NMR(125MHz,DMSO-D6)δ:40.4(C-1),128.0(C-1a),56.6(C-3),22.9(C-4),106.6(C-4a),117 .7(C-5),126.2(C-5a),118.6(C-6),121.1(C-7),111.0(C-8),136.1(C-8a),169.2(-COOH).

[0054] Structural identification data of compound 11

[0055] Light yellow powder, ESI-MS m / z 231.13[M+H] + , 1 H-NMR(700MHz,DMSO-d6)δ:10.92(1H,s,-NH),7.45(1H,br d,J=7.5Hz,H-4),7.34(1H,br d,J=7.5Hz,H-7),7.08(1H,t,J=7.5Hz,H-6),7.00(1H,t,J=7.5Hz,H-5),4.26(1H,d,J=15.4Hz,H-8a),4.20(1 H,d,J=15.4Hz,H-8b),3.77(1H,m,H-11),3.17(1H,dd,J=16.1,4.5Hz,H-12a),2.86(1H,t,J=13.6Hz,H-12b); 13 C-NMR(175MHz,DMSO-d6)δ:126.0(C-2),106.2(C-3),127.3(C-3a),117.7(C-4),118.7(C-5) ,121.3(C-6),111.1(C-7),136.1(C-7a),40.3(C-8),169.4(C-10),56.0(C-11),22.6(C-12).

[0056] Structural identification data of compound 12

[0057] Yellow powder, ESI-MS m / z 200.03[M+H] + , 1 H-NMR (400MHz, CDCl3), δ: 8.28 (1H, br d, J = 8.5Hz, H-5), 8.03 (1H, br d,J=8.5Hz,H-8),7.69(1H,td,J=8.5,1.5Hz,H-7),7.64(1H,d,J=2.8Hz,H-2),7 .47(1H,td,J=8.5,1.5Hz,H-6),7.09(1H,d,J=2.8Hz,H-3),4.46(3H,s,4-OCH3); 13 C-NMR (125MHz, CDCl3) δ: 143.6 (C-2), 104.8 (C-3), 118.6 (C-3a), 157.1 (C-4), 103.4 (C-4a), 1 22.4(C-5),123.8(C-6),129.7(C-7),127.6(C-8),145.3(C-8a),163.7(C-9),59.0(4-OCH3).

[0058] Example 2

[0059] In vitro anti-inflammatory activity evaluation of compounds 1–12 inhibiting the secretion of inflammatory factor TNF-α by RAW264.7 cells

[0060] (1) Experimental methods

[0061] RAW 264.7 in the logarithmic growth phase was plated in a 96-well plate and incubated at 37°C, 5% CO2 for 12–18 hours. Next, the supernatant was removed and 2% FBS medium was added at 80 μl / well for 4 hours. After that, the test compound group (10 μM) and the positive drug group were added at 10 μL / well and incubated for 1 hour. Finally, LPS was added at 10 μL / well and incubated for 24 hours. After the incubation was completed, the supernatant was taken to detect TNF-α. The specific method was performed according to the instructions of the ELISA kit. The OD value was read at 570 nm, a standard curve was drawn, the TNF-α concentration of the sample was obtained, and the inhibition rate was calculated.

[0062] Inhibition rate (%) = (C 模型 -C 样品 ) / C 模型 *100%, C is the absolute concentration of TNF-α.

[0063] (2) Experimental results

[0064] Compound 1-12 showed a strong inhibitory effect on TNF-α, and its TNF-α inhibitory activity was higher than or close to that of the positive control drug dexamethasone. The results are shown in Table 1:

[0065] Table 1 Inhibitory effect of compounds 1-12 on the secretion of TNF-α inflammatory factors by RAW264.7 cells

[0066]

[0067] Example 3

[0068] In vitro anti-inflammatory activity evaluation of compounds 1–12 on the inhibition of NO production in primary mouse peritoneal macrophages

[0069] (1) Experimental methods

[0070] The anti-inflammatory activity of the compounds was evaluated using a lipopolysaccharide (LPS)-induced NO production model in primary mouse peritoneal macrophages. Cells were subcultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2 in an incubator. Experimental groups included a blank control, an LPS group, a drug-treated group, and a positive drug group. The control group received only culture medium, the positive drug group received LPS and 1 μM dexamethasone, and the drug-treated group received LPS and 10 μM of the test sample. Cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours. 100 μL of the supernatant was removed and Griess reagent was added. After 10 minutes, the optical density (OD) was measured at 540 nm using a microplate reader. NO production was calculated using a NO standard curve. The cell growth inhibition rate in each group was determined using the MTT assay.

[0071] (2) Experimental results

[0072] Compounds 4, 5, 7 and 8 can significantly inhibit the release of inflammatory factor NO. Among them, the activity of compounds 5 and 8 is close to that of the positive control drug dexamethasone (Table 2)

[0073] Table 2 Inhibitory effect of compounds 1-12 on NO production

[0074]

[0075]

[0076] Example 4

[0077] In vivo anti-inflammatory activity evaluation of compounds 1, 4, 7-9, and 12 against croton oil-induced mouse otitis model

[0078] (1) Experimental methods

[0079] The in vivo anti-inflammatory activity of the compounds was evaluated using a croton oil-induced mouse otitis model. Mice were randomly divided into a blank control group and a treatment group. The treatment group was given a single subcutaneous injection of 25 mg / kg. One hour later, the mice were sensitized by evenly applying 2% croton oil on both sides of the left ear. The mice were killed 4 hours after sensitization, and the left and right auricles were cut off and punched into ear pieces of equal size. The pieces were weighed, and the difference in mass between the two ears was used as the degree of swelling (Table 3).

[0080] Table 3 Evaluation of the activity of compounds 1, 4, 7-9, and 12 against croton oil-induced mouse otitis model

[0081]

[0082] * p<0.05, ** p<0.01

[0083] (2) Experimental results

[0084] Compounds 8 and 9 could significantly inhibit croton oil-induced ear swelling in mice, and compound 7 could inhibit croton oil-induced ear swelling in mice, which may serve as potential compounds with anti-inflammatory effects.

[0085] Example 5

[0086] In vitro Western blot experiments of compounds 7 and 8

[0087] (1) Experimental methods

[0088] RAW 264.7 cells (3×10 6 The cells were plated in 6-well plates (100 μg / well) and cultured in a 37°C cell culture incubator for an appropriate period of time before being replaced with serum-free culture medium. To detect the protein expression of the NF-κB signaling pathway of the cells, the drugs were added according to the following requirements: Compounds 7 and 8 (2.2, 6.8, 20 μM) were added to RAW 264.7 cells for pretreatment for 2 hours, and then LPS induced cells for 24 hours. Next, cell lysate was added and total protein was extracted using a protein extraction kit, and the protein concentration in the lysate was detected using a protein analysis kit. The protein was transferred to a polyvinylidene fluoride (PVDF) membrane using 8%-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subsequently, the PVDF membrane was blocked in 10% skim milk at room temperature for 2 hours, and specific antibodies were added for detection using a chemiluminescence reagent. GAPDH protein was used as an internal reference.

[0089] (2) Experimental results

[0090] ① Effects of compounds 7 and 8 on the expression of inflammatory mediators in LPS-induced RAW 264.7 cells

[0091] like Figure 1As shown, compounds 7 and 8 inhibited NO production in LPS-stimulated RAW264.7 cells by regulating inducible iNOS in a dose-dependent manner. Compared with the model group, the high-dose groups of compounds 7 and 8 had a very significant inhibitory effect on the expression levels of IL-6 and TNF-α ( * p<0.05, ** p<0.01, *** p<0.001).

[0092] ②Regulatory effects of compounds 7 and 8 on the NF-κB signaling pathway

[0093] like Figure 2 As shown in the figure, after treatment with different concentrations of compounds 7 and 8 (2.2, 6.8 and 20 μM), the expression levels of p-NF-κBp65, p-IκBα and p-IKKα were significantly downregulated in a dose-dependent manner. The results showed that compounds 7 and 8 inhibited LPS-induced inflammation in RAW264.7 cells by inhibiting the NF-κB pathway ( * p<0.05, ** p<0.01, *** p<0.001).

[0094] The above descriptions are merely embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent structural transformations made using the description of the present invention, or directly or indirectly applied to other related technical fields, are included in the scope of patent protection of the present invention.

Claims

1. The following alkaloid compound or a pharmaceutically acceptable salt thereof, 2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the alkaloid compound or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier or excipient.

3. Use of the alkaloid compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing and / or treating inflammation.

4. Use of compound 7 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating inflammation,

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