A limonoid compound, its preparation method and application

Abstract

This invention relates to the field of natural medicines, and particularly to a limonin compound, its preparation method, and its application. This invention provides a limonin compound having the structure shown in any of compounds 1-8. Data from the examples show that the limonin compound provided by this invention can inhibit the assembly of the NLRP3 inflammasome by inhibiting ASC oligomerization, thereby reducing the generation and release of downstream inflammatory factors and exerting an anti-inflammatory effect.

Description

Technical Field

[0001] This invention relates to the field of natural medicines, and in particular to a limonene compound, its preparation method, and its application. Background Technology

[0002] Xiangtian fruit is the fruit of the large-leaved mahogany tree (Swietenia macrophylla). Its effects include a bitter and astringent taste, cool properties, and astringent and antipyretic effects. The seeds are mainly used to treat hyperglycemia and hypertension. Currently, it is circulated on major e-commerce platforms and medicinal herb markets as an edible agricultural product or medicinal material with "three-lowering" effects (lowering blood sugar, blood lipids, and cholesterol). Modern pharmacological studies have shown that Xiangtian fruit extract has bioactivities such as hypoglycemic, lipid-lowering, antioxidant, antitumor, whitening, and insecticidal effects. Its main component is limonene, a class of tetraterpenoid compounds with a variable skeleton.

[0003] Inflammation is a complex biological response to injury, infection, or tissue damage, involving various immune cells, chemical messengers, and other inflammatory mediators. However, exaggerated inflammatory responses can lead to the development of a variety of chronic diseases, including asthma, cardiovascular disease, osteoporosis, cancer, obesity, and bronchitis. Inflammasomes are cytoplasmic polyprotein complexes formed in response to various physiological and pathogenic stimuli. As an important component of the innate immune system, inflammasomes play a crucial role in host defense by recognizing viral infections and triggering autoimmune responses. Among them, the NLRP3 inflammasome is the most widely studied and considered the most typical inflammasome, associated with various human autoinflammatory and autoimmune diseases. Activation of the NLRP3 inflammasome promotes caspase-1 activation, which in turn induces the maturation and release of pro-inflammatory cytokines IL-1β and IL-18, triggering a series of inflammatory responses. Aberrant activation of the NLRP3 inflammasome plays a crucial role in neurological diseases such as multiple sclerosis, Alzheimer's disease, and Parkinson's syndrome; metabolic disorders such as type 2 diabetes and atherosclerosis; and inflammatory diseases such as rheumatoid arthritis, enteritis, and peritonitis. Currently, there are no specific NLRP3 inflammasome inhibitors available clinically. Therefore, further research into safe and effective NLRP3 inflammasome inhibitors is a key strategy for addressing many inflammatory diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a limonoid compound, its preparation method, and its application. The limonoid compound provided by this invention has good anti-inflammatory effects and can exert a therapeutic effect on the inflammatory response of LPS-stimulated THP-1 cells.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a limonene compound having the structure shown in any one of compounds 1 to 8:

[0007] .

[0008] This invention also provides a method for preparing the limonene compounds described in the above technical solution, comprising the following steps:

[0009] (1) Extracting the fruit of the *Syzygium aromaticum* with petroleum ether and then concentrating the extract to obtain the petroleum ether fraction extract;

[0010] (2) The petroleum ether fraction extract was subjected to silica gel column chromatography with gradient elution using an eluent, and fractions A to F were obtained in the order of elution.

[0011] The eluents are, in sequence, petroleum ether, a mixture of petroleum ether and dichloromethane, dichloromethane, a mixture of dichloromethane and ethyl acetate, and ethyl acetate and methanol;

[0012] (3) The fraction E was subjected to silica gel column chromatography and gradient elution was performed using a petroleum ether-ethyl acetate system. The fractions EA-EM were obtained in the order of elution.

[0013] (4) The EG fraction was separated by ODS column chromatography using an acetonitrile-water system for gradient elution, and the EG fraction EGA-EGI was obtained in the order of elution.

[0014] (5) After the EH fraction crystallized, the mother liquor was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 2;

[0015] (6) The EI fraction was separated by ODS column chromatography using a methanol-water gradient elution system, and the EIA-EIF fractions were obtained in the order of elution.

[0016] (7) The EIB fraction was separated by Sphadex LH-20 column chromatography, eluted with methanol, and the fractions EIB1-EIB5 were obtained in the order of elution.

[0017] (8) Component EIB3 was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 8;

[0018] (9) The component EK was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The fractions EKA-EKM were obtained in the order of elution.

[0019] (10) The EKC fraction was separated by Sphadex LH-20 column chromatography, eluted with methanol, and the fractions EKC1-EKC4 were obtained in the order of elution.

[0020] (11) EKC4 was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 4;

[0021] (12) The EM fraction was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The EMA-EMO fraction was obtained in the order of elution.

[0022] (13) The EMC fraction was crystallized, purified, and compound 3 was obtained;

[0023] (14) The EMD fractions were separated by Sphadex LH-20 column chromatography and eluted with methanol to obtain EMD1-EMD4 in the order of elution.

[0024] (15) The fraction EMD2 was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 5;

[0025] (16) The EMG fractions were separated by Sphadex LH-20 column chromatography, eluted with methanol, and obtained in the order of elution as EMG1-EMG5;

[0026] (17) The EMG2 fraction was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 6 and compound 7;

[0027] (18) The fraction F was separated by MCI column chromatography and gradient elution was performed using a methanol-water system. The fractions FA-FR were obtained in the order of elution.

[0028] (19) The fraction FI was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The fractions FIA-FIF were obtained in the order of elution.

[0029] (20) The fraction FIE was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 1.

[0030] Preferably, the extraction method is cold soaking; the cold soaking temperature is 25°C; the time is 30 days, and the extract is collected every 48 hours.

[0031] Preferably, the volume ratio of petroleum ether to dichloromethane in the petroleum ether and dichloromethane mixture is 1:1; and the volume ratio of dichloromethane to ethyl acetate in the dichloromethane and ethyl acetate mixture is 1:1.

[0032] Preferably, in step (3), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate system is 100:0-1:1;

[0033] In step (4), the volume ratio of acetonitrile to water in the acetonitrile-water system is 50:50-100:0;

[0034] In step (5), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 75%.

[0035] Preferably, in step (6), the volume ratio of methanol to water in the methanol-water system is 70:30-100:0; in step (8), the volume concentration of the acetonitrile aqueous solution is 63%; in step (9), the volume ratio of methanol to water in the methanol-water system is preferably 60:40-100:0.

[0036] Preferably, in step (11), the volume concentration of the acetonitrile aqueous solution is 53%;

[0037] In step (11), the volume ratio of methanol to water in the methanol-water system is 50:50-100:0;

[0038] In step (15), the volume ratio of acetonitrile to water in the acetonitrile-water system is 55:45-75:25.

[0039] Preferably, in step (17), the gradient elution procedure is as follows:

[0040] The gradient elution procedure is as follows:

[0041] 0~15 min: The volume ratio of acetonitrile to water in the acetonitrile-water system changes uniformly from 60:40 to 75:25;

[0042] 15~35 min: The volume ratio of acetonitrile to water in the acetonitrile-water system is 75:25;

[0043] In step (18), the volume concentration of the methanol-water system is 10:90-100:0.

[0044] Preferably, in step (19), the volume concentration of the methanol-water system is 40:60-80:20.

[0045] This invention also provides the application of the limonin compounds described in the above technical solutions or the limonin compounds prepared by the above preparation methods in the preparation of anti-inflammatory drugs.

[0046] This invention provides a limonin compound having the structure shown in any one of compounds 1 to 8. Data from the examples show that the limonin compound provided by this invention can inhibit the assembly of the NLRP3 inflammasome by inhibiting ASC oligomerization, thereby reducing the generation and release of downstream inflammatory factors and exerting an anti-inflammatory effect. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0048] Figure 1 For compound 1 1 H NMR spectrum;

[0049] Figure 2 For compound 1 13 CNMR spectrum;

[0050] Figure 3 For compound 2 1 H NMR spectrum;

[0051] Figure 4 For compound 2 13 CNMR spectrum;

[0052] Figure 5 For compound 3 1 H NMR spectrum;

[0053] Figure 6 For compound 3 13 CNMR spectrum;

[0054] Figure 7 For compound 4 1 HNMR spectrum;

[0055] Figure 8 For compound 4 13 CNMR spectrum;

[0056] Figure 9 For compound 5 1 HNMR spectrum;

[0057] Figure 10 For compound 5 13 CNMR spectrum;

[0058] Figure 11 For compound 6 1 HNMR spectrum;

[0059] Figure 12 For compound 6 13 CNMR spectrum;

[0060] Figure 13 For compound 7 1 HNMR spectrum;

[0061] Figure 14 For compound 7 13 CNMR spectrum;

[0062] Figure 15 For compound 8 1 H NMR;

[0063] Figure 16 For compound 8 13 CNMR spectrum;

[0064] Figure 17 The effects of compound 2 on the expression of NLRP3 inflammasome-related proteins (A) and on ASC oligomerization (B). Detailed Implementation

[0065] This invention provides a limonene compound having the structure shown in any one of compounds 1 to 8:

[0066]

[0067] This invention also provides a method for preparing the limonene compounds described in the above technical solution, comprising the following steps:

[0068] (1) Extracting the fruit of the *Syzygium aromaticum* with petroleum ether and then concentrating the extract to obtain the petroleum ether fraction extract;

[0069] (2) The petroleum ether fraction extract was subjected to silica gel column chromatography with gradient elution using an eluent, and fractions A to F were obtained in the order of elution.

[0070] The eluents are, in sequence, petroleum ether, a mixture of petroleum ether and dichloromethane, dichloromethane, a mixture of dichloromethane and ethyl acetate, and ethyl acetate and methanol;

[0071] (3) The fraction E was subjected to silica gel column chromatography and gradient elution was performed using a petroleum ether-ethyl acetate system. The fractions EA-EM were obtained in the order of elution.

[0072] (4) The EG fraction was separated by ODS column chromatography using an acetonitrile-water system for gradient elution, and the EG fraction EGA-EGI was obtained in the order of elution.

[0073] (5) After the EH fraction crystallized, the mother liquor was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 2;

[0074] (6) The EI fraction was separated by ODS column chromatography using a methanol-water gradient elution system, and the EIA-EIF fractions were obtained in the order of elution.

[0075] (7) The EIB fraction was separated by Sphadex LH-20 column chromatography, eluted with methanol, and the fractions EIB1-EIB5 were obtained in the order of elution.

[0076] (8) Component EIB3 was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 8;

[0077] (9) The component EK was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The fractions EKA-EKM were obtained in the order of elution.

[0078] (10) The EKC fraction was separated by Sphadex LH-20 column chromatography, eluted with methanol, and the fractions EKC1-EKC4 were obtained in the order of elution.

[0079] (11) EKC4 was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 4;

[0080] (12) The EM fraction was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The EMA-EMO fraction was obtained in the order of elution.

[0081] (13) The EMC fraction was crystallized, purified, and compound 3 was obtained;

[0082] (14) The EMD fractions were separated by Sphadex LH-20 column chromatography and eluted with methanol to obtain EMD1-EMD4 in the order of elution.

[0083] (15) The fraction EMD2 was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 5;

[0084] (16) The EMG fractions were separated by Sphadex LH-20 column chromatography, eluted with methanol, and obtained in the order of elution as EMG1-EMG5;

[0085] (17) The EMG2 fraction was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 6 and compound 7;

[0086] (18) The fraction F was separated by MCI column chromatography and gradient elution was performed using a methanol-water system. The fractions FA-FR were obtained in the order of elution.

[0087] (19) The fraction FI was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The fractions FIA-FIF were obtained in the order of elution.

[0088] (20) The fraction FIE was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 1.

[0089] Specifically, the reaction conditions of this invention are as follows:

[0090] (1) Extracting the fruit of the *Syzygium aromaticum* with petroleum ether and then concentrating it to obtain the petroleum ether extract.

[0091] In one embodiment of the present invention, in step (1), the extraction method is preferably cold soaking; the temperature of the cold soaking is preferably 25°C, the time is preferably 30 days, and the extract is collected every 48 hours during the cold soaking process; the concentration is preferably vacuum concentration.

[0092] (2) The petroleum ether fraction extract was subjected to silica gel column chromatography and gradient elution was performed using an eluent. The fractions A to F were obtained in the order of elution.

[0093] In one embodiment of the present invention, the eluent is, in sequence, petroleum ether, a mixture of petroleum ether and dichloromethane, dichloromethane, a mixture of dichloromethane and ethyl acetate, ethyl acetate and methanol; the volume ratio of petroleum ether to dichloromethane in the mixture of petroleum ether and dichloromethane is preferably 1:1; the volume ratio of dichloromethane to ethyl acetate in the mixture of dichloromethane and ethyl acetate is preferably 1:1.

[0094] As one embodiment of the present invention, after gradient elution, it is preferable to further combine the fractions after thin-layer chromatography to obtain fractions A to F.

[0095] (3) The fraction E was subjected to silica gel column chromatography and gradient elution was performed using a petroleum ether-ethyl acetate system. The fractions EA-EM were obtained in the order of elution.

[0096] In one embodiment of the present invention, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate system is preferably 100:0 to 1:1.

[0097] As one embodiment of the present invention, after gradient elution, it is preferable to further combine the fractions after thin-layer chromatography to obtain fraction EA-EM.

[0098] (4) The EG fraction was separated by ODS column chromatography using an acetonitrile-water system for gradient elution, and the EG fraction EGA-EGI was obtained in the order of elution.

[0099] In one embodiment of the present invention, the volume ratio of acetonitrile to water in the acetonitrile-water system is preferably 50:50 to 100:0; in another embodiment of the present invention, after gradient elution, it is preferable to further combine the fractions after thin-layer chromatography to obtain fraction EGA-EGI.

[0100] (5) After the EH fraction crystallized, the mother liquor was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 2.

[0101] As one embodiment of the present invention, it is preferred to use natural volatilization to precipitate crystals.

[0102] In one embodiment of the present invention, the volume concentration of acetonitrile in the acetonitrile aqueous solution is preferably 75%.

[0103] (6) The EI fraction was separated by ODS column chromatography using a methanol-water system for gradient elution, and the EIA-EIF fractions were obtained in the order of elution.

[0104] In one embodiment of the present invention, the volume ratio of methanol to water in the methanol-water system is preferably 70:30 to 100:0; in another embodiment of the present invention, after gradient elution, it is preferable to further combine the fractions after thin-layer chromatography to obtain fractions EIA-EIF.

[0105] (7) The EIB fraction was separated by Sphadex LH-20 column chromatography, eluted with methanol, and the fractions EIB1-EIB5 were obtained in the order of elution.

[0106] (8) Component EIB3 was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 8;

[0107] In one embodiment of the present invention, the volume concentration of the acetonitrile aqueous solution is preferably 63%.

[0108] (9) The component EK was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The fractions EKA-EKM were obtained in the order of elution.

[0109] In one embodiment of the present invention, the volume ratio of methanol to water in the methanol-water system is preferably 60:40-100:0.

[0110] (10) The EKC fraction was separated by Sphadex LH-20 column chromatography, eluted with methanol, and the fractions EKC1-EKC4 were obtained in the order of elution.

[0111] (11) EKC4 was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 4.

[0112] In one embodiment of the present invention, the volume concentration of the acetonitrile aqueous solution is preferably 53%.

[0113] (12) The EM fraction was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The EMA-EMO fraction was obtained in the order of elution.

[0114] In one embodiment of the present invention, the volume ratio of methanol to water in the methanol-water system is preferably 50:50 to 100:0.

[0115] (13) The EMC fraction was crystallized, purified, and compound 3 was obtained;

[0116] As one embodiment of the present invention, it is preferred to use natural evaporation to precipitate crystals; the preferred purification method is to dissolve the crystals in methanol and then allow them to recrystallize naturally.

[0117] (14) The EMD fractions were separated by Sphadex LH-20 column chromatography and eluted with methanol to obtain EMD1-EMD4 in the order of elution.

[0118] (15) The fraction EMD2 was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 5.

[0119] In one embodiment of the present invention, the volume ratio of acetonitrile to water in the acetonitrile-water system is 55:45 to 75:25.

[0120] (16) The EMG fractions were separated by Sphadex LH-20 column chromatography, eluted with methanol, and obtained in the order of elution as EMG1-EMG5.

[0121] (17) The EMG2 fraction was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 6 and compound 7.

[0122] As one embodiment of the present invention, the gradient elution procedure is preferably as follows:

[0123] 0~15 min: The volume ratio of acetonitrile to water in the acetonitrile-water system changes uniformly from 60:40 to 75:25;

[0124] 15~35 min: The volume ratio of acetonitrile to water in the acetonitrile-water system is 75:25.

[0125] (18) The fraction F was separated by MCI column chromatography and gradient elution was performed using a methanol-water system. The fraction FA-FR was obtained in the order of elution.

[0126] In one embodiment of the present invention, the volume concentration of the methanol-water system is preferably 10:90-100:0.

[0127] (19) The fraction FI was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The fractions FIA-FIF were obtained in the order of elution.

[0128] In one embodiment of the present invention, the volume concentration of the methanol-water system is preferably 40:60 to 80:20.

[0129] (20) The fraction FIE was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 1.

[0130] In one embodiment of the present invention, the volume ratio of acetonitrile to water in the acetonitrile-water system is 40:60 to 60:40.

[0131] This invention also provides the application of the limonin compounds described in the above technical solutions or the limonin compounds prepared by the above preparation methods in the preparation of anti-inflammatory drugs.

[0132] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0133] Example 1

[0134] 13 kg of *Anacarya paliurus* fruit was crushed and cold-soaked in petroleum ether for 30 days. The extract was collected every 48 hours. The extracts were combined and concentrated under reduced pressure to obtain 3.4 kg of petroleum ether fraction extract. The petroleum ether fraction extract was separated by silica gel column chromatography using a gradient elution of petroleum ether, a 1:1 mixture of petroleum ether and dichloromethane, dichloromethane, a 1:1 mixture of dichloromethane and ethyl acetate, and ethyl acetate and methanol. After identification by thin-layer chromatography (TLC), the fractions were combined to obtain fraction AF.

[0135] Fraction E (140.36 g) was separated by silica gel column chromatography using a petroleum ether-ethyl acetate system (volume ratio of petroleum ether to ethyl acetate 100:0-1:1), gradient elution, and TLC identification followed by merging of similar components to obtain fraction EA-EM. Fraction EG (1.96 g) was separated by ODS column chromatography using a gradient elution (eluting agent: acetonitrile-water, volume ratio of acetonitrile to water 50:50-100:0), and TLC identification followed by merging of similar components to obtain fraction EGA-EGI. Fraction EH (5.22 g) was allowed to evaporate naturally to precipitate crystals. The mother liquor after crystallization was separated by high performance liquid chromatography using 75 vol.% acetonitrile aqueous solution as eluent for 50 min to obtain compound 2 (42.1 min, 11.85 mg).

[0136] EI (7.1 g) was separated by ODS column chromatography using a methanol-water system (methanol:water volume ratio 70:30-100:0) to obtain fractions EIA-EIF; EIB (691.9 mg) was separated by Sphadex LH-20 column chromatography, eluted with methanol to obtain fractions EIB1-EIB5. Fraction EIB3 (240.8 mg) was separated by high performance liquid chromatography, eluted with 63 vol.% acetonitrile aqueous solution for 50 min to obtain compound 8 (25.2 min, 10.02 mg);

[0137] The fraction EK (7.06 g) was separated by ODS column chromatography using a gradient elution in a methanol-water system (methanol:water volume ratio of 60:40-100:0) to obtain fractions EKA-EKM. EKC (930 mg) was separated by Sphadex LH-20 column chromatography using methanol elution to obtain fractions EKC1-EKC4. EKC4 (42 mg) was separated by high-performance liquid chromatography using 53 vol.% acetonitrile aqueous solution elution for 45 min to obtain compound 4 (39.7 min, 6.96 mg).

[0138] The EM fraction (6.5 g) was separated by ODS column chromatography using a gradient elution in a methanol-water system (methanol:water volume ratio of 50:50-100:0) to obtain the EMA-EMO fraction. EMC was allowed to evaporate and crystallize out. The crystals were dissolved in methanol and allowed to evaporate and recrystallize to obtain compound 3 (26.74 mg).

[0139] The EMD fraction (399 mg) was separated by Sphadex LH-20 column chromatography and eluted with methanol to obtain EMD1-EMD4. The EMD fraction EMD2 was separated by high performance liquid chromatography and eluted with an acetonitrile-water system (acetonitrile:water volume ratio of 55:45-75:25) for 40 min to obtain compound 5 (37.1 min, 3.79 mg).

[0140] The EMG fraction (164.6 mg) was separated by Sphadex LH-20 column chromatography and eluted with methanol to obtain EMG1-EMG5. The EMG fraction was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system (0-15 min: acetonitrile:water volume ratio changed from 60:40 to 75:25; 15-35 min: acetonitrile:water volume ratio was 75:25) to obtain compound 6 (20.5 min, 3.16 mg) and compound 7 (31.3 min, 4.29 mg).

[0141] Fraction F (29 g) was separated by MCI column chromatography using a gradient elution with a methanol-water system (methanol:water volume ratio of 10:90-100:0) to obtain fraction FA-FR. Fraction FI (797 mg) was separated by ODS column chromatography using a gradient elution with a methanol-water system (methanol:water volume ratio of 40:60-80:20) to obtain fraction FIA-FIF. Fraction FIE (15 mg) was separated by high performance liquid chromatography using a gradient elution with an acetonitrile-water system (acetonitrile:water volume ratio of 40:60-60:40) for 35 min to obtain compound 1 (30.5 min, 2.49 mg).

[0142] Compound 1 detection information: C 31 H 42 O 11 A white amorphous powder, soluble in solvents such as methanol and chloroform. =−110.0 (CH3OH, 0.1); UV (CH3OH) λ max (log ε ) 195 (3.96) nm; ECD (CH3OH) λmax (Δ ε )240 (+0.32), 293 (−3.07) nm; HR-ESI-MS m / z : 589.2654,[MH]− (C 31 H 41 O 11 , calcd. for589.2654).

[0143] Compound 1 1 H NMR spectrum and 13 CNMR spectra are shown below. Figure 1-2 The NMR data of compound 1 are shown in Table 1.

[0144] Compound 2 detection information: C34H44O10, white amorphous powder, soluble in solvents such as methanol and chloroform.

[0145] = −117.4 (CH3OH, 0.1); UV(CH3OH) λ max (log ε ) 195(4.19) nm; ECD(CH3OH) λmax (Δ ε ) 241 (+0.55), 294 (−2.39) nm; HR-ESI-MS m / z : 635.2823, [M+Na]+(C 34 H 44 O 10Na, calcd. for 635.2826).

[0146] Compound 2 1 H NMR spectrum and 13 CNMR spectra are shown below. Figure 3-4 The NMR data for compound 2 are shown in Table 1.

[0147] Compound 3 detection information: C31H38O12, colorless crystals, soluble in chloroform, slightly soluble in methanol. = −44.0(CH3OH, 0.1); UV(CH3OH) λ max (log ε ) 195 (4.23) nm, ECD (CH3OH) λmax (Δ ε ) 208 (+20.3), 294 (−5.88) nm; HR-ESI-MS m / z : 625.2252, [M+Na]+ (C 31 H 38 O 12 Na, calcd. for 625.2255).

[0148] Compound 3 1 H NMR spectrum and 13 CNMR spectra are shown below. Figure 5-6 The NMR data for compound 3 are shown in Table 2.

[0149] Compound 4 detection information: C29H36O9, white amorphous powder, soluble in solvents such as methanol and chloroform. = −13.8 (CH3OH, 0.1); UV (CH3OH) λ max (log ε ) 211 (4.11) nm; ECD (CH3OH) λmax (Δ ε )202 (+ 0.43), 231 (− 10.21), 259 (+15.68), 296 (− 4.70) nm; HR-ESI-MS m / z : 563.2047, [M+Cl]− (C 29 H 36 O9Cl, calcd. for 563.2042).

[0150] Compound 4 1 H NMR spectrum and 13 CNMR spectra are shown below. Figure 7-8 The NMR data for compound 4 are shown in Table 2.

[0151] Compound 5 detection information: C42H54O19, white amorphous powder, soluble in methanol and chloroform. =+15.6(CH3OH, 0.1); UV(CH3OH) λ max (log ε ) 195 (3.80) nm; ECD (CH3OH) λmax (Δ ε ) 225 (−4.83), 259 (+ 0.35) nm; HR-ESI-MS m / z : 885.3128 [M+Na]+(C 42 H 54 O 19 Na, calcd. for 885.3151).

[0152] Compound 5 1 H NMR spectrum and 13 CNMR spectra are shown below. Figure 9-10 The NMR data for compound 5 are shown in Table 3.

[0153] Compound 6 detection information: C 42 H 54 O 19 White amorphous powder, soluble in methanol and chloroform. =+0.82(CH3OH, 0.1); UV(CH3OH)λ max (log ε )206(3.81)nm; ECD (CH3OH) λmax (Δ ε ) 215 (+ 7.07), 233 (− 0.65), 254 (− 0.85) nm; HR-ESI-MS m / z: 885.3140, [M+Na]+(C 42 H 54 O 19 Na, calcd. for 885.3151).

[0154] Compound 6 1 H NMR spectrum and 13 CNMR spectra are shown below. Figure 11-12 The NMR data for compound 5 are shown in Table 3.

[0155] Compound 7 detection information: White amorphous powder, soluble in solvents such as methanol and chloroform. =+9.13(CH3OH, 0.1); UV(CH3OH) λ max (log ε) 206 (3.80) nm; ECD (CH3OH) λmax (Δ ε 216 (+14.62) nm; HR-ESI-MS m / z : 773.2599 [M+Na]+ (C 36 H 46 O 17 Na, calcd. for 773.2627).

[0156] Compound 7 1 H NMR spectrum and 13 CNMR spectra are shown below. Figure 13-14 The NMR data for compound 7 are shown in Table 4.

[0157] Compound 8 detection information: C36H44O14, white amorphous powder, soluble in solvents such as methanol and chloroform. =−58.2 (CH3OH, 0.1), UV(CH3OH) λmax(log ε ) 205 (3.87) nm; ECD (CH3OH) λmax (Δ ε 234 (+4.06) nm; HR-ESI-MS m / z : 701.2805 [M+H]+ (C 36 H 45 O 14 , calcd. for 701.2803).

[0158] Compound 8 1 H NMR spectrum and 13 CNMR spectra are shown below. Figure 15-16 The NMR data for compound 8 are shown in Table 4.

[0159] Table 1. NMR data of compounds 1 and 2 (CDCl3, 600 MHz)

[0160]

[0161] Table 2. NMR data of compounds 3 and 4 (CDCl3, 600 MHz)

[0162]

[0163] Table 3. NMR data of compounds 5 and 6 (CDCl3, 600MHz)

[0164]

[0165]

[0166] Table 4. NMR data of compounds 7 and 8 (CDCl3, 600MHz)

[0167]

[0168]

[0169] Anti-inflammatory activity screening:

[0170] Healthy THP-1 cells were diluted to 1×10⁻⁶. 7 Cells / mL were induced to adhere to the plate using RPMI-1640 medium containing PMA (0.1 μg / mL), and seeded into 24-well plates. After 12 h, the medium was aspirated, and the drug diluted in serum-free RPMI-1640 medium was added and incubated for 1 h. LPS was added and incubation continued for 16 h. Then, ATP (2.5 mM) was added and ATP was added for 30 min of stimulation. The supernatant was aspirated into 1.5 mL EP tubes and stored at -80°C. The supernatant was centrifuged at 12000 rpm for 10 min and transferred to new EP tubes for testing according to the ELISA kit instructions.

[0171] (1) Remove the kit from the refrigerator and allow it to equilibrate to room temperature. Prepare the washing buffer, standards, biotinylated antibody working solution, and enzyme conjugate working solution.

[0172] (2) Take out the required strips, add 100µL of the centrifuged sample and standard to the well, incubate at 37℃ for 90min, and dilute the sample according to the concentration.

[0173] (3) Discard the liquid, pat dry with absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, and pat dry with absorbent paper. Repeat this washing process 4 times.

[0174] (4) Add 100µL of biotinylated antibody working solution to each well and incubate at 37℃ for 60min.

[0175] (5) Discard the liquid, pat dry with absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, and pat dry with absorbent paper. Repeat this washing process 4 times.

[0176] (6) Add 100µL of enzyme conjugate working solution to each well and incubate at 37℃ for 30 min.

[0177] (7) Discard the liquid, pat dry with absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, and pat dry with absorbent paper. Repeat this washing process 4 times.

[0178] (8) Add 100µL to each well, keep in the dark at 37℃ for 10-20 minutes.

[0179] (9) Add 100µL of stop solution, mix well, and measure the OD value at 450nm using an ELISA reader.

[0180] THP-1 cells were fed at a rate of 1×10 5 2 mL of the drug was seeded into each well of a 6-well plate. 24 h after drug administration, cells were collected and lysed on ice for 30 min using RIPA lysis buffer containing 1% protease inhibitor. After lysis, the cells were centrifuged at 4 °C and 12000 rpm for 10 min. The supernatant was collected to prepare protein samples, and the expression of NLRP3, ASC, and Caspase-1 proteins in the samples was detected by Western blot.

[0181] THP-1 cells were used at a rate of 5 × 10⁻⁶ 5 Cells were seeded at 1 / mL in 96-well plates. Pretreatment with Compound 2 for 1 h was followed by treatment with 1 μg / mL LPS for 12 h, and then with 5 mM NIG for 0.5 h. After removing the supernatant, cells were fixed with 4% paraformaldehyde for 10 min. Cells were then washed three times with PBS + 0.05% Triton solution and incubated overnight at 4°C with ASC antibody. On the second day, after three washes, cells were incubated with labeled goat anti-rabbit IgG (H+L) for 2 h. Cells were then incubated with 4′,6-diamino-2-phenylindole (DAPI) for 10 min to stain the nuclei, and the fluorescence staining was observed using a high-content imaging system. Figure 17 Table 5 shows the IL-1β inhibitory activity of limonene in *Swissia lingua*, representing the effects of compound 2 on the expression of NLRP3 inflammasome-related proteins (A) and on ASC oligomerization (B).

[0182] Table 5. IL-1 of limonene in *Swissonia lactiflora* β Inhibitory activity

[0183]

[0184] From Table 5 and Figure 17 Compound 2 significantly reduced the protein levels of NLRP3, caspase-1, and IL-1β in LPS- and Nigericin-treated THP-1 cells. Immunofluorescence staining experiments indicated that compound 2 inhibited ASC oligomerization during NLRP3 inflammasome activation. Therefore, compound 2 can inhibit the assembly of the NLRP3 inflammasome by inhibiting ASC oligomerization, thereby reducing the production and release of downstream inflammatory factors and exerting an anti-inflammatory effect.

[0185] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A limonoid compound, characterized by, It has the structure shown in Equation 2: 。 2. A method for preparing a limonene compound, characterized in that, Includes the following steps: (1) Extracting the fruit of the *Syzygium aromaticum* with petroleum ether and then concentrating the extract to obtain the petroleum ether fraction extract; (2) The petroleum ether fraction extract was subjected to silica gel column chromatography with gradient elution using an eluent, and fractions A to F were obtained in the order of elution. The eluents are, in sequence, petroleum ether, a mixture of petroleum ether and dichloromethane, dichloromethane, a mixture of dichloromethane and ethyl acetate, and ethyl acetate and methanol; (3) The fraction E was subjected to silica gel column chromatography and gradient elution was performed using a petroleum ether-ethyl acetate system. The fractions EA-EM were obtained in the order of elution. (4) The EG fraction was separated by ODS column chromatography using an acetonitrile-water system for gradient elution, and the EG fraction EGA-EGI was obtained in the order of elution. (5) After the EH fraction crystallized, the mother liquor was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 2; (6) The EI fraction was separated by ODS column chromatography using a methanol-water gradient elution system, and the EIA-EIF fractions were obtained in the order of elution. (7) The EIB fraction was separated by Sphadex LH-20 column chromatography, eluted with methanol, and the fractions EIB1-EIB5 were obtained in the order of elution. (8) Component EIB3 was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 8; (9) The component EK was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The fractions EKA-EKM were obtained in the order of elution. (10) The EKC fraction was separated by Sphadex LH-20 column chromatography, eluted with methanol, and the fractions EKC1-EKC4 were obtained in the order of elution. (11) EKC4 was separated by high performance liquid chromatography and eluted with acetonitrile aqueous solution to obtain compound 4; (12) The EM fraction was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The EMA-EMO fraction was obtained in the order of elution. (13) The EMC fraction was crystallized, purified, and compound 3 was obtained; (14) The EMD fractions were separated by Sphadex LH-20 column chromatography and eluted with methanol to obtain EMD1-EMD4 in the order of elution. (15) The fraction EMD2 was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 5; (16) The EMG fractions were separated by Sphadex LH-20 column chromatography, eluted with methanol, and obtained in the order of elution as EMG1-EMG5; (17) The EMG2 fraction was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 6 and compound 7; (18) The fraction F was separated by MCI column chromatography and gradient elution was performed using a methanol-water system. The fractions FA-FR were obtained in the order of elution. (19) The fraction FI was separated by ODS column chromatography and gradient elution was performed using a methanol-water system. The fractions FIA-FIF were obtained in the order of elution. (20) The fraction FIE was separated by high performance liquid chromatography and gradient elution was performed using an acetonitrile-water system to obtain compound 1; The limonoid compounds, compounds 1-8 having the structures shown below: 。 3. The preparation method according to claim 2, characterized in that, The extraction method is cold soaking; the cold soaking temperature is 25°C, the time is 30 days, and the extract is collected every 48 hours.

4. The preparation method according to claim 2, characterized in that, The volume ratio of petroleum ether to dichloromethane in the petroleum ether and dichloromethane mixture is 1:1; the volume ratio of dichloromethane to ethyl acetate in the dichloromethane and ethyl acetate mixture is 1:

1.

5. The preparation method according to claim 2, characterized in that, In step (3), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate system is 100:0~1:1; In step (4), the volume ratio of acetonitrile to water in the acetonitrile-water system is 50:50 to 100:0; In step (5), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 75%.

6. The preparation method according to claim 2, characterized in that, In step (6), the volume ratio of methanol to water in the methanol-water system is 70:30 to 100:0; in step (8), the volume concentration of the acetonitrile aqueous solution is 63%; in step (9), the volume ratio of methanol to water in the methanol-water system is 60:40 to 100:

0.

7. The preparation method according to claim 2, characterized in that, In step (11), the volume concentration of the acetonitrile aqueous solution is 53%; In step (11), the volume ratio of methanol to water in the methanol-water system is 50:50 to 100:0; In step (15), the volume ratio of acetonitrile to water in the acetonitrile-water system is 55:45 to 75:

25.

8. The preparation method according to claim 2, characterized in that, In step (17), the gradient elution procedure is as follows: The gradient elution procedure is as follows: 0~15 min: The volume ratio of acetonitrile to water in the acetonitrile-water system changes uniformly from 60:40 to 75:25; 15~35 min: The volume ratio of acetonitrile to water in the acetonitrile-water system is 75:25; In step (18), the volume concentration of the methanol-water system is 10:90~100:

0.

9. The preparation method according to claim 2, characterized in that, In step (19), the volume concentration of the methanol-water system is 40:60~80:20.

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