Phenylpropanoid compound as well as preparation method and application thereof

Abstract

The invention relates to a phenylpropanoid compound as well as a preparation method and application thereof, in particular to a phenylpropanoid compound extracted from pleione bulbus pseudobulb as well as a preparation method and application thereof, and belongs to the technical field of medicines. The invention provides a method for preparing 10 new compounds from pleione bulbocodioides pseudobulb as a raw material for the first time, systematically evaluates the anti-neuroinflammation activity of pleione bulbocodioides pseudobulb, and clarifies the application of pleione bulbocodioides pseudobulb in development and treatment of central nervous system diseases. According to the invention, abnormally activated BV-2 microglial cells induced by LPS (Lipopolysaccharide) are adopted, and the NO release amount is taken as an index, so that the effect of the compound 1-10 on inhibiting excessive activation of the microglial cells is preliminarily evaluated. The result shows that the novel compounds 1-10 can inhibit the release of LPS-induced BV-2 microglial cell NO. Therefore, the compound prepared in the invention can be applied to development of drugs for treating central nervous system diseases.

Description

Technical Field

[0001] This invention relates to a class of phenylpropanoid compounds, their preparation methods and applications, specifically to phenylpropanoid compounds extracted from the pseudobulbs of *Gynostemma pentaphyllum*, their preparation methods and applications, belonging to the field of pharmaceutical technology. Background Technology

[0002] Single garlic orchid ( Pleione bulbocodioides *Franch.* Rolfe is a Class II protected plant in China, belonging to the Orchidaceae family and the *Franch.* genus, and is endemic to China. It is a semi-epiperi herb, growing in humus-rich soil under evergreen broad-leaved forests or at the edges of shrub forests, or on moss-covered rocks, at altitudes of 900-3600 meters. It is found in southern Shaanxi, southern Gansu, Anhui, Hubei, Hunan, northern Guangdong, northern Guangxi, Sichuan, Guizhou, northwestern Yunnan, and southeastern Tibet in China. Modern pharmacological studies have shown that *Franch.* Rolfe possesses antitumor, anti-inflammatory, antioxidant, and hepatoprotective activities. Its chemical components mainly include phenylpropanol, bibenzyl, malate, dihydrophenanthrene, and flavonoids. Currently, extraction techniques for *Franch.* Rolfe mainly include solvent extraction, ultrasonic-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, and enzyme-assisted extraction, with solvent extraction being the most common. However, solvent extraction may leave residual organic solvents, affecting safety and quality. Enzyme-assisted extraction offers milder conditions, but enzymes are more expensive, and their efficiency is significantly affected by factors such as pH and temperature, making process optimization difficult. Summary of the Invention

[0003] This invention provides a class of phenylpropanoid compounds extracted from the pseudobulbs of *Gnaphalium affine*, as well as an extraction method and the application of this class of compounds in the preparation of drugs for treating anti-neuroinflammatory diseases.

[0004] A class of phenylpropanoid compounds and their pharmaceutically acceptable salts having the following chemical structural formulas.

[0005] A method for preparing a class of phenylpropanoid compounds includes the following process steps: Single garlic orchid ( Pleione bulbocodioides The pseudobulbs were extracted with ethanol or methanol solution, and the crude extract was recovered. The crude extract was dissolved in water and extracted with organic solvents to obtain extracts of different polarities. The extracts of different polarities were separated by ODS column chromatography with gradient elution using methanol-water or acetonitrile-water mixed solvent as the mobile phase. The methanol-water or acetonitrile-water eluents were further separated by HPLC with gradient elution using methanol and water mixed solvent, acetonitrile and water mixed solvent, n-hexane and anhydrous ethanol mixed solvent, or n-hexane and isopropanol mixed solvent as the mobile phase to obtain compounds 1-10.

[0006] Preferably, the extraction with refluxed ethanol or methanol solution is performed 3 to 5 times, with a material-to-liquid ratio of 1:8 to 1:20 g / mL, wherein the volume concentration of the ethanol solution is 70% to 95% and the volume concentration of the methanol solution is 70% to 90%.

[0007] Preferably, the organic solvent extraction is performed by sequentially extracting with petroleum ether, dichloromethane, chloroform, ethyl acetate, and n-butanol 3 to 5 times at a volume ratio of aqueous phase to organic phase of 1:1 to 1:4, and then recovering the organic solvents under reduced pressure.

[0008] In the above scheme, at least two of petroleum ether, dichloromethane, chloroform, ethyl acetate, and n-butanol are selected for extraction in sequence.

[0009] Preferably, in the gradient elution and HPLC separation, the volume ratio of methanol to water is 1:9 to 9:1; and the volume ratio of acetonitrile to water is 1:9 to 9:1.

[0010] Preferably, the ethyl acetate extract is subjected to ODS chromatography with a gradient elution using methanol-water as the mobile phase; the n-butanol extract is subjected to ODS chromatography with a gradient elution using methanol-water as the mobile phase, wherein the volume ratio of methanol to water is 1:9 to 9:1; and the volume ratio of acetonitrile to water is 1:9 to 9:1.

[0011] Preferably, the methanol-water eluent or acetonitrile-water eluent is separated by HPLC, wherein the volume ratio of methanol to water is 1:9 to 9:1; the volume ratio of acetonitrile to water is 1:9 to 9:1; the volume ratio of n-hexane to anhydrous ethanol is 10:90 to 99:1; and the volume ratio of n-hexane to isopropanol is 10:90 to 99:1.

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned phenylpropanoid compounds.

[0013] A pharmaceutical composition comprising a phenylpropanoid compound having the following chemical structural formula, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0014]

[0015] Another object of the present invention is to provide the use of the above-mentioned phenylpropanoid compounds and their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of medicaments for treating neuroinflammatory diseases.

[0016] The beneficial effects of this invention are as follows: This invention provides for the first time a method for preparing and identifying 10 new compounds using *Gynostemma pentaphyllum* pseudobulbs as raw materials, and systematically evaluates their anti-neuroinflammatory activity, clarifying their application in the development and treatment of drugs for central nervous system diseases. This invention uses LPS-induced abnormally activated BV-2 microglia, with NO release as an indicator, to preliminarily evaluate the inhibitory effect of compounds 1-10 on excessive microglia activation. The results show that the new compounds 1-10 can inhibit LPS-induced NO release from BV-2 microglia. Therefore, the compounds prepared in this invention can be used in the development of drugs for the treatment of central nervous system diseases. Detailed Implementation

[0017] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0018] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0019] A method for preparing a phenylpropanoid compound, comprising the following steps: (1) Single Garlic Orchid ( Pleione bulbocodioides The pseudobulbs were extracted with ethanol or methanol solution, and the crude extract was obtained by recovering the extract. (2) The crude extract obtained in step (1) above is dissolved in water and extracted with organic solvents to obtain extracts of different polarities; (3) The fraction obtained in step (2) above is separated by ODS column chromatography, with methanol-water or acetonitrile-water mixed solvent as the mobile phase for gradient elution; (4) The methanol-water or acetonitrile-water eluent obtained in step (3) above is further separated by HPLC and eluted with a gradient of mobile phases of methanol and water, acetonitrile and water, n-hexane and anhydrous ethanol, or n-hexane and isopropanol to obtain compounds 1-10.

[0020] Preferably, in step (1), the extraction method is to extract with refluxed ethanol solution and refluxed methanol solution 3 to 5 times, with a material-to-liquid ratio of 1:8 to 1:20 g / mL, wherein the volume concentration of ethanol solution is 70% to 95% and the volume concentration of methanol solution is 70% to 90%.

[0021] Furthermore, the extraction method involves heating and refluxing with ethanol solution for extraction and heating and refluxing with methanol solution for extraction 3 to 4 times, with a material-to-liquid ratio of 1:8 to 1:15 g / mL, wherein the volume concentration of the ethanol solution is 75% to 95% and the volume concentration of the methanol solution is 75% to 90%.

[0022] Preferably, in step (2), the organic solvent extraction specifically involves: dissolving the crude extract in water, and extracting it 3 to 5 times sequentially with petroleum ether, dichloromethane, chloroform, ethyl acetate, and n-butanol at a volume ratio of 1:1 to 1:5 between the aqueous phase and the organic phase, and recovering the organic solvents under reduced pressure.

[0023] Further, in step (2), the aqueous phase and organic phase are extracted five times in sequence using petroleum ether, dichloromethane, ethyl acetate and n-butanol, respectively, according to a volume ratio of 1:1 to 1:4, and the organic solvents are recovered under reduced pressure.

[0024] Preferably, in step (3), the volume ratio of methanol to water is 1:9 ~ 9:1; the volume ratio of acetonitrile to water is 1:9 ~ 9:1.

[0025] Further, in step (3), the volume ratio of methanol to water is 2:8 ~ 9:1; the volume ratio of acetonitrile to water is 2:8 ~ 9:1.

[0026] Preferably, in step (4), the volume ratio of methanol to water is 1:9 ~ 9:1; the volume ratio of acetonitrile to water is 1:9 ~ 9:1; the volume ratio of n-hexane to anhydrous ethanol is 10:90 ~ 99:1; and the volume ratio of n-hexane to isopropanol is 10:90 ~ 99:1.

[0027] Further, in step (4), the volume ratio of methanol to water is 2:8 ~ 8:2; the volume ratio of acetonitrile to water is 2:8 ~ 8:2; the volume ratio of n-hexane to anhydrous ethanol is 20:80 ~ 98:2; and the volume ratio of n-hexane to isopropanol is 20:80 ~ 98:2.

[0028] Example 1 (1) 2000 g of pseudobulb of Garlic Orchid was extracted 5 times by heating and reflux with 75% ethanol (dosage: 20 L), and the crude extract was obtained by vacuum recovery. (2) The crude extract of 75% ethanol obtained in step (1) above is dissolved in water and extracted sequentially with petroleum ether and ethyl acetate. Each organic phase is extracted 3 times, and the volume ratio of the aqueous phase to the organic phase is 1:1 each time to obtain extracts of different polar fractions. (3) The petroleum ether extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. (4) The ethyl acetate extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2. (5) The methanol-water (7:3 ~ 9:1) fraction obtained in step (3) above was separated by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min and a mobile phase of acetonitrile:water = 50:50, to obtain a racemic mixture of compounds 1 and 2 (t R =35.9 min (yield 0.0005‰); the mobile phase was acetonitrile:water = 60:40, yielding a racemic mixture of compounds 3 and 4 (t R = 25.9 min (yield 0.0004‰), yielding a racemic mixture of compounds 5 and 6 (t R = 29.1 min (yield 0.0004‰), yielding a racemic mixture of compounds 7 and 8 (t R = 32.2 min (yield 0.0004‰). The racemic mixture of compounds 1 and 2 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (80:20) to give compound 1 (t R = 6.9 min) and compound 2 (t R = 8.5 min (yields of 0.0002‰ each), the racemic mixture of compounds 3 and 4 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (80:20) to give compound 3 (t R = 8.2min) and compound 4 (t R = 9.9 min (yields of 0.0002‰ each), the racemic mixture of compounds 5 and 6 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (80:20) to give compound 5 (t R = 12.5 min) and compound 6 (t R = 14.0 min (yield of 0.0002‰ each), the racemic mixture of compounds 7 and 8 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (80:20) to give compound 7 (t R = 14.8 min) and compound 8 (t R =16.7 min)(yield of 0.0002‰).

[0029] (6) The methanol-water (6:4 ~ 8:2) fraction obtained in step (4) above was separated by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min and a mobile phase of methanol:water = 45:55, to obtain compound 9 (t R = 25.8 min), compound 10 (t R = 32.9 min (yield of 0.0003‰).

[0030] The structures of compounds 1-10 were identified based on their physicochemical properties and spectroscopic data.

[0031] The structural identification data of compound 1 / 2 are as follows: White powder (CH3OH), [α] D 20 +3.9 ( c 0.10, MeOH). HR-ESI-MS yielded quasi-molecular ion peaks. m / z 347.1494 [M+H] + : (calcd. 347.1495 for C 19 H 22 O6), whose molecular formula is presumed to be C. 19 H 22 O6 has an unsaturation degree of 9.

[0032] 1 In H-NMR (600 MHz, CD3OD), a set of symmetrical aromatic hydrogen signals was observed in the low-field region: d H 6.73 (2H,s, H-2, 6); Group 1 meta-hydrogen signals on the benzene ring: d H 6.44 (2H, s, H-2', 6'). A set of characteristic hydrogen signals for a 1,4-benzodioxane fragment: d H 5.01 (1H, dd, J = 8.8, 2.4 Hz, H-7), 4.35 (1H, dd, J = 11.3, 2.4 Hz, H-8a), 3.96 (1H, m, H-8b); 3 methoxy hydrogen signals: d H 3.86 (6H, s, OCH3-3, 5'), 3.82 (3H, s, OCH3-5'); One methylene hydrogen signal is visible in the high-field region. d H 2.55 (2H, q,J = 7.6 Hz, H-7'); 1 methyl hydrogen signal: d H 1.20 (3H, t, J = 7.6 Hz, CH3-8').

[0033] 13 Nineteen carbon signals were obtained from C-NMR (150 MHz, CD3OD), including 12 carbon signals on the benzene ring: d C 150.0 (C-5'), 149.4 (C-3, 5), 145.6 (C-3'), 138.1 (C-1'), 136.9 (C-4), 132.1 (C-4'), 128.7 (C-1), 110.2 (C-2'), 105.7 (C-6'), 105.0 (C-2, 6); 1 hydroxymethyl carbon signal: d C 76.6 (C-7); 1 hydroxymethylene carbon signal: d C 70.4 (C-8); 3 methoxy carbon signals: d C 56.8(O C H3-3, 5), 56.7 (O C H3-5'); 1 methylene carbon signal: d C 29.7 (C-7') and 1 methyl carbon signal: d C 16.3 (C-8'). Combined 1 H NMR and 13 C10 NMR data identified the compound as a 3',7-epoxyneurolipane. Hydrogen and carbon signals were assigned based on HSQC spectral information.

[0034] In the HMBC spectrum d H 3.86 (OC H 3-3, 5) and d C 149.4 (C-3, 5) is relevant; note -OC H 3 is connected at C-3,5 bits; d H 3.82 (OC H 3-5') and d C 150.0 (C-5') related, hint -OC H3 is connected at C-5'; d H 2.55 (H-7') and d C 138.1 (C-1'), 105.7 (C-6'), 110.2 (C-2') are relevant. d H 1.20 (H-8') and d C The related information for 138.1 (C-1') and 29.7 (C-7') indicates the presence of an ethyl group attached at the C-1' position. d H 5.01 (H-7) and d C The connection to 105.0 (C-2, 6) and 128.7 (C-1) indicates that C-7 is connected at C-1. d H 4.35 (H-8a) and d C The 132.1 (C-4') correlation indicates that C-8 is attached at the C-4' position, thus confirming the planar structure of the compound.

[0035] Further calculations using the theoretical B3LYP / 6-31G(d,p) ECD method determined the absolute configurations. Comparing the measured and calculated ECD spectra, the absolute configurations of compounds 1 and 2 were determined to be 7. S and 7 R Literature search revealed that compounds 1 and 2 were novel compounds not previously reported, and they were named 7. S -pleilignanine A and 7 R -pleilignanine A, the NMR signal assignments are shown in Table 1.

[0036] The structural identification data of compounds 3 / 4 are as follows: White powder (CH3OH). [α] D 20 -4.5 ( c 0.10, MeOH). HR-ESI-MS yielded quasi-molecular ion peaks. m / z 375.1804 [M+H] + : (calcd. 375.1808 for C 21 H 26 O6), whose molecular formula is presumed to be C. 21 H 26 O6 has an unsaturation degree of 9.

[0037] 1In H-NMR (600 MHz, CD3OD), a set of symmetrical aromatic hydrogen signals was observed in the low-field region: d H 6.69 (2H,s, H-2, 6); Group 1 meta-coupled hydrogen signals on the benzene ring: d H 6.41 (1H, d, J = 1.9 Hz, H-6'), 6.38 (1H, d, J = 1.9 Hz, H-2'). Characteristic hydrogen signals of a set of 8-substituted 1,4-benzodioxane segments: d H 4.54(1H, d, J = 7.8 Hz, H-7), 4.08 (1H, dq, J = 7.8, 6.3 Hz, H-8), and positions 7 and 8 are in the trans configuration; 3 methoxy hydrogen signals: d H 3.86 (6H, s, OCH3-3, 5'), 3.83 (3H, s, OCH3-5'); Two methylene hydrogen signals are visible in the high-field region: d H 2.48 (2H, t, J = 7.8 Hz, H-7'), 1.61 (2H, q, J = 7.4 Hz, H-8'); 2 methyl hydrogen signals: d H 1.17 (3H, d, J = 6.4 Hz, CH3-9), 0.93 (3H, t, J = 7.4 Hz, CH3-9').

[0038] 13 The C-NMR (150 MHz, CD3OD) spectrum yielded 21 carbon signals, including 12 carbon signals on the benzene ring: d C 149.8 (C-5'), 149.4 (C-3, 5), 145.7 (C-3'), 137.2 (C-4), 136.1 (C-1'), 132.5 (C-4'), 129.3 (C-1), 110.5 (C-2'), 106.2 (C-6'), 106.0 (C-2, 6); 2 hydroxymethyl carbon signals: d C82.5 (C-7), 75.4 (C-8); 3 methoxy carbon signals: d C 56.8 (O C H3-3, 5), 56.7(O C H3-5'); 2 methylene carbon signals: d C 38.9 (C-7'), 25.8 (C-8') and two methyl carbon signals: d C 17.5 (C-9), 14.0 (C-9'). Combined 1 H NMR and 13 C10 NMR data identified the compound as a 3',7-epoxyneurolipane. Hydrogen and carbon signals were assigned based on HSQC spectral information.

[0039] In the HMBC spectrum d H 3.86 (OC H 3-3, 5) and d C 149.4 (C-3, 5) is relevant; note -OC H 3 is connected at C-3,5 bits; d H 3.83 (OC H 3-5') and d C 149.8 (C-5') related, hint -OC H 3 is connected at C-5'; d H 1.17 (C H 3-9) and d C 82.5 (C-7), 75.4 (C-8) are relevant, indicating that -CH3 is connected to the C-8 position; d H 0.93 (H-9') and d C 38.9 (C-7') related, d H 1.61 (H-8') and d C 136.1 (C-1') is relevant. d H 2.48 (H-7') and d C The correlation between 110.5 (C-2') and 106.2 (C-6') indicates the presence of an n-propyl group attached at the C-1' position. dH 4.54 (H-7) and d C The correlation between 106.0 (C-2, 6) and 129.3 (C-1) indicates that C-7 is attached at C-1, thus confirming the planar structure of the compound.

[0040] This compound has two chiral carbons, theoretically allowing for four configurations. However, the coupling constants at the H-7 and H-8 positions indicate a trans configuration, thus suggesting two possible configurations: (7...) S , 8 S (7) R , 8 R Further, the absolute configurations were determined by ECD calculations at the theoretical level of B3LYP / 6-31G(d,p). By comparing the measured and calculated ECD spectra, the absolute configurations of compounds 3 and 4 were determined to be 7. S , 8 S and 7 R , 8 R Literature search revealed that compounds 3 and 4 were novel compounds not previously reported, and they were named (7). S , 8 S ) -pleilignanine B and (7 R , 8 R )-pleilignanine B, the NMR signal attribution is shown in Table 1.

[0041] The structural identification data for compounds 5 and 6 are as follows: White powder (CH3OH). [α] D 20 -7.3 ( c 0.10, MeOH). HR-ESI-MS yielded quasi-molecular ion peaks. m / z 339.1206 [M+Na] + : (calcd. 339.1208 for C 18 H 20 O5), whose molecular formula is presumed to be C. 18 H 20 O5 has an unsaturation degree of 9.

[0042] 1 In H-NMR (600 MHz, CD3OD), a set of symmetrical aromatic hydrogen signals was observed in the low-field region: d H 6.68 (2H, s, H-2, 6); ABX coupled hydrogen signal on the benzene ring: d H 6.74 (1H, d,J = 8.2 Hz, H-5'), 6.70 (1H,d, J = 2.1 Hz, H-2'), 6.64 (1H, dd, J = 8.2, 2.1 Hz, H-6'). Characteristic hydrogen signals of a set of 8-position substituted 1,4-benzodioxane segments: d H 4.52 (1H, d, J = 7.9 Hz, H-7), 4.08 (1H, dq, J = 7.9, 6.3 Hz, H-8), and positions 7 and 8 are in the trans configuration; 2 methoxy hydrogen signals: d H 3.86 (6H, s, OCH3-3, 5); Two methyl hydrogen signals are visible in the high-field region: d H 2.23 (3H, s, CH3-7'), 1.13 (3H, d, J = 6.3 Hz, CH3-9).

[0043] 13 In C-NMR (150 MHz, CD3OD), 18 carbon signals were provided, including 12 carbon signals on the benzene ring: d C 149.4 (C-3, 5), 145.2 (C-3'), 142.7 (C-4'), 137.2 (C-4), 131.8 (C-1'), 129.4(C-1), 122.9 (C-6'), 118.2 (C-2'), 117.5 (C-5'), 106.0 (C-2, 6); Two hydroxymethyl carbon signals: d C 82.6 (C-7), 75.3 (C-8); 2 methoxy carbon signals: d C 56.8 (O C H3-3, 5); 2 methyl carbon signals: d C 20.7 (C-7'), 17.6 (C-9). Combined 1 H NMR and 13 C10 NMR data identified the compound as a 3',7-epoxyneurolipane. Hydrogen and carbon signals were assigned based on HSQC spectral information.

[0044] In the HMBC spectrum dH 3.86 (OC H 3-3, 5) and d C 149.4 (C-3, 5) is relevant; note -OC H 3 is connected at C-3,5 bits; d H 1.13 (C H 3-9) and d C 82.6 (C-7), 75.3 (C-8) are relevant, indicating that -CH3 is connected to the C-8 bit; d H 2.13(C H 3-7') and d C 131.8 (C-1'), 122.9 (C-6'), 118.2 (C-2') are related, indicating that -CH3 is connected at C-1'; d H 4.52 (H-7) and d C The correlation between 106.0 (C-2, 6) and 129.4 (C-1) indicates that C-7 is connected at C-1; by optimizing the correlation parameters in the HMBC spectrum, it was found that... d H 1.13 (C H 3-9) and d C The 142.7 (C-4') correlation determined the planar structure of the compound.

[0045] This compound has two chiral carbons, theoretically allowing for four configurations. However, the coupling constants at the H-7 and H-8 positions indicate a trans configuration, thus suggesting two possible configurations: (7...) S , 8 S (7) R , 8 R Further, the absolute configurations were determined by ECD calculations at the theoretical level of B3LYP / 6-31G(d,p). By comparing the measured and calculated ECD spectra, the absolute configurations of compounds 5 and 6 were determined to be 7. S , 8 S and 7 R , 8 R Literature search revealed that compounds 5 and 6 were novel compounds not previously reported, and they were named (7). S , 8 S ) -pleilignanine C and (7 R , 8 R)-pleilignanine C, the NMR signal assignments are shown in Table 2.

[0046] The structural identification data for compounds 7 and 8 are as follows: White powder (CH3OH). [α] D 20 +2.8 ( c 0.10, MeOH). HR-ESI-MS yielded quasi-molecular ion peaks. m / z 353.1361 [M+Na] + : (calcd. 353.1365 for C 19 H 22 O5), whose molecular formula is presumed to be C. 19 H 22 O5 has an unsaturation degree of 9.

[0047] 1 In H-NMR (600 MHz, CD3OD), a set of symmetrical aromatic hydrogen signals was observed in the low-field region: d H 6.68 (2H,s, H-2, 6); Group 1 para-hydrogen signals on the benzene ring: d H 6.66 (1H, s, H-2'), d H 6.65 (1H, s, H-5'). Characteristic hydrogen signals of a group of 8-substituted 1,4-benzodioxane fragments: d H 4.49 (1H, d, J = 7.8 Hz, H-7), 4.07(1H, dq, J = 7.8, 6.3 Hz, H-8), and positions 7 and 8 are in the trans configuration; 2 methoxy hydrogen signals: d H 3.86 (6H, s, OCH3-3, 5); Three methyl hydrogen signals are visible in the high-field region: d H 2.15 (3H, s, CH3-7'), 2.15 (3H, s,CH3-8'), 1.12 (3H, d, J = 6.3 Hz, CH3-9).

[0048] 13 Nineteen carbon signals were obtained from C-NMR (150 MHz, CD3OD), including 12 carbon signals on the benzene ring: d C149.4 (C-3, 5), 143.1 (C-3'), 142.5 (C-4'), 137.1 (C-4), 130.3 (C-1'), 130.1(C-6'), 129.5 (C-1), 118.6 (C-2'), 118.5 (C-5'), 106.0 (C-2, 6); Two hydroxymethyl carbon signals: d C 82.5 (C-7), 75.4 (C-8); 2 methoxy carbon signals: d C 56.8 (O C H3-3, 5); 3 methyl carbon signals: d C 19.1 (C-8'), 19.1 (C-7'), 17.6 (C-9). Combined 1 H NMR and 13 C10 NMR data identified the compound as a 3',7-epoxyneurolipane. Hydrogen and carbon signals were assigned based on HSQC spectral information.

[0049] In the HMBC spectrum d H 3.86 (OC H 3-3, 5) and d C 149.4 (C-3, 5) is relevant; note -OC H 3 is connected at C-3,5 bits; d H 1.12 (C H 3-9) and d C 82.5 (C-7), 75.4 (C-8) are relevant, indicating that -CH3 is connected to the C-8 position; d H 2.15(C H 3-7') and d C 130.1 (C-6'), 118.6 (C-2') are relevant, indicating that -CH3 is connected at C-1'; d H 2.15 (C H 3-8') and d C 130.3 (C-1'), 118.5 (C-5') are relevant, indicating that -CH3 is connected at C-6'; d H 4.49 (H-7) and dC The correlation between 106.0 (C-2, 6) and 129.5 (C-1) indicates that C-7 is attached at C-1, thus confirming the planar structure of the compound.

[0050] This compound has two chiral carbons, theoretically allowing for four configurations. However, the coupling constants at the H-7 and H-8 positions indicate a trans configuration, thus suggesting two possible configurations: (7...) S , 8 S (7) R , 8 R The absolute configurations were further determined by ECD calculations at the theoretical level of B3LYP / 6-31G(d,p). By comparing the measured and calculated ECD spectra, the absolute configurations of compounds 7 and 8 were determined to be 7. S , 8 S and 7 R , 8 R Literature search revealed that 7 and 8 are novel compounds not previously reported, and they were named (7...). S , 8 S ) -pleilignanine D and (7 R , 8 R The NMR signal attribution of )-pleilignanine D is shown in Table 2.

[0051] The structural identification data of compound 9 are as follows: Colorless oil (CH3OH). HR-ESI-MS yielded a quasi-molecular ion peak. m / z 323.1400 [M+Na] + : (calcd.323.1471 for C 15 H 24 O6Na), its molecular formula is presumed to be C. 15 H 24 O6 has an unsaturation degree of 4.

[0052] 1 H-NMR (600 MHz, DMSO- d In section 6), the low-field region provides one set of benzene ring-symmetric hydrogen signals: d H 6.52 (2H, s, H-2, 6). Two sets of methoxy hydrogen signals are given in the high-field region. d H 3.73 (6H, s, 3, 5-OC H 3); In addition, there are 3 sets of hydrogen-oxygen-linked signals. d H 4.09 (1H, d, J=5.7 Hz, H-7), 3.48 (1H, m, H-8), 3.28 (1H, m, H-9a), 3.11(1H, dd, J = 6.4, 10.9 Hz, H-9b); and one set of oxygen-n-butyl hydrogen signals: d H 3.24 (2H, m, H-1'),1.47 (2H, m, H-2'), 1.32 (2H, m, H-3'), 0.84 (3H, t, J = 7.4 Hz, H-4'). According to the literature [6] H-7 coupling constant (5.7 < 6.0), determine 7th and 8th bits as red.

[0053] 13 C-NMR (150 MHz, DMSO- d 6) provides 15 carbon signals, of which d C 130.2 (C-1), 104.5 (C-2, 6), 147.6 (C-3, 5), and 134.6 (C-4) represent carbon signals on the benzene ring; d C 82.1 (C-7), 75.2 (C-8), and 62.5 (C-9) are carbon hydroxyl signals; in addition, d C 67.9 (C-1'), 31.5 (C-2'), 18.9 (C-3'), and 13.8 (C-4') represent one group of oxygen-bonded n-butyl carbon signals. Hydrogen and carbon signals were assigned based on relevant information from the HSQC spectrum.

[0054] In the HMBC spectrum d H 3.73 (3, 5-OC H 3) with d C 147.6 (C-3, 5) is relevant, indicating that 3, 5-OCH3 is connected at C-3, 5. d H 3.24 (H-1') and d C 18.9 (C-3'), 31.5 (C-2') are relevant. d H 1.47 (H-2') and d C 13.8 (C-4'), 18.9 (C-3'), 67.9 (C-1') are relevant. dH 1.32 (H-3') and d C 13.8 (C-4'), 31.5 (C-2'), 67.9 (C-1') are relevant. d H 0.84 (H-4') and d C 18.9 (C-3'), 31.5 (C-2') are related, suggesting that the side chain is n-butyl and is obtained through... d H 3.24 (H-1') and d C 82.1 (C-7) Related Explanation: The n-butyl group is connected to the C-7 position via an ether bond; through... d H 4.09 (H-7) and d C 104.6 (C-2, 6), 75.2 (C-8) are relevant. d H 3.48 (H-8) and d C Related to 82.1 (C-7) and 62.5 (C-9) d H 3.28 (H-9a), 3.11 (H-9b) and d C The correlation between 75.2 (C-8) and 82.1 (C-7) indicates that this fragment has a C6-C3 structure. Therefore, the planar structure of this compound is determined.

[0055] Further calculations using the theoretical B3LYP / 6-31G(d,p) ECD method determined the absolute configuration. Comparison of the measured and calculated ECD spectra confirmed that the absolute configuration of compound 9 was 7. R , 8 R A literature search revealed that 9 was a previously unreported novel compound, named (7). R , 8 R The NMR signal attribution of )-pleilignanine E is shown in Table 3.

[0056] The structural identification data of compound 10 are as follows: Colorless oil (CH3OH). HR-ESI-MS yielded a quasi-molecular ion peak. m / z 323.1477 [M+Na] + : (calcd.323.1471 for C 15 H 24 O6Na), its molecular formula is presumed to be C.15 H 24 O6 has an unsaturation degree of 4.

[0057] 1 H-NMR (600 MHz, DMSO- d In section 6), the low-field region provides one set of benzene ring-symmetric hydrogen signals: d H 6.53 (2H, s, H-2, 6). Two sets of methoxy hydrogen signals are given in the high-field region. d H 3.73 (6H, s, 3, 5-OC H 3); In addition, there are 3 sets of hydrogen-oxygen-linked signals. d H 4.02 (1H, d, J =6.5 Hz, H-7), 3.52 (1H, m, H-8), 3.48 (1H, m, H-9a), 3.36 (1H, m, H-9b); and one set of hydrogen signals for the hydroxyl group n-butyl: d H 3.20 (2H, m, H-1'), 1.43 (2H, m, H-2'), 1.30 (2H, m, H-3'), 0.83 (3H, t, J = 7.4 Hz, H-4'). Based on the coupling constant of H-7 in the literature (6.5 > 6.0), the 7th and 8th bits are determined to be of the Soviet type.

[0058] 13 C-NMR (150 MHz, DMSO- d 6) provides 15 carbon signals, of which d C 130.1 (C-1), 105.2 (C-2, 6), 147.5 (C-3, 5), and 134.6 (C-4) represent carbon signals on the benzene ring; d C 82.3 (C-7), 74.4 (C-8), and 63.0 (C-9) are carbon pyrolysis signals; in addition, d C 67.6 (C-1'), 31.5 (C-2'), 18.9 (C-3'), and 13.7 (C-4') represent a group of oxygen-bound n-butyl carbon signals. Based on the comparison of C and H spectra, it was determined to be an epimer of compound 9.

[0059] Further calculations using the theoretical B3LYP / 6-31G(d,p) ECD method determined the absolute configuration. Comparison of the measured and calculated ECD spectra confirmed that the absolute configuration of compound 10 was 7. R , 8 S A literature search revealed that 10 was a previously unreported novel compound, named (7). R , 8 S The NMR signal attribution of )-pleilignanine F is shown in Table 3.

[0060] Table 1. NMR data of compounds 1-4 (a: Methanol- d 4)

[0061] Table 2. NMR data of compounds 5-8 (a: Methanol- d 4)

[0062] Table 3. NMR data of compounds 9-10 (b: DMSO- d 6)

[0063] Example 2 (1) 3000 g of pseudobulb of Garlic Orchid was extracted 4 times by heating and reflux with 75% methanol (dosage: 18 L), and the crude extract was obtained by vacuum recovery. (2) The 75% methanol crude extract obtained in step (1) above is dissolved in water and extracted sequentially with petroleum ether and ethyl acetate. Each organic phase is extracted 4 times, and the volume ratio of the aqueous phase to the organic phase is 1:2 each time to obtain extracts of different polar fractions. (3) The petroleum ether extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of acetonitrile-water 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2; (4) The ethyl acetate extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of acetonitrile-water in the ranges of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2. (5) The acetonitrile-water (7:3 ~ 8:2) fraction obtained in step (3) above was separated by HPLC-UV chromatography, detected at 220 nm, with a flow rate of 2 mL / min and a mobile phase of methanol:water = 60:40, to obtain a racemic mixture of compounds 1 and 2 (t R= 25.8 min (yield 0.0008‰); the mobile phase was methanol:water = 70:30, yielding a racemic mixture of compounds 3 and 4 (t R = 30.2 min (yield 0.0006‰), yielding a racemic mixture of compounds 5 and 6 (t R = 33.1 min (yield 0.0006‰), yielding a racemic mixture of compounds 7 and 8 (t R = 36.5 min (yield 0.0007‰). The racemic mixture of compounds 1 and 2 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (90:10) to give compound 1 (t R = 7.2 min) and compound 2 (t R = 8.8 min (yields of 0.0003‰ each), the racemic mixture of compounds 3 and 4 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (90:10) to give compound 3 (t R =6.8 min) and compound 4 (t R = 8.1 min (yield of 0.0003‰ each), the racemic mixture of compounds 5 and 6 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (90:10) to give compound 5 (t R = 10.4 min) and compound 6 (t R = 12.5 min (yield of 0.0002‰ each), the racemic mixture of compounds 7 and 8 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (90:10) to give compound 7 (t R = 11.7 min) and compound 8 (t R =13.5 min) (yield of 0.0002‰).

[0064] (6) The acetonitrile-water (7:3 ~ 8:2) fraction obtained in step (4) above was separated by HPLC-UV chromatography, detected at 220 nm, with a flow rate of 2 mL / min and a mobile phase of methanol:water = 50:50, to obtain compound 9 (t R = 23.1 min), compound 10 (t R = 29.8 min (yield of 0.0005‰).

[0065] The structural identification methods for compounds 1-10 are described in Example 1.

[0066] Example 3 (1) 1000 g of pseudobulb of Garlic Orchid was extracted three times by heating and refluxing with 80% ethanol (dosage: 15 L), and the crude extract was obtained by vacuum recovery. (2) The crude extract of 80% ethanol obtained in step (1) above is dissolved in water and extracted sequentially by petroleum ether and n-butanol. Each organic phase is extracted 5 times, and the volume ratio of the aqueous phase to the organic phase is 1:1 each time to obtain extracts of different polar fractions. (3) The petroleum ether extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. (4) The n-butanol extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7: 3, 8: 2, 9: 1. (5) The methanol-water (7:3 ~ 9:1) fraction obtained in step (3) above was prepared by HPLC-UV chromatography with detection at 230 nm, a flow rate of 2.5 mL / min, and a mobile phase of acetonitrile:water = 60:40, to obtain a racemic mixture of compounds 1 and 2 (t R = 22.1 min (yield 0.0004‰); the mobile phase was acetonitrile:water = 70:30, yielding a racemic mixture of compounds 3 and 4 (t R = 25.3 min (yield 0.0004‰), yielding a racemic mixture of compounds 5 and 6 (t R = 28.2 min (yield 0.0004‰), yielding a racemic mixture of compounds 7 and 8 (t R = 32.3 min (yield 0.0003‰). The racemic mixture of compounds 1 and 2 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (95:5) to give compound 1 (t R = 9.0 min) and compound 2 (t R = 11.2 min (yield of 0.0001‰ each), the racemic mixture of compounds 3 and 4 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (95:5) to give compound 3 (t R =8.2 min) and compound 4 (t R = 10.2 min (yield of 0.0002‰ each), the racemic mixture of compounds 5 and 6 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (95:5) to give compound 5 (t R = 12.9 min) and compound 6 (t R= 15.2 min (yield of 0.0001‰ each), the racemic mixture of compounds 7 and 8 was then separated by chiral column chromatography, eluting with n-hexane:anhydrous ethanol (95:5) to give compound 7 (t R = 14.7 min) and compound 8 (t R = 16.2 min (yield of 0.0001‰).

[0067] (6) The methanol-water (7:3 ~ 9:1) fraction obtained in step (4) above was separated by HPLC-UV chromatography, detected at 230 nm, with a flow rate of 2.5 mL / min and a mobile phase of acetonitrile:water = 60:40, to obtain compound 9 (t R = 20.2min), compound 10 (t R = 24.6 min (yield of 0.0003‰).

[0068] The structural identification methods for compounds 1-10 are described in Example 1.

[0069] Example 4 (1) 2500 g of pseudobulb of Garlic Orchid was extracted three times by heating and refluxing with 90% ethanol (dosage: 12 L), and the crude extract was obtained by vacuum recovery. (2) The crude extract of 90% ethanol obtained in step (1) above is dissolved in water and extracted sequentially by petroleum ether and ethyl acetate. Each organic phase is extracted 4 times, and the volume ratio of the aqueous phase to the organic phase is 1:2 each time to obtain extracts of different polar fractions. (3) The petroleum ether extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. (4) The ethyl acetate extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. (5) The methanol-water (6:4 ~ 9:1) fraction obtained in step (3) above was separated by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 2 mL / min and a mobile phase of acetonitrile:water = 60:40, to obtain a racemic mixture of compounds 1 and 2 (t R =35.6 min (yield 0.0005‰); the mobile phase was acetonitrile:water = 65:35, yielding a racemic mixture of compounds 3 and 4 (t R = 37.3 min (yield 0.0006‰), yielding a racemic mixture of compounds 5 and 6 (t R= 40.8 min (yield 0.0005‰), yielding a racemic mixture of compounds 7 and 8 (t R = 43.2 min (yield 0.0004‰). The racemic mixture of compounds 1 and 2 was then separated by chiral column chromatography, eluting with n-hexane:isopropanol (95:5) to give compound 1 (t R = 10.2 min) and compound 2 (t R = 13.5 min (yield of 0.0002‰ each), the racemic mixture of compounds 3 and 4 was then separated by chiral column chromatography, eluting with n-hexane:isopropanol (95:5) to give compound 3 (t R = 10.9min) and compound 4 (t R = 14.0 min (yield of 0.0002‰ each), the racemic mixture of compounds 5 and 6 was then separated by chiral column chromatography, eluting with n-hexane:isopropanol (95:5) to give compound 5 (t R = 15.2min) and compound 6 (t R = 18.4 min (yields of 0.0002‰ each), the racemic mixture of compounds 7 and 8 was then separated by chiral column chromatography, eluting with n-hexane:isopropanol (95:5) to give compound 7 (t R = 17.6 min) and compound 8 (t R = 19.9 min (yield of 0.0002‰).

[0070] (6) The methanol-water (6:4 ~ 8:2) fraction obtained in step (4) above was separated by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 2 mL / min and a mobile phase of methanol:water = 65:45, to obtain compound 9 (t R = 17.6 min), compound 10 (t R = 20.8 min (yield of 0.0005‰).

[0071] The structural identification methods for compounds 1-10 are described in Example 1.

[0072] Example 5 (1) 3000 g of pseudobulb of Garlic Orchid was extracted 4 times by heating and refluxing with 95% ethanol (dosage: 20L), and the crude extract was obtained by vacuum recovery. (2) The crude extract of 95% ethanol obtained in step (1) above is dissolved in water and extracted sequentially by petroleum ether, ethyl acetate and n-butanol. Each organic phase is extracted 5 times, and the volume ratio of the aqueous phase to the organic phase is 1:1 each time to obtain extracts of different polar fractions. (3) The ethyl acetate extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of acetonitrile-water in the ranges of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. (4) The n-butanol extract obtained in step (2) above was subjected to ODS chromatography and eluted with a gradient of mixed solvents of acetonitrile-water in the ranges of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. (5) The acetonitrile-water (6:4 ~ 9:1) fraction obtained in step (3) above was prepared by HPLC-UV chromatography, with detection at 210 nm, a flow rate of 2 mL / min, and a mobile phase of acetonitrile:water = 55:45, to obtain a racemic mixture of compounds 1 and 2 (t R =30.7 min (yield 0.0005‰); the mobile phase was acetonitrile:water = 50:50, yielding a racemic mixture of compounds 3 and 4 (t R = 33.2 min (yield 0.0005‰), yielding a racemic mixture of compounds 5 and 6 (t R = 36.5 min (yield 0.0005‰), yielding a racemic mixture of compounds 7 and 8 (t R = 40.8 min (yield 0.0004‰). The racemic mixture of compounds 1 and 2 was then separated by chiral column chromatography, eluting with n-hexane:isopropanol (90:10) to give compound 1 (t R = 7.8 min) and compound 2 (t R = 9.6 min (yields of 0.0003‰ each), the racemic mixture of compounds 3 and 4 was then separated by chiral column chromatography, eluting with n-hexane:isopropanol (90:10) to give compound 3 (t R = 9.2 min) and compound 4 (t R = 12.5 min (yield of 0.0003‰ each), the racemic mixture of compounds 5 and 6 was then separated by chiral column chromatography, eluting with n-hexane:isopropanol (90:10) to give compound 5 (t R = 13.4 min) and compound 6 (t R = 15.9 min (yields of 0.0003‰ each), the racemic mixture of compounds 7 and 8 was then separated by chiral column chromatography, eluting with n-hexane:isopropanol (90:10) to give compound 7 (t R = 18.2 min) and compound 8 (t R = 20.3 min (yield of 0.0003‰).

[0073] (6) The acetonitrile-water (6:4 ~ 9:1) fraction obtained in step (4) above was separated by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 2 mL / min and a mobile phase of methanol:water = 60:40, to obtain compound 9 (t R = 24.6 min), compound 10 (t R = 28.2 min (yield of 0.0006‰).

[0074] The structural identification methods for compounds 1-10 are described in Example 1.

[0075] Example 6: Test of the anti-neuroinflammatory activity of compounds 1-10 prepared in Examples 1-5.

[0076] (1) Experimental Principle: Neurodegenerative diseases are a class of diseases characterized by the chronic and progressive loss of neurons, exhibiting high incidence, high disability rate, high mortality rate, and high recurrence rate. In animal models of neurodegenerative diseases and the brains of human patients, a gradually increasing number of activated microglia can be observed. Under normal physiological conditions, microglia are in a neurotrophic state and secrete anti-inflammatory factors, which can protect neurons from external damage. However, under pathological conditions, such as stimulation by exogenous pathogens or endogenous cell debris and abnormally folded proteins, microglia are activated. Activation of microglia releases a variety of pro-inflammatory factors, and the large release of these pro-inflammatory factors further activates microglia, forming a vicious cycle that ultimately leads to neuronal damage and induces neurodegenerative diseases. Therefore, inhibiting pathological neuroinflammation caused by microglia activation is of great significance for the treatment of neurodegenerative diseases. This invention constructs an in vitro screening model for abnormal activation of BV2 microglia by LPS activation, using the release of NO from activated microglia as an indicator to evaluate the anti-neuroinflammatory activity of new phenylpropanoid compounds 1-10.

[0077] (2) Experimental method: 1. Culture of mouse microglia BV-2 All glassware and metal instruments (culture dishes, pipettes, solution dishes, etc.) used in cell culture and model establishment were autoclaved at 121 °C for 30 min to thoroughly remove LPS contamination. A cell culture medium containing 10% fetal bovine serum was prepared using DMEM as a base. Microglia were cultured at approximately 2.5 × 10⁶ cells / day. 5 cells·mL -1The concentration of the active ingredient was used for subculturing in 5% CO2 at 37°C. By the third day, adherent cells occupied approximately 70-80% of the bottom area of ​​the culture dish. The adherent cells were then digested with trypsin and subcultured into another culture dish. BV-2 cells that had been cryopreserved and thawed at -80°C were used as the first generation, and BV-2 cells from the 3rd to 8th generations were selected for experiments.

[0078] 2. Drug preparation method The monomeric compounds from the pseudobulbs of *Gynostemma pentaphyllum* were dissolved in DMSO to prepare a 100 mM stock solution, which was stored at -20°C. Before use, the solution was diluted with DMEM culture medium to 100 μM, 30 μM, 10 μM, and 1 μM, respectively. The final DMSO concentration was <1‰.

[0079] 3. Griess method for detecting the inhibitory effect of compounds on LPS-activated microglia. BV-2 microglia in the logarithmic growth phase were harvested and their density adjusted to 2.0 × 10⁶ cells / year using fresh DMEM medium containing 10% fetal bovine serum. 5 Cells were seeded at 100 μL / well in 96-well plates and cultured at 37 °C in a 5% CO2 incubator. After 24 h of adherent culture, the medium was replaced with serum-free fresh medium, and the cells were treated with the drugs simultaneously. Ten monomeric compounds were administered at doses of 100 μM, 30 μM, 10 μM, and 1 μM in combination with LPS. A blank control was also included. The final concentration of LPS in each treatment group was 100 ng / mL. After culturing for another 24 h following drug administration, the supernatant was collected, and the NO content in the supernatant was determined using the Griess colorimetric method. 2- content.

[0080] 4. Effect of MTT assay on microglial cell viability BV-2 microglia cultured in the logarithmic growth phase were used to adjust the cell density to 2.0 × 10⁶ cells / year using fresh DMDM ​​medium containing 10% fetal bovine serum. 4Cells were seeded at 100 μL / well in 96-well plates and cultured at 37 ℃ in a 5% CO2 incubator. After 24 h of adherent culture, the medium was replaced with fresh medium, and the drugs were added simultaneously. Ten monomeric compounds were administered at doses of 100 μM, 30 μM, 10 μM, and 1 μM in combination with LPS. A blank control was also included. In each treatment group, the final LPS concentration was 100 ng / mL. After cell addition, the cells were cultured for another 24 h, and then MTT solution (10 μL / well) was added to the cell culture medium. The cells were then co-incubated with 0.25 mg / mL MTT at 37 ℃ for 3 h. The culture medium was then removed, and 150 μL of DMSO solution was added. The optical density (OD) value was measured. Data processing was performed using the corresponding software of the microplate reader. The average OD value of three wells for each sample was calculated, and the cell viability (CV%) was calculated using the average value according to the following formula.

[0081] Cell viability % = [mean OD value of sample group / mean OD value of blank control group] × 100% 5. Statistical Methods All data were analyzed using SPSS (27.0) statistical software. Results are expressed as mean ± standard error. To assess overall differences, one-way ANOVA was used to analyze homogeneity of variance between groups, combined with Dunnett's test for inter-group comparisons. Levene's test was used to test homogeneity of variance in multiple samples. p >0.05, the variances are homogeneous, and Dunnett's two-tailed t-test is used to assess the difference in means among multiple groups. p <0.05 indicates unequal variances; therefore, the Dunnett T3 test is used to assess the difference in means among multiple groups.

[0082] 6. IC 50 Calculation method IC was calculated by fitting parameters such as dose and inhibition rate using nonlinear regression. 50 .

[0083] (3) Experimental results: The experimental results are shown in Table 4.

[0084] Table 4. Effects of compounds 1-10 on LPS-activated NO release (%) in BV-2 microglia (Mean ± SEM)

[0085] The results show that the new compound 1(1) prepared in Implementation Regulations 1-5 is... m M, 10 m M, 30 m M, 100 m M), 2(10 m M, 30 m M, 100 m M), 3(1 m M, 10 m M, 30 m M, 100 m M), 4(1 m M, 10 m M, 30 m M, 100 m M), 5(10) m M, 30 m M, 100 m M), 6(10 m M, 30 m M, 100 m M), 7(1 m M, 10 m M, 30 m M, 100 m M), 8(10) m M, 30 m M,100 m M), 9(1) m M, 10 m M, 30 m M, 100 m M), 10(1 m M, 10 m M, 30 m M, 100 m M) can significantly inhibit the release of NO from LPS-induced overactivated BV2 microglia in a concentration-dependent manner.

Claims

1. A class of phenylpropanoid compounds having the following chemical structural formula and their pharmaceutically acceptable salts, characterized in that, 。 2. The method for preparing the phenylpropanoid compound according to claim 1, characterized in that, Single garlic orchid ( Pleione bulbocodioides The pseudobulbs were extracted with ethanol or methanol solution, and the crude extract was recovered. The crude extract was dissolved in water and extracted with organic solvents to obtain extracts of different polarities. The extracts of different polarities were separated by ODS column chromatography with gradient elution using methanol-water or acetonitrile-water mixed solvent as the mobile phase. The methanol-water or acetonitrile-water eluents were further separated by HPLC with gradient elution using methanol and water mixed solvent, acetonitrile and water mixed solvent, n-hexane and anhydrous ethanol mixed solvent, or n-hexane and isopropanol mixed solvent as the mobile phase to obtain compounds 1-10.

3. The method according to claim 2, characterized in that, The extraction is performed 3 to 5 times with reflux of ethanol or methanol solution, with a material-to-liquid ratio of 1:8 to 1:20 g / mL, wherein the volume concentration of the ethanol solution is 70% to 95% and the volume concentration of the methanol solution is 70% to 90%.

4. The method according to claim 2, characterized in that, The organic solvent extraction is performed by sequentially extracting with petroleum ether, dichloromethane, chloroform, ethyl acetate, and n-butanol 3 to 5 times at a volume ratio of aqueous phase to organic phase of 1:1 to 1:4, and then recovering the organic solvents under reduced pressure.

5. The method according to claim 2, characterized in that: In the ODS column chromatography separation, the volume ratio of methanol to water is 1:9 to 9:1; the volume ratio of acetonitrile to water is 1:9 to 9:

1.

6. The method according to claim 2, characterized in that: In the HPLC separation, the volume ratio of methanol to water is 1:9 to 9:1; the volume ratio of acetonitrile to water is 2:8 to 8:2; the volume ratio of n-hexane to anhydrous ethanol is 10:90 to 99:1; and the volume ratio of n-hexane to isopropanol is 10:90 to 99:

1.

7. A pharmaceutical composition, characterized in that, It comprises the compound of claim 1, its pharmaceutically acceptable salt, and its pharmaceutically acceptable carrier.

8. The use of the compound of claim 1 and its pharmaceutically acceptable salt or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating neuroinflammatory diseases.

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