Extraction and separation method and application of anti-inflammatory active compound in leucophyllum angustifolium

Abstract

The present application relates to the technical field of medicine, in particular to an extraction and separation method and application of an anti-inflammatory active compound in Leucophyllum angustifolium. Seven compounds are separated and identified from Leucophyllum angustifolium by a specific extraction and separation method, the separated and identified compounds are classified in the first level, there are six benzyl alcohol compounds, which are phellodenol-A (1), chengzhong wintergreen (2), 2,4-dimethoxy cinnamic acid (4), alternaria alternata phenol (5), alternaria alternata phenol monomethyl ether (6) and alternaria alternata phenol-3,9-dimethyl ether (7); there is one alkaloid compound, which is new sea urchin spirit A (3). Among them, four compounds are separated from the plant of Leucophyllum for the first time, which are phellodenol-A (1), new sea urchin spirit A (3), 2,4-dimethoxy cinnamic acid (4) and alternaria alternata phenol-3,9-dimethyl ether (7).

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the extraction and separation methods and applications of anti-inflammatory active compounds from *Prunus cerasifera*. Background Technology

[0002] *Lysimachia paridiformis* var. *stenophylla* Franch., belonging to the genus *Lysimachia* of the family Primulaceae, is scientifically known as *Lysimachia paridiformis*. The entire plant can be used medicinally. It is mainly distributed in southwestern my country and is a commonly used medicine among ethnic minorities such as the Miao people in Guizhou. It has the effects of dispelling wind, promoting blood circulation, and relieving pain, and is often used to treat rheumatic pain, hemiplegia, limb contractures, infantile convulsions, and traumatic injuries.

[0003] However, current research on the chemical composition of *Ulva lactuca* (a type of medicinal herb) is not yet in-depth, especially regarding its chemical components. These chemical components are the material basis for the traditional efficacy of traditional Chinese medicine. Because traditional Chinese medicine is characterized by "multiple components, multiple targets, and synergistic effects through multiple pathways," basic research on it still faces many challenges. Therefore, to further explore the pharmacologically active substances in *Ulva lactuca*, further research on its chemical composition is needed. Simultaneously, in vitro and in vivo efficacy studies should be conducted in conjunction with metabolomics, network pharmacology, and other disciplines to further isolate various chemical components from *Ulva lactuca*, confirm their efficacy, and determine their application directions. Summary of the Invention

[0004] In order to solve the above-mentioned technical problems in the prior art, the present invention provides a method and application for the extraction and separation of anti-inflammatory active compounds from *Prunus cerasifera*.

[0005] A method for extracting and separating anti-inflammatory active compounds from *Prunus cerasifera*, comprising the following steps (when it is not necessary to obtain a certain compound, the corresponding steps can be omitted):

[0006] (1) Take the whole herb of the narrow-leaved pine tree coarse powder, and extract it by maceration with methanol and water at room temperature using different gradients. Combine the extracts from multiple extractions and concentrate them under reduced pressure until there is no alcohol taste to obtain the total extract of the pine tree. After dispersing the total extract with an appropriate amount of hot water, extract it sequentially with petroleum ether, ethyl acetate and n-butanol, and recover it under reduced pressure to obtain the petroleum ether fraction, ethyl acetate fraction and n-butanol fraction.

[0007] (2) The ethyl acetate fraction was decolorized by MCI column chromatography using a methanol-water system with gradient elution of 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. The resulting fractions were analyzed by TLC and combined to obtain 7 fractions, namely Fr.A to Fr.F.

[0008] (3) The Fr.A fraction was separated and purified by silica gel column chromatography using a dichloromethane-methanol system, with sequential elution using gradients of 100:1, 50:1, 30:1, 10:1, 5:1, and 1:1. The fractions were identified by TLC and separated into 6 components, namely Fr.A1 to Fr.A6. The obtained Fr.A1 to Fr.A6 fractions were then further eluted and separated.

[0009] (4) Fr.A2 was separated and purified by silica gel column chromatography using a dichloromethane-methanol system with sequential elution at a gradient of 50:1, 30:1, 10:1, 5:1, and 1:1. The fractions were identified by TLC and then combined into three fractions, namely Fr.A2-1 to Fr.A2-3. Fr.A2-1 was further separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate system with sequential elution at a gradient of 50:1, 30:1, 10:1, 5:1, and 1:1. The fractions were identified by TLC and then combined to obtain a crude product with a high concentration of compound 1. Compound 1 was then purified by recrystallization.

[0010] (5) Fr.A4 was subjected to silica gel column chromatography and eluted sequentially with a dichloromethane-methanol system at a gradient of 50:1, 30:1, 10:1, 5:1, 3:1, and 1:1 to obtain compound 2;

[0011] (6) The Fr.B fraction was separated and purified by silica gel column chromatography using a dichloromethane-methanol system, with sequential elution using gradients of 50:1, 30:1, 10:1, 5:1, 3:1, and 1:1. After TLC detection, the fractions were combined to obtain 7 components, namely Fr.B1 to Fr.B7. The obtained Fr.B1 to Fr.B7 fractions were further eluted and separated. Specifically, Fr.B1 was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system, with sequential elution using gradients of 50:1, 30:1, 20:1, 10:1, 5:1, and 3:1. After TLC detection, the fractions were combined to obtain 3 components, namely Fr.B1-1 to Fr.B1-3.

[0012] (7) Fr.B1-1 was subjected to silica gel column chromatography and eluted sequentially with a petroleum ether-ethyl acetate system at a gradient of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, and 1:1 to obtain compound 3;

[0013] (8) Fr.B1-2 was purified by Sephadex LH-20 gel column chromatography, eluted with dichloromethane-methanol at a ratio of 1:1, to obtain compound 4;

[0014] (9) The Fr.C fraction was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate system, and compound 5 was obtained by elution with gradients of 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1.

[0015] (10) The Fr.D fraction was subjected to silica gel column chromatography and eluted sequentially with a petroleum ether-ethyl acetate system at gradients of 50:1, 30:1, 15:1, 10:1, 5:1, and 3:1 to obtain compound 6;

[0016] (11) The Fr.E fraction (50g) was subjected to silica gel column chromatography and eluted sequentially with a petroleum ether-ethyl acetate system at gradients of 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1 to obtain compound 7.

[0017] Furthermore, the coarse powder is 60-80 mesh.

[0018] Furthermore, the extraction was carried out at room temperature using methanol-water immersion with different gradients. Specifically, 100%, 90%, and 70% methanol were added sequentially for two extractions each, each lasting 5 days.

[0019] Furthermore, the volume ratio of the methanol-water mixture to the whole plant of *Lysimachia nummularia* is 1:1.5.

[0020] This application describes the isolation and identification of seven compounds from the whole plant of *Phellodenol*: Phellodenol-A (compound 1), holmyl styracifolium (compound 2), neo-sea urchin-A (compound 3), 2,4-dimethoxycinnamic acid (compound 4), albiflorin (compound 5), albiflorin monomethyl ether (compound 6), and albiflorin-3,9-dimethyl ether (compound 7). Compounds 1, 3, 4, and 7 are isolated from the *Phellodenol* genus for the first time.

[0021] The structural formulas of the isolated compounds are as follows:

[0022]

[0023] Compounds 1-7 significantly inhibited nitric oxide production in LPS-induced inflammatory responses in RAW 264.7 cells, exhibiting excellent anti-inflammatory properties and thus suitable for the preparation of anti-inflammatory drugs. Furthermore, compounds 1-7 all possessed certain antitumor activity, showing good inhibitory effects against liver cancer, lung cancer, and ovarian cancer cells, and could be used in the preparation of anticancer drugs.

[0024] Compared with the prior art, the technical effects of this invention are reflected in:

[0025] (1) This application isolated and identified seven compounds from *Pteris vittata*. The compounds were classified into primary categories: six were phenylpropanoids, namely: phellodenol-A (1), holly sibirica (2), 2,4-dimethoxycinnamic acid (4), albiziol (5), albiziol monomethyl ether (6), and albiziol-3,9-dimethyl ether (7); and one was an alkaloid, namely, neo-sea urchin A (3). Four of these compounds were isolated from this genus for the first time: phellodenol-A (1), neo-sea urchin A (3), 2,4-dimethoxycinnamic acid (4), and albiziol-3,9-dimethyl ether (7). The compounds isolated and identified from *Pteris vittata* in this application not only enrich the variety of chemical constituents in the *Pteris* genus but also provide a scientific basis for subsequent research on the chemical constituents and other aspects of *Pteris vittata*.

[0026] (2) The compounds phellodenol-A (1), neo-sea urchin A (3), 2,4-dimethoxycinnamic acid (4), and 3,9-dimethyl ether (7) in this application were isolated from the narrow-leaved pine for the first time, providing new preparation methods and sources for the above compounds. While enriching the preparation routes of the above compounds, it also provides a scientific basis for subsequent research on the chemical composition and other aspects of the wind-chasing umbrella.

[0027] (3) Compounds 1 to 7 isolated from the narrow-leaved pine in this application all have certain antitumor and anti-inflammatory activities, and can be used to prepare anti-inflammatory or anticancer drugs. Attached Figure Description

[0028] Figure 1 For compound 1 1 H-NMR (400MHz, CD3OD) spectrum.

[0029] Figure 2 For compound 1 13 C-NMR (100MHz, CD3OD) spectrum.

[0030] Figure 3 For compound 2 1 H-NMR (600MHz, CD3OD) spectrum.

[0031] Figure 4 For compound 2 13 C-NMR (150MHz, CD3OD) spectrum.

[0032] Figure 5 For compound 3 1 H-NMR (400MHz, CD3OD) spectrum.

[0033] Figure 6 For compound 3 13 C-NMR (100MHz, CD3OD) spectrum.

[0034] Figure 7 For compound 4 1 H-NMR (400MHz, CD3OD) spectrum.

[0035] Figure 8 For compound 4 13 C-NMR (100MHz, CD3OD) spectrum.

[0036] Figure 9 For compound 5 1 H-NMR (600MHz, DMSO-d6) spectrum.

[0037] Figure 10 For compound 5 13 C-NMR (150MHz, DMSO-d6) spectrum.

[0038] Figure 11 For compound 6 1 H-NMR (400MHz, DMSO-d6) spectrum.

[0039] Figure 12 For compound 6 13 C-NMR (100MHz, DMSO-d6) spectrum.

[0040] Figure 13 For compound 7 1 H-NMR (600MHz, CDCl3) spectrum.

[0041] Figure 14 For compound 7 13 C-NMR (150MHz, CDCl3) spectrum.

[0042] Figure 15 The graph shows the significant effects of compounds 1, 3-5, and 7 on NO release.

[0043] Figure 16 This is a significant analytical plot of compound 2 for NO release.

[0044] Figure 17 This is a significant analytical plot of compound 6 for NO release. Detailed Implementation

[0045] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0046] Example 1

[0047] Research on the chemical composition of the Wind Chaser Umbrella

[0048] 1. Instruments and Materials

[0049] 1.1 Source of medicinal materials

[0050] The medicinal material was purchased from Guanxing Town, Yuqing County, Guizhou Province, and identified by Associate Professor Wu Zhikun of Guizhou University of Traditional Chinese Medicine as the whole herb of *Lysimachia paridiformis* var. *stenophylla* Franch., belonging to the genus *Lysimachia* of the family Primulaceae. The sample was naturally air-dried, then pulverized for later use. The original plant sample is currently stored in the Key Laboratory of Traditional Chinese and Ethnic Medicines at Guizhou University of Traditional Chinese Medicine.

[0051] 1.2 Instruments

[0052] The instruments used in this experiment are detailed in Table 1-1.

[0053] Table 1-1 Experimental Instruments

[0054]

[0055] 1.3 Reagents and Fillers

[0056] The reagents and packing materials used in this experiment are detailed in Table 1-2.

[0057] Table 1-2 Experimental Reagents and Fillers

[0058]

[0059]

[0060] Note: The colorimetric reagents used in this experiment are: 10% sulfuric acid ethanol solution and elemental iodine.

[0061] 2 Experimental Methods and Results

[0062] 2.1 Extraction and Separation of *Umbrella Chaser* Herbs

[0063] 35.58 kg of coarse powder (60-80 mesh) of the whole plant of *Lysimachia nummularia* was extracted at room temperature using 100%, 90%, and 70% methanol-water solvents, respectively. Each solvent was used twice, for 5 days each time (the volume ratio of methanol-water to whole plant of *Lysimachia nummularia* was 1:1.5). The extracts were combined and concentrated under reduced pressure until no alcohol odor was detected to obtain the total extract of *Lysimachia nummularia* (6.96 kg, yield 19.56%). The total extract was dispersed in an appropriate amount of hot water and then extracted sequentially with equal volumes of petroleum ether, ethyl acetate, and n-butanol. The petroleum ether fraction (993 g), ethyl acetate fraction (328 g), and n-butanol fraction (4.04 kg) were recovered under reduced pressure.

[0064] The ethyl acetate fraction was decolorized by MCI column chromatography using a methanol-water gradient elution system (50:50, 60:40, 70:30, 80:20, 90:10, 100:0). The resulting fractions were analyzed by TLC and then combined to obtain seven fractions (Fr.A to Fr.F).

[0065] The Fr.A fraction (71g) was separated and purified by silica gel column chromatography using a gradient elution system of dichloromethane-methanol (100:1, 50:1, 30:1, 10:1, 5:1, 1:1). The fractions were identified by TLC and separated into 6 fractions (Fr.A1 to Fr.A6).

[0066] Fr.A2 was separated and purified by silica gel column chromatography using a gradient elution with a dichloromethane-methanol system (50:1, 30:1, 10:1, 5:1, 1:1). TLC analysis revealed that the fractions were combined and separated into three fractions (Fr.A2-1 to Fr.A2-3). Fr.A2-1 was further purified by silica gel column chromatography using a gradient elution with a petroleum ether-ethyl acetate system (50:1, 30:1, 10:1, 5:1, 1:1). TLC analysis revealed that the fractions were combined to obtain a crude product containing a high concentration of compound 1. Compound 1 was then purified by recrystallization.

[0067] Fr.A4 was subjected to silica gel column chromatography and gradient elution with dichloromethane-methanol systems (50:1, 30:1, 10:1, 5:1, 3:1, 1:1) to yield compound 2.

[0068] Fraction B (25g) was separated and purified by silica gel column chromatography using a gradient elution of a dichloromethane-methanol system (50:1, 30:1, 10:1, 5:1, 3:1, 1:1). The fractions were identified by TLC and combined to yield seven fractions (Fr.B1–Fr.B7). Fr.B1 was further purified by silica gel column chromatography using a gradient elution of a petroleum ether-ethyl acetate system (50:1, 30:1, 20:1, 10:1, 5:1, 3:1). The resulting fractions were identified by TLC and combined to yield three fractions (Fr.B1-1–Fr.B1-3). Fr.B1-1 was further purified by silica gel column chromatography using a gradient elution of a petroleum ether-ethyl acetate system (30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1) to yield compound 3.

[0069] Fr.B1-2 was purified by Sephadex LH-20 gel column chromatography, eluted with dichloromethane-methanol (1:1), to give compound 4.

[0070] The Fr.C fraction (11.3 g) was separated and purified by silica gel column chromatography, and compound 5 was obtained by gradient elution with petroleum ether-ethyl acetate system (30:1, 20:1, 10:1, 5:1, 3:1, 1:1).

[0071] Fr.D fraction (13.5 g) was subjected to silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate systems (50:1, 30:1, 15:1, 10:1, 5:1, 3:1) to give compound 6.

[0072] Fr.E fraction (50 g) was subjected to silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate systems (30:1, 20:1, 10:1, 5:1, 3:1, 1:1) to give compound 7.

[0073] 2.2 Identification of Compound Structures

[0074] After separation and purification, the compounds were analyzed by physicochemical properties and methods such as nuclear magnetic resonance (NMR) and single-crystal diffraction (X-Ray), and their structures were identified with the help of micro-spectral databases.

[0075] 1. Structural identification of compound 1

[0076] Compound 1 is a colorless needle-like crystal (methanol). It is readily soluble in DMSO and methanol, slightly soluble in ethyl acetate and dichloromethane, and insoluble in petroleum ether. TLC detection: using dichloromethane-methanol (20:1) as the developing solvent, it exhibits fluorescence at UV wavelengths of 254 and 365 nm after development; after spraying with 10% sulfuric acid ethanol solution and heating at 105℃, no spots appear; after iodine fumes, a single yellow spot (R) appears. f The value is 0.53), and the molecular formula is C. 11 H 10 O4. According to1 H-NMR (400MHz, CD3OD) indicates that δ H 7.36 (1H, s, H-5) and 6.71 (1H, s, H-8) show hydrogen atoms on the benzene ring, δ H 7.83 (1H, d, J = 9.4Hz, H-4) and 6.17 (1H, d, J = 9.4Hz, H-3) indicate the presence of one pair of double bonds. According to... 13 C-NMR (100MHz, CD3OD) data showed that the compound contains 11 carbon atoms. C 131.2 (C-5), 125.3 (C-6), 161.3 (C-7), 102.9 (C-8), 155.8 (C-9), and 112.2 (C-10) indicate carbon signals on the benzene ring, δ C 113.0 (C-3) and 146.2 (C-4) show a pair of double bonds, δ C The presence of a carbonyl group at 163.9 (C-2) suggests that this compound is a phenylpropanoid. Based on the above data, structural analysis and verification using single-crystal diffraction techniques confirmed that the compound is Phellodenol-A. 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0077]

[0078] Datawere recorded in CD3OD on aBrukerAV-400MHz spectrometer.

[0079] The structural formula of compound 1 is shown below:

[0080]

[0081] The crystal structure of compound 1 is shown below:

[0082]

[0083] Crystal data for compound 1: Pn (No. 7), M = 206.19, monoclinic. α=90.00°, β=96.556(3)°, γ=90°, T = 273(2)K, Z = 2, R gt (F) = 0.0512, wR ref (F 2 = 0.1249.

[0084] 2. Structural identification of compound 2

[0085] Compound 2 is a colorless needle-like crystal (methanol). It is readily soluble in methanol, slightly soluble in ethyl acetate, and insoluble in dichloromethane and petroleum ether. TLC detection: using dichloromethane-methanol (10:1) as the developing solvent, it exhibits fluorescence at 254 nm UV light but not at 365 nm; after spraying with 10% sulfuric acid ethanol solution and heating at 105 °C, it shows a single purple spot (R). f The value is 0.51), and the molecular formula is C. 22 H 28 O9. According to 1 H-NMR (600MHz, CD3OD) indicates that δ H 6.76 (4H, s, H-2, 2', 6, 6') shows hydrogen on the benzene ring, δ H 3.88 (12H, s, 3, 3', 5, 5'-OCH3) indicates a methoxy group, and the intensity and number of integrals of its two hydrogen signals suggest a symmetrical structure. Based on... 13 C-NMR (150 MHz, CD3OD) data showed that the compound contains 22 carbon atoms. C 146.3 (C-3, 3', 5, 5'), 136.2 (C-4, 4'), 134.2 (C-1, 1'), and 104.8 (C-2, 2', 6, 6') indicate carbon signals on the benzene ring, δ C The presence of four methoxy groups at 56.8 (3,3',5,5'-OCH3) suggests that this compound is a phenylpropanoid. Based on the data and consistent with literature reports, this compound is identified as Ilex cornuta extract. 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0086]

[0087]

[0088] Datawere recorded in CD3OD on aBrukerAV-600MHz spectrometer.

[0089] The structural formula of compound 2 is shown below:

[0090]

[0091] 3. Structural identification of compound 3

[0092] Compound 3: Colorless needle-like crystals (methanol). Easily soluble in hot methanol, slightly soluble in ethyl acetate, insoluble in petroleum ether. TLC detection: Using dichloromethane-methanol (15:1) as the developing solvent, it fluoresces at 254 nm UV light after development, but not at 365 nm; after spraying with 10% sulfuric acid ethanol solution and heating at 105 °C, it shows a single yellow spot (R). f The value is 0.61), and the molecular formula is C. 19 H 21 N3O2. According to 1 H-NMR (400MHz, CD3OD) indicates that δ H 7.44 (1H, dt, J = 8.1, 1.0 Hz, H-7), 7.24–7.28 (1H, m, H-4), 7.14 (1H, ddd, J = 8.1, 7.0, 1.4 Hz, H-6), and 7.08 (1H, td, J = 7.5, 7.1, 1.4 Hz, H-5) show hydrogen atoms on the benzene ring, δ H 7.22 (1H, s, H-8), 6.12 (1H, dd, J = 17.3, 10.7 Hz, H-17), 5.12–5.14 (1H, m, H-18a), and 5.10 (1H, dd, J = 9.8, 1.1 Hz, H-18b) show two sets of double bonds, δ H 4.23 (1H, q, J = 7.0 Hz, H⁻¹²) shows the hydrogen signal on the nitrogen-carbon junction, δ H 1.56 (3H, s, H-19), 1.55 (3H, s, H-20), and 1.54 (3H, d, J = 7.0 Hz, H-15) show three methyl groups. According to... 13 C-NMR (100MHz, CD3OD) data showed that the compound contains 19 carbon atoms. C 127.3 (C-3a), 119.9 (C-4), 121.2 (C-5), 122.6 (C-6), 112.7 (C-7), and 136.8 (C-7a) indicate carbon signals on the benzene ring, δ C The presence of two carbonyl groups at 162.3 (C-10) and 168.7 (C-13) suggests that this compound is an alkaloid. Based on the data and its consistency with literature reports, this compound is identified as Neo-Sea Urchin A. 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0093]

[0094] Datawere recorded in CD3OD on aBrukerAV-400MHz spectrometer.

[0095] The structural formula of compound 3 is shown below:

[0096]

[0097] 4. Structural identification of compound 4

[0098] Compound 4 is a colorless needle-like crystal (methanol). It is readily soluble in methanol, slightly soluble in chloroform and ethyl acetate, and insoluble in petroleum ether. TLC detection: Using dichloromethane-methanol (10:1) as the developing solvent, it exhibits fluorescence at both 254 and 365 nm wavelengths after development; after spraying with 10% sulfuric acid ethanol solution and heating at 105 °C, it shows a single cinnamon-colored spot (R). f The value is 0.49), and the molecular formula is C. 11 H 12 O4. According to 1 H-NMR (400MHz, CD3OD) indicates that δ H 7.61 (1H, d, J = 8.5 Hz, H-6), 6.50 (1H, d, J = 2.4 Hz, H-3), and 6.46 (1H, dd, J = 8.5, 2.4 Hz, H-5) show hydrogen atoms on the benzene ring, δ H 7.06 (1H, d, J = 12.6 Hz, H-7) and 5.80 (1H, d, J = 12.6 Hz, H-8) show a pair of double bonds, δ H 3.81 (1H, s, 2-OCH3) and 3.80 (1H, s, 4-OCH3) show two methoxy groups. According to... 13 C-NMR (100MHz, CD3OD) data showed that the compound contains 11 carbon atoms. C 163.6 (C-4), 160.3 (C-2), 132.9 (C-6), 118.5 (C-5), 105.5 (C-5), and 98.6 (C-3) indicate carbon signals on the benzene ring, δ C The presence of a carboxyl group at 170.6(-COOH) suggests that this compound is a phenylpropanoid. Based on the above data, structural analysis and verification using single-crystal diffraction techniques confirmed that the compound is 2,4-dimethoxycinnamic acid. 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0099]

[0100] Datawere recorded in CD3OD on aBrukerAV-600MHz spectrometer.

[0101] The structural formula of compound 4 is shown below:

[0102]

[0103] The crystal structure of compound 4 is shown below:

[0104]

[0105] Crystal data for compound 4: P-1 (No. 2), M = 208.21, triclinic. α=79.855(2)°, β=86.800(2)°, γ=80.295(2)°, T = 273(2)K, Z = 2, R gt (F) = 0.0483, wR ref (F 2 = 0.1204.

[0106] 5. Structural identification of compound 5

[0107] Compound 5 is a yellow powder. It is readily soluble in DMSO, sparingly soluble in methanol, and insoluble in ethyl acetate, dichloromethane, and petroleum ether. TLC detection: Using dichloromethane-methanol (10:1) as the developing solvent, it exhibits fluorescence at both 254 and 365 nm UV wavelengths after development; after spraying with 10% sulfuric acid ethanol solution and heating at 105 °C, it shows a single cinnamon-colored spot (R). f The value is 0.53), and the molecular formula is C. 14 H 10 O5. According to 1 H-NMR (600MHz, DMSO-d6) indicates that δ H 7.23 (1H, d, J = 2.1 Hz, H⁻¹⁰), 6.71 (1H, d, J = 2.6 Hz, H⁻²), 6.63 (1H, d, J = 2.5 Hz, H⁻⁴), and 6.36 (1H, d, J = 2.0 Hz, H⁻⁸) show hydrogen atoms on the benzene ring, δ H 2.69 (3H, s, -CH3) shows a methyl group. According to... 13 C-NMR (150 MHz, DMSO-d6) data showed that the compound contains 14 carbon atoms. C138.3 (C-1), 117.5 (C-2), 158.4 (C-3), 101.6 (C-4), 152.6 (C-4a), 97.4 (C-6a), 164.1 (C-7), 100.9 (C-8), 165.4 (C-9), 104.3 (C-10), 138.1 (C-10a), and 109.0 (C-10b) indicate carbon signals on the benzene ring, δ C 164.7 (C-6) shows one carbonyl group, δ C The presence of one methyl group at 25.2(-CH3) suggests that this compound is a phenylpropanoid. Based on the data and its consistency with literature reports, this compound is identified as aleuropein. 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0108]

[0109] Datawere recorded inDMSO-d6on aBrukerAV-600MHz spectrometer.

[0110] The structural formula of compound 5 is shown below:

[0111]

[0112] 6. Structural identification of compound 6

[0113] Compound 6 is a white powder. It is readily soluble in DMSO, soluble in dichloromethane-methanol (1:1), sparingly soluble in methanol, and insoluble in ethyl acetate, dichloromethane, and petroleum ether. TLC detection: Using dichloromethane-methanol (30:1) as the developing solvent, it exhibits fluorescence at UV wavelengths of 254 and 365 nm after development; after spraying with 10% sulfuric acid ethanol solution and heating at 105°C, it shows a single cinnamon-colored spot (R). f The value is 0.41), and the molecular formula is C. 15 H 12 O5. According to 1 H-NMR (400MHz, DMSO-d6) indicates that δ H 7.25 (1H, s, H-10), 6.85 (2H, s, H-2, 4), and 6.38 (1H, s, H-8) indicate hydrogen atoms on the benzene ring, δ H 3.83 (3H, s, -OCH3) shows a methoxy group, and δH 2.72 (3H, s, -CH3) shows a methyl group. According to 13C-NMR (100MHz, DMSO-d6) data show that this compound contains 15 carbon atoms. δC 138.2 (C-1), 116.6 (C-2), 159.7 (C-3), 101.2 (C-4), 152.6 (C-4a), 97.6 (C-6a), 164.1 (C-7), 99.9 (C-8), 165.4 (C-9), 104.6 (C-10), 137.8 (C-10a), and 110.2 (C-10b) indicate carbon signals on the benzene ring. δC 164.6 (C-6) indicates a carbonyl group, δC 55.6 (-OCH3) indicates a methoxy group, and δC 25.2 (-CH3) indicates a methyl group. Based on these findings, it can be preliminarily inferred that this compound is a phenylpropanoid compound. Based on the above data, which is largely consistent with literature reports, the compound was identified as Alternaria alternifolia monomethyl ether. 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0114]

[0115] Datawere recorded inDMSO-d6on aBrukerAV-400MHz spectrometer.

[0116] The structural formula of compound 6 is shown below:

[0117]

[0118] 7. Structural identification of compound 7

[0119] Compound 7 is a white powder. It is readily soluble in dichloromethane and slightly soluble in methanol and petroleum ether. TLC detection: Using petroleum ether-ethyl acetate (5:1) as the developing solvent, it exhibits fluorescence at both 254 and 365 nm UV wavelengths after development; after spraying with 10% sulfuric acid ethanol solution and heating at 105 °C, it shows a single orange spot (R). f The value is 0.47), and the molecular formula is C. 16 H 14 O5. According to 1 H-NMR (600MHz, CDCl3) indicates that δ H 7.29 (1H, d, J = 6.7 Hz, H-10), 6.73 (2H, d, J = 4.4 Hz, H-2, 4), and 6.53 (1H, d, J = 2.5 Hz, H-8) show hydrogen atoms on the benzene ring, δ H 3.91 (3H, s, 9-OCH3) and 3.85 (3H, s, 3-OCH3) show two methoxy groups, δ H2.79 (3H, s, -CH3) shows one methyl group. According to... 13 C-NMR (150 MHz, CDCl3) data showed that the compound contains 15 carbon atoms. C 138.3 (C-1), 117.0 (C-2), 160.1 (C-3), 100.1 (C-4), 153.3 (C-4a), 99.5 (C-6a), 165.5 (C-7), 99.1 (C-8), 166.6 (C-9), 104.7 (C-10), 138.1 (C-10a), and 111.0 (C-10b) indicate carbon signals on the benzene ring, δ C 165.3 (C-6) shows one carbonyl group, δ C 55.7 (3-OCH3) and 55.8 (9-OCH3) show two methoxy groups, δ C The presence of a methyl group at 25.9(-CH3) suggests that this compound is a phenylpropanoid. Based on the data and its consistency with literature reports, this compound is identified as Alternaria alterniflora-3,9-dimethyl ether. 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0120]

[0121] Datawere recorded in CDCl3 on aBrukerAV-600MHz spectrometer.

[0122] The structural formula of compound 7 is shown below:

[0123]

[0124] 2.3 Partial physicochemical constants and spectral data of the compound

[0125] Phellodenol-A(1): Colorless needle-like crystals (methanol). Developed with dichloromethane-methanol (20:1), it shows a single yellow spot after iodine fuming (R). f The value is 0.53. 1 H-NMR (400MHz, CD3OD) δ7.83 (1H, d, J = 9.4Hz, H-4), 7.36 (1H, s, H-5), 6.71 (1H, s, H-8) , 6.17 (1H, d, J = 9.4Hz, H-3), 3.78 (2H, t, J = 6.8Hz, H-2'), 2.87 (2H, t, J = 6.8Hz, H-1'); 13C-NMR (100MHz, CD3OD) δ163.9(C-2), 113.0(C-3), 146.2(C-4), 131.2(C-5), 125.3(C -6), 161.3(C-7), 102.9(C-8), 155.8(C-9), 112.2(C-10), 34.3(C-1'), 62.4(C-2'). Crystal data: Pn(No.7), M=206.19, monoclinic, α=90.00°, β=96.556(3)°, γ=90°, T = 273(2)K, Z = 2, R gt (F) = 0.0512, wR ref (F 2 = 0.1249.

[0126] Huazhong holly extract (2): White needle-like crystals (methanol). Developed in dichloromethane-methanol (10:1), it shows a single purple spot (R) in 10% sulfuric acid ethanol solution. f The value is 0.51. 1 H-NMR (600MHz, CD3OD) δ6.76 (4H, s, H-2, 2', 6, 6'), 4.89 (2H, d, J = 8.2Hz, H-7, 7'), 3.88 (12H, s, 2.0Hz, 3, 3', 5, 5'-OCH3), 3.73 (2H, dd, J=11.2, 3.3Hz, H-9'), 3.65 (2H, dd, J=11.2, 4.6Hz, H-9), 2.34 (2H, q, J=4.3Hz, H-8, 8'); 13 C-NMR (150MHz, CD3OD) δ134.2(C-1), 104.8(C-2), 149.3(C-3), 136.2(C-4), 149. 3(C-5), 104.8(C-6), 84.6(C-7), 55.2(C-8), 61.7(C-9), 134.2(C-1'), 104.8(C-2 '), 149.3(C-3'), 136.2(C-4'), 149.3(C-5'), 104.8(C-6'), 84.6(C-7'), 56.8(C -8')61.7(C-9'), 56.8(3-OCH3), 56.8(5-OCH3), 56.8(3'-OCH3), 56.8(5'-OCH3).

[0127] New Sea Urchin Extract A(3): Colorless needle-like crystals (methanol). Developed in dichloromethane-methanol (15:1), it shows a single yellow spot (R) in 10% sulfuric acid ethanol solution. f The value is 0.61. 1 H-NMR (400MHz, CD3OD) δ7.44 (1H, dt, J=8.1, 1.0Hz, H-7), 7.24~7.28 (1H, m, H-4), 7.22 (1H, s , H-8), 7.14 (1H, ddd, J=8.1, 7.0, 1.4Hz, H-6), 7.08 (1H, td, J=7.5, 7.1, 1.4Hz, H-5), 6.12 (1H , dd, J=17.3, 10.7Hz, H-17), 5.12~5.14 (1H, m, H-18a), 5.10 (1H, dd, J=9.8, 1.1Hz, H-18b), 4. 23 (1H, q, J=7.0Hz, H-12), 1.56 (3H, s, H-19), 1.55 (3H, s, H-20), 1.54 (3H, d, J=7.0Hz, H-15); 13 C-NMR (100MHz, CD3OD) δ146.0(C-2), 104.3(C-3), 127.3(C-3a), 119.9(C-4), 121.2(C-5), 122.6(C-6), 112.7(C-7), 136.8(C-7a), 114.3(C -8), 124.7(C-9), 162.3(C-10), 52.6(C-12), 168.7(C-13), 20.7(C-15 ), 40.5(C-16), 146.2(C-17), 112.6(C-18), 28.1(C-19), 28.2(C-20).

[0128] 2,4-Dimethoxycinnamic acid (4): Colorless needle-like crystals (methanol). Developed in dichloromethane-methanol (10:1), it shows a single cinnamon-colored spot in 10% sulfuric acid-ethanol solution (R). f The value is 0.49. 1 H-NMR (400MHz, CD3OD) δ7.61 (1H, d, J = 8.5Hz, H-6), 7.06 (1H, d, J = 12.6Hz, H-7), 6.50 (1H, d, J = 2.4Hz, H-3) , 6.46 (1H, dd, J=8.5, 2.4Hz, H-5), 5.80 (1H, d, J=12.6Hz, H-8), 3.81 (1H, s, 2-OCH3), 3.80 (1H, s, 4-OCH3); 13C-NMR (100MHz, CD3OD) δ118.5(C-1), 160.3(C-2), 98.6(C-3), 163.6(C-4), 105.5(C-5) , 132.9(C-6), 139.1(C-7), 118.0(C-8), 170.6(-COOH), 55.9(2-OCH3), 55.8(4-OCH3). Crystal data: P-1(No.2), M=208.21, triclinic, α=79.855(2)°, β=86.800(2)°, γ=80.295(2)°, T = 273(2)K, Z = 2, R gt (F) = 0.0483, wR ref (F 2 = 0.1204.

[0129] Alternaria (5): Yellow powder. Developed in dichloromethane-methanol (10:1), it shows a single cinnamon-colored spot in 10% sulfuric acid-ethanol solution (R). f The value is 0.53. 1 H-NMR (600MHz, DMSO-d6) δ 11.75 (1H, s, 7-OH), 10.88 (1H, s, 9-OH), 10.32 (1H, s, 3-OH), 7.23 (1H, d, J = 2.1Hz, H-10), 6.71 (1H, d, J=2.6Hz, H-2), 6.63 (1H, d, J=2.5Hz, H-4), 6.36 (1H, d, J=2.0Hz, H-8), 2.69 (3H, s, -CH3); 13 C-NMR (150MHz, DMSO-d6) δ138.3(C-1), 117.5(C-2), 158.4(C-3), 101.6(C-4), 152.6(C-4a), 164.7(C-6), 97 .4(C-6a), 164.1(C-7), 100.9(C-8), 165.4(C-9), 104.3(C-10), 138.1(C-10a), 109.0(C-10b), 25.2(-CH3).

[0130] Alternaria monomethyl ether (6): white powder. Developed in dichloromethane-methanol (30:1), it shows a single cinnamon-colored spot in 10% sulfuric acid-ethanol solution (R). f The value is 0.41. 1H-NMR (400MHz, DMSO-d6) δ11.72(1H,s,7-OH), 10.94(1H,s,3-OH), 7.25(1H,s,H- 10), 6.85(2H,s,H-2,4), 6.38(1H,s,H-8), 3.83(3H,s,-OCH3), 2.72(3H,s,-CH3); 13 C-NMR (100MHz, DMSO-d6) δ138.2(C-1), 116.6(C-2), 159.7(C-3), 101.2(C-4), 152.6(C-4a), 164.6(C-6), 97.6(C- 6a), 164.1(C-7), 99.9(C-8), 165.4(C-9), 104.6(C-10), 137.8(C-10a), 110.2(C-10b), 55.6(-OCH3), 25.2(-CH3).

[0131] Alternaria-3,9-dimethyl ether (7): white powder. Developed in petroleum ether-ethyl acetate (5:1), it shows a single orange spot in 10% sulfuric acid ethanol solution (R). f The value is 0.47. 1 H-NMR (600MHz, CDCl3) δ11.94 (1H, s, 7-OH), 7.29 (1H, d, J = 6.7Hz, H-10), 6.73 (2H, d, J = 4.4Hz, H -2, 4), 6.53 (1H, d, J = 2.5Hz, H-8), 3.91 (3H, s, 9-OCH3), 3.85 (3H, s, 3-OCH3), 2.79 (3H, s, -CH3); 13 C-NMR (150MHz, CDCl3) δ 138.3 (C-1), 117.0 (C-2), 160.1 (C-3), 100.1 (C-4), 153.3 (C-4a), 165.3 (C-6), 99.5 (C-6a), 165. 5(C-7), 99.1(C-8), 166.6(C-9), 104.7(C-10), 138.1(C-10a), 111.0(C-10b), 55.7(3-OCH3), 55.8(9-OCH3), 25.9(-CH3).

[0132] Example 2

[0133] Anti-inflammatory activity experiment of the compound

[0134] The in vitro anti-inflammatory activity of compounds phellodenol-A (1), Huazhong wintergreen (2), new sea urchin A (3), 2,4-dimethoxycinnamic acid (4), albiziol (5), albiziol monomethyl ether (6), and albiziol-3,9-dimethyl ether (7) on LPS-induced RAW264.7 cells was studied.

[0135] 1. Experimental Principle

[0136] (1) The full English name of CCK-8 is Cell Counting Kit-8. The main chemical component is WST-8, chemically named 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt. Its core group is the same as that of MTT, making it an upgraded version of MTT. In the presence of electron coupling reagents (i.e., when the cells are alive, respiring, and metabolizing energy), it can be oxidized and reduced by NAD+ to form a water-soluble yellow formazan product. The more living cells there are, the more formazan is produced, and the darker the color will be.

[0137] (2) NO is readily oxidized to NO in vivo or in aqueous solutions and other environments. 2- Under acidic conditions, NO can undergo a series of reactions to produce a pink special substance. This special substance has a maximum absorption peak at 540 nm, and the NO concentration can be estimated by the OD value.

[0138] 2. Experimental Procedure

[0139] (1) Cell activity assay of compounds 1-7

[0140] Logarithmically growing RAW264.7 cells were digested with trypsin containing 0.25% EDTA (1×) until the cells were in suspension. The trypsin concentration was then diluted with complete culture medium to stop the digestion. After centrifugation and resuspending, cells were prepared at a density of 1×10⁻⁶ cells / cells using complete culture medium. 5Single-cell suspensions of [number] cells / mL were seeded at 100 μL / well in 96-well plates. 100 μL of PBS buffer was added to the outermost cell culture wells to minimize edge effects. The plates were incubated at 37°C in a 5% CO2 incubator for 20 h. After incubation, 100 μL of culture medium containing different concentrations of the aforementioned compounds was added. The compound concentrations were divided into two groups: Group 1: Compounds 1, 3–7 were prepared at concentrations of 6.25, 12.5, 25, and 50 μM; Group 2: Compound 2 was prepared at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μM. Each concentration was tested in triplicate. Three wells were left untreated as the control group, and no drugs or cells were added as the blank group. After culturing for 20 hours, discard the cell supernatant, add 10 μL of CCK-8 working solution, and continue culturing in an incubator for another 2 hours. Then, measure the absorbance at 450 nm using a microplate reader. Relative cell viability = [OD experiment - OD blank] / [OD normal - OD blank] × 100%.

[0141] (2) Detection of anti-inflammatory activity of compounds 1-7

[0142] RAW264.7 cells in the logarithmic growth phase were used to prepare a culture medium at a density of 1×10⁻⁶ cells. 5 Single-cell suspension (cells / mL) was seeded at 100 μL / well in 96-well plates. 100 μL of PBS buffer was added to the outermost cell culture wells to minimize edge effects. The plates were incubated statically at 37°C with 5% CO2 for 20 h. After incubation, 100 μL of culture medium containing the aforementioned compounds was added. Compounds 1 and 3–7 were added at a concentration of 50 μM, and compound 2 was added at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μM, respectively. Each compound concentration was administered in triplicate. Three wells were left untreated as the control group, and wells without either the drug or cells were left as the blank group. After incubation at 37°C with the added drugs and static incubation at 5% CO2 for 1 h, 1 μg / mL of LPS was added to stimulate inflammation. Finally, the plates were incubated at 37°C with 5% CO2 for 20 h.

[0143] Remove Griess Reagent I and II and allow them to return to room temperature. Prepare a standard curve according to the reagent instructions. Add 50 μL of the cultured cell supernatant to a new 96-well plate along with the standard and sample, again at 50 μL per well. Add Griess Reagent I to each well first, followed by Griess Reagent II. Incubate at 37°C in a 5% CO2 incubator for 15 min. Finally, measure the absorbance at 540 nm using a microplate reader and calculate the concentration of nitric oxide (NO) in the sample based on the standard curve.

[0144] 3. Experimental Results

[0145] (1) The relative viability of RAW 264.7 cells after 20 h of administration of compounds 1, 3-7 at concentrations of 6.25, 12.5, 25, and 50 μM, respectively, is shown in Table 1; the relative viability of RAW 264.7 cells after administration of compound 2 at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μM, respectively, is shown in Table 2. The results in Tables 1 and 2 indicate that compounds 1-7 did not have significant toxic effects on cells at the above concentrations.

[0146] Table 1. Relative viability of compounds 1, 3-7 against RAW 264.7 cells.

[0147]

[0148] Table 2. Relative viability of compound 2 against RAW 264.7 cells.

[0149]

[0150] (2) Figures 15-17 As shown, LPS stimulation of RAW264.7 cells resulted in a highly significant increase in NO release compared to the control group without LPS stimulation. Figure 15 It was found that the NO release level in the LPS group without the addition of compounds 1, 3, 4, 5, and 7 after LPS stimulation reached 14.10 μM. Compared with the LPS group, the experimental groups that continued culturing with compounds 1, 3, 4, 5, and 7 showed that all compounds except compound 3 inhibited NO release. Specifically, when 50 μM of compounds 1, 4, 5, and 7 were added, the NO release levels in the cells decreased to 12.75, 13.66, 12.53, and 13.73 μM, respectively. Figure 16 It was found that the NO release level in the LPS group without compound 2 after LPS stimulation reached 7.11 μM. Compared with the LPS group, the NO production in the experimental group with compound 2 added and continued culture decreased with increasing concentration of compound 2, showing a certain concentration-dependent effect. Specifically, after adding 200, 100, 50, 25, 12.5, and 6.25 μM of compound 2, the NO release level in the cells decreased to 4.93, 6.03, 6.57, 7.31, 7.19, and 7.55 μM, respectively. When the concentration of compound 2 reached 200 μM, its ability to inhibit NO release was more significant. Figure 17It was found that the NO release in the LPS group without compound 6 after LPS stimulation reached 16.52 μM. Compared with the LPS group, the NO production in the experimental group with compound 6 added and continued culture decreased with increasing concentration of compound 6, showing a certain concentration-dependent activity. Specifically, when 50, 25, 12.5, and 6.25 μM of compound 6 were added, the NO release in the cells decreased to 12.70, 16.03, 16.38, and 17.31 μM, respectively. When the concentration of compound 6 reached 50 μM, its ability to inhibit NO release was more significant.

[0151] The above results indicate that compounds 1, 2, 4-7 isolated from *Lysimachia nummularia* all inhibited the production of nitric oxide in LPS-induced inflammatory responses in RAW 264.7 cells, reflecting certain anti-inflammatory properties.

[0152] III. Experiments on the antitumor activity of the compounds

[0153] Compounds phellodenol-A (1), Huazhong wintergreen (2), and new sea urchin A were tested.

[0154] (3), 2,4-dimethoxycinnamic acid (4), co-sporin (5), co-sporin monomethyl ether (6), and co-sporin-3,9-dimethyl ether (7) were used to screen colorectal cancer (SW480, HCT116), liver cancer (HepG2), lung cancer (A549), and ovarian cancer (SK-OV-3) cells for cytotoxic activity, and the present invention will be further illustrated by the following implementation.

[0155] 1. Experimental Principle

[0156] The basic principle of the CCK-8 method is the same as above.

[0157] 2. Experimental Procedure

[0158] Logarithmic growth phase cells were harvested and digested with trypsin containing 0.25% EDTA (1×) until the cells were in suspension. The trypsin concentration was then diluted with serum-containing culture medium to terminate the digestion. Cells were prepared at a density of 2×10⁶ cells / year using complete culture medium. 4Single-cell suspension (cells / mL) was seeded at 100 μL / well in 96-well plates. 100 μL of PBS buffer was added to the outermost cell culture wells to minimize edge effects. Cells were incubated at 37°C with 5% CO2 for 24 hours to observe cell adhesion. Then, 100 μL of culture medium containing the test compound was added. The compound concentration range was set as follows: 50, 20, 10, 5, 2.5, and 1.25 μM, with three replicates for each concentration. Three wells containing only cell culture medium served as a blank control, and wells containing only DMSO culture medium served as a negative control. After 48 hours of culture, the supernatant was discarded, and 100 μL of 10% CCK-8 working solution was added. The mixture was then incubated for another 2 hours. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. All absorbance values ​​were first subtracted from the blank control values, and then compared with the control group values ​​to obtain the relative cell inhibition rate of the experimental group. The cell inhibition rate was calculated as: [OD control - OD experiment] / [OD control - OD negative] × 100%. The concentration at which the drug inhibits cell growth by 50% (IC50) can be determined by comparing the different concentrations of the same drug with the tumor cell growth inhibition rate. 50 Since each compound at the same concentration occupied 3 wells in a 96-well plate, the average concentration was used to treat cells three times, and each experiment was repeated three times. The data obtained are expressed as mean ± standard error (mean ± SEM).

[0159] 3. Experimental Results

[0160] Table 3 shows the IC50 values ​​of the above-mentioned test compounds for SW480, HCT116, HepG2, A549, and SK-OV-3. 50 (Unit: μM)

[0161]

[0162] Note: * indicates the IC50 of the compound. 50 It is superior to the positive control.

[0163] Table 3 shows that compound 3 has an IC50 value for hepatocellular carcinoma cells (HepG2). 50 The values ​​were all superior to the positive control sorafenib; compounds 3, 6, and 7 showed better IC50 values ​​against lung cancer cells (A549). 50 The values ​​were all superior to the positive control; compound 7 showed better IC50 values ​​against ovarian cancer cells (SK-OV-3). 50 The values ​​were all better than the positive control.

[0164] The above results indicate that compounds 1–7 isolated from *Prunus cerasifera* all possess certain antitumor activities.

[0165] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for extracting and separating active compounds from *Prunus cerasifera*, characterized in that, Includes the following steps: (1) Take the whole herb of *Prunus cerasifera* in coarse powder, and extract it by maceration with methanol and water at room temperature using different gradients. Combine the extracts from multiple extractions and concentrate them under reduced pressure until there is no alcohol taste to obtain the total extract of *Prunus cerasifera*. After dispersing the total extract with an appropriate amount of hot water, extract it sequentially with petroleum ether, ethyl acetate and n-butanol, and recover it under reduced pressure to obtain the petroleum ether fraction, ethyl acetate fraction and n-butanol fraction. (2) The ethyl acetate fraction was decolorized by MCI column chromatography using a methanol-water system with gradient elution of 50:50, 60:40, 70:30, 80:20, 90:10, and 100:

0. The resulting fractions were analyzed by TLC and combined to obtain seven fractions, namely Fr. A to Fr. F. (3) The obtained Fr. A to Fr. F were further eluted and separated to obtain different anti-inflammatory compounds from *Prunus angustifolia*. Step (3) specifically involves separating and purifying the Fr. A fraction using silica gel column chromatography. A dichloromethane-methanol system is used, with sequential elution using gradients of 100:1, 50:1, 30:1, 10:1, 5:1, and 1:

1. The fractions are identified by TLC and separated into six components, namely Fr. A1 to Fr. A6. The obtained Fr. A1 to Fr. A6 fractions are then further eluted and separated. The obtained Fr. A1 to Fr. A6 were further separated by elution. Specifically, Fr. A2 was eluted by silica gel column chromatography using a dichloromethane-methanol system with sequential elution gradients of 50:1, 30:1, 10:1, 5:1, and 1:

1. TLC analysis revealed that the fractions were combined and separated into three components, Fr. A2-1 to Fr. A2-3. Fr. A2-1 was further purified by silica gel column chromatography using a petroleum ether-ethyl acetate system with sequential elution gradients of 50:1, 30:1, 10:1, 5:1, and 1:

1. TLC analysis revealed that the fractions were combined to obtain a crude product containing a high concentration of compound 1. This crude product was then purified by recrystallization to obtain compound 1, which is Phellodenol-A, and its specific structural formula is as follows: ; The obtained Fr. A1 to Fr. A6 are further separated by elution. Specifically, Fr. A4 is subjected to silica gel column chromatography using a dichloromethane-methanol system, eluted sequentially with gradients of 50:1, 30:1, 10:1, 5:1, 3:1, and 1:1 to obtain compound 2. Compound 2 is Ilex cornuta extract, and its specific structural formula is as follows: ; Step (3) specifically involves separating and purifying the Fr. B fraction using silica gel column chromatography. A dichloromethane-methanol system is used, with sequential elution using gradients of 50:1, 30:1, 10:1, 5:1, 3:1, and 1:

1. The fractions are identified by TLC and combined into seven components, namely Fr. B1 to Fr. B7. The obtained Fr. B1 to Fr. B7 fractions are further eluted and separated. Specifically, Fr. B1 is subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system, with sequential elution using gradients of 50:1, 30:1, 20:1, 10:1, 5:1, and 3:

1. The resulting fractions are identified by TLC and combined into three components, namely Fr. B1-1 to Fr. B1-3. The obtained Fr. B1-1 to Fr. B1-3 fractions are then further eluted and separated. The obtained Fr.B1-1 to Fr. B1-3 are further eluted and separated. Specifically, Fr. B1-1 is subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system, eluted sequentially with gradients of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, and 1:1 to obtain compound 3. Compound 3 is neo-sea urchin A, and its specific structural formula is as follows: ; The obtained Fr.B1-1 to Fr. B1-3 were further eluted and separated. Specifically, Fr. B1-2 was subjected to Sephadex LH-20 gel column chromatography with dichloromethane-methanol at a ratio of 1:1 to purify and obtain compound 4, which is 2,4-dimethoxycinnamic acid, and its specific structural formula is as follows: ; Step (3) specifically involves separating and purifying the Fr. C fraction using silica gel column chromatography. A petroleum ether-ethyl acetate system is used, with sequential elution using gradients of 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1 to obtain compound 5. Compound 5 is a polysaccharide, and its specific structural formula is as follows: ; Step (3) specifically involves passing the Fr. D fraction through silica gel column chromatography using a petroleum ether-ethyl acetate system, eluting sequentially with gradients of 50:1, 30:1, 15:1, 10:1, 5:1, and 3:1 to obtain compound 6. Compound 6 is a polysaccharide monomethyl ether, and its specific structural formula is as follows: ; Step (3) specifically involves passing the Fr. E fraction through silica gel column chromatography using a petroleum ether-ethyl acetate system, eluting sequentially with gradients of 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1 to obtain compound 7. Compound 7 is aleuropein-3,9-dimethyl ether, and its specific structural formula is as follows: .

2. The extraction and separation method as described in claim 1, characterized in that, The coarse powder is 60-80 mesh.

3. The extraction and separation method as described in claim 1, characterized in that, The extraction process involves room temperature immersion in methanol and water at different gradients. Specifically, 100%, 90%, and 70% methanol are added sequentially, and the extraction is performed twice, each time for 5 days.

4. The extraction and separation method as described in claim 3, characterized in that, The volume ratio of methanol-water to the whole plant of *Lysimachia nummularia* is 1:1.

5.

5. The application of an active compound from *Prunus cerasifera* in the preparation of anti-inflammatory or anticancer drugs, characterized in that, The anti-inflammatory active compound in the narrow-leaved pine tree is Phellodenol-A, whose specific structural formula is as follows: .

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