Two Anti-inflammatory Compounds Extracted from Leucophyllum Texanum and Extraction Method and Application Thereof

Abstract

The present application relates to the technical field of medicine, in particular to two anti-inflammatory compounds extracted from Leptodermum, and the extraction method and application thereof. Two compounds are isolated and identified from Leptodermum for the first time in the present application, which are 2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one (1) and 3,6-diisopropyl-2,5-diketopiperazine (2). Among them, there is one new compound (compound 1), and both compound 1 and compound 2 are isolated from the plants of the genus for the first time. The compound 1 and compound 2 isolated and identified in the present application both have excellent anti-inflammatory properties, and can be used for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to two anti-inflammatory compounds extracted from *Prunus cerasifera* var. *narrow-leaved*, their extraction methods, and applications. 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] Currently, research on the chemical components of *Ulva lactuca* (a type of medicinal herb) is limited, yet these components form the material basis for the traditional efficacy of traditional Chinese medicine. Due to the synergistic effects of traditional Chinese medicine involving multiple components, targets, and pathways, fundamental research on it still faces many challenges. Therefore, to further explore the pharmacologically active substances in *Ulva lactuca*, continued research on its chemical components is necessary. Furthermore, 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 two anti-inflammatory compounds extracted from *Prunus cerasifera*, their extraction methods and applications.

[0005] An anti-inflammatory compound (compound 1) extracted from *Prunus cerasifera* has the following specific structural formula:

[0006]

[0007] The chemical name of compound 1 is 2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one, and its molecular formula is C1. 15 H 12 O6 has a molecular weight of 288.25.

[0008] An anti-inflammatory compound (compound 2) extracted from *Prunus cerasifera* has the following specific structural formula:

[0009]

[0010] The chemical name of compound 2 is 3,6-diisopropyl-2,5-diketopiperazine, and its molecular formula is C2. 10 H 18 N2O2 has a molecular weight of 198.26.

[0011] Compounds 1 and 2 significantly inhibited the production of nitric oxide in LPS-induced inflammatory responses in RAW 264.7 cells, exhibiting excellent anti-inflammatory properties and could be used in the preparation of anti-inflammatory drugs.

[0012] The extraction method for the anti-inflammatory compounds (compound 1 and compound 2) specifically includes the following steps (when it is not necessary to obtain a certain compound, the corresponding steps can be omitted):

[0013] (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.

[0014] (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.

[0015] (3) The Fr.A fraction was separated and purified by silica gel column chromatography. The dichloromethane-methanol system was used for elution with 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 fractions, namely Fr.A1 to Fr.A6.

[0016] (4) Fr.A3 was eluted by silica gel column chromatography with a dichloromethane-methanol system using gradients of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1. The fractions were identified by TLC and separated into three components, namely Fr.A3-1 to Fr.A3-3. Fr.A3-1 was eluted by silica gel column chromatography with a petroleum ether-ethyl acetate system using gradients of 30:1, 20:1, 10:1, 5:1, and 1:1 to obtain compound 2.

[0017] (4) The Fr.C fraction was separated and purified by silica gel column chromatography. Compound 1 was obtained by elution with a petroleum ether-ethyl acetate system at gradients of 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1.

[0018] Furthermore, the coarse powder is 60-80 mesh.

[0019] 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.

[0020] Compared with the prior art, the technical effects of this invention are reflected in:

[0021] (1) This application is the first to isolate and identify two compounds from *Lysimachia nummularia*, namely: 2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one (1) and 3,6-diisopropyl-2,5-diketopiperazine (2). Among them, there is one new compound (compound 1). Both compounds 1 and 2 are isolated from plants of this genus for the first time.

[0022] (2) Compounds 1 and 2, which were isolated and identified from the narrow-leaved pine tree for the first time in this application, both have excellent anti-inflammatory properties and can be used to prepare anti-inflammatory drugs.

[0023] (3) Compound 1 (2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one(1)), which was isolated from *Lysimachia nummularia* for the first time in this application, is a novel compound obtained through extraction, separation, and purification for the first time. It also possesses excellent anti-inflammatory properties and can be used in the preparation of anti-inflammatory drugs. Currently, no relevant research literature has been found to report on Compound 1 or its application in the preparation of anti-inflammatory drugs. Compound 1 is classified as a phenylpropanoid and a diphenyl-α-pyranone. Such compounds are relatively rare in herbal medicines; currently, only four species have been found in the *Lysimachia* genus, all of which were isolated from *Ulva prostrata* medicinal materials. The compound 1 isolated and identified from *Ulva prostrata* in this experiment not only enriches the variety of chemical components in *Lysimachia* genus but also provides a scientific basis for subsequent research on the chemical components and other aspects of *Ulva prostrata*.

[0024] (4) Compound 2 (3,6-diisopropyl-2,5-diketopiperazine(2)) was isolated from *Lysimachia nummularia* for the first time in this application. This first isolation from *Lysimachia nummularia* provides a new preparation method and source for compound 2, enriching the preparation pathways of compound 2 and providing a scientific basis for subsequent research on the chemical composition and other aspects of *Umbrella chaperone*.

[0025] (5) This application studies the chemical composition and quality control of the Chasing Wind Umbrella, which can not only lay the foundation for the development and utilization of the medicinal material resources and the research and development of new products, but also lay a certain foundation for improving the quality control level of the Chasing Wind Umbrella and filling the geographical indication gap. Attached Figure Description

[0026] Figure 1 For compound 1 1 H-NMR (600MHz, DMSO-d6) spectrum.

[0027] Figure 2 For compound 1 13 C-NMR (150MHz, DMSO-d6) spectrum.

[0028] Figure 3 The HMBC (600MHz, DMSO-d6) spectrum of compound 1 is shown.

[0029] Figure 4 The HMQC (600MHz, DMSO-d6) spectrum of compound 1 is shown.

[0030] Figure 5 For compound 1 1 H- 1 H COSY (600MHz, DMSO-d6) spectrum.

[0031] Figure 6 The image shows the DEPT (150MHz, DMSO-d6) spectrum of compound 1.

[0032] Figure 7 The NOESY (600MHz, DMSO-d6) spectrum of compound 1 is shown.

[0033] Figure 8 This is the high-resolution mass spectrum of compound 1.

[0034] Figure 9 Here is the infrared spectrum of compound 1.

[0035] Figure 10 For compound 2 1 H-NMR (600MHz, CD3OD) spectrum.

[0036] Figure 11 For compound 2 13 C-NMR (150MHz, CD3OD) spectrum.

[0037] Figure 12 The graph shows the significant effects of compounds 1 and 2 on NO release. Detailed Implementation

[0038] 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.

[0039] Example 1

[0040] Research on the chemical composition of the Wind Chaser Umbrella

[0041] 1. Instruments and Materials

[0042] 1.1 Source of medicinal materials

[0043] 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.

[0044] 1.2 Instruments

[0045] The instruments used in this experiment are detailed in Table 1-1.

[0046] Table 1-1 Experimental Instruments

[0047]

[0048] 1.3 Reagents and Fillers

[0049] The reagents and packing materials used in this experiment are detailed in Table 1-2.

[0050] Table 1-2 Experimental Reagents and Fillers

[0051]

[0052]

[0053] Note: The colorimetric reagents used in this experiment are: 10% sulfuric acid ethanol solution and elemental iodine.

[0054] 2 Experimental Methods and Results

[0055] 2.1 Extraction and Separation of *Umbrella Chaser* Herbs

[0056] 35.58 kg of coarse powder (60-80 mesh) of the whole plant of *Ulva lactuca* was taken and extracted at room temperature using different gradients of methanol and water (100%, 90%, and 70% methanol were added twice each, for 5 days each time). The extracts were combined and concentrated under reduced pressure until no alcohol odor was detected to obtain the total extract of *Ulva lactuca* (6.96 kg, yield 19.56%). The total extract was dispersed in an appropriate amount of hot water and then extracted successively with 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.

[0057] The ethyl acetate fraction was decolorized by MCI column chromatography using a gradient elution with a methanol-water 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). The Fr.A fraction (71 g) was further purified by silica gel column chromatography using a gradient elution with a dichloromethane-methanol system (100:1, 50:1, 30:1, 10:1, 5:1, 1:1). The fractions were identified by TLC and then combined to form six fractions (Fr.A1 to Fr.A6).

[0058] Fr.A3 was subjected to silica gel column chromatography with gradient elution using a dichloromethane-methanol system (50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1), and identified by TLC as three fractions (Fr.A3-1 to Fr.A3-3). Fr.A3-1 was further subjected to silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system (30:1, 20:1, 10:1, 5:1, 1:1) to yield compound 2.

[0059] The Fr.C fraction (11.3 g) was separated and purified by silica gel column chromatography, and compound 1 was obtained by gradient elution with petroleum ether-ethyl acetate system (30:1, 20:1, 10:1, 5:1, 3:1, 1:1).

[0060] 2.2 Identification of Compound Structures

[0061] After separation and purification, the compounds were analyzed by physicochemical properties and methods such as nuclear magnetic resonance (NMR), infrared (IR) and high-resolution mass spectrometry (HR-ESI-MS), and their structures were identified with the help of micro-spectral databases and SciFinder Scholar.

[0062] 1. Structural identification of compound 1

[0063] Compound 1: White powder. Optical rotation: (c 0.207, pyridine). Easily soluble in DMSO and pyridine; sparingly soluble in methanol; insoluble in ethyl acetate, dichloromethane, and petroleum ether. TLC detection: Using dichloromethane-methanol (10:1) as the developing solvent, fluorescence was observed at UV wavelengths of 254 and 365 nm after development; after spraying with 10% sulfuric acid ethanol solution and heating at 105℃, a single orange-yellow spot appeared (R). f The value is 0.56. According to HR-ESI-MS, the test value m / z is 287.0573 [MH]. - (The calculated value is 287.0561, C) 15 H 11 O6), thus determining the molecular formula as C. 15 H 12 O6, with an unsaturation degree of 10. According to IR spectroscopy, it exhibits good spectral density at 1654.2 and 3419.6 cm⁻¹. -1 The absorption at the respective locations indicates that the compound may contain carbonyl and hydroxyl groups.

[0064] according to 1 H-NMR (600MHz, DMSO-d6) indicates that δ H 11.78 (1H, s, 7-OH), 10.82 (1H, s), and 8.80 (1H, s) may represent three active hydrogen atoms, δ H 7.21 (1H, d, J = 1.5Hz, H-10) and 6.30 (1H, d, J = 1.6Hz, H-8) suggest a possible set of meta-coupled aromatic signals, as indicated by... 1 H- 1 ¹H COSY spectroscopy confirmed this, indicating that the compound may possess a tetrasubstituted aromatic ring system. δ H The 6.86 (1H, s, H⁻⁴) value suggests the presence of one aromatic hydrogen, indicating the possible existence of a five-substituted aromatic ring system in the compound. δ H 3.83(3H, s, -OCH3) may be a single methoxy group, δ H 2.51(3H, s, -CH3) may be a single benzyl group. According to... 13 C-NMR (150MHz, DMSO-d6), DEPT, and HMQC spectra indicate that this compound contains 15 carbon atoms, suggesting the presence of 10 quaternary carbons (including one carbonyl carbon δ). C 165.3), 3 methine carbons [δ C 104.7 (C-10), 101.1 (C-8), 98.3 (C-4)], 1 methoxy carbon [δ C 56.1(-OCH3)], 1 methyl carbon [δ C[15.8(-CH3)]. Based on the compound's degree of unsaturation of 10, the two benzene rings occupy a total of 8 degrees of unsaturation, and the carbonyl group occupies 1 degree of unsaturation. The remaining 1 degree of unsaturation suggests that the compound may have a tricyclic system. Based on the above data and literature review, compound 1 is similar to the reported data of 2-hydroxy-alternariol (2-OH-AOH), suggesting that this compound is a phenylpropanoid compound. Compared to 2-OH-AOH, this compound has one more methoxy group and one less hydroxyl hydrogen.

[0065] By observing the HMBC spectrum and 1 H- 1 According to the H COSY spectrum: 1 H-NMR spectrum δ H 11.78 (1H, s, 7-OH) and 13 C-NMRδ C Long-range correlation signals are present at 97.9 (C-6a), 101.1 (C-8), and 164.1 (C-7); δ H 6.30 (1H, d, J = 1.6 Hz, H⁻⁸) and δ C Long-range correlation signals are present at 97.9 (C-6a), 104.7 (C-10), and 164.1 (C-7), δ H 7.21 (1H, d, J = 1.5Hz, H-10) and δ H The 6.30 (1H, d, J = 1.6 Hz, H-8) correlation indicates a hydroxyl group at C-7, with the carbon atom connected to the 7-OH group having a δ value of 164.1 ppm. C-6a, C-7, C-8, C-9, and C-10 represent five consecutive carbon signals on a tetrasubstituted aromatic ring system (A ring). δ H 7.21 (1H, d, J = 1.5Hz, H-10) and δ C Long-range correlation signals were observed at 97.9 (C-6a), 101.1 (C-8), 110.1 (C-10b), and 164.9 (C-9), indicating a hydroxyl group at C-9. The carbon atom linked to the 7-OH group had a δ value of 164.9 ppm. C-6a, C-7, C-8, C-9, and C-10 represented five consecutive carbons in ring A. δ H 2.51(3H, s, -CH3) and δ C Long-range correlation signals are present at 104.7 (C-10), 110.1 (C-10b), 121.3 (C-1), and 141.9 (C-2), indicating that the two benzene rings are connected by C-10b, and that C-2, C-1, and C-10b represent three consecutive carbon signals on a pentasubstituted aromatic ring system (B ring). δ H 3.83 (3H, s, -OCH3) and δ C149.2(C-3) has a long-range correlation signal; δ H 6.86 (1H, s, H⁻⁴) and δ C Long-range correlation signals were observed at 110.1 (C-10b), 141.9 (C-2), 144.9 (C-4a), and 149.2 (C-3). Combined with the HMQC spectrum, this indicates that the carbon bonded to the methoxy group has a δ value of 149.2 ppm, and C-2, C-3, C-4, C-4a, and C-10b represent five consecutive carbon signals on the B ring. In the NOESY spectrum (see...),... Figure 1 )middle, 1 H-NMR spectrum δ H 2.51(3H, s, -CH3) and δ H The correlation of 7.21 (1H, d, J = 1.5Hz, H-10) indicates that rings A and B are a spatially correlated whole.

[0066] In summary, compound 1 was identified as a phenylpropanoid compound and named 2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one. A search of SciFinder Scholar and a review of relevant literature confirmed compound 1 as a novel compound. The compound's... 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0067]

[0068] Datawere recorded inDMSO on aBrukerAV-600MHzspectrometer.

[0069] The structural formula of compound 1 is shown below:

[0070]

[0071] Compound 1 1 H- 1 H COSY(-), HMBC(→), NOESY The spectral correlation signal is shown below:

[0072]

[0073] 2. Structural identification of compound 2

[0074] Compound 2: White powder. Easily soluble in methanol, slightly soluble in ethyl acetate, insoluble in dichloromethane and petroleum ether. TLC detection: Using dichloromethane-ethyl acetate-formic acid (20:2:1) as the developing solvent, it fluoresces at UV wavelengths of 254 and 365 nm after development; after spraying with 10% sulfuric acid ethanol solution and heating at 105℃, it shows a single dark orange spot (R). f The value is 0.43; the molecular formula is C 10 H 18 N2O2. According to 1 H-NMR (600MHz, CD3OD) indicates that δ H 3.84 (2H, d, J = 3.4 Hz, H-3, 6) shows the hydrogen signal on the nitrogen-carbon junction, δ H 1.07 (6H, d, J = 7.0 Hz, H-8, 8') and 0.97 (6H, d, J = 6.7 Hz, H-9, 9') show four methyl groups. According to... 13 C-NMR (150 MHz, CD3OD) data showed that the compound contains 10 carbon atoms. C 168.8 (C-2, 5) shows two carbonyl groups, δ C The presence of four methyl groups at 16.4 (C-8, 8') and 17.9 (C-9, 9') suggests that this compound is an alkaloid. Based on the data and consistent with literature reports, compound 2 is identified as 3,6-diisopropyl-2,5-diketopiperazine. 1 H-NMR and 13 The C-NMR data are shown in the table below.

[0075]

[0076]

[0077] Datawere recorded in CD3OD on aBrukerAV-600MHzspectrometer.

[0078] The structural formula of compound 2 is shown below:

[0079]

[0080] 2.3 Partial physicochemical constants and spectral data of the compound

[0081] 2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one (1): White powder. Developed in dichloromethane-methanol (10:1), it shows a single orange-yellow spot in 10% sulfuric acid-ethanol solution (R). f The value is 0.58). Optical rotation: (c 0.207, pyridine); IR(KBr)V max 1654.2, 3419.6cm -1 HR-ESI-MS test value m / z 287.0573 [MH] - (The calculated value is 287.0561, C) 15 H 11 O6); 1 H-NMR (600MHz, DMSO-d6) δ11.78 (1H, s, 7-OH), 7.21 (1H, d, J = 1.5Hz, H-10), 6.86 (1H, s, H-4), 6.30 (1H, d, J = 1.6Hz, H-8), 3.83 (3H, s, -OCH3), 2.51 (3H, s, -CH3); 13 C-NMR (150MHz, DMSO-d6) δ121.3(C-1), 141.9(C-2), 149.2(C-3), 98.3(C-4), 144.9(C-4a), 165.3(C-6), 97.9(C-6 a), 164.1(C-7), 101.1(C-8), 164.9(C-9), 104.7(C-10), 138.3(C-10a), 110.1(C-10b), 56.1(-OCH3), 15.8(-CH3).

[0082] 3,6-diisopropyl-2,5-diketopiperazine(2): White powder. Developed in dichloromethane-ethyl acetate-formic acid (20:2:1), it shows a single dark orange spot in 10% sulfuric acid ethanol solution (R). f The value is 0.43. 1 H-NMR (600MHz, CD3OD) δ3.84 (2H, d, J = 3.4Hz, H-3, 6), 2.30 (2H, dq, J = 12.4, 6.1 , 5.7Hz, H-7, 7'), 1.07 (6H, d, J = 7.0Hz, H-8, 8'), 0.97 (6H, d, J = 6.7Hz, H-9, 9'); 13C-NMR (150MHz, CD3OD) δ 168.8 (C-2, 5), 59.8 (C-3, 6), 31.8 (C-7, 7'), 16.4 (C-8, 8'), 17.9 (C-9, 9').

[0083] Example 2

[0084] Anti-inflammatory activity experiments of the two compounds

[0085] In vitro anti-inflammatory activity of compounds 2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one (1) and 3,6-diisopropyl-2,5-diketopiperazine (2) against LPS-induced RAW264.7 cells.

[0086] 1. Experimental Principle

[0087] (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.

[0088] (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.

[0089] 2. Experimental Procedure

[0090] (1) Cell activity assay of compounds 1 and 2

[0091] 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 with 5% CO2 for 20 h. After incubation, 100 μL of culture medium containing different concentrations of the aforementioned compound was added. The compound concentration ranges were 6.25, 12.5, 25, and 50 μM, with three replicates for each concentration. Three wells were left untreated as the normal control group, and three wells were left untreated and untreated as the control group. After 20 h of incubation, the cell supernatant was discarded, and 10 μL of CCK-8 working solution was added. The plates were incubated for another 2 h, and the absorbance was measured at 450 nm using a microplate reader. Relative cell viability = [OD experiment - OD blank] / [OD normal - OD blank] × 100%.

[0092] (2) Detection of anti-inflammatory activity of compounds 1 and 2

[0093] 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 50 μM of the aforementioned compound was added, with three replicates. Three wells were left untreated as the control group, and three wells were left untreated and without cells as the blank group. For the control group, cells were incubated statically at 37°C with 5% CO2 for 1 h, followed by the addition of 1 μg / mL LPS to stimulate inflammation. Finally, the plates were incubated at 37°C with 5% CO2 for 20 h.

[0094] 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.

[0095] 3. Experimental Results

[0096] The relative viability of RAW 264.7 cells after 20 h of administration of compounds 1 and 2 at concentrations of 6.25, 12.5, 25, and 50 μM, respectively, is shown in Table 1. The results indicate that compounds 1 and 2 did not have toxic effects on the cells at the above concentrations.

[0097] Table 1. Relative viability of compounds 1 and 2 against RAW 264.7 cells.

[0098]

[0099] like Figure 12 As shown, LPS stimulation of RAW264.7 cells significantly increased cellular NO release compared to the control group without LPS stimulation. The LPS group without compounds 1 and 2 showed a NO release of 14.10 μM after LPS stimulation. Compared to the LPS group, the experimental groups with compounds 1 and 2 showed significantly reduced NO production. Specifically, the addition of compound 1 (50 μM) reduced cellular NO release to 7.75 μM; the addition of compound 2 (50 μM) reduced cellular NO release to 10.76 μM.

[0100] The above results indicate that compounds 1 and 2 extracted from *Lysimachia nummularia* significantly inhibited the production of nitric oxide in LPS-induced inflammatory responses in RAW264.7 cells, reflecting their excellent anti-inflammatory properties.

[0101] 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. The application of an anti-inflammatory compound extracted from *Prunus cerasifera* in the preparation of anti-inflammatory drugs, characterized in that, The specific structural formula of the compound is as follows: The chemical name of this compound is 3,6-diisopropyl-2,5-diketopiperazine, and its molecular formula is C2. 10 H 18 N2O2 has a molecular weight of 198.

26.

2. A method for extracting anti-inflammatory compounds from *Prunus cerasifera*, characterized in that, Specifically, the steps include the following: (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 Fr. A fraction was separated and purified by silica gel column chromatography using a dichloromethane-methanol system, with gradient elutions of 100:1, 50:1, 30:1, 10:1, 5:1, and 1:

1. The fractions were identified by TLC and separated into 6 fractions, namely Fr. A1 to Fr. A6. (4) Fr. A3 was subjected to silica gel column chromatography with a dichloromethane-methanol system, eluted sequentially with gradients of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, and 1:

1. The fractions were identified by TLC and separated into three components, Fr. A3-1 to Fr. A3-3. Fr. A3-1 was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate system, eluted sequentially with gradients of 30:1, 20:1, 10:1, 5:1, and 1:1 to obtain 3,6-diisopropyl-2,5-diketopiperazine, with the following structural formula: ; (5) The Fr. C fraction was separated and purified by 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, and 1:1 to obtain 2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one, with the following structural formula: .

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

4. The extraction method as described in claim 2, 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.

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