A costunolide compound and a preparation method and application thereof

Abstract

This invention discloses an acetamine-type sesquiterpene compound, its preparation method, and its applications, belonging to the field of natural compound extraction technology. The preparation method involves extracting dried *Elsholtzia ciliata* by reflux with 95% ethanol, followed by petroleum ether extraction to obtain an extract. The extract can be obtained by column chromatography and semi-preparative high-performance liquid chromatography. The method is simple, time-saving, labor-saving, and yields higher results. Furthermore, the obtained acetamine-type sesquiterpene compound exhibits high anti-neuritis activity, showing significant application potential for developing new drugs to treat neuritis-related diseases.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to acetamiprid-type sesquiterpenoid compounds extracted from Elsholtzia ciliata, their preparation methods, and applications. Background Technology

[0002] Purple Elsholtzia ( Elsholtzia argyi ) belongs to the genus Elsholtzia in the family Lamiaceae (Lamiaceae). Elsholtzia This plant, the entire herb, can be used medicinally. It is warm in nature and pungent in taste, possessing diaphoretic, heat-relieving, diuretic, antiemetic, antidiarrheal, and cold-dampness-dispelling effects. Traditional Chinese medicine believes it can be used to treat various symptoms such as colds, fever without sweating, jaundice, leukorrhea, cough, summer heat-induced halitosis, vomiting, and diarrhea. Besides its medicinal value, *Elsholtzia ciliata* can also be used as a food ingredient, with potential applications in aromatic flavoring, functional tea development, and phytoremediation of heavy metal-contaminated soil. Guizhou, as one of the main producing areas of *Elsholtzia ciliata*, may have unique karst topography and climate conditions that induce the production of novel and more bioactive secondary metabolites.

[0003] In recent years, research on the chemical composition and bioactivity of plants in the Lamiaceae family has deepened both domestically and internationally, revealing the diversity and wide range of chemical components and activities within this family. The genus *Elsholtzia* comprises approximately 40 species globally, mainly distributed in East Asia; my country has 33 species, 15 varieties, and 5 forms, with the majority concentrated in southwestern regions such as Yunnan, Guizhou, and Sichuan. Most plants in this genus are herbaceous or semi-shrubby, and the whole plant possesses diaphoretic, dampness-resolving, and diuretic properties, widely used in traditional Chinese medicine to treat common ailments such as colds, heatstroke, acute gastroenteritis, headaches, fever, and rheumatic pain. Some species, such as *Elsholtzia champaca* (Haizhou Elsholtzia),... Elsholtzia splendid It has been included in the Chinese Pharmacopoeia and is one of the original plants of the commonly used Chinese medicine "Xiangru".

[0004] Regarding chemical composition, current research on Elsholtzia plants mainly focuses on volatile oils, flavonoids, phenolic acids, and terpenoids. Modern pharmacological studies have shown that Elsholtzia plants possess various biological activities, including antibacterial, anti-inflammatory, antioxidant, antiviral, antipyretic, analgesic, and immunomodulatory activities. Although some progress has been made in previous studies, systematic research on sesquiterpenoids in Elsholtzia purpurea, especially novel sesquiterpenoids with anti-inflammatory activity, remains relatively weak. Therefore, to further explore new anti-inflammatory active substances from Elsholtzia purpurea, this application conducted an in-depth study of the chemical components of Elsholtzia purpurea and carried out research on the anti-inflammatory activity mechanism, from which a new calamusane-type sesquiterpenoid compound was isolated. Summary of the Invention

[0005] This invention proposes a novel strychnine-type sesquiterpene compound, its preparation method, and its application. This not only solves the problem of complex preparation methods but also provides a foundation for the discovery of new anti-inflammatory drugs.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a novel strychnine-type sesquiterpene compound extracted from Elsholtzia ciliata, the chemical structure of which includes Formula 1, Formula 2 and Formula 3: .

[0007] Secondly, the present invention also discloses a method for preparing a strychnine-type sesquiterpene compound, comprising the following steps: (1) After drying, the purple elm was crushed, extracted with organic solvent, and then extracted by hot reflux and concentrated under reduced pressure to recover the solvent, yielding crude extract. (2) The crude extract was suspended in water, extracted with petroleum ether, and the petroleum ether was recovered to obtain the petroleum ether extract of Elsholtzia ciliata. (3) The petroleum ether extract of Elsholtzia ciliata from step (2) was eluted with a normal-phase silica gel gradient using petroleum ether / ethyl acetate at a volume ratio of 100:0 to 0:100. The fractions that showed fluorescence under UV light and the fractions that showed color development under 5% sulfuric acid ethanol were collected sequentially to obtain fractions Fr.1 to Fr.8. (4) The Fr.5 from step (3) was eluted by gradient elution of RP-C18 reversed-phase column chromatography with methanol / water at a volume ratio of 60:40 to 100:0. Thin-layer chromatography was then used to detect the fluorescence under ultraviolet light and the colorimetric components of 5% sulfuric acid ethanol, respectively, to obtain the Fr.5.1 to Fr.5.6 components. (5) The Fr.5.2 from step (4) was eluted with a normal-phase silica gel gradient using petroleum ether / ethyl acetate at a volume ratio of 100:1 to 1:1. The fractions were then detected by thin-layer chromatography. The fractions that showed fluorescence under UV light and the fractions that showed color development under 5% sulfuric acid ethanol were collected in sequence to obtain the Fr.P5-2a to P5-2d fractions, respectively. (6) The Fr.P5-2b from step (5) was subjected to Sephadex LH-20 column chromatography with dichloromethane / methanol as the eluent at a volume ratio of 1:1, and then detected by thin-layer chromatography. The components that fluoresce under ultraviolet light and the colorimetric components of 5% sulfuric acid ethanol were collected in sequence to obtain the Fr.P5-2b-1 to Fr.P5-2b-6 components respectively. (7) The Fr. P5-2b-3 from step (6) was further purified by semi-preparative HPLC. Methanol / water with a volume ratio of 88:12 was selected as the mobile phase, and the chromatographic peak was collected at a retention time of 28 min to obtain compound 1. (8) The Fr.P5-2c from step (5) was subjected to Sephadex LH-20 column chromatography with dichloromethane / methanol as the eluent at a volume ratio of 1:1, and then detected by thin-layer chromatography. The components that fluoresce under ultraviolet light and the colorimetric components of 5% sulfuric acid ethanol were collected in sequence to obtain the Fr.P5-2c-1 to Fr.P5-2c-6 components respectively. (9) Fr. P5-2c-2 from step (8) was further purified by semi-preparative HPLC. Methanol / water with a volume ratio of 86:14 was selected as the mobile phase. The chromatographic peaks were collected at retention times of 28 min and 35 min to obtain compounds 2 and 3, respectively.

[0008] Furthermore, in step (1), the organic solvent is 95% ethanol, the volume of ethanol used is 3-5 L / kg based on the weight of Elsholtzia ciliata powder, and the reflux extraction is performed at least twice, with each extraction lasting 2 hours.

[0009] Further, the organic solvent in step (2) is petroleum ether, and the crude extract is extracted with petroleum ether 5-6 times in sequence; the volume of water used when adding water for suspension is 2-5 L / kg based on the weight of the crude extract; the volume ratio of petroleum ether to water is 1:1.

[0010] Furthermore, the elution conditions for silica gel column chromatography in step (3) are as follows: gradient elution is performed using petroleum ether / ethyl acetate mixed solvents with volume ratios of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 0:100.

[0011] Furthermore, in step (4), the elution conditions for RP-C18 reversed-phase column chromatography are as follows: gradient elution is performed using methanol / water mixed solvents with volume ratios of 60:40, 70:30, 75:25, 80:20, 85:15, and 90:10 as eluents.

[0012] Furthermore, in step (5), the elution conditions for silica column chromatography in normal phase silica gradient elution are as follows: gradient elution is performed sequentially using a mixture of petroleum ether / ethyl acetate in volume ratios of 100:1, 85:15, 75:25, 65:35, and 55:45.

[0013] Thirdly, this invention provides the application of calamine-type sesquiterpenoid compounds in the preparation of neuritis inhibitors. The calamine-type sesquiterpenoid compounds extracted from Elsholtzia ciliata of this invention exhibit significant anti-inflammatory activity, exerting their anti-neuritis activity by downregulating the release of nitric oxide from BV-2 neurons.

[0014] Fourthly, this invention provides the application of an acetamipane-type sesquiterpene compound in the preparation of anti-inflammatory drugs. The acetamipane-type sesquiterpene compound extracted from Elsholtzia ciliata provided by this invention has significant anti-inflammatory activity and can be used as an anti-inflammatory drug or for the preparation of anti-inflammatory drugs.

[0015] The beneficial effects of this invention, including the purine-type sesquiterpenoid compounds, their preparation method, and their applications, are as follows: (1) The strychnine-type sesquiterpenoids extracted from Elsholtzia splendens in this invention are obtained by reflux extraction with 95% ethanol followed by petroleum ether extraction to obtain an extract. The extract can be obtained by column chromatography and semi-preparative high performance liquid chromatography. The method is simple, time-saving, labor-saving and has a higher yield.

[0016] (2) The strychnine-type sesquiterpenoid compounds extracted from Elsholtzia splendens in this invention are novel structural compounds that have the effect of inhibiting neuroinflammation and have important application prospects for the development of new neuroinflammation drugs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 It is compounds 1, 2, and 3. 1 H-NMR spectrum; where A is the H-NMR spectrum of compound 1. 1 H-NMR spectrum, B represents compound 2. 1 H-NMR spectrum, C represents compound 3 1 H-NMR spectrum; Figure 2 It is compounds 1, 2, and 3. 13 C-NMR spectrum; where A is the C-NMR spectrum of compound 1. 13 C-NMR spectrum, B represents compound 2. 13 C-NMR spectrum, C represents compound 3. 13 C-NMR spectrum; Figure 3 These are the HSQC spectra of compounds 1, 2, and 3, where A is the HSQC spectrum of compound 1, B is the HSQC spectrum of compound 2, and C is the HSQC spectrum of compound 3. Figure 4 These are the HMBC spectra of compounds 1, 2, and 3; where A is the HMBC spectra of compound 1, B is the HMBC spectra of compound 2, and C is the HMBC spectra of compound 3. Figure 5 The middle part contains compounds 1, 2, and 3. 1 H- 1 HCl COSY spectrum, where A represents compound 1. 1 H- 1 H COSY spectrum, B represents compound 2. 1 H- 1 H COSY spectrum, C represents compound 3. 1 H- 1 H COSY spectrum; Figure 6 These are the NOESY spectra of compounds 1, 2, and 3; where A is the NOESY spectrum of compound 1, B is the NOESY spectrum of compound 2, and C is the NOESY spectrum of compound 3. Figure 7 These are the HR-ESI-MS spectra of compounds 1, 2, and 3; where A is the HR-ESI-MS spectrum of compound 1, B is the HR-ESI-MS spectrum of compound 2, and C is the HR-ESI-MS spectrum of compound 3. Figure 8 These are the IR spectra of compounds 1, 2, and 3; where A is the IR spectrum of compound 1, B is the IR spectrum of compound 2, and C is the IR spectrum of compound 3. Figure 9 These are the UV spectra of compounds 1, 2, and 3; where A is the UV spectrum of compound 1, B is the UV spectrum of compound 2, and C is the UV spectrum of compound 3. Figure 10 These are single-crystal spectra of compounds 1 and 2; where A is the single-crystal spectrum of compound 1 and B is the single-crystal spectrum of compound 2. Figure 11 It is compounds 1, 2, and 3. 1 H- 1 H COSY and HMBC correlation diagram; where A represents compound 1. 1 H- 1 Correlation diagram of H COSY and HMBC, B represents compound 2. 1 H- 1 Correlation diagram of H COSY and HMBC, where C represents compound 3. 1 H- 1 H COSY and HMBC related diagrams; Figure 12 These are NOESY correlation graphs for compounds 1, 2, and 3. In the graphs, A represents the NOESY correlation graph for compound 1, B represents the NOESY correlation graph for compound 2, and C represents the NOESY correlation graph for compound 3. Figure 13This is a graph showing the effects of compounds 1, 2, and 3 on the viability of BV-2 cells. The control group in the graph is the blank control group. Figure 14 The inhibitory effects of compounds 1, 2, and 3 on NO production in LPS-induced BV-2 cells are shown in the figure; the control group is the blank control group, and the LPS group is the model group. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0020] The chemical structural formula of a strychnine-type sesquiterpene compound extracted from Elsholtzia ciliata is shown in the figure: .

[0021] Example 1

[0022] A method for preparing an acetamide-type sesquiterpene compound includes the following steps: (1) Take 31 kg of dried whole plant sample of Elsholtzia ciliata, crush it and extract it three times with 95% ethanol at 80℃ (2 hours each time). Combine the extracts and concentrate them under reduced pressure until dry to obtain crude extract. (2) After dispersing the crude extract with water, it was extracted five times with petroleum ether to obtain 362.5 g of Elsholtzia petroleum ether extract; (3) The petroleum ether extract (362g) of Elsholtzia ciliata from step (2) was eluted with a normal-phase silica gel gradient using petroleum ether / ethyl acetate at a volume ratio of 100:0 to 0:100. Thin-layer chromatography was then used to detect the fractions. The fractions that showed fluorescence under UV light and the fractions that showed color development under 5% sulfuric acid ethanol were collected sequentially. The fractions were combined to obtain fractions Fr.1 to Fr.8. Specifically, the petroleum ether / ethyl acetate elution ratios were: 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 0:100. The following combinations of components were collected at a ratio of 90:10, with both fluorescent and ethanolic sulfate components being combined to obtain Fr.1; at 80:20, the same components were collected and combined to obtain Fr.2; at 70:30, the same components were collected and combined to obtain Fr.3; at 60:40, the same components were collected and combined to obtain Fr.4; at 50:50, the same components were collected and combined to obtain Fr.5; at 40:60, the same components were collected and combined to obtain Fr.6; at 30:70, the same components were collected and combined to obtain Fr.7; and at 20:80, the same components were collected and combined to obtain Fr.8. (4) Fr.5 (39g) was eluted by gradient elution using RP-C18 reversed-phase column chromatography with methanol / water at a volume ratio of 60:40 to 100:0. Thin-layer chromatography was then used to detect the fluorescence under UV light and / or the colorimetric fraction obtained from 5% sulfuric acid ethanol, yielding Fr.5.1 to Fr.5.6. Specifically, the gradient ratios of the methanol-water gradient were 60:40, 70:30, 75:25, 80:20, 85:15, and 90:10. Fr.5.1 was obtained at 60:40; Fr.5.2 at 70:30; Fr.5.3 at 75:25; Fr.5.4 at 80:20; Fr.5.5 at 85:15; and Fr.5.6 at 90:10. (5) The Fr.5.2 obtained in step (4) was eluted with a normal-phase silica gel gradient using petroleum ether / ethyl acetate at a volume ratio of 100:1 to 1:1. The fractions that showed fluorescence under UV light and the fractions that showed color development under 5% sulfuric acid ethanol were collected sequentially to obtain the Fr.P5-2a to P5-2d fractions, respectively. Specifically, the gradient ratio of petroleum ether / ethyl acetate eluent was 100:1, 85:15, 75:25, 65:35, and 55:45. The components exhibiting fluorescence and color development with 5% sulfuric acid ethanol at an eluent gradient ratio of 85:15 were combined to obtain component Fr.P5-2a; the components exhibiting fluorescence and color development with 5% sulfuric acid ethanol at an eluent gradient ratio of 75:25 were combined to obtain component Fr.P5-2b; the components exhibiting fluorescence and color development with 5% sulfuric acid ethanol at an eluent gradient ratio of 65:35 were combined to obtain component Fr.P5-2c; and the components exhibiting fluorescence and color development with 5% sulfuric acid ethanol at an eluent gradient ratio of 55:45 were combined to obtain component Fr.P5-2d. (6) The Fr.P5-2b from step (5) was subjected to Sephadex LH-20 column chromatography with dichloromethane / methanol as the eluent at a volume ratio of 1:1, and then detected by thin-layer chromatography. The components that fluoresce under ultraviolet light and the colorimetric components of 5% sulfuric acid ethanol were collected in sequence to obtain the Fr.P5-2b-1 to Fr.P5-2b-6 components respectively. (7) The Fr.P5-2b-3 from step (6) was further purified by semi-preparative HPLC. Methanol / water with a volume ratio of 88:12 was selected as the mobile phase (2 mL / min). The chromatographic peak was collected at a retention time of 28 min to obtain compound 1 (8 mg, tR = 28 min). (8) The Fr.P5-2c from step (5) was subjected to Sephadex LH-20 column chromatography with dichloromethane / methanol as the eluent at a volume ratio of 1:1, and then detected by thin-layer chromatography. The components that fluoresce under ultraviolet light and the colorimetric components of 5% sulfuric acid ethanol were collected in sequence to obtain the Fr.P5-2c-1 to Fr.P5-2c-6 components respectively. (9) The Fr.P5-2c-2 from step (8) was further purified by semi-preparative HPLC. Methanol / water with a volume ratio of 86:14 was selected as the mobile phase (2 mL / min). The chromatographic peaks were collected at retention times of 28 min and 35 min to obtain compound 2 (8 mg, tR = 28 min) and compound 3 (2 mg, tR = 35 min).

[0023] Example 2

[0024] Structural identification of compounds 1, 2, and 3 Compound 1: Yellow needle-like crystals (methanol). α]25 D-12.4° (c 0.050, MeOH), UV (MeOH) l max(log) e 275 (0.37) nm. IR spectroscopy shows that this compound contains hydroxyl groups (3741 cm⁻¹). -1 ) and carbonyl groups (1708 and 1527 cm) -1 ) Phenyl (1526, 1491 cm) −1 ) and double bonds (1397 cm) −1 Functional groups such as [M+Na] are present. HR-ESI-MS spectrum shows the molecular ion peak (m / z) at 407.2178. + (Calculated value C) 24 H 32 O4Na + (407.2193), based on the proton and carbon spectra of this compound, its molecular formula is deduced to be C. 24 H 32 O4 has 9 degrees of unsaturation.

[0025] 1 1H NMR spectrum data (Table 1), four methyl signals [ d H 1.10 (d, J = 4.2 Hz, H-14), 1.09 (d, J = 4.2 Hz, H-15), 0.92 (d, J = 6.6 Hz, H-12) and 0.88 (d, J = 6.6 Hz, H-13)], two oxymethyl groups [δH 5.38 (td, J = 4.8, 2.4 Hz, H-3) and 4.04 (dd, J = 12.0, 6.0 Hz, H-6)], one double-substituted double bond [ d H 7.70 (d, J = 16.2 Hz, H-3'), 6.39 (d, J = 16.2 Hz, H-2')] and five aromatic proton signals [ d H 7.38 (s, H-7'), 7.53 (dd, J = 7.2, 1.8 Hz, H-5', 8'), and 7.39 (dd, J = 7.2, 1.8 Hz, H-6', 9')]. 13 C10 NMR and HSQC spectra showed 24 carbon resonance signals, including 4 methyl groups, 3 methylene groups, 6 methine groups, 2 quaternary carbons, and one cinnamoyloxy group. d C165.6 (C-1'), 134.1 (C-4'), 146.4 (C-3'), 117.1 (C-2'), 129.1 (C-6', 9'), 128.4 (C-5', 8'), and 130.9 (C-7')].

[0026] The above information indicates that compound 1 is a 6 / 5 bispiral skeleton arugula-type sesquiterpene, similar to the known compound (1S,4S,5S,10S)-10-hydroxyacorenone B. The main difference is that compound 1 lacks a double bond at C-7 and has a cinnamyloxy group at C-3. 2D NMR data further clarified this inference. 1 H- 1 H COSY correlations show the existence of two spin-coupled systems, H-1 / H-11 / H-12 / H-13 and H-6 / H-7 / H-8 / H-15. Combined with HMBC correlations, H-14 was observed ( d H 1.10) and H-1 ( d H 2.48) and C-3 ( d C The signal correlation of 77.9) essentially determined the planar structure of compound 1. In the NOESY correlation, the correlations of H-3 / H-4 / H-13, H-4 / H-10, and H-6 / H-8 indicate that the groups H-3, H-4, H-6, H-8, and C-5–C-10 are allocated as... α Orientation, while H-1 is allocated as β Orientation. To further confirm the absolute configuration of compound 1, recrystallization of compound 1 in a methanol / water system at 4°C was attempted, yielding suitable crystals. Subsequently, X-ray diffraction analysis was performed using a copper target, and combined with Flack parameters, the configuration was finally determined to be 1. S ,3 R 4 S 5 S 6 S 8 R In summary, the structure of compound 1 has been determined.

[0027] Table 1. Compound 1 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) data

[0028] Compound 2: Yellow needle-like crystals (methanol), quasi-molecular ion peak (m / z) 449.2282 [M + Na]+ in HR-ESI-MS spectrum (calculated value is C). 26 H 34 O5Na + (449.2298), its molecular formula is speculated to be C 26 H 34 O5, with an unsaturation degree of 10. Detailed analysis of compounds 3 and 1... 1 H and 13 The C1NMR spectral data (Table 2) show that the two compounds have highly similar skeletal structures. The main difference is that compound 3 has an additional set of acetoxy groups in its NMR spectrum [C1″ ( d C 170.0), C 2″ ( d C 21.8), H 2″ ( d H [2.09], suggesting that the hydroxyl group at the C-6 position in compound 3 may be replaced by an acetoxy group. The above inference is based on the interaction between H6 (δH 5.24) and H2″ (δH 2.09) and the carbonyl carbon C1″ ( d C The presence of HMBC correlation (170.0) was confirmed, thus determining the planar structure of compound 3. In the NOESY spectrum, the correlation signals of H3 / H4 / H12, H4 / H10, and H6 / H8 indicate that the relative configuration of compound 3 is consistent with that of compound 1. To further confirm the absolute configuration of compound 3, recrystallization was attempted in a methanol / water system at 4°C, yielding crystals suitable for X-ray diffraction analysis. X-ray single-crystal diffraction analysis using a copper target ultimately determined the absolute configuration of compound 3 to be 1. S ,3 R 4 S 5 S 6 S 8 R .

[0029] Table 2. Compound 2 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) data

[0030] Compound 3: Yellow oily substance; HR-ESI-MS spectrum shows a quasi-molecular ion peak (m / z) of 449.2286 [M+Na]+ (calculated value C). 26 H 34 O5Na +(449.2298), its molecular formula is speculated to be C 26 H 34 O5, with an unsaturation degree of 10. Detailed comparative analysis of the 1D and 2D NMR data of compounds 2 and 3 (Table 3) revealed that compounds 2 and 3 have the same planar structure, with the main difference lying in the chemical shifts of some carbon signals. In compound 2, C4 ( d C 45.1) and C8( d C 39.2) shifts to a higher field, while C1 ( d C 49.3) shifts to a lower field, in compound 3 C4 ( d C 47.0), C 8 ( d C 41.9) and C1 ( d C 46.7). The above results indicate that compounds 2 and 3 form a pair of epimers at the C6 position. Further analysis of the NOESY spectra revealed correlations between H3 / H4 / H12, H4 / H10, and H6 / H14, indicating that the relative configuration of compound 2 is consistent with that of compound 3. Its absolute configuration was determined by comparing the calculated ECD with the experimental ECD, thus confirming that the absolute configuration of compound 3 is 1. S ,3 R 4 S 5 S 6 R 8 R In summary, the structure of compound 3 has been determined.

[0031] Table 3. Compound 3 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) data

[0032] Example 3 The in vitro anti-inflammatory activity of the compound of the present invention Experimental methods 1. Cell Culture Resuscitated mouse BV-2 cells were passaged in DMEM high-glucose medium (containing 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotics) under constant temperature cell culture conditions of 37°C and 5% CO2. Once the BV-2 cells were in good growth condition, cells in the logarithmic growth phase were collected and prepared into a cell suspension. Cells were counted using a hemocytometer, and the cell density was then adjusted to 2.78 × 10⁻⁶ cells / year. 5Cells / mL. Subsequently, the cell suspension was seeded into 96-well plates at a rate of 90 µL / well and incubated at 37°C in a 5% CO2 incubator for 24 h. Three experimental groups were set up in the 96-well plates: a blank control group, a model group, and a drug-treated group. Each experimental group had three replicates. The blank control group and the model group each had one well, and the rest were drug-treated groups.

[0033] 2. MTT assay to determine the effect of compounds on BV-2 cell viability Remove the 96-well plate from the incubator after 24 hours of culture. Dilute all prepared stock solutions of the compound (20 mM) to 400 μM with culture medium, and then add 10 µL / well to the drug treatment group to achieve a final concentration of 40 μM. After incubation at 37°C and 5% CO2 for two hours, add 10 μL of LPS (1 mg / mL LPS stock solution diluted 100 times with culture medium to 10 μg / mL) to each well in both the model group and the drug treatment group. The blank control group only added the same volume of culture medium; continue culturing the cells under the same conditions for 24 hours. After the culture, add 10 μL of MTT staining agent (5 mg / mL) to each well, and then place the 96-well plate in an incubator at 37°C and 5% CO2 for another 4 hours. Subsequently, carefully aspirate the supernatant, add 150 μL of DMSO to each well, and place the 96-well plate in a 37°C constant temperature shaker for 15 minutes until the formazan crystals are completely dissolved. Finally, the absorbance (OD) value of each well was measured using a microplate reader, and the cell viability was calculated according to the formula.

[0034] Calculation formula: Survival rate (%) = (OD value of the drug treatment group - OD value of the blank control group) / (OD value of the model group - OD value of the blank control group) × 100%.

[0035] See results Figure 13 Cell viability was determined by the MTT assay, and compounds 1, 2, and 3 were all non-toxic at a concentration of 40 μM.

[0036] 3. Griess method for detecting the inhibitory effect of compounds on LPS-induced NO release from BV-2 cells Remove the prepared 96-well plates from the incubator and add 10 μL of the test sample compounds 1-3 (final concentration 20 μM) to each well, then incubate for 2 h. Subsequently, add 10 μL of LPS (10 μg / mL) to each well in both the model group and the drug treatment group. After incubation at 37℃ and 5% CO2 for 24 h, aspirate 50 μL of the cell supernatant from each well and add 50 μL each of Griess reagent I and II to the supernatant in each well. Finally, use a microplate reader to detect the absorbance of each well at 540 nm and calculate the inhibition rate of NO production by the compounds. The results are shown in [Figure 1]. Figure 14The experiment was repeated three times, and the average value was taken. Then, compounds with a NO inhibition rate greater than 60% were selected and diluted with culture medium to prepare six samples of different concentrations. The above steps were repeated, and the absorbance values ​​at each concentration were measured. Minocycline was used as a positive control in each experiment. Cell growth curves were plotted with compound concentration on the x-axis and NO inhibition rate on the y-axis. The half-maximal inhibitory concentration (IC50) of the compounds was calculated using the Reed-Muench method. 50 The experiment was repeated three times, and the average value was taken. The results are shown in Table 4.

[0037] Table 4. In vitro anti-inflammatory activity (IC50) of compounds 1, 2, and 3 50 ( m M)

[0038] The results in Table 4 show that compounds 1, 2, and 3 inhibited LPS-induced NO production in BV-2 cells. Further IC50 analysis... 50 Test results show that compound 1 has an IC50 value. 50 The value was 10.14 ± 1.33 μM, and the IC50 of compound 2 was... 50 The IC50 value for compound 3 was 9.93 ± 0.51. 50 The value was 9.72±1.73 μM, which was superior to the positive control drug minocycline (18.89 ± 3.69 μM).

Claims

1. A strychnine-type sesquiterpene compound, characterized in that: Extracted from *Elsholtzia ciliata* (purple-flowered elm) produced in Guizhou, its chemical structural formula is selected from formulas 1, 2, and 3: 。 2. A method for preparing the neo-acorane-type sesquiterpene according to claim 1, characterized in that: Includes the following steps: (1) After drying, the purple elm was crushed, extracted with organic solvent, and then extracted by hot reflux and concentrated under reduced pressure to recover the solvent, yielding crude extract. (2) The crude extract was suspended in water, extracted with petroleum ether, and the solvent was recovered to obtain the petroleum ether extract of Elsholtzia ciliata. (3) The petroleum ether extract of Elsholtzia ciliata from step (2) was eluted with a normal-phase silica gel gradient using petroleum ether / ethyl acetate at a volume ratio of 100:0 to 0:

100. The fractions that showed fluorescence under UV light and the fractions that showed color development under 5% sulfuric acid ethanol were collected sequentially to obtain fractions Fr.1 to Fr.

8. (4) The Fr.5 from step (3) was eluted by gradient elution of RP-C18 reversed-phase column chromatography with methanol / water at a volume ratio of 60:40 to 100:

0. Thin-layer chromatography was then used to detect the fluorescence under ultraviolet light and the colorimetric components of 5% sulfuric acid ethanol, respectively, to obtain the Fr.5.1 to Fr.5.6 components. (5) The Fr.5.2 from step (4) was eluted with a normal-phase silica gel gradient using petroleum ether / ethyl acetate at a volume ratio of 100:1 to 1:

1. The fractions were then detected by thin-layer chromatography. The fractions that showed fluorescence under UV light and the fractions that showed color development under 5% sulfuric acid ethanol were collected in sequence to obtain the Fr.P5-2a to P5-2d fractions, respectively. (6) The Fr.P5-2b from step (5) was subjected to Sephadex LH-20 column chromatography with dichloromethane / methanol as the eluent at a volume ratio of 1:1, and then detected by thin-layer chromatography. The components that fluoresce under ultraviolet light and the colorimetric components of 5% sulfuric acid ethanol were collected in sequence to obtain the Fr.P5-2b-1 to Fr.P5-2b-6 components respectively. (7) The Fr. P5-2b-3 from step (6) was further purified by semi-preparative HPLC. Methanol / water with a volume ratio of 88:12 was selected as the mobile phase, and the chromatographic peak was collected at a retention time of 28 min to obtain compound 1. (8) The Fr.P5-2c from step (5) was subjected to Sephadex LH-20 column chromatography with dichloromethane / methanol as the eluent at a volume ratio of 1:1, and then detected by thin-layer chromatography. The components that fluoresce under ultraviolet light and the colorimetric components of 5% sulfuric acid ethanol were collected in sequence to obtain the Fr.P5-2c-1 to Fr.P5-2c-6 components respectively. (9) Fr. P5-2c-2 from step (8) was further purified by semi-preparative HPLC. Methanol / water with a volume ratio of 86:14 was selected as the mobile phase. The chromatographic peaks were collected at retention times of 28 min and 35 min to obtain compounds 2 and 3, respectively.

3. The method for preparing the strychnine-type sesquiterpene compound as described in claim 2, characterized in that, In step (1), the organic solvent is 95% ethanol, and the volume of ethanol used is 3-5 L / kg based on the weight of Elsholtzia ciliata powder. The reflux extraction is performed at least twice, with each extraction lasting 2 hours.

4. The method for preparing the strychnine-type sesquiterpene compound as described in claim 2, characterized in that, The crude extract in step (2) is extracted with petroleum ether 5-6 times; the volume of water used when suspending the crude extract in water is 2-5 L / kg based on the weight of the crude extract; the volume ratio of petroleum ether to water is 1:

1.

5. The method for preparing the strychnine-type sesquiterpene compound as described in claim 2, characterized in that, In step (3), the elution conditions for silica gel column chromatography are as follows: gradient elution is performed using petroleum ether / ethyl acetate mixed solvents with volume ratios of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 0:100 as eluents.

6. The method for preparing the strychnine-type sesquiterpene compound as described in claim 2, characterized in that, In step (4), the elution conditions for RP-C18 reversed-phase column chromatography are as follows: gradient elution is performed using methanol / water mixed solvents with volume ratios of 60:40, 70:30, 75:25, 80:20, 85:15, and 90:10 as eluents.

7. The method for preparing the strychnine-type sesquiterpene compound as described in claim 2, characterized in that, In step (5), the elution conditions for silica gel column chromatography in normal-phase silica gel gradient elution are as follows: gradient elution is performed sequentially using a petroleum ether / ethyl acetate mixed solvent with volume ratios of 85:15, 75:25, 65:35, and 55:

45.

8. The use of the strychnine-type sesquiterpene compound of claim 1 in the preparation of a neuritis inhibitor.

9. The use of the strychnine-type sesquiterpene compound of claim 1 in the preparation of an anti-inflammatory drug.

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