Phenanthrene type norditerpenoid, preparation method thereof and application of phenanthrene type norditerpenoid in neuroinflammation prevention and treatment medicine

Abstract

The invention relates to a phenanthrene type norditerpenoid, a preparation method thereof and an application of the phenanthrene type norditerpenoid in a medicine for preventing and treating neuroinflammation in the technical field of biological medicines, and the phenanthrene type norditerpenoid is extracted from Leucodendron hainanense and is a norditerpenoid component with a gem-dimethyl 6 / 6 / 6 tricyclic phenanthrene type structure. The extraction method comprises the following steps: firstly, extracting a dried medicinal material of Hainan acalypha australis with ethanol with the mass concentration of 95%, and then filtering, concentrating under reduced pressure and drying to obtain a 95% ethanol extract; adding water to disperse, and sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol to obtain extracts of corresponding parts; and finally, performing chromatographic separation and purification on the ethyl acetate part. The phenanthrene type norditerpenoid disclosed by the invention can be used for inhibiting the release of an inflammatory factor NO of BV-2 neural microglial cells; the invention has important significance on the development of drugs for treating neuroinflammation diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and in particular to new compounds discovered from plants and their applications, specifically a phenanthrene-type diterpenoid extracted and isolated from *Euonymus alatus* hainanensis, its preparation method, and its application in drugs for the prevention and treatment of neuroinflammatory diseases. Background Technology

[0002] Hainan calyx tree ( Blachia siamensis It is a perennial shrub belonging to the genus Blachia in the family Euphorbiaceae. It is mainly distributed in Hainan and Guangdong, China, and there are currently no records of its traditional medicinal use.

[0003] However, there are no reports on the chemical composition of *Euphorbia hainanensis*, either domestically or internationally. Closely related genera of the *Euphorbia* genus include *Trifolium* (*Euphorbia*). Trigonostemon ), genus *Pterocarya* ( Ostodes ), genus *Pterocarya* ( Strophioblachia ) and the genus Croton ( Codiaeum The genus *Euphorbia* has been found to contain a large number of novel secondary metabolites, exhibiting significant pharmacological activities, including antitumor, anti-inflammatory, antibacterial, and antimalarial effects. This suggests that *Euphorbia* species may hold enormous research potential in terms of chemical composition and biological activity. *Euphorbia hainanensis* is distributed in Hainan, China, Cambodia, and Thailand, growing in diverse habitats such as coastal low mountains, sparse coastal forests, ravines, and cliffs. It is characterized by drought and flood tolerance, strong stress resistance, and is a unique plant resource with both ecological adaptability and ornamental value. Currently, the phenanthrene-type norditerpenes obtained from *Euphorbia hainanensis* possess a 6 / 6 / 6 tricyclic structure system and substituents such as gem-dimethyl, along with the plant's unique ecological protection functions, which helps to explore its medicinal value and application in the prevention and treatment of human neuroinflammatory diseases. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by providing a phenanthrene-type diterpenoid, its preparation method, and its application in the prevention and treatment of neuroinflammatory drugs. This type of compound has a significant inhibitory effect on the release of NO from BV-2 microglia.

[0005] This invention provides phenanthrene-type diterpenoids and their pharmaceutically acceptable salts: ; Where R1 is -H, -CH2CH3 or -CH3, R2 is =O, -OH, -OCH3 or -OCH2CH3, R3 is -H, -OH, -OCH3 or halogen, and R4, R5, R6 are =O, -OH, -H, -OCH3 or -OCH2CH3.

[0006] Furthermore, this invention provides three phenanthrene-type diterpenoids, the structures and names of which are as follows: ; Compounds 1-3 were named siamensisoid C (1); siamensisoid D (2); and 9-O-Demethyltrigonostemone (3), respectively.

[0007] The spectroscopic data of compounds 1-3 are as follows: Compound 1 siamensisoid C Orange oily substance (methanol); UV (MeOH) λmax (logє) 215nm (0.89), 248nm (0.73); HR-ESI-MS m / z 327.1226 [M+H] + The molecular formula is C 19 H 18 O 5。

[0008] Compound 2 siamensisoid D Green amorphous solid (methanol); UV (MeOH) λmax (logє) 212nm (1.08), 250 nm (0.69), 265nm (0.62), 352nm (0.32); HR-ESI-MS m / z 365.1003 [M+Na] + The molecular formula is C 19 H 18 O6.

[0009] Table 1: New compounds 1 and 2 of the present invention 1 H-NMR (600MHz) and 13 C-NMR (150MHz) data

[0010] Recorded in Acetone- d 6 . Compound 3 9-O-Demethyltrigonostemone Yellow amorphous solid; HR-ESI-MS m / z 312.1364; Molecular formula is C 19 H 20 O. 1 H-NMR (Acetone- d 6,600 MHz) δ: 7.56 (1H, s, H-1), 6.94 (1H, s, H-5), 7.58 (1H, s, H-11), 8.00(1H, s, H-14), 2.34 (1H, s, H-17), 1.43 (1H, s, H-18), 1.43 (1H, s, H-19), 3.84 (3H, s, 2-OCH3), 4.02 (3H, s, 12-OCH3), 9.22 (1H, s, 7-OH). 13 C-NMR (Acetone- d 6, 150 MHz) δ : 113.7 (C-1), 148.4 (C-2), 198.9 (C-3), 49.7 (C-4), 144.2 (C-5), 105.9 (C-6), 154.1 (C-7), 119.8 (C-8), 132.7 (C-9), 115.2 (C-10), 101.0 (C-11), 158.9 (C-12), 127.4 (C-13), 124.5 (C-14), 16.9 (C-17), 28.5 (C-18), 28.5 (C-19), 55.9 (2-OCH3), 55.9 (12-OCH3). The above compound was isolated from *Euonymus alatus*, and its preparation method includes the following steps: A) The dried medicinal material of Hainan Euonymus alatus was pulverized, extracted with 95% ethanol, filtered, the filtrates were combined, and then concentrated and dried under reduced pressure to obtain a 95% ethanol extract. B) The 95% ethanol extract was dispersed in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether fraction, ethyl acetate fraction and n-butanol fraction; C) The ethyl acetate fraction was subjected to column chromatography on silica gel, eluted with a petroleum ether-ethyl acetate gradient at a volume ratio of (1:0) to (1:1), and the fractions were combined and divided into 14 fractions. The petroleum ether-ethyl acetate eluent was then subjected to MCI and ODS column chromatography, eluted with a methanol-water gradient to obtain a methanol-water eluent. The acetonitrile-water eluent was purified by semi-preparative high performance liquid chromatography, eluted with acetonitrile-water as the mobile phase, to obtain phenanthrene-type norditerpenoid compounds.

[0011] The specific steps for chromatographic separation and purification of the ethyl acetate fraction in step C) above are as follows: 1) The ethyl acetate fraction was subjected to silica gel column chromatography and eluted with a petroleum ether-ethyl acetate gradient at volume ratios of 100:1, 50:1, 25:1, 12:1, 6:1, 3:1, and 1:1. The fractions were combined to obtain 14 fractions. 2) Fractions 6, 7, 8, and 9 were combined and eluted by ODS and MCI with a methanol-water gradient of volume ratios of 3:7, 5:5, 7:3, 9:1, and 1:0. After merging, 6 fractions were obtained. Fraction 3 was purified by semi-preparative high performance liquid chromatography and eluted with acetonitrile-water at a volume ratio of 49:51 to obtain compounds 1 and 2. 3) Fractions 10, 11, 12, 13, and 14 were combined and subjected to silica gel column chromatography, eluted with a dichloromethane-methanol gradient at volume ratios of 100:0, 100:1, 50:1, 25:1, 12:1, 6:1, 3:1, 1:1, and 0:100 to obtain 12 fractions. Fraction 4 was then subjected to petroleum ether-ethyl acetate, eluted with a gradient at volume ratios of 20:1, 10:1, 5:1, 3:1, and 1:1 to obtain seven fractions. Fraction 2 was subjected to MCI and ODS, eluted with a methanol-water gradient at volume ratios of 3:7, 5:5, 7:3, 9:1, and 1:0 to obtain six fractions. Fraction 3 was purified by semi-preparative high-performance liquid chromatography, eluted with acetonitrile-water at a volume ratio of 50:50 to obtain compound 3.

[0012] The application of the phenanthrene-type diterpenoids and their pharmaceutically acceptable salts obtained in this invention in the preparation of drugs for the prevention and treatment of neuroinflammation.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The isolation, purification, and bioactivity studies of novel compounds 1 and 2 from *Euonymus alatus* described in this invention have not been reported in existing journals. This invention provides a method for the extraction, separation, and purification of phenanthrene-type diterpenoids from *Euonymus alatus*. Alcohol extraction, silica gel column chromatography, MCI column chromatography, and semi-preparative HPLC were used for separation and purification, successfully obtaining phenanthrene compounds. The method is simple and rapid, and the compounds obtained by this method have high purity, all greater than 95%. Furthermore, studies have shown that these compounds possess potential anti-inflammatory activity and can be used to prepare anti-inflammatory drugs. Western blot and ROS studies indicate that compounds 1 and 3 can exert anti-inflammatory effects through a dual mechanism of inhibiting the production of pro-inflammatory mediators and enhancing cellular antioxidant defense. Compound 3 outperforms compound 1 in all detection indicators, demonstrating its potential for further development into an anti-inflammatory drug. Attached Figure Description

[0014] Figure 1 For the new compound siamensisoid C 1 H-NMR spectrum.

[0015] Figure 2 For the new compound siamensisoid C 13 C-NMR spectrum.

[0016] Figure 3 The HSQC spectrum of the new compound siamensisoid C is shown.

[0017] Figure 4 The HMBC spectrum of the new compound siamensisoid C is shown.

[0018] Figure 5 The NOESY spectrum of the new compound siamensisoid C.

[0019] Figure 6 For the new compound siamensisoid C 1 H- 1 H COSY spectrum.

[0020] Figure 7 This is the high-resolution mass spectrum of the new compound siamensisoid C.

[0021] Figure 8 The UV spectrum of the new compound siamensisoid C.

[0022] Figure 9 For the new compound siamensisoid D 1 H-NMR spectrum.

[0023] Figure 10 For the new compound siamensisoid D 13 C-NMR spectrum.

[0024] Figure 11 The HSQC spectrum of the new compound siamensisoid D is shown.

[0025] Figure 12 The HMBC spectrum of the new compound siamensisoid D is shown.

[0026] Figure 13 The NOESY spectrum of the new compound siamensisoid D is shown.

[0027] Figure 14 This is the high-resolution mass spectrum of the new compound siamensisoid D.

[0028] Figure 15 The UV spectrum of the new compound siamensisoid D.

[0029] Figure 16 The compound 9-O-Demethyltrigonostemone 1 H-NMR spectrum.

[0030] Figure 17 The compound 9-O-Demethyltrigonostemone 13 C-NMR spectrum.

[0031] Figure 18 This is a high-resolution mass spectrum of compound 9-O-Demethyltrigonostemone.

[0032] Figure 19 Western blotting was used to detect the expression levels of COX-2 and iNOS proteins in the novel compound siamensisoid D and compound 9-O-Demethyltrigonostemone.

[0033] Figure 20 The effects of the new compound siamensisoid D and compound 9-O-Demethyltrigonostemone on the expression of Nrf2, HMOX1, and NQO1 in LPS-treated RAW264.7 cells are presented.

[0034] Figure 21 The effect of the novel compound siamensisoid D on ROS levels in LPS-treated RAW264.7 cells.

[0035] Figure 22 The effect of compound 9-O-Demethyltrigonostemone on ROS levels in LPS-treated RAW264.7 cells. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. Example 1

[0037] Preparation of phenanthrene diterpenes The material was collected in Ledong County, Hainan Province in April 2021, and the specimen is preserved in the Herbarium of Chinese Medicinal Herbs, Department of Pharmacy, Medical School, Yangzhou University.

[0038] Extraction and separation: 17.9 kg of Hainan Euonymus alatus medicinal material was chopped and extracted three times by reflux with 95% ethanol at 85°C. The extracts were combined, concentrated under reduced pressure, and dried to obtain a total extract (766.3 g). The total extract was dispersed in 10 times its volume of distilled water and extracted sequentially with equal volumes of petroleum ether, ethyl acetate, and n-butanol to obtain the petroleum ether fraction (56.2 g), ethyl acetate fraction (75.3 g), and n-butanol fraction (136.2 g). The ethyl acetate fraction was subjected to silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient of (1:0) to (1:1) v / v, and the fractions were combined to obtain 14 fractions. Fractions 6, 7, 8, and 9 were combined and subjected to ODS, eluted with a methanol-water gradient of (3:7, 5:5, 7:3, 9:1, 1:0) v / v, and the fractions were combined to obtain 6 fractions. Fraction 3 was purified by semi-preparative high performance liquid chromatography, eluted with acetonitrile-water at a v / v ratio of 49:51 v / v, to obtain compounds 1 and 2. Fractions 10, 11, 12, 13, and 14 were combined and subjected to silica gel column chromatography, eluted with acetonitrile-water gradients of (100:0, 100:1) v / v, and eluted with acetonitrile-water gradients of (1 ... The mixture was eluted with a dichloromethane-methanol gradient of 50:1, 25:1, 12:1, 6:1, 3:1, 1:1, and 0:100 to obtain 12 fractions. Fraction 4 was then eluted with petroleum ether-ethyl acetate using a gradient of 20:1, 10:1, 5:1, 3:1, and 1:1 (v / v) to obtain seven fractions. Fraction 2 was then eluted with MCI and ODS using a gradient of methanol-water at v / v ratios of 3:7, 5:5, 7:3, 9:1, and 1:0 to obtain six fractions. Fraction 3 was purified by semi-preparative high-performance liquid chromatography and eluted with acetonitrile-water at a v / v ratio of 50:50 to obtain compound 3. Example 2

[0039] Structural identification of new compounds: The novel compound 1 prepared in Example 1 of this invention is an orange oil. High-resolution mass spectrometry yielded a quasi-molecular ion peak at m / z: 327.1226 [M+H]. + (The calculated value is 327.1227), combined with 1 H-NMR and 13 C-NMR determined the molecular formula of the compound to be C 19 H 18 O5, the calculated degree of unsaturation is 11. 1 H, 13C and HSQC show three methyl groups, two methoxy groups, three methine groups, and 11 quaternary carbons (including three carbonyl groups). In the HMBC spectrum, firstly, from H-1 to C-3, C-5, C-7, and Me-2; from H-4 to C-2, C-3, C-6, C-7, C-10, and Me-2; from Me-2 to C-3; and from OMe-3 to C-3, there is an HBMN correlation, forming a six-membered ring A with a methyl group substituted at position 2 and a methoxy group substituted at position 3. Secondly, H-8 shows an HBMN correlation with C-6, C-10, and C-14; forming a six-membered ring B with a carbonyl group substituted at position C-7. Rings A and B share C-5 and C-6. Me-14 correlates with HBMN at C-9 and C-13, forming a six-membered ring C with a methoxy group at position 11, carbonyl carbons at positions 12 and 13, and a gem-dimethyl group at position C-14. Rings B and C share C-9 and C-10. The NOESY diagram shows strong signals at positions 1 and 2, and clear signals at positions 3 and 4, further confirming the substituent positions. Thus, the structure of compound 1 has been determined and named siamensisoid C, a phenanthrene compound with a 6 / 6 / 5 tricyclic structure system. This is the first time it has been isolated from *Euonymus alatus* hainanensis and possesses an ortho-quinone structure.

[0040] Analysis of structure-related signals: .

[0041] The novel compound 2 prepared in Example 1 of this invention is a green amorphous solid. High-resolution mass spectrometry yields a quasi-molecular ion peak at m / z: 365.1003 [M+Na] + (The calculated value is 365.0996), combined with 1 H-NMR and 13 C-NMR determined the molecular formula of the compound to be C 19 H 18 O6, the calculated degree of unsaturation is 11. 1 H, 13 C and HSQC show three methyl groups, two methoxy groups, two methine groups, and twelve quaternary carbons (three carbonyl groups). According to 1 ¹H NMR data revealed that compound 2 was very similar to the isolated compound 1. The difference was that compound 2 had one more hydroxyl group substituted. Analysis of the signal on the ¹H MBC spectrum further confirmed the inference was correct: the C-8 position in compound 2 was substituted with a hydroxyl group, and compound 2 was named siamensisoid D.

[0042] Analysis of structure-related signals: .

[0043] Similarly, the structural formula of compound 3 can be determined: . Example 3

[0044] Screening of in vitro anti-inflammatory activity of phenanthrene-type diterpenoids BV-2 cells were cultured to the logarithmic growth phase, digested, and seeded into 96-well plates. After 24 h, different concentrations of Hainan euryale compounds (dissolved in DMSO) were added for 1 h, followed by the addition of LPS (1 μg / mL) to induce an inflammatory response, and the cells were cultured for another 24 h. 50 μL of the supernatant was reacted with Griess' reagent (solution A+B) for 10 min, and the absorbance at 540 nm was measured to calculate the NO inhibition rate. Blank controls, solvent controls, and an LPS positive control group were included to evaluate the anti-inflammatory effect of the extracts. The NO inhibition rate of each Hainan euryale extract was calculated.

[0045] The calculation formula is: Inhibition rate (%) = [(Model group absorbance - Drug group absorbance) ÷ (Model group absorbance - Blank group absorbance)] × 100% STR = (MG - TG) ÷ (MG - NG) × 100% in: Model group absorbance (MG): The absorbance of the model group Absorbance of blank group (NG): Absorbance of blank group Absorbance of the treatment group (TG): Absorbance of the treatment group Experimental results showed that the phenanthrene-type diterpenoids of this invention could effectively inhibit the release of NO in BV-2 cells, with compound 3 showing significant inhibitory effects (IC50). 50 The value was 11.35 ± 1.055 μM, as shown in Table 2.

[0046] Table 2. Effects of phenanthrene-type norditerpenes on LPS-induced NO release in BV-2 cells

[0047] Table 2 shows that compound 3 is more active than compound 2, while compound 1 is the least active. Based on the structures of the three compounds, it can be inferred that this is because compound 3 has no substituent at the R6 position and no ortho-quinone, while compounds 1 and 2 both have ortho-quinone. The reason why compound 2 is more active than compound 1 may be that compound 2 has an additional hydroxyl group at the R3 position, which introduces a hydroxyl group as a strong hydrogen bond donor, forming an additional hydrogen bond with the target. Example 4

[0048] Western blotting of LPS-induced expression of iNOS, COX-2, Nrf2, HO-1, and NQO1 proteins in RAW264.7 cells. Cell treatment: RAW264.7 cells were seeded on medium-sized dishes and randomly divided into control group, model group, and drug treatment groups at different concentrations. After 24 h of cell culture, the control group was replaced with DMEM high-glucose medium, the model group was replaced with DMEM high-glucose medium containing LPS (1 μg / mL), and the drug treatment groups were replaced with DMEM high-glucose medium containing the specified concentration of each drug + LPS (1 μg / mL). After 16 h of culture, the cells were collected, and total protein was extracted by lysing the cells using Western blotting and IP cell lysis buffer. After quantification using a BCA kit, the protein content was detected by Western blotting.

[0049] Gel preparation: Based on the molecular weight of the protein to be analyzed and the number of separating gels to be prepared, prepare a certain volume of 8-15% separating gel solution. Pour the separating gel into the gap between clean, vertically placed glass plates, leaving approximately 1 cm of space for the stacking gel at the top. Then, slowly add a small amount of double-distilled water to cover the separating gel. Let it stand at room temperature for about 20 minutes until the separating gel solidifies and a clear boundary line appears between the separating gel and the water. Pour off the double-distilled water from the gap between the glass plates and use absorbent paper to blot away any remaining moisture at the top of the gel, being careful not to let the absorbent paper touch the separating gel. Next, prepare a 4% stacking gel solution. Slowly pour it into the gap between the glass plates and quickly insert a clean Teflon comb, being careful not to create air bubbles. Let it stand vertically at room temperature until the stacking gel is completely solidified. Then, place the prepared gel in an electrophoresis tank containing an appropriate amount of electrophoresis buffer.

[0050] Electrophoresis: Remove the comb from the stacking gel, and then add 4-40 µg of sample per well and protein pre-stained marker to the corresponding lane. Connect the electrophoresis apparatus to the power supply. Initially set the voltage to 80 V until the sample enters the separating gel, then adjust the voltage to 120 V and continue electrophoresis. Analyze the progress of electrophoresis based on the markers until the bromophenol blue reaches near the bottom of the separating gel. Turn off the power to end the electrophoresis process.

[0051] Transfer: Cut the gel of the desired molecular weight into bands according to the marker indication range, and transfer them to the buffer solution to soak for 15 minutes. Then place them on an activated PVDF membrane (activation process: activate the PVDF membrane with methanol for 5 seconds, transfer it to ultrapure water for 5 minutes, and then soak it in transfer buffer for 5 minutes). Next, moisten the sponge with transfer buffer and assemble it in the following order: sponge-double filter paper-gel-membrane-double filter paper-sponge. Close the clamp, fix it in the electrophoresis tank, pour in an appropriate amount of transfer buffer, and transfer at a constant voltage of 100 V for a period of time. The specific time depends on the molecular weight of the protein.

[0052] Blocking and antibody incubation: After electroporation, remove the PVDF membrane, wash it in TBST solution for 10 minutes, and then incubate it in a 5% skim milk solution prepared with TBST at room temperature for 2 hours to block non-specific binding sites on the membrane. After blocking, wash the membrane twice with TBST for 10 minutes each time. Then place the membrane in a plastic-sealed bag, add the corresponding primary antibody dilution, and incubate overnight at 4°C to allow for sufficient antigen-antibody binding. The next day, remove the membrane from the plastic-sealed bag, wash it twice with TBST for 10 minutes each time, place it in a new plastic-sealed bag, add HRP-labeled rabbit or mouse secondary antibody to bind the primary antibody, and incubate on a shaker at room temperature for 2 hours.

[0053] Colorimetric reaction: After 2 hours of incubation with secondary antibody, the membrane was washed twice with TBST solution for 10 minutes each time, followed by a final wash with TBS for 5 minutes. Detection was then performed using ECL chemiluminescence immunoassay, and images were taken using a gel imaging system. Western Blot data analysis: The gray value of the target protein was divided by the gray value of the internal control to correct for error. The result represents the relative expression level of the target protein in a given sample.

[0054] (2) Effect of the compound on ROS levels in LPS-treated RAW264.7 cells ROS content assay: RAW264.7 cells were seeded in six-well plates and cultured for 18 hours, followed by incubation with different concentrations of the drug for 1 hour, and then stimulation with LPS for 18 hours. The culture medium was discarded, and the cells were washed with PBS, digested with trypsin for an appropriate time, and then collected in centrifuge tubes. The cells were washed twice with PBS and transferred to 1.5 mL EP tubes. The ROS probe was diluted to an appropriate ratio; 500 μL of ROS probe solution was added to each sample, and the cells were incubated at 37°C in the dark for 25-30 minutes, shaking every 3-5 minutes to ensure sufficient probe binding to the cells. The cells were washed three times with serum-free medium to remove unbound ROS probe; the cells were resuspended and filtered through a nylon mesh to remove cell clumps; the ROS content was detected by flow cytometry, and the mean values ​​were plotted.

[0055] (3) Statistical analysis Data were analyzed using GraphPad Prism. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 (* indicates a significant difference compared to the LPS group). (4) Experimental results Western blot analysis showed that compounds 2 and 3 significantly inhibited LPS-induced expression of iNOS and COX-2 proteins in RAW264.7 cells. Figure 19Compound 3 showed stronger inhibitory effects on iNOS at a concentration of 25 μM than compound 2. Regarding antioxidant effects, both compounds activated the Nrf2 signaling pathway and its downstream effector molecules HO-1 and NQO1, with compound 3 exhibiting a more pronounced activation effect. Figure 20 ).

[0056] ROS analysis results showed that both compounds effectively alleviated LPS-induced oxidative stress, with compound 3 showing a more pronounced effect. Figure 21 , Figure 22 ).

[0057] The comprehensive results indicate that both compounds 2 and 3 exert anti-inflammatory effects through a dual mechanism of inhibiting the production of pro-inflammatory mediators and enhancing cellular antioxidant defense. Furthermore, compound 3 outperformed compound 2 in all detection indicators, demonstrating its potential for further development into an anti-inflammatory drug, particularly for the intervention of neuroinflammatory-related diseases.

[0058] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A phenanthrene-type norditerpene, characterized in that, The structural formula is: ； In the formula, R1 is -H, -CH2CH3 or -CH3, R2 is =O, -OH, -OCH3 or -OCH2CH3, R3 is -H, -OH, -OCH3 or halogen, and R4, R5, R6 are =O, -OH, -H, -OCH3 or -OCH2CH3.

2. The phenanthrene-type diterpene as described in claim 1, characterized in that, It is one of the compounds with the following structural formula; ； Compounds 1-3 were named siamensisoid C (1); siamensisoid D (2); and 9-O-Demethyltrigonostemone (3), respectively.

3. The method for preparing a phenanthrene-type diterpene as described in claim 1, characterized in that, Includes the following steps: A) The dried medicinal material of Hainan Euphorbia hainanensis was pulverized, extracted with 95% ethanol, filtered, the filtrates were combined, and then concentrated and dried under reduced pressure to obtain a 95% ethanol extract. B) Disperse the 95% ethanol extract in water and extract it sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether fraction, ethyl acetate fraction and n-butanol fraction; C) The ethyl acetate fraction was subjected to column chromatography on silica gel, eluted with a petroleum ether-ethyl acetate gradient at a volume ratio of (1:0) to (1:1). The fractions were combined and separated into 14 fractions. The petroleum ether-ethyl acetate eluent was subjected to MCI and ODS column chromatography, eluted with a methanol-water gradient to obtain a methanol-water eluent. The acetonitrile-water eluent was purified by semi-preparative high performance liquid chromatography, eluted with acetonitrile-water as the mobile phase to obtain phenanthrene-type norditerpenoid compounds.

4. The method for preparing a phenanthrene-type diterpene as described in claim 3, characterized in that, The specific steps for chromatographic separation and purification of the ethyl acetate fraction in step C) are as follows: The ethyl acetate fraction is subjected to silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient at volume ratios of 100:1, 50:1, 25:1, 12:1, 6:1, 3:1, and 1:1, and the fractions are combined to obtain 14 fractions; Fractions 6, 7, 8, and 9 are combined and eluted with ODS and MCI, using a methanol-water gradient at volume ratios of 3:7, 5:5, 7:3, 9:1, and 1:0, and the fractions are combined to obtain 6 fractions; Fraction 3 is purified by semi-preparative high performance liquid chromatography, eluted with acetonitrile-water at a volume ratio of 49:51, to obtain compounds 1 and 2; Fractions 10, 11, 12, 13, and 14 were combined and subjected to silica gel column chromatography, eluted with a dichloromethane-methanol gradient at volume ratios of 100:0, 100:1, 50:1, 25:1, 12:1, 6:1, 3:1, 1:1, and 0:100, yielding 12 fractions. Fraction 4 was then subjected to petroleum ether-ethyl acetate, eluted with a gradient at volume ratios of 20:1, 10:1, 5:1, 3:1, and 1:1, yielding seven fractions. Fraction 2 was subjected to MCI and ODS, eluted with a methanol-water gradient at volume ratios of 3:7, 5:5, 7:3, 9:1, and 1:0, yielding six fractions. Fraction 3 was purified by semi-preparative high-performance liquid chromatography, eluted with acetonitrile-water at a volume ratio of 50:50, to obtain compound 3.

5. The use of the phenanthrene diterpenoids and their pharmaceutically acceptable salts as described in claim 1 in the preparation of drugs for the prevention and treatment of neuroinflammatory diseases.

6. The use of a plant extract containing the phenanthrene-type diterpenoids of claim 1 in the preparation of a drug for the prevention and treatment of neuroinflammatory diseases.

Images