A crotan-type diterpene compound and its preparation method and application

Abstract

The present invention discloses a crotan-type diterpene compound, its preparation method, and application, relating to the field of pharmaceutical technology. The present invention extracts a crotan-type diterpene compound of novel structure from sage. The compound has significant inhibitory activity against neuronal inflammation, exhibits excellent anti-neuritis effects, and significantly inhibits inflammatory mediators. It can be developed as a therapeutic drug for neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a crotan-type diterpene compound, a preparation method and an application thereof. Background Art

[0002] Mexican sage ( Salvia leucantha Cav is a species of the genus Salvia of the family Labiatae. Salvia Mexican sage is native to eastern and central Mexico in Central America and has been introduced to my country. Modern pharmacological research shows that Mexican sage improves microcirculation, dilates capillaries, has anti-inflammatory properties, and calms the nerves. It also has memory-enhancing, anti-aging, eye-improving properties, and relieves headaches and neuralgia.

[0003] Literature reports that the secondary metabolites of Salvia plants mainly include sesquiterpenes, diterpenes, triterpenes, flavonoids, phenylpropanoids and other compounds, and abietane-type (Abietane) and cleodane-type (Clerodane) diterpenes are the characteristic chemical components of Salvia.

[0004] Publication No. CN110092797A discloses a class of crotonane-type diterpenoid compounds and their pharmaceutical applications. These crotonane-type diterpenoid compounds extracted from sage are primarily used in cardioprotective drugs. Publication No. CN110003230A discloses crotonane-type diterpenoid compounds, pharmaceutical compositions, and applications thereof. The crotonane-type diterpenoid compounds extracted from sage are primarily used in the preparation of drugs and health foods for the treatment or prevention of type II diabetes and hyperlipidemia.

[0005] With the accelerating aging of the population, neurodegenerative diseases (NDs) have become a global health concern. BV-2 cells are mouse microglia, which are a major component of the central nervous system (CNS). Microglia are key mediators of neuroinflammation. Activation of microglia also leads to increased production of nitric oxide (NO). High levels of NO may lead to neuronal death by inhibiting mitochondrial cytochrome oxidase and mitochondrial respiration in neurons. Chronic microglial activation can lead to neurodegenerative diseases such as Alzheimer's disease. Currently, there are no targeted drugs for the treatment of neurodegenerative diseases, and existing drugs can only temporarily alleviate clinical symptoms but cannot prevent disease progression. Summary of the Invention

[0006] In order to solve the above problems, the present invention proposes a crotonane-type diterpene compound and its preparation method and application. A crotonane-type diterpene compound with a novel structure is extracted from sage, and has good preventive and therapeutic effects on neuritis and neurodegenerative diseases.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A crotan-type diterpene compound, including compounds 1-6 of the following structural formulas: .

[0009] The present invention also discloses a method for preparing a crotonyl diterpene compound, comprising the following steps:

[0010] (1) Crush the dried whole sage herb and extract it with 95% ethanol under reflux for 2-3 times, each time for 1-2 hours. Combine the extracts and concentrate under reduced pressure until there is no alcohol smell. After recovering the solvent, obtain the crude extract of Mexican sage.

[0011] (2) The crude extract is extracted with ethyl acetate to obtain an ethyl acetate extract;

[0012] (3) The extract was eluted with methanol solvent of different gradient concentrations to obtain different fractions; the different fractions were purified to obtain the structural formula compounds 1-6 of the present invention.

[0013] Furthermore, in step (3), the methanol solvents with different gradient concentrations are 60%, 70%, 80%, 90% and 100% methanol solvents, and after elution, five fractions are obtained in sequence, namely Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5.

[0014] Furthermore, the Fr.1 precipitated into block crystals in methanol solvent to obtain compound 1.

[0015] Furthermore, the Fr.2 was sequentially subjected to silica gel column chromatography, Sephadex LH-20 separation, and semi-preparative HPLC liquid phase separation to obtain compound 2, compound 4, and compound 6;

[0016] The elution system for the silica gel column chromatography and Sephadex LH-20 separation is dichloromethane / methanol; the separation system for the semi-preparative HPLC liquid phase separation is methanol / water.

[0017] Furthermore, the Fr.3 was subjected to silica gel column chromatography with an elution system of dichloromethane / methanol, and then to semi-preparative HPLC liquid phase separation with isocratic elution of methanol / water (50:50) to obtain compound 3 and compound 5.

[0018] The present invention also claims to protect the use of a crotan-type diterpenoid compound in anti-neuritis and / or treatment of neurodegenerative diseases.

[0019] The present invention also claims protection for a pharmaceutical composition comprising any one or more of the compounds of the structural formulas 1-6 and a pharmaceutically acceptable carrier.

[0020] The present invention also claims the use of the pharmaceutical composition in the preparation of anti-neuritis drugs and / or drugs for treating neurodegenerative diseases.

[0021] The present invention discloses a crotan-type diterpene compound, its preparation method, and its application. The invention provides a novel structural crotan-type diterpene compound extracted from sage using a specific preparation method. The compound exhibits significant inhibitory activity against neuronal inflammation, exhibits excellent anti-neuritis effects, and significantly inhibits inflammatory mediators. The compound can be developed as a therapeutic drug for neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The effects of compound 3 on proinflammatory cytokines, including (A) the half-inhibitory concentration of compound 3 on NO release; (B) the effects of different concentrations of compound 3 on the inflammatory factor IL-6; (C) the effects of different concentrations of compound 3 on the inflammatory factor IL-1 β (D) Effects of different concentrations of compound 3 on inflammatory factor TNF-α; (E) Effects of different concentrations of compound 3 on inflammatory factor COX-2; (F) Effects of different concentrations of compound 3 on inflammatory factor iNOS; ### P < 0.001 compared with the control group. * P <0.05, ** P <0.01, *** P <0.001 compared with the LPS group;

[0024] Figure 2The anti-inflammatory mechanism of compound 3 of the present invention is shown in Figure 3; wherein (A) the expression of iNOS and COX-2 proteins was detected by Western blot; (B) the relative protein expression of iNOS; (C) the relative protein expression of COX-2; (D) the expression of TLR4, p65 / p-p65, IkBα / p-IkBα, and MYD88 proteins was detected by Western blot; (E) the relative protein expression of TLR4; (F) the relative protein expression of p65 / p-p65; (G) the relative protein expression of IkBα / p-IkBα; and (H) the relative protein expression of MYD88. ### P < 0.001 compared with the control group. * P <0.05, ** P <0.01, *** P <0.001 compared with the LPS group;

[0025] Figure 3 is the H NMR spectrum of compound 1;

[0026] Figure 4 is the C NMR spectrum of compound 1;

[0027] Figure 5 1H–1H COSY pattern of compound 1;

[0028] Figure 6 is the H NMR spectrum of compound 2;

[0029] Figure 7 is the C NMR spectrum of compound 2;

[0030] Figure 8 For compound 2 1 H– 1 H COSY diagram;

[0031] Figure 9 is the H NMR spectrum of compound 3;

[0032] Figure 10 is the C NMR spectrum of compound 3;

[0033] Figure 11 1H–1H COSY pattern of compound 3;

[0034] Figure 12 is the H NMR spectrum of compound 4;

[0035] Figure 13 is the C NMR spectrum of compound 4;

[0036] Figure 14 1H–1H COSY pattern of compound 4;

[0037] Figure 15 is the H NMR spectrum of compound 5;

[0038] Figure 16 is the C NMR spectrum of compound 5;

[0039] Figure 17 For compound 5 1 H– 1 H COSY diagram;

[0040] Figure 18 is the H NMR spectrum of compound 6;

[0041] Figure 19 is the C NMR spectrum of compound 6;

[0042] Figure 20 This is the 1H–1H COSY pattern of compound 6. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0044] Example 1

[0045] A method for preparing a crotonyl diterpene compound comprises the following steps:

[0046] (1) The dried whole herb of Sage (10.0 kg) was crushed and extracted with 95% ethanol under reflux for 3 times, each time for 2 h. The combined extracts were concentrated under reduced pressure until there was no alcohol smell. The solvent was recovered to obtain the crude extract of Sage.

[0047] (2) The crude extract is extracted with ethyl acetate to obtain an ethyl acetate extract;

[0048] (3) The extract was eluted with 15 L each of 60%, 70%, 80%, 90% and 100% methanol solvents. After concentrating the eluent, five fractions were obtained, namely Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5. Fr.1 (5.5 g) was precipitated as block crystals in methanol solvent to obtain compound 1 (124.5 mg). Fr.2 (4.4 g) was purified by silica gel column chromatography (100:1 → 0:100, CH2Cl2 / CH3OH) and Sephadex LH-20 (1:1, CH2Cl2 / CH3OH) and semi-preparative HPLC (55:45, MeOH / H2O) to obtain compound 2 (7.7 mg), compound 4 (4.4 mg) and compound 6 (15.4 mg). Fr.3 (2.5 g) was purified by silica gel column chromatography (100:1→0:100, CH2Cl2 / CH3OH) and semi-preparative HPLC (50:50, MeOH / H2O) to give compound 3 (82 mg) and compound 5 (3.5 mg).

[0049] Example 2

[0050] Compound structure identification

[0051] Compound 1 is a colorless block crystal. Its HR-ESI-MS data showed that the quasi-molecular ion peak m / z 541.1686 [M + Na] + The positive ion peak at (calculated as C 26 H 30 O 11 Na, 541.1680) to determine its molecular formula is C 26 H 30 O 11 .according to 1 H spectrum data showed the presence of glucose signal [ d H 4.27, (d J = 7.8 Hz), H-1ʹ; d H 2.92, m, H-2ʹ; δH3.16, m, H-3ʹ; d H 3.01, m, 58 H-4ʹ; d H 3.06, m, H-5ʹ; d H 3.61, m, H-6ʹa; and d H 3.36, m, H-6ʹb]. In addition, the aglycone 1 H NMR data showed that d H 6.96 (dd, J = 7.8, 2.4 Hz), 6.59 (dd, J = 7.8, 2.4 Hz)、7.73(d, J = 1.8 Hz), there are four olefin protons at 7.85 (s); there is also a singlet methyl d H 1.48 (s). Combined with HMBC data, 13 C NMR spectroscopy revealed the furan ring ( d C 124.1, 109.1, 144.8 and 142.1), α , β Conjugated carbonyl ( d C 170.2, 133.6 and 139.7), γ-lactone fragment ( d C 38.3, 133.3, 74.6 and 177.6) and one glucose unit ( d C 99.4, 73.8, 76.4, 70.3, 76.6 and 61.3). The above information shows that the aglycone part of compound 1 is very similar to de-O-acetylsalvigenolide, and the only difference is that compound 1 has an additional glucose unit. In addition, the coupling constant value ( J = 7.8 Hz) showed that the glucose structure of compound 1 was β-glucose. The above inference was further clarified by 2D NMR data. In the HMBC spectrum, H-1 ( d H 4.27) and C-6 ( d C 74.0) indicated that glucose was linked to C-6 via an ether bond. Therefore, the planar structure of compound 1 was determined. The relative configuration of compound 1 was inferred by NOESY spectroscopy. There was a NOESY correlation between H-19 / H-6 / H-1ʹ / H-3ʹ / H-5ʹ, and a NOESY correlation between H-2ʹ / H-4ʹ / H-6ʹ. The NOESY correlation of H-10 / H-8 indicated that they were in the same orientation. Crystals of compound 1 were obtained in methanol solvent. Finally, the absolute configuration of compound 1 was confirmed by X-ray diffraction with an appropriate Flack parameter [0.15 (1)]. It was retrieved as a new compound from the Scifinder database and named Saleucane A.

[0052] Compound 2 is a colorless oil. Its HR-ESI-MS data showed that the quasi-molecular ion peak m / z 409.1454 [M + Na] + The positive ion peak at (calculated as C 21 H 22 O7Na, 409.1258) to determine its molecular formula is C 21 H 22 O7. 1 H NMR spectrum in d H 5.98 (dd, J = 9.6, 2.4 Hz), 6.31 (m), 5.36 (s) and 6.82 (s) show four olefinic proton signals; d H 5.04 (t, J = 7.8Hz), 5.36(s) and 5.37(s), showing three oxidized methylene signals and one oxygen-containing methylene signal. d H 4.24 (dd, J = 7.8, 2.4 Hz), 4.88 (dd, J = 7.8, 2.4 Hz). Detailed analysis of the 1D and 2D NMR spectra of 2 showed that its structure was similar to that of salvifaricin (Savona et al., 1983). The main difference is that one of the furan rings in salvifaricin is replaced by compound 2. c -lactone substitution. The above inference is obtained by H-16 ( d H 6.82) and C-12 ( d C 77.2), C-13 ( d C 143.7), C-14 ( d C 86.2) and C-15 ( d C 173.1) was further elucidated by HMBC correlation. In addition, from -OMe ( d H 3.31) to C-14 ( d C 86.2) indicates that the methoxy group is located at C-14. In addition, the NOESY correlations of H-10 / H-19 / H-17 indicate that these protons are α In contrast, based on the Me-20 / H-12 correlation in the NOESY spectrum, Me-20 and H-12 are βSubsequently, the absolute configuration of compound 2 was determined by comparing the experimental ECD curve of compound 2 with the calculated ECD curve, and it was named Saleucane B.

[0053] Compound 3, colorless block crystals, its HR-ESI-MS data showed that the quasi-molecular ion peak m / z 359.0890[M + Na] + The positive ion peak at (calculated as C 20 H 16 O5Na, 359.0890) to determine its molecular formula is C 20 H 16 O5. Its NMR data are very similar to those of 3-epi-tilifodiacetate. Subsequently, H-1 ( d H 2.90) to C-6, C-10 ( d C 143.6), C-9 ( d C 130.6) related, H-12 ( d H 6.30) and C-11 ( d C 147.2), C-18 ( d C 124.1), C-17 ( d C 170.4), C-13 ( d C 120.8)、C-14( d C 108.8) and C-16 ( d C 142.4) and H-20 ( d H 2.59) is related to C-4 (δC 137.0), combined with H2-1-H2-2-H-3-H2-20, H-6-H-7, H-5-H-19 and H-14-H-15 1 H- 1 HCOSY correlation showed that the planar structure of compound 3 was similar to that of 3-epi-tilifodiolide. Finally, a high-quality crystal of compound 3 was obtained from its methanol solution, and Cu Ka radiation X-ray single crystal diffraction clearly elucidated that compound 3 was 3 S , 12 SThe configuration of compound 3 was determined [Flack parameter: 0.09 (7)]. Subsequently, the optical rotation of compound 3 ([α]25 D +62.77, c 0.046, MeOH) and 3-epi-tilifodiolide ([α]25 D+139, c 0.046, MeOH) were obtained, and the absolute configuration of 3-epi-tilifodiolide was further corrected to 3. S , 12 S The compound was identified as a new compound by searching the Scifinder database and named Saleucane E.

[0054] Compound 4, colorless oil, was detected by HR-ESI-MS data. m / z 375.0840 [M + Na] + (The calculated value is C 20 H 16 O6Na, 375.0839) and the molecular formula deduced from DEPT spectrum is C 20 H 16 O6. Analysis of the NMR data of 4 shows that it is very similar to tilifodiolide. The main difference is that one of the methylene groups in tilifodiolide is oxidized to an oxygen-containing methyl group in compound 4. d H 4.92) and C-6 ( d C 125.9), C-10 ( d C The HMBC correlation of 148.6) and the 1H-1H COSY correlation of H-1-H2-2-H-3 confirm that the methyl group is at the C-1 position. In the NOESY spectrum, the H-1 / H-2α / H-5 correlations indicate that these protons are oriented in the same direction. The NOESY correlation of H-3 / H-2β indicates that they are oriented in opposite directions. Therefore, Figure 3 As shown in (6a-6d), four seemingly reasonable configurations of compound 4 were deduced. 13 C NMR data and DP4 + The analysis further confirmed that the relative configuration of compound 4 was 6α (1 S *、3 R * and 12 R *). Finally, by comparing its experimental ECD curve with the calculated ECD curve, the absolute configuration of compound 4 was determined to be 1 S , 3 R , 12 R The compound was identified as a new compound by searching the Scifinder database and named Saleucane F.

[0055] Compound 5, yellow oil, m / z 357.1235 [M + H] according to HRESIMS + (C 20 H 21 The calculated value of O6 is 357.1333) and the molecular formula is determined to be C 20 H 20 O6, indicating 11 degrees of unsaturation. Analysis of the 1D NMR data of 5 showed that its structure is similar to that of bacchotricuneatin B. The main difference is that one of the methyl groups in bacchotricuneatin B is replaced by an oxymethylene group in 5. In addition, based on the d H 3.77) to C-8 ( d C 138 75.8)、C-9( d C 125.9), C-10 ( d C 39.8) and C-11 ( d C 29.7), along with the remaining unsaturation, the rare oxetane unit was constructed. Subsequently, NOESY correlations at H-6β / H-10 / H-11 / H-12 indicated that these protons were in a β orientation. In contrast, NOESY correlations at H-6α / H-19 / H-20 indicated that they were in an α orientation. Finally, by comparing its experimental and calculated ECD curves, the absolute configuration of compound 5 was determined. It was named Saleucane G.

[0056] Compound 6, white amorphous powder, based on HRESIMS data m / z [M + H] + 437.1206 (calculated value is C 22 H 22 O8, 437.1207) determined its molecular formula C 22 H 22 O8. A step-by-step comparison of the NMR data of compound 6 with that of Salvileucanthsin A showed that the only difference between the two was the addition of an acetyl group in compound 6. Subsequently, from H-1ʹ ( d H 2.08)、H-7( d H 5.96) and C-2ʹ ( d CAn HMBC correlation of 172.5 indicated that the acetyl group was attached to C-2 via an ether bond. Analysis of the NOESY spectrum of compound 6 revealed that its relative configuration was identical to that of Salvileucanthsin A. Finally, the absolute configuration of compound 6 was determined by comparing its experimental and calculated ECD curves. This compound was identified as a new compound through a Scifinder database search and named Saleucane I.

[0057] The data assignments of compounds 1-6 are shown in Table 1 and Table 2:

[0058]

[0059]

[0060] Example 3

[0061] Evaluation of the anti-neuritis activity of compounds 1-6 of the present invention

[0062] 3.1 Activity screening method

[0063] 3.1.1 Preparation of compound stock solutions

[0064] The isolated compounds 1–6 of the present invention were prepared into 20 mM stock solutions using DMSO and stored in a refrigerator at 4°C.

[0065] 3.1.2 Culture and treatment of BV-2 cells

[0066] BV-2 cells were cultured in a constant-temperature incubator set at 5% CO2 and 37°C using DMEM supplemented with 10% fetal bovine serum. Cell growth was observed and, when the cell density reached above 90%, cells were passaged. The cells were washed once with PBS and digested with 1 mL of trypsin for 2 minutes to detach into single cells. The cells were collected in a centrifuge tube and centrifuged. Fresh culture medium was added to dilute the cells and culture was continued.

[0067] 3.1.3 MTT assay to detect the effects of compounds on BV-2 cell viability

[0068] Cells in logarithmic growth phase were counted and diluted with culture medium to prepare cell suspension, which was evenly plated in 96-well plates (2.5×10 4Cells were plated at 40 μM for 24 hours at 37°C and 5% CO2 in an incubator. Experimental and control groups were set up. The experimental group consisted of 40 μM test samples prepared from the stock solutions of compounds 1–6; the control group consisted of a blank control. 10 μL of test sample was added to each well, with three replicates per well. After a further 24-hour incubation, 10 μL of MTT (5 mg / mL) reagent was added to each group of cells. After incubation at 37°C for 4 hours, the culture medium was removed and 100 μL of DMSO was added to each well. The absorbance (A) of each well was measured at 490 nm using a microplate reader. The calculation formula is as follows: .

[0069] 3.1.4 Evaluation of the inhibitory effect on NO production using the Griess method

[0070] The NO content in the cell culture medium was detected by Griess reagent. BV-2 cells were seeded in 96-well culture plates (2.5×10 4 cells / well) and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Experimental and positive control groups were set up. The experimental group was a test sample with a concentration of 40 μM prepared from the stock solution of compounds 1-6; the positive control group used the NO inhibitor minocycline hydrochloride. 10 μL of the test sample with a final concentration of 40 μM was added to the cells of each experimental group, and 1 μg / mL LPS was added to stimulate the cells. After incubation for 24 hours, according to the instructions of the Griess kit, 50 μL of the cell culture supernatant was aspirated and 50 μL of Griess Reagent Ⅰ and 50 μL of Griess Reagent Ⅱ solution were added to measure the production of NO. The absorbance of the sample was measured at 540 nm. After that, the compounds with better initial activity were selected for IC 50 Activity determination (final concentrations of monomer compounds were (50, 25, 12.5, 6.25, 3.125, 1.5625 m Minocycline was selected as the positive drug. Data were statistically analyzed using Prism and SPSS 26.0 software. P < 0.05 indicated statistical significance. Each group was repeated three times.

[0071] The results showed that the three croton-type diterpenoid compounds 2, 3 and 6 in the present invention exhibited good anti-neuritis activity (IC 50 The half-inhibitory concentrations (CIs) of the compounds of the present invention were 3.29-13.38 μM, and were superior to the positive drug minocycline (18.89 μM), as shown in Table 3. The data in Table 3 indicate that the compounds of the present invention have a strong anti-neuritis effect.

[0072] .

[0073] 3.1.5 Detection of inflammatory gene expression by qPCR

[0074] Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of BV-2 cells. 5 The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The experiment included one blank group, one model group, and three experimental groups. The blank group was treated with DMEM medium; the model group was treated with LPS solution at a final concentration of 1 μg / mL; the experimental groups were treated with the stock solutions of compounds 1-6, with the stock solutions of each compound set at a concentration of 2 μg / mL. m M, 5 m M, 10 m Three concentrations of test sample were added; LPS solution and test sample solution at a final concentration of 1 μg / mL were added to each well and incubated in an incubator for an additional 12 hours. Total cellular RNA was extracted and reverse transcribed. The cDNA of each test sample was amplified using a fluorescent quantitative PCR reaction system. After completion of the reaction, the amplification and melting curves were confirmed, and the Ct values ​​of the test samples were recorded. GAPDH was used as an internal reference, and the expression levels of each gene were calculated based on the Ct values ​​for analysis. The fold change was calculated as 2 - ∆∆Ct. The primer sequences used in the experiment were used. Each data set was replicated three times.

[0075] 3.1.6 Western Blot Detection of NF-κB Signaling Pathway-Related Protein Expression

[0076] BV-2 cells were cultured at 8 × 10 5 The cells were inoculated into 6-well plates at a concentration of 1 μg / well and cultured overnight in a cell culture incubator. The experiment set up 1 blank group, 1 model group and 3 experimental groups. The blank group was added with DMEM culture medium; the model group was added with LPS solution with a final concentration of 1 μg / mL; the experimental group was the stock solution of compound 3, and the stock solution of the compound was set at a concentration of 2 μg / mL. m M, 5 m M, 10 m Each well was added with LPS solution with a final concentration of 1 μg / mL and LPS solution with concentrations of 2, 5, and 10 μg / mL. mM of the test sample solution was added and incubated in an incubator for 24 hours. Cells were harvested after centrifugation, and total protein was extracted using SDS cell lysis buffer containing 1% protease inhibitors PMSF and phosphoprotease inhibitors. Total protein concentration was determined using a BCA assay kit, and the proteins were denatured by heating at 95°C. Proteins were separated by electrophoresis using a 10% SDS-PAGE gel (stack gel voltage 80 V for 30 minutes; separation gel voltage 110 V for 60 minutes) and transferred to a PVDF membrane (constant voltage 120 V for 90 minutes). The membrane was blocked with 10% BSA solution at room temperature for 1 hour. The blocked membrane was then incubated with the specific primary antibody overnight at 4°C. After removal and return to room temperature, the membrane was washed three times with 1× TBST for 5 minutes each. The membrane was then incubated with an HRP-conjugated secondary antibody for 2 hours at room temperature and washed three times with 1× TBST for 5 minutes each. Finally, the membrane was developed using the developer solution prepared according to the ECL chemiluminescence kit instructions. The grayscale values ​​of protein bands were analyzed using ImageJ software. The experiment was repeated three times.

[0077] Real-time fluorescence quantitative PCR (qRT-PCR) was used to determine the effect of compound 3 on the expression of proinflammatory cytokines IL-6, IL-1β, TNF-α, COX-2 and iNOS. Figure 1 and Figure 2 As shown in the figure, after treatment with compound 3 (at concentrations of 2, 5, and 10 μM, respectively), the mRNA levels of IL-6, IL-1β, TNF-α, COX-2, and iNOS were significantly decreased in a dose-dependent manner. These data indicate that compound 3 improves LPS-induced inflammatory response by inhibiting the gene expression of IL-6, IL-1β, TNF-α, COX-2, and iNOS. Figure 1 and Figure 2 Compound 3 is abbreviated as “comp.3” or “compound.3”.

[0078] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute a separate or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0079] Finally, it should be noted that the embodiments disclosed in the present invention are only preferred embodiments of the present invention, which are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A crotan-type diterpene compound, characterized in that: Selected from compound 2, compound 3 or compound 6 of structural formula: .

2. A method for preparing a crotonyl diterpene compound according to claim 1, characterized in that: The following steps are involved: (1) The dried whole herb of sage was crushed and extracted with 95% ethanol under reflux for 2-3 times, each time for 1-2 h. The combined extracts were concentrated under reduced pressure until there was no alcohol smell. The solvent was recovered to obtain the crude extract of Mexican sage. (2) The crude extract is extracted with ethyl acetate to obtain an ethyl acetate extract; (3) The extract was eluted with methanol solvent of different gradient concentrations to obtain different fractions; the different fractions were purified to obtain the structural formula compounds 2, 3 and 6 of the present invention.

3. The method for preparing a crotonyl diterpene compound according to claim 2, wherein: In step (3), the methanol solvents with different gradient concentrations are 60%, 70%, 80%, 90% and 100% methanol solvents, and after elution, five fractions are obtained in sequence, namely Fr.1, Fr.2, Fr.3, Fr.4 and Fr.

5.

4. The method for preparing a crotonyl diterpene compound according to claim 3, wherein: The Fr.2 was sequentially subjected to silica gel column chromatography, Sephadex LH-20 separation, and semi-preparative HPLC liquid phase separation to obtain compound 2 and compound 6; The elution system for the silica gel column chromatography and Sephadex LH-20 separation is dichloromethane / methanol; the separation system for the semi-preparative HPLC liquid phase separation is methanol / water.

5. The method for preparing a crotonyl diterpene compound according to claim 3, wherein: The Fr.3 was subjected to silica gel column chromatography with a dichloromethane / methanol elution system, and then to semi-preparative HPLC liquid phase separation with methanol / water isocratic elution to obtain compound 3.

6. Use of the crotan-type diterpenoid compound according to claim 1 in the preparation of drugs for treating neuritis and / or neurodegenerative diseases.

7. A pharmaceutical composition, characterized in that: Comprising any one or more of the compounds of the structural formula according to claim 1 and a pharmaceutically acceptable carrier.

8. Use of the pharmaceutical composition according to claim 7 in the preparation of anti-neuritis drugs and / or drugs for treating neurodegenerative diseases.

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