Clerodane diterpenoid compound as well as preparation method and application thereof

Abstract

The invention discloses a clerodane diterpenoid compound as well as a preparation method and application thereof, and relates to the technical field of medicines. The clerodane diterpenoid compound with a novel structure is extracted from salvia officinalis, has obvious activity inhibition on nerve cell inflammation, has a good anti-neuritis effect, has an obvious inhibition effect on inflammatory mediators, and can be developed as a medicine for treating 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 and a preparation method and application thereof. Background Art

[0002] Mexican sage (Salvia leucantha Cav) is a plant of the genus Salvia in the family Labiatae. Mexican sage is native to eastern and central Mexico in Central America and has been introduced into my country. Modern pharmacological studies have shown that Mexican sage plants have the effects of improving microcirculation, dilating microvessels, anti-inflammatory, calming the nerves, enhancing memory, anti-aging, improving eyesight, and relieving headaches and neuralgia.

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

[0004] Publication No. CN110092797A discloses a class of crotan-type diterpenoid compounds and their application in pharmaceutical preparation. The crotan-type diterpenoid compounds extracted from sage are mainly used for cardioprotective drugs. Publication No. CN110003230A discloses crotan-type diterpenoid compounds and their pharmaceutical compositions and their applications. The crotan-type diterpenoid compounds extracted from sage in this technical solution are mainly used for preparing drugs and health foods for treating or preventing type II diabetes and hyperlipidemia.

[0005] With the accelerated aging of the population, neurodegenerative diseases (ND) have gradually become a global health problem. BV-2 cells are mouse microglia, which are the main components of the central nervous system (CNS) among glial cells. Microglia are key mediators of neuroinflammation. The activation of microglia also leads to increased production of nitric oxide (NO), producing high levels of NO, which may lead to neuronal death by inhibiting mitochondrial cytochrome oxidase and mitochondrial respiration in neurons. Chronic activation of microglia can lead to neurodegenerative diseases such as Alzheimer's disease. At present, there are no targeted drugs for the treatment of neurodegenerative diseases, and existing drugs can only temporarily relieve their clinical symptoms, but still cannot prevent the progression of the disease. Summary of the invention

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

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

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

[0009]

[0010] The present invention also discloses a method for preparing a crotan-type diterpene compound, comprising the following steps:

[0011] (1) The dried whole sage herb is crushed, and reflux-extracted with 95% ethanol for 2-3 times, each time for 1-2 hours. The extracts are combined and concentrated under reduced pressure until there is no alcohol taste. The solvent is recovered to obtain a crude extract of Mexican sage;

[0012] (2) extracting the crude extract with ethyl acetate to obtain an ethyl acetate extract;

[0013] (3) The extract is eluted with methanol solvent of different gradient concentrations to obtain different fractions; the different fractions are purified to obtain the structural formula compounds 1-6 of the present invention.

[0014] Furthermore, in step (3), the methanol solvents with different gradient concentrations are 60%, 70%, 80%, 90% and 100% methanol solvents, and after elution, 5 fractions are obtained in sequence, namely Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5.

[0015] Furthermore, the Fr.1 precipitates block crystals in methanol solvent to obtain compound 1.

[0016] Further, 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;

[0017] The elution system of the silica gel column chromatography and Sephadex LH-20 separation is dichloromethane / methanol; the separation system of the semi-preparative HPLC liquid phase separation is methanol / water.

[0018] 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.

[0019] The present invention also claims to protect the use of a crotan-type diterpene compound in anti-neuritis and / or treatment of neurodegenerative diseases.

[0020] The present invention also claims protection for a pharmaceutical composition, which comprises any one or more of the compounds of the structural formula 1-6 and a pharmaceutically acceptable carrier.

[0021] The present invention also claims to protect the use of the pharmaceutical composition in the preparation of anti-neuritis drugs and / or drugs for treating neurodegenerative diseases.

[0022] The crotan-type diterpene compound of the present invention and its preparation method and application have the beneficial effects that: the present invention extracts a crotan-type diterpene compound of a novel structural formula from sage by a specific preparation method. The compound of the present invention has obvious active inhibition on nerve cell inflammation, has good anti-neuritis effect, has significant inhibitory effect on inflammatory mediators, and can be developed as a therapeutic drug for neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative labor.

[0024] Figure 1 is the half-inhibitory concentration of compound 3 on NO release ( Figure 1 -A); Effects of different concentrations of compound 3 on inflammatory factor IL-6 ( Figure 1 -B), IL-1β ( Figure 1 -C), TNF-α( Figure 1 -D)、COX-2( Figure 1 -E)、iNOS( Figure 1 -F), among which ### P<0.001 compared with the control group. *P<0.05, **P<0.01, ***P<0.001 compared with the LPS group;

[0025] Figure 2It is the anti-inflammatory mechanism of compound 3 of the present invention; wherein (A) Western blot method is used to detect the expression of iNOS and COX-2 proteins; (B) the relative protein expression of iNOS; (C) the relative protein expression of COX-2; (D) Western blot method is used to detect the expression of TLR4, p65 / p-p65, IkBα / p-IkBα, and MYD88 proteins; (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; (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;

[0026] Figure 3 is the H NMR spectrum of compound 1;

[0027] Figure 4 is the NMR carbon spectrum of compound 1;

[0028] Figure 5 1H–1H COSY pattern of compound 1;

[0029] Figure 6 is the H NMR spectrum of compound 2;

[0030] Figure 7 is the NMR carbon spectrum of compound 2;

[0031] Figure 8 For compound 2 1 H– 1 H COSY diagram;

[0032] Fig. 9 is the H NMR spectrum of compound 3;

[0033] Fig.10 is the NMR carbon spectrum of compound 3;

[0034] Fig.11 1H–1H COSY pattern of compound 3;

[0035] Fig.12 is the H NMR spectrum of compound 4;

[0036] Fig.13 is the NMR carbon spectrum of compound 4;

[0037] Fig.14 1H–1H COSY pattern of compound 4;

[0038] Fig.15 is the H NMR spectrum of compound 5;

[0039] Fig.16 is the NMR carbon spectrum of compound 5;

[0040] Fig.17 For compound 5 1 H– 1 H COSY diagram;

[0041] Fig.18 is the H NMR spectrum of compound 6;

[0042] Fig.19 is the NMR carbon spectrum of compound 6;

[0043] Fig. 20 1H–1H COSY pattern of compound 6. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Example 1

[0046] A method for preparing a crotan-type diterpene compound comprises the following steps:

[0047] (1) The dried whole sage herb (10.0 kg) was crushed and extracted with 95% ethanol under reflux for 3 times, each time for 2 hours. The extracts were combined and concentrated under reduced pressure until there was no alcohol taste. The solvent was recovered to obtain a crude extract of Mexican sage;

[0048] (2) extracting the crude extract with ethyl acetate to obtain an ethyl acetate extract;

[0049] (3) The extract was eluted with 15 L each of 60%, 70%, 80%, 90% and 100% methanol solvents, and the eluent was concentrated to obtain five fractions, 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, CH 2 Cl 2 / CH3OH) and Sephadex LH-20 (1:1, CH2Cl2 / CH 3 OH) and semi-preparative HPLC (55:45, MeOH / H 2O) 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, CH 2 Cl 2 / CH 3 OH) and semi-preparative HPLC (50:50, MeOH / H 2 O) to obtain compound 3 (82 mg) and compound 5 (3.5 mg).

[0050] Example 2

[0051] Compound structure identification

[0052] Compound 1 is a colorless block crystal. Its HR-ESI-MS data shows that the quasi-molecular ion peak m / z541.1686[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 that there was a glucose signal [δ H 4.27, (dJ = 7.8 Hz), H-1′; δ H 2.92,m,H-2′; δH 3.16,m,H-3′; δ H 3.01,m,58H-4′;δ H 3.06,m,H-5′;δ H 3.61,m,H-6′a; and δ H 3.36,m,H-6′b]. In addition, the aglycone 1 H NMR data show that in δ H There are four olefin protons at 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), and 7.85 (s); there is also a single peak methyl δ H 1.48(s). Combined with HMBC data, 13 CNMR spectra revealed that the furan ring (δ C 124.1, 109.1, 144.8 and 142.1), α, β conjugated carbonyl (δ C 170.2, 133.6 and 139.7), γ-lactone fragment (δ C 38.3, 133.3, 74.6 and 177.6) and one glucose unit (δC 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) shows that the glucose structure of compound 1 is β-glucose. The above inference is further clarified by 2D NMR data. In the HMBC spectrum, H-1 (δ H 4.27) and C-6 (δ C 74.0) indicated that glucose was connected 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 spectrum. There was NOESY correlation between H-19 / H-6 / H-1′ / H-3′ / H-5′, and there was NOESY correlation between H-2′ / H-4′ / H-6′. 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 a suitable Flack parameter [0.15(1)]. It was retrieved as a new compound from Scifinder database and named Saleucane A.

[0053] Compound 2 is a colorless oil. Its HR-ESI-MS data show that the quasi-molecular ion peak is m / z 409.1454 [M+Na] + The positive ion peak at (calculated as C 21 H 22 O 7 Na, 409.1258) to determine its molecular formula is C 21 H 22 O 7 . 1 H NMR spectrum at δ H Four olefinic proton signals are shown at 5.98 (dd, J = 9.6, 2.4 Hz), 6.31 (m), 5.36 (s) and 6.82 (s); H Three oxidized methylene signals and one oxygen-containing methylene signal are shown at 5.04 (t, J = 7.8 Hz), 5.36 (s) and 5.37 (s), δ 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 the γ-lactone of compound 2. The above inference was obtained by H-16 (δH 6.82) and C-12 (δ C 77.2), C-13(δ C 143.7), C-14(δ C 86.2) and C-15 (δ C 173.1) was further clarified by HMBC correlation. H 3.31) to C-14 (δ C 86.2) indicated that the methoxy group was located at C-14. In addition, the NOESY correlation of H-10 / H-19 / H-17 indicated that these protons were in the α orientation. In contrast, based on the correlation of Me-20 / H-12 in the NOESY spectrum, Me-20 and H-12 were in the β orientation. Subsequently, by comparing the experimental ECD curve of compound 2 with the calculated ECD curve, the absolute configuration of compound 2 was determined and named Saleucane B.

[0054] Compound 3, colorless block crystals, its HR-ESI-MS data showed that the quasi-molecular ion peak m / z359.0890[M+Na] + The positive ion peak at (calculated as C 20 H 16 O 5 Na, 359.0890) to determine its molecular formula is C 20 H 16 O 5 Its NMR data are very similar to those of 3-epi-tilifodiacetate. Subsequently, HMBC revealed that H-1(δ H 2.90) to C-6, C-10 (δ C 143.6), C-9(δ C 130.6) related, H-12 (δ H 6.30) and C-11(δ C 147.2), C-18(δ C 124.1), C-17(δ C 170.4), C-13(δ C 120.8), C-14(δ C 108.8) and C-16 (δ C 142.4) and H-20 (δ H 2.59) is related to C-4 (δC 137.0), and the combined H 2 -1-H 2 -2-H-3-H 2 -20, H-6-H-7, H-5-H-19 and H-14-H-151 H- 1 HCOSY correlation indicated 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 single-crystal X-ray diffraction with Cu Ka radiation clearly elucidated that compound 3 was 3S, 12S in configuration [Flack parameter: 0.09 (7)]. Subsequently, the optical rotation of compound 3 was obtained ( +62.77, c 0.046, MeOH) and 3-epi-tilifodiolide ([α]25D+139, c 0.046, MeOH), and the absolute configuration of 3-epi-tilifodiolide was further corrected to 3S, 12S. The new compound was retrieved from Scifinder database and named Saleucane E.

[0055] Compound 4, colorless oil, m / z 375.0840 [M+Na] according to its HR-ESI-MS data + (The calculated value is C 20 H 16 O 6 Na, 375.0839) and the molecular formula deduced from DEPT spectrum is C 20 H 16 O 6 Analysis of the NMR data of 4 showed that it was very similar to tilifodiolide. The main difference was that one of the methylene groups of tilifodiolide was oxidized to an oxygen-containing methyl group in compound 4. H 4.92) and C-6 (δ C 125.9), C-10(δ C 148.6) HMBC correlation and H-1-H 2 The 1H-1H COSY correlation of -2-H-3 confirms that the methyl group is at the C-1 position. In the NOESY spectrum, the correlation of H-1 / H-2α / H-5 shows that these protons are in the same direction. The NOESY correlation of H-3 / H-2β shows that they are in opposite directions. Therefore, Figure 3 As shown in (6a-6d), four plausible 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 (1S*, 3R* and 12R*). Finally, by comparing its experimental ECD curve with the calculated ECD curve, the absolute configuration of compound 4 was determined to be 1S, 3R, and 12R. The new compound was retrieved from the Scifinder database and named Saleucane F.

[0056] Compound 5, yellow oil, according to HRESIMS at m / z 357.1235 [M+H] + (C 20 H 21 O 6 The calculated value, 357.1333) determines the molecular formula to be C 20 H 20 O 6 , indicating 11 unsaturations. Analysis of the 1D NMR data of 5 showed that its structure was similar to that of bacchotricuneatin B. The main difference was that one of the methyl groups in bacchotricuneatin B was replaced by an oxymethylene group in 5. In addition, based on the results from H-20 (δ H 3.77) to C-8 (δ C 13875.8), C-9(δ C 125.9), C-10(δ C 39.8) and C-11 (δ C 29.7), together with the remaining one unsaturation, the rare oxetane unit was constructed. Subsequently, the NOESY correlation of H-6β / H-10 / H-11 / H-12 showed that these protons were β-oriented. In contrast, the NOESY correlation of H-6α / H-19 / H-20 showed that they were α-oriented. Finally, the absolute configuration of compound 5 was determined by comparing its experimental ECD curve with the calculated ECD curve. It was named Saleucane G.

[0057] Compound 6, white amorphous powder, based on HRESIMS data m / z [M+H] + 437.1206 (calculated value is C 22 H 22 O 8 , 437.1207) determined its molecular formula C 22 H 22 O 8 The 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. H 2.08), H-7(δ H 5.96) and C-2′(δ C172.5) HMBC correlation indicated that the acetyl group was connected to C-2 via an ether bond. Analysis of the NOESY spectrum of compound 6 showed that its relative configuration was the same as that of Salvileucanthsin A. Finally, by comparing the experimental ECD curve of compound 6 with the calculated ECD curve, the absolute configuration of compound 6 was determined. It was retrieved as a new compound through the Scifinder database and named Saleucane I.

[0058] The data attribution of compounds 1-6 is shown in Table 1 and Table 2:

[0059] Table 1 Compounds 1–6 13 H NMR (600 MHz) data

[0060]

[0061]

[0062] Table 2 Compounds 1–6 13 C NMR (150 MHz) data

[0063]

[0064]

[0065] Example 3

[0066] Evaluation of the anti-neuritis activity of compounds 1-6 of the present invention

[0067] 3.1 Activity screening method

[0068] 3.1.1 Preparation of compound stock solutions

[0069] 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.

[0070] 3.1.2 Culture and treatment of BV-2 cells

[0071] BV-2 cells were cultured in a DMEM medium containing 10% fetal bovine serum in an incubator set at 5% CO. 2 , 37°C. Observe the growth of cells. When the cell density reaches more than 90%, subculture the cells, wash the cells once with PBS, add 1 mL of trypsin to digest for 2 minutes, so that the cells fall off into single cells, collect them in a centrifuge tube, centrifuge, add fresh culture medium to dilute the cells and continue to culture.

[0072] 3.1.3 MTT assay to detect the effects of compounds on BV-2 cell viability

[0073] 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 4 cells / well) and placed in 37°C, 5% CO 2 Incubate in an incubator for 24 hours; set up an experimental group and a control group. The experimental group is a test sample with a concentration of 40μM prepared from the stock solution of compound 1-6; the control group is a blank control group; 10μL of the test sample is added to each well, and 3 replicate wells are set for each sample. After incubation for 24 hours, 10μL of MTT (5mg / mL) reagent is added to each group of cells. After keeping at 37°C for 4 hours, the culture medium is removed and 100μL of DMSO is added to each well. The absorbance A value of each well is measured at 490nm using an enzyme marker. The calculation formula is as follows:

[0074]

[0075] 3.1.4 Evaluation of the inhibitory effect on NO production by Griess method

[0076] 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 placed in 37°C, 5% CO 2 Cultured in an incubator for 24 hours. An experimental group and a positive control group were set up. The experimental group was a test sample with a concentration of 40 μM prepared from the stock solution of compound 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 solutions were added to determine the production of NO. The absorbance of the sample was measured at 540 nm. After that, the compounds with better initial screening activity were selected for IC 50 Activity determination (the final concentrations of monomer compounds were (50, 25, 12.5, 6.25, 3.125, 1.5625 μM), minocycline was selected as the positive drug. Prism and SPSS26.0 software were used for statistical analysis of the data, and P<0.05 was statistically significant; each group of data was repeated three times in parallel.

[0077] The results showed that the three crotonyl diterpenoid compounds 2, 3 and 6 in the present invention showed good anti-neuritis activity (IC 50The half inhibition concentration values ​​are better than that of the positive drug minocycline (18.89 μM), as shown in Table 3. The data in Table 3 show that the compounds of the present invention have a strong anti-neuritis effect.

[0078] Table 3 Inhibition rate and IC of compounds on BV-2 cells 50 (μM)

[0079]

[0080] 3.1.5 qPCR detection of inflammatory gene expression

[0081] Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect BV-2 cells. 5 cells / well) and placed in 37°C, 5% CO 2 The cells were cultured in an incubator for 24 hours. The experiment set up 1 blank group, 1 model group and 3 experimental groups. DMEM culture medium was added to the blank group; LPS solution with a final concentration of 1 μg / mL was added to the model group; the experimental group was the stock solution of compound 1-6, and the stock solution of each compound was set with 3 concentrations of test samples of 2 μM, 5 μM and 10 μM respectively; LPS solution with a final concentration of 1 μg / mL and test sample solution were added to each well, and the cells were placed in an incubator for further culture for 12 hours. Total cell RNA was extracted and reverse transcribed, and the fluorescent quantitative PCR reaction system in the cDNA of each sample to be tested was configured with PCR reaction solution and amplified. After the reaction was completed, the amplification curve and melting curve of the reaction were confirmed, and the Ct value of the sample to be tested was recorded. GAPDH was used as the internal reference in the experiment, and the expression of each gene was calculated and analyzed according to the Ct value. The change factor = 2-ΔΔCt, the primer sequence used in the experiment. The data of each group were repeated three times in parallel.

[0082] 3.1.6 Western Blot detection of the expression of proteins related to the NF-κB signaling pathway

[0083] BV-2 cells were cultured at 8×10 5The cells were inoculated into 6-well plates at a concentration of 1 μg / well, and the plates were placed in a cell culture incubator for overnight culture. The experiment set up 1 blank group, 1 model group and 3 experimental groups, in which DMEM culture medium was added to the blank group; LPS solution with a final concentration of 1 μg / mL was added to the model group; the experimental group was the stock solution of compound 3, and the stock solution of the compound was set with 3 concentrations of test samples of 2 μM, 5 μM, and 10 μM respectively; LPS solution with a final concentration of 1 μg / mL and test sample solutions with concentrations of 2, 5, and 10 μM were added to each well, and the plates were continued to be cultured in an incubator for 24 hours. After centrifugation, the cells were collected, and the total cell protein was extracted with SDS cell lysis buffer containing 1% protease inhibitor PMSF and phosphorylated protease inhibitors. The concentration of total protein was determined using a BCA kit, and the protein was heated at 95°C to denature the protein. 10% SDS-PAGE separation gel was used for electrophoresis to separate proteins (concentration gel voltage was 80V, 30min; separation gel voltage was 110V, 60min), and the separated proteins were transferred to PVDF membrane (constant voltage 120V, 90min). 10% BSA solution was added to block at room temperature for 1h, and then the completely blocked membrane was incubated with specific primary antibody in a 4℃ refrigerator overnight; taken out, after returning to room temperature, the membrane was washed 3 times with 1×TBST solution, each time for 5min; then incubated with HRP-labeled secondary antibody at room temperature for 2h, and then washed 3 times with 1×TBST solution, each time for 5min, and finally developed according to the instructions of the ECL chemiluminescence kit. The experimental results were analyzed by ImageJ software for grayscale value of protein bands. The experiment was repeated three times in parallel.

[0084] 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, after treatment with compound 3 (concentrations of 2, 5, and 10 μM, respectively), the mRNA levels of IL-6, IL-1β, TNF-α, COX-2, and iNOS were significantly reduced in a dose-dependent manner. The above 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”.

[0085] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0086] 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 may still be modified, or some of the technical features may 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: The compounds include the following structural formulas 1-6:

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

3. The method for preparing a crotan-type diterpene compound according to claim 2, characterized in that: In step (3), the methanol solvents with different gradient concentrations are 60%, 70%, 80%, 90% and 100% methanol solvents, and after elution, 5 fractions are obtained in sequence, namely Fr.1, Fr.2, Fr.3, Fr.4 and Fr.

5.

4. The method for preparing a crotan-type diterpene compound according to claim 3, characterized in that: The Fr.1 precipitates block crystals in methanol solvent to obtain compound 1.

5. The method for preparing a crotan-type diterpene compound according to claim 3, characterized in that: 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; The elution system of the silica gel column chromatography and Sephadex LH-20 separation is dichloromethane / methanol; the separation system of the semi-preparative HPLC liquid phase separation is methanol / water.

6. The method for preparing a crotan-type diterpene compound according to claim 3, characterized in that: 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 to obtain compound 3 and compound 5.

7. Use of the crotan-type diterpene compound according to claim 1 in anti-neuritis and / or treatment of neurodegenerative diseases.

8. A pharmaceutical composition, characterized in that: It comprises any one or more of the compounds of structural formula 1-6 according to claim 1, and a pharmaceutically acceptable carrier.

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

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