A diterpene alkaloid compound, and an extraction method and application thereof

Abstract

The application belongs to the field of traditional Chinese medicine extraction, and particularly relates to a diterpene alkaloid type compound separated from delphinium uliginosum huth, an extraction method thereof and application thereof in preparation of anti-inflammatory drugs. The compound is as shown in general formula (I) and (II), or an isomer of the compound, or a pharmaceutically acceptable salt of the compound; and substituents are as recorded in the specification. The diterpene alkaloid compound separated from delphinium uliginosum huth, the isomer of the compound, the pharmaceutically acceptable salt of the compound or a pharmaceutical composition containing the compound can be used for preparation of anti-inflammatory drugs due to the inhibitory effect on NO production in LPS-induced RAW264.7 cells.

Description

[0001] The present application is a divisional application of the Chinese invention patent application with the original application date of August 9, 2021, the application number of 202110907232.5, the invention creation name of a diterpene alkaloid type compound and its extraction method and application, the publication number of CN 113717105A; due to the original application having the problem of single pointed out by the examiner, the applicant proposes a divisional application. TECHNICAL FIELD

[0002] The present application belongs to the field of traditional Chinese medicine extraction, and specifically relates to a diterpene alkaloid type compound separated from delphinium uliginosum, an extraction method thereof and application thereof in preparing anti-inflammatory drugs. BACKGROUND

[0003] Delphinium forrestii var. viride is a Ranunculaceae plant, which is widely distributed in Tibet, Sichuan, Yunnan and other provinces. The plants of this genus were recorded in the 1979 edition of Flora of China that they were used as folk medicine to treat injuries, rheumatism, toothache, enteritis and other symptoms. In addition, they were used as pesticides to kill the larvae of lice, mosquitoes and flies. In recent years, domestic and foreign scholars have reported the chemical constituents and corresponding pharmacological effects of diterpenoid alkaloids in plants of Ranunculaceae, such as Delphinium and Aconitum. Most of the diterpenoid alkaloids isolated from Delphinium are substituted by simple groups such as hydroxyl, methoxyl and acetyl. Some of the compounds isolated from D. forrestii var. viride contain complex groups such as 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)benzoyloxy.Known diterpene alkaloids can often be considered to have one or several of the following effects, such as anti-inflammatory effects (V. N. Yu, T. N. Povetieva, N. I. Suslov, G. N. Zyuz'Kov, A. V. Krapivin, Anti-Inflammatory Activity of Diterpene Alkaloids from Aconitum baikalense, Bulletin of Experimental Biology and Medicine 2014, 156(5) 665-668.), analgesic effects (Wang, D. P., Lou, H. Y., Huang, Hao, X.. J., Liang, G. Y., Yang, A novel franchetine type norditerpenoid isolated from the roots of Aconitum carmichaeli Debx. with potential analgesic activity and less toxicity, Bioorganic and Medicinal Chemistry Letters 2012, 22(13) 4444-4446.), anticancer effects (M. Hazawa; K. Wada; K. Takahashi; T. Mori; N. Kawahara, I. Kashiwakura, Suppressive effects of novel derivatives prepared from Aconitum alkaloids on tumor growth, Investigational New Drugs 2009, 27(2) 111-119.), antiarrhythmic effects (F.. A.H. A. Ulubelen, H. K. Desai, S. W. Pelletier, Norditerpenoid and diterpenoid alkaloids from Turkish Consolida orientalis, Journal of Natural Products 2001, 64(6) 787-789., antibacterial activity (M. Ahmad, W. Ahmad, M. Ahmad, M. Zeeshan, Obaidullah, F. Shaheen, Norditerpenoid alkaloids from the roots of Aconitum heterophyllum Wall with antibacterial activity, J Enzyme Inhib Med Chem 2008, 23(6) 1018-1022.). At present, the research on the anti-inflammatory activity of diterpenoid alkaloids only stays in its effect itself, and the anti-inflammatory mechanism thereof is not further explored. In order to maximize the medicinal value of A. orientalis, it is necessary to further explore the systematic composition of the dried whole plant of A. orientalis. SUMMARY

[0004] The primary object of the present application is to provide a diterpenoid alkaloid type compound.

[0005] The second object of the present application is to provide an extraction method of the diterpenoid alkaloid type compound.

[0006] The third object of the present application is to provide a pharmaceutical composition containing the diterpenoid alkaloid type compound.

[0007] The fourth object of the present application is to provide the use of the above-mentioned diterpenoid alkaloid type compound or the isomer of the compound, the pharmaceutically acceptable salt of the compound or the pharmaceutical composition containing the compound in the preparation of an anti-inflammatory drug.

[0008] To achieve the above objects, the technical solutions of the present application are as follows:

[0009] A diterpenoid alkaloid type compound is a compound as shown in general formula (I), (II) or an isomer of the compound or a pharmaceutically acceptable salt of the compound.

[0010]

[0011] wherein: R1, R2, R3, R4, R5 are hydrogen, hydroxyl, methoxyl, acetyloxy, anthraniloyloxy or 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)benzoyloxy.

[0012] The compound is shown in the following structural formula, and isomers, pharmaceutically acceptable salts of the compound;

[0013]

[0014]

[0015] The pharmaceutically acceptable salt includes sodium salt, potassium salt, ammonia salt, hydrochloride and sulfate.

[0016] The isomers include optical isomers, cis-trans isomers, racemates and mixtures thereof.

[0017] The extraction method of the diterpene alkaloid type compound is to take the dried whole plant of Eustoma grandiflorum as raw material, and obtain the diterpene alkaloid type compound by ethanol extraction.

[0018] Specifically,

[0019] (1) The dried whole plant of Eustoma grandiflorum is taken as raw material, and after being crushed, 0.1-1 times the mass of the raw material of 70%-98% ethanol is added to soak at room temperature for 1-5 times, each time for 5-10 days. The extract is concentrated under reduced pressure to obtain the extract;

[0020] (2) The total extract is dispersed in 2-6 times the mass of water, and the pH of the suspension is adjusted to 2-3 with hydrochloric acid solution. The suspension is extracted with petroleum ether and ethyl acetate for 2-5 times, respectively. Then the pH of the obtained extract is adjusted to 9-11 with ammonia water, and the suspension is extracted with dichloromethane for 2-5 times to obtain the dichloromethane layer extract;

[0021] (3) The dichloromethane layer extract is concentrated and separated by silica gel column chromatography, and eluted with petroleum ether-acetone-diethylamine (100:1:0.1-0:1:0.1) as eluent. The fractions with volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1 are collected and marked as D2, D3 and D5, respectively;

[0022] (4) The fractions D2, D3 and D5 are further purified after concentration to obtain compounds 1-6.

[0023] The specific separation and purification process of the fractions D2, D3 and D5 in step (4) is as follows:

[0024] The fraction D2 is concentrated and separated by silica gel column chromatography, and eluted with petroleum ether-acetone-diethylamine (100:1:0.1-0:1:0.1) as eluent. The fractions with volume ratio of 10:1:0.1 and 8:1:0.1 are collected and marked as D26 and D27, respectively.

[0025] The fraction D26 was concentrated and separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine (50:1:0.1-0:1:0.1, by volume) as eluent, and the fraction with a volume ratio of 5:1 was collected and recorded as D264;

[0026] The fraction D264 was concentrated and purified by preparative HPLC chromatography with methanol-water (75:25, by volume) as mobile phase to obtain compound 3,4;

[0027] The fraction D27 was concentrated and separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine (20:1:0.1-0:1:0.1, by volume) as eluent, and the fraction with a volume ratio of 5:1:0.1 was collected and recorded as D274;

[0028] The fraction D274 was concentrated and purified by preparative HPLC chromatography with methanol-water (75:25, by volume) as mobile phase to obtain compound 2;

[0029] The fraction D3 was concentrated and separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine (100:1:0.1-0:1:0.1, by volume) as eluent, and the fractions with a volume ratio of 5:1:0.1 and 3:1:0.1 were collected and recorded as D37 and D39, respectively;

[0030] The fraction D37 was concentrated and separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine (20:1:0.1-0:1:0.1, by volume) as eluent, and the fraction with a volume ratio of 5:1:0.1 was collected and recorded as D374;

[0031] The fraction D374 was concentrated and purified by preparative HPLC chromatography with methanol-water (80:20, by volume) as mobile phase to obtain compound 6;

[0032] The fraction D39 was concentrated and separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine (20:1:0.1-0:1:0.1, by volume) as eluent, and the fraction with a volume ratio of 5:1:0.1 was collected and recorded as D394;

[0033] The fraction D394 was concentrated and separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine (10:1:0.1-0:1:0.1, by volume) as eluent, and the fraction with a volume ratio of 5:1:0.1 was collected and recorded as D3944;

[0034] The fraction D3944 was concentrated and purified by preparative HPLC chromatography with methanol-water (75:25, by volume) as mobile phase to obtain compound 1;

[0035] The fraction D5 is concentrated and separated by reverse-phase ODS column chromatography eluted with methanol-water (40%-90% in volume fraction) as eluent, and the fraction with 60% in volume fraction is collected and recorded as D52.

[0036] The fraction D52 is concentrated and purified by preparative HPLC chromatography with methanol-water (75:25 in volume ratio) as mobile phase to obtain compound 5.

[0037] A pharmaceutical composition, the composition containing one or more of the diterpene alkaloid type compound, isomers of the compound, and pharmaceutically acceptable salts of the compound.

[0038] A pharmaceutical preparation, the preparation being one or a combination of active ingredients and pharmaceutically acceptable carriers, excipients, diluents; wherein the active ingredient is the compound or the composition.

[0039] The administration route of the preparation is oral or injection administration, and the dosage form is tablet, capsule, powder, syrup or needle.

[0040] Application of a diterpene alkaloid type compound or a pharmaceutical composition or a pharmaceutical preparation, the diterpene alkaloid type compound, the composition, and the pharmaceutical preparation being used for preparing anti-inflammatory drugs.

[0041] The diterpene alkaloid type compound, isomers of the compound, pharmaceutically acceptable salts of the compound, or the pharmaceutical composition has an inhibitory effect on NO production in LPS-induced RAW264.7 cells, and is applied to preparing anti-inflammatory drugs.

[0042] The diterpene alkaloid type compound, the composition, and the pharmaceutical preparation are applied to drugs for preventing and treating rheumatoid arthritis, inflammatory bowel disease or atherosclerosis.

[0043] The present application has the following advantages:

[0044] The diterpene alkaloid type compound obtained by the present application is obtained by extraction from Delphinium uliginosum Huth, and the structure of the compound is confirmed by means of nuclear magnetic resonance, mass spectrometry and the like. It can be seen that part of the diterpene alkaloids separated from Delphinium uliginosum Huth contains relatively complex groups such as 2-(2-methyl-4-oxoquinazoline-3(4H)-yl)benzoyloxy. The obtained diterpene alkaloid type compound or isomer thereof or pharmaceutically acceptable salt thereof or pharmaceutical composition thereof has an inhibitory effect on the production of NO in LPS-induced RAW264.7 cells, and plays an anti-inflammatory role by inhibiting the production of ROS and regulating the NF-κB, MAPK and Nrf2 signaling pathways, and is applied to the preparation of a drug for treating inflammation. The present application further enriches the structural diversity of active substances of Delphinium uliginosum Huth, and on this basis, lays a foundation for subsequent biological activity tests of monomer compounds, provides active lead compounds for new drug development, and also provides a theoretical basis for deep research and development of Delphinium uliginosum Huth medicinal materials. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Effect diagram of different concentrations of compound 6 on the survival rate of RAW264.7 cells in the presence or absence of LPS.

[0046] Figure 2 Effect diagram of compound 6 on the expression of inflammatory cytokines secreted by LPS-induced macrophage RAW264.7. (a) (b) (c) show the expression of inflammatory factors IL-1β, TNF-α and IL-6 in the cell supernatant detected by ELISA kit. ### p<0.001 vs control group, ***p<0.001 vs LPS group.

[0047] Figure 3 Effect diagram of compound 6 on the expression of iNOS, COX-2 and NF-κB p65, MAPKs and Nrf2 signaling pathway related proteins. (a) (b) (c) show the expression of inflammatory factor iNOS, COX-2 protein level and the corresponding gray scale statistical diagram. (d) shows the expression of p-p65, p65, p-IκBα, IκBα detected by western-blot, (e) is the gray scale analysis of (d) diagram, showing the relative expression amount of p-p65, p-IκBα protein. (g) shows the expression of p-p38, p38, p-ERK, ERK, p-JNK, JNK detected by western-blot, (f) is the gray scale analysis of (g) diagram, showing the relative expression amount of p-p38, p-ERK and p-JNK protein. (h) (i) show the protein expression of keap-1, Nrf2, HO-1 and the corresponding gray scale statistical diagram detected by western-blot. ###p<0.001 vs control group, **p<0.01 vs LPS group, ***p<0.001 vs LPS group.

[0048] Figure 4 Effect of compound 6 on NF-κB p65 and Nrf2 nuclear translocation. (a) shows the inhibitory effect of compound 6 on LPS-induced NF-κB p65 nuclear translocation, blue fluorescence is DAPI-labeled nucleus, and red fluorescence is NF-κB p65. (b) shows the promoting effect of compound 6 on LPS-induced Nfr2 nuclear translocation, blue fluorescence is DAPI-labeled nucleus, and red fluorescence is Nrf2.

[0049] Figure 5 Effect of compound 6 on reactive oxygen species and mitochondrial membrane potential level. (a) shows that after treatment with different concentrations of compound 6 for 3 hours, LPS was added, and the cells were treated for 12 hours. The cells were collected, stained with DCFH-DA, and the images were obtained by fluorescence microscopy. (b) shows that the cells were treated according to the above method, and the fluorescence intensity of DCFH-DA was detected by flow cytometry. (c) shows the DCFH-DA fluorescence intensity calculated by Flow jo software. (d) (e) shows the mitochondrial membrane potential level detected by flow cytometry, and the proportion of cells in two states of JC-1 was calculated. ### p<0.001 vs control group, **p<0.01 vs LPS group, ***p<0.001 vs LPS group. DETAILED DESCRIPTION

[0050] The technical solutions of the present application will be further described below in combination with specific embodiments.

[0051] Example 1

[0052] Extraction method of diterpene alkaloid type compounds 1-6 in Delphinium uliginosum Huth:

[0053] (1) Take Delphinium uliginosum Huth with a total dry weight of 10.0 kg as raw material, add 95% ethanol reagent (4 L) with a mass of 0.4 times that of the raw material, and soak at room temperature for 3 times, each time for 7 days. The extract is concentrated under reduced pressure to obtain an extract (360 g).

[0054] (2) Disperse the total extract into water (720 mL) with a mass of 2 times that of the extract, adjust the pH of the suspension to 2 with a hydrochloric acid solution, extract with petroleum ether, ethyl acetate 3 times in turn, adjust the pH of the suspension to 10 with ammonia water, and extract with dichloromethane 3 times to obtain a dichloromethane layer extract (75 g).

[0055] (3) The concentrated solution of dichloromethane layer was dried and mixed, and then separated by 200-300 mesh silica gel column chromatography. Petroleum ether-acetone-diethylamine with a volume ratio of 100:1:0.1-0:1:0.1 was used as eluent for gradient elution at a speed of 1.2 mL / min. Fractions with a volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1 were collected and denoted as D2, D3 and D5, respectively;

[0056] (4) Fractions D2, D3 and D5 were further purified after being concentrated under reduced pressure to obtain 27.2 mg of compound 1, 55 mg of compound 2, 235 mg of compound 3, 53 mg of compound 4, 32.9 mg of compound 5 and 10.7 mg of compound 6. The specific separation and purification process was as follows:

[0057] Fraction D2 was concentrated under reduced pressure to obtain 22.6 g of concentrated solution, which was dried and mixed, and then separated by 200-300 mesh silica gel column chromatography. Petroleum ether-acetone-diethylamine with a volume ratio of 50:1:0.1, 15:1:0.1, 10:1:0.1, 8:1:0.1, 5:1:0.1 and 3:1:0.1 was used as eluent for gradient elution at a speed of 2.5 mL / min. Fractions with a volume ratio of 10:1:0.1 and 8:1:0.1 were collected and denoted as D26 and D27, respectively.

[0058] Fraction D26 was concentrated under reduced pressure to obtain 2.6 g of concentrated solution, which was dried and mixed, and then separated by 200-300 mesh silica gel column chromatography. Petroleum ether-acetone-diethylamine with a volume ratio of 50:1:0.1, 40:1:0.1, 30:1:0.1, 20:1:0.1, 15:1:0.1, 10:1:0.1, 5:1:0.1 and 2:1:0.1 was used as eluent for gradient elution at a speed of 2.5 mL / min. A fraction with a volume ratio of 5:1:0.1 was collected and denoted as D264.

[0059] Fraction D264 was concentrated to obtain 1.7 g, which was purified by preparative HPLC chromatography. The column conditions were SHIMADZU 5 μm C18 20x250 mm, and methanol-water with a volume ratio of 75:25 was used as mobile phase at a flow rate of 8 mL / min. This resulted in 235 mg of compound 3 and 53 mg of compound 4.

[0060] The fraction D27 was concentrated to obtain 1.9 g of concentrated liquid, which was separated by silica gel column chromatography with 200-300 mesh after dry mixing, and gradient elution was performed with petroleum ether-acetone-diethylamine with a volume ratio of 50:1:0.1, 40:1:0.1, 30:1:0.1, 20:1:0.1, 15:1:0.1, 10:1:0.1, 5:1:0.1, and 2:1:0.1 as eluents at a flow rate of 2.5 mL / min, and fractions with a volume ratio of 5:1:0.1 were collected, which were recorded as D274;

[0061] The fraction D274 was concentrated to obtain 335 mg, which was purified by preparative HPLC chromatography under the column conditions of SHIMADZU 5 μm C18 20*250 mm, with methanol-water with a volume ratio of 75:25 as the mobile phase at a flow rate of 8 mL / min, to obtain 55 mg of compound 2;

[0062] The fraction D3 was concentrated to obtain 3 g of concentrated liquid, which was separated by silica gel column chromatography with 200-300 mesh after dry mixing, and gradient elution was performed with petroleum ether-acetone-diethylamine with a volume ratio of 50:1:0.1, 30:1:0.1, 15:1:0.1, 10:1:0.1, 5:1:0.1, and 3:1:0.1 as eluents at a flow rate of 2.5 mL / min, and fractions with a volume ratio of 5:1:0.1 and 3:1:0.1 were collected, which were recorded as D37 and D39, respectively;

[0063] The fraction D37 was concentrated to obtain 2.1 g of concentrated liquid, which was separated by silica gel column chromatography with 200-300 mesh after dry mixing, and gradient elution was performed with petroleum ether-acetone-diethylamine with a volume ratio of 50:1:0.1, 40:1:0.1, 30:1:0.1, 20:1:0.1, 15:1:0.1, 10:1:0.1, 5:1:0.1, and 2:1:0.1 as eluents at a flow rate of 2.5 mL / min, and fractions with a volume ratio of 5:1:0.1 were collected, which were recorded as D374;

[0064] The fraction D374 was concentrated to obtain 40 mg, which was purified by preparative HPLC chromatography under the column conditions of SHIMADZU 5 μm C18 20*250 mm, with methanol-water with a volume ratio of 75:25 as the mobile phase at a flow rate of 8 mL / min, to obtain 10.7 mg of compound 6;

[0065] Fraction D39 was concentrated to give 1.1 g of concentrated liquid, which was dried and separated by column chromatography on a 200-300 mesh silica gel column with petroleum ether-acetone-diethylamine (20:1:0.1, 15:1:0.1, 10:1:0.1, 8:1:0.1, 5:1:0.1, 2:1:0.1, by volume) as eluent in gradient elution at a flow rate of 2 mL / min, and fractions with a volume ratio of 5:1:0.1 were collected and recorded as D394;

[0066] Fraction D394 was concentrated to give 1.1 g of concentrated liquid, which was dried and separated by column chromatography on a 200-300 mesh silica gel column with petroleum ether-acetone-diethylamine (15:1:0.1, 10:1:0.1, 8:1:0.1, 5:1:0.1, 2:1:0.1, by volume) as eluent in gradient elution at a flow rate of 2 mL / min, and fractions with a volume ratio of 5:1:0.1 were collected and recorded as D3944;

[0067] Fraction D3944 was concentrated to give 320 mg, which was purified by preparative HPLC chromatography with a SHIMADZU 5 μm C18 20 x 250 mm column and methanol-water (75:25, by volume) as the mobile phase at a flow rate of 8 mL / min to give 27.2 mg of Compound 1.

[0068] Fraction D5 was concentrated to give 10.5 g, which was separated by column chromatography on a reversed-phase ODS column with methanol-water (40%, 60%, 70%, 90%, by volume) as eluent to give fractions with a volume fraction of 60% and recorded as D52.

[0069] Fraction D52 was concentrated to give 32.9 mg of Compound 5, which was purified by preparative HPLC chromatography with a SHIMADZU 5 μm C18 20 x 250 mm column and methanol-water (75:25, by volume) as the mobile phase at a flow rate of 8 mL / min.

[0070] The physical and chemical constants of the compound are as follows:

[0071] Compound 1: white amorphous powder; HRESIMS m / z: 670.3463 [M+H] + (calcd for C 39 H 48 N3O7, 670.3487), and the molecular formula of Compound 1 was determined to be C 39 H 47 N3O7; (c 0.3, MeOH); 1 H-NMR (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDC13), data in Table 1.

[0072] Compound 2: white amorphous powder; HRESIMS m / z: 684.3623 [M+H] + (calcd for C 40 H 50 N3O7, 684.3643), confirming the molecular formula of compound 2 as C 40 H 49 N3O7; (c 0.3, MeOH); 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13), data in Table 2.

[0073] Compound 3: white amorphous powder; HRESIMS m / z: 698.3767 [M+H] + (calcd for C 41 H 51 N3O7, 698.3799), confirming the molecular formula of compound 3 as C 41 H 50 N3O7; (c 0.4, MeOH); 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13), data in Table 3.

[0074] Compound 4: white amorphous powder; HRESIMS m / z: 569.3210 [M+H] + (calcd for C 32 H 45 N2O7, 569.3221), confirming the molecular formula of compound 4 as C 32 H 44 N3O7; (c 0.2, MeOH); 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13), data in Table 4.

[0075] Compound 5: white amorphous powder; HRESIMS m / z: 436.3045 [M+H] + (calcd for C 25 H 42 NO5, 436.3058), confirming the molecular formula of compound 5 as C 25 H41 NO5; (c 0.5, MeOH); 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13), data in Table 5.

[0076] Compound 6: white amorphous powder; HRESIMS m / z: 316.2261 [M+H] + (calcd for C 20 H 30 NO2, 316.2271), the molecular formula of compound 6 was determined as C 20 H 29 NO2; (c 0.1, MeOH); 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13), data in Table 6.

[0077] Table 1 Carbon and hydrogen spectral data of compound 1

[0078]

[0079] Note: 1 H-NMR, 600 MHz, CDC13; 13 C-NMR, 150 MHz, CDC13.

[0080] Table 2 Carbon and hydrogen spectral data of compound 2

[0081]

[0082] Note: 1 H-NMR, 600 MHz, CDC13; 13 C-NMR, 150 MHz, CDC13.

[0083] Table 3 Carbon and hydrogen spectral data of compound 3

[0084]

[0085]

[0086] Note: 1 H-NMR, 600 MHz, CDC13; 13 C-NMR, 150 MHz, CDC13.

[0087] Table 4 Carbon and hydrogen spectral data of compound 4

[0088]

[0089] Note: 1 H-NMR, 600MHz, CDC13; 13 C-NMR, 150MHz, CDC13.

[0090] Table 5 Carbon and hydrogen spectrum data of compound 5

[0091]

[0092]

[0093] Note: 1 H-NMR, 600MHz, CDC13; 13 C-NMR, 150MHz, CDC13.

[0094] Table 6 Carbon and hydrogen spectrum data of compound 6

[0095]

[0096] Note: 1 H-NMR, 600MHz, CDC13; 13 C-NMR, 150MHz, CDC13.

[0097] By physicochemical constants and modern spectroscopy means (HRESIMS and NMR), combined with literature related data, its structure was identified, and the structures of compounds 1, 2, 3, 4, 5 and 6 are as follows:

[0098]

[0099] Example 2

[0100] (1) The short distance of the light stem of the original material of the total dry weight of 15.0 kg was added to the raw material 0.3 mass times of 95% ethanol reagent (4.5 L), and soaked at room temperature for 3 times, 6 days each time. The extract was concentrated under reduced pressure to obtain the extract (400 g),

[0101] (2) The total extract was dispersed into 2 mass times of water (800 mL), the pH of the suspension was adjusted to 2.5 with hydrochloric acid solution, and then extracted with petroleum ether and ethyl acetate for 3 times, and then the pH of the suspension was adjusted to 10.5 with ammonia water, and extracted with dichloromethane to obtain the dichloromethane layer extract (81 g).

[0102] (3) The dichloromethane layer extraction concentrated liquid is separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine as eluent with a gradient of 100:1:0.1 to 0:1:0.1 by volume ratio, and the fractions with a volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1 are collected and recorded as D2, D3 and D5, respectively;

[0103] (4) The fractions D2, D3 and D5 are further purified after concentration to obtain 29.2 mg of compound 1, 61 mg of compound 2, 272 mg of compound 3, 61 mg of compound 4, 34.9 mg of compound 5 and 10.8 mg of compound 6.

[0104] Example 3

[0105] The light stem short distance delphinium with a total dry weight of 12.0 kg is used as raw material, 0.2 mass times of 95% ethanol reagent (2.4 L) is added, and the raw material is soaked at room temperature for 3 times, each time for 7 days. The extraction liquid is concentrated under reduced pressure to obtain an extract (350 g).

[0106] (2) The total extract is dispersed into 2 mass times of water (700 mL), the pH of the suspension is adjusted to 3 with a hydrochloric acid solution, the suspension is extracted with petroleum ether and ethyl acetate for 3 times in sequence, the pH of the suspension is then adjusted to 11 with ammonia water, and the suspension is extracted with dichloromethane to obtain a dichloromethane layer extract (77 g).

[0107] (3) The dichloromethane layer extraction concentrated liquid is separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine as eluent with a gradient of 100:1:0.1 to 0:1:0.1 by volume ratio, and the fractions with a volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1 are collected and recorded as D2, D3 and D5, respectively;

[0108] (4) The fractions D2, D3 and D5 are further purified after concentration to obtain 28.2 mg of compound 1, 60 mg of compound 2, 265 mg of compound 3, 60 mg of compound 4, 34.1 mg of compound 5 and 10.2 mg of compound 6.

[0109] Example 4

[0110] Study on the effect of the product of the application on the generation of NO by RAW264.7 cells

[0111] RAW264.7 cells are inoculated in a 96-well plate, treated with 20 μM of the compound prepared in Example 1 above for 3 hours, and then incubated with LPS (0.5 μg / mL) for 24 hours. DMSO with or without LPS is used as a vehicle control or a model. Griess reagent is used to measure nitrite accumulation in the culture medium at 540 nm by an enzyme marker. The inhibition rate (%) of the compound treatment group is calculated to evaluate the NO inhibition activity.

[0112] Table 7. Inhibition rate (%) of compound on NO production in RAW264.7 cells

[0113] Compound Inhibition rate (%) Compound 1 12.44 Compound 2 37.20 Compound 3 10.30 Compound 6 99.14

[0114] From the above table, it can be found that compound 6 has strong inhibitory activity.

[0115] Further study on anti-inflammatory effect and mechanism of compound 6 in RAW264.7 cells

[0116] (1) CCK8 method for detecting the effect of compound 6 on cell survival rate

[0117] RAW264.7 cells in logarithmic growth phase were inoculated in 96-well plates at 25000 cells per well and cultured for 12 h. The cells were treated with different concentrations of compound 6 (100, 50, 25, 12.5, 6.25 and 3.125 μmol / L). The cells with the addition of corresponding volume of DMSO were used as blank control. After 24 h, the culture medium was discarded, and 100 μL of culture medium containing 10% CCK8 was added to each well. After 40 min of incubation, the OD value of each well at 450 nm was detected by a microplate reader to calculate the cell survival rate of the experimental group (with the cell survival rate of the blank control group as 100%). Results Figure 1

[0118] It can be seen that compound 6 has no significant effect on the survival rate of RAW264.7 cells. Figure 1

[0119] (2) ELISA method for detecting the inhibitory effect of compound 6 on the production of interleukin 1β (IL-1β), interleukin 6 (IL-6) and TNF-α

[0120] RAW264.7 cells in logarithmic growth phase were inoculated in 96-well plates and cultured for 12 h. The experimental group was treated with different concentrations of compound 6 (5, 10, 20 and 40 μmol / L). After 3 h, LPS was added to the model group and the experimental group to make the final concentration 0.5 μg / mL, while the blank control group was added with the same volume of DMEM medium. After 24 h of continuous culture, the cell supernatant was taken from each well. According to the ELISA kit operation manual, the OD value of each well at 450 nm was detected by a microplate reader to calculate the cytokine content of each group. The results are shown in Figure 2

[0121] It can be seen that compound 6 can inhibit the cytokine content secreted by macrophage RAW264.7 caused by LPS, and the inhibitory effect is dose-dependent. Figure 2

[0122] ​​​​(3) Western Blot detection of the inhibitory effect of compound 6 on the expression of inflammation-related proteins and NF-κB, MAPK, and Nrf2 / HO-1 pathways

[0123] RAW264.7 cells were seeded in 96-well plates and cultured for 12 h. The experimental group was treated with different concentrations of compound 6 (5, 10, and 20 μmol·L -1 ) for 3 h. Then, the model group and the experimental group were added with LPS to a final concentration of 0.5 μg / mL, while the blank control group was added with the same volume of DMEM medium, and cultured for another 24 h. The culture medium was discarded, and the cells were collected and lysed with RIPA. BCA quantification was performed for Western Blot experiment to detect the expression of iNOS, COX-2, and NF-κB, MAPKs, and Nrf2 signaling pathway-related proteins after treatment with different concentrations of compound 6. The exposed bands were analyzed by gray scale analysis using Gel-Pro analyzer. The results are shown in Figure 3

[0124] As can be seen from Figure 3 , compound 6 can exert an anti-inflammatory effect by reducing the expression of iNOS, COX-2, NF-κB, and MAPKs pathway and up-regulating the expression of Nrf2 signaling pathway and downstream-related proteins.

[0125] (4) Immunofluorescence detection of the effect of compound 6 on the nuclear translocation of NF-κB p65 and Nrf2

[0126] RAW264.7 cells were seeded in 24-well plates at a density of 8 x 10 4 cells per well and cultured for 12 h. Then, they were pretreated with DMSO or 6 (20 μM) for 2 h and stimulated with 0.5 μg / mL LPS for 12 h. The cells were fixed with freshly prepared 4% paraformaldehyde for 10 min, washed with PBS for 3 times, and then permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 5% bovine serum albumin (BSA) at room temperature for 1 h, NF-κB p65 (Proteintech, Cat# 10745-1-AP) antibody or Nrf2 (Proteintech, Cat# 16396-1-AP) was added at a dilution of 1:400 and incubated at 4°C overnight. After washing with PBS, the secondary antibody was added at a dilution of 1:400 at room temperature and in the dark for 1 h. Finally, the cells were stained with DAPI at room temperature and in the dark for 5 min. Then, they were washed with PBS and added with anti-fluorescence quenching mounting medium, observed under an immunofluorescence microscope, and photographed to obtain images. The results are shown in Figure 4

[0127] As can be seen from Figure 4 ​​It is known that compound 6 (20 μM) can significantly inhibit the p65 subunit of NF-κB from cytoplasm to nucleus in LPS-activated RAW264.7 cells, and at the same time, compound 6 (20 μM) can promote LPS-induced Nrf2 nuclear translocation.

[0128] (5) Effect of compound 6 on the level of reactive oxygen species and the level of mitochondrial membrane potential

[0129] LPS-induced RAW264.7 cells were collected and centrifuged, and after centrifugation, the cells were washed with PBS three times. Then, according to the operation steps of the kit instructions, DCFH-DA or JC-1 fluorescent dye was added, and the cells were stained at 37°C for 30 min. The stained cells were collected and detected and analyzed using a flow cytometer or a fluorescence microscope, and the results are shown in Figure 5

[0130] As can be seen from Figure 5 , compound 6 can dose-dependently inhibit the level of reactive oxygen species and the level of mitochondrial membrane potential.​

Claims

1. A method for extracting a diterpene alkaloid-type compound, characterized by, Compounds 1~5 are obtained by extracting the dried whole plant of Eritrichium nanocapitatum Maxim. with ethanol; The specific extraction method is as follows: (1) the dried whole plant of Eritrichium nanocapitatum Maxim. is crushed, and then 0.1-1 mass times of the crushed plant is soaked in 70%-98% ethanol at room temperature for 1-5 times, each time for 5-10 days, and then the extract is concentrated under reduced pressure to obtain an extract; (2) the total extract is dispersed in 2-6 mass times of water, and then the pH of the suspension is adjusted to 2-3 with a hydrochloric acid solution, and then the suspension is extracted with petroleum ether and ethyl acetate for 2-5 times, respectively, and then the pH of the suspension is adjusted to 9-11 with ammonia water, and then the suspension is extracted with dichloromethane for 2-5 times to obtain a dichloromethane layer extract; (3) the dichloromethane layer extract is concentrated, and then the concentrated solution is separated by silica gel column chromatography, and then the concentrated solution is eluted with petroleum ether-acetone-diethylamine (100:1:0.1~0:1:0.1) as an eluent, and then the fractions with a volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1 are collected, and then the fractions are denoted as D2, D3 and D5, respectively; (4) the fractions D2, D3 and D5 are further purified after being concentrated to obtain compounds 1~5; the specific separation and purification process of the fraction D2, D3 and D5 in step (4) is as follows: the fraction D2 is concentrated, and then the concentrated solution is separated by silica gel column chromatography, and then the concentrated solution is eluted with petroleum ether-acetone-diethylamine (100:1:0.1~0:1:0.1) as an eluent, and then the fractions with a volume ratio of 10:1:0.1 and 8:1:0.1 are collected, and then the fractions are denoted as D26 and D27, respectively; the fraction D26 is concentrated, and then the concentrated solution is separated by silica gel column chromatography, and then the concentrated solution is eluted with petroleum ether-acetone-diethylamine (50:1:0.1~0:1:0.1) as an eluent, and then the fraction with a volume ratio of 5:1 is collected, and then the fraction is denoted as D264; the fraction D264 is concentrated, and then the concentrated solution is purified by preparative HPLC chromatography with methanol-water (75:25) as a mobile phase to obtain compounds 3 and 4; the fraction D27 is concentrated, and then the concentrated solution is separated by silica gel column chromatography, and then the concentrated solution is eluted with petroleum ether-acetone-diethylamine (20:1:0.1~0:1:0.1) as an eluent, and then the fraction with a volume ratio of 5:1:0.1 is collected, and then the fraction is denoted as D274; the fraction D274 is concentrated, and then the concentrated solution is purified by preparative HPLC chromatography with methanol-water (75:25) as a mobile phase to obtain compound 2; the fraction D3 is concentrated, and then the concentrated solution is separated by silica gel column chromatography, and then the concentrated solution is eluted with petroleum ether-acetone-diethylamine (100:1:0.1~0:1:0.1) as an eluent, and then the fraction with a volume ratio of 3:1:0.1 is collected, and then the fraction is denoted as D39; the fraction D39 is concentrated, and then the concentrated solution is separated by silica gel column chromatography, and then the concentrated solution is eluted with petroleum ether-acetone-diethylamine (20:1:0.1~0:1:0.1) as an eluent, and then the fraction with a volume ratio of 5:1:0.1 is collected, and then the fraction is denoted as D394; The fraction D3944 was concentrated and separated by silica gel column chromatography, eluted with petroleum ether-acetone-diethylamine (10:1:0.1~0:1:0.1 by volume ratio) as eluent gradient, and the fraction with volume ratio of 5:1:0.1 was collected and recorded as D3944; The fraction D3944 was concentrated and purified by preparative HPLC chromatography with methanol-water (75:25 by volume ratio) as mobile phase to obtain compound 1; The fraction D5 was concentrated and separated by reversed-phase ODS column chromatography with methanol-water (40%~90% by volume fraction) as eluent to elute the fraction with volume fraction of 60% and record as D52; The fraction D52 was concentrated and purified by preparative HPLC chromatography with methanol-water (75:25 by volume ratio) as mobile phase to obtain compound 5; The structural formulae of compounds 1~5 are as follows: 。

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