CN113717105B - Diterpene alkaloid compound and extraction method and application thereof - Google Patents
Diterpene alkaloid compound and extraction method and application thereof Download PDFInfo
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- CN113717105B CN113717105B CN202110907232.5A CN202110907232A CN113717105B CN 113717105 B CN113717105 B CN 113717105B CN 202110907232 A CN202110907232 A CN 202110907232A CN 113717105 B CN113717105 B CN 113717105B
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Images
Classifications
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- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- A61K2236/30—Extraction of the material
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Abstract
The invention belongs to the field of traditional Chinese medicine extraction, and in particular relates to a diterpenoid alkaloid compound separated from delphinium acutum, an extraction method thereof and application thereof in preparing anti-inflammatory drugs. The compound is shown in general formulas (I), (II) or an isomer of the compound or a pharmaceutically acceptable salt of the compound; the substituent is described in the specification. The diterpenoid alkaloid compound separated from the short-distance sparrows of the photinia serrulata, the isomer of the compound, the pharmaceutically acceptable salt of the compound or the pharmaceutical composition containing the compound has the effect of inhibiting NO production in RAW264.7 cells induced by LPS, and can be used for preparing anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine extraction, and in particular relates to a diterpenoid alkaloid compound separated from delphinium acutum, an extraction method thereof and application thereof in preparing anti-inflammatory drugs.
Background
The short-distance sparrow (Delphinium forrestii var. Viride) is a plant of the genus delphinium of the family Ranunculaceae, and is widely distributed and mainly produced in Tibet, sichuan, yunnan and other provinces. The 1979 edition of Chinese plant Saint A records that the plants of this genus act as medicines in folk, mainly treat diseases such as traumatic injury, rheumatism, toothache, enteritis, etc., and are also used as pesticides, and kill larvae of lice, mosquitoes and flies. In recent years, scholars at home and abroadThe chemical components and the corresponding pharmacological actions of diterpene alkaloids in plants of the genera Cuachyranthes and aconitum of Ranunculaceae are reported successively. At present, diterpenoid alkaloids separated from delphinium are mostly replaced by hydroxyl, methoxy, acetyl and other groups with simpler structures. And the compound separated from the short-distance delphinium of the phoma contains a part of groups with more complex structures such as 2- (2-methyl-4-oxo quinazoline-3 (4H) -group) benzoyl oxy and the like. Known diterpene alkaloids can often be considered to have one or several of the following actions, such as Anti-inflammatory action (V.N.Yu, T.N.Povetieva, N.I.Suslov, G.N.Zyuz' Kov, a.v. kraphin, anti-Inflammatory Activity of Diterpene Alkaloids from Aconitum baikalense, bulletin of Experimental Biology and Medicine 2014,156 (5) 665-668.), analgesic action (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 Letters2012,22 (13) 4444-4446), anticancer action (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 action (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 action (M.Ahmad, W.Ahmad, M.Ahmad, M.Zeeshan, obaidula h, f.shahen, norditerpenoid alkaloids from the roots of Aconitum heterophyllum Wall with antibacterial activity, J Enzyme Inhib Med Chem 2008,23 (6) 1018-1022). The current research on the anti-inflammatory activity of diterpene alkaloids only stays on the action itself, and no further discussion on the anti-inflammatory mechanism is made. To exert the medicinal value of the short-distance delphinium with the photic stemsAt the maximum, the component research of the dried whole grass of the short-distance delphinium with the light stems is needed to be further explored at present.
Disclosure of Invention
The primary object of the present invention is to provide a diterpene alkaloid compound.
The second object of the present invention is to provide a method for extracting diterpene alkaloid-type compounds.
It is a third object of the present invention to provide a pharmaceutical composition comprising a diterpene alkaloid type compound.
A fourth object of the present invention is to provide the use of the diterpene alkaloid compound or an isomer of the compound, a pharmaceutically acceptable salt of the compound or a pharmaceutical composition containing the compound in the preparation of anti-inflammatory drugs.
In order to achieve the above object, the technical scheme of the present invention is summarized as follows:
a diterpene alkaloid compound which is a compound shown in general formulas (I), (II) or an isomer of the compound or a pharmaceutically acceptable salt of the compound;
wherein: r is R 1 ，R 2 ，R 3 ,R 4 ,R 5 Is hydrogen, hydroxy, methoxy, acetoxy, anthranilyloxy or 2- (2-methyl-4-oxoquinazolin-3 (4H) -yl) benzoyloxy.
The compound is shown in the following structural formula, and isomers of the compound and pharmaceutically acceptable salts of the compound;
the pharmaceutically acceptable salts include sodium salt, potassium salt, ammonia salt, hydrochloride and sulfate.
The isomer comprises: optical isomers, cis-trans isomers, racemates and mixtures thereof.
The diterpenoid alkaloid compound is obtained by taking the short-distance sparrow dried whole herb of the photinia serrulata as a raw material and extracting the whole herb with ethanol.
The method comprises the following steps:
(1) Taking a short-distance delphinium dry whole herb of a photinia serrulata as a raw material, crushing, adding ethanol with the volume fraction of 70% -98% which is 0.1-1 times of the raw material, soaking for 1-5 times at room temperature, each time for 5-10 days, and concentrating the extracting solution under reduced pressure to obtain an extract;
(2) Dispersing the total extract into water with the mass of 2-6 times of that of the total extract, regulating the pH value of the suspension to 2-3 by using hydrochloric acid solution, extracting for 2-5 times by using petroleum ether and ethyl acetate in sequence, regulating the pH value of the suspension to 9-11 by using ammonia water, and extracting the suspension for 2-5 times by using dichloromethane to obtain dichloromethane layer extract;
(3) Separating the dichloromethane layer extraction concentrated solution by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 100:1:0.1-0:1:0.1 as eluent, and collecting fractions with the volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) Fractions D2, D3 and D5 were concentrated and further purified to give compounds 1 to 6.
The specific separation and purification process of the fractions D2, D3 and D5 in the step (4) is as follows:
concentrating the fraction D2, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 100:1:0.1-0:1:0.1 as eluent, and collecting fractions with the volume ratio of 10:1:0.1 and 8:1:0.1, respectively denoted as D26 and D27;
concentrating the fraction D26, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 50:1:0.1-0:1:0.1 as eluent, collecting the fraction with the volume ratio of 5:1, and recording as D264;
concentrating fraction D264, purifying by preparative HPLC chromatography with methanol-water as mobile phase at volume ratio of 75:25 to obtain compounds 3,4;
concentrating the fraction D27, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 20:1:0.1-0:1:0.1 as eluent, and collecting the fraction with the volume ratio of 5:1:0.1, and recording as D274;
concentrating fraction D274, purifying by preparative HPLC chromatography with methanol-water as mobile phase at volume ratio of 75:25 to obtain compound 2;
concentrating the fraction D3, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 100:1:0.1-0:1:0.1 as eluent, and collecting fractions with the volume ratio of 5:1:0.1 and 3:1:0.1, respectively denoted as D37 and D39;
concentrating the fraction D37, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 20:1:0.1-0:1:0.1 as eluent, and collecting the fraction with the volume ratio of 5:1:0.1, and recording as D374;
concentrating fraction D374, purifying by preparative HPLC chromatography with methanol-water as mobile phase at volume ratio of 80:20 to obtain compound 6;
concentrating the fraction D39, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 20:1:0.1-0:1:0.1 as eluent, and collecting the fraction with the volume ratio of 5:1:0.1, and recording as D394;
concentrating the fraction D394, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 10:1:0.1-0:1:0.1 as eluent, and collecting the fraction with the volume ratio of 5:1:0.1, and recording as D3944;
concentrating fraction D3944, purifying by preparative HPLC chromatography with methanol-water as mobile phase at volume ratio of 75:25 to obtain compound 1;
concentrating fraction D5, separating by reversed-phase ODS column chromatography, eluting with 40-90% methanol-water as eluent, collecting 60% fraction, and recording as D52;
fraction D52 was concentrated and purified by preparative HPLC chromatography using a volume ratio of 75:25 methanol-water as mobile phase to give compound 5.
A pharmaceutical composition comprising one or more of said diterpene alkaloid-type compound, an isomer of the compound, and a pharmaceutically acceptable salt of the compound.
A pharmaceutical formulation comprising an active ingredient and one or a combination of pharmaceutically acceptable carriers, excipients, diluents; wherein the active ingredient is the compound or the composition.
The administration route of the preparation is oral administration or injection administration, and the dosage form is as follows: tablets, capsules, powders, syrups or injections.
Use of a diterpenoid alkaloid compound or a pharmaceutical composition or a pharmaceutical preparation, wherein the diterpenoid alkaloid compound, the composition and the pharmaceutical preparation are used for preparing anti-inflammatory drugs.
The diterpene alkaloid compound, the isomer of the compound, the pharmaceutically acceptable salt of the compound or the pharmaceutical composition has the effect of inhibiting NO production in RAW264.7 cells induced by LPS, and is applied to the preparation of anti-inflammatory drugs.
The diterpenoid alkaloid compound, the composition and the pharmaceutical preparation are applied to medicines for preventing and treating rheumatoid arthritis, inflammatory bowel disease or atherosclerosis.
The invention has the advantages that:
the diterpenoid alkaloid compound is obtained by extracting the short-distance sparrow of the photinia, and the structure of the compound is confirmed by using means such as nuclear magnetism, mass spectrum and the like, and part of diterpenoid alkaloid separated from the short-distance sparrow of the photinia contains groups with complex structures such as 2- (2-methyl-4-oxo quinazoline-3 (4H) -yl) benzoyloxy and the like; meanwhile, the diterpenoid alkaloid compound or the isomer thereof or the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof has an anti-inflammatory effect on inhibiting NO production in RAW264.7 cells induced by LPS, and can be applied to preparing medicines for treating inflammation by inhibiting the production of ROS and regulating NF-kappa B, MAPK and Nrf2 signal paths. The invention further enriches the structural diversity of the short-distance sparrow active substances, lays a foundation for carrying out relevant biological activity tests on the subsequently obtained monomer compounds, provides active lead compounds for new drug development, and simultaneously provides theoretical basis for deep research and development of the short-distance sparrow medicinal materials.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of Compound 6 on survival of RAW264.7 cells in the presence or absence of LPS.
FIG. 2 is a graph showing the effect of Compound 6 on the expression of inflammatory cytokines secreted by LPS-induced macrophage RAW 264.7. (a) (b) (c) graphic ELISA kits for detecting the expression of inflammatory factors IL-1 beta, TNF-alpha and IL-6 in cell supernatants. ### p < 0.001vs control group,/p<0.001vs LPS group.
FIG. 3 shows the effect of Compound 6 on expression of iNOS, COX-2 and NF-. Kappa. B p65, MAPKs and Nrf2 signaling pathway-related proteins. (a) (b) and (c) graphical western blot detection of expression of inflammatory factors iNOS, COX-2 protein levels and corresponding grey scale statistical plots. (d) The western-blot is shown to detect the expression of p-p65, p-IκBα and IκBα, and the (e) graph shows the gray scale analysis of the (d) graph, showing the relative expression of p-p65 and p-IκBα proteins. (g) The western blot is shown to detect the expression of p-p38, p-ERK, ERK, p-JNK and JNK, and the (f) graph is the gray analysis condition of the (g) graph, and the relative expression amounts of p-p38, p-ERK and p-JNK proteins are shown. (h) (i) graphically representing protein expression of the western-blot detection keap-1, nrf2 and HO-1 and corresponding gray statistics. ### p < 0.001vs control group, ×p<0.01vs LPS group,/p<0.001vs LPS group.
FIG. 4 is a graph showing the effect of Compound 6 on NF-. Kappa. B p65, nrf2 nuclear translocation. (a) Compound 6 is shown to have an inhibitory effect on LPS-induced nuclear translocation of NF- κ B p65, with DAPI-labeled nuclei for blue fluorescence and NF- κ B p65 for red fluorescence. (b) Compound 6 is shown to have a promoting effect on LPS-induced nuclear translocation of Nfr2, with DAPI-labeled nuclei for blue fluorescence and Nrf2 for red fluorescence.
Figure 5 effect of compound 6 on active oxygen and mitochondrial membrane potential levels. (a) Graphical representation cells were treated with compound 6 at different concentrations for 3 hours and then further LPS was added for 12 hours, except for the control and LPS groups. Cells were collected and stained with DCFH-DA and then imaged by fluorescence microscopy. (b) The cells were treated as described above and the fluorescence intensity of DCFH-DA was measured by flow cytometry. (c) DCFH-DA fluorescence intensity calculated by Flow jo softwareDegree. (d) (e) graphically, measuring mitochondrial membrane potential levels by flow cytometry, and calculating the cell ratio in both JC-1 states. ### p < 0.001vs control group, ×p<0.01vs LPS group,/p<0.001vs LPS group.
Detailed Description
The technical scheme of the invention will be further described with reference to specific embodiments.
Example 1
The extraction method of diterpenoid alkaloid compounds 1-6 in the short-distance sparrow of the photinia serrulata comprises the following steps:
(1) The method comprises the steps of taking short-distance delphinium with a total dry weight of 10.0kg as a raw material, adding 95% ethanol reagent (4L) with the mass of 0.4 times of the raw material, and soaking for 3 times at room temperature for 7 days each time. The extract was concentrated under reduced pressure to obtain an extract (360 g).
(2) The total extract was dispersed in water (720 mL) 2 times by mass, the pH of the suspension was adjusted to 2 with hydrochloric acid solution, extraction was performed 3 times with petroleum ether and ethyl acetate, respectively, the pH of the suspension was adjusted to 10 with ammonia water, and extraction was performed 3 times with dichloromethane to obtain dichloromethane layer extract (75 g).
(3) Separating by 200-300 mesh silica gel column chromatography after dry sample mixing of the dichloromethane layer extraction concentrated solution, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 100:1:0.1-0:1:0.1 as eluent at the eluting speed of 1.2mL/min, and collecting fractions with the volume ratios of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) Fractions D2, D3 and D5 were concentrated under reduced pressure and further purified to give 27.2mg of compound 1, 55mg of compound 2, 235mg of compound 3, 53mg of compound 4, 32.9mg of compound 5 and 10.7mg of compound 6. The specific separation and purification process is as follows:
concentrating the fraction D2 under reduced pressure to obtain 22.6g of concentrated solution, carrying out dry sample mixing, and then carrying out 200-300 mesh silica gel column chromatography separation, wherein petroleum ether-acetone-diethylamine with the 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 is used as eluent for gradient elution, the elution flow rate is 2.5mL/min, and the fractions with the volume ratio of 10:1:0.1 and 8:1:0.1 are collected and respectively marked as D26 and D27;
concentrating fraction D26 under reduced pressure to obtain 2.6g of concentrated solution, carrying out dry sample mixing, separating by 200-300 mesh silica gel column chromatography, and collecting fraction with 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, 2:1:0.1 and petroleum ether-acetone-diethylamine as eluent gradient elution at flow rate of 2.5mL/min and volume ratio of 5:1:0.1, wherein D264 is obtained;
concentrating fraction D264 to obtain 1.7g, purifying by preparative HPLC chromatography under the conditions of SHIMADZU5 μm C, 20X1250 mm and methanol-water with volume ratio of 75:25 as mobile phase at flow rate of 8mL/min to obtain 235mg of compound 3 and 53mg of compound 4;
concentrating the fraction D27 to obtain 1.9g of concentrated solution, carrying out dry sample mixing, separating by 200-300 mesh silica gel column chromatography, and collecting fractions with volume ratios 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 by using petroleum ether-acetone-diethylamine as eluent in a gradient manner, wherein the elution flow rate is 2.5mL/min, and the fraction is marked as D274;
concentrating fraction D274 to obtain 335mg, purifying by preparative HPLC chromatography under the conditions of SHIMADZU5 μm C, 20×250mm with methanol-water as mobile phase at a volume ratio of 75:25 at a flow rate of 8mL/min to obtain 55mg of compound 2;
concentrating the fraction D3 to obtain 3g of concentrated solution, carrying out 200-300 mesh silica gel column chromatography separation after dry sample mixing, and carrying out gradient elution by taking petroleum ether-acetone-diethylamine with the 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 eluent, wherein the elution flow rate is 2.5mL/min, and respectively collecting fractions with the volume ratio of 5:1:0.1 and 3:1:0.1, and marking as D37 and D39;
concentrating the fraction D37 to obtain 2.1g of concentrated solution, carrying out dry sample mixing, separating by 200-300 mesh silica gel column chromatography, and collecting fractions with volume ratios 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 by using petroleum ether-acetone-diethylamine as eluent in a gradient manner, wherein the elution flow rate is 2.5mL/min, and the fraction is marked as D374;
concentrating fraction D374 to obtain 40mg, purifying by preparative HPLC chromatography under the conditions of SHIMADZU5 μm C, 20×250mm with methanol-water as mobile phase at a volume ratio of 75:25 at a flow rate of 8mL/min to obtain 10.7mg of compound 6;
concentrating the fraction D39 to obtain 1.1g of concentrated solution, carrying out dry sample mixing, separating by 200-300 mesh silica gel column chromatography, and carrying out gradient elution by taking petroleum ether-acetone-diethylamine with the volume ratio of 20:1:0.1, 15:1:0.1, 10:1:0.11, 8:1:0.1, 5:1:0.1 and 2:1:0.1 as eluent, wherein the elution flow rate is 2mL/min, and collecting the fraction with the volume ratio of 5:1:0.1, and marking as D394;
concentrating the fraction D394 to obtain 1.1g of concentrated solution, carrying out dry sample mixing, separating by 200-300 mesh silica gel column chromatography, and carrying out gradient elution by taking petroleum ether-acetone-diethylamine with the volume ratio of 15:1:0.1, 10:1:0.11, 8:1:0.1, 5:1:0.1 and 2:1:0.1 as eluent, wherein the elution flow rate is 2mL/min, and collecting the fraction with the volume ratio of 5:1:0.1, and recording as D3944;
concentrating fraction D3944 to obtain 320mg, purifying by preparative HPLC chromatography under the conditions of SHIMADZU5 μm C, 20X1250 mm and methanol-water with volume ratio of 75:25 as mobile phase at flow rate of 8mL/min to obtain 27.2mg of compound 1;
after concentrating the fraction D5, 10.5g of the fraction was eluted with 40%, 60%, 70% and 90% by volume of methanol-water as an eluent by reversed-phase ODS column chromatography, and the fraction 60% by volume was collected and designated as D52.
Fraction D52 was concentrated and purified by preparative HPLC chromatography on a SHIMADZU 5. Mu. m C18, 18X 250mm column with 75:25 by volume methanol-water as mobile phase to give 32.9mg of compound 5.
The physicochemical and constants of the compounds are as follows:
compound 1: white amorphous powder; HRESIMS m/z 670.3463[ M+H ]] + (calcd for C 39 H 48 N 3 O 7 670.3487) and determining that the molecular formula of the compound 1 is C 39 H 47 N 3 O 7 ；
-202.0(c 0.3,MeOH)； 1 H-NMR(600MHz,CDCl 3 ) And 13 C-NMR(150MHz,CDCl 3 ) The data are shown in Table 1.
Compound 2: white amorphous powder; HRESIMS m/z 684.3623[ M+H ]] + (calcd for C 40 H 50 N 3 O 7 684.3643) and determining that compound 2 has a molecular formula of C 40 H 49 N 3 O 7 ；
-143.0(c 0.3,MeOH)； 1 H-NMR(600MHz,CDCl 3 ) And 13 C-NMR(150MHz,CDCl 3 ) The data are shown in Table 2.
Compound 3: white amorphous powder; HRESIMS m/z 698.3767[ M+H ]] + (calcd for C 41 H 51 N 3 O 7 698.3799) and determining that compound 3 has a molecular formula of C 41 H 50 N 3 O 7 ；
-331.5(c 0.4,MeOH)； 1 H-NMR(600MHz,CDCl 3 ) And 13 C-NMR(150MHz,CDCl 3 ) The data are shown in Table 3.
Compound 4: white amorphous powder; HRESIMS m/z 569.3210[ M+H ]] + (calcd for C 32 H 45 N 2 O 7 569.3221) and determining the molecular formula of compound 4 as C 32 H 44 N 3 O 7 ；
-332.0(c 0.2,MeOH)； 1 H-NMR(600MHz,CDCl 3 ) And 13 C-NMR(150MHz,CDCl 3 ) The data are shown in Table 4.
Compound 5: white amorphous powder; HRESIMS m/z 436.3045[ M+H ]] + (calcd for C 25 H 42 NO 5 436.3058) and determining that compound 5 has the molecular formula C 25 H 41 NO 5 ；
-141.0(c 0.5,MeOH)； 1 H-NMR(600MHz,CDCl 3 ) And 13 C-NMR(150MHz,CDCl 3 ) The data are shown in Table 5.
Compound 6: white amorphous powder; HRESIMS m/z 316.2261[ M+H ]] + (calcd for C 20 H 30 NO 2 316.2271) and determining that compound 6 has the molecular formula C 20 H 29 NO 2 ；
-468.5(c 0.1,MeOH)； 1 H-NMR(600MHz,CDCl 3 ) And 13 C-NMR(150MHz,CDCl 3 ) The data are shown in Table 6.
Table 1 carbon and hydrogen spectrum data for compound 1
Note that: 1 H-NMR，600MHz，CDCl 3 ； 13 C-NMR，150MHz，CDCl 3 。
table 2 carbon and hydrogen spectrum data for compound 2
Note that: 1 H-NMR，600MHz，CDCl 3 ； 13 C-NMR，150MHz，CDCl 3 。
TABLE 3 carbon and Hydrogen Spectrum data for Compound 3
Note that: 1 H-NMR，600MHz，CDCl 3 ； 13 C-NMR，150MHz，CDCl 3 。
TABLE 4 carbon and hydrogen Spectrometry data for Compound 4
Note that: 1 H-NMR，600MHz，CDCl 3 ； 13 C-NMR，150MHz，CDCl 3 。
TABLE 5 carbon and hydrogen spectral data for Compound 5
Note that: 1 H-NMR，600 MHz，CDCl 3 ； 13 C-NMR，150 MHz，CDCl 3 。
TABLE 6 carbon and hydrogen Spectrometry data for Compound 6
Note that: 1 H-NMR，600 MHz，CDCl 3 ； 13 C-NMR，150 MHz，CDCl 3 。
by physicochemical constants and modern spectroscopic means (hresis and NMR), in combination with literature-related data, the structure was identified, compound 1, 2, 3,4, 5 and 6 structures, as shown below:
example 2
(1) The method comprises the steps of taking 15.0kg of short-distance delphinium with the total dry weight as a raw material, adding 95% ethanol reagent (4.5L) with the mass times of 0.3 of the raw material, and soaking for 3 times at room temperature for 6 days each time. Concentrating the extractive solution under reduced pressure to obtain extract (400 g),
(2) The total extract was dispersed in 2 mass times of water (800 mL), the pH of the suspension was adjusted to 2.5 with hydrochloric acid solution, extraction was performed 3 times with petroleum ether ethyl acetate in sequence, the pH of the suspension was adjusted to 10.5 with ammonia water, and extraction was performed with dichloromethane to obtain dichloromethane layer extract (81 g).
(3) Separating the dichloromethane layer extraction concentrated solution by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 100:1:0.1-0:1:0.1 as eluent, and collecting fractions with the volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) Fractions D2, D3 and D5 were concentrated and further purified to give 29.2mg of compound 1, 61mg of compound 2, 272mg of compound 3, 61mg of compound 4, 34.9mg of compound 5 and 10.8mg of compound 6.
Example 3
The method comprises the steps of taking short-distance delphinium with 12.0kg of total dry weight as a raw material, adding 95% ethanol reagent (2.4L) with the mass times of 0.2 of the raw material, and soaking for 3 times at room temperature for 7 days each time. The extract was concentrated under reduced pressure to obtain an extract (350 g).
(2) The total extract was dispersed in 2 times by mass of water (700 mL), the pH of the suspension was adjusted to 3 with hydrochloric acid solution, extraction was performed 3 times with petroleum ether ethyl acetate in sequence, the pH of the suspension was adjusted to 11 with aqueous ammonia, and extraction was performed with dichloromethane to obtain a dichloromethane layer extract (77 g).
(3) Separating the dichloromethane layer extraction concentrated solution by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 100:1:0.1-0:1:0.1 as eluent, and collecting fractions with the volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) Fractions D2, D3 and D5 were concentrated and further purified to give 28.2mg of compound 1, 60mg of compound 2, 265mg of compound 3, 60mg of compound 4, 34.1mg of compound 5 and 10.2mg of compound 6.
Example 4
Research on influence of products of the invention on NO generation of RAW264.7 cells
RAW264.7 cells were seeded in 96-well plates, treated with 20. Mu.M of the compound obtained in example 1 above for 3 hours, and then incubated with LPS (0.5. Mu.g/mL) for 24 hours. DMSO with or without LPS was treated as vehicle control or model. Nitrite accumulation in the medium was measured with a microplate reader using Griess reagent at 540 nm. The inhibition (%) of the compound-treated group was calculated to evaluate the NO inhibitory activity.
Table 7 Table of the NO production inhibition (%) value of the Compound inhibiting RAW264.7 cells
| Compounds of formula (I) | Inhibition ratio (%) |
| |
12.44 |
| |
37.20 |
| Compound 3 | 10.30 |
| |
99.14 |
From the above table, it can be seen that compound 6 has a strong inhibitory activity.
Further studies on the anti-inflammatory action and mechanism of the above compound 6 in RAW264.7 cells
(1) CCK8 assay for the influence of Compound 6 on cell viability
RAW264.7 cells in logarithmic growth phase were inoculated at 25000 cells/well into 96-well plates and cultured for 12 hours. Cells were treated with varying concentrations of compound 6 (100, 50, 25, 12.5, 6.25 and 3.125. Mu. Mol/L). Cell wells with corresponding volumes of DMSO added were used as blank. After 24 hours, the culture medium was discarded, 100. Mu.L of 10% CCK 8-containing culture medium was added to each well, and after culturing for 40 minutes, the cell viability of the experimental group (100% of the cell viability of the blank group) was calculated by detecting the OD value of each well at 450nm by using a microplate reader. Results are shown in fig. 1:
as can be seen from fig. 1, compound 6 had no significant effect on the survival of RAW264.7 cells.
(2) ELISA method for detecting inhibition of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and TNF-alpha production by compound 6
RAW264.7 cells in logarithmic growth phase were inoculated in 96-well plates for 12h of culture. The experimental groups were treated with different concentrations of compound 6 (5, 10, 20 and 40. Mu. Mol/L). After 3h, LPS was added to the model and experimental groups to a final concentration of 0.5. Mu.g/mL, and the blank group was added to an equal volume of DMEM medium and incubation was continued for 24h. Cell supernatants were taken from each well, and the OD value of each well at 450nm was measured by an ELISA kit protocol, and the cytokine content of each group was calculated. The results are shown in FIG. 2:
as can be seen from fig. 2, compound 6 can inhibit the cytokine content secreted by macrophage RAW264.7 caused by LPS, and its inhibition exhibits dose dependency.
(3) Inhibition of inflammation-associated protein and NF- κ B, MAPK, nrf2/HO-1 pathway expression by Western Blot detection of Compound 6
RAW264.7 cells were inoculated in 96-well plates and cultured for 12h. The experimental groups used various concentrations of Compound 6 (5, 10 and 20. Mu. Mol.L) -1 ) And (5) processing. After 3h, LPS was added to the model and experimental groups to a final concentration of 0.5. Mu.g/mL, and the blank group was added to an equal volume of DMEM medium and incubation was continued for 24h. The medium was discarded, cells were collected, lysed with RIPA, western Blot experiments were performed with BCA quantification, expression of iNOS, COX-2 and NF- κ B, MAPKs, nrf2 signaling pathway-associated proteins were detected after treatment with different concentrations of Compound 6, and the exposed bands were subjected to grey scale analysis with Gel-Pro analyzer. The results are shown in FIG. 3.
As can be seen from FIG. 3, compound 6 can exert an anti-inflammatory effect by decreasing the expression of iNOS, COX-2, NF-. Kappa.B and MAPKs pathways and up-regulating the Nrf2 signaling pathway and downstream related proteins.
(4) Immunofluorescence method for detecting influence of compound 6 on NF-KB p65 and Nrf2 nuclear translocation
RAW264.7 cells were seeded into 8X 10 per well 4 Cells were cultured in 24-well plates for 12h, then pretreated with DMSO or 6 (20. Mu.M) for 2h, and stimulated with 0.5. Mu.g/mL LPS for 12h. Cells were fixed with freshly prepared 4% paraformaldehyde for 10min, washed 3 times with PBS, and then permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 5% Bovine Serum Albumin (BSA) for 1h at room temperature, 1:400 dilutions of NF-. Kappa. B p65 (Proteintech, cat# 10745-1-AP) antibodies or Nrf2 (Proteintech, cat# 16396-1-AP) were incubated overnight at 4 ℃. After washing by PBSAt room temperature and in the dark at 1: the secondary antibody was added at 400 dilution for 1h. Finally, staining with DAPI was performed for 5min at room temperature and in the dark. Then PBS was washed and added with an anti-fluorescence quenching coverslipping solution, and the mixture was observed and photographed under an immunofluorescence microscope, and an image was obtained. The results are shown in FIG. 4.
As can be seen from fig. 4, compound 6 (20 μm) can significantly inhibit the transfer of the p65 subunit of NF- κb from the cytosol to the nucleus in LPS-activated RAW264.7 cells, while compound 6 (20 μm) can promote LPS-induced Nrf2 nuclear translocation.
(5) Effect of Compound 6 on active oxygen levels and mitochondrial Membrane potential levels
LPS-induced RAW264.7 cells were collected and centrifuged, and after centrifugation, the cells were washed three times with PBS. Then, DCFH-DA or JC-1 fluorescent dye was added according to the procedure of the kit instructions, stained at 37℃for 30min, and stained cells were collected and examined using a flow cytometer or a fluorescence microscope, and the results are shown in FIG. 5.
As can be seen from fig. 5, compound 6 can inhibit active oxygen and mitochondrial membrane potential levels in a dose-dependent manner.
Claims (7)
1. A diterpenoid alkaloid compound, which is characterized in that the compound is a compound shown in the following structural formula, and the compound is pharmaceutically acceptable salt;
2. the method for extracting diterpene alkaloid compound according to claim 1, wherein the diterpene alkaloid compound according to claim 1 is obtained by ethanol extraction from a short-distance delphinium dry whole herb as a raw material;
the method comprises the following specific steps:
(1) Taking a short-distance delphinium dry whole herb of a photinia serrulata as a raw material, crushing, adding ethanol with the volume fraction of 70% -98% which is 0.1-1 times of the raw material, soaking for 1-5 times at room temperature, each time for 5-10 days, and concentrating the extracting solution under reduced pressure to obtain an extract;
(2) Dispersing the total extract into water with the mass of 2-6 times of that of the total extract, regulating the pH value of the suspension to 2-3 by using hydrochloric acid solution, extracting for 2-5 times by using petroleum ether and ethyl acetate in sequence, regulating the pH value of the suspension to 9-11 by using ammonia water, and extracting the suspension for 2-5 times by using dichloromethane to obtain dichloromethane layer extract;
(3) Separating the dichloromethane layer extraction concentrated solution by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 100:1:0.1-0:1:0.1 as eluent, and collecting fractions with the volume ratio of 50:1:0.1, 15:1:0.1 and 10:1:0.1, which are sequentially marked as D2, D3 and D5;
(4) Fraction D3 is concentrated and further purified to give compound 6;
the specific separation and purification process of the fraction D3 in the step (4) is as follows:
concentrating the fraction D3, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 100:1:0.1-0:1:0.1 as eluent, and collecting fractions with the volume ratio of 5:1:0.1 and 3:1:0.1, respectively denoted as D37 and D39;
concentrating the fraction D37, separating by silica gel column chromatography, gradient eluting with petroleum ether-acetone-diethylamine with the volume ratio of 20:1:0.1-0:1:0.1 as eluent, and collecting the fraction with the volume ratio of 5:1:0.1, and recording as D374;
fraction D374 was concentrated and purified by preparative HPLC chromatography using a volume ratio of 80:20 methanol-water as mobile phase to give compound 6.
3. A pharmaceutical composition comprising one or more diterpene alkaloid compounds of claim 1 and pharmaceutically acceptable salts thereof.
4. A pharmaceutical formulation characterized in that the formulation is one or a combination of an active ingredient and a pharmaceutically acceptable carrier, excipient, diluent; wherein the active ingredient is a compound according to claim 1 or a composition according to claim 3.
5. The pharmaceutical formulation of claim 4, wherein: the administration route of the preparation is oral administration or injection administration, and the dosage form is as follows: tablets, capsules, powders, syrups or injections.
6. Use of a diterpenoid alkaloid compound or a pharmaceutical composition or a pharmaceutical preparation, characterized in that the diterpenoid alkaloid compound according to claim 1, the composition according to claim 3 and the pharmaceutical preparation according to claim 4 for preparing anti-inflammatory drugs.
7. The use according to claim 6, characterized in that: use of the diterpenoid alkaloid compound of claim 1, the composition of claim 3 and the pharmaceutical preparation of claim 4 for preparing medicines for preventing and treating rheumatoid arthritis, inflammatory bowel disease or atherosclerosis.
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