A method for preparing a diterpene compound and its application in preparing anti-inflammatory drugs

Abstract

The present invention discloses a preparation method of a diterpene compound and its application in the preparation of an anti-inflammatory drug. The diterpene compound has a structure shown in Formula 1, Formula 2 and Formula 3: #imgabs0#. The preparation method is to use silver edge jade seeds as raw materials, extract and extract with chemical solvents, separate by normal phase column chromatography, and then separate by preparative HPLC. The present invention provides three diterpene compounds with strong anti-inflammatory activity. The IC values ​​of the three compounds for NO are 50 The values ​​are 2.17, 2.23 and 2.14#imgabs1#, respectively, which are stronger than the NOS inhibitor L-monomethylarginine, and can be used as a leading compound for anti-inflammatory drugs. It has a good application prospect in the field of anti-inflammatory drug research and development, and also provides a basis and new ideas for the extraction and medicinal research of such diterpenoid compounds. The preparation method of the three diterpenoid compounds of the present invention is simple, the purity of the three diterpenoid compounds is as high as 98-99%, and the yield is also greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product chemistry, and specifically relates to a preparation method of a diterpene compound and application of the diterpene compound in the preparation of anti-inflammatory drugs. Background Art

[0002] Nitric oxide (NO) has a wide range of important biological regulatory functions and plays an important role in inflammation, tumors, and the cardiovascular system. When immune cells are stimulated by microbial endotoxins, inflammatory mediators, etc., a large amount of induced nitric oxide synthase (iNOS) will be generated to produce NO for immune response. Therefore, inhibiting NO production is a direct indicator of the anti-inflammatory activity of a compound.

[0003] The present invention aims to provide a diterpene compound with strong anti-inflammatory activity and a preparation method thereof. Summary of the invention

[0004] The first object of the present invention is to provide a diterpene compound or a pharmaceutically acceptable salt thereof as an active ingredient for use in the preparation of an anti-inflammatory drug. The second object of the present invention is to provide a method for preparing the diterpene compound.

[0005] The first object of the present invention is achieved by using a diterpene compound or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of an anti-inflammatory drug, wherein the diterpene compound is any one of 3-de-O-acetyleuphornan R, euphornan D and euphornan N.

[0006] The second object of the present invention is achieved by the method for preparing the diterpene compound, which is achieved by the following steps:

[0007] (1) Dry the seeds of the Herba Lysimachiae, crush them into 30-90 mesh, soak them in 85%-99% ethanol or 85%-99% methanol at 55-60 degrees Celsius, and then perform ultrasonic extraction for 5-7 times, each time for 60-80 minutes. Combine the extracts, concentrate them, and obtain Herba Lysimachiae seed extract a;

[0008] (2) Pour the extract a into a separatory funnel, add 2 to 3 times the weight of the extract a of water and 85% to 99% of dichloromethane or 85% to 99% of ethyl acetate, stir evenly and shake, let stand for a long time, extract the organic solvent layer after the solution is separated, repeat 3 times, combine the organic solvent layers, and distill under reduced pressure to obtain the silver edge jade extract b;

[0009] (3) separating the extract b by silica gel column chromatography, gradient eluting with petroleum ether / ethyl acetate in volume ratios of 1:0, 9:1, 7:1, 4:1, 2:1, 1:1 and 0:1, and combining similar components after thin layer chromatography and ultraviolet spectroscopy analysis to obtain 6 components A to F;

[0010] (4) Preparative HPLC purification: Component C (9:1 petroleum ether / ethyl acetate) was separated and purified by preparative HPLC to obtain Compound 1, Compound 2 and Compound 3.

[0011] The beneficial effects of the present invention are as follows: the present invention provides three diterpene compounds 3-de-O-acetyleuphornan R, euphornans E and euphornans N with strong anti-inflammatory activity, the IC50 values ​​of the three compounds for NO are 2.17, 2.23 and 2.14 μM respectively, which are stronger than the NOS inhibitor L-monomethylarginine, and can be used as leading compounds for anti-inflammatory drugs, and have good application prospects in the field of anti-inflammatory drug research, and also provide a basis and new ideas for the extraction and medicinal research of such diterpene compound substances. The preparation method of the three diterpene compounds of the present invention is simple, the purity of the three diterpene compounds is as high as 98% to 99%, and the yield is also greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the compound 1 in Example 1 of the present invention 1 H NMR spectrum;

[0013] Figure 2 is the DEPT spectrum of compound 1 in Example 1 of the present invention;

[0014] Figure 3 is the MS spectrum of compound 1 in Example 1 of the present invention;

[0015] Figure 4 is the compound 2 in Example 1 of the present invention 1 H NMR spectrum;

[0016] Figure 5 is the DEPT spectrum of compound 2 in Example 1 of the present invention;

[0017] Figure 6 is the MS spectrum of compound 2 in Example 1 of the present invention;

[0018] Figure 7 is the compound 3 in Example 1 of the present invention 1 H NMR spectrum;

[0019] Figure 8 is the DEPT spectrum of compound 3 in Example 1 of the present invention;

[0020] Fig. 9 is the MS spectrum of compound 3 in Example 1 of the present invention;

[0021] Fig.10 is the structural formula of the diterpene compound of the present invention. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0023] The present invention will be further described below with reference to the embodiments.

[0024] The present invention provides a diterpene compound or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of an anti-inflammatory drug, wherein the diterpene compound has a structure shown in Formula 1, Formula 2 and Formula 3 ( Fig.10 ):

[0025]

[0026] The dosage form of the anti-inflammatory drug is tablets, creams, capsules, oral liquids, buccal tablets, granules, suspensions, suppositories, injections, powder injections, pills, sustained-release agents, controlled-release agents or targeted preparations.

[0027] The present invention also provides a method for preparing the diterpene compound, which is achieved by the following steps:

[0028] (1) Dry the seeds of the Herba Lysimachiae, crush them into 30-90 mesh, soak them in 85%-99% ethanol or 85%-99% methanol at 55-60 degrees Celsius, and then perform ultrasonic extraction for 5-7 times, each time for 60-80 minutes. Combine the extracts, concentrate them, and obtain Herba Lysimachiae seed extract a;

[0029] (2) Pour the extract a into a separatory funnel, add 2 to 3 times the weight of the extract a of water and 85% to 99% of dichloromethane or 85% to 99% of ethyl acetate, stir evenly and shake, let stand for a long time, extract the organic solvent layer after the solution is separated, repeat 3 times, combine the organic solvent layers, and distill under reduced pressure to obtain the silver edge jade extract b;

[0030] (3) The extract b was separated by silica gel column chromatography, and gradient eluted with petroleum ether / ethyl acetate in volume ratios of 1:0, 9:1, 7:1, 4:1, 2:1, 1:1 and 0:1. After thin layer chromatography and ultraviolet spectrum analysis, similar components were combined to obtain 6 components A~F; among which component A was mainly obtained by eluting with petroleum ether / ethyl acetate 1:0, B was mainly obtained by eluting with 9:1, C was obtained by 7:1, D was obtained by 4:1, E was obtained by 2:1, and F was obtained by 1:1 and 1:0.

[0031] (4) Preparative HPLC purification: Component C was separated and purified by preparative HPLC to obtain Compound 1, Compound 2 and Compound 3.

[0032] In step (4), the mobile phase used in the preparative HPLC is 46% to 97% methanol / water solution or 35% to 84% acetonitrile / water solution, the preparative column is 30×250 mm, 5 μm Yuexu Ultimate XB-C18, the DAD detector wavelengths are 230 nm, 210 nm, 254 nm and 306 nm, the flow rate is 15 to 40 mL / min, and the injection volume is 1.0 to 9.0 mL.

[0033] In step (3), the silica gel column is loaded as follows: extract b is stirred evenly with 84% to 99% dichloromethane, 80 to 100 mesh silica gel is added in an amount of 1 to 3 times the amount of the sample, and the mixture is dried, and 10 to 70 times the amount of the sample is used to load the chromatographic column with 100 to 200 mesh silica gel.

[0034] The Euphorbia marginata (Latin name: Euphorbia marginata) of the Euphorbia genus described in the present invention is not limited by regions and varieties, and can realize the present invention.

[0035] Example 1

[0036] 2.5 kg of Euphorbia marginata seeds produced in Xinyi City, Jiangsu Province were dried and crushed into 60 mesh, and ultrasonically extracted with 93% industrial ethanol at 60 degrees Celsius for 6 times, each time for 70 minutes, and the ethanol solution was concentrated to obtain 0.2 kg of extract.

[0037] Pour the extract into a separatory funnel, add 0.4L of water, stir evenly, add 0.4L of ethyl acetate, shake, and then let it stand fully. Wait for the water and organic solvent to separate and then extract the ethyl acetate. Repeat this process 3 times, combine the ethyl acetate extraction solution, and obtain 0.1Kg of silver-edged emerald extract after reduced pressure distillation. Add 190mL of dichloromethane to the extract to dissolve the extract, then add 0.3Kg of silica gel (80-100 mesh), stir evenly, and dry at room temperature. Weigh 5.0Kg of silica gel (100-200 mesh) and install silica gel column chromatography, and use petroleum ether / ethyl acetate with a volume ratio of 1:0, 9:1, 7:1, 4:1, 2:1, 1:1 and 0:1 for gradient elution to obtain 6 components A to F. Component C (50 g) was separated and purified by preparative HPLC, with the following preparative chromatographic column parameters: 30×250 mm, 5 μm Yuexu Ultimate XB-C18, DAD detector wavelengths of 230 nm, 210 nm, 254 nm and 306 nm, flow rate of 15-40 mL / min, injection volume of 1.0-9.0 mL. Compounds 1-3 were separated from component C. Among them, compound 1 used 85% methanol / water solution as the mobile phase, and the eluent corresponding to the chromatographic peak retention time of 15.4min after each injection was collected, and 359.1mg of 3-de-O-acetyleuphornan R with a purity of 99% was obtained after evaporation; compound 2 used 87% methanol / water solution as the mobile phase, and the eluent corresponding to the chromatographic peak retention time of 20.4min after each injection was collected, and 373.0mg of euphornans E with a purity of 99% was obtained after evaporation; compound 3 used 88% methanol / water solution as the mobile phase, and the eluent corresponding to the chromatographic peak retention time of 23.5min after each injection was collected, and 391.0mg of euphornans N with a purity of 99% was obtained after evaporation.

[0038] 3-de-O-acetyleuphornan R: white amorphous powder, according to ESIMS m / z 635 ([M+Na] + ) It is inferred that its molecular formula is C 33 H 40 O 11 ( Figure 3 ). 1 H NMR (600MHz,CDCl3):δ H:2.25(dd,J=13.1,16.4Hz,H-1a);2.11(m,H-1b);1.96(d,J=8.8Hz,H-2);4.00(d,J=4.6Hz,H-3);6.15(s,H-5);5.53(s,H-7),4.70(d,J=10.7Hz,H-8);1.62(m,H-9);1.46(m,H-11);4.98(m,H-12);2.92(m,H-13);1.00(d,J=6.4Hz,C H 3-16);2.18(s,C H 3-17);1.17(s,C H 3-18);3.60(d,J=12.0Hz,H-19a);4.25(d,J=12.0Hz,H-19b);1.08(d,J=7.2Hz,C H 3-20);7.60-8.04(m,H-2′-H-7′);1.99(s,8-C H 3CO);2.13(s,12-C H 3CO);2.04(s,19-C H 3CO)( Figure 1 )。 13 C NMR(150MHz,CDCl3):δ C :32.2(C-1),32.9(C-2), 76.9(C-3),72.5(C-4),117.0(C-5),137.7(C-6),76.5(C-7),71.0(C-8),25.1(C-9),22.1(C-10),31.0(C-11),69.2(C-12),43.4(C-13),207.4(C-14),73.2(C-15),11.9(C-16),17.7(C-17),24.4(C-18),64.4(C-19),13.3(C-20),165.2(C-1′),129.8(C-2′),129.7(C-3′),129.6(C-4′),128.6(C-5′,C-6′),133.4(C-7′),170.6(8-CH3 C O),20.6(8- C H3CO),170.3(12-CH3 C O),20.7(12- C H3CO),170.3(19-CH3 C O),20.8(19- C H3CO)( Figure 2 )。

[0039] Euphornans E: colorless oil, according to ESIMS m / z 720 ([M+Na] + ) It is inferred that its molecular formula is C 37 H 47 O 12 ( Figure 6 ). 1 H NMR (600MHz,CDCl3):δ H :2.25 (dd, J=13.8, 10.7Hz, H-1a); 2.02 (dd, J=13.8, 10.7Hz, H-1b); 2.12 (m, H-2); 5.30 (d, J=5.5Hz, H-3); 5.64 (s, H-5); 5 .27(s,H-7),4.62(d,J=8.1Hz,H-8);1.47-1.62(m,H-9);1.46(m,H-11);4.98(m,H-12);2.91(m,H-13);0.93(d,J=6.4Hz,C H 3-16); 2.13(s,C H 3-17); 1.35(s,C H 3-18); 3.90 (d, J=11.9Hz, H-19a); 4.41 (d, J=11.9Hz, H-19b); 1.04 (d, J=7.2Hz, C H 3-20); 2.34 (dd, J=13.8, 6.9Hz, H-2′); 1.68 (m, H-3′a); 1.47 (m, H-3′b); 1.17 (d, J=6.9Hz, C H 3-4′); 0.90(d, J=7.4Hz, C H 3-5′); 7.39 (m, H-3″); 8.25 (m, H-4″); 8.78 (m, H-5″); 9.15 (m, H-6″); 2.11 (s, 3-C H 3CO); 2.10(s,7-C H 3CO); 1.78(s,12-C H 3CO) Figure 4 ). 13 C NMR (150MHz, CDCl3):δ C:32.8(C-1),32.6(C-2),76.8(C-3),70.8(C-4),115.2(C-5),139.2(C-6), 76.1(C-7),70.6(C-8),24.6(C-9),22.2(C-10),31.0(C-11),68.9(C-12),43.7(C-13 ),207.0(C-14),73.1(C-15),12.3(C-16),17.6(C-17),24.8(C-18),65.9(C-19),13.1 (C-20),176.1(C-1′),40.9(C-2′),26.9(C-3′),16.2(C-4′),11.5(C-5′),164.9(C-1″ ),126.3(C-2″),123.3(C-3″),137.0(C-4″),153.3(C-5″),150.4(C-6″),169.5(3-CH3 C O),20.8(3- C H3CO),168.9(7-CH3 C O),20.6(7- C H3CO),170.3(12-CH3 C O),20.7(12- C H3CO) Figure 5 ).

[0040] Euphornans N: colorless oil, according to ESIMS m / z 697 ([M+Na] + ) It is inferred that its molecular formula is C 38 H 42 O 11 ( Fig. 9 ). 1 H NMR (600MHz,CDCl3):δ H :2.25(dd,J=13.2,16.3Hz,H-1a);1.97(dd,J=6.1,15.1Hz,H-1b);2.12(m,H-2);4.02(s,H-3);6.18(s,H-5);5.58 (s,H-7),4.74(d,J=10.7Hz,H-8);1.62(m,H-9);1.55(m,H-11);5.05(m,H-12);3.00(m,H-13);1.00(d,J=6.4Hz,C H 3-16); 2.19(s,C H 3-17); 1.34(s,C H3-18); 3.94 (d, J=11.9Hz, H-19a); 4.34 (d, J=11.9Hz, H-19b); 1.05 (d, J=7.2Hz, C H 3-20); 8.07 (m, H-3′); 7.49 (m, H-4′); 7.54 (m, H-5′); 7.49 (m, H-6′); 8.07 (m, H-7′); 7. 99(m,H-3″);7.42(m,H-4″);7.61(m,H-5″);7.42(m,H-6″);7.99(m,H-7″);2.02(s,8-C H 3CO); 1.77(s,12-C H 3CO) Figure 7 ). 13 C NMR (150MHz,CDCl3):δ C :32.2(C-1),32.9(C-2),76.8(C-3), 70.7(C-4),117.0(C-5), 137.8(C-6),76.5(C-7),71.1(C-8),25.3(C-9),22.2(C-10),31.0(C-11),69.0(C-12),43.5(C-13), 207.4(C-14),73.1(C-15),12.0(C-16),17.7(C-17),24.7(C-18),65.1(C-19),13.2(C-20),165.2(C- 1′),130.3(C-2′),129.8(C-3′),128.7(C-4′),132.9(C-5′),128.7(C-6′),129.8(C-7′),166.2(C-1 ″),130.3(C-2″),129.7(C-3″),128.3(C-4″),133.5(C-5″),128.3(C-6″),129.7(C-7″),170.3(8-CH3 C O),21.7(8- C H3CO),170.4(12-CH3 C O), 21.0(12- C H3CO) Figure 8 ).

[0041] Example 2

[0042] 3.7 kg of Euphorbia marginata seeds produced in Kunming, Yunnan Province were dried, crushed to 80 mesh, and ultrasonically extracted with 90% industrial methanol at 60 degrees Celsius for 7 times, each time for 80 minutes, and the methanol solution was concentrated to obtain 0.30 kg of extract.

[0043] Pour the extract into a separatory funnel, add 0.7L of water, stir evenly, add 0.7L of dichloromethane, shake, and then let it stand fully. Wait for the water and organic solvent to separate and then extract the dichloromethane. Repeat this process 3 times, combine the dichloromethane extraction solution, and obtain 0.22Kg of silver-edged emerald green extract after reduced pressure distillation. Add 240mL of dichloromethane to the extract to dissolve the extract, then add 0.4Kg of silica gel (80-100 mesh), stir evenly, and dry at room temperature. Weigh 7.0Kg of silica gel (100-200 mesh) and install silica gel column chromatography, and use petroleum ether / ethyl acetate with a volume ratio of 1:0, 9:1, 7:1, 4:1, 2:1, 1:1 and 0:1 for gradient elution to obtain a total of 6 components A to F. Component D (58 g) was separated and purified by preparative HPLC, with the following preparative chromatographic column parameters: 30×250 mm, 5 μm Yuexu Ultimate XB-C18, DAD detector wavelengths of 230 nm, 210 nm, 254 nm and 306 nm, flow rate of 15-40 mL / min, injection volume of 1.0-9.0 mL. Compounds 1-3 were separated from component C. Among them, compound 1 used 84% methanol / water solution as the mobile phase, and the eluent corresponding to the chromatographic peak retention time of 17.5min after each injection was collected, and 484.1mg of 3-de-O-acetyleuphornan R with a purity of 99% was obtained after evaporation; compound 2 used 86% methanol / water solution as the mobile phase, and the eluent corresponding to the chromatographic peak retention time of 23.6min after each injection was collected, and 481.0mg of euphornans E with a purity of 99% was obtained after evaporation; compound 3 used 87% methanol / water solution as the mobile phase, and the eluent corresponding to the chromatographic peak retention time of 26.5min after each injection was collected, and 498.0mg of euphornans N with a purity of 99% was obtained after evaporation.

[0044] Experimental Example 1 Detection of anti-inflammatory activity of three diterpenoid compounds

[0045] Experimental methods: (1) Cell culture: RAW264.7 cells were cultured in RPMI-1640 medium containing 10% FBS, penicillin (final concentration of 100 U / mL) and streptomycin (final concentration of 100 μg / mL) and cultured at 37°C in an incubator containing 5% CO2. When the cells were 90% confluent, the medium was removed and 2 mL of PBS was added to wash the cells twice. After removing the PBS, 2 mL of 0.25% (w / v) Trypsin-0.53 mM EDTA mixed digestion solution was added and observed under a microscope. When the cells became round, 2 mL of complete medium was quickly added to terminate the digestion. The cells were centrifuged at 800 r / min at 4°C for 5 min. The supernatant was removed and the cells were collected and resuspended in the medium. The cells were cultured in flasks and the medium was changed every other day.

[0046] (2) RAW264.7 cells were treated with compounds 1, 2 and 3 respectively:

[0047] Mouse mononuclear macrophage RAW264.7 cells were inoculated into 96-well plates at a concentration of 2×104 cells / well, and induced with 1μg / mL LPS. The three diterpenoid compounds obtained in Example 1 were prepared into solutions with concentrations of 50μM, 25μM, 12.5μM, 6.25μM, 3.13μM, 1.56μM, and 0.78μM, respectively, to treat RAW264.7 cells, and a drug-free group and an L-NMMA positive drug group were set as controls. After the cells were cultured overnight, the culture medium was taken to detect the NO generation, and the absorbance was measured at 570nm. IC50 was calculated according to the Reed & Muench method. The results are shown in Table 1.

[0048] Table 1 Anti-inflammatory activity test results of compounds 1, 2 and 3

[0049]

[0050] Results: As shown in Table 1, 3-de-O-acetyleuphornan R (compound 1), euphornans E (compound 2) and euphornans N (compound 3) have the activity of inhibiting NO production, and the activities of the three are more than 15 times that of the positive control L-NMMA, indicating that the three diterpene compounds of the present invention have strong anti-inflammatory activity on LPS-induced mouse mononuclear macrophages and have good application prospects in the development of anti-inflammatory drugs.

Claims

1. Use of a diterpene compound or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of an anti-inflammatory drug, characterized in that: The diterpene compound has the structures shown in Formula 1, Formula 2 and Formula 3: 。 2. The application according to claim 1, characterized in that: The dosage form of the anti-inflammatory drug is tablets, creams, capsules, oral liquids, granules, suspensions, suppositories, injections, powder injections, pills, sustained-release agents, controlled-release agents or targeted preparations.

3. The method for preparing the diterpene compound according to claim 1, characterized in that: To do this, follow these steps: (1) Dry the seeds of the Herba Lysimachiae, crush them into 30-90 mesh, soak them in 85%-99% ethanol or 85%-99% methanol at 55-60 degrees Celsius, and then perform ultrasonic extraction for 5-7 times, each time for 60-80 minutes. Combine the extracts, concentrate them, and obtain Herba Lysimachiae seed extract a; (2) Pour the extract a into a separatory funnel, add 2 to 3 times the weight of the extract a of water and 85% to 99% of dichloromethane or 85% to 99% of ethyl acetate, stir evenly and shake, let stand for a long time, extract the organic solvent layer after the solution is separated, repeat 3 times, combine the organic solvent layers, and distill under reduced pressure to obtain the silver edge jade extract b; (3) separating the extract b by silica gel column chromatography, gradient eluting with petroleum ether / ethyl acetate in volume ratios of 1:0, 9:1, 7:1, 4:1, 2:1, 1:1 and 0:1, and combining similar components after thin layer chromatography and ultraviolet spectroscopy analysis to obtain 6 components A to F; (4) Preparative HPLC purification: Component C was separated and purified by preparative HPLC to obtain Compound 1, Compound 2 and Compound 3.

4. The method for preparing a diterpene compound according to claim 3, characterized in that: In step (4), the mobile phase used in the preparative HPLC was 46% to 97% methanol / water solution or 35% to 84% acetonitrile / water solution, and the preparative column was 30×250 mm, 5 μ m's Moonlight Ultimate XB-C 18 , the DAD detector wavelengths were 230 nm, 210 nm, 254 nm and 306 nm, the flow rate was 15~40 mL / min, and the injection volume was 1.0~9.0 mL.

5. The method for preparing a diterpene compound according to claim 3, characterized in that: In step (3), the silica gel column is loaded as follows: extract b is stirred evenly with 84% to 99% dichloromethane, 80 to 100 mesh silica gel is added in an amount of 1 to 3 times the amount of the sample, and the mixture is dried, and 10 to 70 times the amount of the sample is used to load the chromatographic column with 100 to 200 mesh silica gel.

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