Novel diterpenoid compounds, methods of making and using the same

Abstract

The application belongs to the technical field of medicine, and particularly relates to a new diterpenoid compound with anti-inflammatory activity extracted and separated from Tripterospermum chinense and a preparation method and application thereof. Anti-inflammatory active ingredients in Tripterospermum chinense are systematically separated and studied, 18 new diterpenoid compounds are obtained, structural formulas are shown in the following, and structural types include pinane 1-10, isopimaran 11-14, 16-18 and pimarane diterpene 15. The new diterpenoid compounds obtained by separation are structurally identified, and the anti-inflammatory activity of the monomer compounds obtained by separation is evaluated by using a copper sulfate-induced zebrafish model. The concentration gradient anti-inflammatory activity experiment shows that the compounds 1, 6, 8, 10, 11, 12, 13, 14 and 17 have concentration-dependent anti-inflammatory activity, and have important application prospects for preparing treatment of inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a novel diterpenoid compound with anti-inflammatory activity extracted and separated from Radix Psoraleae truncatum, and a preparation method and application thereof. Background Art

[0002] Inflammation is the body's natural protective response to injury, infection, or other stimuli. It is a complex biological process involving multiple cells, chemicals, and signaling pathways. The purpose of inflammation is to eliminate harmful stimuli and protect the body from further damage. While inflammation is an important component of the immune system, excessive or inappropriate inflammatory responses can also lead to tissue damage and disease.

[0003] Twisted money Marmoritis complanata Dunn AL Budantzev is a plant of the genus Achyranthes in the Lamiaceae family. The Tibetan medical classic "Jingzhu Bencao" records that Achyranthes has anti-inflammatory properties. Currently, there are few reports on the chemical components of this plant, and research on its components and their activities is limited. No isolation or activity studies have been conducted on this plant. Summary of the Invention

[0004] To address the shortcomings of the existing technology, the present invention provides novel diterpenoid compounds, their preparation methods, and applications. A systematic investigation of the isolation of anti-inflammatory active components from the genus Psoralea corylifolia yielded 18 novel diterpenoid compounds, including abietane, isopimarane, and pimarane diterpenes. The structures of the isolated novel diterpenoid compounds were identified, and the anti-inflammatory activity of the isolated monomeric compounds was evaluated using a copper sulfate-induced zebrafish model.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] The first aspect of the present invention provides novel diterpenoid compounds, including novel abietane-type diterpenoid compounds 1-10, novel isopimarane-type diterpenoid compounds 11-14, 16-18, and novel pimarane diterpenoid compound 15. The structures of the novel diterpenoid compounds are as follows:

[0007] .

[0008] The second aspect of the present invention provides a method for preparing the novel diterpenoid compound described in the first aspect, comprising the following steps:

[0009] (1) Take the dried aerial part of the Herba Lycopodii, add solvent and reflux extract, combine the filtrate, and concentrate to obtain a crude extract;

[0010] (2) After the crude extract is suspended in water, it is extracted with ethyl acetate and n-butanol in sequence. The filtrates are combined and concentrated under reduced pressure to obtain the ethyl acetate phase extract;

[0011] (3) The ethyl acetate extract was subjected to MCI column chromatography and eluted with a methanol-water gradient to obtain seven fractions Fr.I-VII;

[0012] (4) The Fr.Ⅱ segment was separated by silica gel column chromatography, and 11 eluates were collected after gradient elution using petroleum ether-acetone. The eluates were identified by silica gel thin layer chromatography using a mixed solvent of petroleum ether and acetone with a volume ratio of 2:1 as the developing solvent. The eluates were combined into 12 eluates and concentrated under reduced pressure to obtain subfractions Fr.Ⅱ1-Fr.Ⅱ12.

[0013] (5) The Fr.Ⅱ6 subfraction was subjected to gel chromatography column elution with dichloromethane-methanol and then to reverse phase C 18 Compounds 16 and 17 were separated by chromatographic column and HPLC;

[0014] (6) The Fr.Ⅱ9 subfraction was separated by silica gel column chromatography using a petroleum ether-acetone gradient elution, followed by reversed-phase ODS column and HPLC to obtain compounds 5, 13, 14, and 15;

[0015] (7) The Fr.Ⅱ10 subfraction was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; compounds 1, 2, 3, 7, 8, 10, and 12 were separated using a reversed-phase ODS column and HPLC.

[0016] (8) The Fr.Ⅱ11 subfraction was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; compounds 4, 6, 9, 11, and 18 were separated using a reversed-phase ODS column and HPLC.

[0017] The third aspect of the present invention provides the use of the novel diterpenoid compound described in the first aspect in the preparation of anti-inflammatory drugs.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention provides a previously unreported abietane-type, isopimarane-type and pimarane-type diterpenoid compounds, and further provides a method for extracting the abietane-type and pimarane-type diterpenoid compounds from schizonepeta tenuifolia with simple operation, good reproducibility and high extraction purity.

[0020] (2) The anti-inflammatory activity of the abietane-type and pimarine-type diterpenoid compounds provided by the present invention was evaluated in a copper sulfate-induced zebrafish model and it was found that the compounds had anti-inflammatory effects, which has important application prospects for the preparation of drugs for treating inflammation. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The anti-inflammatory activity of the new diterpenoid compounds in the embodiments of the present application is shown in the following table:

[0022] Figure 2 The anti-inflammatory activity of the new diterpenoid compounds in the embodiments of the present application is shown in the following table:

[0023] Figure 3 The anti-inflammatory activity of the new diterpenoid compounds in the embodiments of the present application is shown in the following table: DETAILED DESCRIPTION

[0024] There are few reports on the chemical composition of the plant, and the research on the components and their pharmacological activities is less. The plant has not been isolated and pharmacologically studied. The present application first isolates new diterpenoid compounds from the plant, and evaluates the anti-inflammatory activity in the copper sulfate-induced zebrafish model. The results of the anti-inflammatory activity evaluation at different concentrations show that most of the compounds determined have concentration-dependent anti-inflammatory activity.

[0025] In a typical embodiment of the present application, a new diterpenoid compound is provided, and the structure of the new diterpenoid compound is as follows:

[0026] The new diterpenoid compound includes new abietane-type diterpenoid compounds 1-10, new isopimarane-type diterpenoid compounds 11-14, 16-18, and new pimarane diterpenoid compounds 15.

[0027] .

[0028] In another typical embodiment of the present application, a preparation method of the new diterpenoid compound is provided, and the preparation method comprises the following steps:

[0029] (1) The dry aboveground parts of the plant are taken and extracted by refluxing with a solvent. The filtrates are combined and concentrated to obtain a crude extract;

[0030] (2) The crude extract is suspended in water and then extracted with ethyl acetate and n-butanol, respectively. The filtrates are combined and concentrated under reduced pressure to obtain an ethyl acetate phase extract;

[0031] (3) The ethyl acetate phase extract is subjected to MCI column chromatography, and 7 fractions Fr.I-Ⅶ are obtained after gradient elution with methanol-water;

[0032] (4) The fraction Fr.Ⅱ is separated by silica gel column chromatography, and 11 eluents are collected after gradient elution with petroleum ether-acetone. The 12 eluents are combined and concentrated under reduced pressure to obtain sub-fractions Fr.Ⅱ1-Fr.Ⅱ12 using a mixture of petroleum ether-acetone (2:1) as the developing agent and silica gel thin layer chromatography.

[0033] (5) The Fr. II 6 sub-fraction was separated by gel chromatography using dichloromethane-methanol as eluent, and then separated by reversed-phase C 18 chromatography column and HPLC to obtain compounds 16 and 17;

[0034] (6) The Fr. II 9 sub-fraction was separated by silica gel column chromatography using petroleum ether-acetone as eluent, and then separated by reversed-phase ODS column and HPLC to obtain compounds 5, 13, 14 and 15;

[0035] (7) The Fr. II 10 sub-fraction was separated by silica gel column chromatography using petroleum ether-ethyl acetate as eluent, and then separated by reversed-phase ODS column and HPLC to obtain compounds 1, 2, 3, 7, 8, 10 and 12;

[0036] (8) The Fr. II 11 sub-fraction was separated by silica gel column chromatography using petroleum ether-ethyl acetate as eluent, and then separated by reversed-phase ODS column and HPLC to obtain compounds 4, 6, 9, 11 and 18.

[0037] In some embodiments of the embodiment, in step (1), the solvent is 95-98V% ethanol aqueous solution, and the added amount of the solvent is 5-10 times the mass of the twining coin, and the number of times of reflux extraction is 2-4 times, and each time of extraction is 1-3 hours;

[0038] Preferably, the solvent is 95V% ethanol aqueous solution, and the number of times of reflux extraction is 2 times, and each time of extraction is 2 hours.

[0039] In some embodiments of the embodiment, in step (3), the MCI filler is GEL CHP20P, and the volume ratio of methanol-water is (3:7)-(10:0).

[0040] In some embodiments of the embodiment, in step (4), the silica gel column chromatography is performed by wet column packing with 200-300 mesh silica gel, and the volume ratio of petroleum ether-acetone is (100:1)-(1:1).

[0041] Preferably, the volume ratio of petroleum ether-acetone is 100:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1 or 1:1.

[0042] In some embodiments of the embodiment, in step (5), the volume ratio of dichloromethane-methanol is (1:1)-(1:5), the eluent of the reversed-phase C18 chromatography column is methanol-water with a volume ratio of (3:7)-(10:0), the mobile phase of the HPLC is methanol-water with a volume ratio of (90:10)-(50:50), and the flow rate is 1.5-2 mL / min.

[0043] In some embodiments of this embodiment, in step (6), the volume ratio of petroleum ether-acetone is (50:1)-(1:1); the eluent of the reverse-phase ODS column is methanol-water with a volume ratio of (4:6)-(10:0); the HPLC is performed twice, the mobile phase of the first HPLC separation is acetonitrile-water with a volume ratio of (72:28)-(70:30), and the flow rate is 1.5-2 mL / min; the mobile phase of the second HPLC separation is methanol-0.1V% FA aqueous solution with a volume ratio of (83:17)-(80:20), and the flow rate is 1.5-2 mL / min.

[0044] In some embodiments of this embodiment, in step (7), the volume ratio of petroleum ether-ethyl acetate is (50:1)-(1:2); the eluent of the reverse-phase ODS column is methanol-water with a volume ratio of (3:7)-(10:0); the mobile phase of the HPLC is methanol-0.1V% FA aqueous solution with a volume ratio of (76:24)-(88:12), and the flow rate is 1.5-2 mL / min.

[0045] In some embodiments of this embodiment, in step (8), the volume ratio of petroleum ether-ethyl acetate is (40:1)-(1:1); the eluent of the reverse-phase ODS column is methanol-water with a volume ratio of (3:7)-(10:0); the mobile phase of the HPLC is methanol-0.1V% FA aqueous solution with a volume ratio of (78:22)-(81:19), and the flow rate is 1.5-2 mL / min.

[0046] In a third typical embodiment of the present application, the use of the above-mentioned novel diterpenoid compound in the preparation of an anti-inflammatory drug is provided.

[0047] Example 1

[0048] 10 kg of dried Twisted Money aerial parts were crushed, and extracted with 95% ethanol aqueous solution by heating reflux for 2 h, repeated twice. The extract was filtered with gauze, and then filtered with filter paper under reduced pressure. Ethanol was recovered under reduced pressure, and a total of 429.77 g of crude extract was obtained. The crude extract was suspended in 2-3 L of water, and extracted with an equal volume of ethyl acetate multiple times until the color of the ethyl acetate phase solution became light. Then, n-butanol was used for extraction 3-5 times. Each phase solvent was combined and concentrated under reduced pressure to obtain about 147 g of ethyl acetate phase extract.

[0049] The 147 g of ethyl acetate phase extract was mixed with about the same amount of MCI packing, which was GEL CHP20P. The column was packed with methanol by wet method, and the height ratio of the mixed packing to the chromatographic packing was about 2:1. Then the elution was started with 30% methanol water as the initial elution gradient, and then 50%, 60%, 70%, 80%, 90% and 100% methanol water were used in turn for elution. Each 500 mL of eluent was collected as a sample, and the eluents were combined and concentrated under reduced pressure to obtain 7 fractions Fr. I-VII.

[0050] Then 27.25 g of Fr. II was separated by silica gel column chromatography. The silica gel column was packed with 200-300 mesh silica gel by wet method, and eluted with petroleum ether: acetone in a volume ratio of 100:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1 and 1:1 in turn. Each 50 mL of eluent was collected, and the eluents were combined into 12 sub-fractions Fr. II 1-12 according to the results of TLC detection with petroleum ether: acetone 2:1 as the developing agent.

[0051] Fr. II 6 (424 mg) was separated by gel chromatography column Sephadex LH-20 (CH2Cl2:MeOH, 1:1) to obtain fractions Fr. II 6a-b, and then Fr. II 6b (106.5 mg) was separated by reverse phase C18 column to obtain fractions Fr. II 6b-1-Fr. II 6b-7. Fr. II 6b-6 was separated by preparative HPLC (MeOH:H2O, 90:10, 1.5 mL / min) to obtain compound 16 (1.25 mg, t R = 14 min) and 17 (1.24 mg, t R = 15.5 min).

[0052] Fr. II 8 (380 mg) was first separated by gel chromatography column Sephadex LH-20 (CH2Cl2:MeOH, 1:1) to obtain three fractions Fr. II 8a-c. Fr. II 8a was then separated by reverse phase ODS column to obtain sub-fractions Fr. II 8a-1-Fr. II 8a-8. Fr. II 8a-5 was separated and purified by preparative HPLC (MeOH:H2O, 79:21, 1.5 mL / min) to obtain compound 7-oxodehydroabietinol (1.6 mg), Sandaracopimarinol (1.8 mg, t R = 47 min).

[0053] Fr. II 9 (4.4 g) was separated by silica gel column chromatography with gradient elution of petroleum ether-acetone (50:1-1:1) to give nine fractions Fr. II 9a-i. Fr. II 9b (337.2 mg) was separated by reverse phase ODS column (MeOH:H2O, 4:6-10:0) to give sub-fractions Fr. II 9b-1- Fr. II 9b-8. Fr. II 9b-4 was separated by preparative HPLC (MeCN:H2O, 72:28, 1.5 mL / min) and then purified (MeOH:H2O (0.1% FA), 83:17, 1.5 mL / min) to give compound 7-oxodehydroabietic acid (5.0 mg), 14 (3.8 mg), (+)-7-oxo-13-epi-pimara-14,15-dien-18-oic acid (11.9 mg). Fr. II 9f was separated by preparative HPLC (MeOH:H2O (0.1% FA), 87:13, 1.5 mL / min) to give compound 5 (3.5 mg, t R = 45 min), 13 (13.3 mg, t R = 23 min), 15 (3.9 mg, t R = 28.5 min), 7a-hydroxysandaracopimaric acid (3.2 mg, t R = 32 min), 8(14),15-sandaracopimaradiene-7a,18-diol (1.7 mg, t R = 38 min), callicapene M3 (3.5 mg, t R = 33.5 min). Fr. II 9c was separated by preparative HPLC to give compound 18-hydroxy-sandaracopimara-8(14),15-dien-7-one (1.2 mg) and enriched compound 13 (20.3 mg).

[0054] Fr. II 10 (4.2 g) was separated by silica gel column chromatography with gradient elution of petroleum ether-ethyl acetate (50:1-1:2) to give eight fractions Fr. II 10a-h. Fr. II 10c was separated by reverse phase C18 to give sub-fractions Fr. II 10c-1- Fr. II 10c-7, and then Fr. II 10 c-4 was separated by HPLC (MeOH:H2O (0.1% FA), 88:12, 1.5 mL / min) to give compound 8 (6.8 mg, t R= 28 min) and compound 13 was enriched. Fr. II 10c-2 was subjected to preparative separation by HPLC (MeOH:H20 (0.1% FA), 78:22, 1.5 mL / min) to give compound 10 (5.2 mg, t R = 18 min). Fr. II 10f was subjected to reverse phase C18 separation to give subfractions Fr. II 10f-1 to Fr. II 10f-7, and Fr. II 10f-4 was subjected to preparative separation by HPLC (MeOH:H20 (0.1% FA), 80:20, 1.5 mL / min) to give compound 1 (5.5 mg, t R = 24 min), 2 (4.3 mg, t R = 22 min), 3 (7.1 mg, t R = 34.5 min), 7 (4.5 mg, t R = 37.5 min). Fr. II 10g was subjected to reverse phase C18 separation to give subfractions Fr. II 10g-1 to Fr. II 10g-6, and Fr. II 10g-3 was subjected to preparative separation by HPLC (MeOH:H20 (0.1% FA), 76:24, 1.5 mL / min) to give compound 12 (9.2 mg, t R = 18 min).

[0055] Fr. II 11 (3.8 g) was subjected to silica gel column chromatography with gradient elution (petroleum ether-ethyl acetate, 40:1 to 1:1) to give eight fractions Fr. II 11a to h. Fr. II 11c was subjected to preparative separation by HPLC (MeOH:H20 (0.1% FA), 81:19, 1.5 mL / min) to give compound 9 (5.0 mg, t R = 44 min). Fr. II 11e was subjected to preparative separation by HPLC (MeOH:H20 (0.1% FA), 81:19, 1.5 mL / min) to give compound 8 (14), 15-sandaracopimaradiene-2,18-diol (9.3 mg, t R = 29.5 min). Fr. II 11g was subjected to reverse phase C18 separation to give fractions Fr. II 11g-1 to Fr. II 11g-6. Fr. II 11g-3 was subjected to preparative separation by HPLC (MeOH:H20 (0.1% FA), 78.5:11.5, 1.5 mL / min) to give compound 11 (30.0 mg, t R= 31.5 min), the fraction was subjected to a second preparative purification to give compound 18 (2.4 mg), Roscorane D (1.8 mg), (1R, 3aR, 5aR, 6R, 9aS, 9bR, 11aR)-6-(hydroxymethyl)-6, 9a, 11a-trimethyl-1, 2, 3a, 5, 5a, 6, 7, 8, 9, 9a, 9b, 10, 11, 11a-tetradecahydrophenanthro[1, 2-b]furan-1-ol (5.1 mg). Fr. II 11 g-4 was subjected to preparative HPLC (MeOH:H2O (0.1% FA), 80:20, 1.5 mL / min) to give compound 4 (5.4 mg, t R = 51 min), 6 (14.1 mg, t R = 40 min), and enriched compound 8 (14), 15-sandaracopimaradiene-2, 18-diol (6.9 mg).

[0056] Compounds 1 and 11 were crystallized by solvent evaporation in anhydrous ethanol and dichloromethane, and compound 10 was crystallized by slow evaporation at room temperature in methanol / water.

[0057] The structures of the new compounds were identified:

[0058] (1) The 18 compounds were subjected to mass spectrometry, NMR spectrometry, optical rotation and infrared spectrometry using a Thermo Fisher Q-Exactive mass spectrometer and a Bruker AV 400 nuclear magnetic resonance spectrometer, a GYROMAT-HP optical rotation meter and a Thermo Nicolet iN10 micro infrared spectrometer, respectively. The preparative HPLC was an Agilent 1200 series high-performance liquid chromatograph, and the semi-preparative chromatographic column was an Eclipse XDB-C18 5 μm (9.4 × 250 mm) column. The data obtained are as follows:

[0059] Compound 1: colorless crystal; - 50.41 ( c 0.83, MeOH), UV (MeOH) 246(0.44) nm; IR ν max 3467, 2956, 1712, 1459, 1384, 1244, 1026 cm -1HRESIMS m / z 333.20670 [M-H] - (calcd for C 20 H 29 O4, 333.20713); 1 H and 13 C NMR data see Tables 1 and 3.

[0060] Compound 2: pale yellow oil; 28.69 ( c 0.22, MeOH), UV (MeOH) 240(0.30) nm; IR ν max 3430, 2933, 2870, 1702, 1652, 1457, 1385, 1237, 1178, 1037cm -1 ; HRESIMS m / z 333.20721 [M-H] - (calcd for C 20 H 29 O4, 333.20713); 1 H and 13 C NMR data see Tables 1 and 3.

[0061] Compound 3: white solid; 16.06 ( c 0.04, MeOH), UV (MeOH) 245(0.44) nm; IR ν max 3365, 2935, 2869, 1663, 1451, 1385, 1237, 1149, 1116, 1075cm -1 ; HRESIMS m / z 335.22141 [M+H] + (calcd for C 20 H 31 O4, 335.22169); 1 H and 13 C NMR data see Tables 1 and 3.

[0062] Compound 4: white solid; 24.81 ( c 0.31, MeOH), UV (MeOH) 206(0.32) nm; IR ν max3267, 2940, 2866, 1711, 1657, 1458, 1384, 1260, 1208, 1161 cm -1 ; HRESIMS m / z 359.21893 [M+Na] + (calcd for C 20 H 32 O4Na, 359.21928); 1 H and 13 CNMR data are listed in Tables 1 and 3.

[0063] Compound 5: white solid; - 52.50 ( c 0.07, MeOH), UV (MeOH) 200(0.33) nm; IR ν max 3318, 2929, 1689, 1460, 1386, 1267, 1187, 1029, 858 cm -1 ;HRESIMS m / z 319.22736 [M-H] - (calcd for C 20 H 31 O3, 319.22787); 1 H and 13 C NMR data are listed in Tables 1 and 3.

[0064] Compound 6: white solid; - 30.38 ( c 0.73, MeOH), UV (MeOH) 200(0.16) nm; IR ν max 3413, 2933, 2610, 1694, 1462, 1383, 1233, 1150, 1123, 1034cm -1 ; HRESIMS m / z 359.21915 [M+Na] + (calcd for C 20 H 32 O4Na, 359.21928); 1 H and 13 CNMR data are listed in Tables 1 and 4.

[0065] Compound 7: white solid; 4.81 ( c0.03, MeOH), UV (MeOH) 201(0.35) nm; IR ν max 3439, 2950, 1696, 1459, 1369, 1236, 1017, 945, 842 cm -1 ;HRESIMS m / z 401.22980 [M+Na] + (calcd for C 22 H 34 O5Na, 401.22985); 1 H and 13 C NMR data are listed in Tables 1 and 4.

[0066] Compound 8: white solid; 20.29 ( c 0.03, MeOH), UV (MeOH) 246(0.44) nm; IR ν max 3424, 2931, 1694, 1458, 1384, 1203, 1152, 1059 cm -1 ; HRESIMSm / z 341.20825 [M+Na] + (calcd for C 20 H 30 O3Na, 341.20872); 1 H and 13 C NMR data are listed in Tables 1 and 4.

[0067] Compound 9: white solid; 5.94 ( c 0.03, MeOH), UV (MeOH) 242(0.27) nm; IR ν max 3432, 2930, 1686, 1458, 1382, 1200, 1027 cm -1 ; HR-ESI-MS m / z317.21228 [M- H] - (calcd for C 20 H 29 O3, 317.21222); 1 H and 13 C NMR data are listed in Tables 1 and 4.

[0068] Compound 10: yellow crystal; 78.18 ( c 0.03, MeOH), UV (MeOH) 200(0.56) nm; IR ν max 3460, 2932, 2622, 1693, 1612, 1468, 1448, 1425, 1387, 1348cm -1 ; HR-ESI-MS m / z 369.16666 [M+Na] + (calcd for C 20 H 26 O5Na, 369.16725); 1 H and 13 CNMR data are shown in Tables 1 and 4.

[0069] Compound 11: colorless crystals; - 25.45 ( c 0.04, MeOH), UV (MeOH) 205(0.60) nm; IR ν max 3423, 2917, 2614, 1681, 1636, 1450, 1381, 1359, 1250, 1153cm -1 ; HRESIMS m / z 357.20279 [M+Na] + (calcd for C 20 H 30 O4Na, 357.20363); 1 H and 13 CNMR data are shown in Tables 2 and 5.

[0070] Compound 12: pale yellow oil; - 4.42 ( c 0.04, MeOH), UV (MeOH) 252(0.16) nm; IR ν max 3445, 2929, 1682, 1603, 1455, 1389, 1238, 1033, 995, 914 cm -1 ; HRESIMS m / z 333.20575 [M+ H] + , calcd for C 20 H 29 O4, 333.20604); 1 H and 13C NMR data see Tables 2 and 5.

[0071] Compound 13: colorless crystals, 5.82 ( c 1.07, MeOH), UV (MeOH) 216 (-0.35) nm; IR ν max 3402, 2961, 1689, 1256, 1006, 784 cm -1 ; HRESIMS m / z 317.21127[M- H] - (calcd for C 20 H 29 O3, 317.21222); 1 H and 13 C NMR data see Tables 2 and 5.

[0072] Compound 14: white solid; 8.72 ( c 0.12, MeOH), UV (MeOH) 201(0.52) nm; IR ν max 3086, 2932, 1696 cm -1 ; HRESIMS m / z 317.20923 [M+ H] + (calcd for C 20 H 29 O3, 317.21112); 1 H and 13 C NMR data see Tables 2 and 5.

[0073] Compound 15: white solid; 7.86 ( c 0.10, MeOH), UV (MeOH) 204(0.58) nm; IR ν max 3396, 2930, 1707, 1634, 1452, 1384, 1259, 1187, 1031, 998cm -1 ; HRESIMS m / z 327.22739 [M+Na] + (calcd for C 20 H 32 O2Na, 327.22945); 1 H and 13 CNMR data see Tables 2 and 6.

[0074] Compound 16: colorless oil; 16.28 ( c 0.02, MeOH), UV (MeOH) 202(0.39) nm; IR ν max 3337, 2918, 1713, 1377, 1201, 1022, 908 cm -1 ; HRESIMS m / z 325.21271 [M+ Na] + (calcd for C 20 H 30 O2Na, 325.21380); 1 H and 13 C NMR data are listed in Tables 2 and 6.

[0075] Compound 17: colorless oil; - 38.42 ( c 0.02, MeOH), UV (MeOH) 202(0.45) nm; IR ν max 3374, 2930, 1702, 1453, 1374, 1258, 1036 cm -1 ; HRESIMS m / z 325.21249 [M+ Na] + (calcd for C 20 H 30 O2Na, 325.21380); 1 H and 13 C NMR data are listed in Tables 2 and 6.

[0076] Compound 18: white solid; - 38.42 ( c 0.02, MeOH), UV (MeOH) 200(0.22) nm; IR ν max 3282, 2936, 1634, 1436, 1382, 1331, 1260, 1089, 1038, 997cm -1 ; HRESIMS m / z 357.23953 [M+ Na] + (calcd for C 21 H 34 O3Na, 357.24002); 1H and 13 CNMR data are shown in Tables 2 and 6.

[0077] (2) X-ray single crystal diffraction measurement was performed on compound 1, 10, 11 using Bruker and Bruker APEX-II CCD single crystal diffractometer, and the data obtained are as follows:

[0078] X-ray single crystal diffraction data of compound 1: C 20 H 30 O4(M = 334.44 g / mol), tetragonal, space group P41212, α = 90 °, β = 90°, gamma = 90°, a = 12.6981(3) Å, b = 12.6981(3) Å, c = 22.7002(8) Å, V = 3660.2(2) Å 3 , T = 150.00 K, Z = 8, μ(Cu Kα) = 0.663 mm -1 , Dcalc = 1.214 g / cm 3 , F (000) = 1456.0, 31556 reflections measured (7.978° ≤ 2θ ≤ 149.236°), 3752 independent reflections [R int = 0.0362, R sigma = 0.0212] were applied for calculations with Flack parameter as -0.01(6), and the final R 1was 0.0815 (I>2σ(I)) and wR 2was 0.1789 (all data).

[0079] X-ray single crystal diffraction data of compound 10: C 20 H 28 O6(M = 364.42 g / mol), orthorhombic, space group P212121, α = 90 °, β = 90°,gamma = 90°, a = 10.5418(4)Å, b = 12.1799(4)Å, c =14.4121(5)Å, V = 1850.49(11)Å 3 , Z = 4, T = 150.00K, μ (CuKα)= 0.787 mm -1 , Dcalc =1.308g / cm 3 , F (000) = 784.0, 12480 reflections measured (9.506°≤2θ≤149.846°),3739 unique [R int = 0.0289, R sigma = 0.0293] which were used in all calculationswith Flack parameter as 0.03(8). The final R 1was 0.0679 (I>2σ(I)) and wR 2was0.1523 (all data).

[0080] X-ray single crystal diffraction data of compound 11 : C 20 H 30 O4(M=334.44 g / mol), tetragonal, spacegroup P43212, α = 90 °, β = 90°, gamma = 90°, a = 9.7644(3)Å, b = 9.7644(3)Å, c = 39.3927(12)Å, V = 3755.8(3)Å 3 , Z = 8, T = 299.00K, μ (MoKα)= 0.081 mm -1 , Dcalc = 1.183g / cm 3 , F(000) = 1456.0, 27355 reflections measured (4.656° < 2Θ < 56.618°), 4654 unique [R int = 0.0846, R sigma = 0.0564] which were used in all calculations with Flack parameter as 0.1(7). The final R I was 0.0678 (I>2σ(I)) and wR 2was 0.1991 (alldata).

[0081] Table 1. NMR Carbon Spectroscopy Data for Compounds 1-10

[0082]

[0083] Recorded in CDCl3 a , in CD3OD b at 400 MHz Chemicals shift ( delta ) areexpressed in ppm.

[0084] Table 2. NMR Carbon Spectroscopy Data for Compounds 11-18

[0085]

[0086] Recorded in CDCl3 a at 400 MHz Chemicals shift ( delta ) are expressed in ppm.

[0087] Table 3. NMR Hydrogen Spectroscopy Data for Compounds 1-5

[0088]

[0089] Recorded in CDCl3 a , in CD3OD b at 400 MHz Chemicals shift ( delta ) areexpressed in ppm.

[0090] Table 4. NMR Hydrogen Spectroscopy Data for Compounds 6-10

[0091]

[0092] Recorded in CDCl3 a , in CD3OD b at 400 MHz Chemicals shift ( delta ) areexpressed in ppm.

[0093] Table 5. Nuclear magnetic resonance hydrogen spectrum data of compounds 11-14

[0094]

[0095] Recorded in CDCl3 a at 400 MHz Chemicals shift ( delta ) are expressed in ppm.

[0096] Table 6. Nuclear magnetic resonance hydrogen spectrum data of compounds 15-18

[0097]

[0098] Recorded in CDCl3 a at 400 MHz Chemicals shift ( delta ) are expressed in ppm.

[0099] Example 2:

[0100] Evaluation of anti-inflammatory activity of copper sulfate-induced zebrafish model

[0101] Healthy inflammation cell fluorescence transgenic zebrafish at 72 hpf were used as experimental animals, and randomly divided into blank control group, model group, compound treatment group and positive control group (20 μM indomethacin), 10 fish in each group, and 3 replicates were set. After adding the sample, each group was placed in a constant temperature incubator at 28.5°C for 2 h. Then the above model group, compound treatment group and positive control group zebrafish were treated with 20 μM CuSO4 for 1 h to model inflammation. After the end, the zebrafish were washed, and then the inflammation reaction of the zebrafish was observed under a fluorescence microscope, the number of inflammatory cells migrating to the lateral line of the zebrafish was calculated, and the data of each group was statistically analyzed using GraphPad software. SZX16 fluorescence microscope and DP2-BSW image acquisition system were used for zebrafish image acquisition, Forma 3111 water jacket type CO2 incubator, and zebrafish breeding equipment.

[0102] Conclusion:

[0103] The in vivo anti-inflammatory activity of diterpenoids was evaluated in transgenic fluorescent zebrafish. Copper sulfate was used to model the experiment, which can stimulate the nerve hypertrophic cells and mechanosensory cells of the lateral line of zebrafish to produce strong acute inflammatory response, thereby causing inflammatory cell infiltration. According to the activity screening results as shown in Table 1, wherein C is the blank control group; M is the model group, 0 μM concentration of sample; P is the positive control group, 20 μM indomethacin; ### indicates comparison with the C group, P < 0.0001; * P < 0.1, ** P < 0.01, *** P < 0.001, ***** P < 0.0001 indicates comparison with the M group; the concentration of compounds 1-18 is 50 μM. It can be seen that among compounds 1-18, some compounds have different degrees of anti-inflammatory activity. Figure 1 Based on the screening activity results and the amount of isolated compounds, five cembranes (1, 6, 7, 8, 10) and five isopimaranes (11, 12, 13, 14, 17) were selected for anti-inflammatory activity evaluation at different concentrations. The results are shown in Table 2.

[0104] Figure 2 As shown in Table 2, wherein C is the blank control group; M is the model group, 0 μM concentration of sample; P is the positive control group, 20 μM indomethacin; ### indicates comparison with the C group, P < 0.0001; * P < 0.1, ** P < 0.01, *** P < 0.001, ***** P < 0.0001 comparison with the M group. It can be seen that at non-toxic concentrations, all compounds except compound 7 have concentration-dependent anti-inflammatory activity and have significant differences compared with the model group. Compounds 1, 8, 10-14, 17 have significant anti-inflammatory activity. Among them, compounds 8, 10, 12, 14 have the best activity, which is comparable to the positive control group.

[0105] The effects of different sample groups on zebrafish inflammatory cells are shown in Table 3. Figure 3 As shown in Table 3, wherein C is the blank control group; M is the model group, 0 μM concentration of sample; P is the positive control group, 20 μM indomethacin; the concentration of compounds 1, 6, 7, 8, 10 is 25 μM as the treatment group. The yellow box shows that compared with the model group, these compounds 1, 6, 8, 10, 11-14, 17 significantly reduce the number of zebrafish inflammatory cells migrating to the lateral line at medium or high concentrations.

[0106] ​The above describes the specific embodiments of the present application in combination with the drawings, but is not a limitation on the protection scope of the present application. Those skilled in the art should understand that various modifications or variations made by those skilled in the art on the basis of the technical solutions of the present application without creative labor are still within the protection scope of the present application.

Claims

1. A novel diterpenoid compound, characterized in that: The structure of the novel diterpenoid compound is as follows: The novel diterpenoid compounds include novel abietane-type diterpenoid compounds 1-10, novel isopimarane-type diterpenoid compounds 11-14, 16-18 and novel pimarane diterpenoid 15; 。 2. A method for preparing the novel diterpenoid compound according to claim 1, characterized in that: The following steps are involved: (1) Take the dried aerial part of the Herba Lycopodii, add solvent and reflux extract, combine the filtrate, and concentrate to obtain a crude extract; (2) After the crude extract is suspended in water, it is extracted with ethyl acetate and n-butanol in sequence. The filtrates are combined and concentrated under reduced pressure to obtain the ethyl acetate phase extract; (3) The ethyl acetate extract was subjected to MCI column chromatography and eluted with a methanol-water gradient to obtain seven fractions, Fr.I-VII; (4) The Fr.Ⅱ segment was separated by silica gel column chromatography, and 11 eluates were collected after gradient elution using petroleum ether-acetone. The eluates were identified by silica gel thin layer chromatography using a mixed solvent of petroleum ether and acetone with a volume ratio of 2:1 as the developing solvent. The eluates were combined into 12 eluates and concentrated under reduced pressure to obtain subfractions Fr.Ⅱ1-Fr.Ⅱ12. (5) The Fr.Ⅱ6 subfraction was subjected to gel chromatography column elution with dichloromethane-methanol and then to reverse phase C 18 Compounds 16 and 17 were separated by chromatographic column and HPLC; (6) The Fr.Ⅱ9 subfraction was separated by silica gel column chromatography using a petroleum ether-acetone gradient elution, followed by reversed-phase ODS column and HPLC to obtain compounds 5, 13, 14, and 15; (7) The Fr.Ⅱ10 subfraction was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; compounds 1, 2, 3, 7, 8, 10, and 12 were separated using a reversed-phase ODS column and HPLC. (8) The Fr.Ⅱ11 subfraction was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; compounds 4, 6, 9, 11, and 18 were separated using a reversed-phase ODS column and HPLC.

3. The preparation method according to claim 2, wherein In step (1), the solvent is a 95-98V% ethanol aqueous solution, and the added mass is 5-10 times the mass of the twisted money. The number of reflux extractions is 2-4 times, and each extraction takes 1-3 hours.

4. The preparation method according to claim 2, wherein In step (3), the MCI filler is GEL CHP20P.

5. The preparation method according to claim 2, wherein: In step (4), the silica gel column chromatography was performed by wet packing with 200-300 mesh silica gel, and the volume ratio of petroleum ether to acetone was (100:1)-(1:1).

6. The preparation method according to claim 2, wherein: In step (5), the volume ratio of dichloromethane to methanol is (1:1) to (1:5); the mobile phase of HPLC is methanol to water in a volume ratio of (90:10) to (50:50); and the flow rate is 1.5-2 mL / min.

7. The preparation method according to claim 2, wherein: In step (6), the volume ratio of petroleum ether to acetone is (50:1) to (1:1); HPLC separation is performed twice, the mobile phase for the first HPLC separation is acetonitrile to water in a volume ratio of (72:28) to (70:30), with a flow rate of 1.5 to 2 mL / min; the mobile phase for the second HPLC separation is methanol to 0.1V% FA aqueous solution in a volume ratio of (83:17) to (80:20), with a flow rate of 1.5 to 2 mL / min.

8. The preparation method according to claim 2, wherein: In step (7), the volume ratio of petroleum ether to ethyl acetate is (50:1) to (1:2); the mobile phase of HPLC is methanol to 0.1V% FA aqueous solution in a volume ratio of (76:24) to (88:12), with a flow rate of 1.5-2 mL / min.

9. The preparation method according to claim 2, wherein: In step (8), the volume ratio of petroleum ether to ethyl acetate is (40:1) to (1:1); the mobile phase of HPLC is methanol to 0.1V% FA aqueous solution in a volume ratio of (78:22) to (81:19), with a flow rate of 1.5-2 mL / min.

10. The preparation method according to claim 2, characterized in that: In step (1), the solvent is 95% ethanol aqueous solution, and the number of reflux extractions is 2 times, each extraction time is 2 hours.

11. The preparation method according to claim 2, characterized in that: In step (4), the volume ratio of petroleum ether to acetone is 100:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, and 1:

1.

12. Use of the novel diterpenoid compound according to claim 1 in the preparation of anti-inflammatory drugs.

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