Novel diterpenoid compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a novel diterpenoid compound which is extracted and separated from lysimachia christinae hance and has anti-inflammatory activity as well as a preparation method and application of the novel diterpenoid compound. According to the invention, systematic separation research is carried out on anti-inflammatory active components in a longtube euphorbia plant to obtain 18 novel diterpenoid compounds, the structural formula is shown in the specification, and the structural types comprise 1-10 parts of abietane, 11-14 parts of isopimarane, 16-18 parts of isopimarane and 15 parts of pimarane diterpenoid. The novel diterpenoid compound obtained through separation is subjected to structure identification, and a copper sulfate induced zebra fish model is adopted to evaluate the anti-inflammatory activity of the monomeric compound obtained through separation. Concentration gradient anti-inflammatory activity experiments show that the compounds 1, 6, 8, 10, 11, 12, 13, 14 and 17 have concentration-dependent anti-inflammatory activity and have important application prospects in preparation of drugs for treating inflammation. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a novel diterpenoid compound with anti-inflammatory activity extracted and separated from Radix Phellodendri ternata, 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 remove harmful stimuli and protect the body from further damage. Inflammation is an important part of the immune system, but excessive or inappropriate inflammatory responses can also lead to tissue damage and disease.

[0003] Marmoritis complanata (Dunn) ALBudantzev is a plant of the genus Marmoritis in the Lamiaceae family, and is used as a folk medicine in Tibet. The Tibetan medical classic "Jingzhu Bencao" records that Marmoritis complanata has anti-inflammatory effects. At present, there are few literatures reporting the chemical components of this plant, and there are few studies on the components and their activities. There is no isolation and activity research on this plant. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a novel diterpenoid compound and its preparation method and application, and to systematically separate and study the anti-inflammatory active components in the genus Psoralea, and obtain 18 novel diterpenoid compounds, including abietane, isopimarane and pimarane diterpenes. The structure of the novel diterpenoid compounds isolated was identified, and the anti-inflammatory activity of the isolated monomer 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, wherein 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 compound 15; the structure of the novel diterpenoid compounds is 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) taking the dried aerial part of the Psoralea corylifolia, adding a solvent for reflux extraction, combining the filtrate, and concentrating to obtain a crude extract;

[0010] (2) After suspending the crude extract in water, extracting with ethyl acetate and n-butanol in sequence, combining the filtrates, and concentrating under reduced pressure to obtain an ethyl acetate phase extract;

[0011] (3) subjecting the ethyl acetate phase extract to MCI column chromatography, and using a methanol-water gradient elution to obtain seven fractions Fr.I-VII;

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

[0013] (5) The Fr.Ⅱ6 subfraction was subjected to gel chromatography, eluted with dichloromethane-methanol, and then eluted with reverse phase C 18 Chromatographic column and HPLC method were used to separate compounds 16 and 17;

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

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

[0016] (8) The Fr.Ⅱ11 subcomponent was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; and 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 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 Psoralea corylifolia 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 THE DRAWINGS

[0021] Figure 1 This is the preliminary screening result of the anti-inflammatory activity of the novel diterpenoid compounds in the examples of the present invention;

[0022] Figure 2 The anti-inflammatory results of the concentration gradient of the novel diterpenoid compound in the embodiment of the present invention;

[0023] Figure 3 This is a diagram showing the effects of different sample groups on zebrafish inflammatory cells in an embodiment of the present invention. DETAILED DESCRIPTION

[0024] At present, there are few literatures reporting the chemical components of Thunbergia truncatula, and few studies on the components and their pharmacological activities. There is no separation and pharmacological study on the plant. The present invention is the first to isolate new diterpenoid compounds from Thunbergia truncatula, and the anti-inflammatory activity was evaluated in a copper sulfate-induced zebrafish model. The results of the concentration gradient anti-inflammatory activity evaluation showed that most of the compounds tested had concentration-dependent anti-inflammatory activity.

[0025] A typical embodiment of the present invention provides a novel diterpenoid compound, the structure of which is as follows:

[0026] 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 compound 15;

[0027]

[0028] Another typical embodiment of the present invention provides a method for preparing the novel diterpenoid compound, comprising the following steps:

[0029] (1) taking the dried aerial part of the Psoralea corylifolia, adding a solvent for reflux extraction, combining the filtrate, and concentrating to obtain a crude extract;

[0030] (2) After suspending the crude extract in water, extracting with ethyl acetate and n-butanol in sequence, combining the filtrates, and concentrating under reduced pressure to obtain an ethyl acetate phase extract;

[0031] (3) subjecting the ethyl acetate phase extract to MCI column chromatography, and using a methanol-water gradient elution to obtain seven fractions Fr.I-VII;

[0032] (4) The Fr.Ⅱ segment components were separated by silica gel column chromatography, and 11 eluents were collected after gradient elution using petroleum ether-acetone; a mixed solvent of petroleum ether and acetone with a volume ratio of 2:1 was used as a developing solvent, and silica gel thin layer chromatography was used for identification, and the eluents were combined into 12 eluents, and concentrated under reduced pressure to obtain sub-fractions Fr.Ⅱ1-Fr.Ⅱ12;

[0033] (5) The Fr.Ⅱ6 subfraction was subjected to gel chromatography, eluted with dichloromethane-methanol, and then eluted with reverse phase C 18 Chromatographic column and HPLC method were used to separate compounds 16 and 17;

[0034] (6) The Fr.Ⅱ9 subcomponent was separated by silica gel column chromatography using a petroleum ether-acetone gradient elution, and then by reversed phase ODS column and HPLC to obtain compounds 5, 13, 14, and 15;

[0035] (7) The Fr.Ⅱ10 subcomponent was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; and compounds 1, 2, 3, 7, 8, 10, and 12 were separated using a reversed phase ODS column and HPLC.

[0036] (8) The Fr.Ⅱ11 subcomponent was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; and compounds 4, 6, 9, 11, and 18 were separated using a reversed-phase ODS column and HPLC.

[0037] In some examples of this embodiment, 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, and the number of reflux extractions is 2-4 times, and each extraction takes 1-3 hours;

[0038] Preferably, the solvent is 95% by volume ethanol aqueous solution, and the reflux extraction is performed twice, with each extraction lasting 2 hours.

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

[0040] In some examples of this embodiment, in step (4), the silica gel column chromatography is wet packed with 200-300 mesh silica gel, and the volume ratio of petroleum ether to acetone is (100:1)-(1:1);

[0041] Preferably, 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, or 1:1.

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

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

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

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

[0046] A third typical embodiment of the present invention provides use of the novel diterpenoid compounds in the preparation of anti-inflammatory drugs.

[0047] Example 1

[0048] 10kg of dried ground part of Psoralea corylifolia was crushed, and extracted with 95% ethanol aqueous solution by heating and refluxing for 2h, repeated twice, the extract was filtered with gauze, and then filtered with filter paper under reduced pressure, and ethanol was recovered under reduced pressure to obtain a total of 429.77g of crude extract. The crude extract was suspended in 2-3L of water, first extracted with an equal volume of ethyl acetate for multiple times until the color of the ethyl acetate phase solution became lighter, and then extracted with n-butanol for 3 to 5 times, and each phase solvent was combined and concentrated under reduced pressure to obtain about 147g of ethyl acetate phase extract.

[0049] 147g of ethyl acetate phase extract was mixed with about the same amount of MCI filler, MCI filler was GEL CHP20P. The column was filled with methanol wet method, the height ratio of the mixed filler to the chromatography filler was about 2:1, and then eluted with 30% methanol water as the initial elution gradient, and then eluted with 50%, 60%, 70%, 80%, 90%, and 100% methanol water in sequence, and the eluent was collected as a portion every 500mL, combined on the plate, and the eluent was concentrated under reduced pressure to obtain 7-segment components Fr.I-VII.

[0050] Then, 27.25 g of Fr.Ⅱ component was separated by silica gel column chromatography. The silica gel column chromatography was packed with 200-300 mesh silica gel wet method, and eluted with petroleum ether:acetone 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, respectively. Every 50 mL of the eluate was collected, and TLC detection was performed using a mixed solvent of petroleum ether:acetone in a ratio of 2:1 as the developing agent. According to the development and color development results, the eluates were combined into 12 portions, concentrated under reduced pressure, and subcomponents Fr.Ⅱ1-12 were obtained.

[0051] Fr.Ⅱ6 (424 mg) was purified by gel chromatography using Sephadex LH-20 (CH 2 Cl 2 :MeOH, 1:1) to separate the components Fr.Ⅱ6a-b, and then Fr.Ⅱ6b (106.5 mg) was separated using a reverse phase C18 column to obtain components Fr.Ⅱ6b-1 to Fr.Ⅱ6b-7. Fr.Ⅱ6b-6 was separated using preparative HPLC (MeOH:H 2 O, 90:10, 1.5 mL / min) to separate compound 16 (1.25 mg, t R =14min) and 17 (1.24mg, t R =15.5min).

[0052] Fr.Ⅱ8 (380 mg) was firstly chromatographed on a Sephadex LH-20 (CH 2 Cl 2 :MeOH, 1:1) to obtain three components Fr.Ⅱ8a~c. Fr.Ⅱ8a was then separated using a reversed phase ODS column to obtain subcomponents Fr.Ⅱ8a-1~Fr.Ⅱ8a-8. Fr.Ⅱ8a-5 was separated using preparative HPLC (MeOH:H 2 O, 79:21, 1.5 mL / min) to separate and purify the compounds 7-oxodehydroabietinol (1.6 mg), Sandaracopimarinol (1.8 mg, t R =47min).

[0053] Fr.Ⅱ9 (4.4 g) was subjected to silica gel column chromatography with petroleum ether-acetone gradient elution (50:1-1:1) to obtain nine components Fr.Ⅱ9a~i. Fr.Ⅱ9b (337.2 mg) was separated by reverse phase ODS column (MeOH:H 2 O, 4:6-10:0) to obtain subfractions Fr.Ⅱ9b-1 to Fr.Ⅱ9b-8. Fr.Ⅱ9b-4 was prepared by HPLC (MeCN:H 2 O, 72:28, 1.5 mL / min) for preparative separation and then purification (MeOH:H 2 O (0.1% FA), 83:17, 1.5 mL / min) to give compounds 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.II9f was purified by preparative HPLC (MeOH:H 2 O (0.1% FA), 87:13, 1.5 mL / min) to separate compound 5 (3.5 mg, t R =45min), 13(13.3mg,t R =23min), 15(3.9mg,t R =28.5min), 7α-hydroxysandaracopimaric acid (3.2mg,t R =32min), 8(14),15-sandaracopimaradiene-7a,18-diol(1.7mg,t R =38min), callicapene M3 (3.5mg,t R =33.5min). Fr.Ⅱ9c was separated and purified by preparative HPLC to obtain compound 18-hydroxy-sandaracopimara-8(14),15-dien-7-one(1.2mg), and enriched with compound 13(20.3mg).

[0054] Fr.Ⅱ10 (4.2 g) was subjected to silica gel column chromatography with petroleum ether-ethyl acetate gradient elution (50:1-1:2) to obtain eight fractions Fr.Ⅱ10a-h. Fr.Ⅱ10c was subjected to reverse phase C18 to obtain subfractions Fr.Ⅱ10c-1-Fr.Ⅱ10c-7, and then Fr.Ⅱ10c-4 was purified by HPLC (MeOH:H 2 O (0.1% FA), 88:12, 1.5 mL / min) was used for preparative separation to obtain compound 8 (6.8 mg, tR =28min) and enriched compound 13. Fr.II10c-2 was purified by HPLC (MeOH:H 2 O (0.1% FA), 78:22, 1.5 mL / min) was used for preparative separation to obtain compound 10 (5.2 mg, t R =18min). Fr.Ⅱ10f was separated by reverse phase C18 to obtain subcomponents Fr.Ⅱ10f-1 to Fr.Ⅱ10f-7. Fr.Ⅱ10f-4 was separated by HPLC (MeOH:H 2 O (0.1% FA), 80:20, 1.5 mL / min) was used for preparative separation to obtain compound 1 (5.5 mg, t R =24min), 2(4.3mg,t R =22min), 3(7.1mg,t R =34.5min), 7(4.5mg,t R =37.5min). Fr.Ⅱ10g was subjected to reverse phase C18 to obtain sub-fractions Fr.Ⅱ10g-1 to Fr.Ⅱ10g-6. Fr.Ⅱ10g-3 was subjected to HPLC (MeOH:H 2 O (0.1% FA), 76:24, 1.5 mL / min) was used for preparative separation to obtain compound 12 (9.2 mg, t R =18min).

[0055] Fr.Ⅱ11 (3.8 g) was subjected to silica gel column chromatography with petroleum ether-ethyl acetate gradient elution (40:1-1:1) to obtain eight fractions Fr.Ⅱ11a-h. Fr.Ⅱ11c was purified by HPLC (MeOH:H 2 O (0.1% FA), 81:19, 1.5 mL / min) was used for preparative separation to obtain compound 9 (5.0 mg, t R =44min). Fr.II11e was purified by HPLC (MeOH:H 2 O (0.1% FA), 81:19, 1.5 mL / min) to prepare and separate compound 8 (14), 15-sandaracopimaradiene-2,18-diol (9.3 mg, t R =29.5min). Fr.Ⅱ11g was separated by reverse phase C18 to obtain components Fr.Ⅱ11g-1 to Fr.Ⅱ11g-6. Fr.Ⅱ11g-3 was separated by HPLC (MeOH:H 2 O (0.1% FA), 78.5:11.5, 1.5 mL / min) was used for preparative separation to obtain compound 11 (30.0 mg, t R=31.5min), the fraction was purified by secondary preparation to obtain compound 18 (2.4mg), Roscorane D (1.8mg), (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 (5.1mg). Fr.Ⅱ11g-4 was purified by HPLC (MeOH:H 2 O (0.1% FA), 80:20, 1.5 mL / min) was used for preparative separation to obtain compound 4 (5.4 mg, t R =51min), 6(14.1mg,t R =40min), and enriched compound 8(14), 15-sandaracopimaradiene-2,18-diol (6.9mg).

[0056] Compounds 1 and 11 are crystals obtained by solvent evaporation method using anhydrous ethanol and dichloromethane, and compound 10 is a crystal obtained by slow evaporation at room temperature using methanol / water as solvent.

[0057] Structural identification of the isolated new compounds:

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

[0059] Compound 1: colorless crystals; (c 0.83,MeOH),UV(MeOH)λ max (logε)246(0.44)nm; IRν max 3467,2956,1712,1459,1384,1244,1026cm -1 ; HRESIMS m / z333.20670[MH] - (calcd for C 20 H29 O 4 ,333.20713); 1 H and 13 C NMR data are shown in Tables 1 and 3.

[0060] Compound 2: pale yellow oil; (c 0.22,MeOH),UV(MeOH)λ max (logε)240(0.30)nm; IRν max 3430,2933,2870,1702,1652,1457,1385,1237,1178,1037cm -1 ; HRESIMS m / z 333.20721[MH] - (calcd for C 20 H 29 O 4 ,333.20713); 1 H and 13 C NMR data are shown in Tables 1 and 3.

[0061] Compound 3: white solid; (c 0.04,MeOH),UV(MeOH)λ max (logε)245(0.44)nm; IRν max 3365,2935,2869,1663,1451,1385,1237,1149,1116,1075cm -1 ; HRESIMS m / z335.22141[M+H] + (calcd for C 20 H 31 O 4 ,335.22169); 1 H and 13 C NMR data are shown in Tables 1 and 3.

[0062] Compound 4: white solid; (c 0.31,MeOH),UV(MeOH)λ max (logε)206(0.32)nm; IRν max 3267,2940,2866,1711,1657,1458,1384,1260,1208,1161cm -1 ; HRESIMS m / z359.21893[M+Na] + (calcd for C 20 H32 O 4 Na,359.21928); 1 H and 13 C NMR data are shown in Tables 1 and 3.

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

[0064] Compound 6: white solid; (c 0.73,MeOH),UV(MeOH)λ max (logε)200(0.16)nm; IRν max 3413,2933,2610,1694,1462,1383,1233,1150,1123,1034cm -1 ; HRESIMS m / z359.21915[M+Na] + (calcd for C 20 H 32 O 4 Na,359.21928); 1 H and 13 C NMR data are shown in Tables 1 and 4.

[0065] Compound 7: white solid; (c 0.03,MeOH),UV(MeOH)λ max (logε)201(0.35)nm; IRν max 3439,2950,1696,1459,1369,1236,1017,945,842cm -1 ;HRESIMS m / z 401.22980[M+Na] + (calcd for C 22 H 34O 5 Na, 401.22985); 1 H and 13 C NMR data are shown in Tables 1 and 4.

[0066] Compound 8: white solid; (c 0.03,MeOH),UV(MeOH)λ max (logε)246(0.44)nm; IRν max 3424,2931,1694,1458,1384,1203,1152,1059cm -1 ;HRESIMS m / z 341.20825[M+Na] + (calcd for C 20 H 30 O 3 Na,341.20872); 1 H and 13 C NMR data are shown in Tables 1 and 4.

[0067] Compound 9: white solid; (c 0.03,MeOH),UV(MeOH)λ max (logε)242(0.27)nm; IRν max 3432,2930,1686,1458,1382,1200,1027cm -1 ; HR-ESI-MS m / z317.21228[MH] - (calcd for C 20 H 29 O 3 ,317.21222); 1 H and 13 C NMR data are shown in Tables 1 and 4.

[0068] Compound 10: yellow crystal; (c 0.03,MeOH),UV(MeOH)λ max (logε)200(0.56)nm; IRν max 3460,2932,2622,1693,1612,1468,1448,1425,1387,1348cm -1 ;HR-ESI-MS m / z369.16666[M+Na] + (calcd for C 20 H 26 O 5Na,369.16725); 1 H and 13 C NMR data are shown in Tables 1 and 4.

[0069] Compound 11: colorless crystals; (c 0.04,MeOH),UV(MeOH)λ max (logε)205(0.60)nm; IRν max 3423,2917,2614,1681,1636,1450,1381,1359,1250,1153cm -1 ; HRESIMS m / z357.20279[M+Na] + (calcd for C 20 H 30 O 4 Na, 357.20363); 1 H and 13 C NMR data are shown in Tables 2 and 5.

[0070] Compound 12: pale yellow oil; (c 0.04,MeOH),UV(MeOH)λ max (logε)252(0.16)nm; IRν max 3445,2929,1682,1603,1455,1389,1238,1033,995,914cm -1 ; HRESIMS m / z333.20575[M+H] + ,calcd for C 20 H 29 O 4 ,333.20604); 1 H and 13 C NMR data are shown in Tables 2 and 5.

[0071] Compound 13: colorless crystals; (c 1.07,MeOH),UV(MeOH)λ max (logε)216(-0.35)nm; IRν max 3402,2961,1689,1256,1006,784cm -1 ; HRESIMS m / z317.21127[MH] - (calcd for C 20 H 29 O 3 ,317.21222);1 H and 13 C NMR data are shown in Tables 2 and 5.

[0072] Compound 14: white solid; (c 0.12,MeOH),UV(MeOH)λ max (logε)201(0.52)nm; IRν max 3086,2932,1696cm -1 ; HRESIMS m / z 317.20923[M+H] + (calcd for C 20 H 29 O 3 ,317.21112); 1 H and 13 C NMR data are shown in Tables 2 and 5.

[0073] Compound 15: white solid; (c 0.10,MeOH),UV(MeOH)λ max (logε)204(0.58)nm; IRν max 3396,2930,1707,1634,1452,1384,1259,1187,1031,998cm -1 ; HRESIMS m / z327.22739[M+Na] + (calcd for C 20 H 32 O 2 Na,327.22945); 1 H and 13 C NMR data are shown in Tables 2 and 6.

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

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

[0076] Compound 18: white solid; (c 0.02,MeOH),UV(MeOH)λ max (logε)200(0.22)nm; IRν max 3282,2936,1634,1436,1382,1331,1260,1089,1038,997cm -1 ; HRESIMS m / z357.23953[M+Na] + (calcd for C 21 H 34 O 3 Na,357.24002); 1 H and 13 C NMR data are shown in Tables 2 and 6.

[0077] (2) Compounds 1, 10, and 11 were subjected to X-ray single crystal diffraction measurements using Bruker and Bruker APEX-IICCD single crystal diffractometers. The data obtained are as follows:

[0078] X-ray single crystal diffraction data of compound 1: C 20 H 30 O 4 (M=334.44g / mol),tetragonal,spacegroup P4 1 2 1 2,α=90°,β=90°,γ=90°, T=150.00K, Z=8, μ(CuKα)=0.663mm -1 ,Dcalc=1.214g / cm3 ,F(000)=1456.0,31556reflections measured(7.978°≤2Θ≤149.236°),3752independent reflections[R int =0.0362,R sigma =0.0212]were applied forcalculations with Flack parameter as-0.01(6),and the final R 1 was 0.0815(I>2σ(I))and wR 2 was 0.1789 (all data).

[0079] X-ray single crystal diffraction data of compound 10: C 20 H 28 O 6 (M=364.42g / mol),orthorhombic,spacegroup P2 1 2 1 2 1 ,α=90°,β=90°,γ=90°, Z=4, T=150.00K, μ(CuKα)=0.787mm -1 ,Dcalc=1.308g / cm 3 ,F(000)=784.0,12480reflections measured(9.506°≤2Θ≤149.846°),3739unique[R int =0.0289,R sigma =0.0293]which were used in all calculations with Flack parameter as 0.03(8).The final R 1 was 0.0679(I>2σ(I))and wR 2 was 0.1523 (all data).

[0080] X-ray single crystal diffraction data of compound 11: C 20 H 30 O 4 (M=334.44g / mol),tetragonal,spacegroup P4 3 2 12,α=90°,β=90°,γ=90°, Z=8, T=299.00K, μ(MoKα)=0.081mm -1 ,Dcalc=1.183g / cm 3 ,F(000)=1456.0,27355reflections measured(4.656°≤2Θ≤56.618°),4654unique[R int =0.0846,R sigma =0.0564]which were used in all calculations with Flackparameter as-0.1(7).The final R 1 was 0.0678(I>2σ(I))and wR 2 was 0.1991(alldata).

[0081] Table 1. C NMR spectra of compounds 1-10

[0082]

[0083]

[0084] Recorded in CDCl 3 a,in CD 3 ODb at 400MHz Chemicals shift(δ)are expressed in ppm.

[0085] Table 2. C NMR spectra of compounds 11-18

[0086]

[0087] Recorded in CDCl 3 a at 400MHz Chemicals shift(δ)are expressed in ppm.

[0088] Table 3. H NMR data of compounds 1-5

[0089]

[0090]

[0091] Recorded in CDCl 3a,in CD 3 ODb at 400MHz Chemicals shift(δ)are expressed in ppm.

[0092] Table 4. H NMR data of compounds 6-10

[0093]

[0094]

[0095] Recorded in CDCl 3 a,in CD 3 ODb at 400MHz Chemicals shift(δ)are expressed in ppm.

[0096] Table 5. H NMR data of compounds 11-14

[0097]

[0098] Recorded in CDCl 3 a at 400MHz Chemicals shift(δ)are expressed in ppm.

[0099] Table 6. H NMR data of compounds 15-18

[0100]

[0101] Recorded in CDCl 3 a at 400MHz Chemicals shift(δ)are expressed in ppm.

[0102] Embodiment 2:

[0103] Evaluation of anti-inflammatory activity in a copper sulfate-induced zebrafish model

[0104] Healthy inflammatory cell fluorescent 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), with 10 fish in each group and 3 replicate wells. After adding the sample, each group was placed in a constant temperature incubator at 28.5°C for 2 hours. Then, 20 μM CuSO 4The zebrafish in the model group, compound treatment group and positive control group were treated for 1 hour to establish the inflammation model. After the treatment, the zebrafish were washed and the inflammatory response 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 were statistically analyzed using GraphPad software. Zebrafish images were acquired using a SZX16 fluorescence microscope and DP2-BSW image acquisition system, and a Forma 3111 water-jacketed CO 2 Incubator, zebrafish breeding equipment.

[0105] in conclusion:

[0106] The in vivo anti-inflammatory activity of diterpenoids was evaluated in transgenic fluorescent zebrafish. The experiment used copper sulfate as a model, which can stimulate the neuromast cells and mechanical sensing cells in the lateral line of zebrafish to produce a strong acute inflammatory response, thereby causing inflammatory cell infiltration. Figure 1 As shown, C is the blank control group; M is the model group, 0 μM concentration sample; P is the positive control group, 20 μM indomethacin; #### indicates comparison with group C, P<0.0001; *P<0.1, **P<0.01, ***P<0.001, ****P<0.0001 indicates comparison with group M; the concentration of compound 1-18 is 50 μM. It can be seen that among compounds 1-18, some compounds have different degrees of anti-inflammatory activity.

[0107] Based on the preliminary screening results and the amount of isolated compounds, five abietane (1, 6, 7, 8, 10) and five isopimarane diterpenes (11, 12, 13, 14, 17) were selected for concentration gradient anti-inflammatory activity evaluation. The results showed that Figure 2 As shown, C is the blank control group; M is the model group, 0μM concentration sample; P is the positive control group, 20μM indomethacin; #### is compared with the C group, P<0.0001; *P<0.1, **P<0.01, ***P<0.001, ****P<0.0001 are compared with the M group. It can be seen that at non-toxic concentrations, except for compound 7, all have concentration-dependent anti-inflammatory activity, and there is a significant difference compared with the model group. Compounds 1, 8, 10-14, and 17 have significant anti-inflammatory activity. Among them, compounds 8, 10, 12, and 14 have the best activity, and their activity is equivalent to that of the positive control group.

[0108] Effects of different sample groups on zebrafish inflammatory cells Figure 3As shown, C is the blank control group; M is the model group, 0μM concentration sample; P is the positive control group, 20μM indomethacin; Compounds 1, 6, 7, 8, 10 are 25μM as the treatment group. The yellow box shows the migrating inflammatory cells. Compared with the model group, these compounds 1, 6, 8, 10, 11-14, 17 significantly reduced the number of zebrafish inflammatory cells migrating to the lateral line at medium or high concentrations.

[0109] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

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) taking the dried aerial part of the Psoralea corylifolia, adding a solvent for reflux extraction, combining the filtrate, and concentrating to obtain a crude extract; (2) After suspending the crude extract in water, extracting with ethyl acetate and n-butanol in sequence, combining the filtrates, and concentrating under reduced pressure to obtain an ethyl acetate phase extract; (3) subjecting the ethyl acetate phase extract to MCI column chromatography, and using a methanol-water gradient elution to obtain seven fractions Fr.I-VII; (4) The Fr.Ⅱ segment components were separated by silica gel column chromatography, and 11 eluents were collected after gradient elution using petroleum ether-acetone; a mixed solvent of petroleum ether and acetone with a volume ratio of 2:1 was used as a developing solvent, and silica gel thin layer chromatography was used for identification, and the eluents were combined into 12 eluents, and concentrated under reduced pressure to obtain sub-fractions Fr.Ⅱ1-Fr.Ⅱ12; (5) The Fr.Ⅱ6 subfraction was subjected to gel chromatography, eluted with dichloromethane-methanol, and then eluted with reverse phase C 18 Chromatographic column and HPLC method were used to separate compounds 16 and 17; (6) The Fr.Ⅱ9 subcomponent was separated by silica gel column chromatography using a petroleum ether-acetone gradient elution, and then by reversed phase ODS column and HPLC to obtain compounds 5, 13, 14, and 15; (7) The Fr.Ⅱ10 subcomponent was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; and compounds 1, 2, 3, 7, 8, 10, and 12 were separated using a reversed phase ODS column and HPLC. (8) The Fr.Ⅱ11 subcomponent was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution; and 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, characterized in that: 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 extraction is 2-4 times, and each extraction is 1-3h; Preferably, the solvent is 95% by volume ethanol aqueous solution, and the reflux extraction is performed twice, with each extraction lasting 2 hours.

4. The preparation method according to claim 2, characterized in that: In step (3), the MCI filler is GEL CHP20P, and the volume ratio of methanol to water is (3:7)-(10:0).

5. The preparation method according to claim 2, characterized in that: In step (4), the silica gel column chromatography is wet packed with 200-300 mesh silica gel, and the volume ratio of petroleum ether to acetone is (100:1)-(1:1); Preferably, 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, or 1:

1.

6. The preparation method according to claim 2, characterized in that: In step (5), the volume ratio of dichloromethane to methanol is (1:1)-(1:5); the eluent of the reverse phase C18 chromatographic column is methanol-water in a volume ratio of (3:7)-(10:0); the mobile phase of HPLC is methanol-water in a volume ratio of (90:10)-(50:50); and the flow rate is 1.5-2 mL / min.

7. The preparation method according to claim 2, characterized in that: In step (6), the volume ratio of petroleum ether to acetone is (50:1)-(1:1); the eluent of the reversed phase ODS column is methanol-water in a volume ratio of (4:6)-(10:0); HPLC is separated twice, and the mobile phase for the first separation of HPLC is acetonitrile-water in a volume ratio of (72:28)-(70:30), and the flow rate is 1.5-2mL / min; the mobile phase for the second separation of HPLC is methanol-containing 0.1V% FA aqueous solution in a volume ratio of (83:17)-(80:20), and the flow rate is 1.5-2mL / min.

8. The preparation method according to claim 2, characterized in that: In step (7), the volume ratio of petroleum ether to ethyl acetate is (50:1)-(1:2); the eluent of the reversed phase ODS column is (3:7)-(10:0) methanol-water; The mobile phase of HPLC was methanol-containing 0.1V% FA aqueous solution in a volume ratio of (76:24)-(88:12), with a flow rate of 1.5-2mL / min.

9. The preparation method according to claim 2, characterized in that: In step (8), the volume ratio of petroleum ether to ethyl acetate is (40:1)-(1:1); the eluent of the reversed phase ODS column is methanol-water in a volume ratio of (3:7)-(10:0); the mobile phase of HPLC is methanol-containing 0.1V% FA aqueous solution in a volume ratio of (78:22)-(81:19), and the flow rate is 1.5-2mL / min.

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

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