Norditerpenoid compounds with anti-inflammatory activity as well as preparation method and application of norditerpenoid compounds

Abstract

The invention discloses a norditerpenoid compound with anti-inflammatory activity and a preparation method and application thereof, the norditerpenoid compound is separated from a secondary metabolite of marine soft coral, and the preparation method is characterized in that the norditerpenoid compound is obtained by separating from the secondary metabolite of marine soft coral, and comprises the following steps: 1) extracting frozen soft coral with acetone, extracting with diethyl ether and water, and concentrating to obtain an extract; (2) carrying out normal-phase silica gel reduced-pressure column chromatography on the crude extract, carrying out gradient elution by taking petroleum ether and ethyl acetate as mobile phases, collecting and combining fractions, carrying out reversed-phase medium-pressure column chromatography on a second fraction, taking a mixed solution of methanol and water as a mobile phase, collecting the fractions, and carrying out high-performance liquid-phase preparation, purification and separation on a third fraction to obtain a compound 1; the sixth fraction is subjected to reversed-phase medium-pressure column chromatography, a mixed solution of methyl alcohol and water serves as a mobile phase, the fourth fraction is subjected to high-performance liquid phase preparation and purification, and a compound 2 is obtained through separation and has the advantage of being good in anti-inflammatory activity.

Description

Technical Field

[0001] The present invention relates to norditerpenoid compounds, and in particular to a norditerpenoid compound with anti-inflammatory activity derived from marine soft coral, and its preparation method and use. Background Art

[0002] Soft corals of the genus Litophyton are widely distributed in the South China Sea and are important sources of bioactive substances with unique structural characteristics. So far, more than 200 secondary metabolites have been isolated from this soft coral. These compounds can be structurally divided into sesquiterpenes, sesquiterpene dimers, diterpenes, norditerpenes, tetraterpenes, meroterpenes, steroids, ceramides, pyrimidines, polypeptides, etc. Diterpenoid compounds derived from soft corals of the genus Litophyton have diverse structures and can be divided into various types, including cembrane type, eunicellane type, serrulatane type, and other types with novel carbon skeletons. Among them, the cembrane type, i.e., cembrane diterpenes, shows the most structural variations and contains a wide range of functional groups. It is worth noting that natural C17 compounds containing a 14-membered ring tetraene are very rare. In order to discover bioactive compounds from soft corals, the inventors collected a soft coral Litophyton brassicum from Xisha Islands in Hainan, analyzed its chemical composition, and isolated two rare norditerpenoid compounds, litobrassins A (1) and B (2), which are new compounds. Currently, there are no research reports on these two norditerpenoid compounds and their functional activities. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a norditerpenoid compound with anti-inflammatory activity, and its preparation method and use.

[0004] The technical solution adopted by the present invention to solve the above technical problem is: a norditerpenoid compound with anti-inflammatory activity, the norditerpenoid compound is isolated from the secondary metabolites of marine soft coral Litophyton brassicum, and its structural formula is shown as at least one of the following:

[0005]

[0006] The preparation method of the above-mentioned norditerpenoid compound with anti-inflammatory activity includes the following steps:

[0007] (1) Extracting the extract: After crushing the frozen soft coral, it is fully soaked and extracted with acetone, and the extract is concentrated under reduced pressure to obtain an extract. The extract is extracted 2-4 times with a mixed solution composed of equal volumes of ether and water, the ether layer extraction solutions are combined, and after concentration under reduced pressure, a crude extract is obtained;

[0008] (2) The crude extract obtained in step (1) was dissolved in ethyl acetate solvent, and then subjected to normal-phase vacuum column chromatography. A petroleum ether-ethyl acetate solution with a volume ratio of (100:1) to (1:1) was used as the mobile phase for gradient elution. The fractions were collected tube by tube and arranged in ascending order of polarity. A total of 11 fractions were obtained by merging;

[0009] (3) The second fraction obtained in step (2) was subjected to medium-pressure reverse-phase column chromatography. A methanol-aqueous solution with a methanol volume content of 65 wt% to 100 wt% was used as the mobile phase for gradient elution. The fractions were collected tube by tube and arranged in descending order of polarity. A total of 5 fractions were obtained by merging;

[0010] (4) The third fraction obtained in step (3) was separated by high-performance liquid chromatography. An acetonitrile-aqueous solution with an acetonitrile volume percentage of 65% was used as the mobile phase for isocratic elution to obtain compound litobrassin A, the chemical structure of which is shown as compound 1 below;

[0011] (5) The sixth fraction obtained in step (2) was subjected to medium-pressure reverse-phase column chromatography. A methanol-aqueous solution with a methanol volume content of 75 wt% to 100 wt% was used as the mobile phase for gradient elution. The fractions were collected tube by tube and arranged in descending order of polarity. A total of 5 fractions were obtained by merging;

[0012] (6) The fourth fraction obtained in step (5) was separated by high-performance liquid chromatography. An acetonitrile-aqueous solution with an acetonitrile volume percentage of 68% was used as the mobile phase for isocratic elution to obtain compound litobrassin B, the chemical structure of which is shown as compound 2 below;

[0013]

[0014] Furthermore, the elution gradient volume ratios of the petroleum ether-ethyl acetate solution in step (2) were 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1 in sequence.

[0015] Furthermore, the elution gradients of the methanol-aqueous solution in step (3) were 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% in sequence.

[0016] Furthermore, the flow rate of the high-performance liquid chromatography separation in step (4) was 2 mL / min, the isocratic elution time was 50 min, the column temperature was room temperature, and the collection wavelength was 200 nm.

[0017] Furthermore, the elution gradients of the methanol-aqueous solution in step (5) were 75%, 80%, 85%, 90%, 95%, and 100% in sequence.

[0018] Furthermore, the flow rate of the high performance liquid chromatography separation described in step (6) is 2 mL / min, the isocratic time is 50 min, and the column temperature is room temperature.

[0019] Use of any one of the above-mentioned norditerpenoid compounds 1 and 2 in the preparation of anti-inflammatory drugs.

[0020] Compared with the prior art, the advantages of the present invention are as follows: The present invention relates to a norditerpenoid compound derived from soft coral, its preparation method and use. Two novel norditerpenoid compounds, litobrassin A (1) and litobrassin B (2), are isolated from the acetone extract of the soft coral Litophyton brassicum collected from the Xisha Islands in the South China Sea. The extract is obtained by extracting the freeze-dried soft coral with an organic reagent, and then the extract is extracted with diethyl ether to obtain a crude extract. The obtained crude extract is separated and purified by normal-phase vacuum column chromatography, medium-pressure reverse-phase column chromatography, and semi-preparative high performance liquid chromatography. The compound has good anti-inflammatory activity, and the norditerpenoid compound can also be used as a lead compound for anti-inflammatory drugs, laying a foundation for the development of new drugs. Description of the Drawings

[0021] Figure 1 High-resolution mass spectrum of compound litobrassin A (1);

[0022] Figure 2 1H nuclear magnetic resonance spectrum of compound litobrassin A (1);

[0023] Figure 3 13C nuclear magnetic resonance spectrum of compound litobrassin A (1);

[0024] Figure 4 DEPT 135° nuclear magnetic resonance spectrum of compound litobrassin A (1);

[0025] Figure 5 Of compound litobrassin A (1) 1 H- 1 H COSY nuclear magnetic resonance spectrum;

[0026] Figure 6 HSQC nuclear magnetic resonance spectrum of compound litobrassin A (1);

[0027] Figure 7 HMBC nuclear magnetic resonance spectrum of compound litobrassin A (1);

[0028] Figure 8 1D NOE nuclear magnetic resonance spectrum of compound litobrassin A (1) (H-7)

[0029] Figure 9 1D NOE NMR spectrum of compound litobrassin A(1)(H-11);

[0030] Figure 10 Compound litobrassin A(1) DP4 + Probability (calculated at the PCM / mPW1PW91 / 6-31+G(d,p) level), isomer 1 is 1R and 4R, isomer 2 is 1R and 4S, isomer 3 is 1S and 4R, isomer 4 is 1S and 4S;

[0031] Figure 11 Experimental and calculated ECD curves of compound litobrassin A(1) (methanol);

[0032] Figure 12 High-resolution mass spectrum of compound litobrassin B(2);

[0033] Figure 13 1H NMR spectrum of compound litobrassin B(2);

[0034] Figure 14 13C NMR spectrum of compound litobrassin B(2);

[0035] Figure 15 DEPT 135° NMR spectrum of compound litobrassin B(2);

[0036] Figure 16 Compound litobrassin B(2) 1 H- 1 H COSY NMR spectrum;

[0037] Figure 17 HSQC NMR spectrum of compound litobrassin B(2);

[0038] Figure 18 HMBC NMR spectrum of compound litobrassin B(2);

[0039] Figure 19 1D NOE NMR spectrum of compound litobrassin B(2)(H-3);

[0040] Figure 20 1D NOE NMR spectrum of compound litobrassin B(2)(H-7);

[0041] Figure 211D NOE NMR spectrum of compound litobrassin B(2)(H-11);

[0042] Figure 22 Measured and calculated ECD curves (methanol) of compound litobrassin B(2);

[0043] Figure 23 Effects of compounds 1 and 2 on NO expression in LPS- and IFN-γ-induced RAW 264.7 macrophages;

[0044] Figure 24 Effects of compounds 1 and 2 on TNF-α production in LPS- and IFN-γ-induced RAW 264.7 macrophages. Detailed implementation mode

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0046] Example 1. Diterpenoid compounds isolated from the soft coral Litophyton brassicum: litobrassin A(1) and litobrassin B(2), the chemical structural formulas are as follows:

[0047]

[0048] Example 2. Preparation method of the diterpenoid compounds in Example 1 above, the specific steps are as follows:

[0049] Step 1. Extraction of secondary metabolites

[0050] After the soft coral sample is vacuum freeze-dried by a freeze dryer and then crushed by a crusher, it is fully soaked in acetone at room temperature for 2 days each time, followed by ultrasonic extraction for 1 hour. The ultrasonic extraction is repeated 4-5 times until the reagent becomes colorless. The extract is filtered to remove the sample residue, and then concentrated under reduced pressure to obtain an extract. The extract is extracted three times with a mixed solution of ether and pure water with a volume ratio of 1:1, and the ether layer extract is combined and concentrated under reduced pressure to obtain 80 g of an ether layer extract.

[0051] Step 2. Separation and preparation of compounds

[0052] (1) After the above-mentioned crude extract is first dissolved in an ethyl acetate solvent, normal-phase vacuum column chromatography is carried out, and a petroleum ether-ethyl acetate solution with a volume ratio of (100:1)-(1:1) is used as the mobile phase for gradient elution. The fractions are collected bottle by bottle and arranged in ascending order of fraction polarity, and 11 fractions are combined; among them, the elution gradient volume ratios of the petroleum ether-ethyl acetate solution are 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1 in turn;

[0053] (2) The second fraction obtained in step (1) was subjected to medium-pressure reversed-phase column chromatography, and gradient elution was carried out using a methanol-aqueous solution as the mobile phase. The fractions were collected tube by tube, arranged in descending order of fraction polarity, and combined to obtain 5 fractions; the elution gradient of the methanol-aqueous solution was 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% (volume percentage of methanol) in sequence;

[0054] (3) The third fraction collected in step (2) was separated by high-performance liquid chromatography. Isocratic elution was carried out using an acetonitrile-aqueous solution as the mobile phase at room temperature. The volume percentage of acetonitrile in the acetonitrile-aqueous solution was 65%, the flow rate was 2 mL / min, the isocratic time was 50 min, and the acquisition wavelength was 200 nm, to obtain compound litobrassin A (1) (4.7 mg), and its chemical structural formula is shown as compound 1 below;

[0055] (4) The 6th fraction obtained in step (2) was subjected to medium-pressure reversed-phase column chromatography, and gradient elution was carried out using a methanol-aqueous solution as the mobile phase. The fractions were collected tube by tube, arranged in descending order of fraction polarity, and combined to obtain 5 fractions; the elution gradient of the methanol-aqueous solution was 75%, 80%, 85%, 90%, 95% and 100% (volume percentage of methanol) in sequence;

[0056] (5) The fourth fraction collected in step (4) was separated by high-performance liquid chromatography. Isocratic elution was carried out using an acetonitrile-aqueous solution as the mobile phase at room temperature. The acetonitrile content in the acetonitrile-aqueous solution was 68%, the flow rate was 2 mL / min, the isocratic time was 50 min, and the acquisition wavelength was 200 nm, to obtain compound litobrassin B (2) (5.8 mg), and its chemical structural formula is shown as compound 2 below;

[0057]

[0058] Example 3. Structure identification of the compound and NMR signal assignment:

[0059] Litobrassin A (1): Colorless oil, C 19 H 30 O 3 , {[α]25D - 8.00 (c 0.5, MeOH)}; UV (MeOH) λ max (logε) 205 (2.36); HRESIMS m / z 289.2180 [M-H 2 O + H] + (calcd for C 19 H 29 O 2, 289.2167); The 1 H and 13 C NMR data are shown in Figure 1 , Figure 2 , Figure 3 and Table 1.

[0060] Table 1. 1 H NMR (600 MHz) and 13 C NMR (150 MHz, CDCl 3 )

[0061]

[0062]

[0063] Litobrassin A (1) was isolated as an optically active colorless oil. Its molecular formula C 19 H 30 O 3 was determined by the HRESIMS ion peak m / z 289.2180 [M-H 2 O + H] + (calcd 289.2167), indicating five degrees of unsaturation. Key 1 H, 13 C nuclear magnetic resonance (NMR) (Table 1), DEPT 135°( Figure 4 ), and heteronuclear single quantum coherence (HSQC) experiments revealed an acetyl group (δ C 170.5 and δ C / δ H 21.8 / 2.00), two trisubstituted double bonds [δ C 127.4 (C-7) / δ H 5.17, δ C 133.6 (C-8), δ C 125.2 (C-11) / δ H 4.99 and δ C 132.2 (C-12)], a disubstituted double bond [δ C 123.2 (C-2) / δ H 5.52, δ C 143.3 (C-3)] / δ H 5.92], a sp 3 -oxo quaternary carbon, a sp 3 -oxo methylene, six sp 3The presence of a methylene and three methyl groups. Comparison of the 1D and 2D NMR spectral data of the cembranoid norditerpenoid litoamentene B lacking an isopropyl side chain obtained from the soft coral Litophyton amentaceum in the South China Sea ( Figure 5 , 6 and 7) revealed that they have the same planar structure. The relatively large coupling constant (J 2,3 = 15.3 Hz) and the 1D NOE correlations from H-7 to H 2 -9( Figure 8 ), H-11 to H 2 -13( Figure 9 ) indicated that the Δ 2,3 , Δ 7,8 and Δ 11,12 double bonds are in the E configuration, respectively. The quantum mechanical nuclear magnetic resonance (QM-NMR) method was used to calculate the relative configuration. Four possible configurations were calculated: 1a (1R,4R), 1b (1R,4S), 1c (1S,4R), and 1d (1S,4S)( Figure 10 ). The DP4 + correlation between the calculated and experimental data was used, and the corresponding DP4 + probability was estimated. Configuration 1c showed a probability of more than 99%( Figure 10 ), and the relative configuration of 1 was deduced to be (1R*,4S*). Finally, the absolute configuration of 1 was determined to be 1S,4R by time-dependent density functional theory TDDFT / ECD calculation( Figure 11 ).

[0064] Litobrassin B (2): Colorless oil; C 17 H 28 O 2 ; {[α]25D - 37.67 (c 0.5, MeOH)}; UV (MeOH) λ max (logε) 205 (2.40); HRESIMS m / z 247.2061 [M-H 2 O + H] + (calcd for C 17 H 27 O, 247.2056); The 1 H and 13 C NMR data of this compound are shown in Figure 12 , Figure 13 , 14 and Table 2.

[0065] Table 2. 1 H NMR (600 MHz) and 13 C NMR (150 MHz, CDCl3 )

[0066]

[0067] Litobrassin B(2) was isolated as a colorless oil. Its molecular formula was determined to be C 17 H 28 O 2 , based on the HRESIMS ion peak at m / z 247.2061 [M−H 2 O+H] + (calcd for C 17 H 27 O, 247.2056), indicating four degrees of unsaturation. The NMR data of compound 2 ( Figure 13 , 14 and 15) were very similar to those of the deisopropyl cembranoid diterpenoids, revealing that they were structural analogs. The differences were the Δ 1,2 at C-2 instead of the Δ 2,3 at C-1 of litoamentene C, and the position of the sp 3 -oxymethylene group in 1 was at C-3 (δ C 77.7), which was further confirmed by 2D NMR spectra ( Figure 16 , 17 and 18). The relative configuration of 2 was determined to be 1Z, 7E, 11E, 3S*, 4S* by the smaller coupling constant (J 1,2 = 5.9 Hz) and the 1D NOE correlations from H-7 to H 2 -9 ( Figure 20 ), from H-11 to H 2 -13 ( Figure 21 ), and from H-3 to H 3 -15 ( Figure 19 ). The absolute configuration of 2 was determined to be 3S, 4S by TDDFT / ECD calculations ( Figure 22 ).

[0068] Example 4. Anti-inflammatory activities of the norditerpenoid compounds 1 and 2 prepared in Example 2 above.

[0069] 1. Experimental samples

[0070] Preparation of the test sample solution: The test samples were the pure compounds 1 and 2 isolated and purified in Example 2 above. An appropriate amount of the sample was accurately weighed and dissolved in DMSO to a concentration of 10 μM. The positive drug used in this experiment was celastrol.

[0071] 2. Experimental methods

[0072] RAW264.7 macrophages were cultured in 96-well plates for 24 hours. For the cytotoxicity part, RAW264.7 cells were incubated with the compound (either Compound 1 or Compound 2) and medium (DMEM medium containing 0.125% DMSO and 10% fetal bovine serum (FBS)) for 24 hours. 20 μL of CCK-8 reagent was added to each well, and the OD value was collected by a microplate reader at 450 nm (calibrated at 650 nm) after 1 hour.

[0073] For the anti-inflammatory activity assay, RAW264.7 macrophages were treated with lipopolysaccharide (LPS, 100 ng / mL) and interferon-γ (IFN-γ, 20 ng / mL) to induce inflammation, and then co-incubated with celastrol (2 μM) for 6 hours or with the test compound (10 μM) for 24 hours. The NO concentration was quantified using Griess reagent. The TNF-α level was measured using an ELISA kit according to the manufacturer's instructions.

[0074] 3. Experimental Results

[0075] Compounds 1 and 2 showed no cytotoxicity to RAW 264.7 cells at a concentration of 10 μM. Given that macrophages are the main producers of inflammatory cytokines, the inventors investigated the effects of Compounds 1 and 2 on the activation of LPS-induced RAW264.7 cells, with particular attention to the release of NO and TNF-α. As expected, LPS stimulation significantly increased the levels of NO and TNF-α in RAW264.7 cells. Celastrol, known for its anti-inflammatory properties, significantly inhibited the LPS-induced activation of these cells as a positive control. Among the tested compounds, Compounds 1 and 2 effectively reduced the LPS-induced NO production ( Figure 23 ). Notably, Compound 2 was able to significantly reduce the LPS-induced TNF-α release ( Figure 24 ). These findings highlight the potential of Compounds 1 and 2 as anti-inflammatory agents, which have varying degrees of inhibitory effects on the production of NO and TNF-α in macrophages after LPS stimulation.

[0076] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions, or substitutions made by those of ordinary skill in the art within the scope of the essence of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A norditerpenoid compound having anti-inflammatory activity, characterized in that The norditerpenoid compound is isolated from the secondary metabolites of the marine soft coral Litophyton brassicum, and its structural formula is shown in at least one of the following:

2. A method for preparing a norditerpenoid compound with anti-inflammatory activity according to claim 1, characterized in that The following steps are involved: (1) Extraction: The frozen soft coral is crushed and then fully soaked in acetone for extraction, and the extract is concentrated under reduced pressure to obtain an extract. The extract is extracted 2-4 times with a mixture of equal volumes of ether and water, and the ether layer extracts are combined and concentrated under reduced pressure to obtain a crude extract; (2) dissolving the crude extract obtained in step (1) in ethyl acetate solvent, performing normal phase vacuum column chromatography, using a petroleum ether-ethyl acetate solution with a volume ratio of (100:1) to (1:1) as the mobile phase for gradient elution, collecting fractions tube by tube, arranging the fractions from small to large in terms of polarity, and combining to obtain 11 fractions; (3) subjecting the second fraction obtained in step (2) to medium-pressure reverse phase column chromatography, using a methanol-water solution with a methanol volume content of 65 wt % to 100 wt % as the mobile phase for gradient elution, collecting fractions tube by tube, arranging the fractions from large to small in terms of polarity, and combining to obtain 5 fractions; (4) The third fraction obtained in step (3) was separated by high performance liquid chromatography, and an acetonitrile-water solution with a volume percentage of 65% acetonitrile was used as the mobile phase for isocratic elution to separate the compound litobrassin A, whose chemical structure is shown in the following compound 1; (5) subjecting the sixth fraction obtained in step (2) to medium-pressure reverse phase column chromatography, using a methanol-water solution with a methanol volume content of 75 wt % to 100 wt % as the mobile phase for gradient elution, collecting fractions tube by tube, arranging the fractions from large to small in terms of polarity, and combining to obtain 5 fractions; (6) The fourth fraction obtained in step (5) was separated by high performance liquid chromatography, and an acetonitrile-water solution with a volume percentage of 68% acetonitrile was used as the mobile phase for isocratic elution to separate the compound litobrassin B, whose chemical structure is shown in the following compound 2; 3. The method for preparing a norditerpenoid compound with anti-inflammatory activity according to claim 2, characterized in that: The elution gradient volume ratios of the petroleum ether-ethyl acetate solution in step (2) are 100:1, 50:1, 20:1, 10:1, 5:1, 2:1 and 1:1, respectively.

4. The method for preparing a norditerpenoid compound with anti-inflammatory activity according to claim 2, characterized in that: The elution gradient of the methanol-water solution in step (3) is 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% respectively.

5. The method for preparing a norditerpenoid compound with anti-inflammatory activity according to claim 2, characterized in that: The flow rate of the HPLC separation in step (4) is 2 mL / min, the isocratic time is 50 min, the column temperature is room temperature, and the collection wavelength is 200 nm.

6. The method for preparing a norditerpenoid compound with anti-inflammatory activity according to claim 2, characterized in that: The elution gradient of the methanol-water solution in step (5) is 75%, 80%, 85%, 90%, 95% and 100% respectively.

7. The method for preparing a norditerpenoid compound with anti-inflammatory activity according to claim 2, characterized in that: The flow rate of the HPLC separation in step (6) is 2 mL / min, the isocratic time is 50 min, the column temperature is room temperature, and the collection wavelength is 200 nm.

8. Use of any one of the norditerpenoid compounds 1 and 2 as claimed in claim 1 in the preparation of anti-inflammatory drugs.

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