Sponge-sourced polyketide enantiomer as well as preparation method and application thereof

Abstract

The invention discloses a sponge-sourced polyketide enantiomer as well as a preparation method and application thereof, and belongs to the technical field of marine organisms and medicines. The invention provides a sponge-sourced polyketolactone enantiomer on one hand, and provides a preparation method and application of the sponge-sourced polyketolactone enantiomer on the other hand. The sponge-sourced polyketide enantiomer disclosed by the invention is sourced from Hippospongia lacne in South China Sea, and the enantiomer is derived from the Hippospongia lacne in South China Sea. The preparation method of the sponge-sourced polyketide enantiomer is simple, and medicine source materials are widely distributed in South China Sea and are easy to collect. The invention provides a novel compound with anti-inflammatory activity for research of marine drugs, and provides a scientific basis for development and utilization of marine medicinal resources in China.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine organisms and medicine, and specifically relates to a polyketide lactone enantiomer derived from sponge and a preparation method thereof. Background Art

[0002] As the second largest species in the ocean, sponges account for about 30% of the marine species biomass. As "marine fossils", sponges have a survival history of 600 million years. During continuous evolution, in order to adapt to extreme environments, they have developed unique chemical defense mechanisms, and their metabolite skeleton types are diverse and their activities are remarkable, making them an excellent "blue drug treasure house". The medicinal source material, Hippospongia hispida Hippospongia lachne is produced in the South China Sea of China, and a variety of highly active polyketide compounds have been isolated from sponges of this genus. Such compounds often have multiple chiral centers, which also provides the possibility for the existence of enantiomers. The separation of enantiomers and the determination of absolute configuration are difficult points in natural product chemistry.

[0003] In the process of developing and utilizing China's marine biological resources to search for marine natural products with biological activities and drug prospects, we found that Hippospongia hispida Hippospongia lachne from the South China Sea of China shows significant inhibitory activity against the levels of NO and IL-6 in LPS-induced RAW 264.7 cells in vitro anti-inflammatory activity tests. Further bioactivity tracking studies found a polyketide lactone enantiomer. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for a polyketide lactone enantiomer derived from sponge and a preparation method thereof.

[0005] The present invention is specifically realized by the following technical solutions: In the first aspect of the present invention, there is provided a polyketide lactone enantiomer derived from sponge, whose chemical structures are shown in Formula I and Formula II: Formula I; Formula II.

[0006] Furthermore, the above polyketide lactone derived from sponge is derived from Hippospongia hispida in the South China Sea Hippospongia lachne .

[0007] In the second aspect of the present invention, there is provided a preparation method for the above polyketide lactone enantiomer derived from sponge, which includes the following steps: The first step is to prepare the total extract Take Hippospongia hispida Hippospongia lachneCut into small pieces and extract three times with 95% ethanol to obtain the total extract; suspend the total extract in water and extract three times with ethyl acetate, then concentrate under reduced pressure to obtain the fat-soluble extract; suspend the fat-soluble extract in 90% methanol-aqueous solution, extract with petroleum ether and then concentrate under reduced pressure to obtain the total petroleum ether extract. Step 2: Separation and purification 1) Separate the total petroleum ether extract by silica gel column chromatography under reduced pressure, use petroleum ether-acetone solution as the eluent for gradient elution, and combine the fractions according to the thin-layer chromatography color development to obtain 14 fractions Fr. A-N. 2) Perform normal-phase silica gel column chromatography on fraction Fr. F, use petroleum ether-acetone solution for gradient elution, and combine the fractions according to the thin-layer chromatography color development to obtain 13 fractions Fr.F1-F13. 3) Perform ODS medium-pressure column chromatography on fraction Fr. F5, use methanol-aqueous solution for gradient elution, and combine the fractions according to the peak elution situation to obtain 13 fractions Fr.F5a-F5m. 4) Perform ODS medium-pressure column chromatography on fraction Fr.F5e, use methanol-aqueous solution for gradient elution, and combine the fractions according to the peak elution situation to obtain 18 fractions Fr. F5ea-F5er. 5) Purify fraction Fr. F5eb by reverse-phase high-performance liquid chromatography to obtain the racemate of the polyketide lactone derived from the sponge. 6) Separate the above racemate by normal-phase chiral high-performance liquid chromatography to obtain an enantiomer of the polyketide lactone derived from the sponge.

[0008] Furthermore, in the separation and purification step of the second step in this method, in 1), the conditions for gradient elution with petroleum ether-acetone solution as the eluent are: elute successively with petroleum ether-acetone solutions with volume ratios of 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, and 1:1.

[0009] Furthermore, in the separation and purification step of the second step in this method, in 2), the conditions for gradient elution with petroleum ether-acetone solution as the eluent are: elute successively with petroleum ether-acetone solutions with volume ratios of 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, and 1:1.

[0010] Furthermore, in the separation and purification step of the second step in this method, in 3), the volume ratio of methanol-aqueous solution for gradient elution is 10% to 100%, and the time is 9 h.

[0011] Furthermore, in the separation and purification step of the second step in this method, in 4), the volume ratio of methanol-aqueous solution for gradient elution is 10% to 100%, and the time is 7 h.

[0012] Furthermore, in the second separation and purification step of this method, in step 5), the conditions for reversed-phase high performance liquid separation are: the eluent is acetonitrile-water solution with a volume ratio of 80:20, and the chromatographic column is YMC-Pack Pro C 18 RS, the flow rate was 2.0 mL / min, the detection wavelength was 224 nm, and the retention time was 15.0 min.

[0013] Furthermore, in the second separation and purification step of the method, in step 6), the conditions for normal phase chiral high performance liquid separation are: the eluent is a n-hexane-isopropanol solution with a volume ratio of 70:30, and the chromatographic column is CHIRALPAK Ò IC00CE-BT016, the flow rate was 1.0 mL / min, the detection wavelength was 226 nm, and the retention times were 28.0 min (Formula I) and 47.0 min (Formula II), respectively.

[0014] The third aspect of the present invention provides the use of the sponge-derived polyketide lactone enantiomers in the preparation of anti-inflammatory drugs, specifically, the sponge-derived polyketide lactone enantiomers all show significant inhibitory activity on LPS-induced NO and IL-6 levels in RAW 264.7 cells.

[0015] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects: The sponge-derived polyketolactone enantiomer provided by the present invention is derived from the hairy horse sponge in the South China Sea. Hippospongia lachne The sponge-derived polyketolactone enantiomers of the present invention are simple to prepare, and the medicinal materials are widely distributed in the South my country Sea and are easy to collect. The present invention provides new compounds with anti-inflammatory activity for marine drug research and provides a scientific basis for the development and utilization of my country's marine medicinal resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of NOESY signal correlation of the polyketolactone of the present invention.

[0017] Figure 2 The lactone segment (4 R , 5 S )-a and (4 S , 5 R )-b's absolute configuration diagram.

[0018] Figure 3 The measured CD spectra and lactone fragments (4 R , 5 S )-a and (4 S , 5 R) Schematic diagram of the calculated ECD spectrum of -b. Detailed implementation mode

[0019] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific content described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0020] The present invention is a pair of polyketide lactone enantiomers isolated from Hymeniacidon perleve collected from the South China Sea of our country. Hippospongia lachne The compounds of the present invention can also be prepared using other sponges or by artificial synthesis methods.

[0021] Example 1: Preparation of the compounds of the present invention First step, preparation of the total extract The Hymeniacidon perleve frozen in a -20 °C refrigerator Hippospongia lachne (wet weight 10 kg) was thawed and cut into small pieces (about 2 cm * 2 cm), and cold-extracted with 95% ethanol (20 L * 3 times) to obtain a total extract (280 g). The total extract was dispersed in water (1 L), extracted with ethyl acetate (1 L) 3 times, and concentrated under reduced pressure to obtain a fat-soluble extract (141 g). The fat-soluble extract was suspended in a 90% methanol-aqueous solution (800 mL), extracted with petroleum ether (800 mL * 3 times) and then concentrated under reduced pressure to obtain a petroleum ether total extract (75 g); Second step, separation and purification 1) The petroleum ether total extract was separated by medium-pressure silica gel column chromatography (VLC), and gradient elution was carried out with petroleum ether-acetone solutions (50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1) as eluents. According to thin-layer chromatography (TLC) color development, the fractions were combined to obtain 14 fractions Fr. A-N, and their effects on the IL-6 level in LPS-induced RAW 264.7 cells (concentration 10 mg / mL) were detected; 2) The fraction Fr. F containing pink spots after color development and with an IL-6 inhibition rate greater than 50% was separated by normal-phase silica gel column chromatography, and gradient elution was carried out with petroleum ether-acetone solutions (50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1). According to TLC color development, the fractions were combined to obtain 13 fractions Fr. F1-F13, and their effects on the IL-6 level in LPS-induced RAW 264.7 cells (concentration 10 mg / mL) were detected; 3) The fraction Fr. F5 which showed pink spots after color development and had an IL-6 inhibition rate greater than 50% was separated by ODS medium-pressure column chromatography, eluted with a methanol-aqueous solution (10%®100%, 9 h) gradient, and the fractions were combined according to the elution profile to obtain 13 fractions Fr. F5a - F5m, and their effects on the IL-6 level in LPS-induced RAW 264.7 cells (concentration 10 mg / mL) were detected; 4) The fraction Fr. F5e which showed pink spots after color development and had an IL-6 inhibition rate greater than 50% was separated by ODS medium-pressure column chromatography, eluted with a methanol-aqueous solution (10%®100%, 7 h) gradient, and the fractions were combined according to the elution profile to obtain 18 fractions Fr. F5ea - F5er, and their effects on the IL-6 level in LPS-induced RAW 264.7 cells (concentration 10 mg / mL) were detected; 5) The fraction Fr. F5eb which showed a single pink spot after color development and had an IL-6 inhibition rate greater than 50% was purified by reverse-phase high performance liquid chromatography. The eluent was an acetonitrile-aqueous solution (80:20), the chromatographic column was YMC-Pack Pro C 18 RS (10 ´ 250 mm, 5 mm), the flow rate was 2.0 mL / min, the detection wavelength was 226 nm, and the retention time was 15.0 min, to obtain a mixture of Formula I and Formula II.

[0022] 6) The above mixture was resolved by normal-phase chiral high performance liquid chromatography. The eluent was a hexane-isopropanol solution (70:30), the chromatographic column was CHIRALPAK Ò IC00CE-BT016 (4.6 ´ 250 mm, 5 mm), the flow rate was 1.0 mL / min, the detection wavelength was 226 nm, and the retention times were 28.0 min (Formula I) and 47.0 min (Formula II) respectively, to obtain the compounds shown in Formula I and Formula II of the present invention.

[0023] The third step, structure identification The molecular formula of the compounds shown in Formula I and Formula II of the present invention is C 11 H 16 O4. By 1D NMR, 2D NMR, HRESIMS, and the calculated ECD method, the chemical structures of the compounds C 11 H 16 O4 shown in Formula I and Formula II of the present invention are as shown in Formula I and Formula II: Formula I; Formula II.

[0024] The compounds C shown in Formula I and Formula II of the present invention11 H 16 The physical and chemical properties and nuclear magnetic resonance data of H O4 are as follows: m / z [M + Na] + 235.0946 (calcd for C 11 H 16 O4Na, 235.0946); α D 20 +5.45 (MeOH, c 0.110); UV (MeOH) λ max (log ε ) 224 (1.1578) nm; CD(MeOH), λ max (D ε ) 220 (5.657) nm.

[0025] Formula II: light yellow oil; HR-ESI-MS m / z [M + Na] + 235.0946 (calcd for C 11 H 16 O4Na, 235.0946); α D 20 -12.50 (MeOH, c 0.040); UV (MeOH) λ max (log ε ) 224 (0.9876) nm; CD (MeOH), λ max (D ε ) 219 (-4.661) nm; The nuclear magnetic resonance spectrum data are shown in Table 1: Table 1 Nuclear magnetic resonance spectrum data of the compounds shown by Formula I and Formula II of the present invention C 11 H 16 O4 (CDCl3, 600 MHz)

[0026] The relative configuration of the lactone fragment in the structure was determined by calculating the coupling constants and analyzing the NOESY correlation signals (as Figure 1 shown, Figure 1 is a schematic diagram of the NOESY signal correlation of polyketide lactone.), H-7 (​​d H The coupling constant with H-8 ( d H 6.16) is 15.9 Hz, determining the configuration of the double bond as 7 E ; the relative configuration is determined by analyzing the NOESY correlation signals as 4 R *,5 S *. The absolute configuration of the lactone fragment is determined by calculating the ECD method. The calculated ECD analysis (as Figure 2 and Figure 3 shown, Figure 2 is the schematic diagram of the absolute configuration of the lactone fragments (4 R , 5 S )-a and (4 S , 5 R )-b in the polyketide lactone of the present invention. Figure 3 is the measured CD spectra of Formula I and Formula II in the polyketide lactone of the present invention and the schematic diagram of the calculated ECD spectra of the lactone fragments (4 R , 5 S )-a and (4 S , 5 R )-b). It shows that the measured CD curve of Formula I is close to the calculated ECD curve of (4 R , 5 S )-a, and the measured CD curve of Formula II is close to the calculated ECD curve of (4 R , 5 S )-b. Thus, the absolute configurations of Formula I and Formula II are determined to be 4 R , 5 S and 4 S ,5 R .

[0027] Example 2: The compound shows a strong inhibitory effect on the levels of NO and IL-6 in LPS-induced RAW 264.7 cells.

[0028] First step, detect the cytotoxic activity of the compound against RAW 264.7 cells Take cells in the logarithmic growth phase, inoculate the cells in a 96-well plate (about 8 × 10 3 cells / well), culture at 37 °C under 5% CO2 for 24 h. In the experimental group, add 20 μL of the test compound with a final concentration of 10 mM, and in the blank group and the non-drug-added group, add 20 μL of complete medium. Set 3 replicates for each group. Continue to culture for 48 h. Discard the medium and wash 3 times with PBS. Add 10 μL of CCK-8 solution to each well, take it out after incubation for 1 h, measure the absorbance at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader, and calculate the cell survival rate according to the following formula:

[0029] A (Drug addition): OD value of the wells containing culture medium, cells, CCK-8 solution and the test sample solution K (Blank): OD value of the wells containing culture medium and CCK-8 solution without cells B (No drug addition): OD value of the wells containing culture medium, cells, CCK-8 solution without the drug solution In the second step, detect the effects of the compound on the levels of NO and IL-6 in LPS-induced RAW 264.7 cells NO level: Seed RAW 264.7 cells in a 96-well plate, add LPS (2 μg / mL) to each well, and after treating with or without the test compound for 24 h, detect the production of NO in the supernatant by the Griess reaction. Measure the absorbance at 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the NO concentration and inhibition rate. The experiment was repeated three times, and the data are the mean ± standard deviation (SD) of three independent experiments

[0030] IL-6 level: Seed RAW 264.7 cells in a 96-well plate, add LPS (2 μg / mL) to each well, and after treating with or without the test compound for 24 h, operate according to the instructions of the ELISA kit, measure the OD value of each group at 450 nm, and calculate the release level and inhibition rate of the IL-6 cytokine according to the OD value. The experiment was repeated three times, and the data are the mean ± SD of three independent experiments

[0031] The experimental results are as follows: Table 2 shows the cytotoxicity of the polyketolide of the present invention against RAW 264.7 and its inhibitory effects on the levels of NO and IL-6 in LPS-induced RAW 264.7 cells

[0032] The present invention provides a pair of novel polyketolide enantiomers with anti-inflammatory activity and their preparation methods for marine drug research, providing a scientific basis for the development and utilization of marine medicinal resources in China

[0033] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, may make some changes or modifications using the above-disclosed technical content into equivalent embodiments with equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention

Claims

1. A sponge-derived polyketide lactone enantiomer, characterized in that, Its chemical structure is shown in Formula I and Formula II: Formula Ⅰ Formula II.

2. A polyketide lactone enantiomer derived from sponge according to claim 1, wherein The sponge-derived polyketide lactone is derived from Hippospongia hispida collected from the South China Sea ( Hippospongia lachne ).

3. A method for preparing a sponge-derived polyketide lactone enantiomer as claimed in claim 1 or 2, characterized in that, It includes the following steps: The first step is to prepare the total extract Extract the Hymeniacidon perleve Hippospongia lachne with ethanol to obtain a total extract; suspend the total extract in water, extract with ethyl acetate, and concentrate under reduced pressure to obtain a fat-soluble extract; suspend the fat-soluble extract in a methanol-aqueous solution, extract with petroleum ether, and concentrate under reduced pressure to obtain a total petroleum ether extract; The second step is separation and purification 1) The total petroleum ether extract is separated by silica gel column chromatography under reduced pressure, and gradient elution is carried out with a petroleum ether-acetone solution as the eluent. According to the thin-layer chromatography color development, the fractions are combined to obtain fractions Fr. A-N; 2) Normal-phase silica gel column chromatography separation is carried out on fraction Fr. F, and gradient elution is carried out with a petroleum ether-acetone solution. According to the thin-layer chromatography color development, the fractions are combined to obtain fractions Fr.F1-F13; 3) ODS medium-pressure column chromatography separation is carried out on fraction Fr. F5, and gradient elution is carried out with a methanol-aqueous solution. According to the peak elution situation, the fractions are combined to obtain fractions Fr.F5a-F5m; 4) ODS medium-pressure column chromatography separation is carried out on fraction Fr.F5e, and gradient elution is carried out with a methanol-aqueous solution. According to the peak elution situation, the fractions are combined to obtain fractions Fr. F5ea-F5er; 5) Fraction Fr. F5eb is purified by reverse-phase high-performance liquid chromatography to obtain the racemate of the polyketide lactone derived from the sponge; 6) The above racemate is resolved by normal-phase chiral high-performance liquid chromatography to obtain a pair of enantiomers of the polyketide lactone derived from the sponge.

4. The preparation method of a polyketide lactone enantiomer derived from sponge according to claim 3, characterized in that, In the second step of separation and purification, in item 1), gradient elution is carried out with a petroleum ether-acetone solution as the eluent, and the volume ratio of the two is 50-1:

1.

5. The preparation method of a sponge-derived polyketide lactone enantiomer according to claim 3, characterized in that, In the second step of separation and purification, in item 2), gradient elution is carried out with a petroleum ether-acetone solution as the eluent, and the volume ratio of the two is 50-1:

1.

6. The preparation method of a polyketide lactone enantiomer derived from sponge according to claim 3, characterized in that, In the second step of separation and purification, in item 3), the volume ratio of the methanol-aqueous solution gradient elution is 10% to 100%.

7. The preparation method of a polyketide lactone enantiomer derived from sponge according to claim 3, characterized in that, In the second step of separation and purification, in item 4), the volume ratio of the methanol-aqueous solution gradient elution is 10% to 100%.

8. A method for preparing a sponge-derived polyketide lactone enantiomer according to claim 3, characterized in that, In the second step of separation and purification, in item 5), the conditions for reverse-phase high-performance liquid separation: the eluent is an acetonitrile-aqueous solution with a volume ratio of 80:

20.

9. The preparation method of a sponge-derived polyketide lactone enantiomer according to claim 3, characterized in that, In the second step of separation and purification, in item 6), the conditions for normal-phase chiral high-performance liquid separation: the eluent is a n-hexane-isopropanol solution with a volume ratio of 70:

30.

10. Use of an enantiomer of a polyketide lactone derived from a sponge as claimed in claim 1 in the preparation of an anti-inflammatory drug.

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