A sponge-derived polyketolactone enantiomer and its preparation method and application

Abstract

A sponge-derived polyketolactone enantiomer and its preparation method and application belong to the field of marine biology and medical technology. The present invention provides a sponge-derived polyketolactone enantiomer on the one hand, and provides a preparation method and application of the sponge-derived polyketolactone enantiomer on the other hand. The sponge-derived polyketolactone enantiomer of 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.

Description

Technical Field

[0001] The invention belongs to the field of marine biology and medical technology, and in particular relates to a sponge-derived polyketolactone enantiomer and a preparation method thereof. Background Art

[0002] Sponges are the second largest species in the ocean, accounting for about 30% of the biomass of marine species. Sponges are "marine fossils" with a 600 million-year history of survival. In the process of continuous evolution, they have developed unique chemical defense mechanisms to adapt to extreme environments. Their metabolites have diverse skeleton types and significant activity, making them an excellent "blue drug treasure trove." Hippospongia lachne Originating from the South China Sea, a variety of highly active polyketides have been isolated from this genus of sponge. These compounds often possess multiple chiral centers, which also provides the possibility of the existence of enantiomers. However, the separation of enantiomers and the determination of absolute configuration are difficult issues in natural medicinal chemistry.

[0003] In the process of developing and utilizing my country's marine biological resources and searching for marine natural products with biological activity and pharmaceutical prospects, we found that the hairy horse sponge in the South China Sea Hippospongia lachne The ethanol extract of the plant showed significant inhibitory activity against LPS-induced NO and IL-6 levels in RAW 264.7 cells in vitro. Further bioactivity follow-up studies revealed a polyketide lactone enantiomer. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for sponge-derived polyketolactone enantiomers and a preparation method thereof.

[0005] The present invention is specifically implemented by the following technical solutions:

[0006] The first aspect of the present invention provides a sponge-derived polyketolactone enantiomer, the chemical structures of which are shown in Formula I and Formula II:

[0007] Formula I;

[0008] Formula II.

[0009] Furthermore, the sponge-derived polyketone lactone is derived from the hairy horse sponge of the South China Sea. Hippospongia lachne .

[0010] A second aspect of the present invention provides a method for preparing the sponge-derived polyketolactone enantiomers, comprising the following steps:

[0011] The first step is to prepare the total extract

[0012] Hairy horse sponge Hippospongia lachne Cut into small pieces, extract three times with 95% ethanol to obtain a total extract; suspend the total extract in water, extract three times with ethyl acetate, and concentrate under reduced pressure to obtain a fat-soluble extract; suspend the fat-soluble extract in 90% methanol-water solution, extract with petroleum ether, and concentrate under reduced pressure to obtain a petroleum ether total extract;

[0013] The second step is separation and purification

[0014] 1) The total petroleum ether extract was separated by vacuum silica gel column chromatography using a petroleum ether-acetone solution as the eluent for gradient elution. The fractions were combined according to the thin layer chromatography color to obtain 14 components Fr. AN;

[0015] 2) Component Fr. F was separated by normal phase silica gel column chromatography using a petroleum ether-acetone gradient elution. Fractions were combined based on the TLC color development to yield 13 components, Fr. F1-F13.

[0016] 3) Component Fr. F5 was separated by ODS medium-pressure column chromatography using a methanol-water gradient elution. Fractions were combined based on peak elution to obtain 13 components, Fr. F5a-F5m.

[0017] 4) Component Fr. F5e was separated by ODS medium-pressure column chromatography using a methanol-water gradient elution. Fractions were combined based on peak elution to obtain 18 components, Fr. F5ea-F5er.

[0018] 5) Component Fr. F5eb was purified by reverse-phase high performance liquid chromatography to obtain a racemic form of the sponge-derived polyketide lactone;

[0019] 6) Resolving the racemate by normal-phase chiral high performance liquid chromatography to obtain an enantiomer of the sponge-derived polyketide lactone.

[0020] Furthermore, in the second separation and purification step of the method, in step 1), the conditions for gradient elution using petroleum ether-acetone solution as the eluent are: elution with petroleum ether-acetone solutions with volume ratios of 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, and 1:1, in sequence.

[0021] Furthermore, in the second separation and purification step of the method, in step 2), the conditions for gradient elution using petroleum ether-acetone solution as the eluent are: elution with petroleum ether-acetone solution with a volume ratio of 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, and 1:1, in sequence.

[0022] Furthermore, in the second separation and purification step of the method, in step 3), the volume ratio of the methanol-water solution gradient elution is 10% to 100%, and the time is 9 hours.

[0023] Furthermore, in the second separation and purification step of the method, in step 4), the volume ratio of the methanol-water solution gradient elution is 10% to 100%, and the time is 7 hours.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0028] 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

[0029] Figure 1 Schematic diagram of NOESY signal correlation of the polyketolactone of the present invention.

[0030] Figure 2 The lactone segment (4 R , 5 S )-a and (4S , 5 R )-b's absolute configuration diagram.

[0031] Figure 3 The measured CD spectra and lactone fragments (4 R , 5 S )-a and (4 S , 5 R )-b is a schematic diagram of the calculated ECD spectrum. DETAILED DESCRIPTION

[0032] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0033] The present invention is a sponge collected from the South my country Sea Hippospongia lachne The present invention can also use other sponges to prepare the compounds of the present invention or use artificial synthesis methods to prepare the compounds of the present invention.

[0034] Example 1: Preparation of compounds of the present invention

[0035] The first step is to prepare the total extract

[0036] The hairy horse sponge was frozen in -20℃ refrigerator Hippospongia lachne Thawed and cut into small pieces (approximately 2 cm x 2 cm) (wet weight 10 kg), cold-extracted with 95% ethanol (20 L x 3 times) to obtain a total extract (280 g). The total extract was dispersed in water (1 L), extracted three times with ethyl acetate (1 L), and concentrated under reduced pressure to obtain a fat-soluble extract (141 g). The fat-soluble extract was suspended in a 90% methanol-water solution (800 mL), extracted with petroleum ether (800 mL x 3 times), and concentrated under reduced pressure to obtain a petroleum ether total extract (75 g).

[0037] The second step is separation and purification

[0038] 1) The petroleum ether total extract was separated by vacuum silica gel column chromatography (VLC) using a petroleum ether-acetone solution (50:1, 25:1, 15:1, 10:1, 5:1, 2:1, and 1:1) as the eluent. Fractions were combined based on thin-layer chromatography (TLC) color development to obtain 14 fractions, Fr. AN. Their effects on LPS-induced IL-6 levels in RAW 264.7 cells (at a concentration of 10 mg / mL) were examined.

[0039] 2) The fraction Fr. F, which displayed a pink spot and an IL-6 inhibition rate greater than 50%, was separated by normal-phase silica gel column chromatography using a petroleum ether-acetone gradient elution (50:1, 25:1, 15:1, 10:1, 5:1, 2:1, and 1:1). Fractions were combined based on TLC color development to obtain 13 fractions, Fr. F1-F13, which were then assayed for their effects on LPS-induced IL-6 levels in RAW 264.7 cells (at a concentration of 10 mg / mL).

[0040] 3) Fraction Fr. F5, which displayed a pink spot and an IL-6 inhibition rate greater than 50%, was separated by ODS medium-pressure column chromatography using a methanol-water gradient elution (10% to 100%, 9 h). Fractions were combined based on peak elution to obtain 13 fractions, Fr. F5a–F5m. Their effects on LPS-induced IL-6 levels in RAW 264.7 cells (at a concentration of 10 mg / mL) were then assessed.

[0041] 4) The fraction Fr. F5e, which displayed a pink spot after color development and an IL-6 inhibition rate greater than 50%, was separated by ODS medium-pressure column chromatography using a methanol-water gradient elution (10% to 100%, 7 h). Fractions were combined based on peak elution to obtain 18 fractions, Fr. F5ea to F5er. Their effects on LPS-induced IL-6 levels in RAW 264.7 cells (at a concentration of 10 mg / mL) were then assessed.

[0042] 5) The fraction Fr. F5eb, which contained 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 using an acetonitrile-water solution (80:20) as eluent and a YMC-Pack Pro C column. 18 RS (10 ´ 250mm, 5 mm), a flow rate of 2.0 mL / min, a detection wavelength of 226 nm, and a retention time of 15.0 min to obtain a mixture of Formula I and Formula II.

[0043] 6) The above mixture was separated by normal phase chiral high performance liquid chromatography, the eluent was n-hexane-isopropanol solution (70:30), and the chromatographic column was CHIRALPAK Ò IC00CE-BT016 (4.6 ´ 250 mm, 5 mm), a flow rate of 1.0 mL / min, a detection wavelength of 226 nm, and retention times of 28.0 min (Formula I) and 47.0 min (Formula II), respectively, were used to obtain the compounds of Formula I and Formula II of the present invention.

[0044] Step 3: Structural Identification

[0045] The molecular formula of the compound represented by formula I and formula II of the present invention is C 11 H 16 O4, 1D NMR, 2D NMR, HRESIMS, calculated ECD method, confirmed the compound C of formula I and formula II of the present invention 11 H 16 The chemical structure of O4 is shown in Formula I and Formula II:

[0046] Formula I;

[0047] Formula II.

[0048] Compound C represented by formula I and formula II of the present invention 11 H 16 The physical and chemical properties and nuclear magnetic resonance data of O4 are as follows:

[0049] Formula I: light yellow oil; HR-ESI-MS 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.

[0050] 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;

[0051] The NMR spectrum data are shown in Table 1:

[0052] Table 1 Compound C of Formula I and Formula II of the present invention 11 H 16 O4 NMR spectrum data (CDCl3, 600 MHz)

[0053]

[0054] The relative configuration of the lactone fragment in the structure was determined by calculating the coupling constant and analyzing the NOESY related signals (e.g. Figure 1 As shown, Figure 1 Schematic diagram of NOESY signal correlation of polyketolactone. ), H-7 ( d H 6.84) and H-8 ( d H 6.16) has a coupling constant of 15.9 Hz, confirming that the double bond configuration is 7 E ; The relative configuration was determined to be 4 by analyzing the NOESY related signals. R *,5 S *. The absolute configuration of the lactone fragment was determined by the ECD method. Figure 2 and Figure 3 As shown, Figure 2 The lactone segment (4 R , 5 S )-a and (4 S , 5 R )-b absolute configuration diagram, Figure 3 The measured CD spectra and lactone fragments (4 R , 5 S )-a and (4 S , 5 R )-b calculated ECD spectrum diagram) shows that the measured CD curve of formula I is consistent with (4 R , 5 S )-a is close to the calculated ECD curve, and the measured CD curve of formula II is close to (4 R , 5 S )-b calculated ECD curves are close, thus determining that the absolute configurations of formula I and formula II are 4 R , 5 S and 4 S ,5 R .

[0055] Example 2: The compound exhibited a strong inhibitory effect on the levels of NO and IL-6 in RAW 264.7 cells induced by LPS.

[0056] The first step is to detect the cytotoxic activity of the compounds against RAW 264.7 cells

[0057] Cells in the logarithmic growth phase were plated in 96-well plates (approximately 8 × 10 3 Each well was incubated at 37°C, 5% CO₂ for 24 hours. 20 μL of the test compound (final concentration: 10 mM) was added to the experimental group, while 20 μL of complete culture medium was added to the blank and untreated groups. Three replicate wells were set up for each group. Culture was continued for 48 hours. The culture medium was discarded, and the cells were rinsed three times with PBS. 10 μL of CCK-8 solution was added to each well. After incubation for 1 hour, the cells were removed and the absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated according to the following formula:

[0058]

[0059] A (drug addition): OD value of the well containing culture medium, cells, CCK-8 solution and sample solution to be tested

[0060] K (blank): OD value of the well containing culture medium and CCK-8 solution but no cells

[0061] B (no drug): OD value of the well containing culture medium, cells, CCK-8 solution, and no drug solution

[0062] The second step was to detect the effects of the compounds on the levels of NO and IL-6 in RAW 264.7 cells induced by LPS.

[0063] NO levels: RAW 264.7 cells were seeded in 96-well plates. LPS (2 μg / mL) was added to each well and treated with or without the test compound for 24 hours. NO production in the supernatant was measured by Griess reaction. Absorbance at 540 nm was measured using a microplate reader, and NO concentration and inhibition rate were calculated. Experiments were performed in triplicate. Data are the mean ± SD of three independent experiments.

[0064] IL-6 levels: RAW 264.7 cells were seeded in 96-well plates. LPS (2 μg / mL) was added to each well. After 24 hours of treatment with or without the test compound, the ELISA kit was performed according to the manufacturer's instructions. The OD value of each group was measured at 450 nm. The IL-6 cytokine release level and inhibition rate were calculated based on the OD value. The experiment was performed in triplicate. Data are the mean ± SD of three independent experiments.

[0065] The experimental results are as follows:

[0066] Table 2 shows the cytotoxicity of the polyketide lactones of the present invention to RAW 264.7 cells and the inhibitory effects on NO and IL-6 levels in LPS-induced RAW 264.7 cells.

[0067]

[0068] The present invention provides a pair of new polyketolactone enantiomers with anti-inflammatory activity and a preparation method thereof for marine drug research, and provides a scientific basis for the development and utilization of my country's marine medicinal resources.

[0069] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

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

2. A method for preparing the sponge-derived polyketolactone enantiomer according to claim 1, characterized in that: The following steps are involved: The first step is to prepare the total extract Hairy horse sponge Hippospongia lachne Extract 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-water solution, extract with petroleum ether, and concentrate under reduced pressure to obtain a petroleum ether total extract; The second step is separation and purification 1) The total petroleum ether extract was separated by silica gel column chromatography under reduced pressure, and gradient elution was performed using a petroleum ether-acetone solution as the eluent. The fractions were combined according to the thin layer chromatography color to obtain component Fr. AN; 2) Component Fr. F was separated by normal phase silica gel column chromatography using a petroleum ether-acetone gradient elution. Fractions were combined based on the TLC color development to obtain components Fr. F1-F13; 3) Component Fr. F5 was separated by ODS medium-pressure column chromatography using a methanol-water gradient elution. Fractions were combined based on peak elution to obtain components Fr. F5a-F5m. 4) Component Fr. F5e was separated by ODS medium-pressure column chromatography using a methanol-water gradient elution. Fractions were combined based on peak elution to obtain components Fr. F5ea-F5er. 5) Component Fr. F5eb was purified by reverse-phase high performance liquid chromatography to obtain a racemic form of the sponge-derived polyketide lactone; 6) Resolving the racemate by normal-phase chiral high performance liquid chromatography to obtain a pair of sponge-derived polyketide lactone enantiomers.

3. The method for preparing a sponge-derived polyketolactone enantiomer according to claim 2, characterized in that: In the second step of separation and purification, in step 1), petroleum ether-acetone solution is used as the eluent for elution, and the volume ratio of the two is 50:1:

1.

4. The method for preparing a sponge-derived polyketolactone enantiomer according to claim 2, wherein: In the second step of separation and purification, in step 2), petroleum ether-acetone solution is used as the eluent for elution, and the volume ratio of the two is 50:1:

1.

5. The method for preparing a sponge-derived polyketolactone enantiomer according to claim 2, wherein: In the second separation and purification step, in step 3), the volume ratio of the methanol-water solution gradient elution is 10% to 100%.

6. The method for preparing a sponge-derived polyketolactone enantiomer according to claim 2, characterized in that: In the second separation and purification step, in step 4), the volume ratio of the methanol-water solution gradient elution is 10% to 100%.

7. The method for preparing a sponge-derived polyketolactone enantiomer according to claim 2, characterized in that: In the second separation and purification step, in step 5), the conditions for reverse-phase high performance liquid separation are: the eluent is acetonitrile-water solution with a volume ratio of 80:

20.

8. The method for preparing a sponge-derived polyketolactone enantiomer according to claim 2, wherein: In the second separation and purification step, 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.

9. Use of the sponge-derived polyketolactone enantiomer according to claim 1 in the preparation of anti-inflammatory drugs.

Images