Gentiopicroside compound, preparation method and application thereof

Abstract

The application discloses a gentiopicroside compound and a preparation method and application thereof. The gentiopicroside compound has a structure shown in formula 1. A new gentiopicroside compound is separated from a thermal conversion product of gentiopicroside, the preparation method is simple and easy to implement, the compound can protect the liver by inhibiting the activities of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase, and can be used as a leading compound of liver-protecting drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to a gentiolactone compound, its preparation method, and its application. Background Technology

[0002] Gentipicroside is a compound belonging to the Gentianaceae family (Gentianaceae). Gentianaceae Gentian ( ) GentianaL Gentiana, a plant species, is listed as an index component in the Chinese Pharmacopoeia. However, gentiopicrin is unstable and easily transforms into other gentiopicrin conversion products during processing and storage. This invention aims to provide a novel gentiolactone compound with hepatoprotective activity derived from gentiopicrin, thereby providing more candidate compounds for hepatoprotective drugs. Summary of the Invention

[0003] The first objective of this invention is to provide a gentiolactone compound, the second objective of this invention is to provide a method for preparing the gentiolactone compound, and the third objective of this invention is to provide applications of the gentiolactone compound.

[0004] The first objective of this invention is achieved by providing a gentiolactone compound having the structure shown in Formula 1:

[0005]

[0006] The second objective of this invention is achieved by the method for preparing the gentiolactone compound, specifically through the following steps:

[0007] 1) Take gentiopicrin monomer, add 5-10 times the amount of pure water, reflux extract at 95-100℃ until no gentiopicrin is found, and concentrate under reduced pressure to obtain extract.

[0008] 2) The extract obtained in step 1) was adsorbed by macroporous adsorption resin and eluted with a gradient of water and methanol mixed solvent of 75:25-100:0. The sugar and lactone fractions were collected separately by TLC monitoring.

[0009] 3) The lactone fraction obtained in step 2) was subjected to silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate solvents at volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:3, and 1:5. The eluent with a petroleum ether-ethyl acetate volume ratio of 2:1 was collected, concentrated under reduced pressure, and then subjected to reversed-phase column chromatography with methanol-water at a volume ratio of 45-60:40-55 to obtain the target gentiolactone compound.

[0010] The third objective of this invention is achieved by using the gentiolactone compound in the preparation of hepatoprotective drugs.

[0011] The beneficial effects of this invention are as follows: This invention isolates a new gentiolactone compound from the thermal conversion product of gentiopicroside. Its preparation method is simple and easy. This compound can protect the liver by inhibiting the activity of aspartate aminotransferase and alanine aminotransferase, and can be used as a lead compound for hepatoprotective drugs. Attached Figure Description

[0012] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the gentiolactone compound prepared in Example 1;

[0013] Figure 2 The carbon NMR spectrum of the gentiolide compound prepared in Example 1;

[0014] Figure 3 The infrared spectrum of the gentiolactone compound prepared in Example 1;

[0015] Figure 4 Two-dimensional nuclear magnetic resonance (COSY) spectrum of the gentiolactone compound prepared in Example 1;

[0016] Figure 5 Two-dimensional nuclear magnetic resonance (HMBC) spectrum of the gentiolactone compound prepared in Example 1;

[0017] Figure 6 The effect of the gentiolactone compound prepared in Example 1 on the survival rate of L-O2 cells in drug-induced liver injury is shown in the figure (NC group is the normal group, AP is the model group, and NAC positive group). p <0.0001 compared to the normal group; * p <0.05 compared with the model group);

[0018] Figure 7 Effects of gentiolactone compounds prepared in Example 1 on ALT levels in L-O2 cells with drug-induced liver injury (NC group: normal group, AP group: model group, NAC-positive group; #### p <0.0001 compared to the normal group; * p <0.05 compared with the model group);

[0019] Figure 8 Effects of gentiolactone compounds prepared in Example 1 on AST levels in L-O2 cells with drug-induced liver injury (NC group: normal group, AP group: model group, NAC-positive group; #### p <0.0001 compared to the normal group; * p <0.05 compared with the model group). Detailed Implementation

[0020] The present invention will be further described below, but this is not intended to limit the invention in any way. Any modifications made based on the present invention are within the scope of protection of the present invention.

[0021] This invention provides a gentiovarisin compound, named Gentiovarisin C ， It has the structure shown in Equation 1:

[0022] .

[0023] The present invention also provides a method for preparing the gentiolactone compound, which is carried out according to the following steps:

[0024] 1) Take gentiopicrin monomer, add 5-10 times the amount of pure water, reflux extract at 95-100℃ until no gentiopicrin is found, and concentrate under reduced pressure to obtain extract.

[0025] 2) The extract obtained in step 1) was adsorbed by macroporous adsorption resin and eluted with a gradient of water and methanol mixed solvent of 75:25-100:0. The sugar and lactone fractions were collected separately by TLC monitoring.

[0026] 3) The lactone fraction obtained in step 2) was subjected to silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate solvents at volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:3, and 1:5. The eluent with a petroleum ether-ethyl acetate volume ratio of 2:1 was collected, concentrated under reduced pressure, and then subjected to reversed-phase column chromatography with methanol-water at a volume ratio of 45-60:40-55 to obtain the target gentiolactone compound.

[0027] The present invention further provides the application of the gentiolactone compound in the preparation of hepatoprotective and anti-inflammatory drugs.

[0028] The present invention further provides a pharmaceutical composition comprising the gentiolactone compound and a pharmaceutically acceptable carrier.

[0029] Example 1

[0030] Take 1000g of gentiopicroside monomer, add 5L of pure water and reflux at 95℃ for more than 10 hours until no gentiopicroside is found, then concentrate under reduced pressure to 1 / 4 of the original volume; the obtained extract is adsorbed by macroporous adsorption resin and eluted with a gradient of water and methanol mixed solvent with a volume ratio of 75:25-100:0, and the sugar and lactone fractions are collected separately by TLC monitoring. The lactone fraction was dissolved in 1.5 times its weight of methanol, mixed with silica gel of equal weight, and then subjected to silica gel column chromatography. The silica gel was 80 mesh and the volume was 6 times the weight of the lactone fraction. The silica gel column was eluted with petroleum ether-ethyl acetate solvents at volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:3, and 1:5. The eluents were collected, concentrated, and analyzed by TLC. Identical fractions were combined. The petroleum ether-ethyl acetate (1:1) fraction was subjected to reversed-phase column chromatography. The reversed-phase column was eluted with a 55% (v / v) methanol aqueous solution to obtain 10 g of colorless needle-like compound Gentiovarisin C, with the molecular formula C. 19 H 18 O6.

[0031] Example 2

[0032] Take 1500g of gentiopicroside monomer, add 10L of pure water and reflux at 98℃ for more than 10 hours until no gentiopicroside is found, then concentrate under reduced pressure to 1 / 3 of the original volume; the obtained extract is adsorbed by macroporous adsorption resin and eluted with a gradient of water and methanol mixed solvent with a volume ratio of 75:25-100:0, and the sugar and lactone fractions are collected separately by TLC monitoring. The lactone fraction was dissolved in 1.8 times its weight of methanol, mixed with 1.5 times its weight of silica gel, and then subjected to silica gel column chromatography. The silica gel was 90 mesh and the volume was 8 times the weight of the lactone fraction. The silica gel column was eluted with petroleum ether-ethyl acetate solvents at volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:3, and 1:5. The eluents were collected, concentrated, and analyzed by TLC. Identical fractions were combined. The petroleum ether-ethyl acetate (1:1) fraction was subjected to reversed-phase column chromatography. The reversed-phase column was eluted with a 60% (v / v) methanol-water solution to obtain 16.2 g of colorless needle-like compound Gentiovarisin C, with the molecular formula C. 19 H 18 O6.

[0033] Example 3

[0034] Take 4000g of gentiopicrin monomer, add 30L of pure water and reflux at 100℃ for more than 10 hours until gentiopicrin is no longer present. Concentrate under reduced pressure to 1 / 3 of the original volume. The resulting extract is adsorbed by macroporous adsorption resin and eluted with a gradient of water and methanol mixed solvent with a volume ratio of 75:25-100:0. The sugar and lactone fractions are collected separately by TLC monitoring. The lactone fraction was dissolved in twice its weight of methanol, mixed with 1.5 times its weight of silica gel, and then subjected to silica gel column chromatography. The silica gel was 100 mesh and the volume was 7.2 times the weight of the lactone fraction. The silica gel column was eluted with petroleum ether-ethyl acetate solvents at volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:3, and 1:5. The eluents were collected, concentrated, and analyzed by TLC. Identical fractions were combined. The petroleum ether-ethyl acetate fraction (2:1) was subjected to reversed-phase column chromatography. The reversed-phase column was eluted with a 50% (v / v) methanol-water solution to obtain 42.3 g of colorless needle-like crystals, Gentiovarisin C, with the molecular formula C. 19 H 18 O6.

[0035] Example 4

[0036] Take 8000g of gentiopicroside monomer, add 80L of pure water and reflux at 100℃ for more than 10 hours until no gentiopicroside is found, and concentrate under reduced pressure to 1 / 4 of the original volume; the obtained extract is adsorbed by macroporous adsorption resin and eluted with a gradient of water and methanol mixed solvent with a volume ratio of 75:25-100:0, and the sugar and lactone fractions are collected separately by TLC monitoring. The lactone fraction was dissolved in twice its weight of methanol, mixed with silica gel of equal weight, and then subjected to silica gel column chromatography. The silica gel was 100 mesh and the volume was 6.5 times the weight of the lactone fraction. The silica gel column was eluted with petroleum ether-ethyl acetate solutions at volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:3, and 1:5. The eluents were collected, concentrated, and analyzed by TLC. Identical fractions were combined. The petroleum ether-ethyl acetate fraction (2:1) was then subjected to reversed-phase column chromatography. The reversed-phase column was eluted with a 45% (v / v) methanol-water solution to obtain 85.5 g of colorless needle-like crystals, Gentiovarisin C, with the molecular formula C. 19 H 18 O6.

[0037] Example 5

[0038] The compound prepared in Example 1 was identified, and the results are as follows:

[0039] Gentiovarisin C is a white, needle-like crystal. Its molecular formula is... 13 C-NMR and HRESIMS data (m / z 387.1094 [M+ Na]) + Determined as C 19 H 18 O6 contains 11 degrees of unsaturation. 13 C-NMR ( Figure 1 )and 1 H-NMR ( Figure 2 The spectrum indicated the presence of 19 carbon signals, consisting of 7 quaternary carbons, 6 methylene groups, and 6 methoxy groups. Infrared spectrum ( Figure 3 The result shows the presence of a lactone carbonyl group (1706 cm). -1 ) and double bonds (1631cm) -1 ).according to 1 H- 1 HCOSY spectrum ( Figure 4 H-3(δ) in ) H 8.09) / H-8(δ H 7.69) / H-10(δ H 7.41), H-6(δ) H 2.98) / H-7(δ H 4.50), H2-6'(δ) H 2.02, 2.06) / H2-7'(δ H 4.32, 4.59) and H-8' (δ H 4.97) / H2-10'(δ H 1.80, 2.14) indicates the presence of four key segments: (C-3 / C-8 / C-10), (C-6' / C-7'), (C-6' / C-7'), and (C-8' / C-10'). H2-6 and C-4 (δ C 137.7) and C-9 (δ C 126.0), H2-7 and C-5 (δ C 137.1) and C-11 (δ C 165.2), H-3 and C-5, C-8 (δ) C 131.2), C-11, H-8 and C-5, H-10 and C-4, C-9 HMBC ( Figure 5 The relevant signals indicate the presence of one unsaturated lactone ring and one benzene ring. H-1(δ) H 4.90) with C-5 and C-8, H2-9' (δ H 2.11, 1.85) and C-8'(δ C 72.0) and C-10'(δ C37.7), H2-10' with C-5 and C-5' (δ) C 66.3), H-3'(δ) H 8.16) and C-4'(δ) C 102.5), C-8', C-11'(δ) C 164.6), H2-6' with C-4', C-5', H2-7' with C-5', C-11' indicate the presence of three additional unsaturated lactone rings. These five cyclic structures are fused from H-3-C-5, H-7-C-5, H-8-C-5, H-6-C-4, H-10-C-4, H-1-C-8, H-1-C-5, H-9'-C-8', H-9'-C-10', H-10'-C-5, H-10'-C-5, H-3'-C-4', H-6'-C5', and H-7'-C-5'. Thus, its planar structure can be determined.

[0040] Example 6

[0041] The compounds prepared in Examples 2, 3, and 4 were measured using the same method as in Example 5. The compounds prepared in Examples 2, 3, 4, and 5 were confirmed to be the gentiovarisin C compound.

[0042] Test Examples: Hepatoprotective and Anti-inflammatory Activities of the Gentianolone Compounds of the Present Invention

[0043] 1. A hepatocyte injury model was established using the human hepatocyte cell line L-O2 after treatment with APAP (10 mM). The effect of the gentiolactone compound prepared in Example 1 on cell viability was then determined. The specific method is as follows:

[0044] With 5×10 4 L-O2 cells were seeded in 96-well plates at a concentration of [number] cells / mL and cultured for 24 h. Cell status was then observed, and cells were cultured with compounds (concentrations: 10, 25, 50 μg / mL) and a positive control (NAC: 0.25 mM), respectively. After 24 h, cell status was observed again, and cells were incubated with the model drug APAP (10 mM) for 24 h. Absorbance (OD) was then measured using the CCK-8 reagent, and cell viability was calculated. The effects of each compound on the survival rate of L-O2 cells in drug-induced liver injury were obtained. Figure 5 As shown.

[0045] Results analysis: Based on the cell viability measured in each group, it can be seen that the cell survival rate in the model group was lower than that in the control group. p<0.0001), indicating successful modeling; compared with the model group, the cell survival rate increased after administration of compounds at concentrations of 10, 25, and 50 μg / mL and 0.25 mM of positive control drug, respectively, indicating that different concentrations of Gentiovarisin C can alleviate hepatocellular damage caused by APAP.

[0046] 2. Based on the above L-O2 cell survival rate results, using 1×10 5 L-O2 cells were seeded at a concentration of [number] cells / mL and cultured in 6-well plates for 24 h. Cell status was then observed, and cells were cultured with the compound (concentration: 20 μM~80 μM) and a positive control (NAC: 0.25 mM) for another 24 h. Cell status was then observed again, and cells were incubated with APAP (10 mM) for 24 h. The supernatant was collected, and cell homogenate was prepared and stored for ALT and AST level detection. This was to evaluate the protective effect of the gentiolactone compound of this invention against APAP-induced drug-induced liver injury in L-O2 cells, i.e., to explore the hepatoprotective effect of this compound. The results are as follows: Figure 6 and 7 As shown.

[0047] Results analysis: Based on the cell activity measured in each group, it can be seen that compared with the blank group, the cell activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was increased in the model group. p <0.0001 indicates successful modeling; compared with the model group, after administration of compounds at concentrations of 10, 25, and 50 μg / mL and a 0.25 mM positive control drug, respectively, the cell activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) decreased ( p The concentration of Gentiovarisin C is <0.05, indicating that different concentrations of Gentiovarisin C can protect the liver by inhibiting the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0048] In summary, the gentiovarisin C compound provided by this invention can protect the liver by inhibiting the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Therefore, this compound can be used in the preparation of hepatoprotective drugs.

Claims

1. A gentiolactone compound having the structure shown in Formula 1: 。 2. The method for preparing the gentiolactone compound according to claim 1, characterized in that, Follow these steps to achieve the following: 1) Take gentiopicrin monomer, add 5-10 times the amount of pure water, reflux extract at 95-100℃ until no gentiopicrin is found, and concentrate under reduced pressure to obtain extract. 2) The extract obtained in step 1) was adsorbed by macroporous adsorption resin and eluted with a gradient of water and methanol mixed solvent of 75:25-100:

0. The sugar and lactone fractions were collected separately by TLC monitoring. 3) The lactone fraction obtained in step 2) was subjected to silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate solvents at volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:3, and 1:

5. The eluent with a petroleum ether-ethyl acetate volume ratio of 2:1 was collected, concentrated under reduced pressure, and then subjected to reversed-phase column chromatography with methanol-water at a volume ratio of 45-60:40-55 to obtain the target gentiolactone compound.

3. The use of the gentiolactone compound of claim 1 in the preparation of hepatoprotective drugs.

4. A pharmaceutical composition comprising the gentiolactone compound of claim 1 and a pharmaceutically acceptable carrier.

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