Lignin compound as well as preparation method and application thereof

Abstract

The invention relates to a lignin compound as well as a preparation method and application thereof. The compound is prepared by the following steps: adding ethanol into whole hay of Italian anchusa, soaking, heating, refluxing and extracting, and concentrating under reduced pressure to obtain an Italian anchusa concentrated solution; sequentially extracting and concentrating the concentrated solution by using petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol to obtain petroleum ether part extract, dichloromethane part extract, ethyl acetate part extract and n-butyl alcohol part extract; carrying out silica gel column chromatography gradient elution and separation on the obtained ethyl acetate part extract to obtain 8 fractions, and carrying out silica gel column chromatography gradient elution on the obtained Fr.2 fraction to obtain 7 components; carrying out semi-preparative liquid chromatography separation on the component Fr.2-4 to obtain the lignin compound at 18.7 minutes; in-vitro anti-inflammatory pharmacodynamic experiments show that the obtained lignin compound has a certain inhibition effect on release of tumor necrosis factors, interleukin 1 beta and interleukin 6, and is applied to potential anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of the separation and purification of Anchusa italica Retz., and is a lignin compound, a preparation method and an application thereof. Background Art

[0002] Inflammation is an important protective mechanism gradually formed by the body in the process of evolution to resist the invasion of foreign pathogens. However, its persistent existence or excessive reaction often induces diseases, which often plagues people's daily lives and can endanger people's lives and health seriously in severe cases. The long-term use of many drugs is likely to cause drug resistance and side effects, so finding anti-inflammatory drugs with high efficiency and low toxicity is the key to treating diseases. The lignin components in Anchusa italica Retz. have strong anti-inflammatory activities. Therefore, developing and utilizing the lignin components of Anchusa italica Retz., further exploring its potential medicinal value, and determining and characterizing the structures and physicochemical properties of its monomer compounds are of great significance for the development and utilization of Anchusa italica Retz. Summary of the Invention

[0003] The purpose of the present invention is to provide a lignin compound, a preparation method and an application thereof. The compound is obtained by adding ethanol to the dried whole herb of Anchusa italica Retz., soaking, heating under reflux for extraction, and concentrating under reduced pressure to obtain the concentrated solution of Anchusa italica Retz.; then successively extracting the concentrated solution with petroleum ether, dichloromethane, ethyl acetate and n-butanol, and concentrating to obtain the petroleum ether fraction extract, dichloromethane fraction extract, ethyl acetate fraction extract and n-butanol fraction extract; subjecting the obtained ethyl acetate fraction extract to gradient elution on a silica gel column chromatography for separation to obtain 8 fractions Fr.1-Fr.8, and then subjecting the obtained Fr.2 fraction to gradient elution on a silica gel chromatography to obtain 7 components Fr.2-1-Fr.2-7; separating the component Fr.2-4 by semi-preparative liquid chromatography to obtain a lignin compound at 18.7 minutes; and subjecting the obtained compound to an in vitro anti-inflammatory pharmacodynamic experiment. The results show that it has a certain inhibitory effect on the release of tumor necrosis factor (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6), so that such compounds in Anchusa italica Retz. are used as potential anti-inflammatory drugs.

[0004] A lignin compound described in the present invention has the following chemical structural formula (Ⅰ):

[0005]

[0006] Wherein: the name of the compound is (4S,5S)-4-(3-hydroxy-4-(4-hydroxy-3,5-dimethoxyphenoxy)phenyl)-5-methyldihydrofuran-2(3H)-one.

[0007] The preparation method of the lignin compound described above is carried out according to the following steps:

[0008] a. Add the dried whole herb of Anchusa italica Retz. to ethanol with a concentration of 70 - 75%, soak it at room temperature for 30 - 50 min, extract it by heating under reflux 3 times, each time for 2 - 2.5 h, combine the extracts and concentrate them under reduced pressure to obtain the concentrated Anchusa italica Retz. solution;

[0009] b. Extract the concentrated Anchusa italica Retz. solution obtained in step a successively with petroleum ether, dichloromethane, ethyl acetate and n - butanol, concentrate the extracts to obtain the petroleum ether extract, dichloromethane extract, ethyl acetate extract and n - butanol extract;

[0010] c. Use dichloromethane and methanol eluents with volume ratios of 100:1, 50:1, 30:1, 15:1, 10:1, 5:1, 2:1 and 1:1 in sequence to perform gradient elution on the ethyl acetate extract obtained in step b through silica gel column chromatography, and separate to obtain 8 fractions Fr.1 - Fr.8;

[0011] d. Use dichloromethane - methanol with a volume ratio of 1:0 - 0:1 to perform gradient elution on fraction Fr.2 obtained in step c through silica gel chromatography, combine the eluents to obtain 7 components Fr.2 - 1 - Fr.2 - 7; Separate component Fr.2 - 4 by semi - preparative liquid chromatography, elute with 30% methanol - water as the eluent at a flow rate of 2 mL / min, collect the eluate, and obtain the compound at 18.7 minutes.

[0012] Use of the lignin - like compound in the preparation of drugs for anti - inflammatory tumor necrosis factor, interleukin - 1β and interleukin - 6.

[0013] Perform in vitro anti - inflammatory pharmacodynamic experiments on the lignin - like compound of the present invention. The in vitro anti - inflammatory pharmacodynamic experiments adopt the CCK - 8 method to measure the NO content released by cells and the levels of inflammatory factors interleukin - 1β (IL - lβ), interleukin - 6 (IL - 6) and tumor necrosis factor (TNF - α) to screen for anti - inflammatory active components. Description of the Drawings

[0014] Figure 1 For the present invention 1 1H - NMR spectrum;

[0015] Figure 2 For the present invention 13 13C - APT spectrum;

[0016] Figure 3 COSY spectrum of the compound of the present invention;

[0017] Figure 4 QC spectrum of the compound of the present invention;

[0018] Figure 5 BC spectrum of the compound of the present invention;

[0019] Figure 6 NOESY spectrum of the compound of the present invention;

[0020] Figure 7 Effect of the compound of the present invention on the levels of TNF-α, IL-1β and IL-6. Compared with LPS-untreated cells, ##p < 0.01; compared with LPS-treated cells, *p < 0.05, **p < 0.01. Detailed implementation manners

[0021] The present invention is not limited by the following examples, and the specific implementation manners can be determined according to the technical solutions of the present invention and the actual situation. All chemical reagents and chemical supplies mentioned in the present invention are well-known and commonly used chemical reagents and chemical supplies in the prior art unless otherwise specified; the percentages in the present invention are mass percentages unless otherwise specified; the solutions in the present invention are aqueous solutions with water as the solvent unless otherwise specified. For example, a hydrochloric acid solution is an aqueous solution of hydrochloric acid; normal temperature and room temperature in the present invention refer to a temperature range of 15°C to 25°C, defined as 25°C.

[0022] Example 1

[0023] a. Add the dried whole herb of Anchusa italica Retz. to ethanol with a concentration of 70%, soak at room temperature for 30 min, and perform heat reflux extraction 3 times, each time for 2 h. Combine the extraction solutions and concentrate under reduced pressure to obtain a concentrated solution of Anchusa italica Retz.;

[0024] b. Extract the concentrated solution of Anchusa italica Retz. obtained in step a with petroleum ether, dichloromethane, ethyl acetate, and n-butanol in sequence, and concentrate to obtain an extract of the petroleum ether fraction, an extract of the dichloromethane fraction, an extract of the ethyl acetate fraction, and an extract of the n-butanol fraction;

[0025] c. Use dichloromethane and methanol eluents with volume ratios of 100:1, 50:1, 30:1, 15:1, 10:1, 5:1, 2:1, and 1:1 in sequence to perform gradient elution on the silica gel column chromatography for the extract of the ethyl acetate fraction obtained in step b, and separate to obtain 8 fractions, Fr.1 - Fr.8;

[0026] d. For the Fr.2 fraction obtained in step c, perform gradient elution on silica gel chromatography with a dichloromethane - methanol volume ratio of 1:0 - 0:1, combine the elution solutions to obtain 7 components, Fr.2 - Fr.2-7; Separate the component Fr.2-4 by semi-preparative liquid chromatography, elute with 30% methanol water as the eluent at a flow rate of 2 mL / min, collect the eluate, and obtain a lignin compound at 18.7 minutes.

[0027] Example 2

[0028] a. Add the dried whole herb of Anchusa italica Retz. to ethanol with a concentration of 75%, soak at room temperature for 40 min, extract by heating under reflux 3 times, 2.5 h each time. Combine the extracts and concentrate under reduced pressure to obtain the concentrated Anchusa italica Retz. solution;

[0029] b. Extract the concentrated Anchusa italica Retz. solution obtained in step a successively with petroleum ether, dichloromethane, ethyl acetate, and n-butanol. Concentrate the extracts to obtain the petroleum ether fraction extract, dichloromethane fraction extract, ethyl acetate fraction extract, and n-butanol fraction extract;

[0030] c. Use dichloromethane and methanol eluents with volume ratios of 100:1, 50:1, 30:1, 15:1, 10:1, 5:1, 2:1, and 1:1 successively to perform gradient elution on the ethyl acetate fraction extract obtained in step b through silica gel column chromatography, and separate to obtain 8 fractions, Fr.1 - Fr.8;

[0031] d. For the Fr.2 fraction obtained in step c, perform gradient elution on silica gel chromatography with dichloromethane - methanol with a volume ratio of 1:0 - 0:1. Combine the eluents to obtain 7 components, Fr.2 - 1 - Fr.2 - 7; Separate the component Fr.2 - 4 by semi-preparative liquid chromatography, elute with 30% methanol - water as the eluent at a flow rate of 2 mL / min, collect the eluate, and obtain lignin compounds at 18.7 minutes.

[0032] Example 3

[0033] a. Add the dried whole herb of Anchusa italica Retz. to ethanol with a concentration of 70%, soak at room temperature for 50 min, extract by heating under reflux 3 times, 2.5 h each time. Combine the extracts and concentrate under reduced pressure to obtain the concentrated Anchusa italica Retz. solution;

[0034] b. Extract the concentrated Anchusa italica Retz. solution obtained in step a successively with petroleum ether, dichloromethane, ethyl acetate, and n-butanol. Concentrate the extracts to obtain the petroleum ether fraction extract, dichloromethane fraction extract, ethyl acetate fraction extract, and n-butanol fraction extract;

[0035] c. Use dichloromethane and methanol eluents with volume ratios of 100:1, 50:1, 30:1, 15:1, 10:1, 5:1, 2:1, and 1:1 successively to perform gradient elution on the ethyl acetate fraction extract obtained in step b through silica gel column chromatography, and separate to obtain 8 fractions, Fr.1 - Fr.8;

[0036] d. The Fr.2 fraction obtained in step c was eluted with dichloromethane-methanol with a volume ratio of 1:0 - 0:1 by silica gel column chromatography with gradient elution, and the eluents were combined to obtain 7 components Fr.2-1 - Fr.2-7; Component Fr.2-4 was separated by semi-preparative liquid chromatography, and the eluent was 30% methanol-water, and the elution was carried out at a flow rate of 2 mL / min. The eluate was collected, and a lignin compound was obtained at 18.7 minutes.

[0037] The obtained compound was subjected to nuclear magnetic resonance hydrogen spectrum ( 1 1H-NMR) and nuclear magnetic resonance carbon spectrum ( 13 13C-APT) analysis. The 1 1H-NMR spectrum is as shown in Figure 1 , and the 13 13C-APT spectrum is as shown in Figure 2 . The two-dimensional spectra are Figure 3 COSY, Figure 4 QC, Figure 5 BC, Figure 6 NOESY as shown; and the obtained compound was subjected to spectral analysis, and the peak assignments are shown in Table 1. The chemical structural formula of the compound can be determined from the data in Table 1, and it is soluble in methanol;

[0038] For Figure 1 and Figure 7 , spectral analysis was carried out, and the peaks of Figure 2 and Figure 3 were assigned respectively, and the chemical shifts and peak types of carbon and hydrogen in the compound structure were obtained, and the assignments are shown in Table 1

[0039] Table 1

[0040]

[0041] Example 4

[0042] The obtained compound was subjected to in vitro anti-inflammatory pharmacodynamic experiments. The in vitro anti-inflammatory pharmacodynamic experiments were carried out by the CCK-8 method:

[0043] A. RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin mixture, and placed in an incubator at 37 °C and 5% CO 2 2 for culture; the cell state was observed daily, and passage was carried out after the density reached 80% - 90% for subsequent experiments; the groups were divided into a blank group, a control group with quercetin, and an experimental group with the compound; RAW264.7 cells in the logarithmic growth phase were diluted and mixed evenly with blank medium to prepare a cell suspension, and at 37 °C and 5% CO 2Incubate under the conditions until the cells adhere and grow to about 80% of the 96-well plate. Aspirate the supernatant, add 100 μL of the drug solution to each well, and after incubating for 24 h, discard the drug solution. In a light-protected environment, prepare the CCK-8 reaction solution according to the formula [V(blank medium)∶V(CCK-8) = 1∶10], add it to the 96-well plate, and incubate in an incubator at 37 °C and 5% CO 2 for 30 min, and detect the optical density (OD) value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader; cell survival rate = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)×100%;

[0044] B. Take cells in the logarithmic growth phase, dilute the cells with blank medium to obtain a cell suspension, and incubate under the conditions of 37 °C and 5% CO 2 until the cells adhere and grow to about 80% of the 96-well plate. Aspirate the supernatant, add 100 μL of the drug solution to each well, and after incubating for 24 h, discard the drug solution. Then add 100 μL of lipopolysaccharide (LPS) at a concentration of 1 μg / mL to the 96-well plate and place it in an incubator at 37 °C and 5% CO 2 for 12 h to establish an inflammatory cell model; observe the cell growth status, collect cells in the logarithmic growth phase, count the cells, inoculate the cells into the 96-well plate so that the number of cells in each well is about 3.0×104, and incubate overnight in the incubator; after 24 h, discard the medium, first add the medium containing the drug at concentrations of 5 μM / mL, 10 μM / mL, and 20 μM / mL for 1 h, and then add lipopolysaccharide (LPS) to a final concentration of 1 μg / mL; after culturing for 24 h, aspirate 50 μL of the cell supernatant from each well of the cell culture plate and add it to a new 96-well plate. Then, add 50 μL / well of room temperature Griess Reagent I to each well; subsequently, add an equal volume of Griess Reagent II; perform the operation in the dark. After shaking the ELISA reader for 30 s, measure the absorbance at 540 nm; the cell culture and drug addition methods are the same as in step A. Collect the supernatant of the cultured cells in each treatment group, and use the ELISA method to measure the contents of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Each step of the experiment strictly follows the methods provided by the ELISA kit for experiment and data processing. The results are shown in Table 2 and Figure 7 ;

[0045] Table 2. Inhibitory effects of compounds and positive drugs on NO production in LPS-stimulated RAW264.7 cells

[0046]

[0047] As can be seen from Table 2, the lignin compounds of the present invention have a certain inhibitory effect on the release of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), so that the lignin compounds are applied as potential anti-inflammatory drugs.

Claims

1. A lignin compound, characterized in that The chemical structural formula (I) of the compound is: Wherein: the name of the compound is (4S, 5S)-4-(3-hydroxy-4-(4-hydroxy-3,5-dimethoxyphenoxy)phenyl)-5-methyldihydrofuran-2(3H)-one.

2. The method for preparing a lignin compound according to claim 1, characterized in that Follow these steps: a. Add 70-75% ethanol to the whole herb of Italian bugloss, soak at room temperature for 30-50 minutes, heat and reflux for extraction 3 times, each time for 2-2.5 hours, combine the extracts and concentrate under reduced pressure to obtain Italian bugloss concentrate; b. Extracting the Italian Bugloss concentrate obtained in step a with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence, and concentrating the extract to obtain a petroleum ether extract, a dichloromethane extract, an ethyl acetate extract and a n-butanol extract; c. The ethyl acetate extract obtained in step b was subjected to gradient elution by silica gel column chromatography using dichloromethane and methanol eluents in volume ratios of 100:1, 50:1, 30:1, 15:1, 10:1, 5:1, 2:1 and 1:1, respectively, to obtain 8 fractions Fr.1-Fr.8; d. The Fr.2 fraction obtained in step c was gradient eluted by silica gel chromatography with dichloromethane-methanol in a volume ratio of 1:0-0:1, and the eluates were combined to obtain 7 components Fr.2-1-Fr.2-7; component Fr.2-4 was separated by semi-preparative liquid chromatography with 30% methanol in water as the eluent and an elution rate of 2 mL / min. The eluate was collected to obtain the compound at 18.7 minutes.

3. Use of the lignin compound according to claim 1 in the preparation of anti-inflammatory tumor necrosis factor, interleukin-1β and interleukin-6 drugs.

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