A stilbene compound, its preparation method and application

Abstract

The present invention provides a stilbene compound, a preparation method thereof and an application. The structural formula of the stilbene compound is: The extraction method of the stilbene compound is as follows: extract the total extract of Caragana stenophylla, disperse the total extract of Caragana stenophylla in water to form a suspension, and then extract the extracts of different solvent parts. Take the ethyl acetate part extract for gradient elution, purify, separate and collect the eluate. It is proved by experiments that the stilbene compound of the present invention has obvious anti-inflammatory effects, especially significant effects in the lead treatment of rheumatoid arthritis. This enables the present invention to prepare anti-inflammatory drugs using Caragana stenophylla as the raw material, which has a wide source, is non-toxic, pollution-free, has a simple preparation process, low cost, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a stilbene compound, a preparation method thereof, and an application thereof. Background Art

[0002] Stilbenoids compounds, also known as stilbene compounds, refer to a general term for substances having a stilbene nucleus or its polymer, generally having a C6-C2-C6 basic structural unit, and were first isolated from plants in 1899. Stilbenoid components are not only considered important substances for protecting plants from fungal and bacterial invasions, but also have a variety of biological activities, and have good effects in aspects such as anti-inflammatory, antiviral, anti-HIV, antioxidant, anti-cancer, cardiovascular protection, neuroprotection, and anti-Alzheimer's disease (AD). Due to their good activities, stilbenoid compounds have attracted much attention from pharmacologists and have potential development prospects.

[0003] Caragana stenophylla Pojark. is a plant of the genus Caragana in the family Leguminosae, and is distributed in arid and semi-arid regions in northern Xinjiang. Its roots, as a herbal medicine with a long application history, have the effects of promoting blood circulation, diuresis, analgesia, and strengthening the body. However, there are few research reports on the chemical constituents of this plant.

[0004] Although prior art reports suggest that the medicinal effects of Caragana stenophylla may mainly come from the stilbenoid components therein, the specific structural types of the effective components are not clear, which poses obstacles to the development of new drugs and is also not conducive to the development and utilization of the medicinal substance Caragana stenophylla. Summary of the Invention

[0005] The purpose of the present invention is to develop and utilize the stilbene monomer compounds in Caragana stenophylla, further explore their potential medicinal value, and determine and characterize the structures and physicochemical properties of the monomer compounds. The present invention has for the first time discovered a new stilbene structural type with anti-inflammatory activity, and the compounds of this type have strong anti-inflammatory activity, providing good materials for the development of new anti-inflammatory drugs.

[0006] To achieve the above object, in one aspect, the present invention provides a stilbene compound, whose chemical name is (2S,3S)-2-(3,5-dihydroxyphenyl)-5,7-dihydroxy-3-((S)-hydroxy(4-hydroxyphenyl)methyl)-4-((4bR,5R,10S,11S)-1,3,8-trihydroxy-5,11-bis(4-hydroxyphenyl)-4b,5,10,11-tetrahydrobenzo[6,7]cyclohepta[1,2,3-cd]benzofuran-10-yl)-2,3-dihydro-1H-inden-1-one, and the structural formula is: Or a salt or other derivative of the said structural formula.

[0007] On the other hand, the present invention also provides a preparation method of a stilbene compound: The said preparation method includes:

[0008] (1) Extract the total extract of Caragana stenophylla.

[0009] (2) After dispersing the total extract of Caragana stenophylla in water to form a suspension, extract it successively with several solvents including ethyl acetate, and concentrate the extraction solutions of each solvent part.

[0010] (3) Take the ethyl acetate part extract for gradient elution, purify and separate the substances obtained after gradient elution by high performance liquid chromatography gradient elution, and collect the eluate.

[0011] Specifically, the method for extracting the total extract of Caragana stenophylla is: crush the roots of Caragana stenophylla and soak them in ethanol, heat and reflux for extraction 3 - 5 times, 1 - 3 hours each time, combine the reflux extraction solutions each time and recover and concentrate under reduced pressure to obtain the total extract of Caragana stenophylla.

[0012] Further, when extracting successively with several solvents including ethyl acetate, the extraction solvents are petroleum ether, chloroform, ethyl acetate, and n-butanol in sequence. Concentrate the extraction solution obtained with ethyl acetate to obtain the ethyl acetate part extract.

[0013] Further, when taking the ethyl acetate part extract for gradient elution and eluting with the gradient elution solution of silica gel column chromatography, the gradient elution solution of silica gel column chromatography includes chloroform and methanol, and the volume ratio of chloroform to methanol is 40:1.

[0014] Further, when purifying and separating the fraction obtained after silica gel column chromatography gradient elution by high performance liquid chromatography gradient elution, the eluate collected is the eluate obtained at the 30.5th minute.

[0015] Alternatively, specifically, the extract of the ethyl acetate fraction is eluted with a gradient eluent of silica gel column chromatography. The gradient eluent of silica gel column chromatography includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:1.

[0016] Furthermore, when the fraction obtained after gradient elution separation by silica gel column chromatography is purified and separated by gradient elution of high performance liquid chromatography, the gradient eluent of silica gel column chromatography includes acetonitrile and water, and the volume ratio of acetonitrile to water is 11:9.

[0017] Furthermore, when the fraction obtained after gradient elution separation by silica gel column chromatography is purified and separated by gradient elution of high performance liquid chromatography, the eluate collected is the eluate obtained at the 23.0 minute mark.

[0018] Experiments have proven that the stilbene compounds of the above structural formula provided by the present invention, or their salts and derivatives, have good anti-inflammatory activity. Therefore, the present invention also claims the use of the compounds in the preparation of anti-inflammatory therapeutic drugs; in particular, it has outstanding effects in the lead treatment of rheumatoid arthritis, and the anti-inflammatory treatment includes the lead treatment of rheumatoid arthritis.

[0019] The present invention has the following beneficial effects or advantages compared with the prior art:

[0020] (1) The stilbene compounds of the present invention have obvious anti-inflammatory effects and can be used as lead drugs for rheumatoid arthritis, and have good application effects in inhibiting the formation of NO products in lipopolysaccharide-stimulated inflammatory cells;

[0021] (2) The extraction and separation method of the stilbene compounds of the present invention uses Caragana stenophylla as the raw material, which has a wide source, is non-toxic and pollution-free, the preparation process is simple, the cost is low, and it is suitable for large-scale popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the structural formula of the compound of the present invention.

[0023] Figure 2 It is the 1 1H NMR spectrum of the compound of the present invention.

[0024] Figure 3 It is the 13 13C NMR spectrum of the compound of the present invention.

[0025] Figure 4Effect of the compound of the present invention on nitric oxide production in LPS-stimulated RAW264.7 cells, where blank represents the blank group, control represents the model group, N-LAME represents the positive control L-NAME, and 0.5 - 10 respectively represent the addition of the compound of the present invention at the corresponding concentration. Specific Embodiment

[0026] Next, the technical solution of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments.

[0027] In the following embodiments, the experimental methods and detection methods are conventional methods unless otherwise specified; the medicaments and materials can be purchased on the market unless otherwise specified; the index data are measured by conventional methods unless otherwise specified.

[0028] Embodiment 1

[0029] This embodiment provides a method for extracting the stilbene compounds of the present invention

[0030] 1. Extract the total extract: Crush the roots of Caragana stenophylla and soak them in ethanol with a volume concentration of 80% for extraction. Heat under reflux for 3 times, 2 hours each time. Combine the reflux extracts each time and recover and concentrate under reduced pressure to obtain the total extract of Caragana stenophylla.

[0031] 2. Extract the extracts of different parts: Disperse the total extract of Caragana stenophylla in water to form a suspension, and then extract it successively with petroleum ether, chloroform, ethyl acetate, and n-butanol. Concentrate the extracts respectively to obtain the petroleum ether fraction extract, chloroform fraction extract, ethyl acetate fraction extract, and n-butanol fraction extract.

[0032] 3. Gradient elute the ethyl acetate fraction extract: Take the ethyl acetate fraction extract and separate it by gradient elution on a silica gel column chromatography to obtain 8 fractions. Among them, the gradient eluent for silica gel column chromatography includes chloroform and methanol, and the volume ratios of chloroform to methanol are 100:0, 100:1, 80:1, 50:1, 40:1, 30:1, 10:1, 5:1 in sequence.

[0033] 4. Purification, separation and collection: The 5th fraction was purified and separated by gradient elution on high performance liquid chromatography, and the eluate was collected. The substance obtained at 30.5 minutes was (2S,3S)-2-(3,5-dihydroxyphenyl)-5,7-dihydroxy-3-((S)-hydroxy(4-hydroxyphenyl)methyl)-4-((4bR,5R,10S,11S)-1,3,8-trihydroxy-5,11-bis(4-hydroxyphenyl)-4b,5,10,11-tetrahydrobenzo[6,7]cyclohepta[1,2,3-cd]benzofuran-10-yl)-2,3-dihydro-1H-inden-1-one, which was collected and reserved for further use.

[0034] Example 2

[0035] This example provides a method for extracting the stilbene compounds of the present invention.

[0036] 1. Extracting the total extract: The same as in Example 1.

[0037] 2. Extracting the extracts of different parts: The total extract of Caragana stenophylla was dispersed in water to form a suspension, and then successively extracted with petroleum ether, ethyl acetate, and n-butanol. The extracts were concentrated respectively to obtain the petroleum ether fraction extract, ethyl acetate fraction extract, and n-butanol fraction extract.

[0038] 3. Gradient elution of the ethyl acetate fraction extract: The ethyl acetate fraction extract was separated by gradient elution on a silica gel column chromatography to obtain 8 fractions. Among them, the gradient elution solution of the silica gel column chromatography included petroleum ether and ethyl acetate, and the volume ratios of petroleum ether to ethyl acetate were 100:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:100 in turn.

[0039] 4. Purification, separation and collection: The 6th fraction was purified and separated by gradient elution on high performance liquid chromatography, and the eluate was collected. The substance obtained at 23.0 minutes was (2S,3S)-2-(3,5-dihydroxyphenyl)-5,7-dihydroxy-3-((S)-hydroxy(4-hydroxyphenyl)methyl)-4-((4bR,5R,10S,11S)-1,3,8-trihydroxy-5,11-bis(4-hydroxyphenyl)-4b,5,10,11-tetrahydrobenzo[6,7]cyclohepta[1,2,3-cd]benzofuran-10-yl)-2,3-dihydro-1H-inden-1-one, which was collected and stored for future use.

[0040] Example 3

[0041] 1. Extraction of total extract: The same as Example 1.

[0042] 2. Extraction of extracts from different parts: The same as Example 2.

[0043] 3. Elution of the ethyl acetate part extract: The ethyl acetate part extract was eluted by silica gel column chromatography. Among them, the gradient eluent for silica gel column chromatography included acetonitrile and water, and the volume ratio of acetonitrile to water was 55:45.

[0044] 4. Purification, separation and collection: The substance obtained by elution was purified and separated by gradient elution on high performance liquid chromatography, and the eluate was collected. The substance obtained at 23.0 minutes was (2S,3S)-2-(3,5-dihydroxyphenyl)-5,7-dihydroxy-3-((S)-hydroxy(4-hydroxyphenyl)methyl)-4-((4bR,5R,10S,11S)-1,3,8-trihydroxy-5,11-bis(4-hydroxyphenyl)-4b,5,10,11-tetrahydrobenzo[6,7]cyclohepta[1,2,3-cd]benzofuran-10-yl)-2,3-dihydro-1H-inden-1-one, which was collected and stored for future use.

[0045] In this example, the target product obtained above was also reacted with a small amount of sodium hydroxide solution respectively to obtain its sodium salt. It was found that the sodium salt had better solubility and stability, which was more conducive to drug administration and anti-inflammatory effects.

[0046] In this example, the target product obtained above was used to obtain its derivative through methoxylation, and it also had anti-inflammatory activity through experiments.

[0047] Example 4

[0048] This example provides an experiment for determining the physicochemical properties of the stilbene compounds of the present invention

[0049] 1. Perform 1 H-NMR imaging on the compounds obtained in Examples 1-3 (excluding salts and derivatives), and the results are as Figure 2 shown

[0050] 2. Perform 13 C-NMR imaging on the compounds obtained in Examples 1-3 (excluding salts and derivatives), and the results are as Figure 3 shown

[0051] 3. Through observation and dissolution tests, it can be seen that the compounds of the present invention are white powders, soluble in chloroform and methanol; take an appropriate amount of the compounds obtained in Examples 1-3, spray with 10% sulfuric acid ethanol test solution, and show orange-yellow after heating at 105 °C; the quasi-molecular ion peak [M+H] + m / z 847.2388 is obtained through the positive ion mode of HR-ESI-MS, and the chemical formula is determined to be C 50 H 39 O 13 (calculated value 847.2391), and the degree of unsaturation Ω is 32

[0052] 4. Perform spectral analysis on Figure 2 and Figure 3 , assign each peak of Figure 2 and Figure 3 , and the peak assignments of Figure 2 and Figure 3 are shown in Table 1; through Figure 2 , Figure 3 and the data in Table 1, it can be seen that the structural formula of the present invention is as Figure 1 shown

[0053] Table 1 Peak assignments of the H-NMR spectrum and 1 C-NMR spectrum of the compounds of the present invention 13

[0054]

[0055]

[0056] Example 5

[0057] This example provides an experiment on the effect of the stilbene compounds of the present invention on the production of nitric oxide (NO) in LPS-stimulated RAW264.7 cells

[0058] ​Cell culture passage: RAW 264.7 cells were added to DMEM medium containing 10% heat-inactivated FBS, penicillin G (100 U / mL), and streptomycin (100 μg / mL), and cultured at 37°C and 5% CO 2 under saturated humidity conditions for 24 h, and passaged once every 3 days.

[0059] Cell viability test: RAW264.7 cells were seeded at 5×10 6 cells / well in a 96-well plate, incubated at 37°C and 5% CO 2 in a cell culture incubator for 4 h. The supernatant was discarded, and the cells were washed 3 times with DMEM medium. Then, 100 μL of DMEM medium containing 10% FBS was added, and 50 μL of LPS (10 ng / mL) and 50 μL of the test compound were added respectively. After further culturing for 24 h, the supernatant was discarded, MTT reagent was added, and the mixture was shaken for 10 min. The absorbance values of each well were measured at 492 nm using an ELISA reader. The cell viability was 98%, indicating that the compound was not toxic to the cells.

[0060] NO test: A total of 7 groups of experiments were set up, namely (blank group), (control group), (N-LAME group), and treatment groups with different concentrations of 0.5, 1, 5, and 10 μM of the sample; RAW264.7 cells were seeded at 5×10 6 cells / well in a 96-well plate, incubated at 37°C and 5% CO 2 in a cell culture incubator for 4 h. The supernatant was discarded, and the cells were washed 3 times with DMEM medium. Then, 100 μL of DMEM medium containing 10% FBS was added, and 50 μL of LPS (10 ng / mL) and 50 μL of the test compound were added respectively. After further culturing, 100 μL of the culture medium supernatant was taken after 24 h, Greiss reagent was added, and the mixture was shaken for 10 min. The complete medium plus the chromogenic agent was used as a blank control, and the absorbance values of each well were measured at 550 nm using an ELISA reader.

[0061] The results are as Figure 4 shown. Compared with the model group, the compound of the present invention had a significant inhibitory effect on nitric oxide production in LPS-stimulated RAW264.7 cells in the concentration range of 0.5 - 10 μM, and the half-maximal inhibitory concentration IC 50 was 6.21 μM respectively. The results showed that the compound of the present invention could significantly inhibit the production of nitric oxide (NO) in LPS-stimulated RAW264.7 cells, and the inhibitory effect was concentration-dependent, indicating that the compound of the present invention had good anti-inflammatory activity.

[0062] As described above, the present invention can be preferably implemented. The above embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A stilbene compound, characterized in that, the stilbene compound has the following structural formula: or a salt of the said structural formula.

2. A preparation method of the stilbene compound according to claim 1, characterized in that, the preparation method includes: (1) Crushing the roots of Caragana stenophylla, soaking them in ethanol with a volume concentration of 80%, heating under reflux for extraction 3 - 5 times, each time for 1 - 3 hours, combining the reflux extracts each time and recovering and concentrating under reduced pressure to obtain the total extract of Caragana stenophylla; (2) Dispensing the total extract of Caragana stenophylla into a suspension with water, and then extracting successively with petroleum ether, chloroform, ethyl acetate, and n - butanol, concentrating the extracts respectively to obtain the petroleum ether fraction extract, chloroform fraction extract, ethyl acetate fraction extract, and n - butanol fraction extract; (3) Taking the ethyl acetate fraction extract and separating it by gradient elution on a silica gel column chromatography to obtain 8 fractions. Among them, the gradient elution solution for silica gel column chromatography includes chloroform and methanol, and the volume ratios of chloroform to methanol are successively 100:0, 100:1, 80:1, 50:1, 40:1, 30:1, 10:1, 5:1; (4) Purifying and separating the 5th fraction by gradient elution on a high - performance liquid chromatography and collecting the eluate. The collected eluate is the eluate obtained at the 30.5 - minute mark.

3. A preparation method of the stilbene compound according to claim 1, characterized in that, the preparation method includes: (1) Crushing the roots of Caragana stenophylla, soaking them in ethanol with a volume concentration of 80%, heating under reflux for extraction 3 - 5 times, each time for 1 - 3 hours, combining the reflux extracts each time and recovering and concentrating under reduced pressure to obtain the total extract of Caragana stenophylla; (2) Dispensing the total extract of Caragana stenophylla into a suspension with water, and then extracting successively with petroleum ether, ethyl acetate, and n - butanol, concentrating the extracts respectively to obtain the petroleum ether fraction extract, ethyl acetate fraction extract, and n - butanol fraction extract; (3) Taking the ethyl acetate fraction extract and separating it by gradient elution on a silica gel column chromatography to obtain 8 fractions. Among them, the gradient elution solution for silica gel column chromatography includes petroleum ether and ethyl acetate, and the volume ratios of petroleum ether to ethyl acetate are successively 100:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:100; (4) Purifying and separating the 6th fraction by gradient elution on a high - performance liquid chromatography and collecting the eluate. The collected eluate is the eluate obtained at the 23.0 - minute mark.

4. Use of the compound according to claim 1 in the preparation of anti - inflammatory therapeutic drugs.

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