CN110156859B - Sinapic acid compound and preparation method and application thereof - Google Patents

Sinapic acid compound and preparation method and application thereof Download PDF

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CN110156859B
CN110156859B CN201910541141.7A CN201910541141A CN110156859B CN 110156859 B CN110156859 B CN 110156859B CN 201910541141 A CN201910541141 A CN 201910541141A CN 110156859 B CN110156859 B CN 110156859B
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glucopyranosyloxy
sinapoyl
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CN110156859A (en
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阿吉艾克拜尔·艾萨
艾达·凯尔库洛夫那
李俊
崔靖雪
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Xinjiang Technical Institute of Physics and Chemistry of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical

Abstract

The invention relates to a sinapic acid compound and a preparation method and application thereof, wherein the sinapic acid compound is prepared from dependent (seed) of Ligusticum wallichiiLepidium sativumLThe seeds are detected and analyzed by adopting a thin layer chromatography method and an analytical high performance liquid chromatography method, extracted by using an organic solvent, then separated by four to five methods of a solvent extraction method, a normal phase silica gel column chromatography method, a reverse phase silica gel column chromatography method, a small pore resin column chromatography method, a Sephadex LH-20 gel column chromatography method and a semi-preparative high performance liquid chromatography method to obtain five new sinapic acid monomeric compounds, and the five monomeric compounds are determined to be the new sinapic acid monomeric compounds by methods of high resolution mass spectrometry, nuclear magnetic resonance spectroscopy and the like, and the structure identification is carried out on the new sinapic acid monomeric compounds. In addition, these sinapinic compounds were tested for anti-inflammatory activity and the results showed that: the five new sinapic acid compounds have anti-inflammatory activity with different degrees, and can be used for preparing anti-inflammatory drugs.

Description

Sinapic acid compound and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicines, in particular to five sinapic acid compounds and a preparation method and application thereof.
Background
Cruciferae (Brassicaceae) has 330 and 3500 plants, one of the most diverse families of plants, and is responsible for vegetable and oil crops. Lepidium sativum (Lepidium sativum) belongs to Lepidium of Brassicaceae, is usually eaten as edible plant in Sinkiang and Tibet areas of China, and its dry mature seed is called Lepidium sativum seed, and is widely used in traditional national medicine in Sinkiang area for treating gastrointestinal diseases, relaxing bowels, expelling intestinal worms, and treating constipation, intestinal parasites, etc. According to reports, the lepidium sativum seeds have multiple biological activities of oxidation resistance, bacteria resistance, inflammation resistance, hyperglycemia resistance, tumor resistance and the like. The chemical components found by predecessors from the seed of Lepidium Sativum include alkaloids, flavones, terpenoids, phenylpropanoids, sterols, sinapic acids, and the like.
Sinapic acid is also called as erucic acid, is naturally present in seeds of crucifers such as Brassica white mustard [ Brassica alba L. ] Boiss ] and Brassica juncea (L.) coss ], has recently been found to have biological activities of scavenging free radicals, inhibiting lipid peroxidation, resisting bacteria, resisting cancer and inflammation, resisting anxiety, improving memory and the like, and has been applied to various fields such as pharmaceutical industry, food, beverage, cosmetics and the like as the biological activities of sinapic acid and derivatives thereof are continuously found. The five novel compounds in the research are sinapic acid compounds discovered by the family Lepidium, and are structurally characterized by comprising sinapic acid structural fragments, the formulas (I) to (IV) are rare natural derivatives of uracil nucleosides of sinapic acid, and the formula (V) is less erucate ester containing sulfo-glucosyl group, which is discovered in nature at present. The research on the chemical components of the Lepidium meyenii Walp seeds can provide reference for determining secondary metabolites in the Lepidium meyenii Walp seeds and also provide scientific basis for disclosing the material basis of the pharmacodynamic action of the Lepidium meyenii Walp seeds.
Macrophages belong to the mononuclear phagocyte system, including migratory macrophages and immobile macrophages, and are widely distributed in various tissues and organs of the body. When macrophages are activated, various substances such as tumor necrosis factor TNF and interleukin IL-1 are released to participate in inflammatory reaction and immune reaction. Macrophages, when activated by Lipopolysaccharide (LPS) and interferon-gamma (interferon-gamma), use L-arginine to synthesize NO under the action of NO synthase. The NO synthesis of macrophage has important significance in killing and guiding microorganisms and tumor cells and other aspects.
The medicinal plants are a huge resource treasury for innovative medicine research, and finding lead compounds from the treasury is a hot spot of natural medicine research. The research aims to further develop and research sinapinic acid compounds of the common burclover seeds so as to discover the sinapinic acid compounds with novel structures, enrich the structural diversity of natural products and provide reserve compounds for high-throughput drug screening. In addition, the influence of the compounds on the NO content in macrophages provides reference for the development of anti-inflammatory drugs.
Disclosure of Invention
The invention aims to provide a sinapic acid compound and a preparation method and application thereof, wherein the sinapic acid compound is detected and analyzed from the whole herb of Lepidium sativum L by adopting a thin layer chromatography and an analytical high performance liquid chromatography, extracted by using an organic solvent, then separated by four to five methods of a solvent extraction method, a normal phase silica gel column chromatography, a reverse phase silica gel column chromatography, a small pore resin column chromatography, a Sephadex LH-20 gel column chromatography and a semi-preparative high performance liquid chromatography to obtain five new sinapic acid monomeric compounds, determined to be new sinapic acid compounds by methods such as a high resolution mass spectrum and a nuclear magnetic resonance spectrum, structurally identified, and subjected to anti-inflammatory activity determination, and the result shows that: the five new sinapic acid compounds have anti-inflammatory activity with different degrees, and can be used for preparing anti-inflammatory drugs.
The sinapic acid compound is characterized in that the structural formula of the compound is as follows:
Figure BDA0002102591940000021
wherein:
the compound of formula (I) is 3 '- (6-sinapoyl-beta-D-glucopyranosyloxy) -5' -sinapoyl-uridine;
the compound of formula (II) is 3 '- [6- (2-dioxyindole-3-acetyl) -beta-D-glucopyranosyloxy ] -5' -sinapoyl-uridine;
the compound of formula (III) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine-3-benzoyl;
the compound of formula (IV) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine;
the compound of formula (V) is; 8-thio-beta-D-glucopyranosyloxy-sinapic acid butyl ester.
The preparation method of the sinapic acid compound comprises the following steps:
a. taking seeds of the Lepidium Sativum L.as a raw material, crushing, percolating, cold-leaching or heating reflux extraction with 5-10 times of 50-99% ethanol aqueous solution, absolute ethanol, pure acetone, 50-99% methanol aqueous solution or absolute methanol at room temperature, and concentrating to obtain crude extract of Lepidium Sativum L.seeds;
b. dispersing the crude extract obtained in the step a by using water, sequentially adding petroleum ether, n-hexane or cyclohexane, ethyl acetate and n-butanol, extracting for 3-5 times, and concentrating the n-butanol extract to obtain an n-butanol extract;
c. separating the n-butanol extract obtained in step b by two or three of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, small pore resin chromatography, Sephadex LH-20 gel column chromatography and preparative high performance liquid chromatography to obtain 3 '- (6-sinapoyl-beta-D-glucopyranosyloxy) -5' -sinapoyl-uridine as the compound of formula (I); the compound of formula (II) is 3 '- [6- (2-dioxyindole-3-acetyl) -beta-D-glucopyranosyloxy ] -5' -sinapoyl-uridine; the compound of formula (III) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine-3-benzoyl; the compound of formula (IV) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine; the compound of formula (V) is 8-thio- β -D-glucopyranosyloxy-sinapic acid butyl ester.
The normal phase silica gel column chromatography in step c is normal pressure or pressurized column chromatography, the filler is normal phase silica gel, the eluant is a mixture of at least two solvents of petroleum ether, cyclohexane or normal hexane, acetone, trichloromethane, dichloromethane or ethyl acetate and methanol, and isocratic elution or gradient elution is adopted.
And c, performing normal-pressure or pressurized column chromatography on the reverse-phase silica gel column used in the step c, wherein an eluant is a methanol water solution or acetonitrile water solution with the volume concentration of 10-100%, and isocratic elution or gradient elution is adopted.
The small-pore resin chromatography used in the step c is normal-pressure or pressurized column chromatography, the eluant is methanol water solution or acetonitrile water solution with the volume concentration of 10-100%, and isocratic elution or gradient elution is adopted.
And c, performing normal-pressure column chromatography on the Sephadex LH-20 gel column chromatography used in the step c, wherein an eluant is methanol, dichloromethane, trichloromethane or a mixture of at least two solvents, and performing isocratic elution or gradient elution.
And c, performing pressure column chromatography by using the preparative high performance liquid chromatography in the step c, wherein an eluent is 40-99% methanol aqueous solution or 30-99% acetonitrile aqueous solution by volume concentration, and performing isocratic elution or gradient elution.
The sinapic acid compound is used for preparing anti-inflammatory drugs.
The sinapic acid compound is obtained by separating and purifying plants, and can also be synthesized by a chemical modification method well known to the technical personnel in the field.
The sinapic acid compound is structurally determined by modern spectrum means such as high-resolution mass spectrum, one-dimensional and two-dimensional nuclear magnetic resonance spectrum and the like, and the structure identification process is as follows:
the compound of formula (I) is 3 '- (6-sinapoyl-beta-D-glucopyranosyloxy) -5' -sinapoyl-uridine, a yellow amorphous powder,
Figure BDA0002102591940000032
(c 0.57, MeOH); UV (MeOH)195,241,328 nm; by the excimer ion peak [ M-H ] in the high resolution mass spectrum]-m/z 817.2305 (calculated 817.2309) was determined to be C37H42O19N2. According to1H，13C NMR and two-dimensional nuclear magnetic resonance data determined that the structural skeleton type is erucic aciduropyhmidine, which is named 3 '- (6-erucyl-beta-D-glucopyranosyloxy) -5' -erucyl-uridine1H and13the C NMR values are given in Table 1[400MHz ] (1H)，100MHz(13C)，DMSO-d6]；
TABLE 1 preparation of compounds of formula (I)1H and13c NMR data [ delta (ppm), J (Hz)]
Figure BDA0002102591940000031
Figure BDA0002102591940000041
A in the table: performing attribution through two-dimensional nuclear magnetic data for overlapping hydrogen spectrum nuclear magnetic signals;
the compound of the formula (II) is 3' - [6- (2-dioxyindole-3-acetyl) -beta-D-glucopyranosyloxy]-beta-D-glucopyranosyloxy]-5' -sinapoyl-uridine, a yellow amorphous powder,
Figure BDA0002102591940000045
UV (MeOH)195,241,328 nm; by the quasi-molecular ion peak [ M-H ] in the high-resolution mass spectrum]-m/z 784.2204 (calculated 784.2206) was determined to be C36H39O17N3(ii) a According to1H，13C NMR and two-dimensional nuclear magnetic resonance data determine that the structural framework type of the compound is dioxoindole-3-acetylerucic acid uracil nucleoside which is named as 3' - [6- (2-dioxoindole-3-acetyl) -beta-D-glucopyranosyloxy]-beta-D-glucopyranosyloxy]-5' -sinapoyl-uridine, which is useful as a food additive1H and13the C NMR values are given in Table 2[400MHz ] (1H)，100MHz(13C)，DMSO-d6]；
TABLE 2 preparation of the compounds of formula (II)1H and13c NMR data [ delta (ppm), J (Hz)]
Figure BDA0002102591940000042
A in the table: the two-dimensional nuclear magnetic data are subjected to attribution for overlapping hydrogen spectrum nuclear magnetic signals;
the compound of formula (III) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine-3-benzoyl, yellow amorphous powder,
Figure BDA0002102591940000046
UV (MeOH)195,241,328 nm; by the excimer ion peak [ M-H ] in the high resolution mass spectrum]-m/z 715.1995 (calculated 715.1992) was determined to be C33H36O16N2. According to1H，13C NMR and two-dimensional nuclear magnetic resonance data confirmed that the structural skeleton type is benzoyloxy erucic acid uracil nucleoside, named 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uracil nucleoside-3-benzoyl, which1H and13the C NMR values are given in Table 3[600MHz ] (1H)，150MHz(13C)，DMSO-d6]；
TABLE 3 preparation of the compounds of formula (III)1H and13c NMR data [ delta (ppm), J (Hz)]
Figure BDA0002102591940000051
A in the table: performing attribution through two-dimensional nuclear magnetic data for overlapping hydrogen spectrum nuclear magnetic signals;
the compound of formula (IV) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine, a yellow amorphous powder,
Figure BDA0002102591940000053
UV (MeOH)195,241,328 nm; by the excimer ion peak [ M-H ] in the high resolution mass spectrum]-m/z 611.1731 (calculated 611.1729) was determined to be C26H32O15N2. According to1H，13C NMR and two-dimensional nuclear magnetic resonance data determined that the structural skeleton type is sinapinic uracil nucleoside, named 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uracil nucleoside, which1H and13the C NMR values are given in Table 4[400MHz ] (1H)，100MHz(13C)，DMSO-d6]；
TABLE 4 preparation of the Compound of formula (IV)1H and13c NMR data [ delta (ppm), J (Hz)]
Figure BDA0002102591940000052
Figure BDA0002102591940000061
A in the table: performing attribution through two-dimensional nuclear magnetic data for overlapping hydrogen spectrum nuclear magnetic signals;
the compound of formula (V) is 8-thio-beta-D-glucopyranosyloxy-erucic acid butyl ester, yellow oil,
Figure BDA0002102591940000062
Figure BDA0002102591940000063
UV (MeOH)195,241,328 nm; by the excimer ion peak [ M-H ] in the high resolution mass spectrum]-m/z 473.1489 (calculated 473.1486) was determined to be C21H30O10And S. According to1H，13C NMR and two-dimensional nuclear magnetic resonance data confirmed that the structural skeleton type is erucate ester glucosinolate, named 8-thio-beta-D-glucopyranosyloxy-butyl erucate, which1H and13the C NMR values are given in Table 5[400MHz ] (1H)，100MHz(13C)，DMSO-d6]；
TABLE 5 preparation of the compound of formula (V)1H and13c NMR data [ delta (ppm), J (Hz)]
Figure BDA0002102591940000064
A in the table: performing attribution through two-dimensional nuclear magnetic data for overlapping hydrogen spectrum nuclear magnetic signals;
drawings
FIG. 1 shows the preparation of the compounds of formula (I) according to the invention1H NMR(400MHz,DMSO-d6) A spectrogram;
FIG. 2 shows the preparation of the compounds of formula (I) according to the invention13C NMR(100MHz,DMSO-d6) A spectrogram;
FIG. 3 shows the preparation of a compound of formula (II) according to the invention1H NMR(400MHz,DMSO-d6) A spectrogram;
FIG. 4 shows the preparation of a compound of formula (II) according to the invention13C NMR(100MHz,DMSO-d6) A spectrogram;
FIG. 5 shows the preparation of a compound of formula (III) according to the invention1H NMR(600MHz,DMSO-d6) A spectrogram;
FIG. 6 shows the preparation of compounds of formula (III) according to the invention13C NMR(150MHz,DMSO-d6) A spectrogram;
FIG. 7 shows the preparation of the compound of formula (IV) according to the invention1H NMR(400MHz,DMSO-d6) A spectrogram;
FIG. 8 shows the preparation of the compound of formula (IV) according to the invention13C NMR(100MHz,DMSO-d6) A spectrogram;
FIG. 9 shows the preparation of a compound of formula (V) according to the invention1H NMR(400MHz,DMSO-d6) A spectrogram;
FIG. 10 shows the preparation of a compound of formula (V) according to the invention13C NMR(100MHz,DMSO-d6) Spectra.
Detailed Description
The reagents used were all analytically pure and the acetonitrile in the HPLC was of HPLC grade (Merck, Germany). Column chromatography normal phase silica gel (100 meshes 200 meshes, 200 meshes 300 meshes): marine industrial production of Qingdao; the silica gel for thin layer chromatography is HSGF 254: production of yellow silica gel development test factory in cigarette platform market; sephadex LH-20 gel: production by the American general electric medical group; small pore resin gel MCI (CHP 20/120): manufactured by mitsubishi corporation; reversed-phase silica gel ODS: manufactured by merck, germany; high performance liquid chromatography (dean, usa) was configured as follows: a P680HPLC pump, an ASI-100 autosampler, a TCC-100 column oven, a UVD170U ultraviolet detector (four wavelengths), a quaternary solvent system, an online degasser, a chameleon chromatographic workstation; preparative high performance liquid chromatography (dean, usa) was configured as follows: a P680HPLC pump, a UVD170U ultraviolet detector (four wavelengths), a quaternary solvent system, an online degasser, a chameleon chromatography workstation; mass spectra were determined using a quadrupole-time-of-flight hybrid mass spectrometer (applied biosystems, usa); the NMR was measured by a Warran NMR spectrometer model Vnmrs 600/400 (Warran, USA).
The family Lepidium chinensis is purchased from Ili area of Uygur autonomous region of Xinjiang Uygur autonomous region, and is identified as Lepidium sativum L by von Thyscrak secondary researchers of Sinkiang ecological geography institute of Chinese academy of sciences, wherein the purchase time is 11 months in 2014.
Example 1
a. Pulverizing 9kg of Lepidium Sativum L, cold soaking in 45L 95% ethanol-water solution at room temperature for 3 times, and evaporating solvent under reduced pressure to obtain crude extract;
b. dispersing the crude extract obtained in the step a by using water, adding petroleum ether, ethyl acetate and n-butanol, sequentially extracting for 3 times, combining n-butanol layers, and then decompressing and evaporating to dryness to obtain an n-butanol layer extract;
c. separating the n-butanol layer extract obtained in step b with normal phase silica gel column, performing gradient elution with chloroform-methanol at volume ratio of 100:1-1:1, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 9 components (Fr.1-Fr.9); separating the component Fr.6 by a reverse phase silica gel column, and performing gradient elution by using methanol-water with the volume ratio of 10-100% to obtain a component Fr.6.1-Fr.6.10; preparing a reverse phase column (C) from the component Fr.6.3185 μm,10 × 150mm) and isocratic elution with 32% acetonitrile-water solution to obtain the compound of formula (v) which is 8-thio- β -D-glucopyranosyloxy-butyl erucate; fr.8 is separated by reverse phase silica gel column, and eluted by methanol-water with volume ratio of 1:9-100:0 to obtain component Fr.8.1-Fr.8.10; mixing the components Fr.8.7-Fr.8.9, and preparing reverse phase column (C)185 μm,10 × 150mm) and isocratic elution with 30% acetonitrile-water solution to obtain 3 ' - (6-sinapoyl-beta-D-glucopyranosyloxy) -5 ' -sinapoyl-uracil nucleoside as compound of formula (I) and 3 ' - [6- (2-dioxoindole-3-acetyl) -beta-D-glucopyranosyloxy) as compound of formula (II)]-beta-D-glucopyranosyloxy]-5 ' -sinapoyl-uridine, the compound of formula (iii) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine-3-benzoyl and the compound of formula (iv) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine.
Example 2
a. Pulverizing 9kg of Lepidium Sativum L, extracting with 60L of 50% ethanol water solution at 80 deg.C under reflux for 3 times, and evaporating the solvent under reduced pressure to obtain crude extract;
b. b, dispersing the crude extract obtained in the step a by using water, sequentially adding n-hexane, dichloromethane and n-butanol, extracting for 4 times, combining n-butanol layers, and evaporating to dryness under reduced pressure to obtain an n-butanol extract;
c. separating the n-butanol extract obtained in step b with normal phase silica gel column, performing gradient elution with petroleum ether-ethyl acetate at volume ratio of 100:1-1:1, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 9 components (Fr.1-Fr.9); separating the component Fr.6 by reverse phase silica gel column chromatography, and performing gradient elution with methanol-water with volume ratio of 10% -100% to obtain component Fr.6.1-Fr.6.10; preparing a reverse phase column (C) from the component Fr.6.3185 μm,10 × 150mm) and isocratic elution with 60% methanol-water solution to give the compound of formula (v) as 8-thio- β -D-glucopyranosyloxy-butyl erucate; fr.8 is separated by reverse phase silica gel column chromatography, and eluted by methanol-water with the volume ratio of 10-100 percent to obtain a component Fr.8.1-Fr.8.10; mixing the components Fr.8.7-Fr.8.9, and preparing reverse phase column (C)185 μm,10 × 150mm) and isocratic elution with 60% strength methanol-water solution to obtain 3 ' - (6-sinapoyl-beta-D-glucopyranosyloxy) -5 ' -sinapoyl-uracil nucleoside as compound of formula (I) and 3 ' - [6- (2-dioxoindole-3-acetyl) -beta-D-glucopyranosyloxy) -as compound of formula (II)]-beta-D-glucopyranosyloxy]-5 ' -sinapoyl-uridine, the compound of formula (iii) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine-3-benzoyl and the compound of formula (iv) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine.
Example 3
a. Pulverizing 9kg of Lepidium Sativum L, percolating with 80L anhydrous ethanol at room temperature for 3 times, and evaporating the solvent under reduced pressure to obtain crude extract of Lepidium Sativum L;
b. b, dispersing the crude extract obtained in the step a with water, sequentially adding cyclohexane, ethyl acetate and n-butanol, extracting for 5 times, combining n-butanol layers, and evaporating under reduced pressure to obtain an n-butanol extract;
c. b, separating the n-butanol extract obtained in the step b by using a normal phase silica gel column, performing gradient elution by using chloroform-methanol with the volume ratio of 100:1-1:1, analyzing fractions by using silica gel Thin Layer Chromatography (TLC), and combining the same fractions to obtain 9 components (Fr.1-Fr.9); separating the component Fr.6 by a small-hole resin column, and performing gradient elution by using methanol-water with the volume ratio of 10-100% to obtain a component Fr.6.1-Fr.6.10; preparing a reverse phase column (C) from the component Fr.6.3185 μm,10 × 150mm) and isocratic elution with 48% acetonitrile-water solution to obtain the compound of formula (v) which is 8-thio- β -D-glucopyranosyloxy-butyl erucate; fr.8 is separated by a reverse phase silica gel column and is eluted by methanol-water with the volume ratio of 10 percent to 100 percent in a gradient way to obtain a component Fr.8.1-Fr.8.10; mixing the components Fr.8.7-Fr.8.9, and preparing a reverse phase column (C)185 μm,10 × 150mm) and isocratic elution with 50% acetonitrile-water solution to obtain 3 ' - (6-sinapoyl-beta-D-glucopyranosyloxy) -5 ' -sinapoyl-uracil nucleoside as compound of formula (I) and 3 ' - [6- (2-dioxoindole-3-acetyl) -beta-D-glucopyranosyloxy) as compound of formula (II)]-beta-D-glucopyranosyloxy]-5 ' -sinapoyl-uridine, the compound of formula (iii) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine-3-benzoyl and the compound of formula (iv) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine.
Example 4
a. Pulverizing 9kg of Lepidium Sativum L, percolating with 63L of pure methanol at room temperature for 3 times, and evaporating to dryness under reduced pressure to obtain crude extract;
b. b, dispersing the crude extract obtained in the step a with water, sequentially adding cyclohexane, ethyl acetate and n-butanol, extracting for 3 times, combining n-butanol layers, and evaporating under reduced pressure to obtain an n-butanol extract;
c. b, separating the n-butanol extract obtained in the step b by using a normal phase silica gel column, performing gradient elution by using cyclohexane-ethyl acetate with the volume ratio of 100:1-1:1, analyzing fractions by using silica gel Thin Layer Chromatography (TLC), and combining the same fractions to obtain 9 components (Fr.1-Fr.9); separating the component Fr.6 by Sephadex LH-20 gel column chromatography, eluting with chloroform-methanol at volume ratio of 1:1 to obtain 8-thio-beta-D-glucopyranosyloxy-butyl sinapinate as compound of formula (V); fr.8 is separated by a reverse phase silica gel column, and is eluted by acetonitrile-water with the volume ratio of 10 percent to 100 percent to obtain a component Fr.8.1-Fr.8.10; the components Fr.8.7-Fr.8.9 are combined and separated by Sephadex LH-20 gel column chromatography and eluted by chloroform-methanol with the volume ratio of 1:1 to obtain the compound of formula (I) which is 3 ' - (6-sinapoyl-beta-D-glucopyranosyloxy) -5 ' -sinapoyl-uracil nucleoside, the compound of formula (II) which is 3 ' - [6- (2-dioxyindole-3-acetyl) -beta-D-glucopyranosyloxy ] -5 ' -sinapoyl-uracil nucleoside, the compound of formula (III) which is 3 ' -beta-D-glucopyranosyloxy-5 ' -sinapoyl-uracil nucleoside-3-benzoyl and the compound of formula (IV) which is 3 ' -beta-D-chitosan-uridine Glucopyranosyloxy-5' -sinapoyl-uridine.
Example 5
a. Pulverizing 9kg of Lepidium Sativum L, extracting with 90L of 90% methanol water solution at 80 deg.C under reflux for 3 times, and evaporating solvent under reduced pressure to obtain crude extract;
b. b, dispersing the crude extract obtained in the step a with water, sequentially adding n-hexane, chloroform and n-butanol, extracting for 4 times, combining n-butanol layers, and evaporating under reduced pressure to obtain an n-butanol extract;
c. separating the n-butanol extract obtained in step b with normal phase silica gel column, performing gradient elution with petroleum ether-acetone at volume ratio of 100:1-1:1, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 9 components (Fr.1-Fr.9); separating the component Fr.6 by Sephadex LH-20 gel column, eluting with dichloromethane-methanol at volume ratio of 1:1 to obtain component Fr.6.1-Fr.6.10; separating the component Fr.6.3 by using a small-hole resin column, and performing gradient elution by using methanol-water with the volume ratio of 10-100% to obtain a compound of a formula (V) which is 8-sulfo-beta-D-glucopyranosyloxy-butyl erucate; fr.8 is separated by Sephadex LH-20 gel column, and is eluted by dichloromethane-methanol with volume ratio of 1:1 to obtain a component Fr.8.1-Fr.8.10;mixing the components Fr.8.7-Fr.8.9, and preparing reverse phase column (C)185 μm,10 × 150mm) and isocratic elution with 40% acetonitrile-water solution to obtain 3 ' - (6-sinapoyl-beta-D-glucopyranosyloxy) -5 ' -sinapoyl-uracil nucleoside as compound of formula (I) and 3 ' - [6- (2-dioxoindole-3-acetyl) -beta-D-glucopyranosyloxy) as compound of formula (II)]-beta-D-glucopyranosyloxy]-5 ' -sinapoyl-uridine, the compound of formula (iii) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine-3-benzoyl and the compound of formula (iv) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine.
Example 6
a. Pulverizing 9kg of Lepidium Sativum L, extracting with 50L of 50% methanol water solution at 80 deg.C under reflux for 3 times, and evaporating solvent under reduced pressure to obtain crude extract;
b. b, dispersing the crude extract obtained in the step a by using water, sequentially adding n-hexane, dichloromethane and n-butanol, extracting for 4 times, combining n-butanol layers, and evaporating under reduced pressure to obtain an n-butanol extract;
c. b, separating the n-butanol extract obtained in the step b by using a normal phase silica gel column, performing gradient elution by using dichloromethane-methanol with the volume ratio of 100:1-1:1, analyzing fractions by using silica gel Thin Layer Chromatography (TLC), and combining the same fractions to obtain 9 components (Fr.1-Fr.9); separating the component Fr.6 by reverse silica gel column chromatography, and performing gradient elution with acetonitrile-water with the volume ratio of 10% -100% to obtain a component Fr.6.1-Fr.6.10; the component Fr.6.3 adopts a preparative reverse phase column (C)185 μm,10 × 150mm) and isocratic elution with 48% methanol-water solution to obtain a compound of formula (v) which is 8-thio- β -D-glucopyranosyloxy-butyl erucate; fr.8 is separated by a small-hole resin column, and is eluted by acetonitrile-water with the volume ratio of 10 percent to 100 percent to obtain a component Fr.8.1-Fr.8.10; mixing the components Fr.8.7-Fr.8.9, and preparing reverse phase column (C)185 μm,10 × 150mm) and isocratic elution with 40% acetonitrile-water solution to obtain 3 ' - (6-sinapoyl-beta-D-glucopyranosyloxy) -5 ' -sinapoyl-uridine as compound of formula (I) and 3 ' - [6- (2-dioxoindole) as compound of formula (II)-3-acetyl) -beta-D-glucopyranosyloxy]-beta-D-glucopyranosyloxy]-5 ' -sinapoyl-uridine, the compound of formula (iii) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine-3-benzoyl and the compound of formula (iv) is 3 ' - β -D-glucopyranosyloxy-5 ' -sinapoyl-uridine.
Example 7
The application of sinapic acid compounds separated from the spatholobus suberectus seeds in preparing anti-inflammatory drugs is exemplified by macrophage strain (RAW264.7 cells);
compounds of formula (i) -formula (v) anti-inflammatory activity assay:
experimental materials:
1.1 sample preparation: the concentration of the compound of formula (I) -formula (V) is 50. mu.M;
1.2 Experimental consumables: materials and reagents: a15 ml centrifuge tube was purchased from Thermo corporation (manufacturing lot: 339605); the 24-well plate was purchased from CORNING corporation (production lot: 3524); the NO detection kit is purchased from Biyuntian (production batch: S0023);
cells for experiments: RAW264.7 cells, adherent cells, HG-DMEM (10% serum + 1% P/S +1mM N-pyruvate supplemented);
2. the experimental steps are as follows:
2.1 culture of RAW264.7 cells in HG-DMEM at 1:5 passages and 1X 10 counts5cells/mL inoculated 24-well plate;
2.2 after 6 hours of adherence, adding the compound to pretreat the cells for 1 hour, then adding LPS (1mg/mL) to stimulate for 18 hours, and collecting 60 microliters of supernatant;
2.3 the following reagents are added in sequence according to the requirements of the Biyuntian Griess kit in the following reagent table 6:
Figure BDA0002102591940000101
Figure BDA0002102591940000111
2.4 preparing a standard curve by using the absorbance value and calculating the inhibition value of the compound on NO release;
3. the experimental results are as follows:
TABLE 7 anti-inflammatory Activity of Compounds of formula (I) -formula (V)
Compound numbering Nitric oxide inhibition rate
Formula (I) 25.0％
Formula (II) 63.2％
Formula (III) 33.6％
Formula (IV) 30.5％
Formula (V) 49.1％
Positive control (Andrographis paniculata injection) 96.6％
4. And (4) conclusion:
4.1 As can be seen from Table 7: the five compounds have certain anti-inflammatory activity on macrophages;
4.2 the anti-inflammatory activity of the formula (II) and the formula (V) is more obvious, and the NO release amount of mouse macrophage can be effectively inhibited.

Claims (3)

1. A sinapic acid compound, characterized in that the compound has the structural formula:
Figure FDA0003625882770000011
wherein:
the compound of formula (I) is 3 '- (6-sinapoyl-beta-D-glucopyranosyloxy) -5' -sinapoyl-uridine;
the compound of formula (II) is 3 '- [6- (2-dioxyindole-3-acetyl) -beta-D-glucopyranosyloxy ] -5' -sinapoyl-uridine;
the compound of formula (III) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine-3-benzoyl;
the compound of formula (IV) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine;
the compound of formula (V) is; 8-thio-beta-D-glucopyranosyloxy-sinapic acid butyl ester.
2. The method for preparing sinapic acid compounds according to claim 1, comprising the following steps:
a. taking seeds of the Lepidium Sativum L.as a raw material, crushing, percolating, cold-leaching or heating reflux extraction with 5-10 times of 50-99% ethanol aqueous solution, absolute ethanol, pure acetone, 50-99% methanol aqueous solution or absolute methanol at room temperature, and concentrating to obtain crude extract of Lepidium Sativum L.seeds;
b. dispersing the crude extract obtained in the step a by using water, sequentially adding petroleum ether, n-hexane or cyclohexane, ethyl acetate and n-butanol, extracting for 3-5 times, and concentrating the n-butanol extract to obtain an n-butanol extract;
c. separating the n-butanol extract obtained in step b by two or three of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, small pore resin chromatography, Sephadex LH-20 gel column chromatography and preparative high performance liquid chromatography to obtain 3 '- (6-sinapoyl-beta-D-glucopyranosyloxy) -5' -sinapoyl-uridine as the compound of formula (I); the compound of formula (II) is 3 '- [6- (2-dioxoindole-3-acetyl) -beta-D-glucopyranosyloxy ] -5' -sinapoyl-uridine; the compound of formula (III) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine-3-benzoyl; the compound of formula (IV) is 3 '-beta-D-glucopyranosyloxy-5' -sinapoyl-uridine; the compound of the formula (V) is 8-sulfo-beta-D-glucopyranosyloxy-butyl sinapinate, wherein the normal phase silica gel column chromatography is normal pressure or pressurized column chromatography, the used filler is normal phase silica gel, the used eluant is petroleum ether, cyclohexane or normal hexane, acetone, trichloromethane, dichloromethane or ethyl acetate and the mixture of at least two solvents in methanol, and isocratic elution or gradient elution is adopted; the reverse phase silica gel column chromatography is normal pressure or pressurized column chromatography, the eluant is methanol water solution or acetonitrile water solution with volume concentration of 10-100%, and isocratic elution or gradient elution is adopted; the small-pore resin chromatography is normal pressure or pressurized column chromatography, the eluant is methanol water solution or acetonitrile water solution with volume concentration of 10-100%, and isocratic elution or gradient elution is adopted; the Sephadex LH-20 gel column chromatography is normal pressure column chromatography, and the eluant is methanol, dichloromethane, chloroform or a mixture of at least two solvents, and isocratic elution or gradient elution is adopted; the high performance liquid chromatography is pressure column chromatography, and the eluent is 40-99% methanol water solution or 30-99% acetonitrile water solution, and isocratic elution or gradient elution.
3. Use of sinapic compounds according to claim 1 for the preparation of anti-inflammatory medicaments.
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