CN110156859A - Mustard seed acid compounds and its preparation method and application - Google Patents

Mustard seed acid compounds and its preparation method and application Download PDF

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CN110156859A
CN110156859A CN201910541141.7A CN201910541141A CN110156859A CN 110156859 A CN110156859 A CN 110156859A CN 201910541141 A CN201910541141 A CN 201910541141A CN 110156859 A CN110156859 A CN 110156859A
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mustard
formula
compound
acid compounds
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CN110156859B (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]
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • 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

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Abstract

The present invention relates to a kind of mustard seed acid compounds and its preparation method and application, the mustard seed acid compounds be from cultivated pepperweed seed (Lepidium sativumLIt is tested and analyzed in seed) using thin layer chromatography and analytic type high performance liquid chromatography, it is extracted with organic solvent, then it is separated by four kinds to five kinds methods in solvent extraction, normal-phase silica gel column chromatography method, reversed-phase silica gel column chromatography, aperture resin column chromatography, Sephadex LH-20 gel filtration chromatography method and semi-preparative high performance liquid chromatography, obtain five new mustard seed acids monomeric compounds, it determines that five monomeric compounds are new mustard seed acid compounds by the methods of high resolution mass spectrum and NMR spectrum, and Structural Identification has been carried out to it.In addition anti-inflammatory activity measurement has been carried out to these mustard seed acid compounds, the results showed that described five new mustard seed acid compounds have different degrees of anti-inflammatory activity, can be used for preparing the purposes in anti-inflammatory drug.

Description

Mustard seed acid compounds and its preparation method and application
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to five mustard seed acid compounds and its preparation method and application.
Background technique
Cruciferae (Brassicaceae) has 330 categories and 3500 kinds of plants, is most one of the section of floristics, main Produce vegetables and oil crops.Cultivated pepperweed seed (Lepidium sativum) belongs to Cruciferae separate row Vegetable spp (Lepidium), Xinjiang of China and Tibet region are edible frequently as edible plant, its dry mature seed is referred to as cultivated pepperweed seed, in Xinjiang Area is widely used in treating gastrointestinal disease in traditional national medicine, soft intestines defaecation, drive away helminth, for treat inhibited defecation, Enteron aisle is infested etc..According to reports, cultivated pepperweed seed has anti-oxidant, antibacterial, anti-inflammatory, antihyperglycemic and a variety of biologies such as antitumor Activity.Forefathers from the chemical component that cultivated pepperweed seed is found include alkaloid, flavones, terpene, Phenylpropanoid Glycosides, sterol and mustard seed acids Compound etc..
Sinapic acid is also known as sinapsis alba acid, is naturally present in some crucifers such as Brassica plants sinapsis alba [Brassica Alba L.) Boiss] and the seed of SEMEN SINAPIS ALBAE [Brassica juncea (L.) coss.] in, be found to have removing in recent years Free radical, anti-lipid peroxidation, antibacterial, anticancer and anti-inflammatory, improve the bioactivity such as memory at antianxiety, with sinapic acid And its bioactivity of derivative is constantly discovered, and the multiple fields such as pharmaceuticals industry, food, beverage, cosmetics have been applied to. Five new compounds in this research are the mustard seed acid compounds of cultivated pepperweed seed discovery, and structure feature is to contain There is sinapic acid structure fragment, formula (I)-formula (IV) is the uridine diphosphate glycoside natural derivative of rare sinapic acid, and formula (V) is The mustard seed acid esters few in number containing thioglucose oxygroup of nature discovery at present.This research is to cultivated pepperweed seed chemistry The research of ingredient can provide reference for the secondary metabolite in clear cultivated pepperweed seed, can also be to disclose its drug action Material base provides scientific basis.
Macrophage belongs to mononuclear phagocyte system, including migrans macrophage and stationarity macrophage, extensively It is distributed in the various tissues and organ of body.When macrophage is activated, release tumor necrosis factor TNF, interleukin I L- 1 equal many kinds of substance, participate in inflammatory reaction and immune response.Macrophage is by lipopolysaccharides (LPS) and interferon-γ When (interferon- γ) is activated, under the action of NO synzyme, NO is synthesized using L-arginine.The NO of macrophage is synthesized It the various aspects such as leads in the killing of microorganism and tumour cell to be of great significance.
Medicinal plant is the huge resource treasure-house of innovation drug research, and therefrom looking for lead compound is researches on natural drugs Hot spot.This research is intended to carry out further Research on Mining to the mustard seed acid compounds of cultivated pepperweed seed, to find to tie Structure novelty mustard seed acid compounds to enrich the structure diversity of natural products, and provide deposit for high-flux medicaments sifting Compound.In addition, providing ginseng by the influence to NO content in such compounds towards macrophages for the exploitation of anti-inflammatory drug It examines.
Summary of the invention
Present invention aims at provide a kind of mustard seed acid compounds and its preparation method and application, the mustard seed acids Compound is from the herb of cultivated pepperweed seed (Lepidium sativum L.) using thin layer chromatography and analytic type efficient liquid phase Chromatography tests and analyzes, and is extracted with organic solvent, then passes through solvent extraction, normal-phase silica gel column chromatography method, reverse phase silica gel column Four in chromatography, aperture resin column chromatography, Sephadex LH-20 gel filtration chromatography method and semi-preparative high performance liquid chromatography Kind is separated to five kinds of methods, is obtained five new mustard seed acids monomeric compounds, is passed through high resolution mass spectrum and nuclear magnetic resonance The methods of wave spectrum determines that five monomeric compounds are new mustard seed acid compounds, and has carried out Structural Identification to it, and to five A mustard seed acid compounds have carried out anti-inflammatory activity measurement, the results showed that described five new mustard seed acid compounds have not With the anti-inflammatory activity of degree, can be used for preparing anti-inflammatory drug.
A kind of mustard seed acid compounds of the present invention, it is characterised in that the structural formula of the compound are as follows:
Wherein:
Formula (I) compound is 3 '-(6- mustard acyl-β-D- glucopyra glycosyloxy) -5 '-mustard acyl-uridine diphosphates Glycosides；
Formula (II) compound is 3 '-[6- (2- dioxindole -3- acetyl group)-β-D- glucopyra glycosyloxy]-β-D- pyrroles Glucopyranoside oxygroup] -5 '-mustard acyl-uridine；
Formula (III) compound is 3 ' -5 '-mustard acyls of-β-D- glucopyra glycosyloxy-uridine -3- benzoyl Base；
Formula (IV) compound is 3 ' -5 '-mustard acyl-uridines of-β-D- glucopyra glycosyloxy；
Formula (V) compound is；Thio-β-D- glucopyra glycosyloxy-mustard seed the acid butyl ester of 8-.
A kind of preparation method of mustard seed acid compounds, follows these steps to carry out:
A, the seed for taking cultivated pepperweed seed is raw material, and it is water-soluble with 5-10 times to measure the ethyl alcohol that volumetric concentration is 50-99% after crushing Liquid, dehydrated alcohol, pure acetone, the methanol aqueous solution that volumetric concentration is 50-99% or anhydrous methanol carry out diacolation at room temperature, cold Extraction takes or heating and refluxing extraction, is concentrated to get the crude extract of cultivated pepperweed seed；
B, the crude extract obtained step a is water-dispersible, sequentially adds petroleum ether, n-hexane or hexamethylene, ethyl acetate, Butanol extraction liquid is concentrated extracting n-butyl alcohol 3-5 times, obtains n-butyl alcohol extract medicinal extract；
C, the n-butyl alcohol extract medicinal extract for obtaining step b is through normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, aperture tree Rouge chromatographs, is separated for two or three in Sephadex LH-20 gel filtration chromatography and preparative high performance liquid chromatography, i.e., Obtaining formula (I) compound is 3 '-(6- mustard acyl-β-D- glucopyra glycosyloxy) -5 '-mustard acyl-uridines；Formula (II) compound is 3 '-[6- (2- dioxindole -3- acetyl group)-β-D- glucopyra glycosyloxy]-β-D- glucopyra glycosyloxies Base] -5 '-mustard acyl-uridine；Formula (III) compound is 3 ' -5 '-mustard acyl-urine of-β-D- glucopyra glycosyloxy Pyrimidine nucleoside -3- benzoyl；Formula (IV) compound is 3 ' -5 '-mustard acyl-uridine diphosphates of-β-D- glucopyra glycosyloxy Glycosides；Formula (V) compound is the thio-β-D- glucopyra glycosyloxy-mustard seed acid butyl ester of 8-.
Normal-phase silica gel column chromatography method used is normal pressure or pressurized column chromatography in step c, and filler used is purification on normal-phase silica gel, used Eluant, eluent is petroleum ether, hexamethylene or n-hexane, acetone, chloroform, methylene chloride or ethyl acetate, at least two in methanol The mixture of solvent, using isocratic elution or gradient elution.
Reversed-phase silica gel column chromatography method used is normal pressure or pressurized column chromatography in step c, and eluant, eluent is volumetric concentration 10- 100% methanol aqueous solution or acetonitrile solution, using isocratic elution or gradient elution.
Aperture resin chromatography method used is normal pressure or pressurized column chromatography in step c, and eluant, eluent is volumetric concentration 10-100% Methanol aqueous solution or acetonitrile solution, using isocratic elution or gradient elution.
Sephadex LH-20 gel filtration chromatography method used is normal pressure column chromatography in step c, and eluant, eluent is methanol, dichloromethane The mixture of alkane, chloroform or at least two solvents, using isocratic elution or gradient elution.
Preparative high performance liquid chromatography used is pressurized column chromatography in step c, and eluant, eluent is the first of volumetric concentration 40-99% The acetonitrile solution of alcohol solution or 30-99%, using isocratic elution or gradient elution.
The mustard seed acid compounds purposes in preparing anti-inflammatory drugs.
A kind of mustard seed acid compounds of the present invention, are obtained by isolating and purifying from plant, can also be through ability The synthesis of chemical modification method known to field technique personnel obtains.
A kind of mustard seed acid compounds of the present invention, using high resolution mass spectrum, a peacekeeping ID NMR speetna etc. Modern spectroscopy means determine that its structure, Structural Identification process are as follows:
Formula (I) compound is 3 '-(6- mustard acyl-β-D- glucopyra glycosyloxy) -5 '-mustard acyl-uridine diphosphates Glycosides, Yellow amorphous powder,(c 0.57,MeOH)；UV(MeOH)195,241,328nm；Pass through its high score Distinguish quasi-molecular ion peak in mass spectrum [M-H]-M/z 817.2305 (calculated value 817.2309) determines that its molecular formula is C37H42O19N2.According to1H,13C NMR and two dimensional NMR data determine that its structural framework type is two sinapic acid uracils Nucleosides is named as 3 '-(6- mustard acyl-β-D- glucopyra glycosyloxy) -5 '-mustard acyl-uridines,1H and13C NMR ownership be shown in Table 1 [400MHz (1H), 100MHz (13C), DMSO-d6]；
1. formula of table (I) compound1H and13C NMR data [δ (ppm), J (Hz)]
A in table: nuclear magnetic signal overlapping is composed for hydrogen, is belonged to by two-dimentional nuclear magnetic data；
Formula (II) compound is 3 '-[6- (2- dioxindole -3- acetyl group)-β-D- glucopyra glycosyloxy]-β-D- pyrroles Glucopyranoside oxygroup] -5 '-mustard acyl-uridine, Yellow amorphous powder,UV(MeOH)195,241,328nm；Pass through quasi-molecular ion in its high resolution mass spectrum Peak [M-H]-M/z 784.2204 (calculated value 784.2206) determines that its molecular formula is C36H39O17N3；According to1H,13C NMR with And two dimensional NMR data determine that its structural framework type is dioxindole -3- acetyl group sinapic acid uridine, name For 3 '-[6- (2- dioxindole -3- acetyl group)-β-D- glucopyra glycosyloxy]-β-D- glucopyra glycosyloxies] -5 '-mustard seed Acyl group-uridine,1H and13C NMR ownership be shown in Table 2 [400MHz (1H), 100MHz (13C), DMSO-d6]；
2. formula of table (II) compound1H and13C NMR data [δ (ppm), J (Hz)]
A in table: nuclear magnetic signal overlapping is composed for hydrogen, is belonged to by two-dimentional nuclear magnetic data；
Formula (III) compound is 3 ' -5 '-mustard acyls of-β-D- glucopyra glycosyloxy-uridine -3- benzoyl Base, Yellow amorphous powder,UV(MeOH)195,241,328nm；Pass through its height Quasi-molecular ion peak [M-H] in Resolution Mass Spectrometry-M/z 715.1995 (calculated value 715.1992) determines that its molecular formula is C33H36O16N2.According to1H,13C NMR and two dimensional NMR data determine that its structural framework type is benzoxy sinapic acid Uridine is named as 3 ' -5 '-mustard acyls of-β-D- glucopyra glycosyloxy-uridine -3- benzoyl,1H With13C NMR ownership be shown in Table 3 [600MHz (1H), 150MHz (13C), DMSO-d6]；
3. formula of table (III) compound1H and13C NMR data [δ (ppm), J (Hz)]
A in table: nuclear magnetic signal overlapping is composed for hydrogen, is belonged to by two-dimentional nuclear magnetic data；
Formula (IV) compound is 3 ' -5 '-mustard acyl-uridines of-β-D- glucopyra glycosyloxy, Yellow amorphous Powder,UV(MeOH)195,241,328nm；By quasi- point in its high resolution mass spectrum Daughter ion peak [M-H]-M/z 611.1731 (calculated value 611.1729) determines that its molecular formula is C26H32O15N2.According to1H,13C NMR and two dimensional NMR data determine that its structural framework type is sinapic acid uridine, are named as 3 '-β-D- pyrans Glucosyloxy -5 '-mustard acyl-uridine,1H and13C NMR ownership be shown in Table 4 [400MHz (1H), 100MHz (13C), DMSO-d6]；
4. formula of table (IV) compound1H and13C NMR data [δ (ppm), J (Hz)]
A in table: nuclear magnetic signal overlapping is composed for hydrogen, is belonged to by two-dimentional nuclear magnetic data；
Formula (V) compound be the thio-β-D- glucopyra glycosyloxy-mustard seed acid butyl ester of 8-, yellow oil, UV(MeOH)195,241,328nm；Pass through quasi-molecular ion in its high resolution mass spectrum Peak [M-H]-M/z 473.1489 (calculated value 473.1486) determines that its molecular formula is C21H30O10S.According to1H,13C NMR with And two dimensional NMR data determine that its structural framework type is mustard seed acid esters glucosinolate, are named as the thio-β-D- of 8- Glucopyra glycosyloxy-mustard seed acid butyl ester,1H and13C NMR ownership be shown in Table 5 [400MHz (1H), 100MHz (13C), DMSO- d6]；
5. formula of table (V) compound1H and13C NMR data [δ (ppm), J (Hz)]
A in table: nuclear magnetic signal overlapping is composed for hydrogen, is belonged to by two-dimentional nuclear magnetic data；
Detailed description of the invention
Fig. 1 is formula (I) compound1H NMR(400MHz,DMSO-d6) spectrogram；
Fig. 2 is formula (I) compound13C NMR(100MHz,DMSO-d6) spectrogram；
Fig. 3 is formula (II) compound1H NMR(400MHz,DMSO-d6) spectrogram；
Fig. 4 is formula (II) compound13C NMR(100MHz,DMSO-d6) spectrogram；
Fig. 5 is formula (III) compound1H NMR(600MHz,DMSO-d6) spectrogram；
Fig. 6 is formula (III) compound13C NMR(150MHz,DMSO-d6) spectrogram；
Fig. 7 is formula (IV) compound1H NMR(400MHz,DMSO-d6) spectrogram；
Fig. 8 is formula (IV) compound13C NMR(100MHz,DMSO-d6) spectrogram；
Fig. 9 is formula (V) compound1H NMR(400MHz,DMSO-d6) spectrogram；
Figure 10 is formula (V) compound13C NMR(100MHz,DMSO-d6) spectrogram.
Specific embodiment
Agents useful for same is that analysis is pure, and acetonitrile is that (Merck KGaA is not public for high-efficiency liquid chromatographic-grade in high performance liquid chromatography Department).Column chromatographs purification on normal-phase silica gel (100-200 mesh, 200-300 mesh): Haiyang Chemical Plant, Qingdao's production；Tlc silica gel is HSGF254: Yantai City's Huang business silica gel development experiments factory production；Sephadex LH-20 gel: General Electric's Medical Group Production；Aperture resin aperture resin gel MCI (CHP 20/120): Mitsubishi Co., Ltd. production；Reverse phase silica gel ODS: moral The production of Merck & Co., Inc., state；High performance liquid chromatography (Dai An company, the U.S.) configuration is as follows: P680HPLC pump, ASI-100 automatic sampling Device, TCC-100 column oven, UVD170U UV detector (four wavelength), quaternary solvent system, on-line degassing machine, chameleon chromatography Work station；Preparative high-performance liquid chromatographic (Dai An company, the U.S.) configuration is as follows: P680HPLC pump, UVD170U UV detector (four Wavelength), quaternary solvent system, on-line degassing machine, chameleon chromatographic work station；Mass spectrum hybridizes mass spectrum with quadrupole rod-flight time Instrument (Applied biosystems) measurement；The nuclear magnetic resonance 600/400 type Nuclear Magnetic Resonance (U.S. watt Varian Vnmrs Li An company) measurement.
The purchase of cultivated pepperweed seed is studied from Xinjiang Uygur Autonomous Regions Ili Prefecture by Chinese Academy of Sciences's Xinjiang ecogeography Institute specimen museum Feng Ying assistant researcher is accredited as the Lepidium sativum L. time buying as in November, 2014.
Embodiment 1
A, the sub- 9kg of cultivated pepperweed seed is taken, cold soaking extracts 3 to the ethanol-water solution for being 95% with 45L concentration after crushing at room temperature Secondary, evaporated under reduced pressure solvent obtains the sub- crude extract of cultivated pepperweed seed；
B, the crude extract obtained step a is water-dispersible, and petroleum ether, ethyl acetate is added, and n-butanol successively carries out extraction 3 Secondary, evaporated under reduced pressure obtains n-butanol layer extract medicinal extract after merging n-butanol layer；
C, the n-butanol layer extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 100:1-1:1's Chloroform-methanol carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography (TLC), merges identical flow point, obtain 9 components (Fr.1-Fr.9)；By component Fr.6 through reversed phase silica gel post separation, with the methanol-water gradient gradient of volume ratio 10%-100% Elution, obtains component Fr.6.1-Fr.6.10；Component Fr.6.3 is used and prepares reversed-phase column (C18, 5 μm, 10 × 150mm) and separation, The acetonitrile-aqueous solution isocratic elution for being 32% with concentration, obtaining formula (V) compound is the thio-β-D- glucopyra glycosyloxy of 8- Base-mustard seed acid butyl ester；Fr.8 is eluted with the methanol-water of volume ratio 1:9-100:0 through reversed phase silica gel post separation, obtains component Fr.8.1-Fr.8.10；Component Fr.8.7-Fr.8.9 is merged, using preparing reversed-phase column (C18, 5 μm, 10 × 150mm) and separation, The acetonitrile-aqueous solution isocratic elution for being 30% with concentration, obtaining formula (I) compound is 3 '-(6- mustard acyl-β-D- pyrans Portugals Grape glycosyloxy) -5 '-mustard acyl-uridine, formula (II) compound be 3 '-[6- (2- dioxindole -3- acetyl group)-β - D- glucopyra glycosyloxy]-β-D- glucopyra glycosyloxy] -5 '-mustard acyl-uridine, formula (III) compound be 3 ' -5 '-mustard acyl-uridine -3- benzoyls of-β-D- glucopyra glycosyloxy and formula (IV) compound are 3 '-β - D- glucopyra glycosyloxy -5 '-mustard acyl-uridine.
Embodiment 2
A, the sub- 9kg of cultivated pepperweed seed is taken, with refluxing extraction at 80 DEG C of ethanol water temperature that 60L concentration is 50% after crushing 3 times, evaporated under reduced pressure solvent obtains the sub- crude extract of cultivated pepperweed seed；
B, the crude extract obtained step a is water-dispersible, sequentially adds n-hexane, methylene chloride, and n-butanol carries out extraction 4 It is secondary, merge n-butanol layer and evaporated under reduced pressure obtains n-butyl alcohol extract medicinal extract；
C, the n-butyl alcohol extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with the stone of volume ratio 100:1-1:1 Oily ether-ethyl acetate carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography (TLC), merges identical flow point, obtain 9 components (Fr.1-Fr.9)；The inverted silica gel column layer of component Fr.6 is isolated, with the methanol-water gradient elution of volume ratio 10%-100%, Obtain component Fr.6.1-Fr.6.10；Component Fr.6.3 is used and prepares reversed-phase column (C18, 5 μm, 10 × 150mm) and separation, with dense The methanol-water solution isocratic elution that degree is 60%, obtaining formula (V) compound is the thio-β-D- glucopyra glycosyloxy-mustard of 8- Sub- acid butyl ester；The inverted silica gel column chromatography separation of Fr.8, is eluted with the methanol-water of volume ratio 10%-100%, obtains component Fr.8.1-Fr.8.10；Component Fr.8.7-Fr.8.9 is merged, using preparing reversed-phase column (C18, 5 μm, 10 × 150mm) and separation, The methanol-water solution isocratic elution for being 60% with concentration, obtaining formula (I) compound is 3 '-(6- mustard acyl-β-D- pyrans Portugals Grape glycosyloxy) -5 '-mustard acyl-uridine, formula (II) compound be 3 '-[6- (2- dioxindole -3- acetyl group)-β - D- glucopyra glycosyloxy]-β-D- glucopyra glycosyloxy] -5 '-mustard acyl-uridine, formula (III) compound be 3 ' -5 '-mustard acyl-uridine -3- benzoyls of-β-D- glucopyra glycosyloxy and formula (IV) compound are 3 '-β - D- glucopyra glycosyloxy -5 '-mustard acyl-uridine.
Embodiment 3
A, the sub- 9kg of cultivated pepperweed seed is taken, dehydrated alcohol seepage pressure effects 3 times at room temperature of 80L, evaporated under reduced pressure solvent are used after crushing Obtain the sub- crude extract of cultivated pepperweed seed；
B, the crude extract obtained step a is water-dispersible, sequentially adds hexamethylene, ethyl acetate, and n-butanol carries out extraction 5 It is secondary, merge n-butanol layer and evaporated under reduced pressure obtains n-butyl alcohol extract medicinal extract；
C, the n-butyl alcohol extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with the chlorine of volume ratio 100:1-1:1 Imitation-carbinol carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography (TLC), merges identical flow point, obtain 9 components (Fr.1-Fr.9)；Component Fr.6 is obtained through aperture resin post separation with the methanol-water gradient elution of volume ratio 10%-100% To component Fr.6.1-Fr.6.10；Component Fr.6.3 is used and prepares reversed-phase column (C18, 5 μm, 10 × 150mm) and separation, with concentration For 48% acetonitrile-aqueous solution isocratic elution, obtaining formula (V) compound is the thio-β-D- glucopyra glycosyloxy-mustard seed of 8- Acid butyl ester；Fr.8 obtains component with the methanol-water gradient elution of volume ratio 10%-100% through reversed phase silica gel post separation Fr.8.1-Fr.8.10；Component Fr.8.7-Fr.8.9 is merged, using preparing reversed-phase column (C18, 5 μm, 10 × 150mm) and separation, The acetonitrile-aqueous solution isocratic elution for being 50% with concentration, obtaining formula (I) compound is 3 '-(6- mustard acyl-β-D- pyrans Portugals Grape glycosyloxy) -5 '-mustard acyl-uridine, formula (II) compound be 3 '-[6- (2- dioxindole -3- acetyl group)-β - D- glucopyra glycosyloxy]-β-D- glucopyra glycosyloxy] -5 '-mustard acyl-uridine, formula (III) compound be 3 ' -5 '-mustard acyl-uridine -3- benzoyls of-β-D- glucopyra glycosyloxy and formula (IV) compound are 3 '-β - D- glucopyra glycosyloxy -5 '-mustard acyl-uridine.
Embodiment 4
A, the sub- 9kg of cultivated pepperweed seed is taken, seepage pressure effects 3 times at room temperature of the pure methanol of 63L are used after crushing, evaporated under reduced pressure solvent man is only Row vegetable seeds crude extract；
B, the crude extract obtained step a is water-dispersible, sequentially adds hexamethylene, ethyl acetate, and extracting n-butyl alcohol 3 times, Merge n-butanol layer and evaporated under reduced pressure obtains n-butyl alcohol extract medicinal extract；
C, the n-butyl alcohol extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with the ring of volume ratio 100:1-1:1 Ethane-ethyl acetate carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography (TLC), merges identical flow point, obtain 9 components (Fr.1-Fr.9)；Component Fr.6 is separated through Sephadex LH-20 gel filtration chromatography, the chloroform-for being 1:1 with volume ratio Methanol elution, obtaining formula (V) compound is the thio-β-D- glucopyra glycosyloxy-mustard seed acid butyl ester of 8-；Fr.8 is through reversed phase Silica gel post separation is eluted with the acetonitrile-water of volume ratio 10%-100%, obtains component Fr.8.1-Fr.8.10；By component Fr.8.7-Fr.8.9 merges, and is separated using Sephadex LH-20 gel filtration chromatography, and volume ratio is chloroform-methanol of 1:1 Elution, obtaining formula (I) compound is 3 '-(6- mustard acyl-β-D- glucopyra glycosyloxy) -5 '-mustard acyl-uridine diphosphates Glycosides, formula (II) compound are 3 '-[6- (2- dioxindole -3- acetyl group)-β-D- glucopyra glycosyloxy]-β-D- glucopyras Glycosyloxy] -5 '-mustard acyl-uridine, formula (III) compound be 3 ' -5 '-mustard seed acyls of-β-D- glucopyra glycosyloxy Base-uridine -3- benzoyl and formula (IV) compound are 3 ' -5 '-mustard acyl-urine of-β-D- glucopyra glycosyloxy Pyrimidine nucleoside.
Embodiment 5
A, the sub- 9kg of cultivated pepperweed seed is taken, is mentioned after crushing with flowing back at 80 DEG C of methanol aqueous solution temperature that the concentration of 90L is 90% It takes 3 times, evaporated under reduced pressure solvent obtains the sub- crude extract of cultivated pepperweed seed；
B, the crude extract obtained step a is water-dispersible, sequentially adds n-hexane, and chloroform extracting n-butyl alcohol 4 times, merges Simultaneously evaporated under reduced pressure obtains n-butyl alcohol extract medicinal extract to n-butanol layer；
C, the n-butyl alcohol extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with the stone of volume ratio 100:1-1:1 Oily ether-acetone carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography (TLC), merges identical flow point, obtain 9 components (Fr.1-Fr.9)；By component Fr.6 through Sephadex LH-20 gel post separation, with volume by volume than the dichloromethane for 1:1 Alkane-methanol elution, obtains component Fr.6.1-Fr.6.10；Component Fr.6.3 is used into aperture resin post separation, uses volume ratio The methanol-water gradient elution of 10%-100%, obtaining formula (V) compound is the thio-β-D- glucopyra glycosyloxy-mustard seed of 8- Acid butyl ester；Fr.8 is through Sephadex LH-20 gel post separation, with volume by volume than being eluted for the methylene chloride-methanol of 1:1, Obtain component Fr.8.1-Fr.8.10；Component Fr.8.7-Fr.8.9 is merged, using preparing reversed-phase column (C18,5μm,10× It 150mm) separates, the acetonitrile-aqueous solution isocratic elution for being 40% with concentration, obtaining formula (I) compound is 3 '-(6- mustard acyls- β-D- glucopyra glycosyloxy) -5 '-mustard acyl-uridine, formula (II) compound be 3 '-[6- (2- dioxindole -3- Acetyl group)-β-D- glucopyra glycosyloxy]-β-D- glucopyra glycosyloxy] -5 '-mustard acyl-uridine, formula (III) Compound is 3 ' -5 '-mustard acyl-uridine -3- benzoyls of-β-D- glucopyra glycosyloxy and formula (IV) compound For 3 ' -5 '-mustard acyl-uridines of-β-D- glucopyra glycosyloxy.
Embodiment 6
A, the sub- 9kg of cultivated pepperweed seed is taken, is mentioned after crushing with flowing back at 80 DEG C of methanol aqueous solution temperature that the concentration of 50L is 50% It takes 3 times, evaporated under reduced pressure solvent obtains the sub- crude extract of cultivated pepperweed seed；
B, the crude extract obtained step a is water-dispersible, sequentially adds n-hexane, methylene chloride, and extracting n-butyl alcohol 4 times, Merge n-butanol layer and evaporated under reduced pressure obtains n-butyl alcohol extract medicinal extract；
C, the n-butyl alcohol extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with the two of volume ratio 100:1-1:1 Chloromethanes-methanol carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography (TLC), merges identical flow point, obtain 9 components (Fr.1-Fr.9)；Component Fr.6 is separated through reversed silica gel column chromatography, is washed with the acetonitrile-water gradient of volume ratio 10%-100% It is de-, obtain component Fr.6.1-Fr.6.10；Component Fr.6.3 is used and prepares reversed-phase column (C18, 5 μm, 10 × 150mm) and separation, with The methanol-water solution isocratic elution that concentration is 48%, obtaining formula (V) compound is the thio-β-D- glucopyra glycosyloxy-of 8- Mustard seed acid butyl ester；Fr.8 is eluted with the acetonitrile-water of volume ratio 10%-100% through aperture resin post separation, obtains component Fr.8.1-Fr.8.10；Component Fr.8.7-Fr.8.9 is merged, using preparing reversed-phase column (C18, 5 μm, 10 × 150mm) and separation, The acetonitrile-aqueous solution isocratic elution for being 40% with concentration, obtaining formula (I) compound is 3 '-(6- mustard acyl-β-D- pyrans Portugals Grape glycosyloxy) -5 '-mustard acyl-uridine, formula (II) compound be 3 '-[6- (2- dioxindole -3- acetyl group)-β - D- glucopyra glycosyloxy]-β-D- glucopyra glycosyloxy] -5 '-mustard acyl-uridine, formula (III) compound be 3 ' -5 '-mustard acyl-uridine -3- benzoyls of-β-D- glucopyra glycosyloxy and formula (IV) compound are 3 '-β - D- glucopyra glycosyloxy -5 '-mustard acyl-uridine.
Embodiment 7
The mustard seed acid compounds purposes in preparing anti-inflammatory drugs of the present invention separated from cultivated pepperweed seed, By taking macrophage strain (RAW264.7 cell) as an example；
Formula (I)-formula (V) compound anti-inflammatory activity test:
Experimental material:
1.1 preparation of samples: formula (I)-formula (V) compound concentration is 50 μM；
1.2 experiment consumptive materials: material and reagent: 15 milliliters of centrifuge tubes are purchased from Thermo company (product batch number: 339605)；24 Orifice plate is purchased from CORNING company (product batch number: 3524)；NO detection kit purchased from green skies company (product batch number: S0023)；
Experiment cell: RAW264.7 cell, cell type are attached cell, and culture medium is HG-DMEM (adding ingredient 10% serum+1%P/S+1mM N-pyruvate)；
2. experimental procedure:
2.1 culture RAW264.7 cells, culture medium HG-DMEM, 1:5 are passed on and are counted 1 × 105Cells/mL inoculation 24 Orifice plate；
2.2 after adherent 6 hours, are added compound pretreatment cell 1 hour, and LPS (1mg/mL) is added afterwards to be stimulated 18 hours After collect 60 microlitres of supernatant；
2.3 sequentially add following reagent table 6 according to the requirement in green skies Griess kit:
2.4 make standard curve with absorbance value and calculate the inhibiting value that compound discharges NO；
3. experimental result:
7. formula of table (I)-formula (V) compound anti-inflammatory activity test
Compound number Nitric oxide inhibiting 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. conclusion:
4.1 as can be seen from Table 7: five compounds towards macrophages have certain anti-inflammatory activity；
The anti-inflammatory activity of 4.2 formulas (II) and formula (V) is more significant, can effectively inhibit the NO burst size of mouse macrophage.

Claims (8)

1. a kind of mustard seed acid compounds, it is characterised in that the structural formula of the compound are as follows:
Wherein:
Formula (I) compound is 3 '-(6- mustard acyl-β-D- glucopyra glycosyloxy) -5 '-mustard acyl-uridines；
Formula (II) compound is 3 '-[6- (2- dioxindole -3- acetyl group)-β-D- glucopyra glycosyloxy]-β-D- pyrans Portugals Grape glycosyloxy] -5 '-mustard acyl-uridine；
Formula (III) compound is 3 ' -5 '-mustard acyls of-β-D- glucopyra glycosyloxy-uridine -3- benzoyl；
Formula (IV) compound is 3 ' -5 '-mustard acyl-uridines of-β-D- glucopyra glycosyloxy；
Formula (V) compound is；Thio-β-D- glucopyra glycosyloxy-mustard seed the acid butyl ester of 8-.
2. a kind of preparation method of mustard seed acid compounds according to claim 1, it is characterised in that follow these steps into Row:
A, the seed for taking cultivated pepperweed seed is raw material, measures ethanol water, the nothing that volumetric concentration is 50-99% with 5-10 times after crushing Diacolation, the cold soaking that water-ethanol, pure acetone, the methanol aqueous solution that volumetric concentration is 50-99% or anhydrous methanol carry out at room temperature mention It takes or heating and refluxing extraction, is concentrated to get the crude extract of cultivated pepperweed seed；
B, the crude extract obtained step a is water-dispersible, sequentially adds petroleum ether, n-hexane or hexamethylene, ethyl acetate, positive fourth Alcohol extracts 3-5 times, and butanol extraction liquid is concentrated, n-butyl alcohol extract medicinal extract is obtained；
C, the n-butyl alcohol extract medicinal extract for obtaining step b is through normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, aperture resin layer It analyses, separated to get arriving for two or three in Sephadex LH-20 gel filtration chromatography and preparative high performance liquid chromatography Formula (I) compound is 3 '-(6- mustard acyl-β-D- glucopyra glycosyloxy) -5 '-mustard acyl-uridines；Formula (II) Compound is 3 '-[6- (2- dioxindole -3- acetyl group)-β-D- glucopyra glycosyloxy]-β-D- glucopyra glycosyloxies] - 5 '-mustard acyls-uridine；Formula (III) compound is 3 ' -5 '-mustard acyl-uracils of-β-D- glucopyra glycosyloxy Nucleosides -3- benzoyl；Formula (IV) compound is 3 ' -5 '-mustard acyl-uridines of-β-D- glucopyra glycosyloxy；Formula (V) compound is the thio-β-D- glucopyra glycosyloxy-mustard seed acid butyl ester of 8-.
3. the preparation method of mustard seed acid compounds according to claim 2, it is characterised in that positive silicon used in step c Gel column chromatography is normal pressure or pressurized column chromatography, and filler used is purification on normal-phase silica gel, and eluant, eluent used is petroleum ether, hexamethylene or just Hexane, acetone, chloroform, methylene chloride or ethyl acetate, the mixture of at least two solvents in methanol, using isocratic elution Or gradient elution.
4. the preparation method of mustard seed acid compounds according to claim 2, it is characterised in that reverse phase silicon used in step c Gel column chromatography is normal pressure or pressurized column chromatography, and eluant, eluent is the methanol aqueous solution or aqueous acetonitrile of volumetric concentration 10-100% Liquid, using isocratic elution or gradient elution.
5. the preparation method of mustard seed acid compounds according to claim 2, it is characterised in that aperture tree used in step c Rouge chromatography is normal pressure or pressurized column chromatography, and eluant, eluent is the methanol aqueous solution or acetonitrile solution of volumetric concentration 10-100%, Using isocratic elution or gradient elution.
6. the preparation method of mustard seed acid compounds according to claim 2, it is characterised in that used in step c Sephadex LH-20 gel filtration chromatography method is normal pressure column chromatography, and eluant, eluent is methanol, methylene chloride, chloroform or at least two The mixture of kind solvent, using isocratic elution or gradient elution.
7. the preparation method of mustard seed acid compounds according to claim 2, it is characterised in that preparation used is high in step c Effect liquid phase chromatogram method is pressurized column chromatography, and eluant, eluent is the methanol aqueous solution of volumetric concentration 40-99% or the acetonitrile water of 30-99% Solution, using isocratic elution or gradient elution.
8. mustard seed acid compounds purposes in preparing anti-inflammatory drugs according to claim 1.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110963918A (en) * 2019-12-13 2020-04-07 山东中医药大学 Sinapic acid derivative and preparation method and application thereof
CN112979722A (en) * 2021-03-03 2021-06-18 山东中医药大学 Sinapoyl sugar ester compound and preparation method and application thereof

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CN101104630A (en) * 2007-03-23 2008-01-16 南京农业大学 Wheat bran flavonoid compounds
CN102633784A (en) * 2012-04-11 2012-08-15 广西医科大学 Two new flavone C-glycoside compounds as well as preparation method and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101104630A (en) * 2007-03-23 2008-01-16 南京农业大学 Wheat bran flavonoid compounds
CN102633784A (en) * 2012-04-11 2012-08-15 广西医科大学 Two new flavone C-glycoside compounds as well as preparation method and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110963918A (en) * 2019-12-13 2020-04-07 山东中医药大学 Sinapic acid derivative and preparation method and application thereof
CN110963918B (en) * 2019-12-13 2022-05-31 山东中医药大学 Sinapic acid derivative and preparation method and application thereof
CN112979722A (en) * 2021-03-03 2021-06-18 山东中医药大学 Sinapoyl sugar ester compound and preparation method and application thereof

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