CN103865962A - Enzymatic preparation method and application of quercetin-3-O-fatty acid ester - Google Patents
Enzymatic preparation method and application of quercetin-3-O-fatty acid ester Download PDFInfo
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- CN103865962A CN103865962A CN201410036289.2A CN201410036289A CN103865962A CN 103865962 A CN103865962 A CN 103865962A CN 201410036289 A CN201410036289 A CN 201410036289A CN 103865962 A CN103865962 A CN 103865962A
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- UNMYRRZMRDRBQM-UHFFFAOYSA-N CC(C1C(O)=C2)C(O)=C(c(cc3O)ccc3O)OC1C=C2O Chemical compound CC(C1C(O)=C2)C(O)=C(c(cc3O)ccc3O)OC1C=C2O UNMYRRZMRDRBQM-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides an enzymatic preparation method of quercetin-3-O-fatty acid ester. The preparation method comprises the steps of: (1) adding 0.01-0.1mol/L quercetin into an anhydrous organic solvent, and carrying out acylation reaction with an acid anhydride or acyl chloride for 3 to 5 hours to obtain quercetin-3,5,7,3',4'-penta fatty acid ester; (2) adding quercetin-3,5,7,3',4'-penta fatty acid ester to an anhydrous organic solvent, and at 20-80 DEG C, under the catalytic action of lipase Lipozyme IM, performing alcoholysis reaction with n-butanol for 30-100 hours to obtain quercetin-3-O-fatty acid ester. The invention relates to the technical field of medicinal chemistry; the preparation method has the characteristics of mild reaction conditions, high yield, short reaction route, simple process, simple post-processing and easiness for industrial production. The corresponding technical problems in the prior art are solved.
Description
Technical field
The present invention relates to pharmaceutical chemistry technical field, refer to especially a kind of enzyme catalysis preparation method and application thereof of Quercetin-3-O-fatty acid ester.
Background technology
Quercetin is a kind of important flavonoid compound, be almost present in the herbal medicine of all tool blood circulation invigorating efficacies, and toxic side effect is low, and extraction and separation process is ripe simple.Quercetin shows significant platelet aggregation inhibitory activity, and the platelet aggregation of ADP, AA, PAF, collagen, thrombin induction is had to obvious restraining effect.But do not have at present the Quercetin class medicine of platelet aggregation-against to carry out clinical use, this is mainly because Quercetin is poorly soluble, become the property of medicine poor, bioavailability is low.By structural modification, 3 of Quercetin hydroxyls are carried out to esterification; introduce fatty acyl group; change molecular configuration and the intermolecular close-packed arrays of large plane; solvent easily enters molecular gap; thereby improve solvability, simultaneously retentive activity group (3 ', 4 ' adjacent two hydroxyls and 7 hydroxyls); can improve into the property of medicine, improve drug effect.
Quercetin-3-O-fatty acid ester is the compound with logical formula I, R=C1-C4 alkyl in formula.The patents such as Zhai Guangyu (Chinese patent 201210113907.X) are take rutin as starting raw material; prepare Quercetin-3-O-acyl ester compounds through hydrolysis under benzyl, acidic conditions, esterification, catalytic hydrogenolysis four-step reaction; reactions steps is many; the benzyl reagent pungency and the toxicity that use are large, and catalytic hydrogenolytic cleavage has certain danger.Therefore need to improve.
Summary of the invention
The present invention proposes a kind of enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester, there is reaction conditions gentleness, productive rate is high, reaction scheme is short, easy and simple to handle, aftertreatment is simple, be convenient to the feature of suitability for industrialized production, has solved corresponding technical problem in prior art.
Technical scheme of the present invention is achieved in that
The enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester, comprises the following steps:
(1) Quercetin is joined in anhydrous organic solvent, the volumetric molar concentration of Quercetin is 0.01～0.1mol/L, under the condition of 10～100 ℃, under the katalysis of basic catalyst, within 3～5 hours, obtain Quercetin-3,5 with acid anhydrides or acyl chlorides acylation reaction, 7,3 ', 4 '-five fatty acid esters;
(2) by described Quercetin-3,5,7,3 ', 4 '-five fatty acid esters join in anhydrous organic solvent, Quercetin-3,5,7, the volumetric molar concentration of 3 ', 4 '-five fatty acid esters is 0.002～0.04mol/L, under the condition of 20～80 ℃, under the katalysis of Lipozyme IM, within 30～100 hours, obtain Quercetin-3-O-fatty acid ester with propyl carbinol generation alcoholysis reaction;
Described anhydrous organic solvent is selected from one or more in acetonitrile, DMF, methylene dichloride and methyl tertiary butyl ether;
Described basic catalyst is selected from triethylamine, pyridine and Na
2cO
3in one or more;
Described Lipozyme IM is selected from the one in Lipozyme IM-20, Lipozyme IM-60;
Described Quercetin-3-O-fatty acid ester is the compound with logical formula I, R=C1-C4 alkyl in formula.
As the improvement to technique scheme, in step (1), the mol ratio of described Quercetin, acid anhydrides or acyl chlorides and basic catalyst is 1:5～15:5～15; In step (2), described Quercetin-3, the mass ratio of 5,7,3 ', 4 '-five fatty acid esters and Lipozyme IM is 1:0.5～3, described Quercetin-3, the mol ratio of 5,7,3 ', 4 '-five fatty acid esters and propyl carbinol is 1:4～12.
As the improvement to technique scheme, in step (1), the volumetric molar concentration of described Quercetin is 0.022mol/L, and temperature of reaction is 25 ℃, and the reaction times is 3.5～4.5 hours; In step (2), Quercetin-3, the volumetric molar concentration of 5,7,3 ', 4 '-five fatty acid esters is 0.008～0.02mol/L, and temperature of reaction is 45 ℃, and the reaction times is 45～96 hours.
As the improvement to technique scheme, in step (1), the mol ratio of described Quercetin, acid anhydrides or acyl chlorides and basic catalyst is 1:10:10; In step (2), described Quercetin-3, the mass ratio of 5,7,3 ', 4 '-five fatty acid esters and Lipozyme IM is 1:1, described Quercetin-3, the mol ratio of 5,7,3 ', 4 '-five fatty acid esters and propyl carbinol is 1:10.
As the improvement to technique scheme, in step (1), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), and while disappearance to Quercetin spot, acylation reaction stops.
As the further improvement to technique scheme, in step (1), after acylation reaction stops, reaction solution washs with aqueous hydrochloric acid, the saturated aqueous sodium carbonate of 1mol/L successively, collected organic layer, and extremely neutral with deionized water wash, add anhydrous Na
2sO
4dehydration, leaves standstill 10～12 hours, filters to get filtrate, and filtrate is at 20kPa, and vacuum rotary steam is removed anhydrous organic solvent and obtained Quercetin-3,5,7,3 ', 4 '-five fatty acid esters at 40～70 ℃.
As the improvement to technique scheme, in step (2), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), to Quercetin-3,5, when 7,3 ', 4 '-five fatty acid ester spots disappear, alcoholysis reaction stops.
As the further improvement to technique scheme, in step (2), remove by filter Lipozyme IM and stop alcoholysis reaction, filtrate is at 20kPa, at 40～70 ℃, vacuum rotary steam is removed anhydrous organic solvent, and gained crude product obtains Quercetin-3-O-fatty acid ester after polymeric amide chromatographic column purifying.
As the improvement to technique scheme, described Quercetin-3-O-fatty acid ester is applied to the medicine of preparing platelet aggregation-against.
The present invention proposes a kind of enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester, and its synthesis route is as follows:
Utilize the synthetic compound with logical formula I of above-mentioned preparation method, R=C1-C4 alkyl in formula.
Specific as follows:
Quercetin-3-O-acetic ester;
Quercetin-3-O-propionic ester;
Quercetin-3-O-butanic acid ester;
The positive valerate of Quercetin-3-O-.
Its structural formula is as follows respectively:
Above-mentioned synthetic Quercetin-3-O-fatty acid ester is carried out to the screening of in vitro and in vivo platelet aggregation inhibitory activity, and result shows that four kinds of Quercetin-3-O-fatty acid esters all have certain restraining effect to the platelet aggregation of ADP, AA, PAF, collagen and thrombin induction.Wherein Quercetin-3-O-acetic ester and Quercetin-3-O-propionic ester platelet aggregation inhibitory activity are all apparently higher than Quercetin.
Adopt after technique scheme, the invention has the beneficial effects as follows:
The method that Quercetin-3-O-fatty acid ester is synthesized in enzyme catalysis of the present invention has mild condition, productive rate is high, step is few, easy and simple to handle, aftertreatment is simple, easier suitability for industrialized production.Synthetic Quercetin-3-O-fatty acid ester has good platelet aggregation inhibitory activity, and Quercetin-3-O-acetic ester and Quercetin-3-O-propionic ester platelet aggregation inhibitory activity be all apparently higher than Quercetin, be expected to be further developed as the medicine of the cardiovascular disorder that treatment platelet aggregation causes.
Quercetin-3-O-fatty acid ester of synthesized of the present invention all has platelet aggregation inhibitory activity, can be applied to the medicine of the cardiovascular disorder that preparation treatment platelet aggregation causes.Quercetin-3-O-fatty acid ester of the present invention or their mixture during for the preparation of said medicine, can be made according to the ordinary method of field of medicaments the drug forms such as injection, tablet, capsule, suppository, film, pill.
Embodiment
Below in conjunction with embodiments of the invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Based on the embodiment in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1:
The enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester, comprises the following steps:
(1) Quercetin is joined in acetonitrile, the volumetric molar concentration of Quercetin is 0.01mol/L, under the condition of 10 ℃, under the katalysis of triethylamine, within 5 hours, obtains Quercetin-3,5,7,3 ', 4 '-five fatty acid esters with acid anhydrides or acyl chlorides acylation reaction;
(2) by described Quercetin-3,5,7,3 ', 4 '-five fatty acid esters join in acetonitrile, Quercetin-3,5,7, the volumetric molar concentration of 3 ', 4 '-five fatty acid esters is 0.002mol/L, under the condition of 20 ℃, under the katalysis of Lipozyme IM-20, within 100 hours, obtain Quercetin-3-O-fatty acid ester with propyl carbinol generation alcoholysis reaction;
Described Quercetin-3-O-fatty acid ester is the compound with logical formula I, R=C1-C4 alkyl in formula.
In step (1), the mol ratio of described Quercetin, acid anhydrides or acyl chlorides and basic catalyst is 1:5:5; In step (2), described Quercetin-3, the mass ratio of 5,7,3 ', 4 '-five fatty acid esters and Lipozyme IM is 1:0.5, described Quercetin-3, the mol ratio of 5,7,3 ', 4 '-five fatty acid esters and propyl carbinol is 1:4.
In step (1), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), and while disappearance to Quercetin spot, acylation reaction stops.
In step (1), after acylation reaction stops, reaction solution washs with aqueous hydrochloric acid, the saturated aqueous sodium carbonate of 1mol/L successively, collected organic layer, and extremely neutral with deionized water wash, add anhydrous Na
2sO
4dehydration, leaves standstill 10 hours, filters to get filtrate, and filtrate is at 20kPa, and vacuum rotary steam is removed anhydrous organic solvent and obtained Quercetin-3,5,7,3 ', 4 '-five fatty acid esters at 40 ℃.
In step (2), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), and to Quercetin-3, when 5,7,3 ', 4 '-five fatty acid ester spots disappear, alcoholysis reaction stops.
In step (2), remove by filter Lipozyme IM and stop alcoholysis reaction, filtrate is at 20kPa, and vacuum rotary steam is removed anhydrous organic solvent at 40 ℃, gained crude product after polymeric amide chromatographic column purifying Quercetin-3-O-fatty acid ester.
Embodiment 2:
The enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester, comprises the following steps:
(1) Quercetin is joined in DMF, the volumetric molar concentration of Quercetin is 0.022mol/L, under the condition of 25 ℃, under the katalysis of pyridine, within 3.5 hours, obtain Quercetin-3,5 with acid anhydrides or acyl chlorides acylation reaction, 7,3 ', 4 '-five fatty acid esters;
(2) by described Quercetin-3,5,7,3 ', 4 '-five fatty acid esters join in DMF, Quercetin-3,5,7,3 ', the volumetric molar concentration of 4 '-five fatty acid esters is 0.008mol/L, under the condition of 45 ℃, under the katalysis of Lipozyme IM-60, within 45 hours, obtain Quercetin-3-O-fatty acid ester with propyl carbinol generation alcoholysis reaction;
Described Quercetin-3-O-fatty acid ester is the compound with logical formula I, R=C1-C4 alkyl in formula.
In step (1), the mol ratio of described Quercetin, acid anhydrides or acyl chlorides and basic catalyst is 1:10:10; In step (2), described Quercetin-3, the mass ratio of 5,7,3 ', 4 '-five fatty acid esters and Lipozyme IM is 1:1, described Quercetin-3, the mol ratio of 5,7,3 ', 4 '-five fatty acid esters and propyl carbinol is 1:10.
In step (1), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), and while disappearance to Quercetin spot, acylation reaction stops.
In step (1), after acylation reaction stops, reaction solution washs with aqueous hydrochloric acid, the saturated aqueous sodium carbonate of 1mol/L successively, collected organic layer, and extremely neutral with deionized water wash, add anhydrous Na
2sO
4dehydration, leaves standstill 10～12 hours, filters to get filtrate, and filtrate is at 20kPa, and vacuum rotary steam is removed anhydrous organic solvent and obtained Quercetin-3,5,7,3 ', 4 '-five fatty acid esters at 50 ℃.
In step (2), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), and to Quercetin-3, when 5,7,3 ', 4 '-five fatty acid ester spots disappear, alcoholysis reaction stops.
In step (2), remove by filter Lipozyme IM and stop alcoholysis reaction, filtrate is at 20kPa, and vacuum rotary steam is removed anhydrous organic solvent at 50 ℃, gained crude product after polymeric amide chromatographic column purifying Quercetin-3-O-fatty acid ester.
Embodiment 3:
The enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester, comprises the following steps:
(1) Quercetin is joined in methyl tertiary butyl ether, the volumetric molar concentration of Quercetin is 0.1mol/L, under the condition of 100 ℃, and Na
2cO
3katalysis under, within 3 hours, obtain Quercetin-3,5,7,3 ', 4 '-five fatty acid esters with acid anhydrides or acyl chlorides acylation reaction;
(2) by described Quercetin-3,5,7,3 ', 4 '-five fatty acid esters join in methyl tertiary butyl ether, Quercetin-3,5,7, the volumetric molar concentration of 3 ', 4 '-five fatty acid esters is 0.04mol/L, under the condition of 80 ℃, under the katalysis of Lipozyme IM-60, within 30 hours, obtain Quercetin-3-O-fatty acid ester with propyl carbinol generation alcoholysis reaction;
Described Quercetin-3-O-fatty acid ester is the compound with logical formula I, R=C1-C4 alkyl in formula.
In step (1), the mol ratio of described Quercetin, acid anhydrides or acyl chlorides and basic catalyst is 1:15:15; In step (2), described Quercetin-3, the mass ratio of 5,7,3 ', 4 '-five fatty acid esters and Lipozyme IM is 1:3, described Quercetin-3, the mol ratio of 5,7,3 ', 4 '-five fatty acid esters and propyl carbinol is 1:12.
In step (1), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), and while disappearance to Quercetin spot, acylation reaction stops.
In step (1), after acylation reaction stops, reaction solution washs with aqueous hydrochloric acid, the saturated aqueous sodium carbonate of 1mol/L successively, collected organic layer, and extremely neutral with deionized water wash, add anhydrous Na
2sO
4dehydration, leaves standstill 10～12 hours, filters to get filtrate, and filtrate is at 20kPa, and vacuum rotary steam is removed anhydrous organic solvent and obtained Quercetin-3,5,7,3 ', 4 '-five fatty acid esters at 40～70 ℃.
In step (2), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), and to Quercetin-3, when 5,7,3 ', 4 '-five fatty acid ester spots disappear, alcoholysis reaction stops.
In step (2), remove by filter Lipozyme IM and stop alcoholysis reaction, filtrate is at 20kPa, and vacuum rotary steam is removed anhydrous organic solvent at 40～70 ℃, gained crude product after polymeric amide chromatographic column purifying Quercetin-3-O-fatty acid ester.
Preparation Example 1
Synthesizing of Quercetin-3-O-acetic ester
(1) Quercetin-3,5,7,3 ', 4 '-pentaacetate synthetic
Quercetin (0.01mol, 3.02g) is dissolved in anhydrous methyl tertiary butyl ether with the concentration of 0.022mol/L, adds triethylamine (0.1mol, 13.94mL), slowly drips diacetyl oxide (0.1mol, 9.44mL), stirring reaction at 25 ℃ of temperature.With GF254 silica gel thin-layer chromatography plate monitoring reaction process, developping agent: ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio).React after 4 hours, Quercetin spot disappears, and acylation reaction is complete.Reaction solution washs with aqueous hydrochloric acid, the saturated aqueous sodium carbonate of 1mol/L successively, collected organic layer, and extremely neutral with deionized water wash, add anhydrous Na
2sO
4dehydration, spends the night, and vacuum concentration obtains Quercetin-3, and 5,7,3 ', 4 '-pentaacetate (4.83g, 94.29%).
(2) Quercetin-3-O-acetic ester is synthetic
Quercetin-3,5,7,3 ', 4 '-pentaacetate (0.005mol, 2.56g) is dissolved in anhydrous methyl tertiary butyl ether with the concentration of 0.009mol/L, add Lipozyme IM(Novozymes Company) (2.56g) and propyl carbinol (0.05mol, 4.57mL), temperature 45 C stirring reaction.With GF254 silica gel thin-layer chromatography plate monitoring reaction process, developping agent: ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), react Quercetin-3 after 45 hours, 5,7,3 ', 4 '-pentaacetate spot disappears, and illustrates that alcoholysis reaction is complete.Remove by filter enzyme termination reaction, remove methyl tertiary butyl ether under reduced pressure, gained sample obtains Quercetin-3-O-acetic ester (1.59g, 92.43%) after polymeric amide chromatographic column purifying.
162～164 ℃ of fusing points.
ESI-MS：345.2[M+H]
+。
1H-NMR（DMSO-d
6，600MHz）δ：7.50(1H,d,J=2.2Hz),7.40(1H,dd,J=2.2,8.5Hz),6.93(1H,d,J=8.5Hz),5.29(1H,d,J=2.0Hz),5.22(1H,d,J=2.0Hz)，2.51(3H,s)。
13c-NMR(DMSO-d
6600MHz): δ: 176.2 (C-4); 175.0 (C=O, ethanoyl), 168.5 (C-7); 161.1 (C-9); 156.7 (C-5), 148.3 (C-4'), 146.2 (C-2); 145.7 (C-3'); 136.1 (C-3), 122.4 (C-1'), 120.8 (C-6'); 116.5 (C-5'); 115.2 (C-2'), 102.9 (C-10), 99.0 (C-6); 94.9 (C-8), 20.8 (CH
3).
Ultimate analysis: C58.84%, H3.79%, composition and chemical formula C
17h
12o
8unanimously.
Preparation Example 2
Synthesizing of Quercetin-3-O-propionic ester
(1) Quercetin-3,5,7,3 ', 4 '-five propionic esters synthetic
According to the synthetic method of step (1) in Preparation Example 1, raw acetic acid acid anhydride is replaced with to propionyl chloride (0.1mol, 8.73mL), can make Quercetin-3,5,7,3 ', 4 '-five propionic esters (5.32g, 91.33%).
(2) Quercetin-3-O-propionic ester is synthetic
According to the synthetic method of step (2) in Preparation Example 1, by Quercetin-3, the concentration that feeds intake of 5,7,3 ', 4 '-five propionic esters replaces with 0.011mol/L, and the reaction times changes 50 hours into.Can make Quercetin-3-O-propionic ester (1.64g, 91.67%).
158～160 ℃ of fusing points.
ESI-MS：359.2[M+H]
+。
1H-NMR（DMSO-d
6，600MHz）δ：0.94（bt，-CH
3），1.69（bt，-CH
2CO-），6.31（d，J=2.2Hz，H6），6.55（d，J=2.2Hz，H8），7.02（d，J=8.2Hz，H5’），7.40（dd，J=2.2Hz，8.2Hz，H6’），7.47（d，J=2.2Hz，H2’）。
13c-NMR(DMSO-d
6600MHz) δ: 178.3 (C-4), 171.2 (C=O, ethanoyl); 166.4 (C-7); 163.9 (C-5), 160.1 (C-5), 147.2 (C-3'); 146.5 (C-4 '); 142.7 (C-2), 133.3 (C-3), 124.4 (C-1'); 120.4 (C-6'); 117.2 (C-5'), 112.6 (C-2'), 105.5 (C-10); 98.3 (C-6); 98.0 (C-8), 27.7 (CH2), 12.8 (CH
3).
Ultimate analysis: C61.07%, H4.08%, composition and chemical formula C
18h
14o
8unanimously.
Preparation Example 3
Synthesizing of Quercetin-3-O-butanic acid ester
(1) Quercetin-3,5,7,3 ', 4 '-five butanic acid esters synthetic
According to the synthetic method of step (1) in Preparation Example 1, raw acetic acid acid anhydride is replaced with to n-butyryl chloride (0.1mol, 10.35mL), can make Quercetin-3,5,7,3 ', 4 '-five butanic acid esters (6.08g, 93.21%).
2) Quercetin-3-O-butanic acid ester is synthetic
According to the synthetic method of step (2) in Preparation Example 1, by Quercetin-3, the concentration that feeds intake of 5,7,3 ', 4 '-five butanic acid esters replaces with 0.02mg/mL, and the reaction times changes 72 hours into.Can make Quercetin-3-O-butanic acid ester (1.75g, 93.77%).
Fusing point 122-124 ℃.
ESI-MS：363.2[M+H]
+。
1H-NMR（DMSO-d
6，600MHz）δ：0.98（bt，-CH
3），1.60（bs，-CH
2-），2.23（bt，-CH
2CO-），6.27（d，J=2.2Hz，H6），6.41（d，J=2.2Hz，H8），6.95（d，J=8.2Hz，H5’），7.23（dd，J=2.2Hz，8.2Hz，H6’），7.35（d，J=2.2Hz，H2’）。
13c-NMR(DMSO-d
6600MHz) δ: 179.6 (C-4); 172.5 (C=O, ethanoyl), 167.8 (C-7); 160.7 (C-5); 158.2 (C-5), 145.7 (C-3'), 144.3 (C-4 '); 139.7 (C-2); 131.4 (C-3), 121.6 (C-1'), 118.4 (C-6'); 116.2 (C-5'); 109.6 (C-2'), 104.8 (C-10), 95.8 (C-6); 96.6 (C-8), 36.9 (CO-CH
2), 18.5 (CH
2), 13.5 (CH
3).
Ultimate analysis: C61.99%, H4.57%, composition and chemical formula C
19h
16o
8unanimously.
Preparation Example 4
Synthesizing of the positive valerate of Quercetin-3-O-
(1) Quercetin-3,5,7,3 ', 4 '-five positive valerates synthetic
According to the synthetic method of step (1) in Preparation Example 1, raw acetic acid acid anhydride is replaced with to n-amyl chloride (0.1mol, 11.94mL), can make Quercetin-3,5,7,3 ', 4 '-five positive valerates (6.70g, 92.79%).
(2) the positive valerate of Quercetin-3-O-is synthetic
According to the synthetic method of step (2) in Preparation Example 1, by Quercetin-3, the concentration that feeds intake of 5,7,3 ', 4 '-five positive valerates replaces with 0.018mg/mL, and the reaction times changes 96 hours into.Can make Quercetin-3-O-butanic acid ester (1.78g, 92.01%).
Fusing point 106-108 ℃.
ESI-MS：387.2[M+H]
+。
1H-NMR（DMSO-d
6，600MHz）δ：0.96（bt，-CH
3），1.47（bs，-CH
2-），2.34（bt，-CH
2CO-），6.28（d，J=2.2Hz，H6），6.49（d，J=2.2Hz，H8），7.05（d，J=8.2Hz，H5’），7.42（dd，J=2.2Hz，8.2Hz，H6’），7.50（d，J=2.2Hz，H2’）。
13c-NMR(DMSO-d
6600MHz) δ: 179.4 (C-4); 174.9 (C=O, ethanoyl), 168.2 (C-7); 166.7 (C-5); 161.5 (C-5), 148.9 (C-3'), 146.7 (C-4 '); 141.9 (C-2); 134.8 (C-3), 129.4 (C-1'), 122.4 (C-6'); 120.8 (C-5'); 111.5 (C-2'), 108.4 (C-10), 96.7 (C-6); 98.1 (C-8), 33.7 (CO-CH
2), 22.2 (CH
2), 13.8 (CH
3).
Ultimate analysis: C61.76%, H4.72%, composition and chemical formula C
20h
18o
8unanimously.
Application Example 1
Quercetin-3-O-fatty acid ester In Vitro Anti PAgT
1 experiment purpose: the inhibiting rate and the IC50 that measure the external Platelet Aggregation in Rabbits to ADP, AA, PAF, collagen, thrombin induction of Quercetin-3-O-fatty acid ester.
2 experiment materials
2.1 laboratory animal: healthy rabbits, male and female dual-purpose, General Hospital of Jinan Military Command Experimental Animal Center, ad lib and drinking-water, room temperature is raised.
2.2 given the test agent: Quercetin-3-O-acetic ester, Quercetin-3-O-propionic ester, Quercetin-3-O-butanic acid ester, the positive valerate of Quercetin-3-O-, Quercetin are dissolved in respectively in dimethyl sulfoxide (DMSO) (DMSO), are respectively made into the solution of 5 concentration gradients.
2.3 other reagent: Sodital, Pu Bosi bio tech ltd, Beijing.ADP, AA, PAF, zymoplasm, collagen, Sigma reagent company.
2.4 laboratory apparatuss: the raw LBY-NJ4A platelet aggregation instrument of Puli, Pulisheng Instruments Co., Ltd., Beijing.
3 experimental techniques
12h fasting before experiment.The intravenous injection anesthesia of Sodital ear source, arteria carotis communis is got blood, with 3.8% Sodium Citrate anti-freezing (volume ratio 9:1).Centrifugation platelet rich plasma (PRP) and platelet poor plasma (PPP), adjusting PRP concentration with PPP is 5 × 10
8cell/mL.
Experiment is made as blank group, Quercetin-3-O-acetic ester group, Quercetin-3-O-propionic ester group, Quercetin-3-O-butanic acid ester group, the positive valerate group of Quercetin-3-O-, Quercetin group.
The mensuration of blank group: platelet aggregation instrument returns to zero with PPP, in PRP, add a certain amount of DMSO(DMSO final concentration to be less than 1% to eliminate the interference of DMSO to experimental result), in 37 ℃ of incubations 5 minutes, add a certain amount of aggregation inducing agent (ADP or AA or PAF or zymoplasm or collagen) solution, magneton records thrombocyte MA in 5min under stirring.
Medicine group (Quercetin-3-O-acetic ester group, Quercetin-3-O-propionic ester group, Quercetin-3-O-butanic acid ester group, the positive valerate group of Quercetin-3-O-, Quercetin group) mensuration: platelet aggregation instrument returns to zero with PPP, (5 gradient final concentrations of medicine are controlled at 300 – 20 μ mol/L in PRP, to add a certain amount of liquid preparing, and guarantee that DMSO final concentration is less than 1% to eliminate the interference of DMSO to experimental result), in 37 ℃ of incubations 5 minutes, a certain amount of aggregation inducing agent (ADP or AA or PAF or zymoplasm or collagen) solution, magneton records thrombocyte MA in 5min under stirring.
Result is calculated: assemble inhibiting rate (%)=[(1 – medicine group aggregation rate/blank group aggregation rate)] × 100.
Drug level (half drug level IC50) when calculating L-Arginine is 50%.
4 experimental results
The inhibiting rate of table 1 Quercetin-3-O-fatty acid ester, the rabbit platelet aggregation of Quercetin In Vitro to ADP, AA, PAF, collagen and thrombin induction
N=6, x ± s, * P < 0.01, with blank comparison.
Blank group aggregation rate (%): ADP is 40.3 ± 3.3; AA is 68.4 ± 3.5; PAF is 72.9 ± 5.2; Collagen is 54.2 ± 3.2; Zymoplasm is 33.2 ± 2.7.
From table 1, Quercetin-3-O-acetic ester, Quercetin-3-O-propionic ester, Quercetin-3-O-butanic acid ester, the positive valerate of Quercetin-3-O-all have restraining effect to the rabbit platelet aggregation of ADP, AA, PAF, collagen and thrombin induction at each dosage, and are concentration dependent relation.
Table 2 Quercetin-3-O-fatty acid ester, Quercetin In Vitro suppress the IC50 of the rabbit platelet aggregation of ADP, AA, PAF, collagen and thrombin induction.
From table 2, the IC50 minimum of Quercetin-3-O-acetic ester, is secondly Quercetin-3-O-propionic ester, illustrates that the In Vitro Anti platelet aggregation activity of these two kinds of quercetin derivatives is all higher than Quercetin.The IC50 of Quercetin-3-O-butanic acid ester and the positive valerate of Quercetin-3-O-is all greater than Quercetin, and its activity is lower than Quercetin.
Conclusion: Quercetin-3-O-acetic ester, Quercetin-3-O-propionic ester, Quercetin-3-O-butanic acid ester, the positive valerate of Quercetin-3-O-all have the effect of In Vitro Anti platelet aggregation, wherein the effect of Quercetin-3-O-acetic ester and Quercetin-3-O-propionic ester In Vitro Anti platelet aggregation is better than Quercetin.
Application Example 2
Platelet aggregation-against test in Quercetin-3-O-fatty acid ester body
1 experiment purpose: the inhibiting rate of measuring the Wistar rat platelet aggregation to ADP, AA, PAF, collagen, thrombin induction in Quercetin-3-O-fatty acid ester body.
2 experiment materials
2.1 laboratory animal: strain: Wistar rat; Grade: SPF level; Weight: 250-300g; Sex: male; Source: Lukang Medical Co., Ltd., Shandong's Experimental Animal Center; Credit number: SCXK Shandong 20080002; Raise: room temperature, ad lib and drinking-water.
2.2 given the test agent: Quercetin-3-O-acetic ester, Quercetin-3-O-propionic ester, Quercetin-3-O-butanic acid ester, the positive valerate of Quercetin-3-O-, Quercetin are dissolved in respectively in physiological saline, add propylene glycol hydrotropy and obtain certain density solution.
2.3 other reagent: Sodital, Pu Bosi bio tech ltd, Beijing.ADP, AA, PAF, zymoplasm, collagen, Sigma reagent company.
2.4 laboratory apparatuss: the raw LBY-NJ4A platelet aggregation instrument of Puli, Pulisheng Instruments Co., Ltd., Beijing.
3 experimental techniques
Wistar rat is divided into 6 groups at random, and 6 every group, 12h fasting before experiment.
Experiment is made as blank group, Quercetin-3-O-acetic ester group, Quercetin-3-O-propionic ester group, Quercetin-3-O-butanic acid ester group, the positive valerate group of Quercetin-3-O-, Quercetin group.Blank group: tail vein injection saline (containing the propylene glycol with Quercetin-3-O-fatty acid ester solution same concentrations).Medicine group: tail vein injection Quercetin-3-O-fatty acid ester or Quercetin, dosage 10mg/kg.
After administration 0.5h, Sodital intraperitoneal injection of anesthesia, heart extracting blood, with 3.8% Sodium Citrate anti-freezing (volume ratio 9:1).Centrifugation platelet rich plasma (PRP) and platelet poor plasma (PPP), adjusting PRP concentration with PPP is 5 × 10
8cell/mL.
Aggregation rate is measured and is carried out according to effect embodiment 1 method.
4 experimental results
The impact of the rat platelet aggregation on ADP, AA, PAF, collagen and thrombin induction in table 3 Quercetin-3-O-fatty acid ester, Quercetin
N=6, x ± s, * P < 0.01, with the comparison of blank group.
Blank group aggregation rate (%): ADP is 45.7 ± 7.2; AA is 59.2 ± 4.4; PAF is 62.3 ± 5.9; Collagen is 50.2 ± 4.4; Zymoplasm is 30.2 ± 1.8.
Data show, Quercetin-3-O-acetic ester, Quercetin-3-O-propionic ester, Quercetin-3-O-butanic acid ester, the positive valerate of Quercetin-3-O-all obviously suppress the rat platelet aggregation of ADP, AA, PAF, collagen and thrombin induction after administration 0.5h.Wherein Quercetin-3-O-acetic ester to press down poly-rate the highest, be secondly Quercetin-3-O-propionic ester, being all greater than pressing down of Quercetin gathers rate.And the poly-rate that presses down of Quercetin-3-O-butanic acid ester and the positive valerate of Quercetin-3-O-is all less than Quercetin.
Conclusion:, Quercetin-3-O-acetic ester, Quercetin-3-O-propionic ester, Quercetin-3-O-butanic acid ester, the positive valerate of Quercetin-3-O-all have the effect of anticoagulant in body, and wherein the effect of Quercetin-3-O-acetic ester and Quercetin-3-O-propionic ester is greater than Quercetin.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. the enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester, is characterized in that, comprises the following steps:
(1) Quercetin is joined in anhydrous organic solvent, the volumetric molar concentration of Quercetin is 0.01～0.1mol/L, under the condition of 10～100 ℃, under the katalysis of basic catalyst, within 3～5 hours, obtain Quercetin-3,5 with acid anhydrides or acyl chlorides acylation reaction, 7,3 ', 4 '-five fatty acid esters;
(2) by described Quercetin-3,5,7,3 ', 4 '-five fatty acid esters join in anhydrous organic solvent, Quercetin-3,5,7, the volumetric molar concentration of 3 ', 4 '-five fatty acid esters is 0.002～0.04mol/L, under the condition of 20～80 ℃, under the katalysis of Lipozyme IM, within 30～100 hours, obtain Quercetin-3-O-fatty acid ester with propyl carbinol generation alcoholysis reaction;
Described anhydrous organic solvent is selected from one or more in acetonitrile, DMF, methylene dichloride and methyl tertiary butyl ether;
Described basic catalyst is selected from triethylamine, pyridine and Na
2cO
3in one or more;
Described Lipozyme IM is selected from the one in Lipozyme IM-20, Lipozyme IM-60;
Described acid anhydrides is diacetyl oxide;
Described acyl chlorides is propionyl chloride, n-butyryl chloride or n-amyl chloride;
Described Quercetin-3-O-fatty acid ester is the compound with logical formula I, R=C1-C4 alkyl in formula.
2. the enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester as claimed in claim 1, is characterized in that: in step (1), the mol ratio of described Quercetin, acid anhydrides or acyl chlorides and basic catalyst is 1:5～15:5～15;
In step (2), described Quercetin-3, the mass ratio of 5,7,3 ', 4 '-five fatty acid esters and Lipozyme IM is 1:0.5～3, described Quercetin-3, the mol ratio of 5,7,3 ', 4 '-five fatty acid esters and propyl carbinol is 1:4～12.
3. the enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester as claimed in claim 1, is characterized in that: in step (1), the volumetric molar concentration of described Quercetin is 0.022mol/L, and temperature of reaction is 25 ℃, and the reaction times is 3.5～4.5 hours;
In step (2), Quercetin-3, the volumetric molar concentration of 5,7,3 ', 4 '-five fatty acid esters is 0.008～0.02mol/L, and temperature of reaction is 45 ℃, and the reaction times is 45～96 hours.
4. the enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester as claimed in claim 1, is characterized in that: in step (1), the mol ratio of described Quercetin, acid anhydrides or acyl chlorides and basic catalyst is 1:10:10;
In step (2), described Quercetin-3, the mass ratio of 5,7,3 ', 4 '-five fatty acid esters and Lipozyme IM is 1:1, described Quercetin-3, the mol ratio of 5,7,3 ', 4 '-five fatty acid esters and propyl carbinol is 1:10.
5. the enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester as claimed in claim 1; it is characterized in that: in step (1); with the GF254 silica gel thin-layer chromatography plate monitoring reaction times; developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5; volume ratio), while disappearance to Quercetin spot, acylation reaction stops.
6. the enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester as claimed in claim 5; it is characterized in that: in step (1); after acylation reaction stops; reaction solution washs with aqueous hydrochloric acid, the saturated aqueous sodium carbonate of 1mol/L successively; collected organic layer; and extremely neutral with deionized water wash, add anhydrous Na
2sO
4dehydration, leaves standstill 10～12 hours, filters to get filtrate, and filtrate is at 20kPa, and vacuum rotary steam is removed anhydrous organic solvent and obtained Quercetin-3,5,7,3 ', 4 '-five fatty acid esters at 40～70 ℃.
7. the enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester as claimed in claim 1, it is characterized in that: in step (2), with the GF254 silica gel thin-layer chromatography plate monitoring reaction times, developping agent is ethyl acetate/acetone/glacial acetic acid (6:6:1.5, volume ratio), to Quercetin-3,5, when 7,3 ', 4 '-five fatty acid ester spots disappear, alcoholysis reaction stops.
8. the enzyme catalysis preparation method of Quercetin-3-O-fatty acid ester as claimed in claim 7, it is characterized in that: in step (2), remove by filter Lipozyme IM and stop alcoholysis reaction, filtrate is at 20kPa, at 40～70 ℃, vacuum rotary steam is removed anhydrous organic solvent, and gained crude product obtains Quercetin-3-O-fatty acid ester after polymeric amide chromatographic column purifying.
9. the enzyme catalysis preparation method of the Quercetin-3-O-fatty acid ester as described in claim 1～8 any one, is characterized in that: described Quercetin-3-O-fatty acid ester is applied to the medicine of preparing platelet aggregation-against.
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| CN104311523A (en) * | 2014-09-30 | 2015-01-28 | 浙江大学 | Method for selectively preparing 3',4'-diester-based catechin |
| CN107593877A (en) * | 2017-10-27 | 2018-01-19 | 湖北工业大学 | A kind of preparation method of compound Quercetin fatty acid ester antistaling agent |
| CN109678832A (en) * | 2019-01-22 | 2019-04-26 | 郑州工业应用技术学院 | One pot synthesis is synthesized the method and its product and application of Quercetin ester by rutin |
| CN115304572A (en) * | 2022-09-05 | 2022-11-08 | 南京林业大学 | Flavonoid fluorescent probe for detecting hydrazine and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104311523A (en) * | 2014-09-30 | 2015-01-28 | 浙江大学 | Method for selectively preparing 3',4'-diester-based catechin |
| CN107593877A (en) * | 2017-10-27 | 2018-01-19 | 湖北工业大学 | A kind of preparation method of compound Quercetin fatty acid ester antistaling agent |
| CN109678832A (en) * | 2019-01-22 | 2019-04-26 | 郑州工业应用技术学院 | One pot synthesis is synthesized the method and its product and application of Quercetin ester by rutin |
| CN115304572A (en) * | 2022-09-05 | 2022-11-08 | 南京林业大学 | Flavonoid fluorescent probe for detecting hydrazine and preparation method and application thereof |
| CN115304572B (en) * | 2022-09-05 | 2023-12-05 | 南京林业大学 | Flavonoid fluorescent probe for detecting hydrazine and preparation method and application thereof |
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