CN105087707B - A kind of method using lipase-catalyzed synthesis dihydromyricetin monoesters compound - Google Patents
A kind of method using lipase-catalyzed synthesis dihydromyricetin monoesters compound Download PDFInfo
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- CN105087707B CN105087707B CN201410222489.7A CN201410222489A CN105087707B CN 105087707 B CN105087707 B CN 105087707B CN 201410222489 A CN201410222489 A CN 201410222489A CN 105087707 B CN105087707 B CN 105087707B
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- lipase
- dihydromyricetin
- monoesters compound
- catalyzed synthesis
- monoesters
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Abstract
The invention discloses a kind of methods using lipase-catalyzed synthesis dihydromyricetin monoesters compound, belong to biocatalysis field.The method of the present invention includes the following steps：Dihydromyricetin and acyl donor are dissolved in organic solvent, add free or immobilized lipase, is reacted 1~2 day in 30~40 DEG C, 200~300r/min shaking tables；After reaction, reaction solution is centrifuged, takes supernatant, solvent is volatilized, obtained solid is dihydromyricetin monoesters compound.First passage of the present invention dissociates or fixed lipase catalyzed synthesizing dihydro myricetin monoesters compound, the esterified positions of product are in 3 minimum hydroxyls of antioxidant activity, its oil-soluble is improved while the antioxidant activity for remaining dihydromyricetin to greatest extent, makes its practical value bigger.The method has the advantages that catalyst is cheap, reaction condition is mild, reaction specificity height, high selectivity, lipase can be used repeatedly, production cost is low etc..
Description
Technical field
The invention belongs to biocatalysis field, it is related to a kind of using lipase-catalyzed synthesis dihydromyricetin monoesters compound
Method.
Background technology
Dihydromyricetin is a kind of flavone aglycone compound, entitled 3,5,7,3', the 4' of chemistry, the bis- hydrogen of 5'- hexahydroxys -2,3-
Flavonols (dihydromyricetin, DMY) also known as dihydromyricetin, Ampeloptin, ampelopsin.Dihydromyricetin
Mass fraction of the element in dry ampelopsis grossdentata cauline leaf may be up to more than 30%.Dihydromyricetin has a variety of pharmacological activity,
As anti-inflammatory, analgesia, cough-relieving, eliminating the phlegm, anti-hypertension, liver protecting and relieve the effect of alcohol, the fat that disappears, hypoglycemic, strengthen immunity, it is anti-oxidant and suppression
Bacterium effect etc. is expected to exploitation into food antioxidant, health food, cosmetics, pharmaceuticals etc..
Dihydromyricetin molecule oil-soluble is poor, limits its dispersibility in oil phase, is unfavorable for its antioxidation
Performance.Some hydroxyl in dihydromyricetin molecule is carried out esterification can improve its oil-soluble, enhance its antioxidant activity, real
With value bigger.
Esterification modification generally use chemical method and enzyme process.Specificity is lacked using chemical method position, may be made with antioxygen
The hydroxyl of change effect is esterified, although increasing lipophile, reduces the antioxidant effect of compound in itself.Enzymatic esterification one
As hydroxyl is esterified using lipase, since enzyme has substrate good specificity, can selectively be esterified a certain hydroxyl
Base, and reaction condition is mild, and by-product is few, has important application value in food service industry.Overwhelming majority enzyme process ester at present
Change the report of chromocor compound for hydroxyl on flavonoid glycoside glycosyl, the esterification of hydroxyl on flavone aglycone is rarely reported.Two
Hydrogen myricetin hydroxyl is connected directly to phenyl ring mostly, due to the sucting electronic effect of phenyl ring, the oxygen atom cloud density of hydroxyl
It reduces, hydroxy esterification reaction is made to become difficult.Have no the report being esterified about dihydromyricetin enzyme process at present.
Containing multiple hydroxyls on dihydromyricetin molecule, antioxidant activity difference is larger, passes through quantum chemistry calculation two
3 hydroxyls (3-OH) of hydrogen myricetin are the most weak hydroxyls of antioxidant activity, so 3-OH is carried out esterification to improve dihydro
Myricetin is oil-soluble while retains its antioxidant activity to the maximum extent.
Enzyme immobilization (Enzyme immobilization) technology is to make free electrodes method using method physically or chemically
In the area of space of restriction, its activity, and the method recycled are kept.The enzyme that enzyme immobilization can improve unit volume is close
The advantages that degree, tolerance enhancing (such as pH value, temperature, organic solvent, noxious material), enzyme to environment are easily recycled.Enzyme is fixed
Change technology is the emerging biometric technology to grow up the 1960s, in the row such as chemical industry, fermenting and producing, the energy, medicine
Industry, practical application effect are notable.
Invention content
The purpose of the present invention is to provide a kind of methods using lipase-catalyzed synthesis dihydromyricetin monoesters compound.It should
Method enhances its oil-soluble while the antioxidant activity for remaining dihydromyricetin to greatest extent, reaction condition is mild,
React specificity height, high selectivity, the advantages such as lipase can be used repeatedly, production cost is low.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method using lipase-catalyzed synthesis dihydromyricetin monoesters compound is with free-fat enzyme or immobilization
Lipase-catalyzed dihydromyricetin and acry radical donor synthesizing dihydro myricetin monoesters compound；The dihydromyricetin monoesters compound
3 hydroxy esterifications for dihydromyricetin.
The free-fat enzyme includes：Penicillium cammenberti lipase (LPC), rhizopus chinensis lipase (Lipase
RC), Pseudomonas cepacia lipase (PCL), porcine pancreatic lipase (PPL), Rhizopus niveus lipase (Lipase RN), extension are green
Mould lipase (PEL), aspergillus niger HGD0823 lipase.The immobilized lipase includes：LipozymeIM TL、
LipozymeIMRM, Novozym435, Immobilized Aspergillus niger HGD0823 lipase.
Penicillium cammenberti lipase (LPC), rhizopus chinensis lipase (Lipase RC), onion are false in above-mentioned lipase
Fluorecens lipase (PCL), porcine pancreatic lipase (PPL), Rhizopus niveus lipase (Lipase RN), penicillium expansum lipase
(PEL)、LipozymeIM TL、LipozymeIMRM, Novozym435 are commercially produced product, can directly be bought from businessman
It obtains.Aspergillus niger HGD0823 lipase is prepared by aspergillus niger HGD0823 fermentations.Aspergillus niger is fixed using absorption method again
HGD0823 lipase obtains Immobilized Aspergillus niger HGD0823 lipase, and the method for absorption method fixed fat enzyme specifically includes as follows
Step：1. thick enzyme powder is dissolved in Glycine-NaOH buffer solution (0.05M, pH9.4), at 30~40 DEG C with 150~
200r/min stirs 1~2h；2. being centrifuged at 4~10 DEG C with 5000r/min, supernatant is taken；3. by 95% second of D4020 resins
After alcohol impregnates for 24 hours, it is washed with deionized water until filtrate is clarified, is added in 2. supernatant；4. 35~40 DEG C, 150
It under~200r/min, after vibrating 4~5h, is filtered with sand core funnel, with above-mentioned wash buffer 3 times, is freeze-dried after suction filtration
To immobilized lipase, in 0~4 DEG C of preservation.
Preferably, the method using lipase-catalyzed synthesis dihydromyricetin monoesters compound includes the following steps：
(1) dihydromyricetin and acry radical donor are dissolved in organic solvent, add free-fat enzyme or immobilized lipase
Enzyme reacts 1~2 day in 30~40 DEG C, 200~300r/min shaking tables.
(2) after reaction, reaction solution is centrifuged, takes supernatant, by solvent recovery, obtained solid is poplar containing dihydro
The product of syphilis monoesters compound.
Acry radical donor described in step (1) includes：Vinyl acetate, vinyl propionate, vinyl butyrate, sad ethylene
Ester, vinyl benzoate, stearic acid vinyl ester.
The molar ratio of dihydromyricetin and acry radical donor is preferably 1 in step (1):20~30.
Organic solvent described in step (1) includes：Acetonitrile, acetone, tetrahydrofuran, the tert-butyl alcohol, dimethyl sulfoxide (DMSO).
The amount of lipase described in step (1) is preferably 250U/mL.
The present invention is had the following advantages that relative to the prior art and effect：
The present invention is to utilize lipase-catalyzed synthesis dihydromyricetin monoesters compound for the first time.It is synthesized relative to conventional chemical methods
Dihydromyricetin monoesters compound, reaction condition of the present invention is mild, processing step is simple, environmental-friendly, repeatable utilizes and continuous
Metaplasia produces easy large-scale industrial production.
The present invention has the characteristics that fatty enzyme-to-substrate and product can be easily separated, and fat using immobilized lipase
The service life of enzyme is long, repeats utilization and continuous production, greatly reduces production cost.
The esterified positions of product of the present invention remain dihydromyricetin in the minimum 3- hydroxyls of antioxidant activity to greatest extent
The antioxidant activity of element, while enhance its oil-soluble.
Specific embodiment
Further detailed description is done to the present invention, but the implementation of the present invention is not limited to this with reference to embodiment.
Formulas I is dihydromyricetin molecular structure, and the antioxidant activity of 6 hydroxyls of dihydromyricetin molecule is different.Point
The difference (△ HOF) of the front and rear generation heat of sub- dehydrogenation can preferably judge the antioxidant activity of flavones and polyphenols, △ HOF values
The smaller free radical for illustrating to be formed after molecule dehydrogenation is more stable, and the antioxidant activity of molecule is stronger.Compare by using △ HOF
6 hydroxyl antioxidant activities of dihydromyricetin.Each hydroxyl △ HOF of dihydromyricetin see the table below：
According to the △ HOF of quantum chemistry calculation in table as a result, 6 hydroxyl oxidation resistances of dihydromyricetin are followed successively by：4'-
OH ＞ 5'-OH ＞ 3'-OH ＞ 7-OH ＞ 5-OH ＞ 3-OH.From 6 hydroxyl oxidation resistance result of calculations of dihydromyricetin molecule
As can be seen that 3 hydroxyl (3-OH) antioxidant activities are most weak.The generation of dihydromyricetin molecule hot (HOF) is 281.3931kJ/
mol。
Embodiment 1
(1) dihydromyricetin is dissolved in 2mL organic solvents, all adds vinyl acetate (dihydromyricetin after dissolving
Element and the initial concentration of vinyl acetate are respectively 20,500mmol/L), add the Immobilized Aspergillus niger HGD0823 fat of 500U
Fat enzyme reacts 24 hours under conditions of 35 DEG C of temperature, shaking speed 200r/min.
(2) after reaction, reaction solution is centrifuged, takes supernatant, by solvent recovery, obtained solid is poplar containing dihydro
The product of syphilis monoesters compound.
Organic solvent used is respectively acetonitrile, acetone, tetrahydrofuran, the tert-butyl alcohol, dimethyl sulfoxide (DMSO) in the present embodiment.It is different
In organic solvent, conversion ratio (by HPLC measured) of the dihydromyricetin in the case where Immobilized Aspergillus niger HGD0823 is lipase-catalyzed
It is shown in Table 1.
Conversion ratio of 1. dihydromyricetin of table in different organic solvents
Embodiment 2
(1) dihydromyricetin is dissolved in 2mL acetonitriles, all dissolving after add vinyl acetate (dihydromyricetin with
The initial concentration of vinyl acetate is respectively 20,500mmol/L), the lipase of 500U is added, is turned in 35 DEG C of temperature, shaking table
It is reacted 24 hours under conditions of fast 200r/min.
(2) after reaction, reaction solution is centrifuged, takes supernatant, by solvent recovery, obtained solid is poplar containing dihydro
The product of syphilis monoesters compound.
The respectively free lipase of lipase used in the present embodiment：Penicillium cammenberti lipase (LPC), Hua Gen
Mould lipase (Lipase RC), Pseudomonas cepacia lipase (PCL), porcine pancreatic lipase (PPL), Rhizopus niveus lipase
(Lipase RN), penicillium expansum lipase (PEL), aspergillus niger HGD0823 lipase；Immobilized lipase：LipozymeIM
TL、LipozymeIMRM, Novozym435, Immobilized Aspergillus niger HGD0823 lipase.Dihydromyricetin is urged in different lipase
Conversion ratio under changing is shown in Table 2 and table 3：
Conversion ratio of 2. dihydromyricetin of table under different free-fat enzymatics
Conversion ratio of 3. dihydromyricetin of table in the case where difference is fixed lipase catalyzed
The product that embodiment 1 and embodiment 2 obtain turns out to be dihydromyricetin 3-OH esterifications through nuclear magnetic resonance (NMR) analysis
Dihydromyricetin monoesters compound (the most weak hydroxy esterification of antioxidant activity, such as Formula II), yield is between 2~40%.
It is 40% or so that Novozym435 lipase yields are used in acetonitrile, is 25% with Immobilized Aspergillus niger HGD0823 lipase yields
Left and right.
1H NMR(600MHz,CD3OD/TMS):δ1.28(s,1H,OH19),1.97(s,3H,H25), 4.20 (d, J=
2.6Hz,1H,H14), 4.46 (d, J=11.3Hz, 2H, H2+H6), 5.23 (d, J=2.6Hz, 1H, H7+H9),5.90(d,2H,
H16),5.89–5.99(d,1H,OH20+OH22), 6.53 (d, J=7.7Hz, 1H, OH21), 6.53 (d, J=7.7Hz, 1H,
OH23).
13C NMR(151MHz,CD3OD/TMS):δ21.70(C25),73.34(C16),83.13(C14),96.46(C6),
97.42(C2),102.05(C4),107.85(C7,C9),128.24(C8),134.50(C11),147.00(C10,C12),164.59
(C5),165.50(C3),168.76(C1),196.71(C15),198.48(C24).
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
- A kind of 1. method using lipase-catalyzed synthesis dihydromyricetin monoesters compound, it is characterised in that：The method is With free or fixed lipase catalyzed dihydromyricetin and acry radical donor synthesizing dihydro myricetin monoesters compound；The dihydro Myricetin monoesters compound is 3 hydroxy esterifications of dihydromyricetin.
- 2. the method according to claim 1 using lipase-catalyzed synthesis dihydromyricetin monoesters compound, feature exists In：The free-fat enzyme includes：Penicillium cammenberti lipase, rhizopus chinensis lipase, Pseudomonas cepacia fat Enzyme, porcine pancreatic lipase, Rhizopus niveus lipase, penicillium expansum lipase；The immobilized lipase includes：LipozymeIM TL、LipozymeIM RM、Novozym 435。
- 3. the method according to claim 2 using lipase-catalyzed synthesis dihydromyricetin monoesters compound, feature exists In：Penicillium cammenberti lipase, rhizopus chinensis lipase, Pseudomonas cepacia lipase, porcine pancreatic lipase, Rhizopus niveus fat Fat enzyme, penicillium expansum lipase, LipozymeIM TL、LipozymeIMRM, Novozym 435 is directly commercially available from businessman.
- 4. the method according to claim 3 using lipase-catalyzed synthesis dihydromyricetin monoesters compound, feature exists In：The method of absorption method fixed fat enzyme includes the following steps：1. thick enzyme powder is dissolved in Glycine-NaOH buffer solution, 1~2h is stirred with 150~200r/min at 30~40 DEG C；2. being centrifuged at 4~10 DEG C with 5000r/min, supernatant is taken；③ After D4020 resins are impregnated for 24 hours with 95% ethyl alcohol, it is washed with deionized water until filtrate is clarified, is added to 2. supernatant In；4. under 35~40 DEG C, 150~200r/min, after vibrating 4~5h, filtered with sand core funnel, use Glycine-NaOH Wash buffer 3 times is freeze-dried after suction filtration and obtains immobilized lipase, in 0~4 DEG C of preservation.
- 5. the method according to claim 1 using lipase-catalyzed synthesis dihydromyricetin monoesters compound, feature exists In including the following steps：(1) dihydromyricetin and acyl donor are dissolved in organic solvent, free-fat enzyme or immobilized lipase are added, in 30 ~40 DEG C, 200~300r/min shaking tables react 1~2 day；(2) after reaction, reaction solution is centrifuged, takes supernatant, solvent is volatilized, obtained solid is containing dihydromyricetin The product of monoesters compound.
- 6. the method according to claim 5 using lipase-catalyzed synthesis dihydromyricetin monoesters compound, feature exists In：Acry radical donor described in step (1) includes：Vinyl acetate, vinyl propionate, vinyl butyrate, sad vinyl acetate, benzene Vinyl formate, stearic acid vinyl ester.
- 7. the method according to claim 5 using lipase-catalyzed synthesis dihydromyricetin monoesters compound, feature exists In：The molar ratio of dihydromyricetin and acyl donor is 1 in step (1):20~30.
- 8. the method according to claim 5 using lipase-catalyzed synthesis dihydromyricetin monoesters compound, feature exists In：Organic solvent described in step (1) includes：Acetonitrile, acetone, tetrahydrofuran, the tert-butyl alcohol, dimethyl sulfoxide (DMSO).
- 9. the method according to claim 5 using lipase-catalyzed synthesis dihydromyricetin monoesters compound, feature exists In：The amount of lipase described in step (1) is 250U/mL.
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| CN105385675A (en) * | 2015-12-31 | 2016-03-09 | 厦门大学 | Immobilization method of candida antarctica lipase B |
| CN113308506B (en) * | 2021-05-28 | 2023-03-21 | 华南理工大学 | Method for synthesizing dihydromyricetin-7-glucoside through biocatalysis |
| CN114807262B (en) * | 2022-05-25 | 2023-07-25 | 江南大学 | Dihydromyricetin acylated derivative and preparation method thereof |
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| CN1673223A (en) * | 2004-03-25 | 2005-09-28 | 广东省农业科学院蚕业与农产品加工研究所 | Dihydromyricitrin fatty ester preparing process |
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Patent Citations (4)
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| CN1673223A (en) * | 2004-03-25 | 2005-09-28 | 广东省农业科学院蚕业与农产品加工研究所 | Dihydromyricitrin fatty ester preparing process |
| CN101367959A (en) * | 2008-10-09 | 2009-02-18 | 中国科学院广州化学研究所 | Uses of tea polysaccharide extract-dihydromyricetin and its esterified matter as anti-oxidant in superpolymer |
| CN101485655A (en) * | 2009-02-12 | 2009-07-22 | 华南理工大学 | Application of dihydromyricetin in preparing medicament for preventing and treating adverse reaction of tumor chemoradiotherapy |
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