CN101869348B - Method for extracting antioxidation active substance from Cichorium endivia L. - Google Patents
Method for extracting antioxidation active substance from Cichorium endivia L. Download PDFInfo
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- CN101869348B CN101869348B CN2010102096068A CN201010209606A CN101869348B CN 101869348 B CN101869348 B CN 101869348B CN 2010102096068 A CN2010102096068 A CN 2010102096068A CN 201010209606 A CN201010209606 A CN 201010209606A CN 101869348 B CN101869348 B CN 101869348B
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Abstract
The invention relates to a method for extracting an antioxidation active substance from Cichorium endivia L., which adopts ethanol extraction, petroleum ether and ethyl acetate extraction for purification, macroporous absorption resin elution, concentration and drying to finally obtain the substance with high antioxidation activity and belongs to the field of natural product extraction. The Cichorium endivia L. extract has high radical-removing, antioxidation and reducing capabilities, the activity of the extract is higher than the activity of water-soluble vitamine E, and an erythrocyte hemolysis experiment indicates that the Cichorium endivia L. extract also has the antioxidation capability at the cell level. The method, which adopts Cichorium endivia L. as raw material to prepare the high-antioxidation active substance, has the advantages of simple technique and equipment and less investment, and is suitable for mass production and applicable to industries such as medicine and food additives, health-care products and cosmetics.
Description
Technical field
The invention belongs to field of natural product extraction, particularly a kind of method of from the cultivation witloof, extracting the antioxidation activity thing.This extract has the natural anti-oxidation activity, can make antioxidant, is used for preventing Food Oxidation, and prevent disease improves immunity of organisms, and the health products for promoting longevity.
Technical background
Free radical and active oxygen in close relations thereof can cause lipid peroxidation, and accelerate the oxidizing process of lipid, thereby destroy taste and the quality that is rich in oil and fat product.In addition, they also can encroach on many important biomolecule in the organism, such as cell membrane, protein and DNA, cause the damage of cell and tissue.Therefore preventing and remove free radical and active oxygen can be to the preservation of food and human healthy of great advantage, and the use of antioxidant then is topmost means of prevention wherein.Present antioxidant is mainly with manually synthesizing the master, because its toxic action is limited in the use significantly.As everyone knows, all contain numerous active components that serve as antioxidant in a lot of vegetables and the fruit, they can remove free radical, singlet-oxygen quenching or chelated metal ions, these effects have not only represented the nutritive value of vegetable and fruit, also point out them to can be developed into potentiality into antioxidant.
Cultivation witloof (Cichorium endivia L.) is composite family Cichorium plant, claims again hare's-lettuce, or bitter chrysanthemum.It is salad auxiliary material or the edible vegetable that is loved by the people in Europe, and China has also had artificial introducing and planting at present.According to bibliographical information (PapettiA, Daglia M, Gazzani G.Anti-and pro-oxidant water soluble activity of Cichorium genusvegetables and effect of thermal treatment.[J] .J Agric Food Chem, 2002,50 (16): 4696-4704 and Papetti A, Daglia M, Grisoli P, et al.Anti-and pro-oxidant activity of Cichorium genus vegetablesand effect of thermal treatment in biological systems.[J] .Food Chemistry, 2006,97 (1): 157-165.), the aqueous solution of Cichorium plant (comprising the cultivation witloof) has removing DPPH, the ability of the free radical such as ROO and OH, and the aqueous solution of cultivation witloof can also prevent CCl
4The rat hepatocytes microsomal membrane oxidative damage of inducing causes the bacterium damage of Gram-positive gold Portugal that the effect of protection is arranged to isopropyl benzene hydroperoxide, can demonstrate,prove thus the antioxidation activity of cultivating witloof.And the research of so far there are no bitter chrysanthemum polyphenoils extraction process report.
Summary of the invention
The purpose of this research provides a kind of low cost, is suitable for method industrialization development, extract anti-oxidation active substance from bitter chrysanthemum
The present invention is achieved through the following technical solutions:
(1) will cultivate witloof and dry in cool place place, and after the pulverizing, add concentration and be 20～100% ethanolic solution and extract, withdrawal ratio 1: 5～1: 30 extracts 1～4h, repeats lixiviate 4 times, filters.
(2) after extract concentrates, be suspended in 30～95% ethanolic solutions, with petroleum ether extraction 1～6 time (ratio of benzinum and suspension is 1: 1～1: 10).
(3) water layer behind the petroleum ether extraction is volatilized to without the alcohol flavor, add the water suspendible, with ethyl acetate extraction 1～4 time (ratio of ethyl acetate and suspension is 1: 1～1: 10).
(4) water layer behind the ethyl acetate extraction is crossed macroporous absorbent resin, water and 20～95% ethanol elutions, and wherein alcohol layer is through concentrated, and vacuum drying obtains anti-oxidation active substance.
The present invention cultivates chicory extract (KJ-I) and comprises the active ingredients such as Kaempferol, Kaempferol-3-O-β-D-Glucose, adenosine.
The present invention cultivates chicory extract can not only effectively remove various free radicals (such as superoxide anion, hydroxyl free and hydrogen peroxide), can also remarkable inhibitory action be arranged and to organizing lipid peroxidation that very strong inhibitory action is arranged to the body erythrocyte hemolysis.
The present invention has the characteristics of antioxidation activity, can be used as the antioxidation that food antioxidant is applied to food, and is applied to the control of disease and the health products of promoting longevity.Simultaneously, this plant resources is abundant, extracts simple.
The specific embodiment
The preparation of 1 cultivation chicory extract (KJ-I)
The present invention is described in further detail by following examples, but the technology contents under the present embodiment is illustrative, rather than determinate, should not limit to protection scope of the present invention with this.
Method step from cultivation witloof extraction anti-oxidation active substance involved in the present invention is as follows:
Cultivation witloof available from food market, Xinfadi, Beijing, dry herb 5.8kg is extracted three (80L * 2h in 95% alcohol reflux, 70L * 1h, 68L * 1h), filter merging filtrate, reduced pressure concentration, gained medicinal extract is suspended in (cumulative volume 3.5L) in 70% ethanolic solution, petroleum ether extraction four (2L, 1.5L, 1.5L, 1.5L), the alcohol layer is evaporated to without the alcohol flavor, adds approximately 2.3L of water suspendible, three (1.5L of ethyl acetate extraction, 1.2L, 1.2L), water layer is crossed macroporous absorbent resin, water and 60% ethanol elution, 60% ethanol partial concentration, vacuum drying.
Determining of 2 extract components
KJ-I is dissolved 5%NaHCO with the 500ml n-butanol
3Extract three times, each 200ml, then the distilled water extraction is 2 times, and each 200ml to neutral, volatilizes n-butanol, obtains some dry products.This dry product separates repeatedly through high performance liquid chromatography, and mass spectrum and nuclear-magnetism are identified, wherein comprised the active ingredients such as Kaempferol, Kaempferol-3-O-β-D-Glucose, adenosine.Structural Identification is as follows:
Kaempferol (kaempferol)
1H NMR (300MHz, DMSO) δ: 12.47 (1H, s, 5-OH), 10.14 (3H, brs, 3,7,4 '-OH), 8.03 (1H, d, J=8.7Hz, H-2 ', 6 '), 6.91 (1H, d, J=8.7Hz, H-3 ', 5 '), 6.42 (1H, s, H-8), 6.17 (1H, s, H-6).
Kaempferol-3-O-β-D-Glucose (kaempferol-3-β-D-glucoside)
1H NMR (300MHz, DMSO) δ: 12.61 (1H, s, 5-OH), 10.20 (3H, brs, 3,7,4 '-OH), 8.03 (1H, d, J=8.7Hz, H-2 ', 6 '), 6.88 (1H, d, J=8.7Hz, H-3 ', 5 '), 6.42 (1H, s, H-8), 6.20 (1H, s, H-6), 5.45 (1H, d, J=7.0Hz, H-1 "), 5.30-3.08 (hydrogen on the glucose).
Adenosine ESI-MS m/z:268[M+H]+, 290[M+Na]+.
1H NMR (300MHz, D
2O) δ: 8.25 (1H, s, H-2), 8.08 (1H, s, H-8), 6.01 (1H, d, J=6.0Hz), 4.77 (1H, H-2 '), 4.42 (1H, m, H-3 '), 4.29 (1H, s, H-4 '), 3.92 (1H, m, H-5 '), (3.80 1H, m, H-5 ').
The measurement result of 3 antioxidation activities
KJ-I is mixed with certain density solution with dimethyl sulfoxide (DMSO) (DMSO) dissolving, with Trolox (water miscible vitamin E) and the positive contrast of Vc (vitamin C), carry out DPPH radicals scavenging test, the test of ORAC oxidation resistance, the reducing power test of good fortune pouring phenol and erythrocyte hemolysis experimental test.Experimental technique is as follows:
(1) DPPH radicals scavenging test
The reaction system of scavenging ability of DPPH free radical test is carried out in 96 orifice plates, and every hole adds certain density bitter chrysanthemum extract or positive control drug 20 μ l, and then adding rapidly final concentration is 150 μ molL
-1DPPH (being dissolved in absolute ethyl alcohol) 180 μ l, adopt ELIASA to detect OD value in 517nm wavelength place behind the concussion mixing, simultaneously with 20 μ lDMSO replacement test fluid as blank.Through the detection of a plurality of time points, no longer change as final measured value with the OD value of test fluid.Concentration (EC when the Green Tea Extract activity reaches the 50%DPPH clearance rate with test fluid
50) express, DPPH clearance rate formula is:
The DPPH clearance rate=(1-ODi/ODf) * 100%
The OD value of DPPH solution when wherein ODi refers to add test fluid, ODf is the OD value of the DPPH solution of blank; Clearance rate according to the test fluid variable concentrations is calculated EC as curve
50
(2) ORAC oxidation resistance test
Preparation 75mM kaliumphosphate buffer, adjust pH to 7.4.Use kaliumphosphate buffer preparation Fluress (final concentration is 61.2nM) and 2,2-azo-two-(2-amidine propane) chlorination dihydro (AAPH) solution (final concentration is 19.1mM), and become finite concentration with the kaliumphosphate buffer dilute sample with positive control drug.Reaction system is carried out in the luciferase target.Concrete operation step is with reference to table 1:
Table 1 ORAC oxidation resistance test operating procedure (unit: μ L)
The fluorescence intensity data of each micropore of experiment gained outputs in the Excel form by software, carries out next step processing.The absolute fluorescence intensity data of each micropore different time points is compared with the fluorescence intensity of its initial time, is converted to relative intensity of fluorescence f, adopts approximate integration to calculate area (AUC) under the fluorescence decline curve with relative intensity of fluorescence, and its formula is
AUC＝0.5×[2×(f
0+f
1+…+f
n-1+f
n)-f
0-f
n]×Δt
F wherein
nThe relative intensity of fluorescence that represents n measuring point, Δ t are the time intervals between adjacent two time points.
By the time meta-relative intensity of fluorescence mapping; area poor under the fluorescence decline curve of area Free Radical when existing without antioxidant under the fluorescence decline curve under the antioxidant action; the oxyradical that is antioxidant is removed ability ORAC value; the ORAC that is called again antioxidant is to compare with the protected area of standard polyphenoils (Trolox) by the protected area under the fluorescence decline curve to draw.The ORAC value is with trolox equivalent (μ mol Trolox equivalent/mg) express.
(3) good fortune is drenched the test of phenol reducing power
Reaction system detects at 96 orifice plates and ELIASA.Method is as follows: the drug component, monomer and the positive drug that are dissolved in DMSO are diluted respectively and are 2g/L.Above reagent solution is got respectively the distilled water that 10 μ L are dissolved in 600 μ L, mixing, and every hole adds 150 μ L in 96 orifice plates, then adds successively good fortune and drenches phenol reagent 12.5 μ L, the Na of distilled water 50 μ L and 1M
2CO
337.5 μ L.Place 37 ℃ of baking oven 2h, use ELIASA in wavelength 725nm place, measure the OD value.The result is with gallic acid equivalant (μ mol gallic acid equivalant/mg) express.
Operating procedure is with reference to table 2:
Table 2 good fortune is drenched phenol reducing power experimental implementation step
(4) erythrocyte hemolysis test
Get approximately 5～10ml of blood from family's rabbit ear central artery, put into the triangular flask jolting 10 minutes of bead, add again PBS, after shaking up with suspension with the centrifugal 5min of 2500rpm, abandon supernatant, repeatedly wash red blood cell 3～4 times, until the supernatant water white transparency.The gained red blood cell is made into 2% red blood cell suspension by its volume with PBS, is for experiment.Prepare 2% red cell suspension, add 37 ℃ in sample and hatch 30min, then add the radical initiator 2 of 50mM, 2-azo-two-(2-amidine propane) chlorination dihydro (AAPH) is hatched 4h for 37 ℃.AAPH can cause erythrocyte membrane phospholipid generation peroxidization, causes EF, discharges hemoglobin.Capable 4000rpm is centrifugal to red cell suspension, gets supernatant, uses ELIASA to measure the OD value under the 545nm wavelength.
Erythrocyte hemolysis rate (%)=A/B * 100%, A: the OD value of sample system, B: complete hemolysis OD value
Above experimental result is with reference to table 3 and 4.
Table 3 sample is removed DPPH free radical, the anti-oxidant test of ORAC and good fortune and is drenched the test of phenol reducing power
Table 4 sample suppresses the erythrocyte hemolysis test
Above result shows, cultivates chicory extract KJ-I and has certain removing free radical, anti-oxidant and reducing power, and the specific activity watermiscible vitamin E is strong, and erythrocyte hemolysis test prompting KJ-I also has oxidation resistance on cellular level.
Claims (4)
1. method of from the cultivation witloof, extracting antioxidative active extractive, it is characterized in that: the step of its extracting method is as follows:
Get the cultivation witloof, dry herb 5.8kg is extracted three times with 95% alcohol reflux, ethanol consumption and extraction time are respectively 80L * 2h, 70L * 1h, 68L * 1h, filter, merging filtrate, reduced pressure concentration, gained medicinal extract are suspended in 70% ethanolic solution, its cumulative volume is 3.5L, petroleum ether extraction four times, the benzinum consumption is respectively 2L, 1.5L, 1.5L, 1.5L the alcohol layer is evaporated to without the alcohol flavor, adding the water suspendible is 2.3L, ethyl acetate extraction three times, consumption is respectively 1.5L, 1.2L, 1.2L, water layer is crossed macroporous absorbent resin, water and 60% ethanol elution, 60% ethanol partial concentration, vacuum drying obtains active ingredient and comprises Kaempferol, the cultivation chicory extract of Kaempferol-3-O-β-D-Glucose and adenosine.
2. a cultivation chicory extract is characterized in that obtaining by the preparation method of claim 1, and its active ingredient comprises Kaempferol, Kaempferol-3-O-β-D-Glucose and adenosine.
3. cultivation chicory extract claimed in claim 2 is as the application of antioxidant from natural food in preparation food, medicine, cosmetics or health products
4. the application of cultivation chicory extract claimed in claim 2 in preparation removing human free radical or antidotal food, medicine, cosmetics or health products.
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| CN102600223A (en) * | 2011-01-25 | 2012-07-25 | 王爱平 | Application of Cichorium endivia L. extract to liver protection or therapeutic agent |
| CN102512491A (en) * | 2012-01-01 | 2012-06-27 | 山东大学威海分校 | Preparation method for meehania urticaefolia preparation and anti-oxidation application thereof |
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| CN1587251A (en) * | 2004-07-20 | 2005-03-02 | 北京化工大学 | Process for preparing chicoric acid from echinacea purpurea |
| CN1660769A (en) * | 2004-12-09 | 2005-08-31 | 西北农林科技大学 | Process for preparing chicoric acid from chicory |
| CN1899341A (en) * | 2006-07-27 | 2007-01-24 | 上海林赛娇生物科技发展有限公司 | American goldenrod herb total flavone extract and its preparing method and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1587251A (en) * | 2004-07-20 | 2005-03-02 | 北京化工大学 | Process for preparing chicoric acid from echinacea purpurea |
| CN1660769A (en) * | 2004-12-09 | 2005-08-31 | 西北农林科技大学 | Process for preparing chicoric acid from chicory |
| CN1899341A (en) * | 2006-07-27 | 2007-01-24 | 上海林赛娇生物科技发展有限公司 | American goldenrod herb total flavone extract and its preparing method and use |
Non-Patent Citations (2)
| Title |
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| 杨文志等.毛菊苣的化学成分.《中国天然药物》.2009,第7卷(第3期), * |
| 秦俊哲等.苦苣菜黄酮超声提取工艺的研究.《陕西科技大学学报》.2010,第28卷(第2期), * |
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