CN103091446B - Lutein ester saponification degree rapid assay methods - Google Patents
Lutein ester saponification degree rapid assay methods Download PDFInfo
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- CN103091446B CN103091446B CN201310038072.0A CN201310038072A CN103091446B CN 103091446 B CN103091446 B CN 103091446B CN 201310038072 A CN201310038072 A CN 201310038072A CN 103091446 B CN103091446 B CN 103091446B
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Abstract
The invention provides a kind of lutein ester saponification degree rapid assay methods, the saponification liquor in pot marigold medicinal extract saponification process is dissolved in tetrahydrofuran, makes sample liquid; Silica gel is stuck with paste on the glass plate after being spread evenly across cleaning-drying, make chromatoplate; Extract sample liquid with microsyringe, chromatoplate dries up after point sample; Be made into developping agent with sherwood oil and ethyl acetate, the chromatoplate after point sample is put into developping agent, launch in dark place, when developping agent arrives chromatoplate top, taking-up chromatoplate dark place is air-dry; If the sample spot on air-dry chromatoplate does not launch, then pot marigold medicinal extract is fully saponified; If the sample spot on air-dry chromatoplate launches, then pot marigold medicinal extract is not fully saponified, continues saponification, the saponification process of duplicate detection pot marigold medicinal extract, until the sample spot on air-dry chromatoplate no longer launches, stops saponification.This detection method utilizes the difference of lutein ester and xenthophylls molecular melting, fast and convenient mensuration lutein ester saponification degree, and measurement result is accurate, Be very effective.
Description
Technical field
The invention belongs to technological field of biochemistry, relate to a kind of method measuring lutein ester saponification degree, particularly a kind of lutein ester saponification degree rapid assay methods.
Background technology
Xenthophylls is the one of carotenoid, is extensively present in natural plant.Because this natural colouring matter has lovely luster, strong coloring force, safety non-toxic, is rich in the features such as nutrition, be more and more subject to people's attention, be widely used in the fields such as food, cosmetics, bird feed.Colorant class (xenthophylls) feed addictive is exactly utilize the natural carotenol be rich in pot marigold, the lutein ester analog derivative in pot marigold medicinal extract is converted into free lutein and absorbs for bird.The technique of current employing is mostly first by medicinal extract alkaline saponification, then mixing is absorbed with absorbent, but be difficult point in the mensuration of saponification process Lutein ester saponification degree, lutein ester saponification degree measures general employing HPLC(high performance liquid chromatography) method, although the method is relatively more accurate, require very high to condition determination, apparatus expensive, standard items are not easily preserved, and measure length consuming time, instruct and have little significance in actual production.How fast and convenient whether mensuration lutein ester fully saponified is a technical barrier in producing.
Summary of the invention
The object of this invention is to provide a kind of lutein ester saponification degree rapid assay methods, not only result is accurate, and requires lower to condition determination, and minute is short.
The technical solution adopted in the present invention is: a kind of lutein ester saponification degree rapid assay methods, utilize lutein ester and the xenthophylls dissolubility in different solvents, adopt the mensuration lutein ester saponification degree that thin layer chromatography is fast and convenient, the concrete steps of this rapid assay methods are:
A) getting the saponification liquor generated in pot marigold medicinal extract saponification process is dissolved in tetrahydrofuran, makes sample liquid;
Cleaning glass plate, dry;
Silica gel is ground to form thick silica gel to stick with paste;
B) silica gel paste is uniformly coated on dried glass plate, dries, make chromatoplate;
C) extract sample liquid with microsyringe, at the silica gel laying surface point sample of chromatoplate, cold wind dries up;
D) sherwood oil and ethyl acetate are made into developping agent, the chromatoplate after point sample are put into the chromatography cylinder being marked with developping agent, developping agent liquid level is lower than sampling point, sample liquid on chromatoplate can not infiltrate in developping agent, launch in dark place, when developping agent arrives chromatoplate top, taking-up chromatoplate dark place is air-dry;
If the sample spot e) on air-dry chromatoplate does not launch, then illustrate that pot marigold medicinal extract is fully saponified, stops the saponification of pot marigold medicinal extract; If the sample spot on air-dry chromatoplate launches, then illustrate that pot marigold medicinal extract is not fully saponified, continue saponification pot marigold medicinal extract, and press the saponification process of above-mentioned steps duplicate detection pot marigold medicinal extract, until the sample spot on air-dry chromatoplate no longer launches, stop saponification.
Rapid assay methods of the present invention utilizes lutein ester and xenthophylls molecule deliquescent difference in a solvent, take tetrahydrofuran as lytic agent, with the mixed liquor of sherwood oil and ethyl acetate for developping agent, adopt the mensuration lutein ester saponification degree that thin layer chromatography is fast and convenient.Through HPLC(high performance liquid chromatography) method contrast, measurement result is accurate, and solve HPLC method very high to condition determination requirement, measure the shortcoming of length consuming time, in actual production, effect is remarkable.
Accompanying drawing explanation
Fig. 1 is the constitutional diagram after fully saponified lutein ester sample spot developping agent process.
Fig. 2 is the constitutional diagram after not fully saponified lutein ester sample spot developping agent process.
Fig. 3 is non-saponification sample spot chromatogram.
Fig. 4 is fully saponified sample spot chromatogram.
Embodiment
Because xenthophylls is exist with the form of lutein ester in lutein extract, therefore first the xenthophylls in pot marigold medicinal extract will carry out saponification before separation and purification, xenthophylls monomer is separated from lutein ester, and the quality of saponification effect can represent by saponification degree, the xenthophylls molecule of free state is very easily dissolved in polar organic solvent, be dissolved in (mg/L): acetone 800, second eyeball 100, benzene 600, chloroform 6000, cyclohexane 50, cyclohexanone 4000, methylene chloride 800, dimethyl methyl phthalein amine 1000, ethanol 300, ethyl acetate 800, ether 2000, hexane 20, isopropyl alcohol 400, methyl alcohol 200, tertiary butyric acid first vinegar 2000, tetrahydrofuran 8000, toluene 5000, xenthophylls molecule dissolubility in tetrahydrofuran is best, in sherwood oil and ethyl acetate, dissolubility is poor, and lutein ester dissolubility in sherwood oil and ethyl acetate is fine, in tetrahydrofuran, dissolubility is poor.The present invention utilizes lutein ester and the xenthophylls dissolubility in different solvents, and adopt the mensuration lutein ester saponification degree that thin layer chromatography is fast and convenient, concrete steps are:
A) get the saponification liquor 1.00g generated in pot marigold medicinal extract saponification process to be dissolved in 10ml tetrahydrofuran, make sample liquid;
Select the glass plate of flat smooth (specification is 10cm × 20cm), use washing lotion cleaning, drying;
Take 7g silica gel, add appropriate 0.1molL
-1bAS fully grinds in mortar, forms thick silica gel and sticks with paste, and when this silica gel paste sends greasy luster, stops grinding;
B) be uniformly coated on dried glass plate by silica gel paste, the overlay thickness that silica gel is stuck with paste is 0.4mm; Glass plate after laying is placed in 100 DEG C of baking ovens to dry, makes chromatoplate, be placed in exsiccator for subsequent use;
C) extract sample liquid with microsyringe, the horizontal line of distance chromatoplate base 2cm contacts laying surface gently, at laying surface point sample, the sample liquid amount of point sample is no more than 300 μ L, and hair-dryer cold wind dries up;
More than 300 μ L, easily there is hangover or diffusion in the amount of liquid of point sample.
D) by volume 4: 1, be that sherwood oil and the ethyl acetate of 60 ~ 90 DEG C is made into developping agent by boiling range, agents useful for same be analyze pure; Chromatoplate after point sample is put into the chromatography cylinder being marked with developping agent, developping agent liquid level is lower than sampling point 1cm, and the sample liquid on chromatoplate can not infiltrate in developping agent, launches in dark place, and when developping agent reaches chromatoplate top, take out chromatoplate, dark place is air-dry;
If the sample spot e) on air-dry chromatoplate does not launch, then illustrate that pot marigold medicinal extract is fully saponified, stops the saponification of pot marigold medicinal extract; If the sample spot on air-dry chromatoplate launches, then illustrate that pot marigold medicinal extract is not fully saponified, continue saponification pot marigold medicinal extract, and press the saponification process of above-mentioned steps duplicate detection pot marigold medicinal extract, until the sample spot on air-dry chromatoplate no longer launches, stop saponification.
Embodiment
Get the saponification liquor 1.00g generated in pot marigold medicinal extract saponification process to be dissolved in 10ml tetrahydrofuran, make sample liquid; The glass plate (specification is 10cm × 20cm) of flat smooth is cleaned by washing lotion, dry; Take 7g silica gel, add appropriate 0.1molL
-1bAS fully grinds in mortar, forms thick silica gel and sticks with paste, and when this silica gel paste sends greasy luster, stop grinding, stuck with paste by silica gel and be uniformly coated on dried glass plate, the overlay thickness that silica gel is stuck with paste is 0.4mm; Glass plate after laying is placed in 100 DEG C of baking ovens to dry, makes chromatoplate, be placed in exsiccator for subsequent use; Extract sample liquid with microsyringe, the horizontal line of distance chromatoplate base 2cm contacts laying surface gently, at laying surface point sample, the amount of liquid of point sample is no more than 300 μ L, and hair-dryer cold wind dries up; By volume 4: 1, be that sherwood oil and the ethyl acetate of 60 ~ 90 DEG C is made into developping agent by boiling range, agents useful for same be analyze pure; Chromatoplate after point sample is put into the chromatography cylinder being marked with developping agent, developping agent liquid level is lower than sampling point 1cm, and the sample liquid on chromatoplate can not infiltrate in developping agent, launches in dark place, and when developping agent reaches chromatoplate top, take out chromatoplate, dark place is air-dry; If the sample spot on air-dry chromatoplate does not launch, as shown in Figure 1, then illustrate that pot marigold medicinal extract is fully saponified, stops the saponification of pot marigold medicinal extract; If the sample spot on air-dry chromatoplate launches, as shown in Figure 2, then illustrate that pot marigold medicinal extract is not fully saponified, continue saponification pot marigold medicinal extract, and press the saponification process of above-mentioned steps duplicate detection pot marigold medicinal extract, until the sample spot on air-dry chromatoplate no longer launches, stop saponification.
Non-saponification pot marigold medicinal extract sample spot extending forwards on chromatoplate goes out very long distance, and fully saponified pot marigold medicinal extract is almost not dynamic on chromatoplate, this is because lutein ester dissolubility in developping agent is good, and xenthophylls monomer is poorly soluble in developping agent, developping agent on chromatoplate upwards dialysis time, non-saponification pot marigold medicinal extract sample liquid containing lutein ester stretches upwards with developping agent, its chromatogram is as Fig. 3, there is a top when sample introduction 0.715min in non-saponification pot marigold medicinal extract sample, peak area accounts for 84.50% of whole peak area.Fully saponified pot marigold medicinal extract sample liquid containing xenthophylls monomer then moves hardly on chromatoplate, its chromatogram is as Fig. 4, fully saponified pot marigold medicinal extract sample is an existing top when sample introduction 3.496min, peak area accounts for 87.15% of whole peak area, near 0.715min, there is not any peak, the whole saponification of lutein ester in medicinal extract is described.Therefore, can use the saponification degree of the extension degree qualitative representation pot marigold medicinal extract of sample liquid on chromatoplate, sample liquid does not stretch, explanation saponification is complete, sample liquid stretches, and illustrate that the saponification of pot marigold medicinal extract is incomplete, the degree that sample liquid stretches can represent the degree of pot marigold medicinal extract saponification.
comparative example
When detecting the saponification degree of pot marigold medicinal extract by HPLC method:
1) preparation of reagents
Normal hexane (analyzing pure), acetone (analyzing pure), absolute alcohol (analyzing pure) and toluene (analyzing pure) mix by 10 ︰ 7 ︰ 6 ︰ 7 by volume, are made into HEAT potpourri;
By 10.00g anhydrous Na
2sO
4(analyzing pure) dissolves, and is settled to 100ml, is made into 10% metabisulfite solution.
2) chromatographiccondition
Chromatographic column: TR-413212(TRACEREXTRSILSI) 3 μm 15 × 04.
Determined wavelength: 486nm.
Mobile phase: normal hexane (chromatographically pure): ethyl acetate (chromatographically pure)=75:25(volume ratio).
Flow velocity: 2.5ml/min, sample size: 25.00 μ l, time: 8min.
3) preparation of sample
3.1 accurately take with precision balance (sensitivity 0.1 milligram) sample liquid made in the unsaponified pot marigold medicinal extract of 30mg and embodiment respectively, put into respectively bottom 50ml volumetric flask, add 15mlHEAT potpourri, shake 3 minutes, sample is all dissolved.
3.2 rotate and within 1 minute, to be placed in 56 DEG C of hot water 10 minutes.
After 3.3 coolings, add 15ml normal hexane in volumetric flask, be then accurately settled to 50ml with 10% metabisulfite solution; Shaking is placed in dark, till limpid in 3 minutes.
3.4, with pipette, extract supernatant liquor 1ml, are placed in 25ml volumetric flask, use normal hexane constant volume.
3.5 are placed in a small test tube, vacuum drying, until liquid all evaporates in test tube with the above-mentioned liquid 1ml of pipette, extract.
V (normal hexane): V (ethyl acetate)=75:25(chromatographically pure is added in 3.6 test tubes after the drying, cross 25 μm of filter membranes) mixed liquor 1ml, shake gently, the sample in test tube all dissolved, filter by solid phase column, get 25 μ l filtrate analysis.
As can be seen from embodiment and comparative example, the inventive method can measure the saponification degree of pot marigold medicinal extract qualitatively, measurement result conforms to HPLC method measurement result, the extension degree qualitative representation pot marigold medicinal extract saponification degree of sample spot on chromatoplate can be used, sample spot does not stretch, and illustrate that saponification is complete, sample spot stretches, illustrate that medicinal extract saponification is incomplete, the degree that sample spot stretches can represent the degree of medicinal extract saponification.
Claims (4)
1. a lutein ester saponification degree rapid assay methods, utilize lutein ester and the xenthophylls dissolubility in different solvents, adopt the mensuration lutein ester saponification degree that thin layer chromatography is fast and convenient, it is characterized in that, the concrete steps of this rapid assay methods are:
A) getting the saponification liquor generated in pot marigold medicinal extract saponification process is dissolved in tetrahydrofuran, makes sample liquid;
Cleaning glass plate, dry;
Take 7g silica gel, add appropriate 0.1molL
-1bAS fully grinds in mortar, forms thick silica gel and sticks with paste;
B) be uniformly coated on dried glass plate by silica gel paste, the overlay thickness that silica gel is stuck with paste is 0.4mm; Glass plate after laying is placed in 100 DEG C of baking ovens to dry, makes chromatoplate;
C) extract sample liquid with microsyringe, at the silica gel laying surface point sample of chromatoplate, cold wind dries up;
D) by volume 4: 1, be that sherwood oil and the ethyl acetate of 60 ~ 90 DEG C is made into developping agent by boiling range; Chromatoplate after point sample is put into the chromatography cylinder being marked with developping agent, developping agent liquid level is lower than sampling point, and the sample liquid on chromatoplate can not infiltrate in developping agent, launches in dark place, and when developping agent arrives chromatoplate top, taking-up chromatoplate dark place is air-dry;
If the sample spot e) on air-dry chromatoplate does not launch, then illustrate that pot marigold medicinal extract is fully saponified, stops the saponification of pot marigold medicinal extract; If the sample spot on air-dry chromatoplate launches, then illustrate that pot marigold medicinal extract is not fully saponified, continue saponification pot marigold medicinal extract, and press the saponification process of above-mentioned steps duplicate detection pot marigold medicinal extract, until the sample spot on air-dry chromatoplate no longer launches, stop saponification.
2. according to lutein ester saponification degree rapid assay methods according to claim 1, it is characterized in that, in described step a), 1.00g saponification liquor is dissolved in 10ml tetrahydrofuran and makes sample liquid.
3., according to lutein ester saponification degree rapid assay methods according to claim 1, it is characterized in that, in described step c), sample liquid is extracted with microsyringe, point sample on the horizontal line of distance chromatoplate base 2cm, the sample liquid amount of point sample is no more than 300 μ L, and hair-dryer cold wind dries up.
4. according to lutein ester saponification degree rapid assay methods according to claim 1, it is characterized in that, in described step d), the chromatoplate after point sample is put into developping agent, developping agent liquid level is lower than sampling point 1cm.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5648564A (en) * | 1995-12-21 | 1997-07-15 | Kemin Industries, Inc. | Process for the formation, isolation and purification of comestible xanthophyll crystals from plants |
| WO1999020587A1 (en) * | 1997-10-21 | 1999-04-29 | Office Of Technology Liaison | Process for extraction and purification of lutein, zeaxanthin and rare carotenoids from marigold flowers and plants |
| CN1436774A (en) * | 2002-02-05 | 2003-08-20 | 海宁凤鸣叶绿素有限公司 | Lutein crystal preparing process from marigold flower |
| CN1810785A (en) * | 2005-12-21 | 2006-08-02 | 华东理工大学 | Prepn of lutein fally ester and lutein |
-
2013
- 2013-01-31 CN CN201310038072.0A patent/CN103091446B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5648564A (en) * | 1995-12-21 | 1997-07-15 | Kemin Industries, Inc. | Process for the formation, isolation and purification of comestible xanthophyll crystals from plants |
| WO1999020587A1 (en) * | 1997-10-21 | 1999-04-29 | Office Of Technology Liaison | Process for extraction and purification of lutein, zeaxanthin and rare carotenoids from marigold flowers and plants |
| CN1436774A (en) * | 2002-02-05 | 2003-08-20 | 海宁凤鸣叶绿素有限公司 | Lutein crystal preparing process from marigold flower |
| CN1810785A (en) * | 2005-12-21 | 2006-08-02 | 华东理工大学 | Prepn of lutein fally ester and lutein |
Non-Patent Citations (4)
| Title |
|---|
| Carotenoi pigments i goldfish (Carassius auratus): I. Composition and distribution of carotenoids;Masahiro Hataa et al;《International Journal of Biochemistry》;19710228;第2卷(第7期);11-19 * |
| 万寿菊中叶黄素及黄酮的提取与纯化工艺研究;李刚刚;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》;20110415(第04期);B016-203 * |
| 薄层扫描法测定万寿菊中叶黄素的含量;张志强 等;《化学世界》;20090930;第50卷(第9期);536-539 * |
| 高纯度叶黄素的制备及其衍生物的合成;申利英;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑 》;20091115(第11期);B024-47 * |
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