CN105403633B - Ceramide content assay method in konjaku - Google Patents
Ceramide content assay method in konjaku Download PDFInfo
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- CN105403633B CN105403633B CN201510718360.XA CN201510718360A CN105403633B CN 105403633 B CN105403633 B CN 105403633B CN 201510718360 A CN201510718360 A CN 201510718360A CN 105403633 B CN105403633 B CN 105403633B
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- Prior art keywords
- ceramide
- extract
- acetic acid
- ethanol
- ethyl acetate
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- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 title claims abstract description 27
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 title claims abstract description 27
- 229940106189 ceramide Drugs 0.000 title claims abstract description 27
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 title claims abstract description 27
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 229920002752 Konjac Polymers 0.000 title claims abstract description 12
- 238000003556 assay Methods 0.000 title claims abstract description 12
- UGAPHEBNTGUMBB-UHFFFAOYSA-N acetic acid;ethyl acetate Chemical compound CC(O)=O.CCOC(C)=O UGAPHEBNTGUMBB-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000469 ethanolic extract Substances 0.000 claims abstract description 10
- 239000002024 ethyl acetate extract Substances 0.000 claims abstract description 10
- 239000012086 standard solution Substances 0.000 claims abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000012467 final product Substances 0.000 claims description 8
- NPRJSFWNFTXXQC-GWGOZDFZSA-N n-[(e,2r,3s)-1,3-dihydroxyoctadec-4-en-2-yl]hexanamide Chemical compound CCCCCCCCCCCCC\C=C\[C@H](O)[C@@H](CO)NC(=O)CCCCC NPRJSFWNFTXXQC-GWGOZDFZSA-N 0.000 claims description 7
- 238000004090 dissolution Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000000105 evaporative light scattering detection Methods 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000012085 test solution Substances 0.000 claims description 4
- 238000001704 evaporation Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 238000000194 supercritical-fluid extraction Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 2
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 239000000463 material Substances 0.000 description 4
- 241001278826 Amorphophallus Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- -1 ceramide Chemical compound 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000010829 isocratic elution Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 241001312219 Amorphophallus konjac Species 0.000 description 1
- 235000001206 Amorphophallus rivieri Nutrition 0.000 description 1
- 241000209524 Araceae Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000000252 konjac Substances 0.000 description 1
- 235000010485 konjac Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses ceramide content assay method in a kind of konjaku：Ceramide acetic acid ethyl acetate extract, ceramide ethanol extract, standard solution are taken respectively, and according to chromatographic condition sample introduction, spectrogram is gathered by high performance liquid chromatography.Ceramide content assay method has the characteristics that sensitive, quick, favorable reproducibility in konjaku provided by the invention.
Description
Technical field
The present invention relates to ceramide content assay method in a kind of konjaku.
Background technology
Konjaku (konjac) belongs to Araeceae (Araceae) Amorphophallus (Amorphophallus) perennial herb
The content of plant, wherein ceramide is very high, up to 0.15%~0.2%.Konjaku is rich in ceramide type material as one kind
Resource plant cause the great interest of people, ceramide (ceramide), i.e. ceramide, be it is a kind of extensively
The signal transduction material being present in eukaryotic cells, as a kind of lipid molecular with the structure composition composition of cell membrane or born of the same parents
Interior free molecular flow forms exist, and participate in adjusting growth, suppression, differentiation, aging and apoptosis of cell etc..In recent years, neural acyl
Amine substance becomes emerging feature Medicines and Health Product, the important activity component of foods and cosmetics, greatly should have
With value.
The content of the invention
The present invention provides ceramide content assay method in a kind of konjaku.
The present invention adopts the following technical scheme that：
Ceramide content assay method in a kind of konjaku：
Standard solution, test solution are taken respectively, according to chromatographic condition sample introduction, are gathered and composed by high performance liquid chromatography
Figure, standard solution include ceramide acetic acid ethyl acetate extract and ceramide ethanol extract, wherein：
Ceramide acetic acid ethyl acetate extract：Precision measures the extract prepared through ethyl acetate technique, is placed in evaporating dish
In, in 70 DEG C of water bath methods, add methanol ultrasonic dissolution and constant volume, cross 0.45 μm of miillpore filter, to obtain the final product；
Ceramide ethanol extract：Precision is measured added with extract of the ethanol entrainer through means of supercritical extraction, adds volume
95% ethanol of fraction dilutes constant volume, then therefrom accurate measurement, adds 95% ethanol of volume fraction to dilute constant volume, crosses 0.45 μm of micropore filter
Film, to obtain the final product；
The preparation of standard solution：Accurately weighed ceramide C6 standard items, put in volumetric flask, add methanol ultrasonic dissolution simultaneously
Be settled to scale, cross 0.45 μm of miillpore filter, to obtain the final product.
Preferably, the chromatographic condition of the high performance liquid chromatography：Mobile phase solvent flow velocity 0.8-1.2ml/min；Acetic acid delays
Fliud flushing-methanol isocratic elution；25~40 DEG C of chromatographic column column temperature；Evaporative light scattering detector.
Preferably, the mobile phase solvent is 0.1% acetic acid of volume fraction：Methanol=7：93.
Preferably, the mobile phase solvent is 36% acetic acid of volume fraction：Methanol=7：93.
Preferably, the mobile phase solvent flow velocity 1ml/min.
Preferably, spectrogram acquisition time is 30 minutes.
Ceramide content assay method has the characteristics that sensitive, quick, favorable reproducibility in konjaku provided by the invention.
Brief description of the drawings
Fig. 1 is acetic acid ethyl acetate extract chromatogram of the present invention；
Fig. 2 is ethanol extract chromatogram of the present invention；
Fig. 3 is ceramide C6 canonical plottings of the present invention.
Embodiment
The following examples are that the present invention is described in further detail.
1 laboratory apparatus and material
1.1 laboratory apparatus
High performance liquid chromatograph (model：U3000；Producer：Thermo Fisher Scientific), evaporative light-scattering inspection
Survey device (model：M100)；Chromatographic column：Kromasil 100-5C18 (5 μm, 250 × 4.6mm)；Methanol (chromatographic grade)；36% second
Acid (analysis is pure)
1.2 material
Acetic acid ethyl acetate extract, ethanol extract.
2 experiment contents
2.1 chromatographic condition
Mobile phase：0.1% acetic acid (36% acetic acid)：Methanol=7：93；Isocratic elution；Gather 30min；35 DEG C of column temperature；Stream
Fast 1ml/min；Evaporative light scattering detector.
Remarks：1st, chromatographic column：Kromasil 100-5C18 (5 μm, 250 × 4.6mm)；Elution system：Acetate buffer solution-
Methanol；Gather 30min；25-40 DEG C of column temperature；Flow velocity 0.8-1.2ml/min；Evaporative light scattering detector
2nd, through comparing Kromasil C18 chromatographic columns, Kromasil 100-5C18 chromatographic columns, Innoval C18 chromatographic columns
The chromatogram of the anti-phase C18 silica gel chromatographic columns of three different bonding patterns can be drawn：Kromasil C18 chromatographic columns with
The peak response of Kromasil 100-5C18 chromatographic columns is consistent, is only had differences in the retention time at each peak.But for through nanometer
Innoval C18 chromatographic columns made of technology, its appearance time and peak response all there are larger with above two chromatographic column
Difference.
The preparation of 2.2 test solutions
(1) ceramide acetic acid ethyl acetate extract：Precision measures the extract that 2mL is prepared through ethyl acetate technique, is placed in
In evaporating dish, in 70 DEG C of water bath methods, add methanol ultrasonic dissolution and be settled to 10mL, cross 0.45 μm of miillpore filter, to obtain the final product.
(2) ceramide ethanol extract：Precision measures 0.2mL added with extraction of the ethanol entrainer through means of supercritical extraction
Thing, adds the dilution of 95% ethanol to be settled to 10mL, then therefrom accurate measurement 2mL, adds the dilution of 95% ethanol to be settled to 10mL, cross 0.45
μm miillpore filter, to obtain the final product.
The preparation of 2.3 standard solutions
Accurately weighed ceramide C6 standard items 5.04mg, put in 10mL volumetric flasks, add methanol ultrasonic dissolution and are settled to
Scale, crosses 0.45 μm of miillpore filter, to obtain the final product.
2.3 assay method
Standard solution, test solution are taken respectively, according to chromatographic condition sample introduction, gather spectrogram.
3 experimental results and conclusion
3.1 chromatographic peaks are pointed out
(1) acetic acid ethyl acetate extract：As shown in Figure 1, from above-mentioned chromatogram, draw in acetic acid ethyl acetate extract and only exist
Ceramide C6.
(2) ethanol extract：As shown in Fig. 2, from above-mentioned chromatogram, draw and ceramide is only existed in ethanol extract
C6。
The assay of ceramide C6 in 3.2 extracting solutions
3.2.1 the drafting of standard curve
As shown in figure 3, accurate measure prepared standard solution, difference 2,6,10,12,14,18 μ L of sample introduction, difference
Linear equation, Y=1.3785X++5.4958 establish numerical value to numerical value and peak area with sample size；R2=0.9985.
3.2.2 in extracting solution ceramide C6 assay result
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of changes, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (1)
- A kind of 1. ceramide content assay method in konjaku, it is characterised in that：Standard solution, test solution are taken respectively, and according to chromatographic condition sample introduction, spectrogram, mark are gathered by high performance liquid chromatography Quasi- product solution includes ceramide acetic acid ethyl acetate extract and ceramide ethanol extract, wherein：Ceramide acetic acid ethyl acetate extract：Precision measures the extract prepared through ethyl acetate technique, is placed in evaporating dish, in 70 DEG C of water bath methods, add methanol ultrasonic dissolution and constant volume, cross 0.45 μm of miillpore filter, to obtain the final product；Ceramide ethanol extract：Precision is measured added with extract of the ethanol entrainer through means of supercritical extraction, adds volume fraction 95% ethanol dilutes constant volume, then therefrom accurate measurement, adds 95% ethanol of volume fraction to dilute constant volume, crosses 0.45 μm of miillpore filter, i.e., ；The preparation of standard solution：Accurately weighed ceramide C6 standard items, put in volumetric flask, add methanol ultrasonic dissolution and constant volume To scale, cross 0.45 μm of miillpore filter, to obtain the final product；The chromatographic condition of the high performance liquid chromatography：Mobile phase solvent flow velocity 0.8-1.2ml/min；25 ~ 40 DEG C of chromatographic column column temperature； Evaporative light scattering detector；The mobile phase solvent is 0.1% acetic acid of volume fraction：Methanol=7：93 or 36% acetic acid of volume fraction：Methanol=7：93；Spectrogram acquisition time is 30 minutes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510718360.XA CN105403633B (en) | 2015-10-28 | 2015-10-28 | Ceramide content assay method in konjaku |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510718360.XA CN105403633B (en) | 2015-10-28 | 2015-10-28 | Ceramide content assay method in konjaku |
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| Publication Number | Publication Date |
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| CN105403633A CN105403633A (en) | 2016-03-16 |
| CN105403633B true CN105403633B (en) | 2018-05-11 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102198231A (en) * | 2011-04-07 | 2011-09-28 | 浙江大学 | Method for extracting ceramide-rich lipid from byproducts of agriculture and forestry by supercritical extraction technology |
| CN104083556A (en) * | 2014-07-15 | 2014-10-08 | 广州中大南沙科技创新产业园有限公司 | Method for extracting konjak ceramide from konjak |
-
2015
- 2015-10-28 CN CN201510718360.XA patent/CN105403633B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102198231A (en) * | 2011-04-07 | 2011-09-28 | 浙江大学 | Method for extracting ceramide-rich lipid from byproducts of agriculture and forestry by supercritical extraction technology |
| CN104083556A (en) * | 2014-07-15 | 2014-10-08 | 广州中大南沙科技创新产业园有限公司 | Method for extracting konjak ceramide from konjak |
Non-Patent Citations (4)
| Title |
|---|
| 四种魔芋中神经酰胺含量比较研究;周浩 等;《化学与生物工程》;20131231;第30卷(第4期);全文 * |
| 神经酰胺类物质的分离纯化及应用;刘仁萍 等;《湖北民族学院学报(自然科学版)》;20090331;第27卷(第1期);全文 * |
| 魔芋中神经酰胺类物质的HPLC-ELSD分析及其含量测定;刘仁萍 等;《中国生物化学与分子生物学报》;20100228;第26卷(第2期);摘要，第190页右栏 * |
| 魔芋神经酰胺的提取及其鞘磷脂含量的测定;刘仁萍 等;《天然产物研究与开发》;20111231;第23卷;摘要，第543页左栏 * |
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| CN105403633A (en) | 2016-03-16 |
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Inventor after: Ma Jinfang Inventor after: Li Haichi Inventor after: Ge Fahuan Inventor after: Zhang Xiangdong Inventor before: Ma Jinfang Inventor before: Lv Haichi Inventor before: Ge Fahuan Inventor before: Zhang Xiangdong |