CN103808855B - A kind of method and HPTLC scanning method detection method extracting oestrone from krill - Google Patents
A kind of method and HPTLC scanning method detection method extracting oestrone from krill Download PDFInfo
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- CN103808855B CN103808855B CN201410034815.1A CN201410034815A CN103808855B CN 103808855 B CN103808855 B CN 103808855B CN 201410034815 A CN201410034815 A CN 201410034815A CN 103808855 B CN103808855 B CN 103808855B
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Abstract
The invention discloses a kind of from krill, extract oestrone method and HPTLC scanning method detection method, take krill as raw material, extracts with methyl alcohol, steaming instrument evaporate to dryness filtrate again, obtaining the crude extract containing oestrone after filtration with revolving.The detection of krill oestrone adopts HPTLC scanning method method, developping agent is volume ratio is phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: the mixed liquor of cyclohexane=78-80:9-11:9-11:2-4:3d:2d, and coloring agent is 5% ferric chloride in aqueous solution.With CAMAG TLC-scanner-III, with 600nm absorbing wavelength Scanning Detction, calculated the content of oestrone by the absorbance detecting sample and standard items, the method is simple to operate, quick, accurate, reliable.
Description
Technical field
The present invention relates to a kind of from krill, extract oestrone method and HPTLC scanning method detection method.
Background technology
Oestrone [1,3,5 (10)-estratriene-3-alcohol-17-ketone or 3-hydroxyl-1,3,5 (10)-estratriene-17-ketone] or E1 are molecular weight is 270.37 daltonian C18 steroid hormones, and its molecular formula is C
18h
22o
2, be the one of female hormone, its physiological action is estrogenic physiological action.
Krill (Euphausiasuperba), is the krill of one way of life in waters, the Antarctica, lives in the mode of trooping, and density only can reach every cubic metre of 10000-30000 sometimes.They are using small phytoplankton as food, and the about 6cm of height, heavily about 2g, MaLS can reach 6 years.They are key species of Antarctic ecosystems, if with biomass energy, they may be the most successful living species on the earth.Because krill reserves are huge, add that the life of colony is easily fished for, so attract a lot of country to develop it.At present, the main purposes of krill is as fish meal.Then euphausia superba sauce is extracted as health products in the state such as Norway, Canada, price about 2000 yuan/kg.Because krill contains a large amount of fluorine (about 2400mgkg
-1), can not directly eat.Thus, take krill as raw material, exploitation natural drug, to its comprehensive utilization, has broad application prospects.
The detection of oestrone adopts the methods such as gas chromatographic technique, liquid chromatography technology, Placenta function, colourimetry to carry out more, but these detection methods have following weak point: testing process length consuming time, costly, may cause environmental pollution etc.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of detection method of krill oestrone simple to operate, quick, accurate, reliable.
The present invention is achieved by the following technical solutions:
A HPTLC scanning method detection method for oestrone, step is as follows:
(1) sample liquid to be detected and 1.0mgmL is got
-1titer 5 μ L point in same GF
254on High Performance Thin silica gel plate, take volume ratio as phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=78-80:9-11:9-11:2-4:3d:2d is developping agent, launches in expansion cylinder, exhibition, apart from being 9.5cm, dries panel after having opened up;
(2) with 5% ferric chloride in aqueous solution dyeing, at being then placed in 103 DEG C-107 DEG C, 8-10 minute is toasted;
(3) scan with CAMAG thin-layer chromatogram scanner-III, scanning wavelength 600nm, calculated the content of oestrone by the absorbance detecting sample and standard items.
Sample liquid is obtained by being dissolved in by the crude extract of oestrone in 5mL methyl alcohol.
The extraction of the crude extract of oestrone, step is as follows:
(1) get dry krill (w) and methyl alcohol (v) with the ratio of 1:8-11 35 DEG C, ultrasonic extraction under the condition of 350w, 40kHz, filter extract;
(2) above-mentioned filtrate being placed in is revolved steaming instrument evaporate to dryness, obtain the crude extract of oestrone;
When launching in expansion cylinder, first by developping agent pre-equilibration 35-45 minute at ambient temperature, put into High Performance Thin plate again, continue to launch at ambient temperature, when opening up apart from when reaching 4.5cm, taking out thin layer plate, under room temperature, drying 10 minutes, again thin layer plate is put into expansion cylinder, the exhibition that is deployed into is apart from being 9.5cm.
Production standard curve:
(1) precision takes 1.0000mg oestrone standard items (J & K company) and is dissolved in 1.0mL methyl alcohol, and obtaining concentration is 1.0mgmL
-1titer;
(2) get the oestrone standard solution 2 μ L, 3 μ L, 4 μ L, 7 μ L, the 8 μ L that configure, point sample, in same silica gel plate, launches by the HPTLC scanning method detection method condition of oestrone, dries;
(3) with after 5% ferric chloride in aqueous solution dyeing, with the scanning of CAMAG thin-layer chromatogram scanner-III, 600nm absorbing wavelength, obtain regression equation Y=-275.7133+742.4928X through thin-layer chromatogram scanner software wincats1.4.1 statistics, relative coefficient r=0.99916, RSD=2.77%.
The High Performance Thin scanning detection method of detection oestrone of the present invention, its degree of accuracy, repeatability, stability, recovery situation, developping agent formula are determined as follows:
1. precision measures
Accurate pipette samples liquid, horizontal point sample 5 μ L on same High Performance Thin silica gel plate, launch, dry, with CAMAG thin-layer chromatogram scanner-III after 5% ferric chloride in aqueous solution dyeing, scan peak area is respectively 1803.8 with 600nm absorbing wavelength, 1651.7,1655.1,1729.3,1775.0, RSD=3.99%.
2. repeatability measures
Accurate pipette samples liquid 5 μ L, difference point sample on five blocks of High Performance Thin silica gel plates, launch, dry, with CAMAG thin-layer chromatogram scanner-III after 5% ferric chloride in aqueous solution dyeing, 1670.0 are respectively with the peak area of 600nm absorbing wavelength scanning, 1688.2,1663.1,1642.1,1655.1, RSD=1.03%.
3. Stability Determination
Get 5 μ L sample liquid point sample on High Performance Thin plate, launch, dry, with CAMAG thin-layer chromatogram scanner-III after 5% ferric chloride in aqueous solution dyeing, scan with 600nm absorbing wavelength, later every 10 minutes run-downs, record each peak area, calculate corresponding RSD value, test of many times proves, more stable in 40-80 minute after dyeing, average RSD=1.53%.Concrete data are as shown in table 1:
Table 1
| 40min | 50min | 60min | 70min | 80min | RSD |
| 776.6 | 774.9 | 758.0 | 755.5 | 749.3 | 1.60% |
| 799.1 | 795.4 | 778.0 | 775.5 | 770.1 | 1.44% |
| 811.1 | 809.3 | 790.7 | 786.3 | 782.1 | 1.54% |
4. recovery test
Accurate absorption 6 μ L sample liquid 3 times, adds 1.554 μ g, 3.107 μ g, 4.661 μ g oestrone standard items respectively, point sample, launches, dries, with CAMAG thin-layer chromatogram scanner-III after dyeing, with the scanning of 600nm absorbing wavelength, calculating average recovery rate is 101.0%, RSD=0.71%.Data are as shown in table 2:
Table 2
| Sample size/μ g | Add standard items amount/μ g | Measured amount/μ g | Recovery % |
| 3.107 | 1.554 | 4.660 | 100.2 |
| 3.107 | 3.107 | 6.270 | 101.4 |
| 3.107 | 4.661 | 7.838 | 101.5 |
5. developping agent formula is determined
When developping agent is set as different formulas, each combination, all carried out ratio debugging repeatedly, separating effect is as shown in table 3:
Table 3
The present invention produce beneficial effect:
Be material extraction oestrone with krill, have the following advantages: krill biomass is large, the material for content pettiness carries out extractions research by strengthening krill radix amount; Krill can carry out interpolation as a kind of nutritious feed and use, and oestrone belongs to the estrogen harmful to biosome, by detecting oestrone content in krill, has the certain significance to the food security research of krill; Take krill as extraction and the detection that raw material carries out oestrone, there is very strong novelty.
Utilize HPTLC scanning method detection method to analyze the content of oestrone, automaticity is high, and sensitivity is strong.Compared with other detection methods, High Performance Thin Layer Chromatography detection method needs less reagent, and this has good protective effect to environment; Whole analyte detection process is very of short duration.
It take volume ratio as phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: the developping agent that the proportioning of cyclohexane=78-80:9-11:9-11:2-4:3d:2d mixes, make the oestrone in sample liquid and other component separating clear, spot circle and clear, standard items point and sample spot accurately corresponding, Rf value is 0.62, is that in krill, oestrone launches the good formula be separated.
Employing ferric trichloride dyes, and avoids and adopts sulfuric acid dye the carbonization thin layer plate caused, make the drawback of thin layer plate blackening, is unfavorable for observing and having considerable influence to absorption values, thus makes quantitative test become difficult.
This detection method is simple, quick, accurate, reliable, stable.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1
Get dry krill 1g, add methyl alcohol 9mL, 35 DEG C, Ultrasonic Heating extracts in the supersonic wave cleaning machine of 350w, 40kHz, extracts 10 minutes, filters extract, filtrate being placed in is revolved steaming instrument evaporate to dryness, obtain the crude extract of oestrone.
Above-mentioned obtained oestrone crude extract is dissolved in 5mL methyl alcohol and makes sample liquid; Precision takes 1.0000mg oestrone standard items (J & K company) and is dissolved in 1.0mL methyl alcohol, and obtaining concentration is 1mgmL
-1titer.
Sample thief liquid and titer 5 μ L point are in same GF
254high Performance Thin silica gel plate (10 × 10cm
2) on, developping agent volume ratio is phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=78-80:9-11:9-11:2-4:3d:2d, developping agent is added expansion cylinder (10 × 12 × 15cm
3), at room temperature pre-equilibration 35 minutes, then put into High Performance Thin plate, continue at room temperature to launch, when opening up apart from when reaching 4.5cm, taking out thin layer plate, drying 10 minutes under room temperature, then thin layer plate is put into expansion cylinder, plate, apart from 9.5cm, after expansion terminates, takes out and dries by exhibition.With 5% ferric chloride aqueous solutions dyeing, toast 8 minutes at being then placed in 105 DEG C, sample liquid and some purple corresponding to standard items.With CAMAG thin-layer chromatogram scanner-III, scan with the absorbing wavelength of 600nm, obtain Rf=0.62, after testing and to calculate oestrone content in krill be 2.61mgg
-1.
Embodiment 2
Get dry krill 1g, add methyl alcohol 11mL, 35 DEG C, Ultrasonic Heating extracts in the supersonic wave cleaning machine of 350w, 40kHz, extracts 15 minutes, by extracting liquid filtering, filtrate being placed in is revolved steaming instrument evaporate to dryness, obtain the crude extract of oestrone.
Above-mentioned obtained oestrone crude extract is dissolved in 5mL methyl alcohol and makes sample liquid, precision takes 1.0000mg oestrone standard items (J & K company) and is dissolved in 1.0mL methyl alcohol, and obtaining concentration is 1mgmL
-1titer.
Sample thief liquid and titer 5 μ L point are in same GF
254high Performance Thin silica gel plate (10 × 10cm
2) on, developping agent volume ratio is phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=78-80:9-11:9-11:2-4:3d:2d, developping agent is added expansion cylinder (10 × 12 × 15cm
3), at room temperature pre-equilibration 40 minutes, then put into High Performance Thin plate, continue at room temperature to launch, when opening up apart from when reaching 4.5cm, taking out thin layer plate, drying 10 minutes under room temperature, then thin layer plate is put into expansion cylinder, plate, apart from 9.5cm, after expansion terminates, takes out and dries by exhibition.With 5% ferric chloride aqueous solutions dyeing, toast 10 minutes at being then placed in 105 DEG C, sample liquid and some purple corresponding to standard items.With CAMAG thin-layer chromatogram scanner-III, scan with the absorbing wavelength of 600nm, obtain Rf=0.62.After testing and to calculate oestrone content in krill be 2.60mgg
-1.
Embodiment 3
Get dry krill 1g, add methyl alcohol 10mL, 35 DEG C, Ultrasonic Heating extracts in the supersonic wave cleaning machine of 350w, 40kHz, extracts 20 minutes, by extracting liquid filtering, filtrate being placed in is revolved steaming instrument evaporate to dryness, obtain the crude extract of oestrone.
Above-mentioned obtained oestrone crude extract is dissolved in 5mL methyl alcohol and makes sample liquid; Precision takes 1.0000mg oestrone standard items (J & K company) and is dissolved in 1.0mL methyl alcohol, and obtaining concentration is 1mgmL
-1titer.
Sample thief liquid and titer 5 μ L point are in same GF
254high Performance Thin silica gel plate (10 × 10cm
2) on, developping agent volume ratio is phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=78-80:9-11:9-11:2-4:3d:2d, developping agent is added expansion cylinder (10 × 12 × 15cm
3), at room temperature pre-equilibration 45 minutes, then put into High Performance Thin plate, continue at room temperature to launch, when opening up apart from when reaching 4.5cm, taking out thin layer plate, drying 10 minutes under room temperature, then thin layer plate is put into expansion cylinder, plate, apart from 9.5cm, after expansion terminates, takes out and dries by exhibition.With 5% ferric chloride aqueous solutions dyeing, toast 10 minutes at being then placed in 105 DEG C, sample liquid and some purple corresponding to standard items.With CAMAG thin-layer chromatogram scanner-III, scan with the absorbing wavelength of 600nm, obtain Rf=0.62.After testing and to calculate oestrone content in krill be 2.59mgg
-1.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (2)
1. the oestrone HPTLC scanning method detection method in krill, it is characterized in that, step is as follows:
(1) sample liquid to be detected and 1.0mgmL is got
-1titer 5 μ L point in same GF
254on High Performance Thin silica gel plate, take volume ratio as phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=78-80:9-11:9-11:2-4:3:2 is developping agent, launches in expansion cylinder, exhibition, apart from being 9.5cm, dries panel after having opened up;
(2) with 5% ferric chloride in aqueous solution dyeing, at being then placed in 103 DEG C-107 DEG C, 8-10 minute is toasted;
(3) scan with CAMAG thin-layer chromatogram scanner-III, scanning wavelength 600nm, calculated the content of oestrone by the absorbance detecting sample and standard items;
Described step 1) in when launching in expansion cylinder, first by developping agent pre-equilibration 35-45 minute at ambient temperature, put into High Performance Thin plate again, continue to launch at ambient temperature, when opening up apart from when reaching 4.5cm, taking out thin layer plate, under room temperature, drying 10 minutes, again thin layer plate is put into expansion cylinder, the exhibition that is deployed into is apart from being 9.5cm.
2. the HPTLC scanning method detection method of oestrone as claimed in claim 1, is characterized in that, described sample liquid is obtained by being dissolved in by oestrone crude extract in 5mL methyl alcohol.
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| CN106198839B (en) * | 2016-07-18 | 2018-01-12 | 山东师范大学 | A kind of method that 2,6 BHTs are extracted from krill and HPTLC scanning method detection method |
| CN107727777B (en) * | 2017-12-04 | 2020-06-30 | 山东师范大学 | Method for extracting β -ecdysone from euphausia superba and efficient thin-layer chromatography scanning detection method |
| CN110954645B (en) * | 2019-09-09 | 2020-09-29 | 山东琪康生物技术有限公司 | Detection method of high-quality Sihuang dysentery stopping granules |
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