CN102351730A - Method for preparing ceramide from konjac fly powder - Google Patents
Method for preparing ceramide from konjac fly powder Download PDFInfo
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- CN102351730A CN102351730A CN2011103279658A CN201110327965A CN102351730A CN 102351730 A CN102351730 A CN 102351730A CN 2011103279658 A CN2011103279658 A CN 2011103279658A CN 201110327965 A CN201110327965 A CN 201110327965A CN 102351730 A CN102351730 A CN 102351730A
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Abstract
The invention relates to a method for preparing ceramide from konjac fly powder. The method is characterized by comprising the following steps that: a) crude extraction of ceramide: taking konjac fly powder as the raw material, extracting konjac fly powder with an ethanol solution and then using petroleum ether for extraction; b) chromatography purification with a silicagel column: injecting silicagel into a column by a wet method, loading the sample by a wet method and using petroleum ether-ethyl acetate as an eluent to carry out gradient elution; and c) chromatography purification with a medium pressure C18 ODS (octadecylselyl) bonded silicagel column: injecting silicagel into a column by a wet method, using ethanol-water as an eluent to carry out gradient elution and drying to obtain ceramide with a purity being higher than 95%. The method has low production cost and is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of method that from fry starch of konjak, prepares ceramide.
Background technology
Konjaku (konjac) belongs to Angiospermae, Monocotyledonae, Araeceae, is the per nnial herb with bulb.According to statistics, there are 26 kinds of Amorphophallus plants in China, and wherein 14 kinds is that China is peculiar, and at present the konjaku ultimate production accounts for globally 1/3rd, and every year is all in continuous increase.Konjaku is Xi Yin, happiness is wet, To Be Protected from Heat, cold-resistant, the crop that is afraid of accumulated water, and it is special that growing environment requires, and the zone of suitable planting is very limited in the worldwide.In China, it mainly is distributed in ground such as Hunan, Sichuan, Hubei, Yunnan, Guizhou, Taiwan.Enshi, the Hubei Province summer does not have the few severe cold of heat, winter, soil subacidity and is rich in the selenium element, and natural condition are richly endowed by nature, be the plantation konjaku one of ideally.The total planting scale in Quanzhou reaches 600,000 mu; Wherein the commodity base is 450,000 mu; Plant 150,000 mu in taro base; Realize 2,500,000,000 yuan of the plantation output values year; Smart (little, pure) powder of konjaku and beverage, food coating, the total processed output of solvable Edible Film reach 7,500,000,000 yuan; Sell all kinds of konjaku products year more than 25,000 tons.At present, the konjaku powder working method of China can be divided into dry method and wet method substantially, and fry starch of konjak is the by product that produces in the konjaku powder production process, accounts for 35%～45% of the preceding Rhizoma amorphophalli powder quality of processing.Because fry starch of konjak contains peculiar smell and antinutritional factor, and the content of konjac glucomanna is low, has restricted its application in food, medicine and other fields, and only just have an appointment every year 85% fry starch of konjak of Hubei Province is not utilized.
Yet, contain a large amount of effective constituent in the fry starch of konjak, approximately contain 50% total reducing sugar, 20% protein, 0.5% fat, 2.5% Mierocrystalline cellulose, 7% mineral substance and 0.15%～0.20% ceramide.Ceramide, promptly N-acyl schwann's sheath amine alcohol is the important composition unit of sphingophospholipid, is one type of important function material in the organism, also is lipid second messenger important in the organism.That ceramide has is antitumor, induce the human colon cancer cell apoptosis, induce the KB cell apoptosis, anti-liver toxicity, reducing blood-fat, pharmacological action such as hypoglycemic; And fat-reducing, to skin preserve moisture, anti-ageing, effect such as whiten, be widely used in medicine, healthcare products and the daily necessities.
The ceramide goods have two types of natural and chemosynthesis, the field of medicaments master with being natural ceramide.In the past; Some developed countries mainly extract abundant ceramide from the ox brain; But since 1986 " mad cow disease ", the security of animality ceramide is extensively queried, and the ceramide goods of animal-origin are replaced by autonomic nerve acid amides goods.Natural ceramide mainly extracts from plants such as soybean, rice, wheat, yeast, konjaku; But the ceramide content in rice bran and the wheat only 0.01%～0.02%; Content in the konjaku powder is about 0.026%; And the content of the ceramide in the fry starch of konjak is 0.15%～0.20%; Be the important source material that ceramide extracts, have very high DEVELOPMENT PROSPECT.
Summary of the invention
The problem that the present invention will solve provides a kind of method that from fry starch of konjak, prepares ceramide.
A kind of method that from fry starch of konjak, prepares ceramide of the present invention is characterized in that comprising following operation steps:
(a) slightly carrying of ceramide: with the fry starch of konjak is raw material, extracts with ethanolic soln, extracts the after-filtration that finishes; Concentrate, reclaim ethanol, then with concentrated solution with petroleum ether extraction 2 times; Merge the sherwood oil phase, reclaim sherwood oil and get brown ceramide medicinal extract, i.e. ceramide sample I;
(b) purification by silica gel column chromatography: with silica gel wet method dress post, and with initial eluent balance chromatography column; Ceramide sample I dissolves with initial eluent, appearance on the wet method; Use petroleum ether-ethyl acetate to carry out gradient elution as eluent, substep is collected effluent liquid, detects effluent liquid with TLC, merges the effluent liquid with identical composition, reclaims solvent, the dry ceramide sample II that gets;
(c) press C in
18Bonded silica gel ODS column chromatography purification: with C
18Bonded silica gel wet method dress post compresses with pressurizing device, and with initial eluent balance chromatography column; Above-mentioned ceramide sample II is dissolved appearance on the wet method with initial eluent; Use alcohol-water to carry out gradient elution as eluent, substep is collected effluent liquid, detects effluent liquid with TLC, merges the effluent liquid with identical composition, reclaims solvent, the dry ceramide sample III that gets; Detect through HPLC-ELSD, the purity of ceramide sample III can arrive more than 95%.
A kind of method that from fry starch of konjak, prepares ceramide of the present invention is characterized in that comprising following operation steps:
(a) slightly carrying of ceramide: with the fry starch of konjak is raw material, and using concentration is that 75%～95% ethanolic soln extracts, and solid-liquid ratio is the solid-to-liquid ratio fry starch of konjak: ethanolic soln 1: 3～5, the temperature of extraction are 60～80 ℃, and extraction time is 1～2h; The extraction after-filtration that finishes; Concentrate; Reclaim ethanol; Then with concentrated solution with 60～90 ℃ of cut petroleum ether extractions 2 times, churning time is 0.5～1.5h, time of repose is 0.5h; Solid-liquid ratio is the volume ratio concentrated solution: sherwood oil 1: 2～4; Merge the sherwood oil phase, reclaim sherwood oil and get brown ceramide medicinal extract, i.e. ceramide sample I;
(b) purification by silica gel column chromatography: with 300～400 order silica gel activatings, activation temperature is 105～110 ℃, and soak time is 1～2h; Behind the silica gel wet method dress post, with initial eluent balance chromatography column; Ceramide sample I dissolves with initial eluent, appearance on the wet method; Applied sample amount is 1～5g crude product/100g silica gel; Use petroleum ether-ethyl acetate to carry out gradient elution then as eluent; The volume ratio of eluent petroleum ether-ethyl acetate was respectively 95: 5,85: 15,75: 25,60: 40,40: 60, and wherein 95: 5 and 85: 15 two kinds of eluent usage quantitys respectively are 1BV, and the usage quantity of back three kinds of eluents respectively is 2BV; Substep is collected effluent liquid; Elution speed is 2～4BV/h, detects effluent liquid with TLC, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample II that gets;
(c) press C in
18Bonded silica gel ODS column chromatography purification: take by weighing proper C
18Bonded silica gel soaks with initial flow mutually, and stirs, and when treating not have bubble in the silica gel, presses in the chromatographic column during wet method is packed into; Compress with pressurizing device then, make the C after the compression
18The operating pressure of bonded silica gel post under normal flow is 5～20bar, and with initial eluent balance chromatography column; Above-mentioned ceramide sample II is dissolved with initial eluent, appearance on the wet method, applied sample amount is 0.1～0.5g sample/100gC
18Bonded silica gel; Use alcohol-water to carry out gradient elution as eluent; The volume ratio of eluent alcohol-water is respectively 90%, 75%, 60%; The amount of every kind of eluent is 2～2.5BV; Elution speed is 1～1.5BV/h; Detect effluent liquid with TLC, substep is collected effluent liquid, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample III that gets; Detect through HPLC-ELSD, the purity of ceramide sample III can arrive more than 95%.
The invention provides a kind of method that from fry starch of konjak, prepares the high purity ceramide, is to utilize fry starch of konjak to be raw material, through pressing C in the silica gel column chromatography technical tie-up
18The bonded silica gel column chromatography technology prepares highly purified ceramide.
1, ceramide slightly carries
With the fry starch of konjak is raw material; Stir extraction 1～2h with 75%～95% (v/v) ethanol in 60～80 ℃; Solid-liquid ratio is 1: 3～1: 5; The extraction after-filtration that finishes; Collect filtrating; Filtrating changes alcohol recovery device over to; Alcoholic strength in recovered alcohol to the concentrated solution is 0～20%; 60～90 ℃ of sherwood oils that add 2～4 times of volumes then in the concentrated solution extract, and stir standing demix behind 0.5～1.5h, collect the ether phase; Extract 2 times; The combined ether phase is concentrated into the medicinal extract shape mutually with ether, gets brown ceramide sample I;
2, purification by silica gel column chromatography ceramide
2.1 the activation of silica gel
Take by weighing 300～400 purpose silica gel in 105～110 ℃ of activation 1～2h.
2.2 wet method dress post
The silica gel that activation is good is put into container, and adds an amount of sherwood oil, stirs along fixed-direction, when treating not have bubble in the silica gel, presses in the chromatographic column during wet method is packed into.And with initial eluent balance chromatography column.
2.3 appearance and wash-out on the wet method
With above-mentioned ceramide crude product I with the initial flow phased soln after appearance on the wet method, applied sample amount is 1～5g crude product/100g silica gel.Use then petroleum ether-ethyl acetate (95: 5,85: 15,75: 25; 60: 40,40: 60, v/v) carry out gradient elution for eluent; 95: 5 and 85: 15 two kinds of eluent usage quantitys respectively are 1BV; The usage quantity of the three kinds of eluents in back respectively is 2BV, and substep is collected effluent liquid, and elution speed is 2～4BV/h; Detect effluent liquid with TLC; Merge effluent liquid, reclaim solvent, the dry ceramide sample II that gets with identical composition.Silica gel column chromatography is reusable behind Mathanol regenerating.
3, middle pressure C
18Bonded silica gel (ODS) column chromatography purification ceramide
3.1 wet method dress post
Take by weighing proper C
18Bonded silica gel soaks with initial flow mutually, and stirs, and when treating not have bubble in the silica gel, presses in the chromatographic column during wet method is packed into.Compress with pressurizing device then, make the C after the compression
18The operating pressure of bonded silica gel post under normal flow is 5～20bar, and with initial eluent balance chromatography column.
3.2 appearance and wash-out on the wet method
Above-mentioned ceramide sample II is dissolved with initial eluent, appearance on the wet method, applied sample amount is 0.1～0.5g sample/100g C
18Bonded silica gel.With alcohol-water (90%; 75%; 60%, v/v) for eluent carries out gradient elution, the amount of every kind of eluent is 2～2.5BV; Substep is collected effluent liquid; Elution speed is 1～1.5BV/h, detects effluent liquid with TLC, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample III that gets.Detect through HPLC-ELSD, the purity of ceramide sample III can arrive more than 95%.The ceramide sample of other low levels upper prop is again recycled.The middle C that presses
18The bonded silica gel chromatography column is reusable behind Mathanol regenerating.
4, detect
The main TLC of use method is qualitative among the present invention detects ceramide with the HPLC-ELSD quantitative analysis.
The TLC qualitative method:
The activation of silica-gel plate: 110 ℃, 30min; Developping agent: chloroform: methyl alcohol: acetic acid=19: 0.9: 0.1 (v/v/v);
Developer I:0.1% alizarin (get the 0.1g alizarin, with after the small amount of ethanol dissolving, it is subsequent use to be settled to 100mL); Colour developing mode: spraying colour developing;
Developer: 10% copper sulfate phosphoric acid solution (getting the 10g anhydrous cupric sulfate, subsequent use) with being settled to 100mL after 10% the phosphoric acid solution dissolving; Colour developing mode: dry by the fire 30min down at 140 ℃ after soaking 3min, observe change in color.
The HPLC quantitative method:
Instrument and chromatographic condition: Waters 2695 separating units+Alltech ELSD3300 light scattering detector; Masslynx 4.0 chromatographic working stations; Chromatographic column: Kromasil C
18Post (250mm * 4.6mm, 5 μ m); Moving phase: methanol (v/v), gradient elution; Column temperature: 30 ℃; Flow velocity: 1mL/min; Sample size: 20 μ L; The temperature of light scattering detector drift tube: 40 ℃, nitrogen flow rate: 1.5L/min, detection sensitivity: 2.
The present invention and prior art are to utilize fry starch of konjak to be raw material, through pressing C in the silica gel column chromatography technical tie-up relatively
18The bonded silica gel column chromatography technology prepares highly purified ceramide.This explained hereafter cost is low, is fit to suitability for industrialized production.
Description of drawings
Accompanying drawing is a fry starch of konjak ceramide preparation technology schema.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail, but the working of an invention mode is not limited thereto.
Embodiment 1
(1) ceramide slightly carries
Get fry starch of konjak 100g in three mouthfuls of Florence flasks; Add 300mL 75% ethanolic soln; Connect prolong; Magnetic agitation is extracted 1h; Using the electric contact thermometer controlled temperature simultaneously is 50 ± 1 ℃; Extraction finishes; Filter; The ethanol extract of ceramide is transferred to be concentrated into alcohol concn in the rotatory evaporator be 10%, concentrated solution is transferred in three mouthfuls of clean Florence flasks, press 60～90 ℃ of sherwood oils that the concentrated solution volume adds 2 times of amounts; Stir 0.5h; Be transferred to and leave standstill 0.5h in the separating funnel, collect the ether phase on upper strata after the layering, extract 2 times.The combined ether phase reclaims sherwood oil and is concentrated into medicinal extract, gets brown ceramide sample I, and weight is 1.31g;
(2) purification by silica gel column chromatography
By applied sample amount is 1～5g crude product/100g silica gel; Take by weighing 300～400 order silica gel 100g in 105～110 ℃ of activation 1h; To activate good silica gel then and put into container; And add an amount of sherwood oil; Stir along fixed-direction, when treating not have bubble in the silica gel, wet method is packed in the silica gel chromatographic column; Silicagel column is 40*190mm, and column volume is about 250mL.Use the flow velocity balance chromatography column of initial eluent 500mL then with 8mL/min.
With above-mentioned ceramide crude product I with the initial flow phased soln after appearance on the wet method; Use petroleum ether-ethyl acetate (v/v) 95: 5,85: 15,75: 25,60: 40,40: 60 to carry out gradient elution respectively for eluent; 95: 5 and 85: 15 two kinds of eluent usage quantitys respectively are 1BV; The usage quantity of the three kinds of eluents in back respectively is 2BV; Substep is collected effluent liquid; Elution speed is 2BV/h; Detect effluent liquid with TLC; Merge effluent liquid with identical composition; Reclaim solvent; The dry ceramide sample II that gets, weight is 0.22g.Silica gel column chromatography is reusable behind Mathanol regenerating;
(3) press C in
18Bonded silica gel column chromatography purification ceramide
By applied sample amount is 0.1～0.5g sample/100g C
18Bonded silica gel takes by weighing C
18Bonded silica gel 100g soaks with initial flow mutually, and stirs, and when treating not have bubble in the silica gel, presses in the chromatographic column during wet method is packed into.Compress with pressurizing device then, make the C after the compression
18The operating pressure of bonded silica gel post under normal flow is 10bar, and with initial eluent balance chromatography column.
Above-mentioned ceramide sample II is dissolved with initial eluent; Appearance on the wet method; With alcohol-water (v/v) 90%, 75%, 60%, for eluent carries out gradient elution, the amount of every kind of eluent is 500mL; Substep is collected effluent liquid; Elution speed is 4mL/min, detects effluent liquid with TLC, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample III that gets.Detect through HPLC-ELSD, the purity of ceramide sample III can arrive 95.0%, and weight is 0.061g.The ceramide sample of other low levels upper prop is again recycled.The middle C that presses
18The bonded silica gel chromatography column is reusable behind Mathanol regenerating.
Embodiment 2
(1) ceramide slightly carries
Get fry starch of konjak 100g in three mouthfuls of Florence flasks; Add 400mL 85% ethanolic soln; Connect prolong; Magnetic agitation is extracted 1.5h; Using the electric contact thermometer controlled temperature simultaneously is 60 ± 1 ℃; Extraction finishes; Filter; The ethanol extract of ceramide is transferred to be concentrated into alcohol concn in the rotatory evaporator be 10%, concentrated solution is transferred in three mouthfuls of clean Florence flasks, press 60～90 ℃ of sherwood oils that the concentrated solution volume adds 3 times of amounts; Stir 1.0h; Be transferred to and leave standstill 0.5h in the separating funnel, collect the ether phase on upper strata after the layering, extract 2 times.The combined ether phase reclaims sherwood oil and is concentrated into medicinal extract, gets brown ceramide sample I, and weight is 1.29g;
(2) purification by silica gel column chromatography
By applied sample amount is 1～5g crude product/100g silica gel; Take by weighing 300～400 order silica gel in 105～110 ℃ of activation 1.5h; To activate good silica gel then and put into container; And add an amount of sherwood oil; Stir along fixed-direction, when treating not have bubble in the silica gel, press in the chromatographic column during wet method is packed into; Silicagel column is 40*190mm, and column volume is about 250mL.Use the flow velocity balance chromatography column of initial eluent 500mL then with 10mL/min.
With above-mentioned ceramide crude product I with the initial flow phased soln after appearance on the wet method; Use petroleum ether-ethyl acetate (v/v) 95: 5,85: 15,75: 25,60: 40,40: 60 to carry out gradient elution respectively for eluent; 95: 5 and 85: 15 two kinds of eluent usage quantitys respectively are 1BV; The usage quantity of the three kinds of eluents in back respectively is 2BV; Substep is collected effluent liquid; Elution speed is 3BV/h; Detect effluent liquid with TLC; Merge effluent liquid with identical composition; Reclaim solvent; The dry ceramide sample II that gets, weight is 0.19g.Silica gel column chromatography is reusable behind Mathanol regenerating;
(3) press C in
18Bonded silica gel column chromatography purification ceramide
By applied sample amount is 0.1～0.5g sample/100g C
18Bonded silica gel takes by weighing C
18Bonded silica gel 100g soaks with initial flow mutually, and stirs, and when treating not have bubble in the silica gel, presses in the chromatographic column during wet method is packed into.Compress with pressurizing device then, make the C after the compression
18The operating pressure of bonded silica gel post under normal flow is 12bar, and with initial eluent balance chromatography column.
Above-mentioned ceramide sample II is dissolved with initial eluent; Appearance on the wet method; With alcohol-water (v/v) 90%, 75%, 60%, for eluent carries out gradient elution, the amount of every kind of eluent is 550mL; Substep is collected effluent liquid; Elution speed is 5mL/min, detects effluent liquid with TLC, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample III that gets.Detect through HPLC-ELSD, the purity of ceramide sample III can arrive 95.7%, and weight is 0.072g.The ceramide sample of other low levels upper prop is again recycled.The middle C that presses
18The bonded silica gel chromatography column is reusable behind Mathanol regenerating.
Embodiment 3
(1) ceramide slightly carries
Get fry starch of konjak 100g in three mouthfuls of Florence flasks; Add 500mL 90% ethanolic soln; Connect prolong; Magnetic agitation is extracted 1.5h; Using the electric contact thermometer controlled temperature simultaneously is 70 ± 1 ℃; Extraction finishes; Filter; The ethanol extract of ceramide is transferred to be concentrated into alcohol concn in the rotatory evaporator be 5%, concentrated solution is transferred in three mouthfuls of clean Florence flasks, press 60～90 ℃ of sherwood oils that the concentrated solution volume adds 4 times of amounts; Stir 1.0h; Be transferred to and leave standstill 0.5h in the separating funnel, collect the ether phase on upper strata after the layering, extract 2 times.The combined ether phase reclaims sherwood oil and is concentrated into medicinal extract, gets brown ceramide sample I, and weight is 1.35g;
(2) purification by silica gel column chromatography
By applied sample amount is 1～5g crude product/100g silica gel; Take by weighing 300～400 order silica gel in 105～110 ℃ of activation 1.5h; To activate good silica gel then and put into container; And add an amount of sherwood oil; Stir along fixed-direction, when treating not have bubble in the silica gel, press in the chromatographic column during wet method is packed into; Silicagel column is 40*190mm, and column volume is about 250mL.Use the flow velocity balance chromatography column of initial eluent 500mL then with 9mL/min.
With above-mentioned ceramide crude product I with the initial flow phased soln after appearance on the wet method; Use petroleum ether-ethyl acetate (v/v) 95: 5,85: 15,75: 25,60: 40,40: 60 to carry out gradient elution respectively for eluent; 95: 5 and 85: 15 two kinds of eluent usage quantitys respectively are 1BV; The usage quantity of the three kinds of eluents in back respectively is 2BV; Substep is collected effluent liquid; Elution speed is 4BV/h; Detect effluent liquid with TLC; Merge effluent liquid with identical composition; Reclaim solvent; The dry ceramide sample II that gets, weight is 0.20g.Silica gel column chromatography is reusable behind Mathanol regenerating;
(3) press C in
18Bonded silica gel column chromatography purification ceramide
By applied sample amount is 0.1～0.5g sample/100g C
18Bonded silica gel takes by weighing C
18Bonded silica gel 100g soaks with initial flow mutually, and stirs, and when treating not have bubble in the silica gel, presses in the chromatographic column during wet method is packed into.Compress with pressurizing device then, make the C after the compression
18The operating pressure of bonded silica gel post under normal flow is 16bar, and with initial eluent balance chromatography column.
Above-mentioned ceramide sample II is dissolved with initial eluent; Appearance on the wet method; With alcohol-water (v/v) 90%, 75%, 60%, for eluent carries out gradient elution, the amount of every kind of eluent is 600mL; Substep is collected effluent liquid; Elution speed is 5mL/min, detects effluent liquid with TLC, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample III that gets.Detect through HPLC-ELSD, the purity of ceramide sample III can arrive 95.2%, and weight is 0.064g.The ceramide sample of other low levels upper prop is again recycled.The middle C that presses
18The bonded silica gel chromatography column is reusable behind Mathanol regenerating.
Embodiment 4
(1) ceramide slightly carries
Get fry starch of konjak 100g in three mouthfuls of Florence flasks; Add 400mL 95% ethanolic soln; Connect prolong; Magnetic agitation is extracted 1.5h; Using the electric contact thermometer controlled temperature simultaneously is 80 ± 1 ℃; Extraction finishes; Filter; The ethanol extract of ceramide is transferred to be concentrated into alcohol concn in the rotatory evaporator be 20%, concentrated solution is transferred in three mouthfuls of clean Florence flasks, press 60～90 ℃ of sherwood oils that the concentrated solution volume adds 4 times of amounts; Stir 1.0h; Be transferred to and leave standstill 0.5h in the separating funnel, collect the ether phase on upper strata after the layering, extract 2 times.The combined ether phase reclaims sherwood oil and is concentrated into medicinal extract, gets brown ceramide sample I, and weight is 1.28g;
(2) purification by silica gel column chromatography
By applied sample amount is 1～5g crude product/100g silica gel; Take by weighing 300～400 order silica gel in 105～110 ℃ of activation 1.5h; To activate good silica gel then and put into container; And add an amount of sherwood oil; Stir along fixed-direction, when treating not have bubble in the silica gel, press in the chromatographic column during wet method is packed into; Silicagel column is 40*190mm, and column volume is about 250mL.Use the flow velocity balance chromatography column of initial eluent 500mL then with 8mL/min.
With above-mentioned ceramide crude product I with the initial flow phased soln after appearance on the wet method; Use petroleum ether-ethyl acetate (v/v) 95: 5,85: 15,75: 25,60: 40,40: 60 to carry out gradient elution respectively for eluent; 95: 5 and 85: 15 two kinds of eluent usage quantitys respectively are 1BV; The usage quantity of the three kinds of eluents in back respectively is 2BV; Substep is collected effluent liquid; Elution speed is 4BV/h; Detect effluent liquid with TLC; Merge effluent liquid with identical composition; Reclaim solvent; The dry ceramide sample II that gets, weight is 0.21g.Silica gel column chromatography is reusable behind Mathanol regenerating;
(3) press C in
18Bonded silica gel column chromatography purification ceramide
By applied sample amount is 0.1～0.5g sample/100g C
18Bonded silica gel takes by weighing C
18Bonded silica gel 100g soaks with initial flow mutually, and stirs, and when treating not have bubble in the silica gel, presses in the chromatographic column during wet method is packed into.Compress with pressurizing device then, make the C after the compression
18The operating pressure of bonded silica gel post under normal flow is 20bar, and with initial eluent balance chromatography column.
Above-mentioned ceramide sample II is dissolved with initial eluent; Appearance on the wet method; With alcohol-water (v/v) 90%, 75%, 60%, for eluent carries out gradient elution, the amount of every kind of eluent is 600mL; Substep is collected effluent liquid; Elution speed is 6mL/min, detects effluent liquid with TLC, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample III that gets.Detect through HPLC-ELSD, the purity of ceramide sample III can arrive 95.1%, and weight is 0.076g.The ceramide sample of other low levels upper prop is again recycled.The middle C that presses
18The bonded silica gel chromatography column is reusable behind Mathanol regenerating.
Claims (4)
1. method that from fry starch of konjak, prepares ceramide is characterized in that comprising following operation steps:
(a) slightly carrying of ceramide: with the fry starch of konjak is raw material, extracts with ethanolic soln, extracts the after-filtration that finishes; Concentrate, reclaim ethanol, then with concentrated solution with petroleum ether extraction 2 times; Merge the sherwood oil phase, reclaim sherwood oil and get brown ceramide medicinal extract, i.e. ceramide sample I;
(b) purification by silica gel column chromatography: with silica gel wet method dress post, and with initial eluent balance chromatography column; Ceramide sample I dissolves with initial eluent, appearance on the wet method; Use petroleum ether-ethyl acetate to carry out gradient elution as eluent, substep is collected effluent liquid, detects effluent liquid with TLC, merges the effluent liquid with identical composition, reclaims solvent, the dry ceramide sample II that gets:
(c) press C18 bonded silica gel ODS column chromatography purification in: with C18 bonded silica gel wet method dress post, compress with pressurizing device, and with initial eluent balance chromatography column; Above-mentioned ceramide sample II is dissolved appearance on the wet method with initial eluent; Use alcohol-water to carry out gradient elution as eluent, substep is collected effluent liquid, detects effluent liquid with TLC, merges the effluent liquid with identical composition, reclaims solvent, the dry ceramide sample III that gets; Detect through HPLC-ELSD, the purity of ceramide sample III can arrive more than 95%.
2. according to the described a kind of method that from fry starch of konjak, prepares ceramide of claim 1, it is characterized in that in the described step (a):
(a) slightly carrying of ceramide: with the fry starch of konjak is raw material, and using concentration is that 75%～95% ethanolic soln extracts, and solid-liquid ratio is the solid-to-liquid ratio fry starch of konjak: ethanolic soln 1: 3～5, the temperature of extraction are 60～80 ℃, and extraction time is 1～2h; The extraction after-filtration that finishes; Concentrate; Reclaim ethanol; Then with concentrated solution with 60～90 ℃ of cut petroleum ether extractions 2 times, churning time is 0.5～1.5h, time of repose is 0.5h; Solid-liquid ratio is the volume ratio concentrated solution: sherwood oil 1: 2～4; Merge the sherwood oil phase, reclaim sherwood oil and get brown ceramide medicinal extract, i.e. ceramide sample I.
3. according to the described a kind of method that from fry starch of konjak, prepares ceramide of claim 1, it is characterized in that in the described step (b):
(b) purification by silica gel column chromatography: with 300～400 order silica gel activatings, activation temperature is 105～110 ℃, and soak time is 1～2h; Behind the silica gel wet method dress post, with initial eluent balance chromatography column; Ceramide sample I dissolves with initial eluent, appearance on the wet method; Applied sample amount is 1～5g crude product/100g silica gel; Use petroleum ether-ethyl acetate to carry out gradient elution then as eluent; The volume ratio of eluent petroleum ether-ethyl acetate was respectively 95: 5,85: 15,75: 25,60: 40,40: 60, and wherein 95: 5 and 85: 15 two kinds of eluent usage quantitys respectively are 1BV, and the usage quantity of back three kinds of eluents respectively is 2BV; Substep is collected effluent liquid; Elution speed is 2～4BV/h, detects effluent liquid with TLC, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample II that gets.
4. according to the described a kind of method that from fry starch of konjak, prepares ceramide of claim 1, it is characterized in that in the described step (c):
(c) press C in
18Bonded silica gel ODS column chromatography purification: take by weighing proper C
18Bonded silica gel soaks with initial flow mutually, and stirs, and when treating not have bubble in the silica gel, presses in the chromatographic column during wet method is packed into; Compress with pressurizing device then, make the C after the compression
18The operating pressure of bonded silica gel post under normal flow is 5～20bar, and with initial eluent balance chromatography column; Above-mentioned ceramide sample II is dissolved with initial eluent, appearance on the wet method, applied sample amount is 0.1～0.5g sample/100g C
18Bonded silica gel; Use alcohol-water to carry out gradient elution as eluent; The volume ratio of eluent alcohol-water is respectively 90%, 75%, 60%; The amount of every kind of eluent is 2～2.5BV; Elution speed is 1～1.5BV/h; Detect effluent liquid with TLC, substep is collected effluent liquid, merges the effluent liquid with identical composition; Reclaim solvent, the dry ceramide sample III that gets; Detect through HPLC-ELSD, the purity of ceramide sample III can arrive more than 95%.
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