CN102351730B - Method for preparing ceramide from konjac fly powder - Google Patents
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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 of preparing ceramide from fry starch of konjak.
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 14Zhong Wei China is peculiar, at present konjaku ultimate production account for global 1/3rd, and every year 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 in worldwide, the region of suitable planting is very limited.In China, it is mainly distributed in the ground such as Hunan, Sichuan, Hubei, Yunnan, Guizhou, Taiwan.The Enshi State of Hubei Province summer is without heat, winter few severe cold, soil subacidity and be rich in selenium element, and natural condition are richly endowed by nature, be plantation konjaku one of ideally.The total planting scale in Quanzhou reaches 600,000 mu, wherein commodity base is 450,000 mu, plant 150,000 mu, taro base, realize 2,500,000,000 yuan of the plantation output values year, konjaku essence (micro-, pure) powder 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 producing in konjaku powder production process, accounts for 35%～45% of the front 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, in fry starch of konjak, contain a large amount of effective constituent, 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, ceramide, is the important composition unit of sphingophospholipid, is the important functional mass of a class in organism, is also important lipid second messenger in organism.That ceramide has is antitumor, induction apoptosis of human colon cancer cells, induction KB cell apoptosis, anti-liver toxicity, reducing blood-fat, the pharmacological action such as hypoglycemic, and fat-reducing, skin is had to the effects such as moisturizing, anti-ageing, whitening, be widely used in medicine, healthcare products and daily necessities.
Ceramide goods have natural and chemosynthesis two classes, and primary at field of medicaments is natural ceramide.In the past, some developed countries mainly extract abundant ceramide from ox brain, but since 1986 " mad cow disease ", the security of animality ceramide is extensively queried, the ceramide goods of animal-origin are replaced by autonomic nerve acid amides goods.Natural ceramide mainly extracts from the plants such as soybean, rice, wheat, yeast, konjaku, but the ceramide content in rice bran and wheat only 0.01%～0.02%, content in konjaku powder is about 0.026%, and the content of ceramide in fry starch of konjak is 0.15%～0.20%, be the important source material that ceramide extracts, there is very high DEVELOPMENT PROSPECT.
Summary of the invention
The problem to be solved in the present invention is to provide a kind of method of preparing ceramide from fry starch of konjak.
A kind of method of preparing ceramide from fry starch of konjak of the present invention, is characterized in that comprising following operation steps:
(a) slightly carrying of ceramide: take fry starch of konjak as raw material, extract with ethanolic soln, filter after extraction, concentrated, reclaim ethanol, then by concentrated solution petroleum ether extraction 2 times, merge sherwood oil phase, reclaim sherwood oil and obtain brown ceramide medicinal extract, i.e. ceramide sample I;
(b) purification by silica gel column chromatography: by silica gel wet method dress post, and with initial eluent balance chromatography column; Ceramide sample I dissolves with initial eluent, wet method loading; With petroleum ether-ethyl acetate, be that eluent carries out gradient elution, substep is collected effluent liquid, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaims solvent, is dried to obtain ceramide sample II;
(c) in, press C
18bonded silica gel ODS column chromatography purification: by 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 to wet method loading with initial eluent; With alcohol-water, be that eluent carries out gradient elution, substep is collected effluent liquid, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaims solvent, is dried to obtain ceramide sample III; Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive more than 95%.
A kind of method of preparing ceramide from fry starch of konjak of the present invention, is characterized in that comprising following operation steps:
(a) slightly carrying of ceramide: take fry starch of konjak as raw material, is that 75%～95% ethanolic soln extracts by concentration, and solid-liquid ratio is solid-to-liquid ratio fry starch of konjak: ethanolic soln 1: 3～5, the temperature of extraction is 60～80 ℃, extraction time is 1～2h; After extraction, filter, concentrated, reclaim ethanol, then by 60～90 ℃ of cut petroleum ether extractions 2 times for concentrated solution, churning time is 0.5～1.5h, and time of repose is 0.5h, solid-liquid ratio is volume ratio concentrated solution: sherwood oil 1: 2～4, merge sherwood oil phase, reclaim sherwood oil and obtain brown ceramide medicinal extract, i.e. ceramide sample I;
(b) purification by silica gel column chromatography: by 300～400 order silica gel activatings, activation temperature is 105～110 ℃, and soak time is 1～2h; After silica gel wet method dress post, with initial eluent balance chromatography column; Ceramide sample I dissolves with initial eluent, wet method loading; Applied sample amount is 1～5g crude product/100g silica gel; Then with petroleum ether-ethyl acetate, be that eluent carries out gradient elution, the volume ratio of eluent petroleum ether-ethyl acetate is respectively 95: 5,85: 15,75: 25,60: 40,40: 60, and wherein 95: 5 and 85: 15 two kinds of eluent usage quantitys are respectively 1BV, and the usage quantity of rear three kinds of eluents is respectively 2BV, substep is collected effluent liquid, elution speed is 2～4BV/h, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample II;
(c) in, press C
18bonded silica gel ODS column chromatography purification: take appropriate C
18bonded silica gel soaks mutually by initial flow, and stirs, and while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column; Then with pressurizing device, compress, make the C after 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, wet method loading, applied sample amount is 0.1～0.5g sample/100gC
18bonded silica gel; With alcohol-water, be that eluent carries out gradient elution, 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, with TLC, detect effluent liquid, substep is collected effluent liquid, merges the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample III; Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive more than 95%.
The invention provides a kind of method of preparing high purity ceramide from fry starch of konjak, is to utilize fry starch of konjak for raw material, by pressing C in silica gel column chromatography technical tie-up
18bonded silica gel column chromatography technology is prepared highly purified ceramide.
1, ceramide slightly carries
Take fry starch of konjak as raw material, with 75%～95% (v/v) ethanol, in 60～80 ℃, stir and extract 1～2h, solid-liquid ratio is 1: 3～1: 5, after extraction, filter, collect filtrate, filtrate proceeds to alcohol recovery device, recovered alcohol to the alcoholic strength in concentrated solution is 0～20%, then in concentrated solution, add 60～90 ℃ of sherwood oils of 2～4 times of volumes to extract, stratification after stirring 0.5～1.5h, collects ether phase, extract 2 times, combined ether phase, is concentrated into medicinal extract shape mutually by ether, obtains brown ceramide sample I;
2, purification by silica gel column chromatography ceramide
The activation of 2.1 silica gel
Take 300～400 object silica gel in 105～110 ℃ of activation 1～2h.
2.2 wet method dress posts
The silica gel having activated is put into container, and add appropriate sherwood oil, along fixed-direction, stir, while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column.And with initial eluent balance chromatography column.
2.3 wet method loading and wash-outs
By wet method loading after initial flow phased soln for above-mentioned ceramide crude product I, applied sample amount is 1～5g crude product/100g silica gel.Then use petroleum ether-ethyl acetate (95: 5,85: 15,75: 25,60: 40,40: 60, v/v) for eluent carries out gradient elution, 95: 5 and 85: 15 two kinds of eluent usage quantitys are respectively 1BV, the usage quantity of rear three kinds of eluents is respectively 2BV, and substep is collected effluent liquid, and elution speed is 2～4BV/h, with TLC, detect effluent liquid, merge the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample II.Silica gel column chromatography is reusable after Mathanol regenerating.
3, middle pressure C
18bonded silica gel (ODS) column chromatography purification ceramide
3.1 wet method dress posts
Take appropriate C
18bonded silica gel soaks mutually by initial flow, and stirs, and while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column.Then with pressurizing device, compress, make the C after compression
18the operating pressure of bonded silica gel post under normal flow is 5～20bar, and with initial eluent balance chromatography column.
3.2 wet method loading and wash-outs
Above-mentioned ceramide sample II is dissolved with initial eluent, wet method loading, 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, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample III.Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive more than 95%.The ceramide sample of other low levels again upper prop is recycled.Middle pressure C
18bonded silica gel chromatography column is reusable after Mathanol regenerating.
4, detect
In the present invention, the main TLC of use method is qualitative detects ceramide with HPLC-ELSD quantitative analysis.
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 0.1g alizarin, with after a small amount of dissolve with ethanol, be settled to 100mL standby); Colour developing mode: spraying colour developing;
Developer: 10% copper sulfate phosphoric acid solution (get 10g anhydrous cupric sulfate, it is standby that the phosphoric acid solution with 10% is settled to 100mL after dissolving); Colour developing mode: dry 30min after soaking 3min at 140 ℃, observe the variation of color.
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/water (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 compared with the prior art, is to utilize fry starch of konjak for raw material, by pressing C in silica gel column chromatography technical tie-up
18bonded silica gel column chromatography technology is prepared highly purified ceramide.This explained hereafter cost is low, is applicable to suitability for industrialized production.
Accompanying drawing explanation
Accompanying drawing is fry starch of konjak ceramide preparation technology schema.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but working of an invention mode is not limited to this.
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, with electric contact thermometer, control temperature is 50 ± 1 ℃ simultaneously, extract complete, filter, the ethanol extract of ceramide is transferred to in rotatory evaporator, to be concentrated into alcohol concn be 10%, concentrated solution is transferred in three mouthfuls of clean Florence flasks, by concentrated solution volume, add 60～90 ℃ of sherwood oils of 2 times of amounts, stir 0.5h, be transferred to standing 0.5h in separating funnel, after layering, collect the ether phase on upper strata, extract 2 times.Combined ether phase, reclaims sherwood oil and is concentrated into medicinal extract, obtains brown ceramide sample I, and weight is 1.31g;
(2) purification by silica gel column chromatography
By applied sample amount, be 1～5g crude product/100g silica gel, take 300～400 order silica gel 100g in 105～110 ℃ of activation 1h, then the silica gel having activated is put into container, and add appropriate sherwood oil, along fixed-direction, stir, while there is no bubble in silica gel, wet method packs in silica gel chromatographic column, silicagel column is 40*190mm, and column volume is about 250mL.Then use initial eluent 500mL with the flow velocity balance chromatography column of 8mL/min.
By wet method loading after initial flow phased soln for above-mentioned ceramide crude product I, use respectively petroleum ether-ethyl acetate (v/v) 95: 5,85: 15,75: 25,60: 40,40: 60 to carry out gradient elution for eluent, 95: 5 and 85: 15 two kinds of eluent usage quantitys are respectively 1BV, the usage quantity of rear three kinds of eluents is respectively 2BV, substep is collected effluent liquid, elution speed is 2BV/h, with TLC, detect effluent liquid, merge the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample II, weight is 0.22g.Silica gel column chromatography is reusable after Mathanol regenerating;
(3) in, press C
18bonded silica gel column chromatography purification ceramide
By applied sample amount, be 0.1～0.5g sample/100g C
18bonded silica gel, takes C
18bonded silica gel 100g, soaks mutually by initial flow, and stirs, and while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column.Then with pressurizing device, compress, make the C after 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, wet method loading, 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, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample III.Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive 95.0%, and weight is 0.061g.The ceramide sample of other low levels again upper prop is recycled.Middle pressure C
18bonded silica gel chromatography column is reusable after 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, with electric contact thermometer, control temperature is 60 ± 1 ℃ simultaneously, extract complete, filter, the ethanol extract of ceramide is transferred to in rotatory evaporator, to be concentrated into alcohol concn be 10%, concentrated solution is transferred in three mouthfuls of clean Florence flasks, by concentrated solution volume, add 60～90 ℃ of sherwood oils of 3 times of amounts, stir 1.0h, be transferred to standing 0.5h in separating funnel, after layering, collect the ether phase on upper strata, extract 2 times.Combined ether phase, reclaims sherwood oil and is concentrated into medicinal extract, obtains brown ceramide sample I, and weight is 1.29g;
(2) purification by silica gel column chromatography
By applied sample amount, be 1～5g crude product/100g silica gel, take 300～400 order silica gel in 105～110 ℃ of activation 1.5h, then the silica gel having activated is put into container, and add appropriate sherwood oil, along fixed-direction, stir, while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column, silicagel column is 40*190mm, and column volume is about 250mL.Then use initial eluent 500mL with the flow velocity balance chromatography column of 10mL/min.
By wet method loading after initial flow phased soln for above-mentioned ceramide crude product I, use respectively petroleum ether-ethyl acetate (v/v) 95: 5,85: 15,75: 25,60: 40,40: 60 to carry out gradient elution for eluent, 95: 5 and 85: 15 two kinds of eluent usage quantitys are respectively 1BV, the usage quantity of rear three kinds of eluents is respectively 2BV, substep is collected effluent liquid, elution speed is 3BV/h, with TLC, detect effluent liquid, merge the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample II, weight is 0.19g.Silica gel column chromatography is reusable after Mathanol regenerating;
(3) in, press C
18bonded silica gel column chromatography purification ceramide
By applied sample amount, be 0.1～0.5g sample/100g C
18bonded silica gel, takes C
18bonded silica gel 100g, soaks mutually by initial flow, and stirs, and while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column.Then with pressurizing device, compress, make the C after 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, wet method loading, 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, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample III.Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive 95.7%, and weight is 0.072g.The ceramide sample of other low levels again upper prop is recycled.Middle pressure C
18bonded silica gel chromatography column is reusable after 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, with electric contact thermometer, control temperature is 70 ± 1 ℃ simultaneously, extract complete, filter, the ethanol extract of ceramide is transferred to in rotatory evaporator, to be concentrated into alcohol concn be 5%, concentrated solution is transferred in three mouthfuls of clean Florence flasks, by concentrated solution volume, add 60～90 ℃ of sherwood oils of 4 times of amounts, stir 1.0h, be transferred to standing 0.5h in separating funnel, after layering, collect the ether phase on upper strata, extract 2 times.Combined ether phase, reclaims sherwood oil and is concentrated into medicinal extract, obtains brown ceramide sample I, and weight is 1.35g;
(2) purification by silica gel column chromatography
By applied sample amount, be 1～5g crude product/100g silica gel, take 300～400 order silica gel in 105～110 ℃ of activation 1.5h, then the silica gel having activated is put into container, and add appropriate sherwood oil, along fixed-direction, stir, while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column, silicagel column is 40*190mm, and column volume is about 250mL.Then use initial eluent 500mL with the flow velocity balance chromatography column of 9mL/min.
By wet method loading after initial flow phased soln for above-mentioned ceramide crude product I, use respectively petroleum ether-ethyl acetate (v/v) 95: 5,85: 15,75: 25,60: 40,40: 60 to carry out gradient elution for eluent, 95: 5 and 85: 15 two kinds of eluent usage quantitys are respectively 1BV, the usage quantity of rear three kinds of eluents is respectively 2BV, substep is collected effluent liquid, elution speed is 4BV/h, with TLC, detect effluent liquid, merge the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample II, weight is 0.20g.Silica gel column chromatography is reusable after Mathanol regenerating;
(3) in, press C
18bonded silica gel column chromatography purification ceramide
By applied sample amount, be 0.1～0.5g sample/100g C
18bonded silica gel, takes C
18bonded silica gel 100g, soaks mutually by initial flow, and stirs, and while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column.Then with pressurizing device, compress, make the C after 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, wet method loading, 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, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample III.Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive 95.2%, and weight is 0.064g.The ceramide sample of other low levels again upper prop is recycled.Middle pressure C
18bonded silica gel chromatography column is reusable after 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, with electric contact thermometer, control temperature is 80 ± 1 ℃ simultaneously, extract complete, filter, the ethanol extract of ceramide is transferred to in rotatory evaporator, to be concentrated into alcohol concn be 20%, concentrated solution is transferred in three mouthfuls of clean Florence flasks, by concentrated solution volume, add 60～90 ℃ of sherwood oils of 4 times of amounts, stir 1.0h, be transferred to standing 0.5h in separating funnel, after layering, collect the ether phase on upper strata, extract 2 times.Combined ether phase, reclaims sherwood oil and is concentrated into medicinal extract, obtains brown ceramide sample I, and weight is 1.28g;
(2) purification by silica gel column chromatography
By applied sample amount, be 1～5g crude product/100g silica gel, take 300～400 order silica gel in 105～110 ℃ of activation 1.5h, then the silica gel having activated is put into container, and add appropriate sherwood oil, along fixed-direction, stir, while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column, silicagel column is 40*190mm, and column volume is about 250mL.Then use initial eluent 500mL with the flow velocity balance chromatography column of 8mL/min.
By wet method loading after initial flow phased soln for above-mentioned ceramide crude product I, use respectively petroleum ether-ethyl acetate (v/v) 95: 5,85: 15,75: 25,60: 40,40: 60 to carry out gradient elution for eluent, 95: 5 and 85: 15 two kinds of eluent usage quantitys are respectively 1BV, the usage quantity of rear three kinds of eluents is respectively 2BV, substep is collected effluent liquid, elution speed is 4BV/h, with TLC, detect effluent liquid, merge the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample II, weight is 0.21g.Silica gel column chromatography is reusable after Mathanol regenerating;
(3) in, press C
18bonded silica gel column chromatography purification ceramide
By applied sample amount, be 0.1～0.5g sample/100g C
18bonded silica gel, takes C
18bonded silica gel 100g, soaks mutually by initial flow, and stirs, and while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column.Then with pressurizing device, compress, make the C after 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, wet method loading, 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, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample III.Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive 95.1%, and weight is 0.076g.The ceramide sample of other low levels again upper prop is recycled.Middle pressure C
18bonded silica gel chromatography column is reusable after Mathanol regenerating.
Claims (2)
1. a preparation method for konjak ceramide, is characterized in that comprising following operation steps:
(a) slightly carrying of ceramide: take fry starch of konjak as raw material, at a certain temperature, with the ethanolic soln of certain volume, extract, after extraction, filter, concentrated, reclaim ethanol, then by concentrated solution petroleum ether extraction 2 times, combined ether phase, reclaims sherwood oil and obtains brown ceramide medicinal extract, i.e. ceramide sample I;
Described alcohol concn is 75%～95%, and the temperature of extraction is 60～80 ℃, and extraction time is 1～2h, and feed liquid mass volume ratio is 1:3～5g/ml;
Described sherwood oil is 60～90 ℃ of cuts, and solid-liquid ratio is mass volume ratio 1:2～4g/ml, and churning time is 0.5～1.5h, and time of repose is 0.5h, extracts 2 times;
(b) purification by silica gel column chromatography: by silica gel wet method dress post, and with initial eluent balance chromatography column, ceramide sample I is dissolved with initial eluent, wet method loading; With petroleum ether-ethyl acetate, be that eluent carries out gradient elution, substep is collected effluent liquid, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaims solvent, is dried to obtain ceramide sample II; Volume ratio
Described silica gel is 300～400 orders, activation temperature is 105～110 ℃, soak time is 1～2h, after silica gel wet method dress post, with initial eluent balance chromatography column, by wet method loading after initial flow phased soln for above-mentioned ceramide crude product I, applied sample amount is 1～5g crude product/100g silica gel, then use sherwood oil: ethyl acetate volume ratio is 95:5, 85:15, 75:25, 60:40, 40:60, for eluent carries out gradient elution, two kinds of eluent usage quantitys of 95:5 and 85:15 are respectively 1BV, the usage quantity of rear three kinds of eluents is respectively 2BV, substep is collected effluent liquid, elution speed is 2～4BV/h, with TLC, detect effluent liquid, merge the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample II,
(c) in, press C
18bonded silica gel ODS column chromatography purification: by 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 to wet method loading with initial eluent; With alcohol-water, be that eluent carries out gradient elution, substep is collected effluent liquid, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaims solvent, is dried to obtain ceramide sample III; Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive more than 95%.
2. ceramide preparation method according to claim 1, is characterized in that:
In described in step (c), press C
18bonded silica gel column chromatography dress column method is: take appropriate C
18bonded silica gel soaks mutually by initial flow, and stirs, and while there is no bubble in silica gel, wet method packs in middle pressure chromatographic column; Then with pressurizing device, compress, make the C after 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, wet method loading, applied sample amount is 0.1～0.5g sample/100g C
18bonded silica gel, take alcohol concn as 90%, 75%, 60% for eluent carries out gradient elution, and the amount of every kind of eluent is 2～2.5BV, substep is collected effluent liquid, elution speed is 1～1.5BV/h, with TLC, detects effluent liquid, merges the effluent liquid with identical composition, reclaim solvent, be dried to obtain ceramide sample III; Through HPLC-ELSD, detect, the purity of ceramide sample III can arrive more than 95%.
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| CN201110327965.8A CN102351730B (en) | 2011-10-22 | 2011-10-22 | Method for preparing ceramide from konjac fly powder |
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| CN105232366B (en) * | 2015-09-19 | 2018-07-06 | 湖南大湘西魔芋有限公司 | A kind of konjak ceramide sponge gourd water moisture-keeping crease-shedding liquid and preparation method thereof |
| CN106008253B (en) * | 2016-05-25 | 2018-09-04 | 湖南华诚生物资源股份有限公司 | A method of extracting high-purity ceramide from rice bran |
| CN108623492A (en) * | 2017-03-18 | 2018-10-09 | 成都希福生物科技有限公司 | A method of the high efficiency extraction ceramide from konjaku |
| CN108947864A (en) * | 2018-06-08 | 2018-12-07 | 三原利华生物技术有限公司 | A kind of preparation method of ceramide |
| CN109265501A (en) * | 2018-11-15 | 2019-01-25 | 派能生物科技（深圳）有限公司 | A kind of method and application for extracting ceramide from pineapple |
| CN110438177A (en) * | 2019-08-12 | 2019-11-12 | 三原利华生物技术有限公司 | A method of ceramide is prepared from apple juicing pomace |
| CN111499537B (en) * | 2020-05-12 | 2020-11-06 | 深圳市三也生物科技有限公司 | Refining and purifying method of plant-derived ceramide extract |
| CN113416184B (en) * | 2021-07-22 | 2023-09-22 | 南京师范大学 | Method for extracting isosaftoside, vitexin and alkaloid from konjak |
| JP7273427B1 (en) * | 2021-12-24 | 2023-05-15 | 雪国アグリ株式会社 | Water-soluble konjac potato extract and method for producing the same |
| CN114807258B (en) * | 2022-04-13 | 2024-06-11 | 广东海洋大学 | Method for extracting ceramide from red rice bran |
| CN116462605A (en) * | 2023-03-09 | 2023-07-21 | 四川晗晨生物科技有限公司 | Extraction method of plant-derived ceramide |
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| CN101885747A (en) * | 2010-06-12 | 2010-11-17 | 暨南大学 | Separation and purification method for cerebroside and ceramide type compound |
| CN102070681A (en) * | 2009-12-22 | 2011-05-25 | 成都希福食品有限公司 | Konjak ceramide and extraction method thereof |
| CN102190689A (en) * | 2010-08-16 | 2011-09-21 | 成都希福生物科技有限公司 | Purification method for ceramide in amorphophalms konjac |
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| CN101914119A (en) * | 2010-08-16 | 2010-12-15 | 成都希福食品有限公司 | Preparation method of konjak ceramide reference sample |
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| CN102070681A (en) * | 2009-12-22 | 2011-05-25 | 成都希福食品有限公司 | Konjak ceramide and extraction method thereof |
| CN101885747A (en) * | 2010-06-12 | 2010-11-17 | 暨南大学 | Separation and purification method for cerebroside and ceramide type compound |
| CN102190689A (en) * | 2010-08-16 | 2011-09-21 | 成都希福生物科技有限公司 | Purification method for ceramide in amorphophalms konjac |
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