CN111499537B - Refining and purifying method of plant-derived ceramide extract - Google Patents

Abstract

The invention relates to a method for refining and purifying a crude plant-derived ceramide extract, which adopts a refining and purifying process of salting out, primary high-molecular magnetic microsphere separation, secondary high-molecular magnetic microsphere separation and crystallization, overcomes the defect that the existing purifying method can only obtain low-purity ceramide, can provide high-purity ceramide with the purity of 99 percent or higher, and can be directly used in the fields of cosmetics and medicines. The purification method adopts a means of combining secondary crystallization and specific magnetic polymer microsphere adsorption, can obviously improve the product purity, effectively reduces the product loss, and is suitable for industrial production.

Description

Refining and purifying method of plant-derived ceramide extract

Technical Field

The invention belongs to the technical field of plant extract purification and separation, and particularly relates to a combined refining and purification method of a plant-derived ceramide crude product.

Background

Ceramides are sphingoid substances formed by condensing sphingosine and long-chain fatty acids through amido bonds, and the molecular formula of the ceramides generally has amide groups, polyhydroxy groups, long-chain alkyl chains with different lengths and other groups, so that the ceramides have hydrophilic and lipophilic amphiphilic characteristics. Ceramide has effects of barrier, moisture retention, whitening, anti-aging, anti-tumor and the like, and is widely applied to the fields of medicine, food and cosmetics. The ceramide can be divided into natural ceramide and chemically synthesized ceramide, and the natural ceramide is generally extracted from plants, animals and zymophyte. The cosmetic application is mainly based on natural ceramide products.

Among the above sources, ceramides derived from plants such as wheat, rice, soybean, etc. have high safety, and can well meet the requirements of pharmaceutical industry and cosmetic market. However, because of the low content of ceramide derived from plants and the hydrophilic and lipophilic properties, extraction and separation are very difficult, and at present, it is difficult to obtain high-purity ceramide by means of organic solvent extraction, column chromatography separation and the like. At present, the product with the purity of 50-70% can be obtained by adopting the common organic solvent extraction and column chromatography separation method in general.

Among plant sources, konjak contains abundant ceramide. A method for separating and extracting ceramide from konjak has been widely developed. For example, the patent of application No. 201410040856.1 discloses a method for extracting and separating ceramide from konjac flying powder, which comprises the steps of firstly extracting the ceramide from konjac by ultrasonic oscillation with 95% ethanol, concentrating the extract, then extracting with supercritical CO2, and extracting the extract with supercritical CO2 for the second time to finally obtain a product with the content of 73.8%; the application of patent No. 201210133207.7 adopts 70% ethanol solution for ultrasonic extraction of konjak ceramide, the extract is concentrated and then extracted with petroleum ether for 3 times, and then purified by silica gel column chromatography, with petroleum ether: eluting with ethyl acetate solvent, concentrating the eluate, purifying by silica gel column chromatography again, eluting with ethanol: water is used as a solvent for elution, and the eluent is concentrated and then is centrifuged by a centrifuge and is frozen and dried in vacuum to prepare a sample, so that the problems of complex process, high production cost and the like are solved. Other related patents may also be listed below.

CN102190689 provides a method for purifying ceramide crude product extracted from rhizoma Amorphophalli. Purifying by silica gel column chromatography twice: and (3) carrying out dry loading, carrying out gradient elution by using an organic solvent, and carrying out column chromatography twice by virtue of first silica gel column chromatography and second silica gel column chromatography to purify ceramide.

CN102070681 discloses konjak ceramide and an extraction method thereof, and the method comprises the following steps: (1) soaking konjac byproducts or/and wastes serving as raw materials in 45-85% (V/V) ethanol for 1-2 hours, collecting leachate, transferring the leachate into a distillation recovery device, standing for layering, and collecting an oil phase; concentrating the water phase under reduced pressure and/or freeze drying, extracting with ethyl acetate, purifying, and collecting oil phase; mixing all oil phases, and concentrating to obtain paste; (2) dissolving the concentrate with 45-80% (V/V) ethanol, extracting with ethyl acetate, extracting with petroleum ether, mixing the residual liquid of ethyl acetate and petroleum ether, drying, concentrating, and removing solvent to obtain crude product containing 0.5-20% of konjak ceramide.

CN102351730 discloses a method for preparing ceramide from konjac flying powder, which is characterized by comprising the following steps: a) crude extraction of ceramide: extracting konjac flying powder serving as a raw material with an ethanol solution, and then extracting with petroleum ether; b) silica gel column chromatography purification: loading silica gel into column by wet method, loading sample by wet method, and performing gradient elution by using petroleum ether-ethyl acetate as eluent; c) medium pressure C18 bonded silica gel ODS column chromatography: loading columns by a wet method, and loading samples by the wet method; gradient eluting with ethanol-water as eluent, and drying to obtain 95% ceramide.

CN102675139 discloses a method for extracting ceramide from rhizoma Amorphophalli flying powder, which comprises eight procedures of selecting materials, comprehensively extracting, concentrating, extracting, gradient eluting, drying, gradient eluting again and drying; the extraction rate of ceramide extracted from the konjac flying powder reaches 75%, and the purity reaches 95%.

CN201710162448 discloses a method for efficiently extracting ceramide from konjak. The method comprises the following steps: coarse extraction, peculiar smell removal, extraction purification and chromatography purification. The crude extraction is carried out on the konjak by-product by ethanol with different volume fractions for multiple times of ultrasonic extraction, filtrate obtained by filtration is distilled to recover ethanol to obtain crude extract concentrated solution, and the recovered ethanol can be used as an extraction solvent again for use after the density of the recovered ethanol is adjusted, so that the solvent consumption is reduced. The extraction purification and chromatographic purification are to utilize the hydrophilic and lipophilic characteristics of ceramide, to extract and elute the crude extract with organic solvents with different polarities, to collect the liquid with the same components, to concentrate and dry to obtain ceramide sample.

CN201710999197 discloses a method for separating and purifying konjak ceramide by molecular distillation, which comprises preheating a konjak ceramide supercritical CO2 fluid extract in a preheater, adding the preheated konjak ceramide supercritical CO2 fluid extract into a molecular distillation system through a feeder, performing secondary molecular distillation treatment on konjak ceramide under appropriate separation and purification conditions under a high vacuum condition, and finally separating to obtain a high-purity ceramide sample.

CN201410336065 discloses a method for extracting konjak ceramide from konjak, which comprises the following steps: (1) crushing konjak into fine powder, adding ethanol with the material-liquid ratio of 1.5: 500-; (2) adding the konjak particles prepared in the step (1) into an extraction kettle of supercritical CO2 extraction equipment, heating the extraction kettle, a separation kettle I and a separation kettle II, refrigerating a refrigerator and a storage tank, opening a carbon dioxide gas cylinder when the temperature of the extraction kettle is 30-70 ℃, the temperature of the separation kettle I is 40-65 ℃, and the temperature of the separation kettle II is 30-60 ℃, pressurizing the extraction kettle and the separation kettle through a high-pressure pump, closing carbon dioxide when the pressure of the extraction kettle is 20-40MPa, the pressure of the separation kettle I is 6-18MPa, and the pressure of the separation kettle II is 4-7MPa, opening a circulating valve, performing extraction circulation, discharging materials from the separation kettle I and the separation kettle II respectively every 30min, and performing circulation extraction for 1-2 h.

According to the prior art, although a number of different extraction processes are disclosed, the purity of the obtained product is generally lower than 95%, obviously not satisfying the field requirements of pharmaceuticals and cosmetics, but only the food field. Moreover, even if the prior art achieves more than 90% purity, it generally comes at the expense of a higher product loss rate, with a lower extraction rate.

In the current extraction separation method, the utilization of magnetic polymer microspheres is more and more common. However, the magnetic polymer microspheres have the disadvantages of non-specific adsorption and insufficient surface functional groups, so that the magnetic polymer microspheres have poor universality on target products during product separation. In order to improve the separation efficiency of a certain product, microsphere matrix construction and surface function modification which are specifically adsorbed according to the structure and group characteristics of the product to be separated are required, so that the application of the magnetic polymer microsphere separation method is limited. The prior art does not report the separation application of the magnetic polymer microspheres in the aspect of konjak ceramide.

Therefore, in order to overcome the drawbacks of the prior art that the extraction and one or more silica gel column chromatography combined ceramide purification methods are adopted (specifically, for example, due to the hydrophilic and lipophilic properties of ceramide, the extraction efficiency is low and a large amount of loss is caused), while the purity can be improved to more than 90% by adopting two or more column chromatography purification methods, the purification method has the disadvantages of long purification time, large solvent consumption, low separation efficiency and the like besides more product loss caused by multiple chromatography, and is not suitable for industrial production), it is necessary to provide a separation and purification method suitable for crude plant-derived ceramide extract products such as konjak and the like, so as to overcome the defects of the prior art.

Disclosure of Invention

In view of the above problems in the prior art, the primary object of the present invention is to provide a method for refining and purifying crude ceramide extract extracted from plant-derived materials, such as konjac, wheat, rice, soybean, etc., preferably konjac.

Another object of the present invention is to provide a high purity ceramide separated from a plant-derived extract, which can be directly used in the fields of cosmetics and pharmaceuticals, and which has a purity of not less than 98%, preferably a HPLC purity of not less than 99%.

The purification method adopts the means of combination of salting out, specific adsorption separation of magnetic microspheres and crystalline phase, mainly utilizes the specific surface modification of different properties of the high molecular magnetic microspheres for adsorption and purification, can obviously improve the purity and separation efficiency of the product, effectively reduces the loss of the product, and is suitable for industrial production.

Wherein, the source or extraction method of the crude plant-derived ceramide raw material is known in the art, and includes but is not limited to organic solvent extraction, distillation, chromatographic separation and other one or more purification means.

Preferably, the raw material purity of the crude plant-derived ceramide extract is not lower than 70% in order to ensure the purity of the final product. When the purity of the ceramide in the crude extract is lower than 70%, a high-purity ceramide product with the purity of more than 98% cannot be obtained by the method.

Further, when the purity of ceramide in the crude extract is less than 70%, the crude extract can be further purified to a purity of more than 70% by the above-mentioned conventional purification methods well known in the art, such as extraction or column chromatography. Conventional purification means such as specific extraction operation and column chromatography purification are well known in the art.

Optionally, the method is also suitable for separating and purifying the crude ceramide extracted by a fermentation method.

The crude product of the fermentation method is a ceramide crude product including glycosyl ceramide derivatives extracted from the bacterial wall of a Sphingomonas sp strain or a yarrowia lipolytica strain, and the fermentation method and the primary extraction method thereof are known in the prior art.

In order to achieve the above object, the technical solution of the present invention is as follows.

In a first aspect, the present invention provides a method for purifying a plant-derived ceramide extract such as konjak, comprising the steps of S1 to S7:

step S1: salting out: dissolving the crude product of the konjak ceramide with an ethanol solution, adding an inorganic sodium salt, adjusting the pH value to weak acidity with hydrochloric acid, evaporating and concentrating, stopping heating when the ceramide begins to be salted out, cooling to room temperature, standing at 0-5 ℃, cooling and crystallizing for 8-12h to obtain the ceramide solid in the form of the sodium salt.

Specifically, weighing a crude product of konjak ceramide, dissolving the crude product of konjak ceramide by using an ethanol solution until the weight of the crude product is 1-5wt%, adding inorganic sodium salt to enable the concentration of the inorganic sodium salt to reach 30-50g/L, adjusting the pH value to be 5.5-6 by using hydrochloric acid, stirring for 0.5-1h, and then evaporating and concentrating to enable the crude product to reach a saturated state; stopping heating when the ceramide begins to be salted and separated out, standing at the temperature of 0-5 ℃, and cooling and crystallizing for 8-12h to obtain ceramide solid in a sodium salt form; filtering and collecting filter cakes, and combining the filtrate with the next batch of raw materials for recycling.

Wherein, the purity of the crude product of konjak ceramide is 70-95%, preferably 80-95%, more preferably 85-95%.

Wherein, the volume concentration of the ethanol solution is preferably 65-75%.

Among them, the inorganic sodium salt is preferably selected from sodium chloride.

Further, when the crude konjak ceramide is too low (for example, less than 80%), a purification pretreatment such as crystallization, silica gel column chromatography, or the like is required; otherwise, the crude product has low purification efficiency and cannot obtain a high-purity product.

Step S2: drying and drying the filter cake after suction filtration, heating and stirring the filter cake at 40-50 ℃ by using an ethanol solution for dissolving, adjusting the mass concentration of the solid to be 1-5%, and dropwise adding hydrochloric acid to adjust the pH to 4-5.

Wherein the volume concentration of the ethanol solution is 50-90% (v/v).

Step S3: primary adsorption and purification:

under the condition of stirring, adding 10-30wt% of macromolecular magnetic microsphere A with carboxyl surface modified into the konjak ceramide solution; slowly stirring for 1-2h to make the ceramide molecules fully adsorbed with the macromolecular magnetic microspheres A; and after adsorption, applying a magnetic field for 10-20min to perform solid-liquid separation, continuously adsorbing the mother liquor subjected to microsphere separation for 1-3 times by using magnetic polymer microspheres, combining the polymer magnetic microspheres A, and leaching and washing by using deionized water at room temperature to obtain the polymer magnetic microspheres A-ceramide composite microspheres.

Wherein, the magnetic field action comprises the adoption of magnetic acting force such as an electromagnetic field, a magnet and the like. The ceramide molecules are separated out along with the macromolecular magnetic microspheres A under the action of a magnetic field through magnetic separation. For example, the supernatant is separated by allowing the magnetic beads to settle by the action of an electromagnetic field or by the action of a strong magnet at the bottom of the reaction vessel.

Wherein, the polymer magnetic microsphere A is a polyvinyl alcohol/poly glycidyl methacrylate based composite polymer magnetic microsphere. The preparation method of this particular magnetic microsphere is described below.

Step S4: dispersing the separated composite microspheres in petroleum ether, desorbing for 15-30min under shaking table vibration or rapid stirring, applying external magnetic field to separate out magnetic microspheres A, collecting petroleum ether solution containing konjak ceramide, and evaporating to recover solvent.

Further, the separated magnetic microspheres A can be desorbed for 1-2 times by the same method, the solution is collected, the petroleum ether solution is combined, and the solvent is recovered by rotary evaporation, so that the first purified konjak ceramide is obtained.

Step S5: performing secondary adsorption, namely adding the obtained konjak ceramide solid into 3-10 times of 50-90% methanol solution in volume ratio by mass for dissolving, and dropwise adding sodium hydroxide to adjust the pH value to 9-11; adding the aminated or aminated surface-modified macromolecular magnetic microsphere B under stirring, slowly stirring and adsorbing for 1-2h, then carrying out solid-liquid separation under the action of a magnetic field, collecting the magnetic microsphere B, repeatedly adsorbing the solution after microsphere separation for 1-2 times, combining the microspheres B, and washing with deionized water at room temperature to obtain the macromolecular magnetic microsphere B-ceramide composite microsphere.

Wherein the volume fraction of the methanol aqueous solution is 50-90%.

Wherein the mass fraction of the added macromolecular magnetic microspheres B is 10-30 wt%.

Wherein the macromolecule magnetic microsphere B is a magnetic microsphere with a surface coated with mesoporous silicon dioxide.

Preferably, sodium hydroxide is added dropwise to adjust the pH to be 9.5-10.5, so as to avoid alkaline hydrolysis of the glycoside units in the ceramide containing the glycoside units under the strong alkaline condition.

Step S6: dispersing the composite microspheres in 75-90% ethanol solution by volume, dropwise adding hydrochloric acid to adjust the pH to 6-7, adding a proper amount of ethyl acetate, rapidly stirring or shaking for 10-15min, then applying an external magnetic field to separate the magnetic microspheres B, and collecting the solution part; desorbing the magnetic microsphere B by the same method for 1-2 times, combining the collected solutions, recovering the solvent by rotary evaporation, and vacuum drying the obtained solid at 50-60 ℃.

Wherein the volume of the ethyl acetate is 5-20%, preferably 10-20% of the ethanol solution.

Step S7: under the condition of heating and stirring in a water bath, dissolving the dried solid with a proper amount of acetone, cooling to room temperature, standing at 0-2 ℃ for crystallization for 8-12h to obtain white crystals, filtering, drying to obtain purified konjak ceramide, and measuring the HPLC purity to be more than 99%, wherein the purification loss rate is lower than 20%.

Wherein the heating temperature of the water bath is 50-55 ℃.

The conditions for measuring HPLC purity of konjak ceramide in the present invention are as follows.

Chromatographic conditions mobile phase: 0.1% acetic acid methanol 10: 90; the column temperature is 35 ℃; the flow rate is 0.8-1 mL/min.

Illustratively, an HPLC column, a Kromasil C18 column or a C18 reverse phase column; the elution system uses the above acetic acid buffer solution-methanol.

In the method of the present invention, the polymer magnetic microsphere A is prepared through the following steps S1-S2.

S1: preparing magnetic polyvinyl alcohol/polyglycidyl methacrylate-based microspheres:

heating and dissolving polyvinyl alcohol, adding nano ferroferric oxide magnetic particles and poly glycidyl methacrylate in a boiling water bath, stirring and uniformly mixing, adding liquid paraffin and tween, stirring, then adding hydrochloric acid and glutaraldehyde solution, stirring and carrying out crosslinking reaction for 15-30min, then centrifugally separating out microspheres, washing with absolute ethyl alcohol, and carrying out vacuum drying to obtain the magnetic polyvinyl alcohol/poly glycidyl methacrylate based microspheres.

Wherein the concentration of the dissolved polyvinyl alcohol is 5-15 wt%.

Wherein the mass ratio of the polyvinyl alcohol to the ferroferric oxide is 10: 0.5-2; the mass ratio of the polyvinyl alcohol to the poly glycidyl methacrylate is 10: 1-3.

Wherein the total mass of the liquid paraffin and the Tween is 10-30 times of that of the polyvinyl alcohol, and the mass ratio of the liquid paraffin to the Tween is preferably not less than 1.

Wherein, the tween is selected from tween 60 or tween 80.

Wherein the concentration of the glutaraldehyde solution is 20-30 wt%.

Wherein, the concentration of the hydrochloric acid is preferably not less than 1 mol/L.

S2: surface carboxyl modification:

dispersing the polyvinyl alcohol/poly glycidyl methacrylate-based microspheres in acetonitrile, sequentially adding methacrylic acid, methylene bisacrylamide and azodiisobutyronitrile, uniformly dispersing, heating in a water bath at 75-80 ℃ for reaction, performing magnetic field separation after the reaction is finished, and washing with absolute ethyl alcohol to obtain the polymer magnetic microspheres A with surface carboxyl modified.

Wherein the dosage ratio of the polyvinyl alcohol/poly glycidyl methacrylate based microspheres to the acetonitrile is 1g:5-20 mL.

Wherein the mass ratio of the microspheres to the methacrylic acid to the methylene bisacrylamide to the azobisisobutyronitrile is 10:3-5:0.1-0.5: 0.1-0.3.

Wherein the reaction time is 4-8h by heating in water bath.

Wherein the washing times of the absolute ethyl alcohol are 2-5 times.

In the method of the present invention, the polymer magnetic microsphere B is prepared through the following steps S1-S2:

step S1: weighing mesoporous silica microspheres, ultrasonically dispersing the mesoporous silica microspheres in deionized water, and uniformly stirring to form a suspension; weighing a ferrous chloride and ferric chloride mixed raw material with a molar ratio of 1:2, dissolving the mixed raw material in a proper amount of deionized water, uniformly stirring, adding the mixture into a silica particle suspension, adding PVP (polyvinylpyrrolidone), starting mechanical stirring, adding concentrated ammonia water in batches under the condition of heating in a water bath at 75-80 ℃, and continuously stirring and reacting for 1-2 hours after the addition is finished; and cooling to room temperature after the reaction is finished, centrifugally or magnetically separating the microspheres, washing the microsphere particles with deionized water, and drying in vacuum to obtain the magnetic microspheres with the surfaces coated with the mesoporous silica.

Wherein, the particle size of the mesoporous silicon dioxide microsphere is preferably 0.1-0.3 μm.

Wherein the dispersion ratio of the silicon dioxide microspheres to the deionized water is 1g: 15-30 g.

Wherein the mass ratio of the silicon dioxide microspheres to the ferrous chloride/ferric chloride mixed raw material is 1: 2-3.

Wherein the mass ratio of the silicon dioxide microspheres to the PVP is 10: 0.5-2.

Wherein, the ammonia water used is preferably 25 to 30 wt%; the mass amount of the ammonia water is 1-1.5 times of that of the ferrous chloride/ferric chloride mixed raw material.

Step S2: surface modification: dispersing the magnetic microsphere coated with the silicon dioxide, ethylenediamine and 3- (trimethoxysilyl) propyl-2-methyl-2-acrylate coupling agent in methanol, reacting at 35-40 ℃ for 6-12h, separating the microsphere by an external magnetic field, and washing with methanol and deionized water in sequence to obtain the amino-modified macromolecular magnetic microsphere B.

Wherein the mass ratio of the magnetic microspheres to the ethylenediamine is 5-10: 1.

Wherein the mass ratio of the magnetic microspheres to the 3- (trimethoxysilyl) propyl-2-methyl-2-acrylate is 3-5: 1.

Wherein the dosage ratio of the methanol to the magnetic microspheres is 20-30ml:1 g.

Among them, the reaction time is preferably 8 to 12 hours.

In a second aspect, the invention also provides a high-purity konjak ceramide purified by the method, wherein the purity of the high-purity konjak ceramide purified by the method is more than 99%.

In a third aspect, the invention also provides application of the high-purity konjak ceramide in the fields of cosmetics and medicines.

Compared with the prior art, the invention has the following beneficial effects:

(1) the method adopts the refining and purifying process of salting out, primary polymer magnetic microsphere separation, secondary polymer magnetic microsphere separation and crystallization, fully utilizes the characteristics of simplicity of crystallization and purification and high efficiency of magnetic microsphere separation, overcomes the defect that the existing purification method can only obtain low-purity konjak ceramide (usually less than 95 percent) by single or twice column chromatography, achieves the HPLC purity of about 99 percent or more, and can meet the purity requirement in the fields of cosmetics and even medicines.

Wherein, the salting-out step utilizes the characteristic that the konjak ceramide molecule contains a plurality of hydroxyl groups, and can be separated out in a sodium salt form under a specific pH value range, thereby increasing the water solubility and the operability of crystal separation; the chromatographic purity of konjak ceramide is initially improved by a salting-out crystallization impurity removal method, and the impurity content is reduced for the next step of specific adsorption and separation of the macromolecular magnetic microspheres.

(2) The surface of the polymer magnetic microspheres A and B modified by surface groups contains a large number of carboxyl, amino, mesoporous silica surface groups or PVA free polar hydroxyl groups and other specific groups, and under the condition of specific acidic or alkaline PH, each magnetic microsphere can be reversibly grafted on the magnetic microsphere by virtue of the specific adsorption effect of the surface groups and the hydroxyl, long-chain hydrophobic groups and other groups on the surface of the ceramide molecules through hydrogen bond action, hydrophobic van der Waals force and the like, so that the enrichment of the ceramide molecules is realized. Moreover, the magnetic microspheres A and the magnetic microspheres B with different surface modification groups are respectively adsorbed, so that although certain purification loss is generated, the interference of the magnetic microspheres A and the magnetic microspheres B on the non-specific adsorption of impurities is favorably improved, the grafting rate is high, the capturing and purifying efficiency is high, and the purifying efficiency is obviously improved.

After reversible adsorption is realized, the adsorption effect of surface groups and ceramide molecules is reduced by changing the property and the pH value of a solution in which the magnetic microspheres are positioned, and the ceramide is released from the magnetic microspheres by adding an excellent soluble solvent, so that a purified product can be conveniently obtained compared with silica gel column chromatography.

(3) Compared with the prior art, the method has high purification efficiency, and can obtain more than 99% of high-purity products; the post-treatment is convenient, the magnetic microspheres can be recycled after simple treatment, and the adopted solvent can be recovered, thereby realizing the effect of environmental protection.

(4) The purification method has wide range of applicable substrates, raw materials can be obtained by various methods for extracting crude products of the konjak ceramide, and the crude products can be obtained by common extraction, supercritical CO2 extraction process, molecular distillation extraction and other methods; in addition, the condition is mild, high temperature and distillation operation are avoided, the possibility of oxidizing ceramide is reduced to the greatest extent, the generation of new impurities is inhibited, and the market demand of high-purity ceramide can be met.

Detailed Description

The present invention will be described in detail with reference to specific examples, but the scope of the present invention is not limited to these examples in any way.

Preparation example 1

Preparing surface carboxyl modified macromolecule magnetic microsphere A

1) Preparation of magnetic polyvinyl alcohol/poly glycidyl methacrylate composite base microsphere

Heating and dissolving 40g of polyvinyl alcohol in 400mL of water, adding 4g of nano ferroferric oxide magnetic particles and 5g of poly glycidyl methacrylate under the condition of boiling water bath, mechanically stirring for 0.5h, uniformly mixing, adding 600mL of liquid paraffin and 200g of Tween 80 when cooling to 60 ℃, stirring for 4h, then adding 10mL of 2mol/L hydrochloric acid solution and 40mL of 30wt% glutaraldehyde solution, stirring for reaction for 30min, centrifugally separating out microspheres, washing for 2-3 times by using absolute ethyl alcohol, and then drying in vacuum to obtain the magnetic polyvinyl alcohol/poly glycidyl methacrylate composite base microspheres with the particle size range of 1-5 mu m.

2) Surface carboxyl modification: ultrasonically dispersing 50g of the polyvinyl alcohol/poly glycidyl methacrylate-based microspheres in 500ml of acetonitrile, sequentially adding 20g of methacrylic acid, 2g of methylene bisacrylamide and 1g of azodiisobutyronitrile, stirring and dispersing uniformly, heating and reacting for 6 hours in a water bath at 75-80 ℃ under slow stirring, separating the microspheres by a magnetic field, fully washing with absolute ethyl alcohol to obtain polymer magnetic microspheres A with surface carboxyl modified, and drying for later use.

Preparation example 2

Preparing surface amino modified macromolecule magnetic microsphere B

1) Weighing 10g of mesoporous silica microspheres with the particle size of about 0.1 mu m, ultrasonically dispersing the mesoporous silica microspheres in 310g of deionized water, and uniformly stirring to obtain a suspension; weighing 25g of a ferrous chloride and ferric chloride mixed raw material with a molar ratio of 1:2, dissolving the raw material in 50ml of deionized water, adding the mixture into the silica particle suspension after uniformly stirring, and slowly dropwise adding 1g of PVP under the stirring condition; starting mechanical stirring, adding 30g of concentrated ammonia water (30 wt%) in batches under the heating condition of 75 ℃ water bath, and continuously stirring and reacting for 2 hours after the adding is finished; and cooling to room temperature after the reaction is finished, centrifugally or magnetically separating the microspheres, washing the microsphere particles with deionized water, and drying in vacuum to obtain the magnetic microspheres with the surfaces coated with the mesoporous silica.

2) Dispersing 4g of the silicon dioxide coated magnetic microsphere, 0.5g of ethylenediamine and 1.1g of 3- (trimethoxysilyl) propyl-2-methyl-2-acrylate coupling agent in 100mL of methanol, stirring and reacting at 37 ℃ for 10h, adding a magnetic field to separate the microsphere, washing with methanol and deionized water in sequence to obtain the amino modified macromolecular magnetic microsphere B, and drying for later use.

Example 1

The refining and purifying method of the konjak ceramide crude product comprises the following steps:

1) 10g of crude konjak ceramide (crude konjak powder obtained by supercritical carbon dioxide fluid extraction and molecular distillation in the example, and extracted according to the extraction method of patent 201710999197.8) with the purity of about 75% is dissolved to 2wt% by using 50% ethanol, 18g of sodium chloride is added, the pH value is adjusted to 5.5-6 by using hydrochloric acid, and the mixture is stirred for 0.5h and then evaporated and concentrated to reach the saturated state. Stopping heating when the ceramide solution begins to separate out solids, cooling to room temperature, standing at the temperature of 1-3 ℃, cooling and crystallizing for 12 hours to obtain salified solids; filtering to obtain solid, mixing the filtrate with the next raw materials, and recycling. Drying the filter cake, heating and stirring the filter cake for dissolving by using 350ml of ethanol solution with the volume ratio of 60% at the temperature of 40 ℃, and dropwise adding hydrochloric acid to adjust the pH value to 4.

2) Adding 52g of the carboxyl surface modified polymer magnetic microspheres A obtained in the preparation example into the konjak ceramide solution under stirring; slowly stirring for 2h to ensure that the ceramide molecules are fully adsorbed with the macromolecular magnetic microspheres A; and then performing solid-liquid separation by using a strong magnet for 15min to obtain magnetic microspheres A, continuously and repeatedly adsorbing the mother liquor after the microspheres are separated by using the magnetic polymer microspheres for 1 time according to the operation, combining the polymer magnetic microspheres A, and washing the polymer magnetic microspheres A by using deionized water at room temperature to obtain the polymer magnetic microspheres A-ceramide composite microspheres.

3) Dispersing the washed composite microspheres in 420ml of petroleum ether, desorbing for 25min under the shaking condition of a shaking table, applying an external magnetic field to separate the magnetic microspheres A, and collecting the petroleum ether solution; and (3) repeating the desorption operation of the magnetic microspheres A for 2 times according to the same method, collecting the solution, combining the petroleum ether solution and carrying out rotary evaporation to recover the petroleum ether solvent.

4) Dissolving the obtained solid by 85ml of a methanol solution with a volume ratio of 65%, dropwise adding sodium hydroxide to adjust the pH value to 10, and adding about 16.5g of the macromolecular magnetic microsphere B prepared in the preparation example 2 under the stirring condition; slowly stirring and adsorbing for 1.5h, then carrying out solid-liquid separation under the action of a magnetic field, collecting the magnetic microspheres B, repeatedly adsorbing the solution after the microspheres are separated for 1 time, combining the microspheres B, and washing with purified water at room temperature to obtain the macromolecular magnetic microsphere B-ceramide composite microspheres.

5) Dispersing the obtained composite microspheres in 220ml of 75% ethanol solution, dropwise adding hydrochloric acid to adjust the pH value to 6, adding 25ml of ethyl acetate, rapidly stirring for 10min, then applying an external magnetic field to separate the magnetic microspheres B, and collecting the solution; desorbing the magnetic microspheres B for 1 time by the same method, combining the collected solutions, and performing rotary evaporation to recover the solvent; a ceramide solid was obtained.

6) Vacuum drying the obtained solid at 50 deg.C, dissolving with acetone under heating and stirring in 55 deg.C water bath, cooling to room temperature, standing at 0-2 deg.C for crystallization for 10 hr to obtain white crystal, filtering, oven drying to obtain high purity rhizoma Amorphophalli ceramide crystal; HPLC purity was measured at 99.07%.

Further, evaporating and drying the filtrate mother liquor, and recovering residual solids for recycling as crude products. The total purification yield of this example was about 83%.

Example 2

1) 10g of crude konjak ceramide (obtained by extracting crude konjak ceramide with ethanol solvent and purifying by column chromatography in the example, and extracted from konjak powder according to the extraction method of patent 201710162448.7) with purity of about 90% is dissolved in 650ml of 65% ethanol, 20g of sodium chloride is added, the pH is adjusted to 5.5-6 with hydrochloric acid, and the mixture is stirred for 0.5h and then evaporated and concentrated to reach a saturated state. Stopping heating when the ceramide solution begins to separate out solids, cooling to room temperature, standing at the temperature of 1-2 ℃, cooling and crystallizing for 12 hours to obtain salified solids; filtering to obtain solid, mixing the filtrate with the next raw materials, and recycling. Drying the filter cake, heating and stirring the filter cake to dissolve the filter cake by using 450ml of ethanol solution with the volume ratio of 70% at 40 ℃, and dropwise adding hydrochloric acid to adjust the pH value to 4.5.

2) Adding 80g of the polymer magnetic microspheres A obtained in the preparation example 1 into the konjak ceramide solution under the stirring condition; slowly stirring for 2h to ensure that the ceramide molecules are fully adsorbed with the macromolecular magnetic microspheres A; and then performing solid-liquid separation by using a strong magnet for 20min to obtain magnetic microspheres A, continuously and repeatedly adsorbing the mother liquor after the microspheres are separated by using the magnetic polymer microspheres for 2 times according to the operation, combining the polymer magnetic microspheres A, and washing the polymer magnetic microspheres A by using deionized water at room temperature to obtain the polymer magnetic microspheres A-ceramide composite microspheres.

3) Dispersing the washed composite microspheres in 550ml of petroleum ether, desorbing for 30min under the condition of rapid stirring, then applying an external magnetic field to separate the magnetic microspheres A, and collecting the petroleum ether solution; and (3) repeating the desorption operation of the magnetic microspheres A for 2 times according to the same method, collecting the solution, combining the petroleum ether solution and carrying out rotary evaporation to recover the petroleum ether solvent.

4) Diluting and dissolving the obtained solid by 120ml of 80 volume percent methanol solution, dropwise adding sodium hydroxide to adjust the pH value to 10, and adding about 30g of the macromolecular magnetic microsphere B prepared in the preparation example 2 under the stirring condition; slowly stirring and adsorbing for 2h, then carrying out solid-liquid separation under the action of a magnetic field, collecting the magnetic microspheres B, repeatedly adsorbing the solution after microsphere separation for 2 times, combining the microspheres B, and washing with purified water at room temperature to obtain the macromolecular magnetic microsphere B-ceramide composite microspheres.

5) Dispersing the obtained composite microspheres in 350ml of 75% ethanol solution, dropwise adding hydrochloric acid to adjust the pH value to 6.5, adding 50ml of ethyl acetate, quickly stirring for 15min, then applying an external magnetic field to separate the magnetic microspheres B, and collecting the solution; desorbing the magnetic microspheres B for 2 times by the same method, combining the collected solutions, and performing rotary evaporation to recover the solvent; a ceramide solid residue was obtained.

6) And (3) carrying out vacuum drying on the obtained solid residue at 50 ℃, then dissolving the solid residue in acetone under the conditions of heating in a water bath at 55 ℃ and stirring, cooling to room temperature, standing at 0-2 ℃ for crystallization for 12 hours to obtain white crystals, filtering, drying and drying to obtain the high-purity konjak ceramide, wherein the HPLC purity is 99.61%.

Further, evaporating and drying the filtrate mother liquor, and recycling residual solids as crude raw materials for recycling.

This example gives 8.2g of ceramide solid in total purification yield of about 91%.

Comparative example 1

The purification was carried out by the method of example 1 under the conditions except that the crude product purity was adjusted to 60%, and the HPLC purity of the obtained konjak ceramide was 96.73%, which could not satisfy the purity requirements in the fields of cosmetics and pharmaceuticals.

Comparative example 2

Purification was carried out by the method of example 1 except that the salting-out crystallization operation was not included in step 1), and the HPLC purity of the obtained konjak ceramide was 98.01%.

Wherein step 1) of this comparative example operates as follows:

10g of crude konjak ceramide (same as example 1) was dissolved in 350ml of 60 vol% ethanol solution at 40 ℃ with heating and stirring, and then pH was adjusted to 4 by dropwise addition of hydrochloric acid.

Comparative example 3

Purification was carried out by the method of example 1 except for not including the steps 4) to 5) (i.e., not including the adsorption operation of the polymeric magnetic microspheres B), and the HPLC purity of the obtained konjak ceramide was 96.06%.

Comparative example 4

The procedure of example 1 was followed except that steps 2) to 3) (i.e., the adsorption operation without the polymeric magnetic microspheres A) were not included and steps 1) and 4) were slightly different, and the HPLC purity of the obtained konjak ceramide was 95.13%.

In this comparative example, the steps of example 1, steps 1) and 4) were adapted and replaced by the following steps 1) to 2):

1) 10g of crude konjak ceramide (same as example 1) was dissolved in 50 vol% ethanol to 2wt%, 18g of sodium chloride was added, the pH was adjusted to 5.5 to 6 with hydrochloric acid, and the mixture was evaporated and concentrated to a saturated state. Stopping heating when the ceramide solution begins to separate out solids, cooling to room temperature, standing at the temperature of 1-3 ℃, cooling and crystallizing for 12 hours to obtain salified solids; filtering to obtain solid, mixing the filtrate with the next raw materials, and recycling. And drying the filter cake.

2) Diluting the obtained solid with 80ml of a 65% methanol solution by volume, dropwise adding sodium hydroxide to adjust the pH to 10, and adding about 16.5g of the macromolecular magnetic microsphere B prepared in the preparation example 2 under stirring; slowly stirring and adsorbing for 1.5h, then carrying out solid-liquid separation under the action of a magnetic field, collecting the magnetic microspheres B, repeatedly adsorbing the solution after the microspheres are separated for 1 time, combining the microspheres B, and washing with purified water at room temperature to obtain the macromolecular magnetic microsphere B-ceramide composite microspheres.

Comparative example 5

The purification was carried out by the salting-out method of example 1 and the crystallization method of step 6) only without the operation of the polymeric magnetic microspheres of steps 2 to 5 of example 1, and it was found that the HPLC purity of the obtained konjak ceramide was not increased but decreased to 67.31% because the salting-out step introduced a large amount of sodium chloride impurities without the acidification treatment and washing removal.

In order to avoid the influence of the salting-out step, when the crude product is crystallized by the crystallization method of step 6) alone, the HPLC purity of the konjak ceramide is measured to rise only to 87.62%, which is caused by the fact that the extract contains a large amount of organic molecular impurities having similar physicochemical properties and is difficult to remove by simple crystallization.

In conclusion, the invention utilizes the amphipathy of hydrophilic and oleophilic konjak ceramide substance due to the hydrophobic chain and a large number of hydroxyl groups contained in the molecule, salt forming treatment is carried out in the purification step to increase the water solubility, the method combining salt forming salting-out, specific modified magnetic microsphere specific adsorption and crystallization is adopted, and the specific macromolecule magnetic microspheres with different properties are used for secondary adsorption, so that the product purity is obviously improved, and the purity requirement of medicines and cosmetics is met.

The above embodiments do not limit the technical solutions of the present invention, and a person skilled in the art may modify the technical solutions described in the above embodiments without departing from the scope of the technical solutions of the present invention.

Claims (8)

1. A method for refining and purifying a plant-derived ceramide extract, wherein the plant-derived ceramide is konjak ceramide, and is characterized by comprising the following steps:

s1: dissolving the crude product of rhizoma Amorphophalli ceramide extract with ethanol solution, adding inorganic sodium salt, adjusting pH to 5-6 with hydrochloric acid, evaporating for concentration, cooling when ceramide begins to salt out, standing at 0-5 deg.C, cooling, and crystallizing for 8-12 hr to obtain solid; filtering and collecting a filter cake;

s2: drying and drying the filtered filter cake, heating and stirring the dried filter cake at 40-50 ℃ by using an ethanol solution for dissolving, adjusting the mass concentration to 1-5%, and dropwise adding hydrochloric acid to adjust the pH value to 4-5;

wherein the volume concentration of the ethanol solution is 50-90%;

s3: primary adsorption and purification:

adding 10-30wt% of carboxyl surface modified polymer magnetic microspheres A into the konjak ceramide solution under stirring; slowly stirring for 1-2h to make the ceramide molecules fully adsorbed with the macromolecular magnetic microspheres A; then applying a magnetic field for 10-20min to perform solid-liquid separation, continuously and repeatedly adsorbing the mother liquor after microsphere separation for 1-3 times by using magnetic polymer microspheres, combining the polymer magnetic microspheres A, and washing with deionized water at room temperature to obtain polymer magnetic microspheres A-ceramide composite microspheres;

wherein, the polymer magnetic microsphere A is a polyvinyl alcohol/poly glycidyl methacrylate based composite polymer magnetic microsphere;

s4: dispersing the separated macromolecule magnetic microsphere A-ceramide composite microsphere in a proper amount of petroleum ether, desorbing for 15-30min under the condition of shaking or rapid stirring by a shaking table, then applying a magnetic field to separate the magnetic microsphere A, collecting the petroleum ether solution containing the konjak ceramide, and evaporating and recovering the solvent to obtain a ceramide component;

s5: secondary adsorption:

adding the obtained konjak ceramide into 3-10 times of methanol aqueous solution, and dropwise adding sodium hydroxide to adjust the pH to be alkaline; adding an aminated or aminated surface-modified macromolecular magnetic microsphere B under stirring, slowly stirring and adsorbing for 1-2h, then carrying out solid-liquid separation under the action of a magnetic field, collecting the magnetic microsphere B, repeatedly adsorbing the solution after microsphere separation for 1-2 times, combining the microspheres B, and washing with deionized water at room temperature to obtain a macromolecular magnetic microsphere B-ceramide composite microsphere;

wherein the macromolecule magnetic microsphere B is a magnetic microsphere with a surface coated with mesoporous silicon dioxide;

s6: dispersing the macromolecular magnetic microsphere B-ceramide composite microsphere in 75-90% ethanol solution by volume, dropwise adding hydrochloric acid to adjust the pH to 6-7, adding a proper amount of ethyl acetate, quickly stirring or shaking for 10-15min, then applying an external magnetic field to separate the magnetic microsphere B, and collecting the solution; desorbing the magnetic microsphere B repeatedly for 1-2 times by the same method, combining the collected solutions, recovering the solvent by rotary evaporation, and vacuum-drying the obtained solid at 50-60 ℃;

s7: under the condition of heating and stirring in a water bath, dissolving the dried solid with a proper amount of acetone, cooling to room temperature, standing at 0-2 ℃ for crystallization for 8-12h to obtain white crystals, filtering, drying and drying to obtain the konjak ceramide with the purity of more than 99%.

2. The method of claim 1, wherein the surface-modified polymeric magnetic microspheres A are prepared by the following steps:

s1: preparing magnetic polyvinyl alcohol/polyglycidyl methacrylate-based microspheres:

heating and dissolving polyvinyl alcohol by using deionized water, adding nano ferroferric oxide magnetic particles and polyglycidyl methacrylate under the condition of boiling water bath, uniformly stirring, adding liquid paraffin and tween, and stirring; then adding hydrochloric acid and glutaraldehyde solution, stirring for crosslinking reaction for 15-30min, centrifuging to separate out microspheres, washing with absolute ethyl alcohol, and vacuum drying to obtain magnetic polyvinyl alcohol/polyglycidyl methacrylate-based microspheres;

wherein the mass ratio of the polyvinyl alcohol to the ferroferric oxide is 10: 0.5-2;

wherein the total mass of the liquid paraffin and the Tween is 10-30 times of that of the polyvinyl alcohol;

wherein the concentration of the glutaraldehyde solution is 20-30 wt%;

s2: surface carboxyl modification:

dispersing the polyvinyl alcohol/poly glycidyl methacrylate-based microspheres in acetonitrile, sequentially adding methacrylic acid, methylene bisacrylamide and azodiisobutyronitrile, uniformly dispersing, heating in a water bath at 75-80 ℃ for reaction, separating by a magnetic field after the reaction is finished, and washing by absolute ethyl alcohol to obtain surface carboxyl modified polymer magnetic microspheres A;

wherein the dosage ratio of the polyvinyl alcohol/poly glycidyl methacrylate based microspheres to the acetonitrile is 1g:5-20 mL;

wherein the reaction time is 4-8h by heating in water bath.

3. The method according to claim 2, wherein the mass ratio of the polyvinyl alcohol to the polyglycidyl methacrylate in step S1 is 10: 1-3; in step S2, the mass ratio of the microspheres to the methacrylic acid to the methylene bisacrylamide to the azobisisobutyronitrile is 10:3-5:0.1-0.5: 0.1-0.3.

4. The method of claim 1, wherein the surface-modified magnetic polymer microspheres B are prepared by the following steps:

s1: weighing mesoporous silica microspheres, ultrasonically dispersing the mesoporous silica microspheres in deionized water, and uniformly stirring to form a suspension; weighing a ferrous chloride and ferric chloride mixed raw material with a molar ratio of 1:2, dissolving the mixed raw material in a proper amount of deionized water, uniformly stirring, adding the mixture into a silica particle suspension, adding PVP, starting mechanical stirring, adding concentrated ammonia water in batches under the water bath heating condition of 75-80 ℃, and continuously stirring and reacting for 1-2 hours after the addition is finished; cooling to room temperature after the reaction is finished, centrifugally or magnetically separating the microspheres, washing the microsphere particles with deionized water, and drying in vacuum to obtain magnetic microspheres with surfaces coated with mesoporous silica;

wherein the dispersion ratio of the silicon dioxide microspheres to the deionized water is 1g: 15-30 mL;

wherein the concentration of the used ammonia water is 25-30 wt%;

s2: surface modification: dispersing the magnetic microsphere coated with the silicon dioxide, ethylenediamine and 3- (trimethoxysilyl) propyl-2-methyl-2-acrylate coupling agent in methanol, reacting at 35-40 ℃ for 6-12h, separating the microsphere by an external magnetic field, and washing with methanol and deionized water in sequence to obtain an amino-modified macromolecular magnetic microsphere B;

wherein the mass ratio of the magnetic microspheres to the ethylenediamine is 5-10: 1;

wherein the mass ratio of the magnetic microspheres to the 3- (trimethoxysilyl) propyl-2-methyl-2-acrylate is 3-5: 1;

wherein the reaction time in the step S2 is 8-12 h.

5. The method according to claim 4, wherein in step S1, the mass ratio of the silica microspheres to the ferrous chloride/ferric chloride mixed raw material is 1:2-3, and the mass ratio of the silica microspheres to the PVP is 10: 0.5-2; in step S2, the dosage ratio of methanol to magnetic microspheres is 20-30mL:1 g.

6. The method according to claim 1, wherein in step S1, the purity of the crude konjak ceramide is 70-95%; the volume concentration of the ethanol solution is 65-75%; wherein the inorganic sodium salt is selected from sodium chloride, and the content of the inorganic sodium salt in the solution is 30-50 g/L.

7. The method of claim 1, wherein in step S5, the volume fraction of the aqueous methanol solution used is 50-90%; the mass fraction of the polymer magnetic microspheres B in the solution is 10-30 wt%; adjusting pH to 9-11 with sodium hydroxide.

8. The method of claim 1, wherein in step S6, the volume of ethyl acetate is 5-20% of the ethanol solution; in step S7, the water bath heating temperature is 50-55 ℃.