CN108743530A - A kind of preparation method of Spine Date Seed jujubosideB liposome micro emulsion - Google Patents
A kind of preparation method of Spine Date Seed jujubosideB liposome micro emulsion Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of Spine Date Seed jujubosideB liposome micro emulsion, using spina date seed as raw material, raw material concentrates this method through rudimentary alcohol extracting, liquid water-oil separating after extraction；Spina date seed total saposins are collected in aqueous nonpolarity bigger serface macroreticular resin selective absorption spina date seed total saposins, elution；β-glucosaccharase is added in spina date seed total saposins and carries out enzymatic conversion structural modification, semi-synthetic Spine Date Seed jujubosideB improves Spine Date Seed jujubosideB content in total saposins；Gained converted product is chromatographed through purification on normal-phase silica gel or reversed column chromatography obtains high-purity Spine Date Seed jujubosideB；Spine Date Seed jujubosideB, auxiliary material lecithin, isopropyl myristate, normal propyl alcohol is taken to be mixed and made into Spine Date Seed jujubosideB liposome micro emulsion, this method overcomes the drawbacks such as existing Spine Date Seed jujubosideB extraction and separation technology yield is low, purity is low；The yield of Spine Date Seed jujubosideB is improved, the use of microemulsion formulation is more advantageous to human body and fully absorbs, and improves bioavilability.
Description
Technical field
The present invention relates to a kind of preparation methods of Spine Date Seed jujubosideB liposome micro emulsion.
Background technology
Spina date seed（Semen Ziziphi Spinosae）It is Rhamnaceae（Rhamnaceae）Plant wild jujube（Ziziphous jujuba）Dry mature seed, the time of the year when autumn changes into winter harvests ripening fruits, removes pulp and nucleocapsid, collects seed, dry it is obtained,
Main product is common sedative hypnotic drug in Chinese medicine medicine in northwest, northeast, many provinces and regions in North China and some southern areas,
There is long usage history in China.Spina date seed major function is nourishing the liver, and calming heart is calmed the nerves, arrest sweating；Restlessness of asrhenia type and insomnia is controlled, is palpitated with fear
Palpitation, polydipsia, abnormal sweating.
Spina date seed main chemical compositions are jujuboside, are the main component of its tranquilizing soporific, jujuboside medicine generation
Dynamics research shows that spina date seed is metabolized in vivo, first Saponin A be converted into Spine Date Seed jujubosideB, Spine Date Seed jujubosideB into
One step is converted into glucoside member and plays tranquilizing effect.Spine Date Seed jujubosideB, English name are Jujuboside B, are that one kind reaching agate
Alkane type triterpenoid saponins compound, chemical formula C52H84O21, molecular weight 1045.22；Spine Date Seed jujubosideB Central nervous system
And its mediator, quenched, receptor and electro physiology etc. have specific effect.
Spine Date Seed jujubosideB has central nervous system tranquilizing and allaying excitement, reduces blood pressure, resists myocardial ischemia, reducing blood lipid and prevention
The effects that atherosclerosis, anti peroxidation of lipid, protection brain tissue；Spine Date Seed jujubosideB can obviously inhibit the work of normal mouse
Dynamic number, inhibits the central excitation of amphetamine to act on, reduces the coordinated movement of various economic factors of rat, be obviously prolonged yellow Jackets threshold dose
Mouse sleep time significantly reduces convulsions rate caused by pentylenetetrazol.
Spine Date Seed jujubosideB blood brain barrier transmissivity is higher than Saponin A, and Saponin A cannot not only lead in vivo
Cross blood-brain barrier（BBB）, and be difficult to have corresponding binding site with GABAA receptors in vitro.After Saponin A hydrolysis, water
Solution is at Spine Date Seed jujubosideB, then is hydrolyzed into jujuboside glucoside member, hydrolysate jujubogenin can penetrate BBB and with
GABAA receptors in conjunction with and play the effect of tranquilizing soporific, Saponin A has more carbohydrate ligands, with the extra spina date seed of sugar
Saponin(e B, blood brain barrier transmissivity are less than Spine Date Seed jujubosideB, therefore Spine Date Seed jujubosideB bioavilability higher.
The application early-stage study Spine Date Seed jujubosideB microemulsion formulation penetrates the effect of blood-brain barrier.Method uses efficient liquid
Phase chromatography measures main active wild jujube after Spine Date Seed jujubosideB micro emulsion and the same content of dispersion administration of Spine Date Seed jujubosideB capsule
Contents of the benevolence saponin(e B in brain tissue, comparing should in Spine Date Seed jujubosideB capsule and jujuboside microemulsion formulation Mice brain tissues
The changes of contents of ingredient.As a result in microemulsion formulation, Spine Date Seed jujubosideB content increases significantly in Mice brain tissues, is capsule
2-3 times of agent.Conclusion acid saponin(e benevolence saponin(e B microemulsion formulations greatly improve the molten reduced performance jujuboside of its lipophilic liver,
The effect of first pass effect in gastrointestinal metabolic, lipophilicity higher is more easy to penetrate blood-brain barrier, enhance brain tonic, antitoxic heart-soothing and sedative.
Saponin A content is 0.09% or so, and Spine Date Seed jujubosideB content is 0.04% or so, from medicinal material spina date seed
Extraction Spine Date Seed jujubosideB isolates and purifies that Research Literature is less, and spina date seed total saposins extracting method is on the high side, Spine Date Seed jujubosideB extraction
Method is mainly that silica gel column chromatography combination reversed phase column chromatography method separation prepares Spine Date Seed jujubosideB；Or by chemical synthesis or
Semi-synthetic, enzymatic conversion synthesizes Spine Date Seed jujubosideB；Or by ultrasonic refluxing extraction, that there are recovery rates is low for these methods, purity is low
The problem of.
Invention content
Present invention aims at a kind of preparation method of Spine Date Seed jujubosideB liposome micro emulsion is provided, this method uses β-Portugal
Part glucoside in grape glucosidase directionally hydrolyzing spina date seed total saposins is allowed to be converted into Spine Date Seed jujubosideB, improves Spine Date Seed jujubosideB
Yield, this method improve 1-2 times than extracting yield in crude drug in whole.
The step of the method for the present invention, is as follows：
（1）Using spina date seed as raw material, the methanol solution or ethanol solution circumfluence distillation of mass concentration 50-70% are used after crushing
2-3 times, 80-85 DEG C of Extracting temperature, each extraction time 1-2h, filtering, merging extracting solution, reduced pressure recycling organic solvent,
Obtain Wild jujube seeds extract；
（2）In step（1）The water of 2-3 times of its volume is added in Wild jujube seeds extract, obtains extract suspension, adds extraction
Solution is placed at 1-3 DEG C and stands for 24 hours, abandon oil reservoir and obtain after heating stirring dissolving by the sodium chloride of object suspension quality 5-10%
Clarify water layer liquid；
（3）By step（3）Middle water layer liquid by large pore resin absorption column adsorb spina date seed total saposins, loading efflux be salt and
Impurity, washed resin column to efflux are colourless, and the ethanol solution of volumetric concentration 70-75% is then used to parse spina date seed total saposins,
Flow velocity 1-1.5 column volumes/hour is parsed, about 3-4 column volume of elution volume collects eluent, and recycling ethyl alcohol is concentrated and dried
Spina date seed total saposins；
（4）By step（3）Middle spina date seed total saposins 10-12g, N-Acetyl-D-glucosamine 10-15g, β-glucosaccharase 20-50 μ g
It is put into reaction kettle, adds pure water 200mL, it is 5.5-6 to adjust pH value with glacial acetic acid after mixing；Then nitrogen protection, 50-60 DEG C
Lower confined reaction 6-10h, carries out the semi-synthetic of Spine Date Seed jujubosideB, increases the temperature to 100 DEG C, enzyme deactivation 10min after reaction,
Pour out reaction solution；
（5）Use step（4）The extracting n-butyl alcohol reaction solution of 1-2 times of reaction solution volume 2-3 times is collected and merges n-butanol layer, positive fourth
Alcohol layer master contains Spine Date Seed jujubosideB, and revolving recycling n-butanol obtains enzymatic conversion reaction product；
（6）Using 200-400 mesh silica gel and chloroform wet method dress post, step is taken（5）Middle enzymatic conversion reaction product dry method loading uses
Chloroform-methanol-aqueous solution elution, chloroform-methanol-aqueous solution is chloroform:Methanol:Water 7-5 by volume:1.2-1:0.2-0.1
Ratio be mixed to prepare, collect the eluent containing Spine Date Seed jujubosideB, concentration and recovery organic solvent, concentrate acetone-methanol is molten
Liquid crystallizes, dry Spine Date Seed jujubosideB；
（7）By step（6）Spine Date Seed jujubosideB is prepared into the Spine Date Seed jujubosideB aqueous solution that mass concentration is 15%-25%, by following
Mass ratio weighs：Jujuboside aqueous solution 10%-15%, isopropyl myristate 34-38%, normal propyl alcohol 22-26%, lecithin
27-31%；Lecithin is dissolved with normal propyl alcohol first, adds isopropyl myristate, after stirring evenly, Spine Date Seed jujubosideB is added
Aqueous solution is stirred when being added dropwise, is stirred for after having added 1 hour, and Spine Date Seed jujubosideB liposome micro emulsion is made.
The macroreticular resin is ADS-1600, and aperture is 80-100 μm, and specific surface area is more than 1000.
The β-glucosaccharase enzyme activity 300U/g.
Above-mentioned steps（6）It replaces with：By step（5）Middle enzymatic conversion reaction product water dissolution is at sample solution, using filler
SMB MCI GEL wet method dress posts, loading after pure water equilibrium, gradient elution reversed phase chromatography, eluant, eluent are methanol aqueous solution, the first ladder
The volume ratio for spending first alcohol and water in eluant, eluent methanol aqueous solution is 30-40:70-60 elutes impurity, the second gradient methanol aqueous solution
The volume ratio of middle first alcohol and water is 50-60:50-40 collects eluent, concentration, and concentrate is crystallized with acetone-methanol solution, is done
It is dry to obtain Spine Date Seed jujubosideB.
The acetone-methanol solution is acetone and methanol 5-8 by volume:1-1.5 ratio be mixed to prepare.
Advantage of the present invention and technique effect are as follows：
The present invention overcomes the drawbacks such as existing Spine Date Seed jujubosideB extraction and separation technology yield is low, purity is low；Using salting out method so that carrying
It takes liquid no longer to be emulsified because oil components are higher, preferable oil-water separation can be reached after refrigeration, using nonpolar big ratio
Surface area macroreticular resin selective absorption jujuboside, adsorbance is 2 times of tradition D101 resins, while sloughing salt and miscellaneous
Matter is eluted by purifying resin and collects spina date seed total saposins, part sugar in β-glucosaccharase directionally hydrolyzing spina date seed total saposins
Glucosyl group is converted to Spine Date Seed jujubosideB, the semi-synthetic wild jujube of Hydrolyze method on glucoside, such as Saponin A positioning hydrolysis glucoside chain
Benevolence saponin(e B is allowed to be converted into Spine Date Seed jujubosideB, improves 2-3 times of Spine Date Seed jujubosideB yield；By purification on normal-phase silica gel chromatography or reversely
Column chromatography is further purified to obtain high-purity Spine Date Seed jujubosideB；Spina date seed total saposins, Saponin A content are higher, so in people
In body pharmacokinetics, Saponin A need to after enteron aisle metabolic water solution the more preferable curative effect of competence exertion, in monomer saponin, spina date seed
Saponin A is easily converted into saponin(e B, and saponin(e B molecular weight is less than saponin A, it is easier to blood-brain barrier is penetrated, with list in galenic pharmacy method
Body is prepared into for bulk pharmaceutical chemicals preparation gained micro emulsion and obtains injection drug effect prepared by spina date seed total saposins of the injection than complicated component
It is safe and reliable and be more easy to blood-brain barrier, bioavilability is improved, gained microemulsion formulation human body of being then more convenient for is prepared and fully absorbs
It utilizes, 2-4 times of bioavailability of drugs can be improved.
Specific implementation mode
Below by embodiment, invention is further described in detail, but the scope of the present invention is not limited in described
Hold.
Embodiment 1：Steps are as follows for the preparation method of this Spine Date Seed jujubosideB liposome micro emulsion：
（1）Using spina date seed as raw material, using the methanol solution circumfluence distillation 2 times of mass concentration 50%, Extracting temperature 80 after crushing
DEG C, each extraction time 2h, filtering merges extracting solution, and recycling organic solvent is concentrated under reduced pressure, obtains Wild jujube seeds extract；
（2）In step（1）The water of 2 times of its volume is added in Wild jujube seeds extract, obtains extract suspension, adds extract
Solution is placed at 1 DEG C and stands for 24 hours after heating stirring dissolving by the sodium chloride of suspension quality 5%, abandons oil reservoir and obtains clarification water layer
Liquid；
（3）By step（3）Middle water layer liquid passes through macroporous absorbent resin ADS-1600（Aperture is 80-100 μm）Column adsorbs spina date seed
Total saposins, loading efflux are salt and impurity, and washed resin column to efflux is colourless, then uses the second of volumetric concentration 70%
Alcoholic solution parses spina date seed total saposins, 1 column volume of parsing flow velocity/hour, and about 4 column volumes of elution volume are collected eluent, returned
It receives ethyl alcohol and is concentrated and dried to obtain spina date seed total saposins；
（4）By step（3）Middle spina date seed total saposins 10g, N-Acetyl-D-glucosamine 15g, β-glucosaccharase（Enzyme activity 300U/g）
20 μ g are put into reaction kettle, add pure water 200mL, it is 5.5 to adjust pH value with glacial acetic acid after mixing；Then nitrogen protection, 60 DEG C
Lower confined reaction 6h, carries out the synthesis of Spine Date Seed jujubosideB, increases the temperature to 100 DEG C, enzyme deactivation 10min after reaction, pours out anti-
Answer liquid；
（5）Use step（4）The extracting n-butyl alcohol reaction solution of 1 times of reaction solution volume 3 times is collected and merges n-butanol layer, n-butanol layer
Main to contain Spine Date Seed jujubosideB, revolving recycling n-butanol obtains enzymatic conversion reaction product；
（6）Using 200 mesh silica gel and chloroform wet method dress post, step is taken（5）Middle enzymatic conversion reaction product dry method loading, wherein enzyme turn
The mass ratio for changing reaction product and silica gel is 1:15；Using chloroform-methanol-aqueous solution elution, chloroform-methanol-aqueous solution is chlorine
It is imitative:Methanol:Water by volume 7:1:0.1 ratio is mixed to prepare, and is collected the eluent of the part containing Spine Date Seed jujubosideB, is concentrated back
Organic solvent is received, concentrate is crystallized with acetone-methanol solution, dry Spine Date Seed jujubosideB, content 92%, wherein acetone-first
Alcoholic solution is acetone and methanol by volume 5:1 ratio is mixed to prepare, Spine Date Seed jujubosideB content 94.3%, yield 0.11%（It presses
Crude drug in whole meter）；
（7）By step（6）Spine Date Seed jujubosideB is prepared into the Spine Date Seed jujubosideB aqueous solution that mass concentration is 15%, by following quality
Than weighing：Jujuboside aqueous solution 10%, isopropyl myristate 38%, normal propyl alcohol 22%, lecithin 30%；First lecithin is used
Normal propyl alcohol dissolves, and adds isopropyl myristate, and after stirring evenly, Spine Date Seed jujubosideB aqueous solution is added, is stirred when being added dropwise
It mixes, is stirred for after having added 1 hour, Spine Date Seed jujubosideB liposome micro emulsion is made.
Embodiment 2：Steps are as follows for the preparation method of this Spine Date Seed jujubosideB liposome micro emulsion：
（1）Using spina date seed as raw material, using the ethanol solution circumfluence distillation 3 times of mass percent concentration 60%, extraction after crushing
85 DEG C of temperature, each extraction time 1h, filtering merge extracting solution, and recycling organic solvent is concentrated under reduced pressure, obtains Wild jujube seeds extract；
（2）In step（1）The water of 3 times of its volume is added in Wild jujube seeds extract, obtains extract suspension, adds extract
Solution is placed at 2 DEG C and stands for 24 hours after heating stirring dissolving by the sodium chloride of suspension quality 8%, abandons oil reservoir and obtains clarification water layer
Liquid；
（3）By step（3）Middle water layer liquid passes through macroporous absorbent resin ADS-1600（Aperture is 80-100 μm）Column adsorbs spina date seed
Total saposins, loading efflux are salt and impurity, and washed resin column to efflux is colourless, then uses the second of volumetric concentration 75%
Alcoholic solution parses spina date seed total saposins, 1.2 column volumes of parsing flow velocity/hour, and about 3 column volumes of elution volume collect eluent,
Recycling ethyl alcohol is concentrated and dried to obtain spina date seed total saposins；
（4）By step（3）Middle spina date seed total saposins 12g, N-Acetyl-D-glucosamine 12g, β-glucosaccharase（Enzyme activity 300U/g）
30 μ g are put into reaction kettle, add pure water 200mL, it is 6 to adjust pH value with glacial acetic acid after mixing；Then at nitrogen protection, 55 DEG C
Confined reaction 8h, carries out the synthesis of Spine Date Seed jujubosideB, increases the temperature to 100 DEG C after reaction, and enzyme deactivation 10min pours out reaction
Liquid；
（5）Use step（4）The extracting n-butyl alcohol reaction solution of 2 times of reaction solution volume 2 times is collected and merges n-butanol layer, n-butanol layer
Main to contain Spine Date Seed jujubosideB, revolving recycling n-butanol obtains enzymatic conversion reaction product；
（6）Using 300 mesh silica gel and chloroform wet method dress post, step is taken（5）Middle enzymatic conversion reaction product dry method loading, wherein enzyme turn
The mass ratio for changing reaction product and silica gel is 1:20；Using chloroform-methanol-aqueous solution elution, chloroform-methanol-aqueous solution is chlorine
It is imitative:Methanol:Water by volume 5:1.2:0.2 ratio is mixed to prepare, and collects the eluent of the part containing Spine Date Seed jujubosideB, concentration
Organic solvent is recycled, concentrate is crystallized with acetone-methanol solution, dry Spine Date Seed jujubosideB, content 93%, wherein acetone-
Methanol solution is acetone and methanol by volume 6:1.2 ratio is mixed to prepare, Spine Date Seed jujubosideB content 96.3%, yield
0.11%（Based on crude drug in whole）；
（7）By step（6）Spine Date Seed jujubosideB is prepared into the Spine Date Seed jujubosideB aqueous solution that mass concentration is 25%, by following quality
Than weighing：Jujuboside aqueous solution 12%, isopropyl myristate 34%, normal propyl alcohol 26%, lecithin 28%；First lecithin is used
Normal propyl alcohol dissolves, and adds isopropyl myristate, and after stirring evenly, Spine Date Seed jujubosideB aqueous solution is added, is stirred when being added dropwise
It mixes, is stirred for after having added 1 hour, Spine Date Seed jujubosideB liposome micro emulsion is made.
Embodiment 3：Steps are as follows for the preparation method of this Spine Date Seed jujubosideB liposome micro emulsion：
（1）Using spina date seed as raw material, using the methanol solution circumfluence distillation 2 times of mass percent concentration 70%, extraction after crushing
83 DEG C of temperature, each extraction time 2h, filtering merge extracting solution, and recycling organic solvent is concentrated under reduced pressure, obtains Wild jujube seeds extract；
（2）In step（1）The water of 2.5 times of its volume is added in Wild jujube seeds extract, obtains extract suspension, adds extraction
Solution is placed at 3 DEG C and stands for 24 hours, abandon oil reservoir and clarified after heating stirring dissolving by the sodium chloride of object suspension quality 10%
Water layer liquid；
（3）By step（3）Middle water layer liquid passes through macroporous absorbent resin ADS-1600（Aperture is 80-100 μm）Column adsorbs spina date seed
Total saposins, loading efflux are salt and impurity, and washed resin column to efflux is colourless, then uses the second of volumetric concentration 72%
Alcoholic solution parses spina date seed total saposins, 1.5 column volumes of parsing flow velocity/hour, and about 3 column volumes of elution volume collect eluent,
Recycling ethyl alcohol is concentrated and dried to obtain spina date seed total saposins；
（4）By step（3）Middle spina date seed total saposins 11g, N-Acetyl-D-glucosamine 10g, β-glucosaccharase（Enzyme activity 300U/g）
45 μ g are put into reaction kettle, add pure water 200mL, it is 5.5 to adjust pH value with glacial acetic acid after mixing；Then nitrogen protection, 50 DEG C
Lower confined reaction 10h, carries out the semi-synthetic of Spine Date Seed jujubosideB, increases the temperature to 100 DEG C, enzyme deactivation 10min after reaction,
Go out reaction solution；
（5）Use step（4）The extracting n-butyl alcohol reaction solution of 1 times of reaction solution volume 2 times is collected and merges n-butanol layer, n-butanol layer
Main to contain Spine Date Seed jujubosideB, revolving recycling n-butanol obtains enzymatic conversion reaction product；
（6）Using 400 mesh silica gel and chloroform wet method dress post, step is taken（5）Middle enzymatic conversion reaction product dry method loading, wherein enzyme turn
The mass ratio for changing reaction product and silica gel is 1:15；Using chloroform-methanol-aqueous solution elution, chloroform-methanol-aqueous solution is chlorine
It is imitative:Methanol:Water by volume 6:1:0.1 ratio is mixed to prepare, and is collected the eluent of the part containing Spine Date Seed jujubosideB, is concentrated back
Organic solvent is received, concentrate is crystallized with acetone-methanol solution, dry Spine Date Seed jujubosideB, content 92%, wherein acetone-first
Alcoholic solution is acetone and methanol by volume 8:1.5 ratio is mixed to prepare, Spine Date Seed jujubosideB content 96.5%, yield 0.10%；
（7）By step（6）Spine Date Seed jujubosideB is prepared into the Spine Date Seed jujubosideB aqueous solution that mass concentration is 25%, by following quality
Than weighing：Jujuboside aqueous solution 15%, isopropyl myristate 34%, normal propyl alcohol 23%, lecithin 28%；First lecithin is used
Normal propyl alcohol dissolves, and adds isopropyl myristate, and after stirring evenly, Spine Date Seed jujubosideB aqueous solution is added, is stirred when being added dropwise
It mixes, is stirred for after having added 1 hour, Spine Date Seed jujubosideB liposome micro emulsion is made.
Embodiment 4：Steps are as follows for the preparation method of this Spine Date Seed jujubosideB liposome micro emulsion：
（1）Using spina date seed as raw material, using the ethanol solution circumfluence distillation 2 times of mass percent concentration 55%, extraction after crushing
82 DEG C of temperature, each extraction time 2h, filtering merge extracting solution, and recycling organic solvent is concentrated under reduced pressure, obtains Wild jujube seeds extract；
（2）In step（1）The water of 2 times of its volume is added in Wild jujube seeds extract, obtains extract suspension, adds extract
Solution is placed at 1.5 DEG C and stands for 24 hours, abandon oil reservoir and obtain primary water after heating stirring dissolving by the sodium chloride of suspension quality 6%
Layer liquid；
（3）By step（3）Middle water layer liquid passes through macroporous absorbent resin ADS-1600（Aperture is 80-100 μm）Column adsorbs spina date seed
Total saposins, loading efflux are salt and impurity, and washed resin column to efflux is colourless, then uses the second of volumetric concentration 70%
Alcoholic solution parses spina date seed total saposins, and 1.2 column volumes of parsing flow velocity/hour collect eluent, and recycling ethyl alcohol is concentrated and dried sour
Jujube kernel total saposins；
（4）By step（3）Middle spina date seed total saposins 10g, N-Acetyl-D-glucosamine 15g, β-glucosaccharase（Enzyme activity 300U/g）
30 μ g are put into reaction kettle, add pure water 200mL, it is 6 to adjust pH value with glacial acetic acid after mixing；Then at nitrogen protection, 60 DEG C
Confined reaction 6h, carries out the semi-synthetic of Spine Date Seed jujubosideB, increases the temperature to 100 DEG C, enzyme deactivation 10min after reaction, pours out anti-
Answer liquid；
（5）Use step（4）The extracting n-butyl alcohol reaction solution of 1 times of reaction solution volume 3 times is collected and merges n-butanol layer, n-butanol layer
Main to contain Spine Date Seed jujubosideB, revolving recycling n-butanol obtains enzymatic conversion reaction product；
（6）By step（5）Middle enzymatic conversion reaction product water dissolution is at sample solution, using filler SMB MCI GEL（Japan three
Water chestnut）Wet method dress post, loading after pure water equilibrium, gradient elution reversed phase chromatography, eluant, eluent is methanol aqueous solution, first gradient eluant, eluent
The volume ratio of first alcohol and water is 30 in methanol aqueous solution:70, impurity is eluted, the body of first alcohol and water in the second gradient methanol aqueous solution
Product is than being 50:50, eluent, concentration are collected, concentrate is crystallized with acetone-methanol solution, and wherein acetone-methanol solution is acetone
With methanol by volume 6:1.1 ratio is mixed to prepare, dry Spine Date Seed jujubosideB, content 98.9%, yield 0.14%；
（7）By step（6）Spine Date Seed jujubosideB is prepared into the Spine Date Seed jujubosideB aqueous solution that mass concentration is 21%, by following quality
Than weighing：Jujuboside aqueous solution 10%, isopropyl myristate 34%, normal propyl alcohol 25%, lecithin 31%；First lecithin is used
Normal propyl alcohol dissolves, and adds isopropyl myristate, and after stirring evenly, Spine Date Seed jujubosideB aqueous solution is added, is stirred when being added dropwise
It mixes, is stirred for after having added 1 hour, Spine Date Seed jujubosideB liposome micro emulsion is made.
Claims (5)
1. a kind of preparation method of Spine Date Seed jujubosideB liposome micro emulsion, which is characterized in that steps are as follows：
（1）Using spina date seed as raw material, the methanol solution or ethanol solution circumfluence distillation of mass concentration 50-70% are used after crushing
2-3 times, 80-85 DEG C of Extracting temperature, each extraction time 1-2h, filtering, merging extracting solution, reduced pressure recycling organic solvent,
Obtain Wild jujube seeds extract；
（2）In step（1）The water of 2-3 times of its volume is added in Wild jujube seeds extract, obtains extract suspension, adds extraction
Solution is placed at 1-3 DEG C and stands for 24 hours, abandon oil reservoir and obtain after heating stirring dissolving by the sodium chloride of object suspension quality 5-10%
Clarify water layer liquid；
（3）By step（3）Middle water layer liquid by large pore resin absorption column adsorb spina date seed total saposins, loading efflux be salt and
Impurity, washed resin column to efflux are colourless, and the ethanol solution of volumetric concentration 70-75% is then used to parse spina date seed total saposins,
Flow velocity 1-1.5 column volumes/hour is parsed, eluent is collected, recycling ethyl alcohol is concentrated and dried to obtain spina date seed total saposins；
（4）By step（3）Middle spina date seed total saposins 10-12g, N-Acetyl-D-glucosamine 10-15g, β-glucosaccharase 20-50 μ g
It is put into reaction kettle, adds pure water 200mL, it is 5.5-6 to adjust pH value with glacial acetic acid after mixing；Then nitrogen protection, 50-60 DEG C
Lower confined reaction 6-10h, carries out the semi-synthetic of Spine Date Seed jujubosideB, increases the temperature to 100 DEG C, enzyme deactivation 10min after reaction,
Pour out reaction solution；
（5）Use step（4）The extracting n-butyl alcohol reaction solution of 1-2 times of reaction solution volume 2-3 times is collected and merges n-butanol layer, revolving
N-butanol is recycled, enzymatic conversion reaction product is obtained；
（6）Using 200-400 mesh silica gel and chloroform wet method dress post, step is taken（5）Middle enzymatic conversion reaction product dry method loading uses
Chloroform-methanol-aqueous solution elution, chloroform-methanol-aqueous solution is chloroform:Methanol:Water 7-5 by volume:1.2-1:0.2-0.1
Ratio be mixed to prepare, collect the eluent containing Spine Date Seed jujubosideB, concentration and recovery organic solvent, concentrate acetone-methanol is molten
Liquid crystallizes, dry Spine Date Seed jujubosideB；
（7）By step（6）Spine Date Seed jujubosideB is prepared into the Spine Date Seed jujubosideB aqueous solution that mass concentration is 15%-25%, by following
Mass ratio weighs：Jujuboside aqueous solution 10%-15%, isopropyl myristate 34-38%, normal propyl alcohol 22-26%, lecithin
27-31%；Lecithin is dissolved with normal propyl alcohol first, adds isopropyl myristate, after stirring evenly, Spine Date Seed jujubosideB is added
Aqueous solution is stirred when being added dropwise, is stirred for after having added 1 hour, and Spine Date Seed jujubosideB liposome micro emulsion is made.
2. the preparation method of Spine Date Seed jujubosideB liposome micro emulsion according to claim 1, it is characterised in that：Macroporous absorption
Resin model is ADS-1600.
3. the preparation method of Spine Date Seed jujubosideB liposome micro emulsion according to claim 1, it is characterised in that：β-glucose
Glucoside enzyme enzyme activity 300U/g.
4. the preparation method of Spine Date Seed jujubosideB liposome micro emulsion according to claim 1, which is characterized in that step（6）It replaces
It is changed to：By step（5）Middle enzymatic conversion reaction product water dissolution is pure using filler SMB MCI GEL wet method dress posts at sample solution
Loading after water balance, gradient elution reversed phase chromatography, eluant, eluent are methanol aqueous solution, first in first gradient eluant, eluent methanol aqueous solution
The volume ratio of alcohol and water is 30-40:70-60 elutes impurity, and the volume ratio of first alcohol and water is in the second gradient methanol aqueous solution
50-60:50-40 collects eluent, concentration, and concentrate is crystallized with acetone-methanol solution, dry Spine Date Seed jujubosideB.
5. the preparation method of Spine Date Seed jujubosideB liposome micro emulsion according to claim 1 or 4, it is characterised in that：Acetone-
Methanol solution is acetone and methanol 5-8 by volume:1-1.5 ratio be mixed to prepare.
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| WO2021056805A1 (en) * | 2019-09-26 | 2021-04-01 | 泰州医药城国科化物生物医药科技有限公司 | Wild jujube seed flavone and phospholipid composite as well as preparation method therefor and application thereof |
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