CN109497561A - A kind of vitamin B12The preparation method of nano liposomes - Google Patents
A kind of vitamin B12The preparation method of nano liposomes Download PDFInfo
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- CN109497561A CN109497561A CN201811598382.7A CN201811598382A CN109497561A CN 109497561 A CN109497561 A CN 109497561A CN 201811598382 A CN201811598382 A CN 201811598382A CN 109497561 A CN109497561 A CN 109497561A
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- 239000002502 liposome Substances 0.000 title claims abstract description 123
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 229930003231 vitamin Natural products 0.000 title description 8
- 235000013343 vitamin Nutrition 0.000 title description 8
- 239000011782 vitamin Substances 0.000 title description 8
- 229940088594 vitamin Drugs 0.000 title description 8
- 150000003722 vitamin derivatives Chemical class 0.000 title description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 38
- 239000011715 vitamin B12 Substances 0.000 claims abstract description 35
- 150000002632 lipids Chemical class 0.000 claims abstract description 20
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 19
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims abstract description 18
- 229940083466 soybean lecithin Drugs 0.000 claims abstract description 18
- 239000003960 organic solvent Substances 0.000 claims abstract description 17
- 229930003270 Vitamin B Natural products 0.000 claims abstract description 15
- 239000011720 vitamin B Substances 0.000 claims abstract description 15
- 235000019156 vitamin B Nutrition 0.000 claims abstract description 15
- 239000012736 aqueous medium Substances 0.000 claims abstract description 13
- 239000004094 surface-active agent Substances 0.000 claims abstract description 13
- 239000012528 membrane Substances 0.000 claims abstract description 10
- 238000002604 ultrasonography Methods 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- 238000001132 ultrasonic dispersion Methods 0.000 claims abstract description 5
- 239000006071 cream Substances 0.000 claims abstract description 3
- 238000001914 filtration Methods 0.000 claims abstract description 3
- 239000012982 microporous membrane Substances 0.000 claims abstract description 3
- 230000003381 solubilizing effect Effects 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 31
- 210000004027 cell Anatomy 0.000 claims description 13
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 10
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 10
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 8
- 239000012535 impurity Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims description 5
- 210000002706 plastid Anatomy 0.000 claims description 5
- 239000006227 byproduct Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 2
- 239000002245 particle Substances 0.000 abstract description 11
- 238000005538 encapsulation Methods 0.000 abstract description 8
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- 238000001727 in vivo Methods 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 2
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- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 34
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 20
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- 238000002474 experimental method Methods 0.000 description 13
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- 239000000243 solution Substances 0.000 description 12
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 10
- 238000000227 grinding Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 6
- 238000010298 pulverizing process Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 235000021588 free fatty acids Nutrition 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 238000000643 oven drying Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 229930003779 Vitamin B12 Natural products 0.000 description 4
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- 150000002168 ethanoic acid esters Chemical class 0.000 description 4
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- 235000019163 vitamin B12 Nutrition 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical class CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
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- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
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- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
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- 230000008859 change Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 210000003405 ileum Anatomy 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
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- 239000007788 liquid Substances 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- -1 that is Substances 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- 239000011709 vitamin E Substances 0.000 description 2
- 229940046009 vitamin E Drugs 0.000 description 2
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- 208000031845 Pernicious anaemia Diseases 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000000823 artificial membrane Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
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- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 210000002011 intestinal secretion Anatomy 0.000 description 1
- 229940029329 intrinsic factor Drugs 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/15—Vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/045—Organic compounds containing nitrogen as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/05—Organic compounds containing phosphorus as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
- A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Engineering & Computer Science (AREA)
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Abstract
The invention belongs to functional health-care food fields, and in particular to a kind of vitamin B12The preparation method of nano liposomes.Vitamin B of the present invention12Nano liposomes are prepared by film-ultrasonic dispersion, and soybean lecithin and cholesterol are mixed in organic solvent evaporated under reduced pressure in preparation process and form lipid membrane, vitamin B is then added12, aqueous media and surfactant solubilizing lipids film and ultrasound are at cream, then through ultrasonic cell disrupte, low-temperature centrifugation and filtering with microporous membrane, obtain vitamin B12Nano liposomes.VB prepared by the present invention12Nano liposomes particle size dispersion is uniform, stability is high, VB12Liposome encapsulation is up to 90% or more；It can effectively avoid VB12By generating destruction when alimentary canal, and it can control VB12Release time in vivo makes the VB wrapped up12The slow release in alimentary canal, effectively shortens VB12The residence time improves intestinal absorption rate in enteron aisle.
Description
Technical field
The invention belongs to functional health-care food fields, and in particular to a kind of vitamin B12The preparation side of nano liposomes
Method.
Background technique
Vitamin B12(VB12) it is unique a kind of a kind of intestinal secretion object (castle's intrinsic factor) to be needed to help just be absorbed
Vitamin.Its residence time in enteron aisle is long, takes around three hours (most of water soluble vitamins only need several seconds)
It can just be absorbed.Vitamin B12Main Physiological Function be participate in manufacture erythrocyte, prevent pernicious anaemia；Prevent brain refreshing
It is subjected to destroying.Vitamin B12For the acicular crystal of light red, soluble easily in water and ethyl alcohol, under 4.5~5.0 mild acid conditions of pH value
It is most stable.
Liposome is a kind of novel form of similar microencapsulation, the film that drug encapsulation is formed in lipoids bilayer
Superfine sphere carrier formulation, is generally made of phosphatide and cholesterol made by centre, since its structure is similar to biomembrane, therefore
Also known as " artificial membrane ".Liposome has amphipathic characteristic, i.e. hydrophily and hydrophobicity after balancing in water.Liposome has
Many outstanding characteristics, such as drug can be brought to cell into, and the poison with cell targeted, compatibility, reduction drug
Property, increase the stability of drug and the features such as avoid tolerance.With new technology, the generation of new material, a kind of lipid of single chamber
Body, that is, nano liposomes are devised.
There are a small number of patent applications about vitamin nano liposome in the country.Chinese patent CN108601734A is disclosed
A kind of preparation method and purposes of capsaicine-vitamin E prodrug liposome；Chinese patent CN105878266A discloses a kind of water solubility
The preparation method and applications of vitamin liposome；Chinese patent CN106256345A discloses a kind of vitamin C liposome group
Close the preparation method of object.But it is domestic at present not about containing only vitamin B12Nano-lipid preparation application.
The traditional preparation method of liposome is film shake method, lipid organic solvent such as chloroform, ethyl alcohol etc. is dissolved,
After even mixing, after so that the solvent in round-bottomed flask is volatilized using Rotary Evaporators, uniform lipid can be formed in bottle wall
Film then under the conditions of temperature more than critical-temperature, is intended to aqueous solution and the lipid film mixing of encapsulating substance, is shaken with hand or so
It is dynamic, the lipid film in bottle wall is shaken, lipid can automatically form liposome (MLV) in aqueous solution.Nowadays there are many going back
Method for preparing lipidosome, such as alcohol injection, inverted evaporation method, film hydration method etc..
Summary of the invention
The object of the present invention is to provide a kind of vitamin Bs12Nano liposomes and preparation method thereof, the vitamin B12It receives
Mizhi plastid is prepared by film-ultrasonic dispersion.Vitamin B prepared by the present invention12Nano liposomes particle size dispersion
Uniformly, stability is high, VB12Liposome encapsulation is up to 90% or more；Vitamin B prepared by the present invention12Nano liposomes can have
Effect avoids VB12By generating destruction when alimentary canal, to successfully arrive at ileum, and VB can control12Release in vivo
Time makes the VB wrapped up12The slow release in alimentary canal, effectively shortens VB12The residence time in enteron aisle, to improve intestines
Road absorptivity.
The present invention is achieved through the following technical solutions above-mentioned purpose, a kind of vitamin B12The preparation side of nano liposomes
Method, the vitamin B12Nano liposomes are prepared by film-ultrasonic dispersion, are included the following steps:
1) by soybean lecithin, cholesterol is mixed in organic solvent, and evaporated under reduced pressure forms smooth lipid membrane；
2) vitamin B is added12, aqueous media and surfactant solubilizing lipids film and ultrasound at cream, then through ultrasonic thin
Born of the same parents crush, and by low-temperature centrifugation and filtering with microporous membrane, obtain vitamin B12Nano liposomes.
Preferably, vitamin B of the present invention12Its further technical solution of the preparation method of nano liposomes are as follows:
1) soybean lecithin and cholesterol are weighed in a round bottom flask, then pours into organic solvent in round-bottomed flask,
It is spread sufficiently under Ultrasonic Conditions, is evaporated organic solvent in Rotary Evaporators, is formed in round-bottomed flask inner wall smooth
Lipid membrane, be put into vacuum oven and be dried in vacuo；It is described that organic solvent is made to be evaporated processing in Rotary Evaporators
Rotary Evaporators temperature is less than or equal to 20 DEG C in the process；
2) vitamin B is then weighed12It is added in round-bottomed flask with surfactant, adds aqueous media, place it in
It is spread under the conditions of water bath sonicator, gained mixed liquor is poured in small beaker, pulverized in instrument and crush in cell, then by gained
Product is centrifuged, and is removed lower layer's impurity, is finally filtered with 0.22 μm of miillpore filter, obtained filtrate i.e. vitamin B12It receives
Mizhi plastid.
Preferably, the vitamin B12VB in nano liposomes12Concentration is 1-3mg/ml, wherein being represented with aqueous media
Vitamin B12The volume of nano liposomes；The further preferably described vitamin B12VB in nano liposomes12Concentration is 2mg/
ml；
Preferably, the surfactant is polyvinylpyrrolidone (PVP-K15)；The vitamin B12Nano liposomes
In every addition 20ml aqueous media need polyvinylpyrrolidone PVP-K15 weight be 0.01-0.03g, further preferably
0.02g；Vitamin B is represented with aqueous media12The volume of nano liposomes calculates, the vitamin B12In nano liposomes
The concentration of polyvinylpyrrolidone (PVP-K15) is 0.5mg/ml-1.5mg/ml；
Preferably, the weight ratio of the soybean lecithin and cholesterol is 5: 1；Further preferably every 40mg VB12It needs
Add 0.4g soybean lecithin and 0.08g cholesterol；
Preferably, the organic solvent is chloroform；
Preferably, the aqueous media is aqueous phosphate buffer, pH 7.0-7.2.
Preferably, the cell pulverizes condition are as follows: the work gap 2.5s/ 2.5s, power 350W, 15min.
Preferably, the centrifugal condition are as follows: 4 DEG C, 6000r/min, 15min.
The present invention is mainly on the basis of traditional film shake method, for the first time by VB12It is encapsulated in nanometer grade liposome,
And optimization is made to its technical process and parameter, VB is improved to reach12Nano liposomes stability and encapsulation rate and biological utilisation
The effect of degree, specific effect can be found in case study on implementation hereinafter.Using the VB of the technique preparation after optimization12Nano liposomes can be effective
Avoid VB12By generating destruction when alimentary canal, to successfully arrive at ileum, and VB can control12When release in vivo
Between, make the VB wrapped up12The slow release in alimentary canal, effectively shortens VB12The residence time in enteron aisle, to improve enteron aisle
Absorptivity.
The present invention has the advantage that
1) the present invention provides a kind of film-ultrasonic dispersions to prepare vitamin B12The method of nano liposomes；
2) present invention is by adjusting VB12Addition opportunity (being added after film forming), additive amount and its surfactant PVP-K15
Additive amount, make the vitamin B prepared12Nano liposomes particle size dispersion is uniform, stability is high, VB12Liposome encapsulation can
Up to 90% or more；
3) prepared by the present invention to can effectively avoid VB12By generating destruction when alimentary canal, and it can control VB12In body
Interior release time makes the VB wrapped up12The slow release in alimentary canal, effectively shortens VB12The residence time in enteron aisle, from
And improve intestinal absorption rate.
Detailed description of the invention
Fig. 1 is VB prepared by technique one12Partial size, the PDI distribution map of liposome；
Fig. 2 is VB prepared by technique one12The Zeta potential figure of liposome；
Fig. 3 is VB prepared by technique two12Partial size, the PDI distribution map of liposome；
Fig. 4 is VB prepared by technique two12The Zeta potential figure of liposome；
Fig. 5 is VB12Concentration is partial size, the PDI distribution map of 2mg/mL liposome；
Fig. 6 is VB12Concentration is the Zeta potential figure of 2mg/mL liposome；
Fig. 7 is VB12Concentration is partial size, the PDI distribution map of 3mg/mL liposome；
Fig. 8 is VB12Concentration is the Zeta potential figure of 3mg/mL liposome；
Fig. 9 is the VB that PVP-K15 additional amount is 0.01g12Partial size, the PDI distribution map of liposome；
Figure 10 is the VB that PVP-K15 additional amount is 0.01g12The Zeta potential figure of liposome；
Figure 11 is the VB that PVP-K15 additional amount is 0.03g12Partial size, the PDI distribution map of liposome；
Figure 12 is the VB that PVP-K15 additional amount is 0.03g12The Zeta potential figure of liposome；
Figure 13 is that ultramicro grinding power is VB under 400W12Partial size, the PDI distribution map of liposome；
Figure 14 is that ultramicro grinding power is VB under 400W12The Zeta potential figure of liposome；
Figure 15 is that ultramicro grinding power is VB under 300W12Partial size, the PDI distribution map of liposome；
Figure 16 is that ultramicro grinding power is VB under 300W12The Zeta potential figure of liposome；
Figure 17 is VB12Canonical plotting.
Specific embodiment
In order to make the objectives, technical solutions and advantages of the present invention clearer, With reference to embodiment, to this
Invention is further described.It should be understood that these descriptions are merely illustrative, and it is not intended to limit the scope of the invention.
1 VB of embodiment12Add the influence to liposome stability after adduction film forming before film forming
1.1 experimental materials:
Soybean lecithin: BR；Sinopharm Chemical Reagent Co., Ltd..
Cholesterol: AR；Sinopharm Chemical Reagent Co., Ltd..
Chloroform: AR；Sinopharm Chemical Reagent Co., Ltd..
Vitamin B12: AR；Sigma company.
Polyvinylpyrrolidone (PVP-K15): GR；Sinopharm Chemical Reagent Co., Ltd..
1.2 experimental methods:
1.2.1 technique one: VB12Preparation (the VB of nano liposomes12Add before film forming)
1) 0.4g soybean lecithin and 0.08g cholesterol are weighed respectively in a round bottom flask, then by 20mg vitamin B12
It is dissolved in 20ml chloroform and pours into round-bottomed flask, spread it sufficiently under ultrasonic conditions, make in Rotary Evaporators organic
Solvent is evaporated (optimum temperature is for 20 DEG C and following), after forming smooth lipid membrane in round-bottomed flask inner wall, by products obtained therefrom
It is put into vacuum oven and is dried in vacuo for 24 hours.
2) it weighs 0.02g PVP-K15 surfactant in a round bottom flask, adds 20ml phosphate buffer (PBS
(pH 7.0-7.2)), it places it under the conditions of water bath sonicator and spreads, gained mixed liquor is poured in small beaker, in cell ultra micro
(work the gap 2.5s/ 2.5s, power 350W, 15min) is crushed in crusher, be then centrifuged products obtained therefrom (4 DEG C,
6000r/min, 15min), lower layer's impurity is removed, is finally filtered with 0.22 μm of miillpore filter, obtained filtrate ties up life
Plain B12 nano liposomes.
1.2.2 technique two: VB12Preparation (the VB of nano liposomes12Add after film forming)
1) 0.4g soybean lecithin and 0.08g cholesterol are weighed respectively in a round bottom flask, then pour into 20ml chloroform
It in round-bottomed flask, spreads it sufficiently under ultrasonic conditions, so that organic solvent is evaporated (optimum temperature in Rotary Evaporators
20 DEG C and it is following), after forming smooth lipid membrane in round-bottomed flask inner wall, products obtained therefrom is put into vacuum in vacuum oven
Drying is for 24 hours.
2) 20mg vitamin B12 and 0.02g PVP-K15 surfactant are weighed in a round bottom flask, adds 20ml phosphorus
Acid buffer (PBS (pH 7.0-7.2)) places it under the conditions of water bath sonicator and spreads, gained mixed liquor is poured on small beaker
In, pulverized in instrument in cell and crush (work the gap 2.5s/ 2.5s, power 350W, 15min), then by products obtained therefrom into
Row centrifugation (4 DEG C, 6000r/min, 15min) removes lower layer's impurity, is finally filtered, is obtained with 0.22 μm of miillpore filter
Filtrate, that is, vitamin B12 nano liposomes.
1.2.3 the VB of different process preparation12The characterization of liposome
To the VB of different process preparation12The stability of liposome is studied, including partial size, polydispersity coefficient
The measurement of (Polydispersity index, PDI), surface potential (Zeta potential).Using laser particle analyzer measurement partial size point
Cloth and PDI.Zeta point is measured using Malvern zeta potential instrument.The above experimentation is both needed to carry out at 25 DEG C, experiment weight
Again three times.
1.3 experimental result
Fig. 1 is VB prepared by technique one12Partial size, the PDI distribution map of liposome；Fig. 2 is VB prepared by technique one12Lipid
The Zeta potential figure of body；As can be seen from Figure, 1mg/mL VB is enclosed12Liposome, its average grain diameter is 90.43nm, PDI
Numerical value is 0.240, and Zeta potential is that (for Zeta potential in 30~60mV or -30~-60mV, charged particle is more steady by -31.2mV
It is fixed).Add VB before can be seen that film forming by the analysis of stability indicator12Prepared liposomal samples, it is more steady to belong to performance
It is fixed, disperse more uniform.
Fig. 3 is VB prepared by technique two12Partial size, the PDI distribution map of liposome；Fig. 4 is VB prepared by technique two12Lipid
The Zeta potential figure of body.As can be seen from Figure, 1 mg/mL VB is enclosed12Liposome, its average grain diameter is 105.5nm, PDI
Numerical value is 0.235, and Zeta potential is -37.3mV.Add VB after can be seen that film forming by the analysis of stability indicator12Prepared
Liposomal samples, it is more stable to also belong to performance, disperses more uniform.
In summary it analyzes, liposomal particle size prepared by liposomal samples technique one prepared by technique two is larger, therefore
Add VB after film forming12More evenly, system is more more stable, therefore preparation side according to the present invention for prepared liposomal particle size dispersion
Add VB after being all made of film forming in method12Preparation process.
The VB of the different addition concentration of embodiment 212Influence to liposome stability
2.1 experimental materials:
Soybean lecithin: BR；Sinopharm Chemical Reagent Co., Ltd..
Cholesterol: AR；Sinopharm Chemical Reagent Co., Ltd..
Chloroform: AR；Sinopharm Chemical Reagent Co., Ltd..
Vitamin B12: AR；Sigma company.
Polyvinylpyrrolidone (PVP-K15): GR；Sinopharm Chemical Reagent Co., Ltd..
2.2 experimental methods:
2.2.1 the VB of different addition concentration12The preparation of nano liposomes
1) 0.4g soybean lecithin and 0.08g cholesterol are weighed respectively in a round bottom flask, then pour into 20ml chloroform
It in round-bottomed flask, spreads it sufficiently under ultrasonic conditions, so that organic solvent is evaporated (optimum temperature in Rotary Evaporators
20 DEG C and it is following), after forming smooth lipid membrane in round-bottomed flask inner wall, products obtained therefrom is put into vacuum in vacuum oven
Drying is for 24 hours.
2) 40mgVB12 and 60mgVB12 and 0.02g PVP-K15 surfactant are weighed respectively in two above-mentioned round bottoms
In flask, then it is each 20ml phosphate buffer (PBS (pH 7.0-7.2)) is added, place it under the conditions of water bath sonicator and spread, will
Gained mixed liquor is poured in small beaker, crushed in cell ultramicro grinding instrument (the work gap 2.5s/ 2.5s, power 350W,
15min), then products obtained therefrom is centrifuged (4 DEG C, 6000r/min, 15min), lower layer's impurity is removed, finally with 0.22 μm
Miillpore filter is filtered, and the filtrate respectively obtained is respectively the VB12 nano liposomes of 2mg/mL and 3mg/mL.
2.2.2 the VB of different addition concentration12The characterization of nano liposomes
To the VB of different addition concentration12The stability of nano liposomes is studied, including partial size, polydispersity coefficient
The measurement of (Polydispersity index, PDI), surface potential (Zeta potential).Using laser particle analyzer measurement partial size point
Cloth and PDI.Zeta point is measured using Malvern zeta potential instrument.The above experimentation is both needed to carry out at 25 DEG C, experiment weight
Again three times.
2.3 experimental result
Fig. 5 is VB12Concentration is partial size, the PDI distribution map of 2mg/mL liposome；Fig. 6 is VB12Concentration is 2mg/mL lipid
The Zeta potential figure of body encloses 2mg/mL VB as can be seen from Figure12Liposome, its average grain diameter is 157.6nm, PDI
Numerical value is 0.361, and Zeta potential is -33.0mV.It can be seen that VB by the analysis of stability indicator12Concentration is 2 mg/mL rouge
The partial size of plastid sample is apparently higher than 1mg/mL VB12Liposome, thus can determine whether to properly increase concentration can be improved and contain object
Content, but stability is slightly poor.
Fig. 7 is VB12Concentration is partial size, the PDI distribution map of 3mg/mL liposomal samples；
Fig. 8 is VB12Concentration is that the Zeta potential figure of 3mg/mL liposomal samples encloses 3mg/mL as can be seen from Figure
VB12Liposome, its average grain diameter is 154.4nm, and PDI numerical value is 0.497, and Zeta potential is -35.9mV.Refer to by stability
Target analysis is as can be seen that 3 mg/mL VB12Although liposomal samples, partial size increase, but stability relative reduction, point
It dissipates uneven.
In summary it analyzes, encloses 2mg/mL VB12Liposome may have reached the upper limit, if improving VB again12
Concentration, the stability of liposome will receive restriction, comprehensively considers, and select 2mg/ml as VB12Add concentration.
Influence of the surfactant (PVP-K15) of the different addition concentration of embodiment 3 to liposome stability
3.1 experimental materials:
Soybean lecithin: BR；Sinopharm Chemical Reagent Co., Ltd..
Cholesterol: AR；Sinopharm Chemical Reagent Co., Ltd..
Chloroform: AR；Sinopharm Chemical Reagent Co., Ltd..
Vitamin B12: AR；Sigma company.
Polyvinylpyrrolidone (PVP-K15): GR；Sinopharm Chemical Reagent Co., Ltd..
3.2 experimental methods:
3.2.1 the preparation of the nano liposomes of the PVP-K15 of different addition concentration
1) 0.4g soybean lecithin and 0.08g cholesterol are weighed respectively in a round bottom flask, then pour into 20ml chloroform
It in round-bottomed flask, spreads it sufficiently under ultrasonic conditions, so that organic solvent is evaporated (optimum temperature in Rotary Evaporators
20 DEG C and it is following), after forming smooth lipid membrane in round-bottomed flask inner wall, products obtained therefrom is put into vacuum in vacuum oven
Drying is for 24 hours.
2) 0.01g PVP-K15 and 0.03g PVP-K15 and 40mgVB12 is weighed respectively in two above-mentioned round-bottomed flasks
In, then it is each 20ml phosphate buffer (PBS (pH 7.0-7.2)) is added, place it under the conditions of water bath sonicator and spread, by gained
Mixed liquor is poured in small beaker, is pulverized in instrument in cell and is crushed (the work gap 2.5s/ 2.5s, power 350W, 15min),
Then products obtained therefrom is centrifuged (4 DEG C, 6000r/min, 15min), lower layer's impurity is removed, finally with 0.22 μm of miillpore filter
It is filtered, the filtrate respectively obtained is respectively the VB12 nano-lipid of PVP-K15 containing 0.01g and 0.03g PVP-K15
Body.
3.2.2 the characterization of the nano liposomes of the PVP-K15 of different addition concentration
The nano liposomes stability of the PVP-K15 of different addition concentration is studied, including partial size, polydispersity coefficient
The measurement of (Polydispersity index, PDI), surface potential (Zeta current potential).Using laser particle analyzer measurement partial size point
Cloth and PDI.Zeta point is measured using Malvern zeta potential instrument.The above experimentation is both needed to carry out at 25 DEG C, experiment weight
Again three times.
3.3 experimental results:
Fig. 9 is the 2mg/mL VB that PVP-K15 additional amount is 0.01g12Partial size, the PDI distribution map of liposome；Figure 10 is
PVP-K15 additional amount is the 2mg/mL VB of 0.01g12The Zeta potential figure of liposome；As can be seen from Figure, PVP-K15 additional amount is
The 2mg/mL VB of 0.01g12Liposome, its average grain diameter is 97.37nm, and PDI numerical value is 0.308, surface Zeta potential
For -24.6mV；
Figure 11 is the 2mg/mL VB that PVP-K15 additional amount is 0.03g12Partial size, the PDI distribution map of liposome；Figure 12 is
PVP-K15 additional amount is the 2mg/mL VB of 0.03g12The Zeta potential figure of liposome；PVP-K15 additional amount is as can be seen from Figure
The 2mg/mL VB of 0.03g12Partial size, PDI, the surface Zeta potential of liposome are respectively 82.32nm, 0.352, -25.8mV.It is real
Apply the 2mg/mL VB that PVP-K15 additional amount in example 2 is 0.02g12The partial size of liposome be 157.6nm, PDI numerical value is
0.361, Zeta potential is -33.0mV.
In summary the liposomal samples stability that 0.01g and 0.03g PVP-K15 is added in analysis reduces, comprehensive performance drop
It is low.So selecting PVP-K15 additional amount is 0.02g.
Influence of the different Ultrasonic Pulverization power of embodiment 4 to liposome stability
4.1 experimental materials:
Soybean lecithin: BR；Sinopharm Chemical Reagent Co., Ltd..
Cholesterol: AR；Sinopharm Chemical Reagent Co., Ltd..
Chloroform: AR；Sinopharm Chemical Reagent Co., Ltd..
Vitamin B12: AR；Sigma company.
Polyvinylpyrrolidone (PVP-K15): GR；Sinopharm Chemical Reagent Co., Ltd..
4.2 experimental methods:
4.2.1 the preparation of the nano liposomes of different Ultrasonic Pulverization power
1) 0.4g soybean lecithin and 0.08g cholesterol are weighed respectively in a round bottom flask, then pour into 20ml chloroform
It in round-bottomed flask, spreads it sufficiently under ultrasonic conditions, so that organic solvent is evaporated (optimum temperature in Rotary Evaporators
20 DEG C and it is following), after forming smooth lipid membrane in round-bottomed flask inner wall, products obtained therefrom is put into vacuum in vacuum oven
Drying is for 24 hours.
2) 0.02g PVP-K15 and 40mgVB12 is weighed respectively in two above-mentioned round-bottomed flasks, then each addition 20ml
Phosphate buffer (PBS (pH 7.0-7.2)) places it under the conditions of water bath sonicator and spreads, gained mixed liquor is poured on small beaker
In, it is pulverized in instrument in cell and crushes (the work gap 2.5s/ 2.5s, 15min), Ultrasonic Pulverization power is respectively set to
400W, 350W, 300W, ultrasonic time are set as 30min, be then centrifuged products obtained therefrom (4 DEG C, 6000r/min,
15min), lower layer's impurity is removed, is finally filtered with 0.22 μm of miillpore filter, the filtrate respectively obtained is respectively ultrasonic powder
The VB12 nano liposomes of broken power 400W, 350W, 300W.
4.2.2 the characterization of the nano liposomes of different Ultrasonic Pulverization power
The nano liposomes stability of different Ultrasonic Pulverization power is studied, including partial size, polydispersity coefficient
The measurement of (Polydispersity index, PDI), surface potential (Zeta potential).Using laser particle analyzer measurement partial size point
Cloth and PDI.Zeta point is measured using Malvern zeta potential instrument.The above experimentation is both needed to carry out at 25 DEG C, experiment weight
Again three times.
4.3 experimental result
Figure 13 is that ultramicro grinding power is VB under 400W12Partial size, the PDI distribution map of liposome；Figure 14 is to pulverize function
Rate is VB under 400W12The Zeta potential figure of liposome；As seen from the figure, the power of ultramicro grinding is VB under 400W12The grain of liposome
Diameter, PDI distribution, Zeta potential are respectively 119.2nm, 0.308, -22.4mV；
Figure 15 is that ultramicro grinding power is VB under 300W12Partial size, the PDI distribution map of liposome；Figure 16 is to pulverize function
Rate is VB under 300W12The Zeta potential figure of liposome；As seen from the figure, the power of ultramicro grinding is VB under 300W12The grain of liposome
Diameter, PDI distribution, Zeta potential are respectively 146.3nm, 0.436, -28.8mV；By Fig. 9 in embodiment 3 it is found that other conditions phase
With in situation, the power of ultramicro grinding is VB under 350W12Partial size, PDI distribution, the Zeta potential of liposome are respectively 157.6nm,
0.361, -33.0mV.From stability indicator as can be seen that the VB obtained under 350W ultrasound condition12Liposomal particle size disperses more
Uniformly, system is more more stable.Therefore choosing Ultrasonic Pulverization power is 350W.
5 vitamin B of embodiment12The entrapment efficiency determination of nano liposomes
5.1 experimental materials:
VB12Liposomal samples: self-control (using the technological parameter after optimization)
5.2 experimental methods:
5.2.1 VB12Liposomal samples preparation
1) 0.4g soybean lecithin and 0.08g cholesterol are weighed respectively in a round bottom flask, then pour into 20ml chloroform
It in round-bottomed flask, spreads it sufficiently under ultrasonic conditions, so that organic solvent is evaporated (optimum temperature in Rotary Evaporators
20 DEG C and it is following), after forming smooth lipid membrane in round-bottomed flask inner wall, products obtained therefrom is put into vacuum in vacuum oven
Drying is for 24 hours.
2) 40mg vitamin B12 and 0.02g PVP-K15 surfactant are weighed in a round bottom flask, adds 20ml phosphorus
Acid buffer (PBS (pH 7.0-7.2)) places it under the conditions of water bath sonicator and spreads, gained mixed liquor is poured on small beaker
In, pulverized in instrument in cell and crush (work the gap 2.5s/ 2.5s, power 350W, 15min), then by products obtained therefrom into
Row centrifugation (4 DEG C, 6000r/min, 15min) removes lower layer's impurity, is finally filtered, is obtained with 0.22 μm of miillpore filter
Filtrate, that is, vitamin B12 nano liposomes.
5.2.2 dialysis measures VB12The encapsulation rate of nano liposomes
VB12Canonical plotting it is as shown in figure 17, take 3mL 2mg/mL VB12Liposome solutions are put into bag filter, will
In its PBS for being put into 300mL (pH 7.0-7.2), magnetic agitation takes 10mL dialysis sample measurement absorbance value every 30min, takes 6
After secondary dialysis sample, 10mL dialysis sample is taken to measure its absorbance value every 60min.Free VB is calculated after it is stablized12。3mL
Blank liposome replaces VB12Liposome is as blank control.According to VB12Standard curve can be calculated VB12Nano liposomes
Encapsulation rate.
5.3 experimental results:
The absorbance that each period measures such as the following table 1:
The dialysis sample absorbance value of table 1
After 30min, measuring dialysis sample absorbance at 361nm is 0.265, according to gained VB before12Standard curve Y=
20.198x+0.021 calculates to obtain 2mg/mL VB12Liposome encapsulation is 90.94%.
6 vitamin B of embodiment12The digestion of nano liposomes simulated gastrointestinal tract
6.1 experimental materials:
Pepsin: AR；Sinopharm Chemical Reagent Co., Ltd.
Pancreatin: AR；Sinopharm Chemical Reagent Co., Ltd.
Concentrated hydrochloric acid: AR；Sinopharm Chemical Reagent Co., Ltd.
VE acetic acid esters: AR；Sigma company
VB12Liposomal samples: self-control (same to 5.2.1)
6.2 experimental methods:
6.2.1 liposomal samples simulated gastrointestinal tract digests
Take 1.5mL, 2mg/mlVB12Liposomal samples and 13.5mL physiological saline (contain 140 mmol/L, 5mmol/L KCl,
150 μm of ol/L BHT mixing, stirring 10min is to uniform.System pH is adjusted to 2.0 with HCl solution, and 1mL concentration is added and is
The pepsin of 3.2g/L, simulate the gastric juice digest 1h.Then, system pH is adjusted to 7.5 with NaOH solution, and 4.5mL intestines is added
Liquid digestive juice (pancreatin containing 4.76g/L and 5.16g/L NaTDC), simulated intestinal fluid digest 2h.Whole experiment process is in nitrogen
It is carried out under atmosphere, the temperature of system is constant at 37 DEG C, and continues to stir.
6.2.2 the measurement of free fatty acid (FFA) burst size
In the 2h of simulated intestinal fluid digestion, using pH-stat method, it is molten that 0.1mol/L NaOH is added constantly into system
Liquid makes pH maintain 7.5, and records the amount of NaOH consumed by the passage in experimentation with digestion time.Using blank rouge
Plastid is as control.
Phosphatidylcholin is constantly hydrolyzed under the action of pancreatic lipase and releases FFA, it is considered that a molecule phosphatide
Phthalein choline is hydrolyzed into a molecule FFA and a molecule lysophosphatide.Therefore, according to the following formula, the volume according to consumption NaOH, can count
Calculate the amount of the FFA obtained in the system.
In formula, mIPThe gross mass (g) of phosphatidyl choline；
MIPThe average molecular weight (g/mol) of phosphatidyl choline；
CNaOHThe concentration (mol/L) of NaOH solution when titration；
The volume (mL) of consumed NaOH solution when VNaOH- digestion time t
6.2.3 biology can give the measurement of rate
Postdigestive sample is fully transferred in centrifuge tube, and 10000r/min is centrifuged 30min at 4 DEG C.Sample quilt
It is divided into two parts, upper layer is the transparent micella layer for being loaded with nutrient, and lower layer is then indigested sample, cholate and free rouge
The fine and close insoluble substance that fat acid is formed.Detect VB in micella layer12Content, calculate its micella rate and biology can be to rate.
6.3 experimental results:
2 simulated gastrointestinal tract of table digests result
7 vitamin B of embodiment12The degree of oxidation measurement of phosphatide in nano liposomes
7.1 experimental materials:
Trichloroacetic acid: AR；Shanghai Aladdin biochemical technology limited liability company
2- thiobarbituricacidα-: AR；Sinopharm Chemical Reagent Co., Ltd.
Concentrated hydrochloric acid: AR；Sinopharm Chemical Reagent Co., Ltd.
VE acetic acid esters: AR；Sigma company
VB12Liposomal samples: self-control (same to 5.2.1)
7.2 experimental methods:
VB12The degree of oxidation of nano liposomes is reflected by malonaldehyde (MDA) content, and malonaldehyde (MDA) is given below
The measuring method of content:
1) configuration of TBA-TCA-HCl solution: take trichloroacetic acid (TCA) 15g, 2- thiobarbituricacidα- (TBA) 0.370g,
Concentrated hydrochloric acid 2.1g (1.6mL) is dissolved in deionized water, and 80 DEG C of stirred in water bath dissolutions are settled to 100mL with deionized water after cooling,
Matching while using.
2) measurement of malonaldehyde (MDA) content: take 1M1 nano liposomes (the VE acetic acid esters of addition different content) in 10mL
In scale test tube, TBA-TCA-HCl solution 5mL is added, then whirlpool concussion mixing reacts 30min in boiling water bath, uses ice water
Bath is cooling rapidly, is settled to 10mL with TBA-TCA-HCl solution, and 2500r/min is centrifuged 5min after mixing, takes supernatant, with
TBA-TCA-HCl solution is the light absorption value under blank determination 532nm wavelength.
7.3 experimental results:
Absorbance value under 3 532nm wavelength of table
As seen from Table 3, under 4 DEG C of refrigerator of storage condition, it is added to the VB of VE acetic acid esters12Liposome is at 532nm
Absorbance value be less than not plus VE acetic acid esters VB12Liposome, absorbance value is compared with blank liposome absorbance value compared to inclined
Greatly, it may be possible to due to by VB12The influence of certain substance after degradation, in later experiments, we will change adding for vitamin E
Add sequence, or addition vitamin C protects liposome in aqueous solution, avoids VB12Directly directly contacted with other vitamins,
And reduce VB12With the oxidation resistance of other antioxidants.
Although embodiments of the present invention are described in detail, it should be understood that, without departing from of the invention
In the case where spirit and scope, embodiments of the present invention can be made with various changes, replacement and change.
Claims (10)
1. a kind of vitamin B12The preparation method of nano liposomes, it is characterised in that: the vitamin B12Nano liposomes pass through
Film-ultrasonic dispersion is prepared, and includes the following steps:
1) by soybean lecithin, cholesterol is mixed in organic solvent, and evaporated under reduced pressure forms smooth lipid membrane；
2) vitamin B is added12, aqueous media and surfactant solubilizing lipids film and ultrasound at cream, then through ultrasonic cell powder
It is broken, by low-temperature centrifugation and filtering with microporous membrane, obtain vitamin B12Nano liposomes.
2. preparation method according to claim 1, it is characterised in that: specific steps are as follows:
1) soybean lecithin and cholesterol are weighed in a round bottom flask, then pours into organic solvent in round-bottomed flask, in ultrasound
It is spread sufficiently under the conditions of wave, is evaporated organic solvent in Rotary Evaporators, and smooth rouge is formed in round-bottomed flask inner wall
Matter film, is put into vacuum oven and is dried in vacuo；It is described so that organic solvent is evaporated treatment process in Rotary Evaporators
Middle Rotary Evaporators temperature is less than or equal to 20 DEG C；
2) vitamin B is then weighed12It is added in round-bottomed flask with surfactant, adds aqueous media, place it in water-bath
It is spread under ultrasound condition, gained mixed liquor is poured in small beaker, pulverized in instrument and crush in cell, then by products obtained therefrom
It is centrifuged, removes lower layer's impurity, be finally filtered with 0.22 μm of miillpore filter, obtained filtrate i.e. vitamin B12Nanometer rouge
Plastid.
3. preparation method according to claim 2, it is characterised in that: the vitamin B12VB in nano liposomes12Concentration
For 1-3mg/ml, wherein representing vitamin B with aqueous media12The volume of nano liposomes.
4. preparation method according to claim 3, it is characterised in that: the vitamin B12VB in nano liposomes12Concentration
For 2mg/ml.
5. preparation method according to claim 2, it is characterised in that: the surfactant is polyvinylpyrrolidone；
The vitamin B12It is 0.01-0.03g that every addition 20ml aqueous media, which needs polyvinylpyrrolidone weight, in nano liposomes.
6. preparation method according to claim 5, it is characterised in that: the vitamin B12Every addition in nano liposomes
It is 0.02g that 20ml aqueous media, which needs polyvinylpyrrolidone weight,.
7. preparation method according to claim 2, it is characterised in that: the mass ratio of the soybean lecithin and cholesterol is
5∶1。
8. preparation method according to claim 2, it is characterised in that: the aqueous media is aqueous phosphate buffer,
PH range is 7.0-7.2.
9. preparation method according to claim 2, it is characterised in that: the cell pulverizes condition are as follows: in power
15min is handled under conditions of 350W, in the process every work 2.5s gap 2.5s.
10. preparation method according to claim 2, it is characterised in that: the centrifugal condition are as follows: be in revolving speed at 4 DEG C
15min is centrifuged under conditions of 6000r/min.
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| CN110122564A (en) * | 2019-04-25 | 2019-08-16 | 华南理工大学 | A kind of probiotics liposome and preparation method thereof |
| CN112545994A (en) * | 2020-11-19 | 2021-03-26 | 河南科技大学 | Synchronous preparation method of multi-chamber and single-chamber liposome with small particle size and high stability |
| CN114532535A (en) * | 2022-01-05 | 2022-05-27 | 齐鲁工业大学 | Preparation method of curcumin nano-liposome |
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| CN110122564A (en) * | 2019-04-25 | 2019-08-16 | 华南理工大学 | A kind of probiotics liposome and preparation method thereof |
| CN112545994A (en) * | 2020-11-19 | 2021-03-26 | 河南科技大学 | Synchronous preparation method of multi-chamber and single-chamber liposome with small particle size and high stability |
| CN112545994B (en) * | 2020-11-19 | 2022-10-14 | 河南科技大学 | Synchronous preparation method of multi-chamber and single-chamber liposome with small particle size and high stability |
| CN114532535A (en) * | 2022-01-05 | 2022-05-27 | 齐鲁工业大学 | Preparation method of curcumin nano-liposome |
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