CN106880049A - Application of the n 3PUFA phosphatide in chronic fatigue syndrome product is improved - Google Patents
Application of the n 3PUFA phosphatide in chronic fatigue syndrome product is improved Download PDFInfo
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- CN106880049A CN106880049A CN201710130672.8A CN201710130672A CN106880049A CN 106880049 A CN106880049 A CN 106880049A CN 201710130672 A CN201710130672 A CN 201710130672A CN 106880049 A CN106880049 A CN 106880049A
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- epa
- dha
- phosphatide
- chloroform
- eluent
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- 206010008874 Chronic Fatigue Syndrome Diseases 0.000 title claims abstract description 21
- 208000029766 myalgic encephalomeyelitis/chronic fatigue syndrome Diseases 0.000 title claims abstract description 21
- 241000238366 Cephalopoda Species 0.000 claims abstract description 26
- 241000251511 Holothuroidea Species 0.000 claims abstract description 25
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 22
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 22
- 210000004681 ovum Anatomy 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 62
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 42
- 239000003480 eluent Substances 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 20
- 239000000741 silica gel Substances 0.000 claims description 20
- 229910002027 silica gel Inorganic materials 0.000 claims description 20
- 229960001866 silicon dioxide Drugs 0.000 claims description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 150000002632 lipids Chemical class 0.000 claims description 18
- -1 DHA phosphatid ylcholines Chemical class 0.000 claims description 17
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 12
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 12
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 238000004587 chromatography analysis Methods 0.000 claims description 8
- 229940067626 phosphatidylinositols Drugs 0.000 claims description 8
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 6
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000011161 development Methods 0.000 claims description 6
- 230000018109 developmental process Effects 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 229910052740 iodine Inorganic materials 0.000 claims description 6
- 239000011630 iodine Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000003760 magnetic stirring Methods 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 229940067605 phosphatidylethanolamines Drugs 0.000 claims description 6
- 229910052710 silicon Inorganic materials 0.000 claims description 6
- 239000010703 silicon Substances 0.000 claims description 6
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 6
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 4
- SWXVUIWOUIDPGS-UHFFFAOYSA-N diacetone alcohol Natural products CC(=O)CC(C)(C)O SWXVUIWOUIDPGS-UHFFFAOYSA-N 0.000 claims description 4
- 230000000937 inactivator Effects 0.000 claims description 4
- 229940067606 lecithin Drugs 0.000 claims description 4
- 235000010445 lecithin Nutrition 0.000 claims description 4
- 239000000787 lecithin Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 150000008105 phosphatidylcholines Chemical class 0.000 claims description 4
- 150000003905 phosphatidylinositols Chemical class 0.000 claims description 4
- 150000008106 phosphatidylserines Chemical class 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 3
- 239000000287 crude extract Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims 2
- ZZVUWRFHKOJYTH-UHFFFAOYSA-N diphenhydramine Chemical compound C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1 ZZVUWRFHKOJYTH-UHFFFAOYSA-N 0.000 claims 2
- 125000001095 phosphatidyl group Chemical group 0.000 claims 2
- 238000012790 confirmation Methods 0.000 claims 1
- 206010016256 fatigue Diseases 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- 230000002929 anti-fatigue Effects 0.000 abstract description 2
- 238000009661 fatigue test Methods 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000000284 extract Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102000015439 Phospholipases Human genes 0.000 description 2
- 108010064785 Phospholipases Proteins 0.000 description 2
- 150000004075 acetic anhydrides Chemical class 0.000 description 2
- 230000001476 alcoholic effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 239000004927 clay Substances 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 125000004494 ethyl ester group Chemical group 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 238000013517 stratification Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 206010003591 Ataxia Diseases 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 240000009212 Coccoloba uvifera Species 0.000 description 1
- 235000003913 Coccoloba uvifera Nutrition 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 238000010162 Tukey test Methods 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001977 ataxic effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 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 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012109 statistical procedure Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
- A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
- C07F9/103—Extraction or purification by physical or chemical treatment of natural phosphatides; Preparation of compositions containing phosphatides of unknown structure
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a kind of application of n 3PUFA phosphatide in chronic fatigue syndrome product is improved.EPA phosphatide is specifically extracted from sea cucumber, the extraction DHA phosphatide from squid ovum, and the specific preparation of each component is carried out to EPA and DHA phosphatide, finally the EPA and DHA phosphatide to each component carries out anti-fatigue test checking.The present invention prepares EPA phosphatide by raw material of sea cucumber, and DHA phosphatide is prepared by raw material of squid ovum, then carries out Component seperation and conversion, and the purity of obtained PC, PE, PS and PI is more than 90%.The present invention is demonstrated by zoopery, EPA the and DHA phosphatide of obtained each component can greatly extend mouse and be run the time in tired instrument, i.e. EPA and DHA phosphatide can be obviously improved fatigue resistance of the chronic fatigue syndrome mouse under moderate strength, illustrate that EPA and DHA phosphatide can effectively prevent, improve or treat chronic fatigue syndrome.
Description
Technical field
The invention belongs to screening bioactive compoundses technical field, it is related to n-3PUFA phosphatide improving chronic fatigue syndrome system
Application in product, and in particular to sea cucumber phosphatide and squid lecithin are in prevention, improvement or treatment chronic fatigue syndrome imbalance system
Application in product.
Background technology
Chronic fatigue syndrome (chronic fatigue syndrome, CFS), is also called chronic fatigue immune function barrier
Hinder syndrome, be a kind of Special Manifestations of sub-health state.With long-term serious sense of fatigue (at least more than half a year) for protrusion is faced
Bed performance, and with insomnia, decrease of memory, bone open-minded flesh pain and various Spirit nerve symptoms of disease, but without other organic and essences
The a complex set of dysfunction syndrome group that godhead disease is characterized.National survey shows CFS crowd's incidence of disease about 0.007%
~2.8%, most of patient age was at 30~50 years old.This disease is predicted as 21 century by American Centers for Disease Control and Prevention
Influence one of subject matter of human health, although without recent life danger, but have a strong impact on the physical and mental health of patient, make
Decline into quality of life, then influence social productive forces and labour, take medical resource, therefore caused the extensive of the whole society
Concern.
The therapeutic effect of the medicine for the treatment of chronic fatigue syndrome is limited at present, and cures the symptoms, not the disease, it is also possible to cause pair
Effect, people tend to select to take functional component or functional food with preventing, slowing down or treat chronic fatigue syndrome
Slow down ataxic purpose to reach, therefore exploitation safely and effectively has prevention, slows down or treat chronic fatigue syndrome
Functional component turn into an urgent demand.
DHA and EPA are two kinds of common n-3 series unrighted acids, and EPA/DHA products in the market are mainly
Ethyl ester type and glycerine ester type, and ethyl ester type EPA/DHA not only digests in human body and absorbs relatively difficult, thereby increases and it is possible to there is safety
Hidden danger, therefore glycerine ester type EPA/DHA enjoys consumers welcomed.In recent years there are some researches show being present in the unsaturated lipid in phosphatide
Relative to glycerine ester type unrighted acid, not only oxidation stability is more preferable, and digests and assimilates speed faster, adds for fat acid
There is phosphatide improvement fat metabolism, strengthen immunity isoreactivity, therefore the phosphatide rich in EPA/DHA to be likely to become instead of sweet in itself
The novel lipid health product of grease type EPA/DHA.
The content of the invention
It is an object of the invention to provide a kind of application of n-3PUFA phosphatide in chronic fatigue syndrome product is improved.Tool
Body extracts EPA phosphatide from sea cucumber, DHA phosphatide is extracted from squid ovum, and carry out the specific of each component to EPA and DHA phosphatide
Prepare, finally the EPA and DHA phosphatide to each component carries out anti-fatigue test checking.
To reach above-mentioned purpose, the concrete technical scheme that the present invention takes is：
Application of the n-3PUFA phosphatide in chronic fatigue syndrome product is improved.
Further, described n-3PUFA phosphatide includes EPA phosphatide and DHA phosphatide.
Further, the EPA phosphatide is EPA phosphatid ylcholines（EPA-PC）, EPA phosphatidyl-ethanolamines（EPA-PE）、
EPA phosphatidylserines（EPA-PS）, EPA phosphatidylinositols（EPA-PI）One or more mixing.
Further, the DHA phosphatide is DHA phosphatid ylcholines（DHA-PC）, DHA phosphatidyl-ethanolamines（DHA-PE）、
DHA phosphatidylserines（DHA-PS）With DHA phosphatidylinositols（DHA-PI）In one or more mixing.
Further, the preparation method of the EPA phosphatide is comprised the following steps：
1）After sea cucumber is crushed through vacuum freeze drying, powder is made；
2）Sea cucumber dry powder is taken, is extracted in the mixed solution of chloroform and methyl alcohol overnight, after filtering, extracted with Klorvess Liquid, it is quiet
Layering is put, chloroform layer is collected, through being concentrated under reduced pressure to give sea cucumber lipid CE；
3）After by the activated dress post of silica gel, silicagel column is balanced with chloroform；Again by step 2）The sea cucumber lipid crude extract for obtaining is used
After chloroform dissolving, it is poured slowly into silicagel column, is eluted with chloroform, chloroform-methanol mixed liquor, acetone and methyl alcohol successively, receives
Collection eluent, eluent carries out thin-layer silicon offset plate chromatography (TLC), and concentrated under reduced pressure after being confirmed with iodine and Dittmer reagent colour developments
Removal residual organic solvent is to obtain sea cucumber phosphatide；
Wherein, the eluent for collecting chloroform-methanol is EPA phosphatidyl-ethanolamines（EPA -PE）, collect methyl alcohol eluent be
EPA phosphatid ylcholines（EPA-PC）；
4）Acetic acid/SAS buffer solution is put into heat collecting type constant-temperature heating magnetic stirring apparatus and is preheated, Serine is molten
In wherein, above-mentioned steps 3 are added）In EPA-PC, be eventually adding PLD, the stirring reaction at 40 DEG C, add HCl, inactivation
Enzyme, reaction terminating with n-hexane-isopropyl alcohol extracting product, that is, obtains EPA phosphatidylserines（EPA -PS）；
5）By step 2）Middle sea cucumber lipid CE, adds acetone, trimethylamine and acetic anhydride, after cold filtration, is washed with acetone
It is de-, obtain EPA phosphatidylinositols（EPA-PI）.
Further, the preparation method of the DHA phosphatide is comprised the following steps：
1）After squid ovum is crushed through vacuum freeze drying, powder is made；
2）Squid ovum dry powder is taken, is extracted overnight in chloroform and methyl alcohol mixed solution, after filtering, extracted with Klorvess Liquid, it is quiet
Layering is put, chloroform layer is collected, through being concentrated under reduced pressure to give squid ovum lipid CE；
3）After by the activated dress post of silica gel, silicagel column is balanced with chloroform；Again by step 2）The lipid of the squid ovum for obtaining slightly is carried
After thing is dissolved with chloroform, it is poured slowly into silicagel column, is washed with chloroform, chloroform-methanol mixed liquor, acetone and methyl alcohol successively
It is de-, collect eluent, eluent carries out thin-layer silicon offset plate chromatography (TLC), and subtracts after being confirmed with iodine and Dittmer reagent colour developments
Pressure concentration removal residual organic solvent is to obtain squid lecithin；
Wherein, the eluent for collecting chloroform-methanol is DHA phosphatidyl-ethanolamines（DHA-PE）, the eluent for collecting methyl alcohol is DHA
Phosphatid ylcholine（DHA-PC）；
4）Acetic acid/SAS buffer solution is put into heat collecting type constant-temperature heating magnetic stirring apparatus and is preheated, Serine is molten
In wherein, above-mentioned steps 3 are added）In DHA-PC, be eventually adding PLD, the stirring reaction at 40 DEG C, add HCl, inactivation
Enzyme, reaction terminating with n-hexane-isopropyl alcohol extracting product, that is, obtains DHA phosphatidylserines（DHA-PS）；
5）By step 2）Middle squid ovum lipid CE, adds acetone, trimethylamine and acetic anhydride, after cold filtration, uses acetone
Wash-out, obtains DHA phosphatidylinositols（DHA-PI）.
Further, described improvement chronic fatigue syndrome product is food, feed or medicine；Wherein, the EPA or
Person is total addition mass fraction of DHA phosphatide in 0.5%-2%.
Beneficial effects of the present invention：The present invention prepares EPA phosphatide by raw material of sea cucumber, and DHA is prepared by raw material of squid ovum
Phosphatide, further carries out Component seperation and conversion, and the purity of obtained PC, PE, PS and PI is more than 90%.The present invention passes through
Zoopery is demonstrated, EPA the and DHA phosphatide of obtained each component can greatly extend mouse and be run in tired instrument
Time, i.e. EPA and DHA phosphatide can be obviously improved fatigue resistance of the chronic fatigue syndrome mouse under moderate strength, and
PS best results；Illustrate that EPA and DHA phosphatide can effectively prevent, improve or treat chronic fatigue syndrome.
Brief description of the drawings
Fig. 1 is that EPA and DHA phosphatide is run the result of experiment to mouse under moderate strength in embodiment 3.
Specific embodiment
Hereinafter the present invention is described in detail using specific embodiment and with reference to accompanying drawing.
Embodiment 1：The preparation of sea cucumber EPA phosphatide
（1）After commercially available North Atlantic Ocean melon ginseng is crushed through vacuum freeze drying, clay into power（200 mesh）.
Second step, takes sea cucumber dry powder 10kg, adds 100L chloroform/methanols (2:1, v/v), stir, filtering is used
0.88% Klorvess Liquid extracting, stratification is collected chloroform layer, is extracted 3 times, merges concentrate, through being concentrated under reduced pressure to give
Sea cucumber or squid ovum lipid CE；
3rd step, weighs silica gel 200g, adds 5% water, and 4h, room temperature cooling are activated at 120 DEG C.It is quiet using wet method dress post
Put after after silica gel sedimentation completely, compression leg is carried out with 2 times of column volume chloroforms, make the silica gel in post more closely knit.Take 50g sea cucumber or
After squid ovum CE sample is with chloroform dissolving less of trying one's best, the solution is shifted with dropper, be uniformly added into along chromatography column wall.
After 5 column volumes are first suitably washed with chloroform, with chloroform-methanol 9:1 mixed liquor washes 2 column volumes, then with 1 cylinder of acetone
Product, is finally eluted with methyl alcohol, collects eluent, and eluent carries out thin-layer silicon offset plate chromatography (TLC), and solvent is chloroform/first
Alcohol/water (80:15:1, v/v) removal residual organic solvent concentrated under reduced pressure is after being confirmed, and with iodine and Dittmer reagent colour developments
Obtain sea cucumber lipid-soluble extract.Wherein, the eluent for collecting chloroform-methanol is EPA-PE, and the eluent for collecting methyl alcohol is EPA
-PC。
Sample identification use TLC methods, silica gel plate is placed in 110 DEG C of baking ovens and activates 20min, after point sample with chloroform/
Methanol/water (80:15:1, v/v) for solvent launches, after expansion is finished, drying, by developer (10% sulfuric acid-second
Alcoholic solution) uniformly it is sprayed on dry lamellae, 110 DEG C of baking oven 5min are placed in, until being showed on TLC plates
Purple dot.
4th step, by the buffer solution of 200ml（20 mM acetic acid/SAS, containing 50 mM CaCl2）It is put into heat collecting type
Preheated in constant-temperature heating magnetic stirring apparatus, the Serine of 20g is dissolved in wherein, add the EPA-PC in 10g step 3, most
Add the 40 U/mL phospholipase Ds of 3g afterwards, the stirring reaction at 40 DEG C after reaction 6h, adds appropriate HCl, inactivator, instead
Should terminate, use n-hexane：Isopropanol（4:1, v/v）Extract product, i.e. EPA-PS.
5th step, 100 milliliters of acetone, 0.1 are added by the sea cucumber in 10 grams of step 2 or squid ovum lipid CE
Milliliter trimethylamine and 2 grams of acetic anhydrides, 40 DEG C of stirring reactions 2 hours, cold filtration, after 80 milliliters of acetone are washed in three times, with rotation
The mode drying to obtain product of rotatable evaporation, its product is EPA-PI.
Embodiment 2：The preparation of squid ovum DHA phosphatide
（1）After commercially available sea grape is crushed through vacuum freeze drying, clay into power（200 mesh）.
Second step, takes squid ovum dry powder 10kg, adds 100L chloroform/methanols (2:1, v/v), stir, filtering is used
0.88% Klorvess Liquid extracting, stratification is collected chloroform layer, is extracted 3 times, merges concentrate, through being concentrated under reduced pressure to give
Squid ovum lipid CE；
3rd step, weighs silica gel 200g, adds 5% water, and 4h, room temperature cooling are activated at 120 DEG C.It is quiet using wet method dress post
Put after after silica gel sedimentation completely, compression leg is carried out with 2 times of column volume chloroforms, make the silica gel in post more closely knit.Take the squid ovum of 50g
After CE sample is with chloroform dissolving less of trying one's best, the solution is shifted with dropper, be uniformly added into along chromatography column wall.First use chlorine
Imitate after suitably washing 5 column volumes, with chloroform-methanol 9:1 mixed liquor washes 2 column volumes, then with 1 column volume of acetone, finally
Eluted with methyl alcohol, collect eluent, eluent carries out thin-layer silicon offset plate chromatography (TLC), and solvent is chloroform/methanol/water
(80:15:1, v/v) the removal residual organic solvent that is concentrated under reduced pressure after being confirmed, and with iodine and Dittmer reagent colour developments is to obtain sea cucumber
Lipid-soluble extract.Wherein, the eluent for collecting chloroform-methanol is DHA-PE, and the eluent for collecting methyl alcohol is DHA-PC.
Sample identification use TLC methods, silica gel plate is placed in 110 DEG C of baking ovens and activates 20min, after point sample with chloroform/
Methanol/water (80:15:1, v/v) for solvent launches, after expansion is finished, drying, by developer (10% sulfuric acid-second
Alcoholic solution) uniformly it is sprayed on dry lamellae, 110 DEG C of baking oven 5min are placed in, until being showed on TLC plates
Purple dot.
4th step, by the buffer solution of 200ml（20 mM acetic acid/SAS, containing 50 mM CaCl2）It is put into heat collecting type
Preheated in constant-temperature heating magnetic stirring apparatus, the Serine of 20g is dissolved in wherein, add the DHA-PC in 10g step 3, most
Add the 40 U/mL phospholipase Ds of 3g afterwards, the stirring reaction at 40 DEG C after reaction 6h, adds appropriate HCl, inactivator, instead
Should terminate, use n-hexane：Isopropanol（4:1, v/v）Extract product, i.e. DHA-PS.
5th step, by the squid ovum lipid CE in 10 grams of step 2 add 100 milliliters of acetone, 0.1 milliliter
Trimethylamine and 2 grams of acetic anhydrides, 40 DEG C of stirring reactions 2 hours, cold filtration, after 80 milliliters of acetone are washed in three times, with rotary
The mode drying to obtain product of evaporation, its product is DHA-PI.
Embodiment 3：Zoopery
1. material and method
（1）Animal feeding and behavior analysis
Experimental animal uses Balb/c mouse, and male, 22 ± 0.5g of body weight has purchased from Beijing dimension tonneau China experimental animal technology
Limit company.Animal House humidity 65 ± 15%, 23 ± 2 DEG C of room temperature, 12:12 h light and shades replace, and mouse can freely ingest and drink water.It is small
Mouse is divided into 10 groups by mouse after adaptability is fed one week by body weight：Normal group, model control group, DHA-PC,
DHA-PE, DHA-PS, DHA-PI and EPA-PC, EPA-PE, EPA-PS, EPA-PI group, every group 8, feeding is corresponding respectively
Feed.Feed is formulated and is improved with reference to AIN-93G rodents, the DHA+EPA of experimental group addition 0.5%,
Per day entry with statistics food ration, record changes of weight, continuous nursing 3 weeks are every other day weighed.Mouse is every other day forced to exist
Continuously run 2 h in the rotary-type tired instrument of YLS- 10B under condition (in 25 r/min, 3s, 1.5 mA difficulty), most
Afterwards one time when time for being exhausted to power of record running of the mouse in the rotary-type tired instrument of YLS- 10B, and pluck eyeball and take blood, put to death
Mouse.Power exhausts the standard of judgement：Time of having a rest 30s after mouse is in long-time electric shock, continuous rest 5 times, are judged as in 5min
Power exhausts state.
（2）Statistical procedures
Experimental data represented with `x ± SEM, and Student ' s t test and are carried out using the softwares of SPSS 18.0
Tukey ' s test are analyzed, with P ＜ 0.05 for there were significant differences.
1. experimental result
The running time of sea cucumber and squid lecithin to mouse under moderate strength is as shown in figure 1, model group is right relative to normal
For according to group, after various forms of EPA and DHA phosphatide are intervened, compared with model group, mouse time of being run in tired instrument is obvious
Extend, this various forms of EPA and DHA phosphatide of prompting can significantly improve chronic fatigue syndrome mouse medium strong
Fatigue resistance under degree, has further pointed out various forms of EPA and DHA phosphatide can prevent, improve or treat chronic tired
Labor syndrome, has significant effect to prevention, improvement or treatment chronic fatigue syndrome.
Claims (7)
- Application of the 1.n-3PUFA phosphatide in chronic fatigue syndrome product is improved.
- 2. application as claimed in claim 1, it is characterised in that described n-3PUFA phosphatide includes EPA phosphatide and DHA phosphatide.
- 3. application as claimed in claim 2, it is characterised in that the EPA phosphatide is EPA phosphatid ylcholines, EPA phosphatidyl second Hydramine, EPA phosphatidylserines, one or more mixing of EPA phosphatidylinositols.
- 4. application as claimed in claim 2, it is characterised in that the DHA phosphatide is DHA phosphatid ylcholines, DHA phosphatidyl second One or more mixing in hydramine, DHA phosphatidylserines and DHA phosphatidylinositols.
- 5. application as claimed in claim 2, it is characterised in that the preparation method of the EPA phosphatide is comprised the following steps：1）After sea cucumber is crushed through vacuum freeze drying, powder is made；2）Sea cucumber dry powder is taken, is extracted in the mixed solution of chloroform and methyl alcohol overnight, after filtering, extracted with Klorvess Liquid, it is quiet Layering is put, chloroform layer is collected, through being concentrated under reduced pressure to give sea cucumber lipid CE；3）After by the activated dress post of silica gel, silicagel column is balanced with chloroform；Again by step 2）The sea cucumber lipid crude extract for obtaining is used After chloroform dissolving, it is poured slowly into silicagel column, is eluted with chloroform, chloroform-methanol mixed liquor, acetone and methyl alcohol successively, receives Collection eluent, eluent carries out thin-layer silicon offset plate chromatography, and residual with the removal that is concentrated under reduced pressure after iodine and the confirmation of Dittmer reagent colour developments Organic solvent is stayed to obtain sea cucumber phosphatide；Wherein, the eluent for collecting chloroform-methanol is EPA phosphatidyl-ethanolamines, and the eluent for collecting methyl alcohol is EPA phosphatidyl courages Alkali；4）Acetic acid/SAS buffer solution is put into heat collecting type constant-temperature heating magnetic stirring apparatus and is preheated, Serine is molten In wherein, above-mentioned steps 3 are added）In EPA phosphatid ylcholines, be eventually adding PLD, the stirring reaction at 40 DEG C, add HCl, inactivator, reaction terminating with n-hexane-isopropyl alcohol extracting product, that is, obtain EPA phosphatidylserines；5）By step 2）Middle sea cucumber lipid CE, adds acetone, trimethylamine and acetic anhydride, after cold filtration, is washed with acetone It is de-, obtain EPA phosphatidylinositols.
- 6. application as claimed in claim 2, it is characterised in that the preparation method of the DHA phosphatide is comprised the following steps：1）After squid ovum is crushed through vacuum freeze drying, powder is made；2）Squid ovum dry powder is taken, is extracted overnight in chloroform and methyl alcohol mixed solution, after filtering, extracted with Klorvess Liquid, it is quiet Layering is put, chloroform layer is collected, through being concentrated under reduced pressure to give squid ovum lipid CE；3）After by the activated dress post of silica gel, silicagel column is balanced with chloroform；Again by step 2）The lipid of the squid ovum for obtaining slightly is carried After thing is dissolved with chloroform, it is poured slowly into silicagel column, is washed with chloroform, chloroform-methanol mixed liquor, acetone and methyl alcohol successively It is de-, collect eluent, eluent carries out thin-layer silicon offset plate chromatography, and concentrated under reduced pressure after being confirmed with iodine and Dittmer reagent colour developments Removal residual organic solvent is to obtain squid lecithin；Wherein, the eluent for collecting chloroform-methanol is DHA phosphatidyl-ethanolamines, and the eluent for collecting methyl alcohol is DHA phosphatidyl courages Alkali；4）Acetic acid/SAS buffer solution is put into heat collecting type constant-temperature heating magnetic stirring apparatus and is preheated, Serine is molten In wherein, above-mentioned steps 3 are added）In DHA phosphatid ylcholines, be eventually adding PLD, the stirring reaction at 40 DEG C, add HCl, inactivator, reaction terminating with n-hexane-isopropyl alcohol extracting product, that is, obtains DHA phosphatidylserines；5）By step 2）Middle squid ovum lipid CE, adds acetone, trimethylamine and acetic anhydride, after cold filtration, uses acetone Wash-out, obtains DHA phosphatidylinositols.
- 7. application as claimed in claim 2, it is characterised in that described improvement chronic fatigue syndrome product is food, raises Material or medicine；Wherein, total addition mass fraction of the EPA or DHA phosphatide is in 0.5%-2%.
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| CN108309991A (en) * | 2018-05-03 | 2018-07-24 | 中国海洋大学 | Application of the phosphatide rich in DHA in improving acute kidney injury product |
| CN111514152A (en) * | 2020-05-18 | 2020-08-11 | 中国海洋大学 | Application of n-3PUFA phosphatidyl glycoside in preparation for improving blood brain barrier damage |
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