CN107266601A - The preparation method and applications of one Polysaccharides From Laminaria Japonica chromic compound - Google Patents
The preparation method and applications of one Polysaccharides From Laminaria Japonica chromic compound Download PDFInfo
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- laminarin
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 42
- 150000004676 glycans Chemical class 0.000 title claims abstract description 23
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 17
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 241000015177 Saccharina japonica Species 0.000 title abstract 2
- 239000011651 chromium Substances 0.000 claims abstract description 37
- DBTMGCOVALSLOR-DEVYUCJPSA-N (2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](CO)O[C@H](O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-DEVYUCJPSA-N 0.000 claims abstract description 34
- 229920001543 Laminarin Polymers 0.000 claims abstract description 34
- 239000005717 Laminarin Substances 0.000 claims abstract description 32
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229910052804 chromium Inorganic materials 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 10
- 239000002955 immunomodulating agent Substances 0.000 claims abstract description 7
- 230000002584 immunomodulator Effects 0.000 claims abstract description 7
- 229940121354 immunomodulator Drugs 0.000 claims abstract description 7
- 230000003712 anti-aging effect Effects 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 41
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- 235000019441 ethanol Nutrition 0.000 claims description 17
- 238000001556 precipitation Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000000502 dialysis Methods 0.000 claims description 7
- 241000512259 Ascophyllum nodosum Species 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 229910021556 Chromium(III) chloride Inorganic materials 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 241001466453 Laminaria Species 0.000 claims description 4
- 102000004142 Trypsin Human genes 0.000 claims description 4
- 108090000631 Trypsin Proteins 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
- 239000011636 chromium(III) chloride Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 238000013019 agitation Methods 0.000 claims description 2
- 239000008346 aqueous phase Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 238000010612 desalination reaction Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- AZHSSKPUVBVXLK-UHFFFAOYSA-N ethane-1,1-diol Chemical compound CC(O)O AZHSSKPUVBVXLK-UHFFFAOYSA-N 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 238000009413 insulation Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 238000013517 stratification Methods 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims 1
- 230000003647 oxidation Effects 0.000 abstract description 7
- 238000007254 oxidation reaction Methods 0.000 abstract description 7
- 241001465754 Metazoa Species 0.000 abstract description 6
- 230000007365 immunoregulation Effects 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 5
- 235000013402 health food Nutrition 0.000 abstract description 5
- 230000000694 effects Effects 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 241000699670 Mus sp. Species 0.000 description 11
- 210000000952 spleen Anatomy 0.000 description 11
- 102000016938 Catalase Human genes 0.000 description 10
- 108010053835 Catalase Proteins 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 8
- 150000004804 polysaccharides Polymers 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000013522 chelant Substances 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 208000033065 inborn errors of immunity Diseases 0.000 description 5
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 5
- 101710088194 Dehydrogenase Proteins 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 102000019197 Superoxide Dismutase Human genes 0.000 description 4
- 108010012715 Superoxide dismutase Proteins 0.000 description 4
- 229910021645 metal ion Inorganic materials 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 210000004988 splenocyte Anatomy 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
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- 230000023852 carbohydrate metabolic process Effects 0.000 description 3
- QSWDMMVNRMROPK-UHFFFAOYSA-K chromium(3+) trichloride Chemical compound [Cl-].[Cl-].[Cl-].[Cr+3] QSWDMMVNRMROPK-UHFFFAOYSA-K 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 210000001541 thymus gland Anatomy 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- 229910014033 C-OH Inorganic materials 0.000 description 2
- 229910014570 C—OH Inorganic materials 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 2
- 239000011609 ammonium molybdate Substances 0.000 description 2
- 229940010552 ammonium molybdate Drugs 0.000 description 2
- 235000018660 ammonium molybdate Nutrition 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 230000037356 lipid metabolism Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 description 1
- LJHJYZOTLWDQCW-UHFFFAOYSA-N 2-[(2,3-dinitrophenyl)hydrazinylidene]propanoic acid Chemical compound OC(=O)C(C)=NNC1=CC=CC([N+]([O-])=O)=C1[N+]([O-])=O LJHJYZOTLWDQCW-UHFFFAOYSA-N 0.000 description 1
- 102000016918 Complement C3 Human genes 0.000 description 1
- 108010028780 Complement C3 Proteins 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 230000002180 anti-stress Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- ZCDOYSPFYFSLEW-UHFFFAOYSA-N chromate(2-) Chemical class [O-][Cr]([O-])(=O)=O ZCDOYSPFYFSLEW-UHFFFAOYSA-N 0.000 description 1
- 229960000359 chromic chloride Drugs 0.000 description 1
- 229910001430 chromium ion Inorganic materials 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
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- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-N hydroperoxyl Chemical compound O[O] OUUQCZGPVNCOIJ-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000000091 immunopotentiator Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
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- 241000894007 species Species 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
-
- 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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Materials Engineering (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Botany (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses the preparation method and applications of a Polysaccharides From Laminaria Japonica chromic compound, by laminarin after purification, self-assembling reaction forms complex to the laminarin chromic compound under certain condition with chromium, and is formed through isolating and purifying.Active animal experiment shows that laminarin chromic compound can improve the oxidation resistance and immunoregulation capability of mouse body.Thus available for preparation anti-aging, the medicine and health food of immunomodulator.
Description
Technical field
The invention belongs to field of medicaments, and in particular to the preparation method and applications of one Polysaccharides From Laminaria Japonica-chromic compound.
Background technology
Chromium is trace element necessary to normal carbohydrate and lipid metabolism, and trivalent chromium participates in glycometabolism, is to maintain
The indispensable element of glucose tolerance of animal and human normal, it is also carried in addition to reduction blood glucose with stronger
Height is immunized and anti-stress effect.It is to use inorganic chromate salt preparation that usual animal, which mends chromium, but can not reach expected effect.
Also there is enhancing to be immunized for sea-tangle and algal polysaccharides, antitumor action.More than the 80% of biological species is contained in ocean,
It is the huge repository of bio-diversity, sea-tangle is a kind of higher edible algae of distributed more widely, nutritive value, and China sea-tangle
Aboundresources.Research shows, laminarin (BSP) on it is normal and hypoimmunity mice immunologic function have significantly affect.
BSP determines each group mouse thymus, spleen index after intraperitoneal injection, and peripheral white blood cell, the T of spleen, B cell increase
Grow the change of ability, the splenocyte generation abilities of IL- 2, and serum and splenocyte haemolysis cellulose content etc..As a result BSP
100mg/ (kgd) × 10d can significantly improve hypoimmunity mice thymus gland, spleen index and peripheral white blood cell, moreover it is possible to carry
High normal mouse thymus gland, spleen index；It is obviously promoted T, B cell proliferation ability and the splenocyte production of normal and hypoimmunity mice spleen
Raw IL-2 abilities；BSP can also increase the content of normal and hypoimmunity mice serum and splenocyte hemolysin.Conclusion：BSP is one
Immunomodulator is planted, there is facilitation to normal and hypoimmunity mice immunologic function.
Organic ligand will be varied widely with its performance after metal ion formation chelate, and it not only can more preferably play original
There is the function of part and metal ion, and assign the properties such as its high bioavailability.May also have other functions, very
Many cancer therapy drug, antibiotic and metal-chelating and the synthesis application of polyferose also demonstrate this point.If in consideration of it, will
Very big improvement will also occur for laminarin and metal ion-chelant, its performance.It is fine because polysaccharide has abundant hydroxyl
Part, and it is good central ion that chromium, which is free 3d tracks, as long as condition will properly form stable chelate.This project
Plan chelates to form laminarin-chromium complex with laminarin and chromium, studies its effect for improving immunity, to develop new height
The small immunopotentiator of effect, safety, adverse reaction lays the foundation, and is that abundant development and utilization sea-tangle resource is offered reference.
The content of the invention
It is an object of the invention to provide the preparation method and applications of one Polysaccharides From Laminaria Japonica-chromic compound.
Laminarin-chromic compound of the present invention is to be gone kelp raw polysaccharide with Svage methods by enzymatic isolation method
Protein purification, by the laminarin after purification in different temperature, different pH value, different reaction time and Cr3+Self assembly
Reaction obtains laminarin-chromic compound.Detected through infrared spectrometer, laminarin is coordinated to form complex compound with chromium.Animal lives
Property experiment show that laminarin-chromic compound can improve the oxidation resistance and immunoregulation capability of mouse body, it is thus available
In preparation anti-aging, the medicine and health food of immunomodulator.
The preparation method and applications of laminarin-chromic compound of the present invention, comprise the following steps：
（1）Kelp raw polysaccharide is dissolved in water through removing protein, and -- alcohol precipitation -- dry, and obtains after purification by dialysis -- alcohol precipitation -- concentrated frozen
Laminarin；
（2）Purified laminarin is dissolved by heating in water.CrCl is slowly added under the conditions of certain pH3The aqueous solution is extremely
Untill just there is muddiness.Reaction solution is taken out, centrifugation takes supernatant, alcohol precipitation is centrifuged again, takes precipitation, with absolute ethyl alcohol, acetone
It is freeze-dried after washing, obtains the crude product of laminarin-chromic compound；
（3）Laminarin-chromic compound crude product is dissolved in suitable quantity of water, is freeze-dried, obtains after dialysis, alcohol precipitation, centrifugation
Laminarin-chromic compound after purification, the wherein content of Cr elements are 4.2wt%；
（4）The sample of dried over anhydrous and KBr are mixed, tabletting after grinding, with infrared spectrum analysis laminarin and laminarin-
The architectural difference of Cr complexs proves that laminarin and Cr have been coordinated.And applied to anti-oxidant and immunoregulation capability activity test
Research.
Laminarin-chromic compound is applied to the activity test research of oxidation resistance and immunoregulation capability, as a result
Display laminarin-chromic compound significantly improves the oxidation resistance and immunoregulation capability of mouse body.
Beneficial effects of the present invention：1st, laminarin-chromic compound not yet has synthesis and with raising mouse body at present
Oxidation resistance and immunoregulation capability activity research report, the present invention can be used for preparing anti-aging, immunomodulator
In medicine and health food.Its great advantage of this new compound is that laminarin is natural extracts, can medicine-food two-purpose；
It is to maintain animal and human normal physiological activity and chromium is trace element necessary to normal carbohydrate and lipid metabolism
Indispensable element, the two formed chelate after its performance vary widely, it not only can more preferably play original part with
The function of metal ion, part and metal ion can also produce synergy, significantly improve the oxidation resistance of body and are immunized
Regulating power, compared with existing similar drugs, nontoxic pair is small, has no adverse reaction, and can be used for a long time.2nd, it is anti-ageing available for preparing
Always, the medicine of immunomodulator, it can also be used to prepare health food.3rd, raw material is easy to get, and complex preparation technology is simple, inexpensive,
Convenient for production, Development volue is high.Can for exploitation Novel series efficiently, the small anti-aging of safety, adverse reaction, immunomodulator
Medicine and health food lay the foundation, and are that the research and development of active polysaccharide-metallo-chelate lay the foundation.
Brief description of the drawings
Fig. 1 is the infrared absorpting light spectra of laminarin；
Fig. 2 is the infrared absorpting light spectra of laminarin-Cr complexs；
Fig. 3 is index and spleen index comparison diagram before and after mouse feeding medicine laminarin, laminarin-Cr complexs.
Embodiment
First, embodiment 1
The purification of 1 kelp raw polysaccharide
The preparation of trypsin solution:1g trypsase is taken, with pH5.8 NaHPO3-NaH2PO3Buffer solution, precise volume setting is extremely
100ml, enzyme liquid final concentration of l0mg/ml, pH5.98.
Kelp raw polysaccharide liquid（10g→100ml）With trypsin solution with 1（100ml）：0.4 (40ml) ratio mixing, 37
0.2 (28ml) times of volume pH7.0 10wt%H is added after DEG C water-bath 5h2O2Reaction is stayed overnight, next day, and the sevage for adding 42ml is molten
Liquid, is placed on magnetic agitation instrument acutely concussion 20min, is fitted into after being demulsified in centrifuge tube with 4000r/min and pours into separatory funnel
Stratification, discards lower floor's organic phase and intermediate protein layer.Take again upper strata aqueous phase add 42ml sevage solution, repeat with
Upper operation is untill intermediate interface is without foreign protein.（2-3 times or so）
The absolute ethyl alcohol of two volumes amount is added in polysaccharide filtrate after deproteinized, centrifuge tube is poured into 3500 after standing 12 h
Precipitation is taken after r/min rotating speed centrifugation 10min, precipitation is respectively cleaned 3 times with 95wt% ethanol, absolute ethyl alcohol, acetone respectively.Alcohol
Filter residue adds appropriate distilled water after heavy washing, is moved it into bag filter and is clipped with dialysis clamp with 5ml liquid-transfering guns after fully redissolving
Two ends, put it into distilled water 24 h that dialyse.Dialysis finishes the ethanol alcohol precipitation that two volumes amount is added in backward liquid glucose, centrifugation
Take its sediment fraction to be put into freeze drier after equally being washed with ethanol and acetone to dry, it is pure to obtain laminarin after drying
Product, are fitted into measuring cup and place it in drier and save backup.
The preparation of 2 laminarins-chromic compound
The laminarins of 2.0 g after purification are taken to be dissolved in 100ml deionized waters, then 70 DEG C of stirring in water bath are added dropwise 1mol/L
CrCl3（It is approximately equivalent to 0.5g CrCl3）With 1mol/L NaOH, the pH9 of solution is maintained, muddy appearance has been dropped to, then be slightly added dropwise
Stop being added dropwise after several drops, then allow reaction solution to continue 70 DEG C of water-bath insulation 1h.Take out reaction solution, be cooled to after room temperature pour into from
8min is centrifuged with 6000r/min rotating speeds in heart pipe, supernatant is taken, after adding two volumes amount 95wt% ethanol for a period of time again
Centrifugation, this takes precipitation, precipitation to be freeze-dried after being washed with absolute ethyl alcohol, acetone, obtains the thick of laminarin-chromic compound
Product.
Load bag filter desalination after laminarin-chromic compound crude product is completely dissolved with a small amount of deionized water, dialyse 24h
Afterwards, take out the solution in bag filter and add the 95wt% ethanol of two volumes amount, place matches somebody with somebody laminarin-chromium for a period of time
Compound is centrifuged after fully separating out, and is collected precipitation, is washed with absolute ethyl alcohol, acetone, and finally freeze-drying obtains sea-tangle after purification
Polysaccharide-chromic compound, the wherein content of Cr elements are 4.2wt%.
3 laminarin chromic compound infrared spectrum analysis
Tabletting after laminarin-chromic compound of dried over anhydrous and KBr are mixed, ground, analyzes sea-tangle many with infrared spectrometer
The architectural difference of sugar and laminarin-Cr complexs, as a result as depicted in figs. 1 and 2.
1st, it can be obtained by the collection of illustrative plates of purifying laminarin：
3420.28cm-1, O-H or N-H stretching vibration；1635.67cm-1, C=O, C=NR, C=C, N=N, N=O stretching vibration；
1225.45cm-1, may be the stretching vibration of C-O keys in phenol；1072.65cm-1 、1033.37cm-1 May be C-O keys in alcohol
Stretching vibration.
2nd, the collection of illustrative plates of purifying laminarin-chromic compound can be obtained：
3436.63cm-1It is the typical absorption peak of hydroxyl, compared with purifying laminarin absworption peak, complex compound absorption band is omited
Micro- to broaden, less, wave number increases Strength Changes, slightly red shift, it may be possible to Cr3+Formed with laminarin mid-molecule point hydroxyl oxygen
Coordinate bond, absworption peak is moved to high wave number direction.1635.54cm-1Had no compared with purifying laminarin absworption peak much
Difference, is essentially coincided, therefore oxygen, nitrogen-atoms herein does not have and Cr3+Coordination.1072.65cm-1 、1033.37cm-1It is C-OH keys
Typical absorption peak, compared with purifying laminarin absworption peak, the absworption peak of complex compound is 1038.78cm in wave number-1 Occur,
And 1072.65cm-1 、1033.37cm-1Two peaks disappear, and may be the dissociation of the H in the C-OH keys in alcohol, C-O keys O is by coordination institute
Cause.
It can be seen that laminarin and Cr3+Complex reaction is there occurs, polysaccharide-chromium complex is formd.
The mouse immune regulation activity of 4 laminarins-chromic compound
4.1 animal packets and processing
This Setup Experiments blank group, laminarin group, inorganic chromium group, the high, medium and low dosage group of laminarin-Cr complexs, totally six
Individual group, every group of 10 mouse.Laminarin group dosage is 100mg/kg, inorganic chromium group dosage 10mg/kg, laminarin chromium
Low dose group dosage is 25mg/kg, and middle dose group dosage is 50mg/kg, and high dose group dosage is 100mg/kg.Daily
Gastric infusion 1 time, each 0.2ml, successive administration 9 days.
The measure of 4.2 immunocompetence indexs
This experiment collects blood by eyeball excise blood taking method, and 5000r/min centrifugations separation serum refrigeration in 10 minutes is standby, will try
Agent box each component takes out from box, balance to room temperature（18~25 DEG C）.Determination of erythrocyte superoxide dismutase activity uses xanthine oxidase
Chemical-enzyme method；The measuring principle of catalase is stopped for the reaction of catalase breaks hydrogen peroxide by adding ammonium molybdate,
Remaining hydrogen peroxide produces flaxen complex compound with ammonium molybdate, and its content is surveyed with this；Lactic acid dehydrogenase activity, which is determined, then to be led to
Cross using pyruvic acid and DNPH reaction generation pyruvic acid dinitrophenylhydrazone, it shows brownish red in the basic conditions,
Therefore can be tried to achieve by colorimetric method；Complement C_3, C4 activity then employ immunoturbidimetry to determine.
By kit measurement, kit is purchased from Nanjing and builds up biotechnology research institute, and operation is carried out by each specification.
The index and spleen index of 4.3 mouse
The index and spleen index experimental result of each experimental mice of table 1
As shown in Table 1：Polysaccharide-Cr high doses group, polysaccharide-Cr middle dose groups index and spleen index are than blank group, polysaccharide group, inorganic chromium
Group is high.Each dosage group index and spleen index of polysaccharide-Cr is higher than blank group, polysaccharide group.
4.4 superoxide dismutase（SOD）Assay
Table 2：Laminarin-Cr complexs are to mice serum superoxide dismutase（SOD）The influence of activity
As shown in Table 2：Each dosage group SOD contents of polysaccharide-Cr are higher than blank group, polysaccharide group and chromium group.
4.5 catalase（CAT）Vitality test
Table 3：Laminarin-Cr complexs are to mice serum catalase（CAT）The influence of vigor
As shown in Table 3：Each dosage group CAT vigor of polysaccharide-Cr is higher than blank, polysaccharide group and inorganic chromium group.
4.6 lactic dehydrogenase（LDH）Determination of activity
Table 4：Laminarin-Cr complexs are to mice serum lactic dehydrogenase（LDH）The influence of activity
As shown in Table 4：Polysaccharide-Cr is high, middle dose group LDH activity is higher than blank group, polysaccharide group and chromium group.
4.7 complement C3, complement C4Assay
Table 5：Laminarin-Cr complexs are to mice serum complement C3, complement C4The influence of assay
As shown in Table 5：Each dosage group C of polysaccharide-Cr3Content is lower than chromium group, polysaccharide group.C4Changes of contents regularity is poor, can
It can be experimental error or other reasonses, need further research.
5 conclusions
Method is combined by trypsase-sevage to purify laminarin, and refined laminarin is made.Laminarin and trivalent chromium
Ion is complexed into laminarin-Cr complexs under weak basic condition, using infrared spectrometer analysis product coordination structure；This reality
Test by superoxide dismutase in kit measurement mice serum（SOD）, lactic dehydrogenase（LDH）, catalase
（CAT）, complement C3And C4Activity, with the immunoregulatory activity of this Primary Study laminarin-Cr complex.As a result show, sea
Band polysaccharide is synthesized with chromium trichloride (1mol/L) under the conditions of pH value 9 or so, 70 DEG C of reaction temperature, and laminarin chromium is made.Exempt from
Epidemic disease regulation activity experiment finds that index and spleen index, CAT, SOD, LDH content are equal after mouse stomach administration laminarin-chromium complex
It is improved to some extent, illustrates that laminarin chromic compound can improve the oxidation resistance and immunological regulation energy of mouse body
Power.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (3)
1. the preparation method of one Polysaccharides From Laminaria Japonica-chromic compound, it is characterised in that：Laminarin is with chromium under certain condition from group
Reaction cartridge formation complex, specifically includes following steps：
（1）The purification of kelp raw polysaccharide
（A）The preparation of trypsin solution：1g trypsase is taken, with pH5.8 NaHPO3-NaH2PO3Buffer solution, it is accurate fixed
Hold to 100ml, enzyme liquid final concentration of l0mg/ml, pH5.98；
（B）Take after the trypsin solution mixing of kelp raw polysaccharide liquid and 40ml that 100ml concentration is 100mg/ml, 37 DEG C of water-bath 5h
Add 28ml pH7.0 10wt%H2O2Solution reaction is stayed overnight, next day, is added 42ml sevage solution, is placed on magnetic agitation
20min is acutely shaken on instrument, is fitted into after being demulsified in centrifuge tube with 4000r/min and pours into stratification in separatory funnel, discard down
Layer organic phase and intermediate protein layer, then take upper strata aqueous phase to add 42ml sevage solution, operation above is repeated until middle boundary
Untill face is without foreign protein；
（C）In step（B）In polysaccharide filtrate after deproteinized add two volumes amount absolute ethyl alcohol, stand 12 h after pour into from
Heart pipe after 3500 r/min rotating speed centrifugation 10min to take precipitation, and precipitation uses 95wt% ethanol, absolute ethyl alcohol, acetone respectively
Each to clean 3 times, filter residue adds appropriate distilled water after alcohol precipitation washing, is moved into after fully redissolving in bag filter, is put into dialysis in distilled water
24 h, dialysis finishes the ethanol alcohol precipitation that two volumes amount is added in backward liquid glucose, centrifuging and taking its sediment fraction equally with ethanol and
It is put into freeze drier and dries after acetone washing, laminarin sterling is obtained after drying；
（2）The preparation of laminarin-chromic compound
The laminarins of 2.0 g after purification are taken to be dissolved in 100ml deionized waters, 1mol/L is added dropwise in 70 DEG C of water-baths while stirring
CrCl3Solution, has dropped to muddy appearance, then i.e. stopping dropwise addition being added dropwise after 3-5 drops, the pH9 of solution is maintained during dropwise addition, then
Allow reaction solution to continue 70 DEG C of water-bath insulation 1h, take out reaction solution, be cooled to after room temperature and pour into centrifuge tube with 6000r/min rotating speeds
8min is centrifuged, supernatant is taken, two volumes amount 95wt% ethanol is added and is centrifuged again after for a period of time, take precipitation, precipitate with anhydrous
It is freeze-dried after ethanol, acetone washing, obtains the crude product of laminarin-chromic compound；
（3）The purifying of laminarin-chromic compound
Load after laminarin-chromic compound crude product is completely dissolved with a small amount of deionized water after bag filter desalination, dialysis 24h,
Take out the solution in bag filter and add the 95wt% ethanol of two volumes amount, place makes laminarin-chromic compound for a period of time
Centrifuged after fully separating out, collect precipitation, wash with absolute ethyl alcohol, acetone, be finally freeze-dried obtain laminarin after purification-
Chromic compound.
2. laminarin-chromic compound made from preparation method as claimed in claim 1, it is characterised in that：Sea after purification
Content with Cr elements in polysaccharide-chromic compound is 4.2wt%.
3. laminarin-chromic compound as claimed in claim 2 is preparing anti-aging, the medicine of immunomodulator and health care food
Application in product.
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