CN106943437A - A kind of Microcystis aeruginosa ethanol extract and preparation method and application - Google Patents
A kind of Microcystis aeruginosa ethanol extract and preparation method and application Download PDFInfo
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- CN106943437A CN106943437A CN201710126577.0A CN201710126577A CN106943437A CN 106943437 A CN106943437 A CN 106943437A CN 201710126577 A CN201710126577 A CN 201710126577A CN 106943437 A CN106943437 A CN 106943437A
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- microcystis aeruginosa
- ethanol extract
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- ethanol
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- 241000192710 Microcystis aeruginosa Species 0.000 title claims abstract description 85
- 239000000469 ethanolic extract Substances 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 230000003064 anti-oxidating effect Effects 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 8
- 235000013402 health food Nutrition 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- 241000195493 Cryptophyta Species 0.000 claims description 23
- 239000006228 supernatant Substances 0.000 claims description 22
- 238000001556 precipitation Methods 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 11
- 238000002137 ultrasound extraction Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 5
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims 1
- 239000000155 melt Substances 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 230000036542 oxidative stress Effects 0.000 abstract description 14
- 229910052760 oxygen Inorganic materials 0.000 abstract description 14
- 239000001301 oxygen Substances 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 12
- 230000032683 aging Effects 0.000 abstract description 9
- 238000001727 in vivo Methods 0.000 abstract description 8
- 230000003712 anti-aging effect Effects 0.000 abstract description 6
- 239000003963 antioxidant agent Substances 0.000 abstract description 5
- 230000003078 antioxidant effect Effects 0.000 abstract description 5
- 235000006708 antioxidants Nutrition 0.000 abstract description 5
- 231100000252 nontoxic Toxicity 0.000 abstract description 3
- 230000003000 nontoxic effect Effects 0.000 abstract description 3
- 241000244203 Caenorhabditis elegans Species 0.000 description 28
- 210000002977 intracellular fluid Anatomy 0.000 description 21
- 238000012360 testing method Methods 0.000 description 16
- 239000000243 solution Substances 0.000 description 13
- -1 oxygen radical Chemical class 0.000 description 12
- 230000004083 survival effect Effects 0.000 description 12
- 230000003647 oxidation Effects 0.000 description 6
- 238000007254 oxidation reaction Methods 0.000 description 6
- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- SRUWWOSWHXIIIA-UKPGNTDSSA-N Cyanoginosin Chemical compound N1C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](C)[C@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C(=C)N(C)C(=O)CC[C@H](C(O)=O)N(C)C(=O)[C@@H](C)[C@@H]1\C=C\C(\C)=C\[C@H](C)[C@@H](O)CC1=CC=CC=C1 SRUWWOSWHXIIIA-UKPGNTDSSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 108010067094 microcystin Proteins 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 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 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
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- 230000005284 excitation Effects 0.000 description 2
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- 230000003834 intracellular effect Effects 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003094 microcapsule Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 235000016425 Arthrospira platensis Nutrition 0.000 description 1
- 240000002900 Arthrospira platensis Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 1
- 241000791981 Chroococcaceae Species 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000192701 Microcystis Species 0.000 description 1
- 241000192709 Microcystis sp. Species 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 230000010757 Reduction Activity Effects 0.000 description 1
- 241000244200 Rhabditida Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- BUIMWOLDCCGZKZ-UHFFFAOYSA-N n-hydroxynitramide Chemical compound ON[N+]([O-])=O BUIMWOLDCCGZKZ-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 229940082787 spirulina Drugs 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to field of marine biotechnology, there is provided a kind of Microcystis aeruginosa ethanol extract and preparation method and application.The Microcystis aeruginosa ethanol extract safety non-toxic that the present invention is provided, activity in vivo oxygen radical (ROS) removing can be promoted, reduce the level of activity in vivo oxygen radical (ROS), it can alleviate the early ageing phenomenon as caused by oxidative stress simultaneously, with significant anti-oxidant and anti-aging effects, it can be applied to prepare health food and medicine with anti-oxidation function.
Description
Technical field
The invention belongs to field of marine biotechnology, more particularly to a kind of Microcystis aeruginosa ethanol extract and preparation method thereof with
Using.
Background technology
The organism of the mankind is made up of all multielements, and the height of human body oxidation resistance level is constituted depending on these
Element and its composition.Such as selenium is the essential trace element of living organism, while be also antioxidant important in vivo, it is right
Maintaining health, anti-aging has important effect.Free radical is the freedom in the metabolite of human normal, organism
Base is mainly oxygen radical.Oxygen radical is that the class produced by oxygen receives electron deficiency in reduction has elevated chemical reaction
The oxy radical of activity, mainly has superoxide radical, hydroxyl radical free radical, singlet oxygen, peroxy radical and hyponitric acid free
Base etc..In in physiological conditions, body can remove internal oxygen radical by effective enzyme and non-enzyme system, so that at it
In physiology dynamic equilibrium.When there is certain factor to make, oxygen radical generation is excessive to exceed eliminating activity intravenousY or eliminating activity intravenousY
Weaken, internal oxygen free radicals is higher than equalization point, oxygen radical can be by making with DNA, protein and unrighted acid
With the peroxidating of DNA oxidative damages, protein cross and lipid being caused, so as to trigger inflammation, cause molecular degradation, body
Aging or the disease for suffering from aging correlation.Therefore, aid in improving body by screening the material with anti-oxidation characteristics
Oxidation resistance is to improve one of the oxidation resistance of body, direct, effective, convenient important method of anti-aging.
Microalgae be individually present in the water body environments such as a class Yu Haiyang, lake, river or into chain, bulk exist it is slender
Born of the same parents' microalgae.Microalgae has great bio-diversity, there are about 200000~800000 kinds of microalgaes, wherein about 35000 kinds
It is described, more than the 15000 kinds new compounds produced from algal biomass have carried out chemical identification.Microalgae produce and
There is the active component contained Important Economic to be worth and social value, in the side such as medicine, health food, feed, chemical industry and environmental protection
Face is widely used.But the microalgae category studied at present is narrower, and the application study of most of microalgae is concentrated mainly on
In terms of efficient oil-producing, the application study in terms of medicine, health food is concentrated mainly on spirulina, chlorella and purple ball at present
The several frequently seen microalgae such as algae, for other microalgaes, then relative reporter is less.
Microcystis kutz Cyanophyta Cyanophyceae Chroococcales Chroococcaceae, cell is spherical in shape, by most cell bags in colloid
It is middle to form irregular colony.Colony's tool is colourless, soft and has deliquescent glue quilt, and spherical in shape, Long Circle is in irregular shape netted
Or window-like, it is microcosmic or visually visible.((Microcystin, MC) is that Microcystis aeruginosa breaks out a kind of produced liver to Microcystin
Toxin, is the ring-type heptapeptide compound that a class has bioactivity, is capable of the activity of strong inhibition phosphoprotein phosphatase, is also simultaneously
Strong liver neoplasm accelerator.The research to Microcystis aeruginosa is concentrated mainly on Microcystin aspect at present, to its anti-oxidant side
The bioactivity research in face is then rarely reported.Therefore active development has the micro-capsule algae extract of antioxidation will be to aging phase
The prevention and treatment of related disorders are of great importance.
The content of the invention
To solve problems of the prior art, the invention provides a kind of Microcystis aeruginosa ethanol extract.The present invention is carried
The Microcystis aeruginosa ethanol extract of confession has significant antioxidation, can be applied to prepare the health food with anti-oxidation function
And medicine.
Technical scheme will be further described in detail below reflect and description.
A kind of Microcystis aeruginosa ethanol extract, its preparation method comprises the following steps：
S1, the wet algae of fresh Microcystis aeruginosa is taken, be to freeze 12 hours under conditions of -20 DEG C~-40 DEG C in temperature, room temperature is melted
12 hours, multigelation 3~5 times centrifuged 5~15min under conditions of rotating speed is 5000r/min~10000r/min, will be upper
Clear liquid and precipitation are separated, and take precipitation standby；Supernatant is Microcystis aeruginosa intracellular fluid；
S2, the EtOH Sonicate that addition volume fraction is 70%~99% into the precipitation described in step S1 are extracted 2~4 times,
5~15min is centrifuged under conditions of rotating speed is 5000r/min~10000r/min, supernatant and precipitation are separated, supernatant is taken
It is dried in vacuo, by the one-level water dissolves of the product after vacuum drying, produces Microcystis aeruginosa ethanol extract.
Preferably, add ethanol by the solid-to-liquid ratio 1g/mL of wet algae and ethanol in step S2 and carry out ultrasonic extraction.
Preferably, the frequency of ultrasonic extraction is 35~40kHz in step S2, and temperature is 4 DEG C.
Preferably, the time of each ultrasonic extraction is 10~30min in step S2.
Microcystis aeruginosa (Microcystis sp.) of the present invention is provided by Inst. of Hydrobiology, Chinese Academy of Sciences, its
Numbering in Chinese Academy of Sciences's algae kind storehouse is FACHB-1349.
The present inventor has carried out substantial amounts of experiment during studying microalgae, has been surprisingly found that Microcystis aeruginosa intracellular
Liquid and the Microcystis aeruginosa ethanol extract being made to extracting remaining residue progress EtOH Sonicate after Microcystis aeruginosa intracellular fluid to extract are equal
With significant antioxidation.
Tested in whole animal level with oxidative stress model and aging correlation model, result of the test shows this hair
The Microcystis aeruginosa ethanol extract of bright offer can significantly extend the life span of Caenorhabditis elegans under oxidative stress conditions, improve
Its survival rate；Meanwhile, it can promote activity in vivo oxygen radical (ROS) removing, significantly reduce activity in vivo oxygen radical
(ROS) level.The Microcystis aeruginosa ethanol extract that the present invention is provided has significant antioxidation, and process of the test does not find micro-
Capsule algae ethanol extract has toxic action to Caenorhabditis elegans, illustrates the Microcystis aeruginosa ethanol extract safety that the present invention is provided
It is nontoxic, it can be applied to prepare health food and medicine with anti-oxidation function.
Microcystis aeruginosa ethanol extract of the present invention can be shared individually or with other active components, as solely or mainly activity into
Divide and be applied to prepare health food and medicine with anti-oxidation function, the formulation of the health food and medicine is preferably piece
Agent, capsule, parenteral solution, oral liquid, granule, pill, powder and pill etc..
Antioxidation of the present invention refer to under pathology or physiological condition because occur response to oxidative stress produced
Raw change is the aging of principal character and the preventive and therapeutic effect of diseases associated with senescence.
Compared with prior art, the invention has the advantages that：
(1) the Microcystis aeruginosa ethanol extract that the present invention is provided can promote activity in vivo oxygen radical (ROS) removing, show
Reduction activity in vivo oxygen radical (ROS) level is write, with significant antioxidation.
(2) the Microcystis aeruginosa ethanol extract that the present invention is provided can extend the life of Caenorhabditis elegans under oxidative stress conditions
The time is deposited, its survival rate is improved, with significant anti-aging and antioxidation.
(3) preparation method for the Microcystis aeruginosa ethanol extract that the present invention is provided has the cycle short, and organic solvent consumption is few, work
Skill is simple, safety non-toxic, the features such as stability and high efficiency, is adapted to industrialized production.
Brief description of the drawings
Influence of Fig. 1 Microcystis aeruginosas ethanol extract to Caenorhabditis elegans survival rate under oxidative stress conditions.
Influence of Fig. 2 Microcystis aeruginosas ethanol extract to ROS levels in Caenorhabditis elegans body.
Influence of Fig. 3 Microcystis aeruginosas intracellular fluid to Caenorhabditis elegans survival rate under oxidative stress conditions.
Influence of Fig. 4 Microcystis aeruginosas intracellular fluid to ROS levels in Caenorhabditis elegans body.
Influence of Fig. 5 Microcystis aeruginosas intracellular fluid to SOD vigor in Caenorhabditis elegans body.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
Test method used in the embodiment of the present invention, unless otherwise specified, is conventional method；Used material
Material, reagent, unless otherwise specified, are the reagent and material commercially obtained.
In the embodiment of the present invention, Microcystis aeruginosa is provided by Inst. of Hydrobiology, Chinese Academy of Sciences, and it is light in the Chinese Academy of Sciences
Numbering in algae kind storehouse is FACHB-1349.
S Basal solution：Weigh 5.8gNaCl, 1.3g K2HPO4·3H2O and 6.0g KH2PO4It is placed in reagent bottle, plus
Enter 750mL deionized waters, be settled to 1L after being completely dissolved, sterilize 30min, be cooled to 40-50 DEG C it is standby.
S Medium solution：The S Basal solution after 1L sterilizings is taken, 1mL 5.0mg/mL cholesterol ethanol is added dropwise molten
Liquid, while add change to shake, makes it fully dissolve (solution switch to micro emulsion by water white transparency transparent in vain, no obvious sediment), then successively
Add 3mL 1M CaCl2Solution, 3mL 1M MgSO4, 10mL 1M potassium citrate solutions (pH=6.0) and 10mL trace elements are molten
Liquid, room temperature is standby.
The preparation of the Microcystis aeruginosa ethanol extract of embodiment 1
S1, the wet algae of fresh Microcystis aeruginosa is taken, is to freeze 12 hours under conditions of -30 DEG C in temperature, room temperature is melted 12 hours,
Multigelation 4 times, centrifuges 10min under conditions of rotating speed is 8000r/min, supernatant and precipitation is separated, take precipitation standby；
S2, to add volume fraction by the solid-to-liquid ratio 1g/mL of wet algae and ethanol into the precipitation described in step S1 be 85%
EtOH Sonicate is extracted 3 times, and the frequency of ultrasonic extraction is 40kHz, and temperature is 4 DEG C, and the time of ultrasound is 20min every time, in rotating speed
To centrifuge 10min under conditions of 8000r/min, supernatant and precipitation are separated, takes supernatant to be dried in vacuo, vacuum is done
Product after dry one-level water dissolves, it is that (A represents Microcystis aeruginosa to 1gA/mL to adjust it relative to the concentration of Microcystis aeruginosa (wet algae) weight
Wet algae), produce Microcystis aeruginosa ethanol extract.
In addition, taking step S1 supernatants isolated after centrifuging, plus one-level water to adjust it relative to Microcystis aeruginosa (wet algae)
The concentration of weight is 2gA/mL (A represents the wet algae of Microcystis aeruginosa), produces Microcystis aeruginosa intracellular fluid.
The preparation of the Microcystis aeruginosa ethanol extract of embodiment 2
S1, the wet algae of fresh Microcystis aeruginosa is taken, is to freeze 12 hours under conditions of -20 DEG C in temperature, room temperature is melted 12 hours,
Multigelation 5 times, centrifuges 8min under conditions of rotating speed is 10000r/min, supernatant and precipitation is separated, take precipitation standby；
S2, to add volume fraction by the solid-to-liquid ratio 1g/mL of wet algae and ethanol into the precipitation described in step S1 be 70%
EtOH Sonicate is extracted 3 times, and the frequency of ultrasonic extraction is 35kHz, and temperature is 4 DEG C, and the time of ultrasound is 30min every time, in rotating speed
To centrifuge 10min under conditions of 8000r/min, supernatant and precipitation are separated, takes supernatant to be dried in vacuo, vacuum is done
Product after dry one-level water dissolves, it is that (A represents Microcystis aeruginosa to 1gA/mL to adjust it relative to the concentration of Microcystis aeruginosa (wet algae) weight
Wet algae), produce Microcystis aeruginosa ethanol extract.
The preparation of the Microcystis aeruginosa ethanol extract of embodiment 3
S1, the wet algae of fresh Microcystis aeruginosa is taken, is to freeze 12 hours under conditions of -40 DEG C in temperature, room temperature is melted 12 hours,
Multigelation 3 times, centrifuges 15min under conditions of rotating speed is 8000r/min, supernatant and precipitation is separated, take precipitation standby；
S2, to add volume fraction by the solid-to-liquid ratio 1g/mL of wet algae and ethanol into the precipitation described in step S1 be 90%
EtOH Sonicate is extracted 4 times, and the frequency of ultrasonic extraction is 40kHz, and temperature is 4 DEG C, and the time of ultrasound is 15min every time, in rotating speed
To centrifuge 15min under conditions of 5000r/min, supernatant and precipitation are separated, takes supernatant to be dried in vacuo, vacuum is done
Product after dry one-level water dissolves, it is that (A represents Microcystis aeruginosa to 1gA/mL to adjust it relative to the concentration of Microcystis aeruginosa (wet algae) weight
Wet algae), produce Microcystis aeruginosa ethanol extract.
Influence of the Microcystis aeruginosa ethanol extract of embodiment 4 to Caenorhabditis elegans survival rate under oxidative stress conditions
Paraquat, scientific name methyl viologen is a kind of strong oxidizer, can induce a large amount of generation active oxygen radicals in vivo
(ROS) body is in oxidative stress environment, and then early ageing phenomenon occur.
Microcystis aeruginosa ethanol extract made from Example 1, the wild of adult initial stage is added to by final concentration 100mgA/mL
In type Caenorhabditis elegans (N2) (being provided by Univ Minnesota-Twin Cities USA's Caenorhabditis elegans hereditary information collection), control
Group adds isometric S Medium solution, is placed in after 20 DEG C of culture 24h, the paraquat for adding final concentration of 50mM is aoxidized
Modeling is coerced, the ratio of Caenorhabditis elegans survival is then counted every 12h, until it is all dead.Caenorhabditis elegans
Survival rate is represented with survivorship curve, is as a result analyzed with Kaplan-Meier methods.As a result it is as shown in Figure 1.
Fig. 1 results show that Microcystis aeruginosa ethanol extract can extend the existence of Caenorhabditis elegans under oxidative stress conditions
Time, improve its survival rate.Result of the test shows that the Microcystis aeruginosa ethanol extract that the present invention is provided can be alleviated by oxidative stress
Caused early ageing phenomenon, with significant anti-oxidant and anti-aging effects.
Influence of the Microcystis aeruginosa ethanol extract of embodiment 5 to ROS levels in Caenorhabditis elegans body
24 well culture plates are taken, if test group and control group, every group sets two holes, be 1mL per hole final volume, test group adds together
Microcystis aeruginosa ethanol extract, Microcystis aeruginosa ethanol made from L4 phases Wild-type C. elegans (N2) and embodiment 1 after stepization
The final concentration 100mgA/mL of extract, control group add synchronize after L4 phases Wild-type C. elegans (N2) and with experiment
The isometric S Medium solution of group Microcystis aeruginosa ethanol extract, is placed in after 20 DEG C of culture 24h, and paraquat is added per hole to dense eventually
Spend for 2mM, continue to cultivate after 48h, collect each group Caenorhabditis elegans, it is homogenized under condition of ice bath respectively, by homogenate
5min is centrifuged under the conditions of 4 DEG C, 10000g, supernatant is collected, final concentration of 50 μM of DCFH-DA probes are added into supernatant
(it is purchased from Sigma companies, No. CAS：2044-85-1) solution, is launched using fluorescence microplate reader in 485nm excitation wavelengths and 538nm
Fluorescence intensity under wavelength, room temperature detection 2h, is surveyed once per 10min.As a result F check analyses are used, as a result as shown in Figure 2.
Fig. 2 results are shown, compared with control group, after being fed through Microcystis aeruginosa ethanol extract, ROS in Caenorhabditis elegans body
Level is remarkably decreased.Result of the test shows that the Microcystis aeruginosa ethanol extract that the present invention is provided can promote internal ROS removing, drops
Low internal ROS levels, with significant oxidation resistance.
Influence of the Microcystis aeruginosa intracellular fluid of embodiment 6 to Caenorhabditis elegans survival rate under oxidative stress conditions
Microcystis aeruginosa intracellular fluid made from Example 1, the wild type show at adult initial stage is added to by final concentration 300mgA/mL
In beautiful hidden rhabditida (N2), control group adds isometric S Medium solution, is placed in after 20 DEG C of culture 24h, adds final concentration
Oxidative stress modeling is carried out for 50mM paraquat, the ratio of Caenorhabditis elegans survival is then counted every 12h, until its is complete
Portion is dead.The survival rate of Caenorhabditis elegans is represented with survivorship curve, is as a result analyzed with Kaplan-Meier methods.As a result such as
Shown in Fig. 3.
Fig. 3 results show that Microcystis aeruginosa intracellular fluid can extend the life span of Caenorhabditis elegans under oxidative stress conditions,
Improve its survival rate.Result of the test shows that the Microcystis aeruginosa intracellular fluid that the present invention is provided can alleviate the early ageing as caused by oxidative stress
Phenomenon, with significant anti-oxidant and anti-aging effects.
Influence of the Microcystis aeruginosa intracellular fluid of embodiment 7 to ROS levels in Caenorhabditis elegans body
24 well culture plates are taken, if test group and control group, every group sets two holes, be 1mL per hole final volume, test group adds together
Microcystis aeruginosa intracellular fluid made from L4 phases Wild-type C. elegans (N2) and embodiment 1 after stepization, Microcystis aeruginosa intracellular fluid
Final concentration 100mgA/mL, control group add synchronize after L4 phases Wild-type C. elegans (N2) and with test group Microcystis aeruginosa
The isometric S Medium solution of intracellular fluid, is placed in after 20 DEG C of culture 24h, and paraquat is added per hole to final concentration of 2mM, is continued
Cultivate after 48h, collect each group Caenorhabditis elegans, it is homogenized under condition of ice bath respectively, by homogenate in 4 DEG C, 10000g
Under the conditions of centrifuge 5min, collect supernatant, final concentration of 50 μM of DCFH-DA probe solutions added into supernatant, using glimmering
Light ELIASA fluorescence intensity under 485nm excitation wavelengths and 538nm launch wavelengths, room temperature detection 2h, one is surveyed per 10min
It is secondary.As a result F check analyses are used, as a result as shown in Figure 4.
Fig. 4 results are shown, compared with control group, after being fed through Microcystis aeruginosa intracellular fluid, ROS levels in Caenorhabditis elegans body
It is remarkably decreased.Result of the test shows that the Microcystis aeruginosa intracellular fluid that the present invention is provided can promote internal ROS removing, and reduction is internal
ROS levels, with significant oxidation resistance.
Influence of the Microcystis aeruginosa intracellular fluid of embodiment 8 to SOD vigor in Caenorhabditis elegans body
24 well culture plates are taken, if test group and control group, every group sets two holes, be 1mL per hole final volume, test group adds together
Microcystis aeruginosa intracellular fluid made from L4 phases Wild-type C. elegans (N2) and embodiment 1 after stepization, Microcystis aeruginosa intracellular fluid
Final concentration 100mgA/mL, control group add synchronize after L4 phases Wild-type C. elegans (N2) and with test group Microcystis aeruginosa
The isometric S Medium solution of intracellular fluid, is placed in after 20 DEG C of culture 72h, collects each group Caenorhabditis elegans, it is existed respectively
It is homogenized under condition of ice bath, homogenate is centrifuged into 5min under the conditions of 4 DEG C, 10000g, collect supernatant, is examined using total SOD activity
Test agent box (is purchased from green skies biotechnology and grinds make internal disorder or usurp institute, production code member：S0101) determine in Caenorhabditis elegans homogenate supernatant
SOD vigor.As a result it is as shown in Figure 5.
Fig. 5 results are shown, compared with control group, and Microcystis aeruginosa intracellular liquid energy significantly improves SOD in Caenorhabditis elegans body
Vigor.Result of the test shows that the Microcystis aeruginosa intracellular fluid that the present invention is provided can significantly improve internal SOD vigor, with significant
Oxidation resistance.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (5)
1. a kind of Microcystis aeruginosa ethanol extract, it is characterised in that the preparation method of the Microcystis aeruginosa ethanol extract includes as follows
Step：
S1, the wet algae of fresh Microcystis aeruginosa is taken, be to freeze 12 hours under conditions of -20 DEG C~-40 DEG C in temperature, it is small that room temperature melts 12
When, multigelation 3~5 times centrifuges 5~15min, by supernatant under conditions of rotating speed is 5000r/min~10000r/min
With precipitation separation, take precipitation standby；
S2, the EtOH Sonicate that addition volume fraction is 70%~99% into the precipitation described in step S1 are extracted 2~4 times, are being turned
Speed is 5~15min of centrifugation under conditions of 5000r/min~10000r/min, and supernatant and precipitation are separated, take supernatant to carry out
Vacuum drying, by the one-level water dissolves of the product after vacuum drying, produces Microcystis aeruginosa ethanol extract.
2. Microcystis aeruginosa ethanol extract according to claim 1, it is characterised in that the system of the Microcystis aeruginosa ethanol extract
Ethanol is added by the solid-to-liquid ratio 1g/mL of wet algae and ethanol carry out ultrasonic extraction in Preparation Method, in step S2.
3. Microcystis aeruginosa ethanol extract according to claim 1, it is characterised in that the system of the Microcystis aeruginosa ethanol extract
In Preparation Method, the frequency of ultrasonic extraction is 35~40kHz in step S2, and temperature is 4 DEG C.
4. Microcystis aeruginosa ethanol extract according to claim 1, it is characterised in that the system of the Microcystis aeruginosa ethanol extract
In Preparation Method, the time of each ultrasonic extraction is 10~30min in step S2.
5. Microcystis aeruginosa ethanol extract according to claim 1 is preparing health food and medicine with anti-oxidation function
In application.
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