CN106954853A - A kind of grid algae water extract and preparation method and application - Google Patents
A kind of grid algae water extract and preparation method and application Download PDFInfo
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- 241000195493 Cryptophyta Species 0.000 title claims abstract description 123
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 82
- 239000000284 extract Substances 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 238000001556 precipitation Methods 0.000 claims abstract description 46
- 239000006228 supernatant Substances 0.000 claims abstract description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 35
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 14
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 14
- 235000006708 antioxidants Nutrition 0.000 claims abstract description 14
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 7
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 238000010438 heat treatment Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 16
- 239000000155 melt Substances 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims 1
- 241000244203 Caenorhabditis elegans Species 0.000 abstract description 23
- 230000036542 oxidative stress Effects 0.000 abstract description 17
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- 238000012360 testing method Methods 0.000 abstract description 10
- 235000013402 health food Nutrition 0.000 abstract description 8
- 238000001727 in vivo Methods 0.000 abstract description 7
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- 230000032683 aging Effects 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 abstract description 2
- 239000003960 organic solvent Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 11
- 210000002977 intracellular fluid Anatomy 0.000 description 8
- 239000000469 ethanolic extract Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 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 5
- 210000004027 cell Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000002604 ultrasonography Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 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
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- 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
- 238000005516 engineering process Methods 0.000 description 2
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- 244000144977 poultry Species 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 238000001291 vacuum drying Methods 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
- 241000195627 Chlamydomonadales Species 0.000 description 1
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 1
- 241000195633 Dunaliella salina Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000195663 Scenedesmus Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
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- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
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- 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
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
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- 230000000243 photosynthetic effect Effects 0.000 description 1
- 230000004962 physiological condition 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
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229940082787 spirulina Drugs 0.000 description 1
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- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 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
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
-
- 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- 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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Alternative & Traditional Medicine (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to field of marine biotechnology, there is provided a kind of grid algae water extract and preparation method and application.The preparation method of grid algae water extract of the present invention is that, by the fresh wet algae multigelation of grid algae, supernatant is removed in centrifugation；Precipitation plus ethanol is taken to carry out ultrasonic extraction, supernatant is removed in centrifugation；Precipitation is taken, add water carry out heating extraction after being washed with water, and produces.The preparation method that the present invention is provided is simple and easy to apply, and organic solvent consumption is few, is adapted to industrialized production.Tested in whole animal level with oxidative stress model and aging correlation model, result of the test shows that the grid algae water extract that the present invention is provided can extend the life span of Caenorhabditis elegans under oxidative stress conditions, improve survival rate, activity in vivo oxygen radical (ROS) removing can be promoted, reduce the level of activity in vivo oxygen radical (ROS), with significant anti-oxidant and anti-aging effects, can be applied to prepare has anti-oxidant and/or anti-senescence function health food and medicine.
Description
Technical field
The invention belongs to field of marine biotechnology, more particularly to a kind of grid algae water extract and preparation method thereof with should
With.
Background technology
Microalgae refers to could to distinguish the small algae group of its form under the microscope, be in water ecosystem it is main just
The level producer.Microalgae has great bio-diversity and genetic diversity, is adapted to various types of environmental conditions, generally
Exist with unicellular, many cells colony or elongated filamentous form, it is widely distributed in biosphere.About exist on the earth
The microalgae of 200000~800000 kinds of different classes, wherein about 35000 kinds have been described, but only sub-fraction microalgae exists at present
Related in scientific research, and it is then less that development and application is obtained in production practices.
Microalgae have it is nutritious, with great bio-diversity and genetic diversity, be not take up arable land, biomass
Greatly, strong environmental adaptability, growth cycle is short, can carry out photoautotrophy, photosynthetic efficiency using solar energy, carbon dioxide and water
High the features such as.With the progress of society and science and technology, global food, resource and energy shortage pressure increasingly increase, microalgae resource
Research become study hotspot.But in terms of the application study of major part microalgae all concentrates on efficient oil-producing at present, in medicine, protect
Application study in terms of health food is less, and it is several often to be concentrated mainly on spirulina, chlorella, Dunaliella salina and purple ball algae etc.
The microalgae seen, other microalgaes are in medicine, the application study in terms of health food then relatively fewer report.
Grid algae, also known as scenedemine, are a category of Chlorophyta Chlorococcale Shan Zao sections, generally by 4~8 cells or 16~32
Individual cell composition sizing colony, seldom to be unicellular.Cell is often ellipse or spindle, and 1~2 row or many are lined up with major axis
Row.Grid algae is common planktonic algae in fresh water, and pole happiness is bred in nutritious hydrostatic, and breeding is rapid, strong adaptability, from
Body Synthetic Oil ability is strong, and numerous species have in stronger patience, grid frustule to organic pollution containing abundant albumen
Matter and vitamin, are the good feeds of fish, are such as breeded fish using organic sewage oxidation pond, can obtain high yield, prolific frond
Also the feed of poultry can be made.But research and application at present to grid algae is concentrated mainly on sewage disposal, bioenergy and poultry
In terms of feed, the bioactivity research of its anti-oxidant aspect is then rarely reported.Therefore, exploitation grid algae has as health food
There is very big application prospect.
The content of the invention
To solve problems of the prior art, the invention provides a kind of grid algae water extract.What the present invention was provided
Grid algae water extract is by the way that by the fresh wet algae multigelation of grid algae, supernatant is removed in centrifugation；Take precipitation plus ethanol to carry out ultrasound to carry
Take, supernatant is removed in centrifugation；Precipitation is taken, the progress heating extraction that added water after being washed with water is made, with significant anti-oxidant and anti-aging
Effect, can be applied to prepare has anti-oxidant and/or anti-senescence function health food and medicine.
Technical scheme will be further described in detail below reflect and description.
A kind of grid algae water extract, its preparation method comprises the following steps：
S1, the fresh wet algae of grid algae is taken, be to freeze 12 hours under conditions of -20 DEG C~-40 DEG C in temperature, room temperature melts 12
Hour, multigelation 3~5 times centrifuges 5~15min, by supernatant under conditions of rotating speed is 5000r/min~10000r/min
Liquid and precipitation are separated, and take precipitation standby；Supernatant is grid algae 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, takes precipitation standby
With；
Take supernatant to be dried in vacuo, by the one-level water dissolves of the product after vacuum drying, produce the extraction of grid algae ethanol
Thing；
S3, precipitation 1~3 time described in step S2 is washed with one-stage water wash, be 5000r/min~10000r/min's in rotating speed
Under the conditions of centrifuge 5~15min, by supernatant and precipitation separate, remove supernatant, into precipitation add one-level water temperature be 70 DEG C
Extracted 3~5 times under conditions of~100 DEG C, 30~60min every time, under conditions of rotating speed is 5000r/min~10000r/min
5~15min is centrifuged, supernatant and precipitation are separated, supernatant is taken, produces grid algae water 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.
Preferably, add one-level water with the solid-to-liquid ratio 1g/mL of one-level water by wet algae in step S3 and carry out heating extraction.
Grid algae (Scenedesmus sp.) of the present invention is provided by Inst. of Hydrobiology, Chinese Academy of Sciences, and it is in
Numbering in the academy of sciences of state algae kind storehouse is FACHB-16.
Inventor has carried out substantial amounts of experiment during studying microalgae, has been surprisingly found that not only grid
Algae intracellular fluid and grid algae ethanol extract have significant anti-oxidant and anti-aging effects, are extracted grid algae intracellular fluid and grid algae second
Residue after alcohol extracting thing add water carry out the obtained grid algae water extract of heating extraction equally have it is significant anti-oxidant and anti-ageing
Old effect, and effect is more notable.
Tested in whole animal level with oxidative stress model and aging correlation model, result of the test shows this hair
The grid algae water extract of bright offer can significantly extend the life span of Caenorhabditis elegans under oxidative stress conditions, improve it and deposit
Motility rate, and effect during the final concentration of 80mgA/mL of grid algae water extract is significantly better than final concentration of 40mgA/mL, 120mgA/
Effect during mL.Grid algae water extract in Caenorhabditis elegans body ROS levels influence experiment result of the test show, this hair
The grid algae water extract of bright offer can promote activity in vivo oxygen radical (ROS) removing, significantly reduce activity in vivo oxygen free
The level of base (ROS).The grid algae water extract that the present invention is provided has significant anti-oxidant and anti-aging effects, can be applied to system
It is standby that there is anti-oxidant and/or anti-senescence function health food and medicine.
Grid algae water extract of the present invention can be shared individually or with other active components, should as solely or mainly active component
There is anti-oxidant and/or anti-senescence function health food and medicine for preparing, the formulation of the health food and medicine is excellent
Elect tablet, capsule, parenteral solution, oral liquid, granule, pill, powder and pill etc. as.
Anti-oxidant, anti-aging effects of the present invention refer to anti-because occurring oxidative stress under pathology or physiological condition
Change that should be produced 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 grid algae water extract that the present invention is provided can promote activity in vivo oxygen radical (ROS) removing, significantly drop
Low activity in vivo oxygen radical (ROS) level, with significant antioxidation.
(2) when the grid algae water extract that the present invention is provided can extend the existence of Caenorhabditis elegans under oxidative stress conditions
Between, its survival rate is improved, with significant anti-aging and antioxidation.
(3) preparation method for the grid algae water extract that the present invention is provided has the cycle short, and organic solvent consumption is few, technique letter
The features such as list, safety non-toxic, stability and high efficiency, it is adapted to industrialized production.
Brief description of the drawings
Influence of Fig. 1 grid algae water extracts to Caenorhabditis elegans survival rate under oxidative stress conditions.
Influence of Fig. 2 grid algae water extracts to ROS levels in Caenorhabditis elegans body.
Influence of Fig. 3 grid algae intracellular fluids to Caenorhabditis elegans survival rate under oxidative stress conditions.
Influence of Fig. 4 grid algae ethanol extracts to Caenorhabditis elegans average life span under natural conditions.
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, algae is provided grid by Inst. of Hydrobiology, Chinese Academy of Sciences, and it is in Chinese Academy of Sciences's fresh water
Numbering in algae kind storehouse is FACHB-16.
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 grid algae water extract of embodiment 1
S1, the fresh wet algae of grid algae is taken, be to freeze 12 hours under conditions of -20 DEG C in temperature, room temperature melts 12 hours, instead
Freeze thawing 5 times again, centrifuges 15min under conditions of rotating speed is 8000r/min, supernatant and precipitation is separated, take and precipitate 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 80%
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 30min every time, in rotating speed
To centrifuge 15min under conditions of 8000r/min, supernatant and precipitation are separated, take precipitation standby；
S3, precipitation 2 times described in step S2 is washed with one-stage water wash, centrifuged under conditions of rotating speed is 8000r/min
15min, supernatant and precipitation are separated, supernatant is removed, and one-level is added by wet algae and the solid-to-liquid ratio 1g/mL of one-level water into precipitation
Water is extracted 4 times under conditions of temperature is 100 DEG C, each 45min, and 15min is centrifuged under conditions of rotating speed is 8000r/min,
Supernatant and precipitation are separated, supernatant is taken, plus it is 1gA/mL that one-level water, which adjusts it relative to the concentration of grid algae (wet algae) weight,
(A represents the wet algae of grid algae), produces grid algae water extract.
In addition, step S1 supernatants isolated after centrifuging are taken, plus one-level water adjusts it relative to grid algae (wet algae) weight
The concentration of amount is 2gA/mL (A represents the wet algae of grid algae), produces grid algae intracellular fluid.
The supernatant for taking step S2 isolated after centrifuging is dried in vacuo, by the one-level water of the product after vacuum drying
Dissolving, the concentration for adjusting it relative to grid algae (wet algae) weight is 1gA/mL (A represents the wet algae of grid algae), produces the extraction of grid algae ethanol
Thing.
The preparation of the grid algae water extract of embodiment 2
S1, the fresh wet algae of grid algae is taken, be to freeze 12 hours under conditions of -30 DEG C in temperature, room temperature melts 12 hours, instead
Freeze thawing 4 times again, centrifuges 5min under conditions of rotating speed is 10000r/min, supernatant and precipitation is separated, take and precipitate 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 3 times, and the frequency of ultrasonic extraction is 35kHz, and temperature is 4 DEG C, and the time of ultrasound is 20min every time, in rotating speed
To centrifuge 10min under conditions of 10000r/min, supernatant and precipitation are separated, take precipitation standby；
S3, precipitation 3 times described in step S2 is washed with one-stage water wash, centrifuged under conditions of rotating speed is 10000r/min
15min, supernatant and precipitation are separated, supernatant is removed, and one-level is added by wet algae and the solid-to-liquid ratio 1g/mL of one-level water into precipitation
Water is extracted 3 times under conditions of temperature is 90 DEG C, each 30min, and 15min is centrifuged under conditions of rotating speed is 10000r/min,
Supernatant and precipitation are separated, supernatant is taken, plus it is 1gA/mL that one-level water, which adjusts it relative to the concentration of grid algae (wet algae) weight,
(A represents the wet algae of grid algae), produces grid algae water extract.
The preparation of the grid algae water extract of embodiment 3
S1, the fresh wet algae of grid algae is taken, be to freeze 12 hours under conditions of -40 DEG C in temperature, room temperature melts 12 hours, instead
Freeze thawing 3 times again, centrifuges 15min under conditions of rotating speed is 5000r/min, supernatant and precipitation is separated, take and precipitate 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 4 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 15min under conditions of 5000r/min, supernatant and precipitation are separated, take precipitation standby；
S3, precipitation 2 times described in step S2 is washed with one-stage water wash, centrifuged under conditions of rotating speed is 10000r/min
15min, supernatant and precipitation are separated, supernatant is removed, and one-level is added by wet algae and the solid-to-liquid ratio 1g/mL of one-level water into precipitation
Water is extracted 5 times under conditions of temperature is 70 DEG C, each 60min, and 10min is centrifuged under conditions of rotating speed is 10000r/min,
Supernatant and precipitation are separated, supernatant is taken, plus it is 1gA/mL that one-level water, which adjusts it relative to the concentration of grid algae (wet algae) weight,
(A represents the wet algae of grid algae), produces grid algae water extract.
Influence of the grid algae water 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.
Grid algae water extract made from Example 1, is added respectively by final concentration 40mgA/mL, 80mgA/mL, 120mgA/mL
The Wild-type C. elegans (N2) entered to adult initial stage (are protected by Univ Minnesota-Twin Cities USA's Caenorhabditis elegans hereditary information
Tibetan center is provided) in, control group adds isometric S Medium solution, is placed in after 20 DEG C of culture 24h, adds final concentration of
50mM paraquat carries out oxidative stress modeling, and the ratio of Caenorhabditis elegans survival is then counted every 12h, until its whole
It 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 as schemed
Shown in 1.
Fig. 1 results show that grid algae water extract can extend the life span of Caenorhabditis elegans under oxidative stress conditions,
Its survival rate is improved, and as the grid final concentration of 80mgA/mL of algae water extract, effect is the most notable.Result of the test shows the present invention
The grid algae water extract of offer can alleviate the early ageing phenomenon as caused by oxidative stress, make with significant anti-oxidant and anti-aging
With.
Influence of the grid algae water 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
Grid algae water extract made from L4 phases Wild-type C. elegans (N2) and embodiment 1 after stepization, grid algae water extract
Final concentration 100mgA/mL, control group add synchronize after L4 phases Wild-type C. elegans (N2) and with test group grid algae water
The isometric S Medium solution of extract, 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, the DCFH-DA probes that final concentration of 50 μM are added into supernatant (are purchased from Sigma public
Department, No. CAS：2044-85-1) solution, detects glimmering using fluorescence microplate reader under 485nm excitation wavelengths and 538nm launch wavelengths
Luminous intensity, 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 grid algae water extract, ROS levels in Caenorhabditis elegans body
It is remarkably decreased.Result of the test shows that the grid algae water extract that the present invention is provided can promote internal ROS removing, and reduction is internal
ROS levels, with significant oxidation resistance.
Influence of the grid algae intracellular fluid of embodiment 6 to Caenorhabditis elegans survival rate under oxidative stress conditions
Grid algae intracellular fluid made from Example 1, as at the beginning of final concentration 200mgA/mL, 600mgA/mL are added separately to adult
In the Wild-type C. elegans (N2) of phase, control group adds isometric S Medium solution, is placed in 20 DEG C of culture 24h
Afterwards, the paraquat for adding final concentration of 50mM carries out oxidative stress modeling, then counts Caenorhabditis elegans survival every 12h
Ratio, until it is all dead.The survival rate of Caenorhabditis elegans is represented with survivorship curve, is as a result entered with Kaplan-Meier methods
Row analysis.As a result it is as shown in Figure 3.
Fig. 3 results show that liquid energy enough extends the life span of Caenorhabditis elegans under oxidative stress conditions in grid gonidium, carries
Its high survival rate, and as the grid final concentration of 600mgA/mL of algae intracellular fluid, effect is the most notable.Result of the test shows that the present invention is carried
Liquid energy enough alleviates the early ageing phenomenon as caused by oxidative stress in the grid gonidium of confession, with significant anti-oxidant and anti-aging effects.
Influence of the grid algae ethanol extract of embodiment 7 to Caenorhabditis elegans average life span under natural conditions
Final concentration of 100mgA/mL embodiment is added into the L4 phases Wild-type C. elegans (N2) after synchronization
Grid algae ethanol extract made from 1, control group adds isometric S Medium solution, 20 DEG C of cultures is placed in, then every 1 day
The survival condition of Caenorhabditis elegans is counted, until it is all dead.The life-span of Caenorhabditis elegans is represented with survivorship curve, is tied
Fruit is analyzed with Kaplan-Meier methods.As a result it is as shown in Figure 4.
Fig. 4 results are shown, compared with control group, and grid algae ethanol extract mutually can extend beautiful hidden bar line under natural conditions
The average life span of worm.Result of the test shows that the grid algae ethanol extract that the present invention is provided has the effect of anti-aging process, i.e.
With significant anti-aging effects.
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 (6)
1. a kind of grid algae water extract, it is characterised in that the preparation method of the grid algae water extract comprises the following steps：
S1, the fresh wet algae of grid algae 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, and takes precipitation standby；
S3, precipitation 1~3 time described in step S2 is washed with one-stage water wash, in the condition that rotating speed is 5000r/min~10000r/min
5~15min of lower centrifugation, by supernatant and precipitation separate, remove supernatant, into precipitation add one-level water temperature be 70 DEG C~
Extracted 3~5 times under conditions of 100 DEG C, 30~60min every time, under conditions of rotating speed is 5000r/min~10000r/min from
5~15min of the heart, supernatant and precipitation are separated, supernatant is taken, produces grid algae water extract.
2. grid algae water extract according to claim 1, it is characterised in that the preparation method of the grid algae water extract
In, add ethanol by the solid-to-liquid ratio 1g/mL of wet algae and ethanol in step S2 and carry out ultrasonic extraction.
3. grid algae water extract according to claim 1, it is characterised in that the preparation method of the grid algae water extract
In, the frequency of ultrasonic extraction is 35~40kHz in step S2, and temperature is 4 DEG C.
4. grid algae water extract according to claim 1, it is characterised in that the preparation method of the grid algae water extract
In, the time of each ultrasonic extraction is 10~30min in step S2.
5. grid algae water extract according to claim 1, it is characterised in that the preparation method of the grid algae water extract
In, add one-level water with the solid-to-liquid ratio 1g/mL of one-level water by wet algae in step S3 and carry out heating extraction.
6. grid algae water extract according to claim 1 is being prepared with anti-oxidant and/or anti-senescence function health care food
Application in product and medicine.
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