CN105853342A - Use of polyphenol extract from spirulina as tyrosinase inhibitor - Google Patents
Use of polyphenol extract from spirulina as tyrosinase inhibitor Download PDFInfo
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- CN105853342A CN105853342A CN201610326240.XA CN201610326240A CN105853342A CN 105853342 A CN105853342 A CN 105853342A CN 201610326240 A CN201610326240 A CN 201610326240A CN 105853342 A CN105853342 A CN 105853342A
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- spirulina
- polyphenol
- extract
- polyphenol extract
- tyrosinase inhibitor
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- 235000016425 Arthrospira platensis Nutrition 0.000 title claims abstract description 86
- 240000002900 Arthrospira platensis Species 0.000 title claims abstract description 86
- 229940082787 spirulina Drugs 0.000 title claims abstract description 86
- 239000000284 extract Substances 0.000 title claims abstract description 79
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 79
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 77
- 101710147108 Tyrosinase inhibitor Proteins 0.000 title claims abstract description 20
- 230000000694 effects Effects 0.000 claims abstract description 20
- 238000004108 freeze drying Methods 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims description 30
- 229940074391 gallic acid Drugs 0.000 claims description 15
- 235000004515 gallic acid Nutrition 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 15
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 12
- 230000001629 suppression Effects 0.000 claims description 12
- 241000195493 Cryptophyta Species 0.000 claims description 8
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- 239000000203 mixture Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 241001593750 Turcica Species 0.000 claims description 4
- 238000002481 ethanol extraction Methods 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 230000002829 reductive effect Effects 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 208000037259 Amyloid Plaque Diseases 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims description 2
- 102000003425 Tyrosinase Human genes 0.000 abstract description 8
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- 238000011160 research Methods 0.000 abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 6
- 230000002087 whitening effect Effects 0.000 abstract description 6
- 235000012055 fruits and vegetables Nutrition 0.000 abstract description 4
- 239000004480 active ingredient Substances 0.000 abstract 2
- 235000019441 ethanol Nutrition 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 239000007787 solid Substances 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 32
- 108090000790 Enzymes Proteins 0.000 description 32
- 229940088598 enzyme Drugs 0.000 description 32
- 239000000758 substrate Substances 0.000 description 14
- 230000005764 inhibitory process Effects 0.000 description 13
- 239000003814 drug Substances 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 8
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 6
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 239000002994 raw material Substances 0.000 description 6
- XKZQKPRCPNGNFR-UHFFFAOYSA-N 2-(3-hydroxyphenyl)phenol Chemical compound OC1=CC=CC(C=2C(=CC=CC=2)O)=C1 XKZQKPRCPNGNFR-UHFFFAOYSA-N 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 241000199919 Phaeophyceae Species 0.000 description 4
- 241000195474 Sargassum Species 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 241000192700 Cyanobacteria Species 0.000 description 3
- 241000218628 Ginkgo Species 0.000 description 3
- 235000011201 Ginkgo Nutrition 0.000 description 3
- 235000008100 Ginkgo biloba Nutrition 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- -1 polyphenol compounds Chemical class 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000206588 Gracilaria tenuistipitata Species 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 241000631640 Ishige okamurae Species 0.000 description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000206572 Rhodophyta Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000003064 anti-oxidating effect Effects 0.000 description 2
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical class O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 229960004502 levodopa Drugs 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
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- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 description 1
- 241001212149 Cathetus Species 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 108010053210 Phycocyanin Proteins 0.000 description 1
- 235000011158 Prunus mume Nutrition 0.000 description 1
- 244000018795 Prunus mume Species 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 241000593522 Sargassum thunbergii Species 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 241000893983 Ulva linza Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
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- 230000003078 antioxidant effect Effects 0.000 description 1
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- 150000001558 benzoic acid derivatives Chemical class 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000000640 hydroxylating effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- BEJNERDRQOWKJM-UHFFFAOYSA-N kojic acid Chemical class OCC1=CC(=O)C(O)=CO1 BEJNERDRQOWKJM-UHFFFAOYSA-N 0.000 description 1
- 229960004705 kojic acid Drugs 0.000 description 1
- WZNJWVWKTVETCG-UHFFFAOYSA-N kojic acid Natural products OC(=O)C(N)CN1C=CC(=O)C(O)=C1 WZNJWVWKTVETCG-UHFFFAOYSA-N 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000008099 melanin synthesis Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Chemical class CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 235000003724 spirulina extract Nutrition 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- 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
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Tropical Medicine & Parasitology (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses the use of a polyphenol extract from spirulina as a tyrosinase inhibitor, and the polyphenol extract from spirulina is used as one of active ingredients or the only active ingredient for preparation of the tyrosinase inhibitor. The method of preparing the polyphenol extract from spirulina comprises the following steps: ethyl alcohol with volume concentration of 80% is used to extract spirulina, spirulina polyphenolic is obtained and then decompressed and concentrated at 40 DEG C, and solid polyphenol extract from spirulina is obtained after freeze-drying. The polyphenol extracted from spirulina for the first time can inhibit the activity of tyrosinase, and can not only provide reliable theoretical basis for the application of polyphenol in such fields as skin whitening, medical treatment and preservation of fruits and vegetables, but also provide a new course towards comprehensive use of spirulina resources and research on the safe and economical tyrosinase inhibitor.
Description
Technical field
The present invention relates to the technical field of biological preparation, particularly relate to the purposes as tyrosinase inhibitor of the polyphenol extract in a kind of spirulina.
Background technology
Seeweed polyphenol be a kind of main from cyanophyceae, chlorella, red algae and Brown algae four Large Marine Ecosystem algae separation and Extraction there is antibacterial, antioxidation, anti-swollen
The general name of the extensive bioactive polyphenol compounds such as tumor, antiviral and blood pressure lowering.Iwai have studied brown algae polyphenols as a kind of natural origin
After phlorose enzyme inhibitor is taken in by mice, in Mice Body, blood sugar level is controlled, and can effectively prevent the generation of diabetes；Lu Hongyu et al. leads to
Cross extraction and hyperfiltration technique extracts 4 kinds of polyphenol extracts from full edge Alga Sgrgassi Enerves, and test the oxidation resistance of 4 kinds of extracts and to sw579
The impact of cell proliferation, the total antioxidant capacity of result display brown algae polyphenols extract and molecular size range negative correlation, the suppression to tumor cell is made
With with concentration positive correlation；The research such as Wei Yuxi finds that sargassum thunbergii polyphenol can disturb the activity of thrombin and reduce Ca inside and outside platelet2+Transport,
Anticoagulant effect is notable.Research shows, seeweed polyphenol exists researching value in food and medicine field.
Tryrosinase energy hydroxylating list phenol substrate and oxidation diphenol substrate, be a kind of active center oxidoreductase of containing double-core copper ion.Black
Element builds up and fruit and vegerable brown stain, skin splash and melanoma can be caused to be formed, and tyrosinase activity size is with Melanin productions in organism
Closely related.Typical tyrosinase inhibitor has kojic acid, polyphenol compound, benzaldehyde and benzoate analog derivative etc., extensively should
For studying fruit and vegetable fresh-keeping agent, white-making ingredient and biological insecticides.Spirulina is a kind of ancient filamentous cyanobacteria, rich in phycocyanin, spiral shell
Rotation algae polyphenol, micromolecular polysaccharide isoreactivity composition.China's spirulina aboundresources, is spirulina raw material big producing country, but China's spirulina is main
Simply utilize, and active component Exploitation Depth is inadequate.
In view of this, the present inventor's research and the polyphenol extract that devises in a kind of spirulina as the purposes of tyrosinase inhibitor, this case by
This produces.
Summary of the invention
It is an object of the invention to provide the purposes as tyrosinase inhibitor of the polyphenol extract in a kind of spirulina, carry from spirulina raw material
Take out spirulina polyphenol, and it is carried out tyrosinase inhibition test, provide experimental basis to the exploitation for spirulina resource, with
Time work out a kind of natural origin economy, safety tyrosinase inhibitor.
To achieve these goals, the present invention solves its technical problem and is adopted the technical scheme that:
A kind of polyphenol extract in spirulina as the purposes of tyrosinase inhibitor, using spirulina polyphenol extract as one of active component or
Unique active component is used for preparing tyrosinase inhibitor.
As the optimal way of embodiment, using spirulina polyphenol extract as one of active component or unique active component for anti-senile plaque or
The antistaling agent of suppression food brown stain.
As the optimal way of embodiment, the preparation method of described spirulina polyphenol extract: the ethanol extraction spiral using volumetric concentration to be 80%
Algae, obtains spirulina polyphenolic extract, then by spirulina polyphenolic extract in 40 DEG C of concentrating under reduced pressure, the spirulina obtaining solid-state after lyophilization is many
Phenol extraction thing.
As the optimal way of embodiment, the assay method of spirulina polyphenol content: use spectrophotography, with forint phenol reagent in extract
Polyphenol colour developing, determine polyphenol content: ultra-pure water dissolves 0.110g gallic acid, use 100mL volumetric flask constant volume, obtain gallic acid standard
Stock solution；10,20,30,40 and 50 μ g mL it are diluted to respectively again with Galla Turcica (Galla Helepensis) gallic acid Standard Stock solutions-1Serial solution, with
Ultra-pure water does blank, takes 1.0mL diluent respectively and is placed in color comparison tube, adds 5.0mL forint phenol solution, reacts 6min, adds 4.0
ML 7.5%Na2CO3Solution, mixing, after lucifuge places 1h, under wavelength 765nm, measure light absorption value, make standard curve；Spirulina
Polyphenol content is according to standard curve colorimetric determination.
The polyphenol that the present invention extracts first from the spirulina suppression to tyrosinase activity, will be applied to skin-whitening for it, medical treatment, fruit and vegerable are protected
The field such as fresh provides reliable theoretical foundation, also will provide new for comprehensive utilization spirulina resource and research safety, economic tyrosinase inhibitor
Approach.
Accompanying drawing explanation
Fig. 1 is the standard curve of gallic acid of the present invention；
Fig. 2 is the spirulina polyphenol extract of the present invention inhibitory action to tryrosinase diphenol enzyme activity；
Fig. 3 is the spirulina polyphenol extract of the present invention mensuration to tryrosinase diphenol enzyme level mechanism；Wherein, the spiral corresponding to straight line 1-5
The concentration of algae polyphenol is respectively 0,0.67,1.33,2.00 and 2.67mg mL-1；
Fig. 4 is that spirulina polyphenol extract of the present invention is to tryrosinase diphenol enzyme level type and the mensuration of inhibition constant；Wherein, straight line 1-5 institute
The concentration of corresponding spirulina polyphenol is respectively 0,0.67,1.33,2.00 and 2.67mg mL-1。
Detailed description of the invention
1 materials and methods
1.1 material
1.1.1 reagent is provided by god health product company limited of Fujian Province six with medicine Spirulina powder；Forint phenol reagent, gallic acid, tryrosinase, purchase
From Sigma company；Natrium carbonicum calcinatum, purchased from Shantou Xilong Chemical Factory；Ethanol, the experiment material such as levodopa is purchased from the examination of Shanghai traditional Chinese medicines chemistry
Agent company limited.
1.1.2 instrument ultraviolet spectrophotometer, GBC instrument company of Australia；Thermostat water bath, Hong Ke instrument plant of Jintan City of Jiangsu Province；FE20K
Acidometer, Shanghai prunus mume (sieb.) sieb.et zucc. Teller-torr benefit Instrument Ltd.；Vacuum freeze drier, Zhengzhou instrument and equipment tomorrow company limited.
1.2 method
1.2.1 the extraction of spirulina polyphenol is with Spirulina powder as raw material, uses 80% ethanol extraction aldehydes matter, then by spirulina polyphenolic extract in
40 DEG C of concentrating under reduced pressure, obtain the spirulina polyphenol extract of solid-state after lyophilization.
1.2.2 the mensuration of spirulina polyphenol content uses spectrophotography, develops the color the polyphenol in extract with forint phenol reagent, determines polyphenol content.
Ultra-pure water dissolves 0.110g gallic acid, 100mL volumetric flask constant volume, obtains gallic acid Standard Stock solutions.Again with Galla Turcica (Galla Helepensis) Galla Turcica (Galla Helepensis) acidity scale
Quasi-stock solution is diluted to 10,20,30,40 and 50 μ g mL respectively-1Serial solution, do blank with ultra-pure water, take 1.0mL respectively
Diluent is placed in color comparison tube, adds 5.0mL forint phenol solution, reacts 6min, adds 4.0mL 7.5%Na2CO3Solution, mixing,
After lucifuge places 1h, under wavelength 765nm, measure light absorption value, make standard curve.Spirulina polyphenol content is according to standard curve colorimetric determination.
1.2.3 spirulina polyphenol extract is to inhibition to enzyme of the inhibitory action extract of tryrosinase two phenolase: by immobilized substrate concentration and enzyme
Concentration, measures the relative surplus enzyme under different effect substrate concentration and lives.Spirulina polyphenol extract is dissolved in dimethyl sulfoxide (DMSO), makes not
Effector solution with concentration.0.5mmol·L-1L-DOPA substrate solution and 2.89 μ g mL-1Enzyme liquid all uses 50mmol L-1PH 6.8 phosphoric acid
Buffer.In 3mL reaction system, it is initially charged 0.1mL effector solution, adds 2.8mL in advance 30 DEG C of waters bath with thermostatic control
The substrate solution of 10min, is eventually adding 0.1mL enzyme liquid, quickly mixes.Setting mensuration wavelength as 475nm, the light measuring reaction system is close
Angle value is with the growth straight line in response time, extinction coefficient epsilon=3700L mol-1·cm-1, by enzyme activity, calculate semi-inhibit rate IC50Value.
The extract inhibiting mechanism to enzyme: by immobilized substrate concentration, changes enzyme amount, measures the enzyme activity under different effect substrate concentration.Containing 2.8
mL 0.5mmol·L-1In the survey live body system of L-DOPA substrate, measuring enzyme amount is 1.15,1.73,2.31,2.89 and 3.46 μ g mL-1Time, no
With the reaction rate of enzyme under concentration spirulina polyphenol extract effect, it is judged that the extract mechanism of action to enzyme.
Extract is to the suppression type of enzyme, inhibition constant: by immobilized enzyme concentration, changes the amount of substrate, measures the enzyme under different effect substrate concentration
Vigor.Fixing enzyme amount is 2.89 μ g mL-1, it is respectively 0 in spirulina polyphenol extract concentration, 0.67,1.33,2.00 and 2.67mg mL-1
Survey live body system in, measuring L-DOPA concentration is 0.3,0.4,0.5,0.6 and 0.7mmol L-1Time enzyme reaction speed.Pass through
Lineweave-Burk double-reciprocal plot, calculates and compares kinetic parameter KmAnd Vm, it is judged that suppression type.
2 results and analysis
2.1 total phenol content in spirulina polyphenol extract
Folin-Ciocalteus reaction normal curve such as Fig. 1 of gallic acid.Gallic acid mass concentration is at 0～50 μ g mL-1Time, light absorption value
A765Good linear relationship is had with concentration of standard solution.The equation of linear regression of gallic acid standard working curve is Y=0.00123X-0.0086, phase
Close coefficients R2=0.9996 (Y is light absorption value, and X is gallic acid concentration).Standard working curve formula according to gallic acid and spirulina solution
Light absorption value under 765nm wavelength, show that the polyphenol content in spirulina extract is 35.05mg g-1。
The impact on tryrosinase vigor of the 2.2 spirulina polyphenol extracts
Fixing L-DOPA concentration and enzyme concentration, measure variable concentrations spirulina polyphenol extract to Inhibition of Tyrosinase Activity.As shown in Figure 2, cheese ammonia
The reaction rate of acid enzyme declines along with the increase of spirulina polyphenol concentration, illustrates that spirulina polyphenol extract has inhibitory action to tryrosinase vigor,
Semi-inhibit rate IC50For 6.23mg mL-1。
The 2.3 spirulina polyphenol extracts inhibiting mechanism to tryrosinase
Immobilized substrate L-DOPA concentration, changes and surveys enzyme amount in live body system, measures the relation of enzyme amount and enzyme activity.From the figure 3, it may be seen that along with spiral
The increase of algae polyphenol extract concentration, straight slope reduces successively, and straight-line intersection is initial point, illustrates that spirulina polyphenol extract is to tryrosinase
Process of inhibition reversible, effective enzyme amount in the addition not minimizing system of spirulina polyphenol extract, but the enzyme activity of tryrosinase be suppressed, its
Catalytic efficiency reduces.
2.4 spirulina polyphenol extracts are to the suppression type of tryrosinase and inhibition constant
Immobilized enzyme concentration, changes the amount of substrate, measures the impact on enzyme activity of the variable concentrations spirulina polyphenol extract.As shown in Fig. 4 (a), with
When extract acts on tryrosinase, the reaction rate of enzyme and DOPA concentration make Lineweaver-Burk double reciprocal plot, obtain intersection point and are positioned at second
One group of straight line of quadrant.This shows, along with the rising of spirulina polyphenol extract concentration, KmValue becomes big, VmValue diminishes, and its suppression type is
Mixing competitive type.Spirulina polyphenol extract is made Fig. 4 (b), Fig. 4 (c) by slope and vertical axis intercept with Fig. 4 (a) cathetus, obtains inhibition constant KI
And KISIt is respectively 1.94mg mL-1With 6.83mg mL-1。
2.5 Sargassum extracts and the comparison to tyrosinase inhibitory action of the Chinese medicine whitening composition
Cyanophyceae (spirulina), chlorella (Enteromorpha linza), red algae (true Gracilaria tenuistipitata) and Brown algae (Ishige okamurae Yendo) and Chinese medicine (Folium Ginkgo, Radix Glycyrrhizae) carry
Take thing and the enzyme kinetics parameter of tryrosinase is shown in Table 1.As shown in Table 1, the extract of six kinds of separate sources is to the process of inhibition of tryrosinase all
Reversible.By comparing semi-inhibit rate IC50Value, can obtain six kinds of extracts and be followed successively by the inhibition of tryrosinase: Ishige okamurae Yendo extract > edge pipe
Entermorpha extract > Radix Glycyrrhizae extract > spirulina polyphenol extract > Folium Ginkgo extract > true Gracilaria tenuistipitata extract.
Table 1 natural extract is to tryrosinase diphenol enzyme level effectiveness comparison
Table 1Comparison of inhitory effect of natural extracts on diphenolase of tyrosinase
3 discuss
Melanin deposition can cause skin splash and fruit and vegerable brown stain, and its growing amount is relevant with the activity of tryrosinase.Separation and Extraction from natural plants
Go out safe and efficient tyrosinase inhibitor and be applied to develop the product such as preserving fruit and vegetable utilizing, cosmetics whitening and be always study hotspot, but about sea
The research report that tryrosinase is suppressed by algae active component is less.
Rich in multiple nutritional components and bioactive substance in spirulina, because there is removing toxic substances, strengthening the merits such as immune, antibacterial, antioxidation, antitumor
Imitate and enjoy favor.Research spirulina-active composition becomes new focus for development function food, cosmetics and medicine.China is through recent decades
Development become spirulina raw material big producing country of the world, but the spirulina raw material of China is main to be simply utilized as, and product finishing stage is inadequate.
Herein with Spirulina powder as raw material, utilize ethanol to extract polyphenols from Spirulina powder, study its suppression to tryrosinase vigor
Effect, and compare Sargassum active component and the inhibition of Chinese medicine whitening composition.Knowable to result of study, the spirulina of 80% ethanol extraction is many
In phenol extraction thing, spirulina polyphenol content is relatively low, and polyphenol content is 35.05mg g-1；Spirulina polyphenol extract has inhibitory action to tryrosinase,
Semi-inhibit rate (IC to two phenolase50) it is 6.29mg mL-1.Extract is reversible to the suppression of tryrosinase two phenolase, shows that spirulina polyphenol carries
Take thing and can reduce enzyme catalysis efficiency, and do not reduce effective enzyme amount.Spirulina polyphenol extract is mixed type to enzyme level type, inhibition constant KI=
1.94mg·mL-1、KIS=6.83mg mL-1, show that spirulina polyphenol extract can be combined with resolvase and enzyme-substrate complex simultaneously, and with trip
Higher from the binding ability of enzyme；The relatively kinetic parameter of four kinds of Sargassum extracts and Chinese medicine whitening composition understands, Sargassum extract suppression tyrosine
Enzymatic activity, and the Chinese medicine extract whitening agent (Radix Glycyrrhizae, Folium Ginkgo extract) that the inhibition of extracting section thing is better than on market, this is for continuing to grind
Study carefully other active component in spirulina and the inhibitory action of tryrosinase is provided possibility.
The suppression to tyrosinase activity of the spirulina polyphenol extract, the fields such as skin-whitening, medical treatment, preserving fruit and vegetable utilizing that will be applied to for it provide can
The theoretical foundation leaned on, also will provide new way for comprehensive utilization spirulina resource and research safety, economic tyrosinase inhibitor.
The above, only present pre-ferred embodiments, therefore the scope that the present invention implements can not be limited according to this, i.e. according to the scope of the claims of the present invention
And the equivalence change made of description with modify, all should still belong in the range of the present invention contains.
Claims (4)
1. the polyphenol extract in a spirulina is as the purposes of tyrosinase inhibitor, it is characterised in that: using spirulina polyphenol extract as activity
One of composition or unique active component are used for preparing tyrosinase inhibitor.
Polyphenol extract in a kind of spirulina the most as claimed in claim 1 is as the purposes of tyrosinase inhibitor, it is characterised in that: with spirulina
Polyphenol extract is used for anti-senile plaque or the antistaling agent of suppression food brown stain as one of active component or unique active component.
Polyphenol extract in a kind of spirulina the most as claimed in claim 1 is as the purposes of tyrosinase inhibitor, it is characterised in that: described spiral
The preparation method of algae polyphenol extract: the ethanol extraction spirulina using volumetric concentration to be 80%, obtains spirulina polyphenolic extract, then by spiral
Algae polyphenolic extract, in 40 DEG C of concentrating under reduced pressure, obtains the spirulina polyphenol extract of solid-state after lyophilization.
Polyphenol extract in a kind of spirulina the most as claimed in claim 1 is as the purposes of tyrosinase inhibitor, it is characterised in that: spirulina is many
The assay method of phenol content: use spectrophotography, develops the color to the polyphenol in extract with forint phenol reagent, determines polyphenol content: ultrapure water-soluble
Solve 0.110g gallic acid, use 100mL volumetric flask constant volume, obtain gallic acid Standard Stock solutions；Store up by Galla Turcica (Galla Helepensis) gallic acid standard again
Standby solution is diluted to 10,20,30,40 and 50 μ g mL respectively-1Serial solution, do blank with ultra-pure water, take 1.0mL respectively dilute
Release liquid to be placed in color comparison tube, add 5.0mL forint phenol solution, react 6min, add 4.0mL 7.5%Na2CO3Solution, mixing, keeps away
After light places 1h, under wavelength 765nm, measure light absorption value, make standard curve；Spirulina polyphenol content is according to standard curve colorimetric determination.
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| CN111944165A (en) * | 2020-07-15 | 2020-11-17 | 汕头大学 | Polyphenol tyrosinase inhibitor and extraction method and application thereof |
| CN114469836A (en) * | 2022-03-03 | 2022-05-13 | 广州市沐家健康产业有限公司 | Whitening preparation containing honey enzyme and seaweed polyphenol as well as preparation method and application of whitening preparation |
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| CN111944165B (en) * | 2020-07-15 | 2022-04-01 | 汕头大学 | Polyphenol tyrosinase inhibitor and extraction method and application thereof |
| CN114469836A (en) * | 2022-03-03 | 2022-05-13 | 广州市沐家健康产业有限公司 | Whitening preparation containing honey enzyme and seaweed polyphenol as well as preparation method and application of whitening preparation |
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