CN109627201A - A method of extracting phycocyanobilin - Google Patents
A method of extracting phycocyanobilin Download PDFInfo
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- CN109627201A CN109627201A CN201910030734.7A CN201910030734A CN109627201A CN 109627201 A CN109627201 A CN 109627201A CN 201910030734 A CN201910030734 A CN 201910030734A CN 109627201 A CN109627201 A CN 109627201A
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- INPDFIMLLXXDOQ-UHFFFAOYSA-N Phycocyanobilin Natural products CCC1=C(C)C(=CC2=NC(=C/c3[nH]c(C=C/4C(C(C(N4)=O)C)=CC)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O INPDFIMLLXXDOQ-UHFFFAOYSA-N 0.000 title claims abstract description 80
- 108010072011 phycocyanobilin Proteins 0.000 title claims abstract description 77
- NNMALANKTSRILL-ZUTFDUMMSA-N 3-[(2z,5z)-2-[[3-(2-carboxyethyl)-5-[(z)-[(3z,4r)-3-ethylidene-4-methyl-5-oxopyrrolidin-2-ylidene]methyl]-4-methyl-1h-pyrrol-2-yl]methylidene]-5-[(4-ethyl-3-methyl-5-oxopyrrol-2-yl)methylidene]-4-methylpyrrol-3-yl]propanoic acid Chemical compound O=C1C(CC)=C(C)C(\C=C/2C(=C(CCC(O)=O)C(=C/C3=C(C(C)=C(\C=C/4\C(\[C@@H](C)C(=O)N\4)=C/C)N3)CCC(O)=O)/N\2)C)=N1 NNMALANKTSRILL-ZUTFDUMMSA-N 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 33
- 239000000047 product Substances 0.000 claims abstract description 115
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 56
- 239000007788 liquid Substances 0.000 claims abstract description 56
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 40
- 238000002156 mixing Methods 0.000 claims abstract description 31
- 239000006228 supernatant Substances 0.000 claims abstract description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 108010053210 Phycocyanin Proteins 0.000 claims abstract description 22
- 239000000843 powder Substances 0.000 claims abstract description 19
- 238000010992 reflux Methods 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000001035 drying Methods 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- 239000000243 solution Substances 0.000 claims description 36
- 239000012528 membrane Substances 0.000 claims description 22
- 239000000706 filtrate Substances 0.000 claims description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 230000002378 acidificating effect Effects 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 150000001298 alcohols Chemical class 0.000 claims 2
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 10
- 241000588724 Escherichia coli Species 0.000 abstract description 8
- 235000016425 Arthrospira platensis Nutrition 0.000 abstract description 7
- 240000002900 Arthrospira platensis Species 0.000 abstract description 7
- 229940082787 spirulina Drugs 0.000 abstract description 7
- 230000014509 gene expression Effects 0.000 abstract description 4
- 235000011167 hydrochloric acid Nutrition 0.000 description 20
- 235000019441 ethanol Nutrition 0.000 description 19
- 229910021642 ultra pure water Inorganic materials 0.000 description 11
- 239000012498 ultrapure water Substances 0.000 description 11
- 239000011888 foil Substances 0.000 description 9
- 230000000873 masking effect Effects 0.000 description 9
- 241000195493 Cryptophyta Species 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000000703 high-speed centrifugation Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 4
- 229960001231 choline Drugs 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical class N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 3
- 239000000460 chlorine Substances 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000004792 oxidative damage Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 102000006410 Apoproteins Human genes 0.000 description 2
- 108010083590 Apoproteins Proteins 0.000 description 2
- 241001062009 Indigofera Species 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 235000021466 carotenoid Nutrition 0.000 description 2
- 150000001747 carotenoids Chemical class 0.000 description 2
- 229930002875 chlorophyll Natural products 0.000 description 2
- 235000019804 chlorophyll Nutrition 0.000 description 2
- 239000001752 chlorophylls and chlorophyllins Substances 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 101150075668 pcyA gene Proteins 0.000 description 2
- 230000029553 photosynthesis Effects 0.000 description 2
- 238000010672 photosynthesis Methods 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 101100083210 Cyanophora paradoxa cpcA gene Proteins 0.000 description 1
- 108010074122 Ferredoxins Proteins 0.000 description 1
- 208000009139 Gilbert Disease Diseases 0.000 description 1
- 208000022412 Gilbert syndrome Diseases 0.000 description 1
- 241000406668 Loxodonta cyclotis Species 0.000 description 1
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 241000206572 Rhodophyta Species 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 108010004469 allophycocyanin Proteins 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- -1 phycocyanobilin Heme Chemical class 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/36—Oxygen or sulfur atoms
- C07D207/38—2-Pyrrolones
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A method of extracting phycocyanobilin, comprising the following steps: alcohol solution is added into phycocyanin powder, is protected from light and is heated to reflux, obtains the first product；First product is centrifugated, supernatant is taken, obtains the second product；It will be rotated after the filtering of second product, obtain third product；It is centrifuged after third product is filtered, takes supernatant, chloroform and water is added, stood, collected lower liquid, obtain the 4th product；Hydrochloric acid solution is added into the 4th product, then collects lower liquid, obtains the 5th product；Hydrochloric acid solution is added toward the 5th product, stands after mixing, then collects lower liquid, obtain the 6th product；By the chloroform drying in the 6th product, phycocyanobilin is obtained.Compared with naturally extracting phycocyanobilin from spirulina, the method for said extracted phycocyanobilin, the period is short, and technique is simpler.Compared with fermenting and producing phycocyanobilin in the Escherichia coli from expression phycocyanobilin, the method for said extracted phycocyanobilin improves the extraction efficiency of phycocyanobilin.
Description
Technical field
The present invention relates to marine organisms medical material tech field more particularly to a kind of methods for extracting phycocyanobilin.
Background technique
Phycocyanobilin (Phycocyanobilin, PCB) is to be present in one of red algae and cyanobacteria photosynthesis complementary colors
Element.It seemingly with bilirubinoid, is to be connected by methene in straight chain by 4 pyrrole rings, molecular weight is about 588.69g/mol.They
Referred to as phycocyanin (phycocyanin) or allophycocyanin in conjunction with soluble protein in vivo, are dissolved in dilute salting liquid.
They play absorption and transmitting luminous energy in photosynthesis.
As the derivative of bilirubin, phycocyanobilin has been demonstrated there is anti-oxidant, scavenging hydroxyl and oxygen at present
Free radical protects biomembrane from multiple efficacies such as oxidative damage and anti-inflammatory.Current research is demonstrate,proved it has been found that phycocyanobilin exists
It can be restored into the cell by ferroheme reductase and be converted into bilirubin, and then feedback inhibition NADPH as caused by oxidative damage
The overexpression of oxidizing ferment plays good treatment for diseases as caused by oxidative damage such as diabetes, Gilbert syndromes
Effect.Food medicine office, the U.S. (FDA) approved is by phycocyanin (phycocyanobilin and apoprotein are combined together) for skin
The photosensitive treatment of skin cancer, breast cancer.Photosensitive treatment also patent applied for of the phycocyanin for atherosclerosis simultaneously.Simultaneously
Phycocyanobilin can be used as fluorescence probe and natural pigment, have wide use in terms of biological detection and functional food preparation
On the way.
It is naturally extracted from spirulina and specifically includes that the culture of frustule, is collected by centrifugation, the broken and phycocyanobilin of frond
Extracting, purification etc..Wherein the isolation and purification method of phycocyanobilin mainly has: two step extraction process of hot methanol cracking process and methanol.
Hot methanol cracking process is the thioether bond slowly opened between phycocyanobilin and apoprotein using hot methanol (70-75 DEG C), from
And it extracts and obtains 2/3 or so free phycocyanobilin.But it is a variety of due to also containing Chlorophylls and Carotenoids etc. in spirulina
Pigment, this method often can only obtain the not high phycocyanobilin of purity.And two step extraction process of methanol be first with cold ethanolic extraction,
Most Chlorophylls and Carotenoids are removed, phycocyanobilin does not dissolve in cold ethyl alcohol at this time, and then reusable heat methanol extracts, this
Sample can obtain the phycocyanobilin of purity higher (80% or so).The phycocyanobilin of higher purity needs further thin-layer chromatography
Or preparative high-efficient liquid phase chromatogram purification can obtain.
Fermentation is extracted phycocyanobilin and is specifically included that by cloning in algae in recombination bacillus coli by expressing phycocyanobilin
Heme oxidase gene ho1 and ferredoxin oxide-reductase gene pcyA, by above-mentioned ho1 gene and pcyA gene
(phycocyanobilin synthesis key gene) is cloned on expression vector, is then imported in Escherichia coli and is carried out heterogenous expression, is produced
Phycocyanobilin is recombinated, then that the Escherichia coli after conversion are broken, chloroform extraction and gel permeation chromatography obtain the recombination
Phycocyanobilin.
However, traditional extracting cycle natural from spirulina is longer, higher cost and technique is more complex, and from recombination
The phycocyanobilin amount of fermenting and producing is very little in Escherichia coli, is not able to satisfy some requirement of experiment.
Summary of the invention
In consideration of it, it is necessary to provide a kind of method for extracting phycocyanobilin, the method for the extraction phycocyanobilin relative to from
Natural extracting cycle is shorter in spirulina, and technique is relatively simple, compared to the algae of the fermenting and producing from recombination bacillus coli
The amount of lifting of the method for blue Choline, phycocyanobilin is higher.
A method of extracting phycocyanobilin, comprising the following steps:
Alcohol solution is added into phycocyanin powder, after mixing, is protected from light and is heated to reflux 5h-10h, obtains the first production
Object；
First product is centrifugated under conditions of temperature is 4 DEG C, the first supernatant is taken, obtains the second product；
Second product is filtered, filtrate is rotated to 5mL-20mL, third product is obtained；
The third product is filtered, filtrate is centrifuged, takes the second supernatant, chloroform and water is added, is uniformly mixed
After stand, collect the first lower liquid, obtain the 4th product；
Hydrochloric acid solution is added into the 4th product, stands after mixing, then collects the second lower liquid, obtains the 5th
Product；
The hydrochloric acid solution is added toward the 5th product, stands after mixing, then collects third lower liquid, obtains
To the 6th product；
By the chloroform drying in the 6th product, the phycocyanobilin is obtained.
In one embodiment, alcohol solution is added into phycocyanin powder, after mixing, is protected from light and is heated to reflux
5h-10h, in the step of obtaining the first product, the mass volume ratio of the phycocyanin powder and methanol is 1:50g/mL-1:10g/
mL。
In one embodiment, alcohol solution is added into phycocyanin powder, after mixing, is protected from light and is heated to reflux
5h-10h, in the step of obtaining the first product, being protected from light the temperature being heated to reflux is 70 DEG C -80 DEG C.
In one embodiment, first product is centrifugated under conditions of temperature is 4 DEG C, takes the first supernatant
Liquid, in the step of obtaining the second product, the revolving speed of centrifuge separation is 8000 × g-13000 × g, and the time of centrifugation is 5min-
20min。
In one embodiment, second product is filtered, filtrate is rotated to 5mL-20mL, third product is obtained
In step, second product is filtered using 0.45 μm of filter membrane and 20mL syringe.
In one embodiment, the third product is filtered, filtrate is centrifuged, take the second supernatant, chlorine is added
In the step of imitative and water, stands after mixing, collects the first lower liquid, obtains four products, the third product is used
0.45 μm of filter membrane and the filtering of 20mL syringe.
In one embodiment, the third product is filtered, filtrate is centrifuged, take the second supernatant, chlorine is added
In the step of imitative and water, stands after mixing, collects the first lower liquid, obtains four products, second supernatant, chlorine
Imitative and water volume ratio is 1:1:2-1:2:4.
In one embodiment, the hydrochloric acid solution is added toward the 5th product, stands, then collects after mixing
Third lower liquid in the step of obtaining six products, collects lower liquid using the 50mL centrifuge tube of masking foil package.
It in one embodiment, will in the step of chloroform in the 6th product being dried up, obtaining the phycocyanobilin
6th product is deposited in the container packed tightly with sealed membrane, and sealed membrane is offered several holes, is passed through nitrogen and is dried up chloroform.
In one embodiment, the alcohol solution is that methanol, ethyl alcohol, acidic methanol, methanol aqueous solution or ethyl alcohol are water-soluble
Liquid.
Compared with naturally extracting phycocyanobilin from spirulina, the method for said extracted phycocyanobilin passes through past algae indigo plant egg
It is added after alcohol solution is protected from light and is heated to reflux, then is centrifugated in white powder end, after taking the first supernatant liquid filtering, mistake again after revolving
Filter, is then centrifuged for separating, takes the second supernatant, and after chloroform and water is added, and collects the first lower liquid, and hydrochloric acid solution is added
Afterwards, the second lower liquid is collected, collects third lower liquid after hydrochloric acid solution is added again, then algae is can be obtained into chloroform drying
Blue Choline, the period is short, and technique is relatively simple and instant experiment effect i.e. may be implemented.
Compared with fermenting and producing phycocyanobilin in the Escherichia coli from expression phycocyanobilin, said extracted phycocyanobilin
Method improves the extraction efficiency of phycocyanobilin.
Detailed description of the invention
Fig. 1 is the flow chart of the method for the extraction phycocyanobilin of an embodiment；
Fig. 2 is the uv-visible absorption spectroscopy of the phycocyanobilin extracted；
Fig. 3 is the fluorescence emission spectrum of the phycocyanobilin extracted.
Specific embodiment
In order to be more clear the objectives, technical solutions, and advantages of the present invention, with reference to the accompanying drawings and embodiments, to this hair
It is bright to be further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and do not have to
It is of the invention in limiting.It is described in the present invention to be fixedly connected, including be directly fixedly connected and fix indirectly.
Referring to FIG. 1, the method for the extraction phycocyanobilin of an embodiment, comprising the following steps:
S10, alcohol solution is added into phycocyanin powder, after mixing, is protected from light and is heated to reflux 5h-10h, obtain
One product.
Wherein, phycocyanin powder is deposited in -27 DEG C of refrigerator.
In one embodiment, the mass volume ratio of phycocyanin powder and alcohol solution can be 1:50g/mL-1:10g/
mL。
In one embodiment, alcohol solution be methanol, ethyl alcohol, acidic methanol, methanol aqueous solution or ethanol water or
The alcohol solution of other various concentration.Alcohol solution and phycocyanin reaction, alcohol solution are used for the cracking of phycocyanin.
In one embodiment, phycocyanin powder and alcohol solution are mixed using being vortexed.
In one embodiment, being protected from light the temperature being heated to reflux can be 70 DEG C -80 DEG C.
In one embodiment, the device being heated to reflux is encased using masking foil and is protected from light.
S20, the first product is centrifugated under conditions of temperature is 4 DEG C, takes the first supernatant, obtains the second product.
In one embodiment, the revolving speed of centrifuge separation can be 8000 × g-13000 × g, and the time of centrifugation can be
5min-20min。
It in one embodiment, further include the revolving speed 8000 by the first supernatant under conditions of temperature is 4 DEG C in S20
× g-13000 × g, the operation of centrifugation time 5min-20min take third supernatant to get the second product.
S30, the second product is filtered, filtrate is rotated to 5mL-20mL, third product is obtained.
In one embodiment, the second product is filtered using 0.45 μm of filter membrane and 20mL syringe.
In one embodiment, revolving is rotated using Rotary Evaporators.
S40, third product is filtered, filtrate is centrifuged, take the second supernatant, chloroform and water is added, be uniformly mixed
After stand, collect the first lower liquid, obtain the 4th product.
In one embodiment, third product is filtered using 0.45 μm of filter membrane and 20mL syringe.
In one embodiment, the volume ratio of the second supernatant, chloroform and water is 1:1:2-1:2:4.
In one embodiment, time of repose 5min-20min.
S50, hydrochloric acid solution is added into the 4th product, stands after mixing, then collect the second lower liquid, obtain
5th product.
In one embodiment, hydrochloric acid solution is the mixed solution of 100 μ L concentrated hydrochloric acids and 10mL ultrapure water.
In S50, hydrochloric acid solution, which is added, can not only stablize phycocyanobilin, but also can play demulsification.
In one embodiment, the time stood after mixing is 5min.
S60, hydrochloric acid solution is added toward the 5th product, stands after mixing, then collect third lower liquid, obtain the
Six products.
In one embodiment, hydrochloric acid solution is the mixed solution of 100 μ L concentrated hydrochloric acids and 10mL ultrapure water.
In one embodiment, third lower liquid is collected using the 50mL centrifuge tube of masking foil package.
S70, the chloroform in the 6th product is dried up, obtains phycocyanobilin.
In one embodiment, the 6th product is deposited in the container packed tightly with sealed membrane, if sealed membrane is offered
Dry hole is passed through nitrogen and dries up chloroform.Specifically, sealed membrane can be pricked to be formed using needle.
After phycocyanobilin is prepared in S70, it is placed in -80 DEG C of ultra low temperature freezers and saves.
Compared with naturally extracting phycocyanobilin from spirulina, the method for said extracted phycocyanobilin passes through past algae indigo plant egg
It is added after alcohol solution is protected from light and is heated to reflux, then is centrifugated in white powder end, after taking the first supernatant liquid filtering, mistake again after revolving
Filter, is then centrifuged for separating, takes the second supernatant, and after chloroform and water is added, and collects the first lower liquid, and hydrochloric acid solution is added
Afterwards, the second lower liquid is collected, collects third lower liquid after hydrochloric acid solution is added again, then algae is can be obtained into chloroform drying
Blue Choline, the period is short, and technique is relatively simple and instant experiment effect i.e. may be implemented.
Compared with fermenting and producing phycocyanobilin in the Escherichia coli from expression phycocyanobilin, said extracted phycocyanobilin
Method improves the extraction efficiency of phycocyanobilin.
It is below specific embodiment part.
Embodiment 1
(1) 4g phycocyanin powder is taken out from -27 DEG C of refrigerators, 100mL acidic methanol is added, be vortexed and mix, then turn
Enter in methanol eddy device, device is encased with masking foil, and 78 DEG C of water-bath 8h, end of reaction obtains the first product.
(2) by the first product at 4 DEG C, high speed centrifugation 20min under the conditions of 13000 × g.Take the first supernatant, similarity condition
Under be centrifuged again primary, obtain the second product.
(3) 0.45 μm of filter membrane of the second product and 20mL syringe are filtered, filtrate is steamed with Rotary Evaporators are outstanding, until remaining
About 10mL liquid, obtains third product.
(4) third product is used again 0.45 μm of filter membrane and 20mL syringe to filter, filtrate is at 4 DEG C, 13000 × g condition
Lower high speed centrifugation 20min.The second supernatant is taken, 10mL chloroform and 20mL ultrapure water is added, is transferred to 125mL separatory funnel, sufficiently
Mixing stands 10min, collects the first lower liquid and obtains the 4th product.
(5) mixing liquid including 100 μ L concentrated hydrochloric acids and 10mL ultrapure water is added into the 4th product, is transferred to liquid separation leakage
Bucket, stands 5min after being sufficiently mixed, and collects the second lower liquid with clean 50mL beaker, obtains the 5th product.
(6) mixing liquid including 100 μ L concentrated hydrochloric acids and 10mL ultrapure water is added toward the 5th product, is transferred to separatory funnel,
5min is stood after being sufficiently mixed, and third lower liquid is then collected using the centrifuge tube that clean drying 50mL masking foil wraps up,
Obtain the 6th product.
(7) centrifugation nozzle is packed tightly with sealed membrane, needle bundle opens several holes, then passes to nitrogen and dries up chloroform, obtains algae
Blue Choline.Then gained phycocyanobilin is placed in -80 DEG C of ultra low temperature freezers and is saved.
In embodiment 1,2g phycobniliprotein powder obtains 100mgPCB by operation.The ultraviolet-visible light of obtained PCB is inhaled
It is as shown in Figure 2 to receive spectrum.Phycocyanobilin has absorption peak in acidic methanol solution at 680nm, 390nm, wherein at 680nm
Absorption peak be phycocyanobilin characteristic absorption peak, the absorption peak at 380nm is the characteristic absorption peak of azoles.
Fig. 3 is the fluorescence emission spectrum of PCB.The methanol solution of phycocyanobilin is excited with the wavelength of 680nm, in the left side 800nm
There is the last one emission peak on the right side, and 730nm or so has a weak emission peak, this may be tripe systems elephant phycocyanobilin in methanol solution institute
Caused by the ratio accounted for is different.
Embodiment 2
(1) 2g phycocyanin powder is taken out from -27 DEG C of refrigerators, 100mL ethyl alcohol is added, be vortexed and mix, be then transferred to back
It flows in device, device is encased with masking foil, and 70 DEG C of water-bath 10h, end of reaction obtains the first product.
(2) by the first product at 4 DEG C, high speed centrifugation 5min under the conditions of 13000 × g.The first supernatant is taken, under similarity condition
It is centrifuged again once, obtains the second product.
(3) 0.45 μm of filter membrane of the second product and 20mL syringe are filtered, filtrate is steamed with Rotary Evaporators are outstanding, until remaining
About 5mL liquid, obtains third product.
(4) third product is used again 0.45 μm of filter membrane and 20mL syringe to filter, filtrate is at 4 DEG C, 13000 × g condition
Lower high speed centrifugation 20min.The second supernatant is taken, 10mL chloroform and 20mL ultrapure water is added, is transferred to 125mL separatory funnel, sufficiently
Mixing stands 20min, collects the first lower liquid and obtains the 4th product.
(5) mixing liquid including 100 μ L concentrated hydrochloric acids and 10mL ultrapure water is added into the 4th product, is transferred to liquid separation leakage
Bucket, stands 5min after being sufficiently mixed, and collects the second lower liquid with clean 50mL beaker, obtains the 5th product.
(6) mixing liquid including 100 μ L concentrated hydrochloric acids and 10mL ultrapure water is added toward the 5th product, is transferred to separatory funnel,
5min is stood after being sufficiently mixed, and third lower liquid is then collected using the centrifuge tube that clean drying 50mL masking foil wraps up,
Obtain the 6th product.
(7) centrifugation nozzle is packed tightly with sealed membrane, needle bundle opens several holes, then passes to nitrogen and dries up chloroform, obtains
40mg phycocyanobilin.Then gained phycocyanobilin is placed in -80 DEG C of ultra low temperature freezers and is saved.
Embodiment 3
(1) 10g phycocyanin powder is taken out from -27 DEG C of refrigerators, 100mL methanol is added, be vortexed and mix, be then transferred to first
In alcohol reflux device, device is encased with masking foil, and 80 DEG C of water-bath 5h, end of reaction obtains the first product.
(2) by the first product at 4 DEG C, high speed centrifugation 20min under the conditions of 8000 × g.The first supernatant is taken, under similarity condition
It is centrifuged again once, obtains the second product.
(3) 0.45 μm of filter membrane of the second product and 20mL syringe are filtered, filtrate is steamed with Rotary Evaporators are outstanding, until remaining
About 20mL liquid, obtains third product.
(4) third product is used again 0.45 μm of filter membrane and 20mL syringe to filter, filtrate is at 4 DEG C, 13000 × g condition
Lower high speed centrifugation 20min.The second supernatant is taken, 20mL chloroform and 40mL ultrapure water is added, is transferred to 125mL separatory funnel, sufficiently
Mixing stands 5min, collects the first lower liquid and obtains the 4th product.
(5) mixing liquid including 100 μ L concentrated hydrochloric acids and 20mL ultrapure water is added into the 4th product, is transferred to liquid separation leakage
Bucket, stands 5min after being sufficiently mixed, and collects the second lower liquid with clean 50mL beaker, obtains the 5th product.
(6) mixing liquid including 100 μ L concentrated hydrochloric acids and 20mL ultrapure water is added toward the 5th product, is transferred to separatory funnel,
5min is stood after being sufficiently mixed, and third lower liquid is then collected using the centrifuge tube that clean drying 50mL masking foil wraps up,
Obtain the 6th product.
(7) centrifugation nozzle is packed tightly with sealed membrane, needle bundle opens several holes, then passes to nitrogen and dries up chloroform, obtains
200mg phycocyanobilin.Then gained phycocyanobilin is placed in -80 DEG C of ultra low temperature freezers and is saved.
The above is only the preferred embodiment of the present invention, it is noted that for those skilled in the art,
Without departing from the principles of the invention, several improvements and modifications can also be made, these improvements and modifications also should be regarded as this hair
Bright protection scope.
Claims (10)
1. a kind of method for extracting phycocyanobilin, which comprises the following steps:
Alcohol solution is added into phycocyanin powder, after mixing, is protected from light and is heated to reflux 5h-10h, obtains the first product；
First product is centrifugated under conditions of temperature is 4 DEG C, the first supernatant is taken, obtains the second product；
Second product is filtered, filtrate is rotated to 5mL-20mL, third product is obtained；
The third product is filtered, filtrate is centrifuged, takes the second supernatant, chloroform and water is added, it is quiet after mixing
It sets, collects the first lower liquid, obtain the 4th product；
Hydrochloric acid solution is added into the 4th product, stands after mixing, then collects the second lower liquid, obtains the 5th production
Object；
The hydrochloric acid solution is added toward the 5th product, stands after mixing, then collects third lower liquid, obtains the
Six products；
By the chloroform drying in the 6th product, the phycocyanobilin is obtained.
2. extracting the method for phycocyanobilin as described in claim 1, which is characterized in that alcohols is added into phycocyanin powder
Solution, after mixing, in the step of being protected from light and be heated to reflux 5h-10h, obtaining the first product, the phycocyanin powder and methanol
Mass volume ratio be 1:50g/mL-1:10g/mL.
3. extracting the method for phycocyanobilin as described in claim 1, which is characterized in that alcohols is added into phycocyanin powder
Solution, after mixing, in the step of being protected from light and be heated to reflux 5h-10h, obtaining the first product, being protected from light the temperature being heated to reflux is
70℃-80℃。
4. as described in claim 1 extract phycocyanobilin method, which is characterized in that will first product temperature be 4
In the step of being centrifugated under conditions of DEG C, taking the first supernatant, obtain the second product, the revolving speed of centrifuge separation is 8000 × g-
13000 × g, the time of centrifugation are 5min-20min.
5. extracting the method for phycocyanobilin as described in claim 1, which is characterized in that filter second product, will filter
In the step of liquid is rotated to 5mL-20mL, obtains third product, second product uses 0.45 μm of filter membrane and 20mL syringe
Filtering.
6. extracting the method for phycocyanobilin as described in claim 1, which is characterized in that filter the third product, will filter
Liquid is centrifuged, and the second supernatant is taken, and chloroform and water is added, stands after mixing, is collected the first lower liquid, is obtained the 4th
In the step of product, the third product is filtered using 0.45 μm of filter membrane and 20mL syringe.
7. extracting the method for phycocyanobilin as described in claim 1, which is characterized in that filter the third product, will filter
Liquid is centrifuged, and the second supernatant is taken, and chloroform and water is added, stands after mixing, is collected the first lower liquid, is obtained the 4th
In the step of product, the volume ratio of second supernatant, chloroform and water is 1:1:2-1:2:4.
8. extracting the method for phycocyanobilin as described in claim 1, which is characterized in that the salt is added toward the 5th product
In the step of acid solution is stood after mixing, is then collected third lower liquid, is obtained six products, using tinfoil paper paper bag
The 50mL centrifuge tube wrapped up in collects lower liquid.
9. extracting the method for phycocyanobilin as described in claim 1, which is characterized in that blow the chloroform in the 6th product
In the step of doing, obtaining the phycocyanobilin, the 6th product is deposited in the container packed tightly with sealed membrane, sealed membrane is opened up
There are several holes, is passed through nitrogen and dries up chloroform.
10. extracting the method for phycocyanobilin as described in claim 1, which is characterized in that the alcohol solution is methanol, second
Alcohol, acidic methanol, methanol aqueous solution or ethanol water.
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| CN115364214A (en) * | 2022-08-23 | 2022-11-22 | 福州大学 | Photosensitizer for photodynamic therapy and preparation method and application thereof |
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Application publication date: 20190416 Assignee: Yantai Zhigong Biomedical Technology Co.,Ltd. Assignor: YANTAI INSTITUTE OF COASTAL ZONE RESEARCH, CHINESE ACADEMY OF SCIENCES Contract record no.: X2023210000165 Denomination of invention: A Method for Extracting Phycocyanobilin Granted publication date: 20210720 License type: Common License Record date: 20231026 |