CN102516246B - Method for extracting and purifying high-purity sepiapterin from silkworm body - Google Patents
Method for extracting and purifying high-purity sepiapterin from silkworm body Download PDFInfo
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- CN102516246B CN102516246B CN201110387345.3A CN201110387345A CN102516246B CN 102516246 B CN102516246 B CN 102516246B CN 201110387345 A CN201110387345 A CN 201110387345A CN 102516246 B CN102516246 B CN 102516246B
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- sepiapterin
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- silkworm body
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- VPVOXUSPXFPWBN-VKHMYHEASA-N sepiapterin Chemical compound N1C(N)=NC(=O)C2=C1NCC(C(=O)[C@@H](O)C)=N2 VPVOXUSPXFPWBN-VKHMYHEASA-N 0.000 title claims abstract description 49
- 229940126478 sepiapterin Drugs 0.000 title claims abstract description 49
- 241000255789 Bombyx mori Species 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 21
- VPVOXUSPXFPWBN-UHFFFAOYSA-N L-sepiapterin Natural products N1=C(N)NC(=O)C2=C1NCC(C(=O)C(O)C)=N2 VPVOXUSPXFPWBN-UHFFFAOYSA-N 0.000 title claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000049 pigment Substances 0.000 claims abstract description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 229920002678 cellulose Polymers 0.000 claims abstract description 7
- 239000001913 cellulose Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 238000009835 boiling Methods 0.000 claims abstract description 5
- 239000000706 filtrate Substances 0.000 claims abstract description 4
- 229920005654 Sephadex Polymers 0.000 claims description 6
- 239000012507 Sephadex™ Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 230000008719 thickening Effects 0.000 claims description 2
- 210000001835 viscera Anatomy 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 3
- 238000005406 washing Methods 0.000 abstract 4
- 238000001035 drying Methods 0.000 abstract 3
- 239000003480 eluent Substances 0.000 abstract 3
- 239000012530 fluid Substances 0.000 abstract 2
- 238000001914 filtration Methods 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 6
- 238000000605 extraction Methods 0.000 description 5
- FNKQXYHWGSIFBK-RPDRRWSUSA-N sapropterin Chemical compound N1=C(N)NC(=O)C2=C1NC[C@H]([C@@H](O)[C@@H](O)C)N2 FNKQXYHWGSIFBK-RPDRRWSUSA-N 0.000 description 5
- 239000012535 impurity Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000219146 Gossypium Species 0.000 description 2
- 230000000274 adsorptive effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- DGGUVLXVLHAAGT-XINAWCOVSA-N 7,8-dihydroneopterin 3'-triphosphate Chemical compound N1CC([C@H](O)[C@H](O)COP(O)(=O)OP(O)(=O)OP(O)(O)=O)=NC2=C1N=C(N)NC2=O DGGUVLXVLHAAGT-XINAWCOVSA-N 0.000 description 1
- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001599018 Melanogaster Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005360 mashing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 230000007830 nerve conduction Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003195 pteridines Chemical class 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 229960004617 sapropterin Drugs 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a method for extracting and purifying high-purity sepiapterin. The method comprises the following steps of: preparing 50% alcohol for a yellow silkworm body wall into a homogenate at a ratio of material to liquid being 1:20; boiling for 10 minutes; centrifuging for 30 minutes at 4 DEG C at a rotating speed of 15000rpm; taking supernatant fluid; filtering the supernatant fluid; concentrating the filtrate to a certain volume, and then separating through an ECTEOLA cellulose column; washing with pure water; after completely separating pigment for above 5cm, washing with 0.01M acetic acid; after concentrating and drying an eluent containing a target pigment stripe, dissolving in a defined amount of pure water; purifying by utilizing a SephadexG-25-150 column; washing with pure water; after concentrating and drying the eluent, dissolving in a defined amount of pure water; further purifying by utilizing a phosphocellurose column; washing with pure water; and concentrating and drying the eluent, thereby obtaining the high-purity sepiapterin. The method provided by the invention has the advantages of mature technology, low cost, simple process, high output, high purity of the extracted sepiapterin and the like. The method is suitable for the large-scale industrialized production of the high-purity sepiapterin.
Description
Technical field
The present invention relates to a kind of extraction purification technique of important compound, specifically a kind of method of extracting purifying height Sepiapterine from silkworm body.
Background technology
Sepiapterine (sepiapterin) is a kind of pteridine compounds of yellow, and molecular formula is C
9h
11n
5o
3, its structure is as follows:
Although Sepiapterine is distributed in many organisms, content is atomic under normal circumstances.For Mammals, Sepiapterine is one of precursor substance generating tetrahydrobiopterin (BH4).BH4 is the important coenzyme that catalysis generates the enzyme of the nerve conduction mediators such as Dopamine HCL, serotonin.Experimentation on animalies and clinical study prove in a large number, and supplementing BH4 has obvious result for the treatment of to BH4 defective type disease, and supplementing Sepiapterine also has good therapeutic action to some disease.Yellow silkworm is a kind of mutant of silkworm, and it is mainly because excessive Sepiapterine and deamination product thereof are deposited on the dermal cell layer of body wall that its larva body colour is yellow.The present invention just research creation of the valuable Biological resources based on reclaim natural Sepiapterine using yellow silkworm as purifying forms.
Mainly contain from the method for extracting purifying Sepiapterine in organism at present:
1. Tsusue M and Akino M. Yellow pterins in mutunt
lemonof silkworm and mutant
sepiaof
D. melanogaster. Zoological Magazine (Tokyo) 1965,74:336-341.
2. Krivi G. G and Brown G. M. Purification and properties of the enzymes from
Drosophila melanogaster that catalyze the synthesis of sepiapterin from dihydroneopterin triphosphate. Biochemical Genetics, 1979, 17:371-390.
The method of the extraction Sepiapterine of describing in above document is not that purity is not high, and step is difficult to control, and is exactly complicated operation, and preparation amount is little, can not large-scale industrial production, thereby all can not extract the Sepiapterine product that is purified into a large amount of high purities (more than 99%).
Summary of the invention
The invention provides a kind of method of extracting purifying high-purity sepiapterin from silkworm body, the Sepiapterine purity that present method is extracted reaches more than 99%.
The technical solution used in the present invention is as follows for achieving the above object:
A method of extracting purifying high-purity sepiapterin from silkworm body, is characterized in that, comprises the following steps:
(1), dissect silkworm body, reject as far as possible viscera tissue, preserve body wall;
(2), take a certain amount of silkworm body wall, be cut into fragment with scissors, according to solid-liquid ratio 1:(18-22) to add concentration be the ethanol of 45-55%, with organizing crushing mechanism to become homogenate at a high speed, in 95-105 DEG C of boiling water bath, heats 8-12min;
(3), by heating after homogenate carry out centrifugation, rotating speed is 15000rpm, centrifuging temperature is 3-5 DEG C, centrifugation time 28-32min, collect supernatant liquor;
(4), supernatant liquor filters by filter paper, at 25-30 DEG C, reduced vacuum concentrated filtrate is to the 1/5-1/7 of original volume;
(5), concentrated solution is through ECTEOLA cellulose column absorption, first uses pure water wash-out pillar, after pigment is separated spacing to exceed 5cm completely, with 0.01M acetic acid wash-out pillar, collects the elutriant containing Sepiapterine pigment band;
(6), elutriant is concentrated into after dry and is dissolved in appropriate pure water, obtain yellow solution;
(7), yellow solution is through Sephadex G-25-150 column purification, uses pure water wash-out, collects pigment elutriant;
(8), repeating step (6);
(9), yellow solution is further purified with phosphocellurose column, use pure water wash-out, collect pigment elutriant;
(10), finally again step (9) gained pigment elutriant is concentrated into dryly, obtains highly purified Sepiapterine.
While adding ethanol in step (2), silkworm body wall fragment and ethanol solid-liquid ratio be 1:20, the ethanolic soln that added ethanol is 50%.
The centrifuging temperature of step (3) is 4 DEG C, and the thickening temperature of step (4) is 30 DEG C.
Step (4) is carried out to the operation of step (10) darkroom at room temperature.
Process characteristic of the present invention is described as follows:
1, adopt solvent extraction method to extract Sepiapterine, in multiple organic solvent, the extracting effect of ethanol is best, when alcohol concn is 50%, and the extraction effect the best to Sepiapterine when solid-liquid ratio is 1:20, so both avoid the wasting of resources, and can extract to greatest extent again Sepiapterine; In boiling water, boil 10min and just the pigment in yellow silkworm body wall can be extracted more completely, economical and energy saving effect is very obvious.
2, step (3) operating under dark room temperature environment is afterwards because Sepiapterine is very unstable, light intensity in separation and Extraction purge process and temperature are all likely oxidized to other materials, so operation greatly reduces the oxidized probability of Sepiapterine under dark room temperature environment.
3, step (2) and (3) are in order to remove the insolubles in body wall.
4, ECTEOLA Mierocrystalline cellulose, as a kind of anionite, can separate with other pigments according to the polarity of material and the large young pathbreaker's Sepiapterine of adsorptive power, can remove most of protein and fatty impurity simultaneously, has played the effect of separation and preliminary purification Sepiapterine.First use pure water wash-out pillar, in the time that exceeding 5cm, two pigment striation widthses use again 0.01M acetic acid wash-out pillar, until Sepiapterine pigment elutes post completely, such gradient elution method not only greatly reduces disengaging time, improve separating effect, and make Sepiapterine pigment band more concentrated, collection containing the elutriant of Sepiapterine because of small volume easily concentrated.
5, Sephadex G-25-150 has molecular sieve effect, can remove macromole impurity residual in Sepiapterine solution, purifying Sepiapterine.
6, phosphorylated cotton is as a kind of cationite, macromolecular substance is had to stronger adsorptive power, can remove through the separation and purification of ECTEOLA cellulose column and Sephadex G-25-150 post still residual a small amount of macromole impurity, be further purified Sepiapterine, finally can obtain the Sepiapterine that purity is greater than 99%.
In the present invention, not only price is lower for the reagent such as ethanol used, acetic acid, and all belong to nontoxic reagent, more easily from pigment, volatilize and remove, all can reuse for separating of chromatography column stopping composition such as the ECTEOLA Mierocrystalline cellulose of purifying, Sephadex G-25-150, phosphorylated cottons simultaneously, greatly reduce extraction cost.Utilize this experimental system to extract Sepiapterine lower than the cost of the synthetic Sepiapterine of chemical industry, medical science security is higher.
Beneficial effect of the present invention:
Technical maturity of the present invention, have with low cost, operation is simple, output is large, the Sepiapterine purity high (more than 99%) of extracting, waits clear superiority, be applicable to large-scale industrial production high purity Sepiapterine.
Embodiment
Embodiment
5 age yellow silkworm larva body wall 10g, add 50% ethanol according to solid-liquid ratio 1:20, homogenate 10min in high-speed tissue mashing machine, in 100 DEG C of boiling water baths, heat 10min, 15000rpm, 4 DEG C, centrifugal 30min, collect supernatant liquor, supernatant liquor filters by filter paper, filtrate is evaporated to 30ml with rotary evaporator, concentrated solution is added on ECTEOLA cellulose column (4 × 25cm), first use pure water wash-out pillar, after separating spacing to exceed 5cm completely, pigment uses 0.01M acetic acid wash-out pillar, the elutriant that collection contains Sepiapterine pigment band, elutriant is concentrated into dry, be dissolved in 10ml pure water, obtain yellow solution, yellow solution is joined on Sephadex G-25-150 post (2.6 × 36cm), pure water wash-out, collect pigment elutriant, elutriant is concentrated into dry, be dissolved in 10ml pure water, obtain yellow solution, yellow solution is joined on phosphocellurose column (2.1 × 35cm), pure water wash-out, collect pigment elutriant, elutriant is concentrated into dry, be dissolved in 3ml pure water, detect through HPLC, the Sepiapterine content that repeats to test gained for three times is respectively 0.358 mg, 0.369 mg and 0.372 mg, purity is all greater than 99%.
Claims (4)
1. a method of extracting purifying high-purity sepiapterin from silkworm body, is characterized in that, comprises the following steps:
(1), dissect silkworm body, reject as far as possible viscera tissue, preserve body wall;
(2), take a certain amount of silkworm body wall, be cut into fragment with scissors, according to solid-liquid ratio 1:(18-22) to add concentration be the ethanol of 45-55%, with organizing crushing mechanism to become homogenate at a high speed, in 95-105 DEG C of boiling water bath, heats 8-12min;
(3), by heating after homogenate carry out centrifugation, rotating speed is 15000rpm, centrifuging temperature is 3-5 DEG C, centrifugation time 28-32min, collect supernatant liquor;
(4), supernatant liquor filters by filter paper, at 25-30 DEG C, reduced vacuum concentrated filtrate is to the 1/5-1/7 of original volume;
(5), concentrated solution is through ECTEOLA cellulose column absorption, first uses pure water wash-out pillar, after pigment is separated spacing to exceed 5cm completely, with 0.01M acetic acid wash-out pillar, collects the elutriant containing Sepiapterine pigment band;
(6), elutriant is concentrated into after dry and is dissolved in appropriate pure water, obtain yellow solution;
(7), yellow solution is through Sephadex G-25-150 column purification, uses pure water wash-out, collects pigment elutriant;
(8), repeating step (6);
(9), yellow solution is further purified with phosphocellurose column, use pure water wash-out, collect pigment elutriant;
(10), finally step (9) gained pigment elutriant is concentrated into dryly again, obtains highly purified Sepiapterine, purity reaches more than 99%.
2. a kind of method of extracting purifying high-purity sepiapterin from silkworm body according to claim 1, is characterized in that: while adding ethanol in step (2), silkworm body wall fragment and ethanol solid-liquid ratio be 1:20, the ethanolic soln that added ethanol is 50%.
3. a kind of method of extracting purifying high-purity sepiapterin from silkworm body according to claim 1, is characterized in that: the centrifuging temperature of step (3) is 4 DEG C, the thickening temperature of step (4) is 30 DEG C.
4. a kind of method of extracting purifying high-purity sepiapterin from silkworm body according to claim 1, is characterized in that: step (4) is carried out to the operation of step (10) darkroom at room temperature.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110387345.3A CN102516246B (en) | 2011-11-30 | 2011-11-30 | Method for extracting and purifying high-purity sepiapterin from silkworm body |
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|---|---|---|---|
| CN201110387345.3A CN102516246B (en) | 2011-11-30 | 2011-11-30 | Method for extracting and purifying high-purity sepiapterin from silkworm body |
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| Publication Number | Publication Date |
|---|---|
| CN102516246A CN102516246A (en) | 2012-06-27 |
| CN102516246B true CN102516246B (en) | 2014-08-06 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1449442A (en) * | 2000-08-31 | 2003-10-15 | 第一三得利制药株式会社 | Process for producing biopterins |
-
2011
- 2011-11-30 CN CN201110387345.3A patent/CN102516246B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1449442A (en) * | 2000-08-31 | 2003-10-15 | 第一三得利制药株式会社 | Process for producing biopterins |
Non-Patent Citations (2)
| Title |
|---|
| purification and indentification of a yellow pteridine characteristic of the larval colour of the kiuki mutant of the silkworm, bombyx mori;TOSHIO MAZDA et al;《Insect Biochem.》;19801231;第10卷;357-358 * |
| TOSHIO MAZDA et al.purification and indentification of a yellow pteridine characteristic of the larval colour of the kiuki mutant of the silkworm, bombyx mori.《Insect Biochem.》.1980,第10卷357-362. |
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