CN102650621B - Method for identifying sepiapterin extracted from silkworm body - Google Patents
Method for identifying sepiapterin extracted from silkworm body Download PDFInfo
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- CN102650621B CN102650621B CN201210133303.1A CN201210133303A CN102650621B CN 102650621 B CN102650621 B CN 102650621B CN 201210133303 A CN201210133303 A CN 201210133303A CN 102650621 B CN102650621 B CN 102650621B
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Abstract
The invention discloses a method for identifying sepiapterin extracted from a silkworm body. According to the method, a high performance liquid chromatography is utilized, and the chromatographic conditions are that ODSC18 (oxide dispersion strengthened copper 18) is used as a chromatographic column, methanol-0.006% of acetic acid mixed liquor is used as a mobile phase, the flow velocity is 1ml/min, the column temperature is 25 DEG C, the sample size is 20 mul, and the detection wavelength is 420mn. Extracted pigment is analyzed according to the chromatographic conditions, and a target product-sepiapterin can be effectively identified. The method disclosed by the invention has the advantages of high precision, simplicity, accuracy, good repeatability, good stability and the like, and a simple, efficient and accurate method is provided for identifying the sepiapterin extracted from the silkworm body.
Description
Technical field
The invention belongs to the authenticate technology field of kind of pigment, relate in particular to a kind of authentication method of the sepiapterin extracting from silkworm body.
Background technology
Sepiapterin (sepiapterin) is a kind of pteridine compounds of yellow, and molecular formula is C
9h
11n
5o
3, be distributed in many biosomes, but content is atomic under normal circumstances.For mammal, sepiapterin 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, serotonin.Zooperies and clinical research prove in a large number, and supplementing BH4 has obvious result for the treatment of to BH4 deficiency disease, and supplementing sepiapterin also has good result for the treatment of to some disease.Yellow silkworm is a kind of mutant of silkworm, the deamination product that its larva body wall contains a large amount of sepiapterin pigments, sepiapterin and a small amount of lactochrome, and therefore yellow silkworm is the precious resources that reclaims natural sepiapterin.We utilize solvent extraction method and column chromatography that pigment separated, purifying in yellow silkworm body are obtained to two kinds of different pigments.At present the authentication method of sepiapterin and deamination product thereof is only had to paper chromatography and UV-Visible absorption spectrum.Wherein paper chromatography running program complexity, although can carry out simple qualitative analysis to sepiapterin according to Rf value, can not determine the purity of extracted sepiapterin and the number of content; Ultraviolet absorption spectroscopy and high performance liquid chromatography can be carried out qualitative and quantitative analysis to material, and high performance liquid chromatography has higher accuracy by contrast.High efficiency liquid phase as a kind of important chromatographic process have that resolution is high, speed is fast, repeatability is high, automation mechanized operation, analytical precision are high, chromatographic column can Reusability etc. advantage, it can not only carry out qualitative analysis to material, also can determine accurately the content of material simultaneously, but the research of also by high performance liquid chromatography, the sepiapterin extracting from silkworm body not being identified at present.The present invention is just based on by high performance liquid chromatography, the research that separates the kind of two kinds of pigments that obtain from yellow silkworm body surface and identify being created and formed.
Summary of the invention
The object of the invention is to carry that a kind of operation of arch precision is high, easy, accurate, reproducible, good stability, the authentication method of the sepiapterin effectively extracting from silkworm body.
To achieve these goals, the present invention adopts following technical scheme:
An authentication method for the sepiapterin extracting from silkworm body, is characterized in that, comprises the following steps:
(1), the body wall of silkworm body is added to 50% ethanol homogenate according to solid-liquid ratio 1:20, boil 10min left and right, centrifugal, concentrated, separate and obtain two kinds of pigments through ECTEOLA cellulose column, be pigment A and pigment B, respectively by two kinds of pigments successively through Sephadex G-25-150 post and phosphocellurose column purifying, finally obtain the high purity powdered form of two kinds of pigments;
(2), the high purity powdered form of the pigment of sepiapterin standard items and two kinds of extractions is dissolved in respectively in 2ml ultrapure water, use 0.45um membrane filtration, filtrate is for examination;
(3), chromatographic condition: by ODS C18 chromatographic column; selecting the mixed liquor of the methyl alcohol of ultrapure water, concentration >99.9% and the glacial acetic acid of concentration >99.9% is mobile phase; by mobile phase 0.45um membrane filtration; ultrasonic degas 20min; flow velocity is 1ml/min; column temperature is 25 ℃, sample size 20ul, and detection wavelength is 420nm;
(4), according to the chromatographic condition balanced system in (3), balance to baseline wander is less than 0.01mV/min, noise is less than 0.001mV; After system balancing is good, sample introduction is analyzed the pigment of sepiapterin standard items and two kinds of extractions respectively, by the retention time of sample, sepiapterin is carried out to identification and analysis.
A kind of described method that the sepiapterin extracting from silkworm body is identified, is characterized in that: the ODS C18 chromatographic column specification of step (3) is for being 250mm × 4.6mm, and filler granularity is 5um.
A kind of described method that the sepiapterin extracting from silkworm body is identified, is characterized in that: the volume ratio of the ultrapure water of step (3), 99.9% methyl alcohol, 99.9% glacial acetic acid is 79.5:20:0.5.
Beneficial effect of the present invention:
1, the present invention's mobile phase used is methyl alcohol-0.006% acetic acid mixed liquor, volume ratio is 20:80, the retention time of two kinds of pigments that extract from silkworm body in the time using this mobile phase is obviously different, according to the retention time of standard specimen sepiapterin, can effectively carry out identification and analysis to the pigment extracting from silkworm body;
2, the present invention's column temperature used is 25 ℃, because the intensity in chromatographic process and temperature affect the stability of sepiapterin, can make it be oxidized to other materials, so column temperature is set as to 25 ℃, can effectively prevent that sepiapterin is oxidized in the process of stratographic analysis;
3, detection wavelength used in the present invention is 420nm, and according to the ultraviolet-visible absorption spectroscopy of sepiapterin, sepiapterin has absorption maximum crest at 269nm and 420nm place, and therefore the present invention selects one of maximum absorption wavelength of sepiapterin 420nm for detecting wavelength;
4, the present invention operates that precision is high, easy, accurate, reproducible, good stability, can effectively carry out qualitative and quantitative analysis to the sepiapterin extracting from silkworm body.
Accompanying drawing explanation
Fig. 1 is the HPLC chromatogram of sepiapterin standard items;
Fig. 2 is the HPLC chromatogram of pigment A;
Fig. 3 is the HPLC chromatogram of pigment B.
embodiment,
5 age yellow silkworm larva body wall 10g, add 50% ethanol according to solid-liquid ratio 1:20, separate and obtain two kinds of pigments through ECTEOLA cellulose column, respectively by two kinds of pigments successively through Sephadex G-25-150 post and phosphocellurose column purifying, finally obtain the high purity powdered form of two kinds of pigments: pigment A and pigment B.Pigment A, pigment B and sepiapterin standard items are dissolved in respectively in appropriate ultrapure water, use 0.45um membrane filtration.With ODS C18 chromatographic column (250mm × 4.6mm, 5um), take methyl alcohol-0.006% acetic acid mixed liquor (volume ratio 20:80) as mobile phase, be 1ml/min at flow velocity, column temperature is 25 ℃, sample size 20ul, the chromatographic condition that detection wavelength is 420nm carries out identification and analysis to sepiapterin standard specimen, pigment A and pigment B respectively.Result is as shown below, and the retention time (Fig. 2) of pigment A is consistent with sepiapterin standard specimen (Fig. 1), and the retention time of pigment B and sepiapterin standard specimen completely different (Fig. 3) can illustrate that pigment A is sepiapterin.Meanwhile, the mensuration of precision, stability and repeatability has been carried out in this research, and the relative standard deviation recording is respectively 0.17%, 0.18%, 3.46%, and result shows that precision of the present invention, stability and repeatability are good.
The present embodiment high performance liquid chromatograph used is Shimadzu LC-20AT type, and Ultraviolet Detector is SPD-20AV;
The present embodiment chromatographic column used is Shimadzu ODS C18 (250mm × 4.6mm, 5um);
The present embodiment mobile phase used is methyl alcohol-0.006% acetic acid mixed liquor, volume ratio is 20:80, the retention time of two kinds of pigments that extract from silkworm body in the time using this mobile phase is obviously different, according to the retention time of standard specimen sepiapterin, can effectively carry out identification and analysis to the pigment extracting from silkworm body;
The flow velocity of the present embodiment mobile phase used is 1ml/min;
The present embodiment column temperature used is 25 ℃, because the intensity in chromatographic process and temperature affect the stability of sepiapterin, can make it be oxidized to other materials, so column temperature is set as to 25 ℃, can effectively prevent that sepiapterin is oxidized in the process of stratographic analysis;
The detection wavelength that the present embodiment uses is 420nm, and according to the ultraviolet-visible absorption spectroscopy of sepiapterin, sepiapterin has absorption maximum crest at 269nm and 420nm place, and therefore the present embodiment selects one of the maximum absorption wavelength of sepiapterin 420nm for detecting wavelength.
Claims (1)
1. an authentication method for the sepiapterin extracting from silkworm body, is characterized in that, comprises the following steps:
(1), the body wall of silkworm body is added to 50% ethanol homogenate according to solid-liquid ratio 1:20, boil 10min left and right, centrifugal, concentrated, separate and obtain two kinds of pigments through ECTEOLA cellulose column, be pigment A and pigment B, respectively by two kinds of pigments successively through Sephadex G-25-150 post and phosphocellurose column purifying, finally obtain the high purity powdered form of two kinds of pigments;
(2), the high purity powdered form of the pigment of sepiapterin standard items and two kinds of extractions is dissolved in respectively in 2ml ultrapure water, use 0.45um membrane filtration, filtrate is for examination;
(3), chromatographic condition: by ODS C18 chromatographic column, selecting the mixed liquor of the methyl alcohol of ultrapure water, concentration >99.9% and the glacial acetic acid of concentration >99.9% is mobile phase, by mobile phase 0.45um membrane filtration, ultrasonic degas 20min, flow velocity is 1ml/min, column temperature is 25 ℃, sample size 20ul, and detection wavelength is 420nm; Described ODS C18 chromatographic column specification is 250mm × 4.6mm, and filler granularity is 5um; The volume ratio of described ultrapure water, 99.9% methyl alcohol, 99.9% glacial acetic acid is 79.5:20:0.5;
(4), according to the chromatographic condition balanced system in (3), balance to baseline wander is less than 0.01mV/min, noise is less than 0.001mV; After system balancing is good, sample introduction is analyzed the pigment of sepiapterin standard items and two kinds of extractions respectively, by the retention time of sample, sepiapterin is carried out to identification and analysis.
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| CN109946412B (en) * | 2017-12-21 | 2021-10-15 | 上海产业技术研究院 | Kit for detecting spectrum of humoral pterin and use thereof |
| CN111505179B (en) * | 2020-04-07 | 2021-07-13 | 厦门大学 | Method for detecting biopterin in marine water body |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008128049A2 (en) * | 2007-04-11 | 2008-10-23 | Biomarin Pharmaceutical Inc. | Methods of administering tetrahydrobiopterin, associated compositions, and methods of measuring |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008128049A2 (en) * | 2007-04-11 | 2008-10-23 | Biomarin Pharmaceutical Inc. | Methods of administering tetrahydrobiopterin, associated compositions, and methods of measuring |
Non-Patent Citations (4)
| Title |
|---|
| Purification and identification of a yellow pteridine characteristic of the larval colour of the kiuki mutant of the silkworm, bombyx mori;Toshio Mazda等;《Insect Biochem》;19801231;第10卷(第4期);第357页右栏第7-34行和第368页图1 * |
| ToshioMazda等.Purificationandidentificationofayellowpteridinecharacteristicofthelarvalcolourofthekiukimutantofthesilkworm bombyx mori.《Insect Biochem》.1980 |
| 崔盼盼等.高效液相色谱-荧光分析法测定人体尿液中的墨蝶呤.《南昌大学学报(理科版)》.2011,第35卷(第1期), |
| 高效液相色谱-荧光分析法测定人体尿液中的墨蝶呤;崔盼盼等;《南昌大学学报(理科版)》;20110228;第35卷(第1期);第60页第1节和第61页第2.1节 * |
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