CN102590378B - Method for detecting content of swainsonine in locoweed endophytic fungi - Google Patents
Method for detecting content of swainsonine in locoweed endophytic fungi Download PDFInfo
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- CN102590378B CN102590378B CN 201210024222 CN201210024222A CN102590378B CN 102590378 B CN102590378 B CN 102590378B CN 201210024222 CN201210024222 CN 201210024222 CN 201210024222 A CN201210024222 A CN 201210024222A CN 102590378 B CN102590378 B CN 102590378B
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Abstract
The invention relates to a method for detecting the content of swainsonine in locoweed endophytic fungi. The method comprises the steps: pre-treating an endophytic fungi hyphae sample to prepare a test solution; using a high performance liquid chromatograph evaporative light-scattering detector to detect the swainsonine in the locoweed endophytic fungi; taking a mixed solution of acetonitrile andammonium acetate buffer solution as a mobile phase, wherein the volume ratio of acetonitrile to ammonium acetate is 0.2-2 to 0.2-2; separating the swainsonine from other materials in the test sample by an HILIC (Hydrophilic Interaction Chromatography) column; and constructing a map by logarithm of swainsonine in different concentrations and logarithm of a corresponding response peak area so as toobtain a standard curve of the swainsonine. According to the method, the swainsonine in the endophytic fungi Undifilum oxytropis can be effectively separated, and the method can be directly applied to the quantitative analysis of the swainsonine. The method is good in separation effect, strong in specificity and high in accuracy, and the analysis method is reliable.
Description
Technical field
The invention belongs to technical field of analytical chemistry, relate to the separation determination of spherosin in the endogenetic fungus, specifically is a kind of method of measuring spherosin content in the loco weed endogenetic fungus.
Background technology
Spherosin (SW) is western pyridine alkaloid in the indoles, western pyridine in chemistry 1,2,8-trihydroxy by name-8-hydrogen-indoles.SW is the main toxic component in the loco weed (general name of pulse family whin genus and Astragalus poisonous plant).Loco weed is worldwide widely distributed, can cause poisoning after animal searches for food in a large number, even dead.
Spherosin is not only a kind of plant-derived toxin, and it also has the effect that suppresses growth of tumour cell and transfer.Because SW is a kind of extremely strong alpha-Mannosidase inhibitor, can not only suppress the expression of tumor cell surface glycoprotein, also can suppress the growth of tumour cell and transfer, cell death inducing, stimulate the immune system of body, strengthen the ability of killing tumour cell.Therefore SW also is used as the essential reserve medicine of antineoplastic.
Discover that plant pathogenic fungi beans rhizoctonia, insect pathogenic fungus Metarhizium anisopliae can produce SW.2000, the source that confirms SW first and the endogenetic fungus in the loco weed of Harris etc. were closely related; 2003, Braun etc. from 3 kinds of loco weeds of the U.S. (
Astragalus mollissimus, Oxytropis lambertii, O. sericea) root, stem, leaf, flower, seed in all be separated to
EmbellisiaThe endogenetic fungus that belongs to, and these endogenetic fungus can produce SW, find the high loco weed of endogenetic fungus separation rate, and its spherosin content is also high.2008, Yu Yongtao etc. also isolated the endogenetic fungus that can produce spherosin from loco weeds such as Chinese glacier whin, Astragalus strictus, hair lobe whin
Undifilum oxytropis
At present thin-layered chromatography and vapor-phase chromatography are mainly adopted in the detection of spherosin.Thin-layered chromatography is the qualitative detection at SW, because its sensitivity is low, is unsuitable for quantitative detection; Vapor-phase chromatography is the effective ways of present quantitative measurement spherosin.But before with the gas chromatographic detection spherosin, need carry out pre-treatment to sample with derivatization reagent.Derivative reaction complexity, time-consuming.And derivative reaction requires very harsh to reaction conditions.
Because spherosin self lacks luminophore, use high performance liquid chromatography-uv detection method and be difficult to spherosin is detected, thereby limited the application of traditional high performance liquid chromatography aspect the detection spherosin.Evaporative light-scattering detector can realize chemical substance low volatility, that do not have luminophore is detected as a kind of common detector.The present invention adopts high performance liquid chromatography-evaporative light-scattering to detect (HPLC-ELSD) method and can directly the spherosin in the sample accurately be measured without pre-column derivatization.
Summary of the invention
At above-mentioned problems of the prior art and defective, the object of the present invention is to provide a kind of method of measuring spherosin content in the loco weed endogenetic fungus fast and accurately.
The technical scheme that realizes the foregoing invention purpose is: a kind of method that detects spherosin in the loco weed endogenetic fungus, it is characterized in that, and comprise the following steps:
1) with endogenetic fungus mycelia sample through pre-service, be prepared into test solution;
2) detecting with the high performance liquid chromatograph evaporative light-scattering detector, is the acetonitrile of 0.2 ~ 2 ︰ 0.2 ~ 2 with volume ratio: the mixed liquor of ammonium acetate buffer is the phase that flows, through the HILIC post, with the spherosin in the sample to be checked and other separating substances;
3) map with the logarithm of variable concentrations spherosin and the logarithm of corresponding response peak area, obtain the typical curve of spherosin.
Mycelia sample pretreatment technology of the present invention is: at first adopt methyl alcohol to extract the mycelia sample, extract volatilizes the back with the acetic acid dissolving, exchanges 1 h through the cation exchange resin column dynamic circulation; With the impurity in the deionized water rinsing resin; Use weak aqua ammonia wash-out resin column again, collect the ammoniacal liquor eluent, concentrated volatilizing; Last methyl alcohol dissolving is got supernatant after centrifugal, volatilizes stand-by.
The testing conditions of high performance liquid chromatograph evaporative light-scattering detector ELSD of the present invention: yield value is 150, flow rate of carrier gas 25 psi, and drift tube temperature: 55 ℃, 36 ℃ in atomizer, sample size 20 μ L.
The present invention adopts chromatographic column Waters XBridge HILIC(specification 150mm * 4.6mm, 3.5 μ m).
The mobile phase preferred proportion of the present invention is acetonitrile: volume ratio 1 ︰ 1 of ammonium acetate buffer.
During flowing mutually, the present invention also adds the ammoniacal liquor that accounts for the phase cumulative volume 0.02% that flows.
The selected ammonium acetate buffer concentration of the present invention is 5 mmol/L.
Analyze through HPLC-ELSD, the retention time of spherosin on chromatogram is 5.3 minutes.
According to the said spherosin HPLC analytical method of the present invention, it is the loco weed endogenetic fungus of need being measured SW content
Undifilum oxytropisSample detection behind the 30 times of-FEL4-F5 pretreatment sample concentration dilutions.
Adopt the present invention to measure the content of spherosin in the loco weed endogenetic fungus, the sensing range of its spherosin is 15.625～250 μ g/mL.This method is highly sensitive, and specificity is strong, favorable reproducibility, and analytical approach is reliable.
Description of drawings
Fig. 1 is the HPLC-ELSD chromatogram of spherosin standard specimen.
Fig. 2 is the loco weed endogenetic fungus
Undifilum oxytropisThe spherosin HPLC-ELSD chromatogram that records among-the FEL4-F5.
Embodiment
Following specific embodiment will the invention will be further described by reference to the accompanying drawings.
Embodiment 1: the drafting of spherosin typical curve
1) preparation of SW standard solution: accurately take by weighing the SW standard items, with the phased soln that flows, gradient dilution.
2) experimental apparatus and chromatographic condition: Waters 1525 type high performance liquid chromatographs, binary pump; It is 150 that Water 2424 evaporative light-scattering detector, parameter arrange yield value, flow rate of carrier gas 25 psi, 55 ℃ of drift tube temperatures, 36 ℃ in atomizer, sample size 20 μ L; Waters XBridge HILIC chromatographic column (specification 150mm * 4.6mm, 3.5 μ m); The control of chromatographic fractionation system and record are finished by Breeze 2.0 workstations.Flow and be made up of acetonitrile and ammonium acetate buffer, volume ratio is 1 ︰ 1, and the concentration of ammonium acetate is 5 mmol/L, adds the ammoniacal liquor of cumulative volume 0.02%.The flow velocity of the phase that flows is 0.5 ml/min.The spherosin standard specimen the results are shown in Figure 1.
3) map with the logarithm of variable concentrations SW and the logarithm of corresponding response peak area, obtain the typical curve of SW.The SW concentration that adopts this method to measure shows good linear relationship in 15.625～250 μ g/mL concentration ranges.Obtaining equation of linear regression is Y=1.0641X+2.5679, linear regression coeffficient R
2=0.9981.
Embodiment 2 endogenetic fungus
Undifilum oxytropisThe detection of-FEL4-F5 sample
1) The pretreatment: get
Undifilum oxytropisThe dry mycelia 0.5g of-FEL4-F5 adds 50 mL methyl alcohol in extraction apparatus, 60 ℃ are extracted 24 h.Methanol extract liquid concentrated volatilize, residue dissolves with the acetic acid solution of 10 mL, 2 %, and this solution is joined in the exchange column that contains the high AG 50W of 5 cm resin cation, makes its dynamic circulation exchange 1 h, makes the abundant combination of spherosin and resin cation; Use the impurity in the 100 mL deionized water rinsing resins then; Use the ammonia spirit wash-out resin column of 100 mL, 1 mol/L again, collect the ammoniacal liquor eluent, concentrate and volatilize, dissolve with 2 mL methyl alcohol, get supernatant and it is volatilized stand-by after centrifugal.Dilute (30 times W/V), the results are shown in Figure 2 with the phase 15mL that flows before the sample introduction.
2) experimental apparatus and chromatographic condition: with embodiment 1.
The detection of the endogenetic fungus SW content that embodiment 3 different loco weeds separate
The processing of endogenetic fungus sample is with embodiment 2, and experimental apparatus and chromatographic condition are with embodiment 1.Obtain endogenetic fungus through HPLC-ELSD detection
Undifilum oxytropisMiddle SW content such as following table:
| The loco weed kind | The place of production | Endogenetic fungus | SW content (μ g/g) |
| O.ochrocepala | The Ningxia Haiyuan County | FEL4-F5 | 4963.14±35.55 |
| A.strictus | Rikaze, Tibet | FEL5-G3 | 3077.64±27.33 |
| O.glabra | The Minqin, Gansu | FEL1b-B4 | 2043.20±22.66 |
| O.kansuensis | The Gansu God blessings | FEL3-E9 | 2334.41±48.25 |
Claims (1)
1. a method that detects spherosin content in the loco weed endogenetic fungus is characterized in that, comprises the following steps:
1) with endogenetic fungus mycelia sample through pre-service, be prepared into test solution;
Described mycelia sample pretreatment is at first to adopt methyl alcohol to extract the mycelia sample, and extract volatilizes the back with the acetic acid dissolving, exchanges 1 h through the cation exchange resin column dynamic circulation; With the impurity in the deionized water rinsing resin; Use weak aqua ammonia wash-out resin column again, collect the ammoniacal liquor eluent, concentrated volatilizing; Last methyl alcohol dissolving is got supernatant after centrifugal, volatilizes stand-by;
2) detecting with the high performance liquid chromatograph evaporative light-scattering detector, is the acetonitrile of 0.2 ~ 2 ︰ 0.2 ~ 2 with volume ratio: the mixed liquor of ammonium acetate buffer is the phase that flows, through the HILIC post, with the spherosin in the sample to be checked and other separating substances;
The parameter of described high performance liquid chromatograph evaporative light-scattering detector is set to yield value: 150; Flow rate of carrier gas: 25 psi; Drift tube temperature: 55 ℃; Atomizer temperature: 36 ℃;
Described ammonium acetate buffer concentration is 5 mmol/L;
Describedly also add the ammoniacal liquor that accounts for the phase cumulative volume 0.02% that flows in flowing mutually;
The flow velocity of described mobile phase is 0.5 ml/min;
3) map with the logarithm of variable concentrations spherosin and the logarithm of corresponding response peak area, obtain the typical curve of spherosin.
2. the method for spherosin content in the detection loco weed endogenetic fungus according to claim 1 is characterized in that described HILIC post is Waters XBridge HILIC, and specification is 150mm * 4.6mm, 3.5 μ m.
3. the method for spherosin content in the detection loco weed endogenetic fungus according to claim 1 is characterized in that flowing is the acetonitrile of volume ratio 1 ︰ 1 mutually: the mixed liquor of ammonium acetate buffer.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104031849A (en) * | 2014-07-07 | 2014-09-10 | 西北农林科技大学 | Method for directional separation and authentication of swainsonine fungal endophyte Undifilum oxytropis produced through oxytropis kansuensis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104977374B (en) * | 2015-07-15 | 2017-06-16 | 成霈 | A kind of method for detecting spherosin content in loco weed extract and capsule |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1999021858A1 (en) * | 1997-10-24 | 1999-05-06 | Glycodesign Inc. | Synthesis of swainsonine salts |
| CN101270118A (en) * | 2007-03-22 | 2008-09-24 | 贺武利 | Technique for spherosinin purification with continuous column chromatography |
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| JP4514280B2 (en) * | 2000-04-05 | 2010-07-28 | 株式会社前川製作所 | Symbiotic bacteria, screening methods for symbiotic bacteria, plants artificially introduced with symbiotic bacteria, seeds obtained from plants, plants grown from seeds, hybrid plants, methods for introducing symbiotic bacteria into plants |
| CN1786706B (en) * | 2004-12-10 | 2010-12-01 | 天津天士力制药股份有限公司 | Cyclovirobuxine D raw medicine and method for determining its content in preparation by chromatography |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999021858A1 (en) * | 1997-10-24 | 1999-05-06 | Glycodesign Inc. | Synthesis of swainsonine salts |
| CN101270118A (en) * | 2007-03-22 | 2008-09-24 | 贺武利 | Technique for spherosinin purification with continuous column chromatography |
Non-Patent Citations (2)
| Title |
|---|
| HPLC分离甘肃棘豆中苦马豆素;童德文 等;《西北农业学报》;20010228;第10卷(第2期);6-8 * |
| 童德文 等.HPLC分离甘肃棘豆中苦马豆素.《西北农业学报》.2001,第10卷(第2期),6-8. |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104031849A (en) * | 2014-07-07 | 2014-09-10 | 西北农林科技大学 | Method for directional separation and authentication of swainsonine fungal endophyte Undifilum oxytropis produced through oxytropis kansuensis |
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