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Method for detecting content of swainsonine in locoweed endophytic fungi

A technology of swainsonine and endophytic fungi, which is applied in the field of analytical chemistry, can solve the problems of limited application, lack of luminescent groups in swainsonine, and difficult detection of swainsonine, and achieve strong specificity and high sensitivity , good reproducibility

Inactive Publication Date: 2013-09-11
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Due to the lack of luminescent groups in swainsonine itself, it is difficult to detect swainsonine by HPLC-UV detection, which limits the application of traditional high performance liquid chromatography in the detection of swainsonine

Method used

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  • Method for detecting content of swainsonine in locoweed endophytic fungi

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1: the drafting of swainsonine standard curve

[0025] 1) Preparation of SW standard solution: Accurately weigh SW standard, dissolve in mobile phase, and serially dilute.

[0026] 2) Experimental equipment and chromatographic conditions: Waters 1525 high performance liquid chromatography, binary pump; Waters 2424 evaporative light scattering detector, parameter setting gain value of 150, carrier gas flow rate of 25 psi, drift tube temperature of 55 °C, atomization 36°C, injection volume 20 μL; Waters XBridge? HILIC chromatographic column (specification 150mm×4.6mm, 3.5μm); the control and recording of the chromatographic separation system are completed by the Breeze 2.0 workstation. The mobile phase was composed of acetonitrile and ammonium acetate buffer, the volume ratio was 1:1, the concentration of ammonium acetate was 5 mmol / L, and 0.02% ammonia water was added to the total volume. The flow rate of the mobile phase was 0.5 ml / min. For the results of t...

Embodiment 2

[0028] Example 2 Endophytic fungi Undifilum oxytropis - Detection of FEL4-F5 samples

[0029] 1) Pretreatment of samples: take Undifilum oxytropis - 0.5 g of dried mycelium of FEL4-F5, add 50 mL of methanol to the extractor, and extract at 60 °C for 24 h. The methanol extract was concentrated and evaporated to dryness, and the residue was dissolved in 10 mL of 2% acetic acid solution, and the solution was added to an exchange column containing a 5 cm high AG 50W cationic resin, and it was dynamically circulated for 1 h to make swainsonine Fully combine with the cationic resin; then wash the impurities in the resin with 100 mL of deionized water; then elute the resin column with 100 mL of 1 mol / L ammonia solution, collect the ammonia eluate, concentrate and evaporate to dryness, and dissolve with 2 mL of methanol , and the supernatant was taken after centrifugation and evaporated for later use. Dilute (30 times, W / V) with 15 mL of mobile phase before injection, see the res...

Embodiment 3

[0031] Example 3 Detection of SW content of endophytic fungi isolated from different locoweeds

[0032] The treatment of endophytic fungi samples is the same as in Example 2, and the experimental equipment and chromatographic conditions are the same as in Example 1. Endophytic fungi detected by HPLC-ELSD Undifilum oxytropis The SW content in the medium is as follows:

[0033] Loco species place of origin Endophytic fungi SW content (μg / g) O. ochrocepala Ningxia Haiyuan County FEL4-F5 4963.14±35.55 A. strictus Shigatse, Tibet FEL5-G3 3077.64±27.33 O. glabra Gansu Minqin FEL1b-B4 2043.20±22.66 O. kansuensis Gansu FEL3-E9 2334.41±48.25

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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 and ammonium 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 to obtain 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 [0001] The invention belongs to the technical field of analytical chemistry, and relates to the separation and determination of swainsonine in endophytic fungi, in particular to a method for determining the content of swainsonine in locoweed endophytic fungi. Background technique [0002] Swainsonine (SW) is an indolizidine alkaloid with the chemical name 1,2,8-trihydroxy-8-hydro-indolizidine. SW is the main toxic component in locoweed (a general term for poisonous plants of the legume genus Oxytropis and Astragalus). Locoweeds are widely distributed around the world, and animals can cause poisoning and even death after eating a large amount of them. [0003] Swainsonine is not only a plant-derived toxin, but it also has the effect of inhibiting the growth and metastasis of tumor cells. Because SW is a very strong α-mannosidase inhibitor, it can not only inhibit the expression of glycoproteins on the surface of tumor cells, but also inhibit the growth and m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
Inventor 王建华杨国栋王妍李金成耿果霞
Owner NORTHWEST A & F UNIV
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