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A kind of extraction and high performance liquid phase detection method of Antarctic krill β-ecdysterone

An Antarctic krill and ecdysone technology, which is applied in the fields of food and chemical industry, can solve the problems of high price of enzyme-linked immunization, complicated extraction steps, low accuracy of results, etc., and achieves accurate and reliable peak area quantification, good detection effect, and detection limit. low effect

Inactive Publication Date: 2020-09-04
SHANDONG NORMAL UNIV
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Problems solved by technology

[0004] β-ecdysone was discovered for the first time in dew grass. Because of its rich content in plants, there are many researches on β-ecdysone in plants. For the extraction of β-ecdysone in plants, water boiling and ethanol extraction are mostly used. And through column chromatography or sherwood oil degreasing and purification, the extraction steps are complicated and time-consuming; and for the detection of β-ecdysone in plants, there are currently thin-layer scanning methods, radioimmunoassay methods, liquid chromatography, etc., as disclosed in CN103837615A A method for the simultaneous determination of plant ecdysterone substances by high performance liquid chromatography was developed. Xu Lirong disclosed the extraction, purification process and quality standard research of β-ecdysterone in Dewweed Grass. Wang Qiang et al. -The content determination of ecdysterone (thin-layer chromatography and high-performance liquid phase), but described method still has the shortcomings such as pretreatment is comparatively loaded down with trivial details, sample consumption is big, result accuracy is low
The β-ecdysone in animals has been reported more in insects and crustaceans decapod. Most of them use animal serum or lymph fluid, and then extract, purify, and dissolve with the prepared lysate for enzyme-linked immunoassay or high-efficiency liquid Phase, liquid chromatography-tandem mass spectrometry detection, such as Zhao Peng et al. disclosed the research of high performance liquid chromatography-triple quadrupole mass spectrometry analysis of 20-hydroxyecdysterone in the lymph and muscle of marine crustaceans, but its operation is relatively complicated. ELISA is expensive and time-consuming in existing tests
[0005] At present, there is no report about the method of extracting β-ecdysone from Antarctic krill and its analysis and detection

Method used

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  • A kind of extraction and high performance liquid phase detection method of Antarctic krill β-ecdysterone
  • A kind of extraction and high performance liquid phase detection method of Antarctic krill β-ecdysterone

Examples

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Embodiment 1

[0037] Example 1: Extraction of Antarctic krill β-ecdysone and detection by high performance liquid chromatography

[0038] 1, the extraction of Antarctic krill β-molting hormone, the steps are as follows:

[0039] (1) Take the frozen Antarctic krill, thaw it in 5% normal saline for 3 hours, absorb the water from the thawed Antarctic krill, and cut it into pieces;

[0040] (2) Take shredded Antarctic krill (w) and methanol (v) with a ratio of 1:5-8, ultrasonically extract under the conditions of 35°C, 300w, and 40kHz, extract 3 times for 30min each time, and filter the extract , combining the 3 extracts;

[0041] (3) Place the above-mentioned filtrate in a rotary evaporator and evaporate to dryness, and the rotary evaporation temperature is 50° C. to obtain the crude extract of β-ecdysone;

[0042] (4) Completely dissolve the crude extract with 40ml of methanol, add an equal volume of n-hexane, ultrasonically mix, centrifuge at 3000rpm at 4°C for 30min, discard the supernata...

Embodiment 2

[0050] Example 2: Extraction and HPLC detection of Antarctic krill β-ecdysone

[0051] Frozen Antarctic krill, thawed in 5% normal saline, thawed for 3 hours, blotted dry, took 100g of thawed Antarctic krill, chopped the tissue, put it into a 1000ml beaker, added 500ml of methanol, and heated it at 25℃, 250w, 40kHz Ultrasonic heating and extraction in an ultrasonic cleaning machine, each extraction was 30min, repeated 3 times, the extracts were combined and filtered, and the filtrate was evaporated to dryness in a rotary evaporator at an evaporation temperature of 50°C to obtain a crude extract of β-ecdysone.

[0052] Completely dissolve the crude extract with 40ml of methanol, add an equal volume of n-hexane, mix it with ultrasound, centrifuge at 3000rpm at 4°C for 30min, discard the supernatant, repeat three times, put the lower layer solution in a -20°C refrigerator, take the supernatant, Impurities were removed, and the supernatant was evaporated to 3 ml in a rotary evapor...

Embodiment 3

[0055] Example 3: Extraction of Antarctic krill β-ecdysone and detection by high performance liquid chromatography

[0056] Frozen Antarctic krill, thawed in 5% normal saline, thawed for 3 hours, blotted dry, took 100g of thawed Antarctic krill tissue, cut it into pieces, put it into a 1000ml beaker, added 800ml of methanol, and heated it under 30℃, 300w, 40kHz ultrasonic waves Ultrasonic heating and extraction in a cleaning machine, each extraction for 30 minutes, repeated 3 times, combined extracts, and filtered, the filtrate was evaporated to dryness in a rotary evaporator at an evaporation temperature of 50°C to obtain a crude extract of β-ecdysone.

[0057] Completely dissolve the crude extract with 40ml of methanol, add an equal volume of n-hexane, mix it with ultrasound, centrifuge at 3000rpm at 4°C for 30min, discard the supernatant, repeat three times, put the lower layer solution in a -20°C refrigerator, take the supernatant, Impurities were removed, and the supernat...

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Abstract

The invention discloses extraction and high-performance liquid detection methods of beta-ecdysone in euphausia superba. Fresh frozen euphausia superba is taken as a material, subjected to unfreezing and tissue cutting treatment, ultrasonic extraction with methanol and degreasing with n-hexane and frozen at subzero 20 DEG C for removal of other impurities, and an extract containing beta-ecdysone isobtained; a high-performance liquid detection method is adopted for detecting the beta-ecdysone in the euphausia superba under the conditions of a UV / Visible detector, a YMC-PACK ODS-A250*4.6 mml.D.Schromatographic column, mobile phase methanol-water, isocratic elution and detection wavelength 248 nm, the peak area is tracked and integrated by Breeze software, and the content of the beta-ecdysone in the euphausia superba is obtained. The operation method is simple and quick and the result is accurate and reliable.

Description

technical field [0001] The invention relates to an extraction and high-efficiency liquid phase detection method of Antarctic krill β-molting hormone, belonging to the technical fields of food and chemical industry. Background technique [0002] β-ecdysterone, also known as β-ecdysterone, 20-hydroxyecdysterone; molecular formula is C 27 h 44 o 7 , with a molecular weight of 480.63 Daltons, is a light yellow crystalline powder. In animals and plants, β-ecdysone participates in the regulation of many aspects such as molting, reproduction, lifespan, diapause and immune response, and plays an important role in maintaining basic physiological functions. β-ecdysone acts on the human body to promote cell growth and division, accelerate collagen synthesis, stimulate dermal cell division, and promote skin regeneration. At the same time, a large number of animal experiments have proved that β-ecdysone can eliminate cholesterol in the body, promote protein, carbohydrate, and lipid m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/14G01N30/88
CPCG01N30/06G01N30/14G01N30/88G01N2030/146G01N2030/8813
Inventor 韩香凝刘代成
Owner SHANDONG NORMAL UNIV
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