CN105403633B - Ceramide content assay method in konjaku - Google Patents

Abstract

The invention discloses a method for measuring the content of ceramide in konjac: taking ceramide ethyl acetate extract, ceramide ethanol extract and standard product solution respectively, injecting samples according to chromatographic conditions, and collecting spectrograms through high-performance liquid chromatography. The method for determining the content of ceramide in konjac provided by the invention has the characteristics of sensitivity, quickness and good reproducibility.

Description

Determination method of ceramide content in konjac

technical field

The invention relates to a method for determining the content of ceramide in konjac.

Background technique

Konjac (konjac) belongs to the perennial herbaceous plant of the genus Amorphophallus of the family Araceae, and the content of ceramide is very high, which can reach 0.15% to 0.2%. Konjac has aroused great interest as a resource plant rich in ceramides. Ceramide, namely N-fatty acyl sphingosine, is a signal transduction widely present in eukaryotic cells. As a lipid molecule, it exists as a structural component of the cell membrane or as a free molecule in the cell, and participates in the regulation of cell growth, inhibition, differentiation, aging and apoptosis. In recent years, ceramides have become important active ingredients of emerging functional medicine and health products, food and cosmetics, and have great application value.

Contents of the invention

The invention provides a method for determining the content of ceramide in konjac.

The present invention adopts following technical scheme:

A kind of ceramide content assay method in konjac:

Take the standard solution and the test solution respectively, inject samples according to the chromatographic conditions, and collect the spectrum through high-performance liquid chromatography. The standard solution includes ceramide ethyl acetate extract and ceramide ethanol extract, wherein:

Ceramide ethyl acetate extract: Precisely measure the extract prepared by the ethyl acetate process, place it in an evaporating dish, evaporate to dryness in a water bath at 70°C, add methanol for ultrasonic dissolution and constant volume, pass through a 0.45 μm microporous membrane, that is have to;

Ceramide ethanol extract: Precisely measure the supercritically extracted extract with ethanol entrainer, add volume fraction 95% ethanol to dilute to volume, then precisely measure from it, add volume fraction 95% ethanol to dilute to volume, pass 0.45 μm microporous membrane, that is;

Preparation of the standard solution: Accurately weigh the standard ceramide C6, put it in a volumetric flask, add methanol for ultrasonic dissolution and set the volume to the mark, and pass through a 0.45 μm microporous membrane to obtain the product.

Preferably, the chromatographic conditions of the high performance liquid chromatography: mobile phase solvent flow rate 0.8-1.2ml/min; acetate buffer-methanol isocratic elution; chromatographic column temperature 25-40°C; evaporative light scattering detector.

Preferably, the mobile phase solvent is 0.1% acetic acid:methanol=7:93 by volume fraction.

Preferably, the mobile phase solvent is 36% acetic acid:methanol=7:93 by volume fraction.

Preferably, the flow rate of the mobile phase solvent is 1ml/min.

Preferably, the spectrum acquisition time is 30 minutes.

The method for determining the content of ceramide in konjac provided by the invention has the characteristics of sensitivity, quickness and good reproducibility.

Description of drawings

Fig. 1 is ethyl acetate extract liquid chromatogram of the present invention;

Fig. 2 is ethanol extract chromatogram of the present invention;

Fig. 3 is a standard curve diagram of ceramide C6 of the present invention.

Detailed ways

The following examples are further detailed descriptions of the present invention.

1 Experimental instruments and materials

1.1 Experimental Instruments

High-performance liquid chromatography (model: U3000; manufacturer: Thermo Fisher Scientific), evaporative light scattering detector (model: M100); chromatographic column: Kromasil 100-5C18 (5μm, 250×4.6mm); methanol (chromatographic grade); 36% acetic acid (analytical pure)

1.2 Materials

Ethyl acetate extract, ethanol extract.

2 Experimental content

2.1 Chromatographic conditions

Mobile phase: 0.1% acetic acid (36% acetic acid):methanol=7:93; isocratic elution; collection 30min; column temperature 35°C; flow rate 1ml/min; evaporative light scattering detector.

Remarks: 1. Chromatographic column: Kromasil 100-5C18 (5μm, 250×4.6mm); elution system: acetate buffer-methanol; collection 30min; column temperature 25-40℃; flow rate 0.8-1.2ml/min; evaporation light scatter detector.

2. After comparing the chromatograms of Kromasil C18 chromatographic column, Kromasil 100-5C18 chromatographic column, and Innoval C18 chromatographic column, three reversed-phase C18 silica gel chromatographic columns with different bonding methods can be drawn: Kromasil C18 chromatographic column and Kromasil 100-5C18 chromatographic column The peak responses of the two peaks are consistent, with only differences in the retention times of the peaks. However, for the Innoval C18 chromatographic column made by nanotechnology, its peak time and peak response are quite different from the above two chromatographic columns.

2.2 Preparation of the test solution

(1) Ceramide ethyl acetate extract: Precisely measure 2mL of the extract prepared by ethyl acetate process, put it in an evaporating dish, evaporate to dryness in a water bath at 70°C, add methanol to dissolve it ultrasonically and dilute to 10mL, pass through 0.45 μm microporous membrane, ready to use.

(2) Ceramide ethanol extract: Accurately measure 0.2mL of the supercritically extracted extract with ethanol entrainer, add 95% ethanol to dilute to 10mL, then accurately measure 2mL from it, add 95% ethanol to dilute to Make up to 10mL, pass through a 0.45μm microporous membrane, and obtain.

2.3 Preparation of standard solution

Accurately weigh 5.04mg of ceramide C6 standard substance, put it in a 10mL volumetric flask, add methanol to dissolve it ultrasonically, adjust the volume to the mark, and pass through a 0.45μm microporous membrane to obtain the product.

2.3 Determination method

Take the standard solution and the test solution respectively, inject samples according to the chromatographic conditions, and collect the spectra.

3 Experimental results and conclusions

3.1 Identification of chromatographic peaks

(1) Ethyl acetate extract: as shown in Figure 1, from the above chromatogram, it can be concluded that only ceramide C6 exists in the ethyl acetate extract.

(2) Ethanol extract: as shown in Figure 2, from the above chromatogram, it can be concluded that only ceramide C6 exists in the ethanol extract.

3.2 Determination of the content of ceramide C6 in the extract

3.2.1 Drawing of standard curve

As shown in Figure 3, accurately measure the prepared standard solution, inject 2, 6, 10, 12, 14, and 18 μL respectively, and establish a linear equation based on the logarithm of the injection volume and the logarithm of the peak area, Y =1.3785X++5.4958; R ² =0.9985.

3.2.2 Determination results of ceramide C6 content in the extract

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (1)

1. a method for assaying ceramide content in konjac, is characterized in that: Take the standard solution and the test solution respectively, inject samples according to the chromatographic conditions, and collect the spectrum through high-performance liquid chromatography. The standard solution includes ceramide ethyl acetate extract and ceramide ethanol extract, wherein: Ceramide ethyl acetate extract: Precisely measure the extract prepared by the ethyl acetate process, place it in an evaporating dish, evaporate to dryness in a water bath at 70°C, add methanol for ultrasonic dissolution and constant volume, pass through a 0.45 μm microporous membrane, that is have to; Ceramide ethanol extract: Precisely measure the supercritically extracted extract with ethanol entrainer, add 95% ethanol to dilute to volume, then precisely measure from it, add 95% ethanol to dilute to volume, pass 0.45 μm microporous membrane, that is; Preparation of the standard solution: Accurately weigh the standard ceramide C6, put it in a volumetric flask, add methanol for ultrasonic dissolution and set the volume to the mark, pass through a 0.45 μm microporous membrane to obtain the product; The chromatographic conditions of the high performance liquid chromatography: mobile phase solvent flow rate 0.8-1.2ml/min; chromatographic column column temperature 25 ~ 40 ℃; evaporative light scattering detector; The mobile phase solvent is 0.1% acetic acid:methanol=7:93 or 36% acetic acid:methanol=7:93; Spectrum acquisition time was 30 minutes.