CN108226350B

CN108226350B - Method for determining ginsenoside in rat plasma

Info

Publication number: CN108226350B
Application number: CN201810330352.1A
Authority: CN
Inventors: 马梅伍
Original assignee: Kanglong Huacheng Beijing Biotechnology Co ltd
Current assignee: Kanglong Huacheng (Beijing) Biotechnology Co.,Ltd.
Priority date: 2018-04-13
Filing date: 2018-04-13
Publication date: 2020-09-15
Anticipated expiration: 2038-04-13
Also published as: CN108226350A

Abstract

The invention relates to detection of drug metabolism in blood plasma, in particular to a method for detecting five ginsenosides in rat blood plasma, which is characterized by comprising the following steps: after precipitation of the protein with methanol, extraction with dichloromethane, purification with a solid phase extraction column, detection of Ra1, Rb1, Rh1, Re and Ro in plasma was achieved by gas chromatography. Compared with the prior art, the method has the advantages that: the method has the advantages of high accuracy, high sensitivity and good repeatability, is suitable for simultaneous quantitative detection of the five ginsenosides in the blood plasma, and can be used for research on metabolic pathways of the ginsenosides in the blood plasma.

Description

Method for determining ginsenoside in rat plasma

Technical Field

The invention relates to a method for detecting ginsenoside in rat plasma, which comprises the simultaneous quantitative detection of five ginsenosides Ra1, Rb1, Rh1, Re and Ro.

Background

Ginseng has wide pharmacological activity on cardiovascular endocrine and immune systems of central nervous systems, and hydrolysis products of ginseng soap in intestinal tracts are considered as main pharmacological active ingredients in Asian ginseng, American ginseng and pseudo-ginseng, however, the action mechanisms of ginseng and American ginseng are still not clear, and evaluation of the exposure level of organisms to soap and shake ingredients after the ginseng preparation is taken helps to uncover the pharmacodynamic substance basis of ginseng soap and shake ingredients.

In recent years, the application of the traditional Chinese medicine pharmacokinetics research in the aspect of explaining the basis and action mechanism of the pharmacodynamics substances of the traditional Chinese medicine compound is more and more extensive, and the dynamic change rule of the processes of absorption, distribution, metabolism, excretion and the like of a single traditional Chinese medicine and the compound traditional Chinese medicine of the effective components of the traditional Chinese medicine after entering the organism through various ways is quantitatively described by utilizing the kinetics principle and the processing method, so that the traditional Chinese medicine pharmacokinetics research plays an important role in the aspects of explaining the action mechanism of the traditional Chinese medicine, scientific connotation design, preferable administration scheme, promotion of research and development of new.

The five ginsenosides Ra1, Rb1, Rh1, Re and Ro are representative ingredients of ginseng and represent part of the drug effect of ginseng. In the prior art, the method for researching the pharmacokinetics of the ginsenoside in the plasma mainly adopts liquid chromatography-tandem mass spectrometry, the instrument cost of the method is higher, and the research report of the five ginsenosides by adopting gas chromatography is not provided.

Disclosure of Invention

The invention aims to provide a method for accurately and simultaneously detecting five ginsenosides (Ra1, Rb1, Rh1, Re and Ro) in rat plasma.

The purpose of the invention is realized by the following technical scheme:

a method for detecting ginsenoside in rat plasma comprises the following steps:

(1) taking rat plasma, placing the rat plasma in a centrifuge tube, adding methanol to precipitate protein, carrying out vortex and centrifugation, taking supernatant, placing the supernatant in another clean centrifuge tube, adding dichloromethane, carrying out vortex and centrifugation;

(2) activating an Oasis HLB column, adding the supernatant, eluting with methanol, collecting eluate, concentrating, redissolving, and sampling;

(3) detection conditions are as follows: a chromatographic column: DB-35 MS; temperature rising procedure: the initial temperature is 60 ℃, the temperature is increased to 160 ℃ at the speed of 50 ℃/min, and then the temperature is increased to 220 ℃ at the speed of 20 ℃; and detecting by using an FID detector.

Further, the protein precipitation step is as follows: 100 μ L rat plasma was taken, placed in a 2mL centrifuge tube, 200 μ L methanol was added, vortexed for 1min, and centrifuged.

Further, the extraction steps are as follows: the supernatant after protein precipitation was taken and placed in another clean 2mL centrifuge tube, 500. mu.L of dichloromethane was added, vortexed for 1min, and centrifuged.

Further, the conditions of the above-mentioned two centrifugations were 1000g for 3 min.

Further, the activation steps are as follows: activating an Oasis HLB column by adopting methanol, water and dichloromethane in sequence, adding the extracted supernatant, eluting by adopting 3mL of methanol, and collecting the eluent.

Further, the concentration and redissolution steps are as follows: the eluent is taken, nitrogen is blown to be nearly dry, and the residue is re-dissolved by methanol.

Further, the chromatographic conditions were as follows: DB-35MS, 30m × 0.25mm, 0.25 μm; temperature rising procedure: the initial temperature is 60 ℃, the temperature is increased to 160 ℃ at the speed of 50 ℃/min, and then the temperature is increased to 220 ℃ at the speed of 20 ℃; the temperature of a sample inlet is 250 ℃; carrier gas: nitrogen with purity more than or equal to 99.999 percent; the flow rate is 1.2 mL/min; the sample volume is 1 mu L; no split-flow sample introduction.

Further, the detection conditions were as follows: and a FID detector, wherein the detection temperature is 250 ℃, and the flow rates of hydrogen and air are 40 mL/min and 400mL/min respectively.

Further, the ginsenosides include Ra1, Rb1, Rh1, Re and Ro.

Compared with the prior art, the invention has the advantages that: high accuracy, high sensitivity, good repeatability and good recovery rate of the added standard, and the range is 85.5-109%. In conclusion, the method is suitable for simultaneously and quantitatively detecting five ginsenosides in blood plasma, and can be used for the study of ginsenoside metabolic pathways.

Drawings

FIG. 1 is a gas chromatogram of five ginsenoside standards, wherein 1-5 are Ra1, Rb1, Rh1, Re and Ro, respectively.

Detailed Description

The invention is further described below with reference to specific examples, but the invention is not limited thereto.

Main experimental materials, instruments and reagents:

gas chromatograph with FID detector (agilent); oasis HLB column (Waters).

Ginsenoside Ra1, Rb1, Rh1, Re and Ro, the purity is more than or equal to 98 percent; SD rat; other reagents were all commercially available analytical grade.

Example 1

100 μ L rat plasma was taken, placed in a 2mL centrifuge tube, 200 μ L methanol was added, vortexed for 1min, and centrifuged. The supernatant was taken and placed in another clean 2mL centrifuge tube, 500. mu.L of dichloromethane was added, vortexed for 1min, and centrifuged. The conditions of the two centrifugations are 1000g and 3 min. Activating an Oasis HLB column by adopting methanol, water and dichloromethane in sequence, adding the extracted supernatant, eluting by adopting 3mL of methanol, and collecting the eluent. The eluent is taken, nitrogen is blown to be nearly dry, and the residue is re-dissolved by methanol.

The chromatographic conditions were as follows: DB-35MS, 30m × 0.25mm, 0.25 μm; temperature rising procedure: the initial temperature is 60 ℃, the temperature is increased to 160 ℃ at the speed of 50 ℃/min, and then the temperature is increased to 220 ℃ at the speed of 20 ℃; the temperature of a sample inlet is 250 ℃; carrier gas: nitrogen with purity more than or equal to 99.999 percent; the flow rate is 1.2 mL/min; the sample volume is 1 mu L; no split-flow sample introduction.

The detection conditions were as follows: and a FID detector, wherein the detection temperature is 250 ℃, and the flow rates of hydrogen and air are 40 mL/min and 400mL/min respectively.

Chromatograms of the five materials were recorded and quantified by standard curve method.

Example 2

Drawing of standard curve

Adding ginsenoside reference substance solutions with different volumes into a centrifugal tube respectively, drying in water bath, adding blank plasma to prepare plasma samples containing ginsenoside with different concentrations, analyzing by adopting the pretreatment-analysis method of the embodiment 1 to obtain a chromatogram, recording peak areas, and drawing a standard curve by taking the peak areas as vertical coordinates and the concentrations as horizontal coordinates. The regression curves of the ginsenosides Ra1, Rb1, Rh1, Re and Ro are as follows in sequence: y 0.057x +0.027, y 0.007x +0.215, y 0.098x-0.125, y 0.105x-0.024, and y 0.0127x + 0.114.

Recovery rate of the process

To verify the recovery rate of the method, sample-loading recovery experiments were performed at three levels, high, medium and low, respectively. The added material was then extracted and quantified. Three repeats are performed at the high, medium and low levels. The results show that the recovery rate is between 87% and 105% in three levels of 4, 20 and 100 mug/mL.

The foregoing is directed to the preferred embodiment of the present invention and is not intended to limit the invention to the specific embodiment described. It will be apparent to those skilled in the art that various modifications, equivalents, improvements and the like can be made without departing from the spirit of the invention, and these are intended to be included within the scope of the invention.

Claims

1. A method for simultaneously detecting five ginsenosides in rat plasma is characterized in that the five ginsenosides are Ra1, Rb1, Rh1, Re and Ro, and the method comprises the following specific steps:

(2) taking an Oasis HLB column, sequentially activating the Oasis HLB column by adopting methanol, water and dichloromethane, adding the supernatant, eluting by adopting 3mL of methanol, collecting eluent, taking the eluent, blowing the eluent to be nearly dry by using nitrogen gas, redissolving the residue by adopting the methanol, and injecting a sample;

(3) detection conditions are as follows: a chromatographic column: DB-35MS, 30m × 0.25mm, 0.25 μm; temperature rising procedure: the initial temperature is 60 ℃, the temperature is increased to 160 ℃ at the speed of 50 ℃/min, and then the temperature is increased to 220 ℃ at the speed of 20 ℃; the temperature of a sample inlet is 250 ℃; carrier gas: nitrogen with purity more than or equal to 99.999 percent; the flow rate is 1.2 mL/min; the sample volume is 1 mu L; no split-flow sample introduction; detecting by using an FID detector, wherein the detection temperature is 250 ℃, and the flow rates of hydrogen and air are 40 mL/min and 400mL/min respectively;

wherein the protein precipitation step is as follows: taking 100 mu L of rat plasma, placing the rat plasma in a 2mL centrifuge tube, adding 200 mu L of methanol, vortexing for 1min, and centrifuging;

wherein the conditions of the two centrifugations are 1000g and 3 min.