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Method for detecting metabolite relative to psoralea corylifolia hepatotoxicity and application thereof

A technology for metabolites and hepatotoxicity, applied in the field of drug metabolism and toxicity research, can solve the problems of active metabolites such as strong electrophilicity, inability to separate, identify, and unstable existence, so as to facilitate early warning and drug toxicity Effect

Active Publication Date: 2014-12-31
TIANJIN UNIV OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The active metabolites cannot exist stably due to their strong electrophilicity, and cannot be separated and identified by conventional means.

Method used

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  • Method for detecting metabolite relative to psoralea corylifolia hepatotoxicity and application thereof
  • Method for detecting metabolite relative to psoralea corylifolia hepatotoxicity and application thereof
  • Method for detecting metabolite relative to psoralea corylifolia hepatotoxicity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 IC 50 shift experiment

[0074] experimental method:

[0075] (+) NADPH group: HLMs 25μL (4mg / ml), NADPH 50μL (1mM), psoralen solution 25μL (0-200μM);

[0076] (-) NADPH group: HLMs 25 μL (4 mg / ml), PBS 50 μL (Ph7.4), psoralen solution 25 μL (0-200 μM);

[0077] After the above two groups of reaction solutions were pre-incubated at 37°C for 30 minutes, take out 20 μL each and add 80 μL LCYP450s substrate, 1.0 mM NADPH100 ​​μL and incubate for 15 minutes, then add 2 times the volume of ice stop solution (containing 75 ng / ml carbamazepine ice methanol solution) to stop the reaction, vortex After centrifugation, the supernatant was taken and analyzed by LC / MS / MS to obtain the concentration of metabolites of each probe substrate.

[0078] Experimental results: see figure 1 and Table 1.

[0079] Table 1 IC of psoralen on CYP450s under (+) / (-)NADPH condition 50 value

[0080]

[0081] In the presence of NADPH, psoralen is a product metabolized by CYP1A2,...

Embodiment 2

[0083] Example 2 Identification and detection of RMs produced by incubation of psoralen in liver microsomes

[0084] experimental method:

[0085] 1 mg / ml HLMs, 50 mM psoralen, 5 mM GSH 50 μL each, incubate at 37 °C for 90 min, add 400 μL ice methanol to terminate the reaction, vortex for 10 s, centrifuge at 14000 r / min for 10 min, take the supernatant N 2 Blow dry, redissolve in 100 μL methanol-water (1:1), and analyze the sample by LC / MS / MS.

[0086] LC and MS Conditions

[0087] Liquid phase conditions:

[0088] Chromatographic column: Acquity UPLC BEH C18 column (1.7μm, 2.1mm×100mm);

[0089] Solvent system: made of 0.1% CH 3 Composition of COOH (A) and 100% ACN (B), using gradient elution:

[0090] 0-7min: 5%-80% B (volume percentage), the balance is A; 7-10min: 5% B, the balance is A.

[0091] Full-scan mass spectrometry conditions: ion mode: ES+; capillary voltage: 3.20kV; cone voltage: 20.0V; source temperature: 110°C; desolvation temperature: 350°C; collision ...

Embodiment 3

[0115] Example 3 Psoralen epoxides induce hepatotoxicity in psoralen mice

[0116] Test drug

[0117] (1) ABT: The administration dose is 100mg / kg, the administration solution is prepared with physiological saline, and administered by intraperitoneal injection;

[0118] (2) BSO: The dosage is 650mg / kg, the drug solution is prepared with physiological saline, and administered by intraperitoneal injection;

[0119] (3) Psoralen: The dosage is 300mg / kg, and 0.5% sodium carboxymethyl cellulose is prepared as a drug solution, and administered by intragastric administration.

[0120] animal grouping

[0121] There are 8 groups of Kunming mice at 6-9 weeks, and the groups and the number of animals in each group are as follows:

[0122] (1) Control group (Control): 10 rats;

[0123] (2) ABT group: 10;

[0124] (3) Psoralen group: 20 rats;

[0125] (4) Psoralen+ABT group: 20 rats;

[0126] (5) BSO group: 30;

[0127] (6) BSO+ABT group: 10;

[0128] (7) Psoralen+BSO group: 20...

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Abstract

The invention provides a method for detecting metabolite relative to psoralea corylifolia hepatotoxicity. The metabolite is psoralen and / or a dihydrodiol metabolite of isopsoralen; the method comprises acquisition of psoralen and / or the dihydrodiol metabolite of isopsoralen, a tandem mass spectrum is used, and a multiple reaction monitoring method is used for detecting the metabolite. The provided method in the application for monitoring hepatotoxicity of psoralen, isopsoralen, a composition containing psoralen and / or isopsoralen provides basis for safe medication and early warning of medicine toxicity.

Description

technical field [0001] The invention relates to the field of drug metabolism and toxicity research, in particular to a method for detecting hepatotoxic metabolites produced by psoralen and its application. Background technique [0002] As the most important drug metabolism organ, the liver is also the main target organ of drug toxicity. In the process of drug use, the liver injury caused by the drug itself or its metabolites, or due to the hypersensitivity of the special constitution to the drug is called drug-induced liver injury (Drug-induced Liver Injury, DILI), according to the statistics of the World Health Organization , DILI has risen to the fifth leading cause of death worldwide. More and more reports have shown that many commonly used traditional Chinese medicines and even some traditional tonics have liver toxicity, such as psoralen, evodia, and Polygonum multiflorum, etc. Liver injury caused by traditional Chinese medicines was mostly detected in clinical cases ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 何新俸珊马叶涛颜晶晶翟毅然
Owner TIANJIN UNIV OF TRADITIONAL CHINESE MEDICINE
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