The method used to study the inhibitory effect of pqq on ages

A technology of inhibition and CEL, applied in the field of biological analysis, which can solve the problems of time-consuming, complicated operation, residual mass spectrometer, etc.

Active Publication Date: 2021-03-23
JIANGSU INST OF NUCLEAR MEDICINE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] The detection methods of CML and CEL reported in the literature mainly include: (1) Enzyme-linked immunosorbent assay (ELISA), which has the disadvantage of high non-specificity;
(2) High-performance liquid chromatography combined with fluorescence detection method, using o-phthalaldehyde or FMOC-Cl pre-column derivatization, takes a long time and has large interference factors
(3) Gas chromatography-mass spectrometry (GC-MS), the operation process is relatively complicated
(4) Liquid chromatography-tandem mass spectrometry detection method, sample pretreatment and ion-pairing reagents are used in the mobile phase, which is easy to remain in the mass spectrometer, resulting in a decrease in sensitivity
The UPLC-MS / MS established in the present invention combined with solid-phase extraction method to study the changes of PQQ to the plasma AGEs content of AD model mice caused by D-gal injury has not been reported in the literature

Method used

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  • The method used to study the inhibitory effect of pqq on ages
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  • The method used to study the inhibitory effect of pqq on ages

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 The research method of the inhibitory effect of PQQ on AGEs, the steps are:

[0040] (1) Preparation of standard solution and internal standard solution: Accurately weigh appropriate amounts of CML and CEL standard products, dissolve them in 70% methanol to prepare CML and CEL standard stock solutions with a concentration of 200 μg / mL, and weigh appropriate amounts of [ 2 h 2 ] - CML and [ 2 h 4 ]–CEL standard product, dissolved in 70% methanol solution to prepare a 50μg / mL internal standard stock solution, and stored in a refrigerator at 4°C; pipette an appropriate amount of CML and CEL standard solution, accurately add the internal standard solution, and dilute with water to determine The concentration of the series standard solution is about 1-800 ng / mL, and the concentration of the internal standard solution is 100 ng / mL.

[0041] (2) Plasma sample pretreatment: Take 50 µl plasma sample, add 400 µL borax buffer solution (pH 9.2, 0.2M), add 100 µL 100mM ...

Embodiment 2

[0055] Embodiment 2 The impact of different solid-phase extraction cartridges on the recovery rate of CML and CEL

[0056] Plasma samples containing high, medium and low concentrations of CML and CEL were added to the blank plasma respectively. Different solid-phase extraction cartridges were used to process plasma samples, and the recoveries were determined. The results are shown in Table 1.

[0057] Table 1 Effect of different SPE cartridges on the recovery of CML and CEL

[0058]

[0059] The experimental results show that the recovery rate of ordinary ODS-C18 solid-phase extraction cartridges is low, and Styre Screen® H2P is polymerized from hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene, and has strong adsorption for polar compounds ability. Compared with ODS-C18 solid phase extraction, the recovery rate is slightly improved. The Sepax CUALD solid phase extraction column uses silica gel as a filler, which is bonded with different covalent groups to fo...

Embodiment 3

[0060] Example 3 Effect of PQQ on plasma CML, CEL, protein-bound CML and protein-bound CEL

[0061] The plasma samples of each group were processed and determined according to the above method. For the measurement results, see Figure 5. After 6 weeks of D-gal-induced modeling, plasma free CML, CEL, protein-bound CML and protein-bound CEL increased in varying degrees compared with the control group, and the plasma CML content decreased significantly after administration of donepezil and PQQ; at the same time, PQQ can also Reduced plasma CEL and protein-bound CML; donepezil had no effect on both; however neither PQQ nor donepezil had any effect on plasma protein-bound CEL. It provides a new basis for the study of the mechanism of PQQ in aging diseases.

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Abstract

The invention relates to a method for researching an inhibiting effect of PQQ (pyrroloquinoline quinone) on AGEs. The method is a researching result of the inhibiting effect of PQQ and later period glycosylation end product AGEs after the method for searching on reaction of PQQ, Lys and Arg and belongs to the technical field of bioanalysis. N epsilon-(carboxymethyl)lysine (CML) and N epsilon-(carboxyethyl)lysine (CEL) are two of the most primary monomers of AGEs. The invention optimizes and improves the method for determining CML and CEL, adopts n-butanol / HCl derivative reaction, solid-phase extraction and UPLC-MS / MS for detecting the contents of CML and CEL in plasma and has the characteristics of high efficiency, accuracy and sensitivity; the established method is adopted for analyzing the change of CML, CEL, protein-binding CML and protein-binding CEL after a D-gal (D-galactose) induced aging rat is intervened with a certain amount of donepezil (Don) and PQQ, so as to supply a new basis for pathogenesis and drug targets of degenerative diseases.

Description

technical field [0001] The research method of the inhibitory effect of PQQ on AGEs is following the research method of the reaction behavior of PQQ (pyrroloquinoline quinone) with Lys (lysine) and Arg (arginine). The result of product AGEs inhibition belongs to the technical field of bioanalysis. Background technique [0002] Senile dementia (Alzheimer disease, AD) is a common disease of the elderly. It is a brain disease mainly manifested by primary mental retardation. Its pathogenesis is still unclear. According to previous assumptions, AD results from the accumulation of Aβ protein and the formation of amyloid in the brain. Therefore, drugs that inhibit the formation of amyloid are under continuous research and development, but the results of clinical trials have no significant effect on the treatment of AD. To effectively prevent and treat AD, new drug targets and mechanisms of action need to be discovered. [0003] Advanced glycation end products (AGEs) are chronic n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/027
Inventor 顾晓波周杏琴蔡刚明张荣军俞惠新包建东
Owner JIANGSU INST OF NUCLEAR MEDICINE
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