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Method for detecting bufadienolide component in musk Tongxin dropping pill in rat plasma and application of bufadienolide component

A technology of bufadienolactone and detection method, which is applied in the direction of measuring devices, material separation, and analysis materials, etc., can solve the problems of complex substrate components, long detection time, and baseline drift of blank serum, and is beneficial to batch detection and detection The effect of shortened time and convenient operation

Pending Publication Date: 2022-08-05
INNER MONGOLIA CONBA PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the retention time of all the chromatographic peaks of the five kinds of bufadienolactone components detected in this paper is greater than 70min, and the detection time is long. For quantification, the quantitative value cannot be judged directly by the retention time and peak area of ​​the MRM chromatogram; the base composition of blank serum is complex, which has a great impact on the detection results of bufadienolactone components

Method used

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  • Method for detecting bufadienolide component in musk Tongxin dropping pill in rat plasma and application of bufadienolide component
  • Method for detecting bufadienolide component in musk Tongxin dropping pill in rat plasma and application of bufadienolide component
  • Method for detecting bufadienolide component in musk Tongxin dropping pill in rat plasma and application of bufadienolide component

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Animal feeding, administration and preparation of standard solution

[0044] 1. Animal feeding: SPF grade SD rats, weighing 200g-250g, male. The temperature is 20~26℃, the relative humidity is 40~70%, and the light and dark are alternated for 12h. Free access to water and standard block maintenance diets were provided. Before the test, the animals were acclimated in the experimental animal room for 5 days. Fasting for 12 hours before blood collection, can not help but water.

[0045] 2. Administration: SD rats were weighed, the dosage was calculated, and 6.4 g / kg was administered by oral gavage respectively, and the blank group was given a solvent control.

[0046] 3. Blood collection: add heparin sodium as an anticoagulant in the blood collection tube. The blank blood sample was collected from abdominal aorta, 14 rats, each at least 5 mL, and blood was collected on the same day. Positive blood samples were collected from the ocular venous sinus. The bl...

Embodiment 2

[0048] Example 2: Establishment of detection method 1

[0049] 1. Plasma test sample processing

[0050] Take 200 μL of rat plasma of Shexiang Tongxin Dropping Pills in Example 1, add 5 μL of tinidazole solution with a concentration of 2 μg / mL, mix well, add 1 mL of methanol, vortex for 5 min, and centrifuge at 10,000 r / min at 4°C for 10 min; The supernatant was blown to near dryness with nitrogen at room temperature, reconstituted by adding 300 μL of methanol, vortexed for 5 min, and centrifuged at 10000 r / min at 4°C for 5 min. The supernatant was filtered through a membrane to prepare a plasma test sample.

[0051] 2. Standard curve preparation

[0052] The standard solution of bufadienolide was added to the blank plasma to prepare a series of plasma standard curve samples; the method of step (1) was used to replace the plasma standard curve samples by gavage of the rat plasma of Shexiang Tongxin Dropping Pills to prepare standard curve samples. Curve plasma samples; injec...

Embodiment 3

[0075] Example 3: Establishment of detection method 2

[0076] 1. Plasma test sample processing

[0077] Take 100 μL of rat plasma of Shexiang Tongxin Dropping Pill by gavage in Example 1, add 2.5 μL of tinidazole solution with a concentration of 1 μg / mL, mix well, add 0.3 mL of methanol, vortex for 7 min, and centrifuge at 8000 r / min at 2°C for 5 min; Take the supernatant, blow it to near dryness with nitrogen at room temperature, add 200 μL of methanol to reconstitute, vortex for 5 min, centrifuge at 8000 r / min at 2°C for 5 min, take the supernatant filter membrane, and prepare a plasma test sample.

[0078] 2. Standard curve preparation

[0079] The standard solution of bufadienolide was added to the blank plasma to prepare a series of plasma standard curve samples; the method of step (1) was used to replace the plasma standard curve samples by gavage of the rat plasma of Shexiang Tongxin Dropping Pills to prepare standard curve samples. Curve plasma samples; inject the s...

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Abstract

The invention provides a method for detecting bufadienolide components of musk Tongxin dripping pills in rat plasma and application of the method, and particularly relates to a method for qualitatively and quantitatively detecting arenobufogenin, telocinobufagin, deacetylcinobufagin, bufalin and resibufogenin in the rat plasma by adopting ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The method comprises the following specific steps: adding an internal standard substance into intragastric musk heart-dredging dropping pill rat plasma, and carrying out methanol protein precipitation; and the detection is carried out through UPLC-MS / MS. Wherein the detection limit of bufalin is 1.00 ng / mL, and the quantification limit of bufalin is 3.00 ng / mL; the detection limit of arenobufogenin, telocinobufagin and resibufogenin is 2.00 ng / mL, and the quantification limit is 6.00 ng / mL; the detection limit of the deacetylated cinobufagin is 3.00 ng / mL, and the quantification limit of the deacetylated cinobufagin is 9.00 ng / mL. According to the method, detection can be completed within 15 min, quantitative calculation is completed through the retention time and the peak area ratio of an MRM chromatogram in combination with a standard curve, operation is more convenient, interference of endogenous substances in plasma is reduced, and the method can be used for pharmacokinetic research of the musk Tongxin dripping pills.

Description

technical field [0001] The invention relates to a method for detecting compound traditional Chinese medicine components in rat plasma and use thereof, in particular to the detection of bufadienolide components in Shexiang Tongxin Dropping Pills in plasma. Background technique [0002] Shexiang Tongxin Dropping Pill is a traditional Chinese medicine compound used for stable angina pectoris with exertional coronary heart disease. The whole recipe includes toad venom, artificial musk, Salvia miltiorrhiza, total saponins from ginseng stems and leaves, artificial bezoar, bear bile powder and seven herbs of borneol. Studies have shown that Shexiang Tongxin Dropping Pills works by reducing endothelin-1 (ET-1), C-reactive protein (CRP) and tumor necrosis factor alpha (TNF-α) in the blood, while increasing the level of nitric oxide in the blood. Protects endothelial cells from atherosclerotic damage. In addition, Shexiang Tongxin Dropping Pills can reduce harmful esters in the huma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/34
CPCG01N30/88G01N30/34G01N2030/8822G01N2030/027Y02A50/30
Inventor 叶剑锋姚建标宋永标许照李学臻麻懿文朱林娜徐斌李绵葵吴飞群
Owner INNER MONGOLIA CONBA PHARMA CO LTD
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