Reversed-phase HPLC (High Performance Liquid Chromatography) fingerprint detection method for snake venom of Changbai Mountain

A Agkistrodon halys, fingerprint technology, applied in the field of medicine, can solve the problems of poor peak shape and separation, low sensitivity, cumbersome operation, etc., and achieve the effect of high specificity, good reproducibility, and strong operability

Pending Publication Date: 2022-07-01
JINZHOU AHON PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has poor reproducibility, low sensitivity and cumbersome operation
[0006] Andrei ("New RP-HPLC Method for Separation of Naja haje haje Venom and Studies of its Bactericidal Effect." Acta Medica Marisiensis 62.1 (2016).) disclose a kind of RP-HPLC method for separating cobra venom, but the method detection consumes Long time, poor peak shape and resolution

Method used

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  • Reversed-phase HPLC (High Performance Liquid Chromatography) fingerprint detection method for snake venom of Changbai Mountain
  • Reversed-phase HPLC (High Performance Liquid Chromatography) fingerprint detection method for snake venom of Changbai Mountain
  • Reversed-phase HPLC (High Performance Liquid Chromatography) fingerprint detection method for snake venom of Changbai Mountain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 The detection method of the fingerprint of venom of white-browed pit viper in Changbai Mountain

[0065] sample

[0066] Test article: Changbai Mountain white-browed pit viper venom (batch number: 20171207-4)

[0067] Control substance: Changbai Mountain white-browed pit viper snake venom control substance (self-made)

[0068] (1) Preparation of test substance and reference substance solution: Take the test substance of the venom of Changbai Mountain white-eyed pit viper and the reference substance of Changbai mountain pit viper venom, dissolve and dilute it with water to make a solution containing 10 mg per 1 ml, centrifuge at 3000rpm and 8°C 15 minutes, take the supernatant, filter, get the test solution and reference solution;

[0069] (2) Fingerprint detection: inject 20 μl of the test solution and the reference solution into the liquid chromatograph, record the chromatogram, and use the Chinese medicine chromatographic fingerprint similarity evaluati...

Embodiment 2

[0079] Embodiment 2 Screening of test solution preparation conditions

[0080] sample

[0081] Test article: Changbai Mountain white-browed pit viper venom (batch number: 20171207-4)

[0082] (1) Preparation of test substance and reference substance solution: except that the following conditions are changed, other methods are the same as in step (1) of Example 1.

[0083]

[0084] (2) Fingerprint detection: same as step (2) in Example 1.

[0085] Result analysis

[0086] The sample stacking control chromatogram is shown in the appendix figure 1 , the results show that the solvent is pure water or 0.1% TFA solution, and the chromatographic behavior of the test solution prepared under the above centrifugation conditions is basically no difference.

[0087] Experimental results

[0088] Considering the simplicity of the experiment, the preparation method of the snake venom test solution was determined as follows: about 20 mg of snake venom + water → 3000 rpm, centrifugati...

Embodiment 3

[0089] Example 3 Selection of chromatographic column

[0090] experimental method

[0091] sample

[0092] Test article: Changbai Mountain white-browed pit viper venom (batch number: 20171207-4)

[0093] (1) preparation of test substance and reference substance solution: with step (1) of embodiment 1

[0094] (2) Fingerprint detection: the chromatographic conditions are adjusted as follows, and the rest are the same as the step (2) of Example 1

[0095] Chromatographic conditions:

[0096] ①Agilent C 18 Column (4.6×250mm, 5um; pore size 300A), 1ml / min, 20μl, 25℃, 280nm, mobile phase: A-0.1%TFA, B-acetonitrile+0.1%TFA, 10%~90%B, 60min ; Sample pan temperature: 8°C.

[0097] ②Agilent C 8 Column (4.6×250mm, 5um; pore size 300A), 1ml / min, 20μl, 25℃, 280nm, mobile phase: A-0.1%TFA, B-acetonitrile+0.1%TFA, 10%~90%B, 60min ; Sample pan temperature: 8°C.

[0098] Result analysis

[0099] The experimental results see Figures 2 to 3 , C 8 with C 18 The chromatographic beha...

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Abstract

The invention belongs to the technical field of medicines, and particularly relates to a Changbai Mountain white agkistrodon halys venom fingerprint detection method, which comprises the following steps: (1) preparation of a test solution and a reference solution: respectively taking a Changbai Mountain white agkistrodon halys venom test solution and a Changbai Mountain white agkistrodon halys venom reference solution, dissolving with a solvent, diluting, centrifuging, taking supernate, and filtering; and (2) fingerprint detection: respectively injecting the test sample and the reference substance solution obtained in the step (1) into a liquid chromatograph, recording chromatograms, and calculating the similarity by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The detection method for the snake venom fingerprint spectrum of the Changbai Mountain white agkistrodon halys has the advantages of strong operability, good reproducibility and high specificity, can be used as a standard method for detecting and identifying the snake venom of the Changbai Mountain white agkistrodon halys, and can provide more comprehensive quality information of the snake venom of the Changbai Mountain white agkistrodon halys.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for detecting the venom fingerprint of Changbai Mountain white-browed pit viper. Background technique [0002] The venom of Changbai Mountain Pit Viper (Agkistrodon, halys (pallas)) is collected from the parotid glands of the Changbai Mountain Pit Viper (Agkistrodon, halys (pallas)), and the venom of the snake is collected by the method of extrusion stimulation, and is obtained by freeze-drying. The main components are proteins, polypeptides and enzymes. Active substances are essentially proteins composed of amino acids. The composition of snake venom is very complex. Different snake species, subspecies, and even the venom secreted by the same snake due to different seasons, origins, genders, and snake ages, there are still certain differences in the toxic components. [0003] Fingerprints originate from fingerprint identification and are often used in trad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/14G01N30/54G01N30/74G01N30/88
CPCG01N30/02G01N30/06G01N30/14G01N30/54G01N30/74G01N30/88G01N2030/027G01N2030/062G01N2030/8809
Inventor 王艳秋靳美霞杜宏明聂丽
Owner JINZHOU AHON PHARM CO LTD
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