Immobilized enzyme based method for rapidly evaluating inhibition of activity of marine natural product cyclooxygenase 2

A technology of immobilized enzymes and natural products, which is applied in the field of rapid screening of COX-2 enzyme inhibitors and rapid screening of COX-2 inhibitors in natural products, which can solve problems such as cumbersome operations, difficult sample preparation, and restrictions on rapid screening of active ingredients , to achieve the effect of mild conditions, high research value, fast and convenient recycling and reuse

Active Publication Date: 2018-11-06
OCEANOGRAPHIC INSTR RES INST SHANDONG ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional chemical research methods of marine natural products have problems such as low discovery efficiency of active substances, cumbersome operations, difficult sample preparation,

Method used

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  • Immobilized enzyme based method for rapidly evaluating inhibition of activity of marine natural product cyclooxygenase 2
  • Immobilized enzyme based method for rapidly evaluating inhibition of activity of marine natural product cyclooxygenase 2
  • Immobilized enzyme based method for rapidly evaluating inhibition of activity of marine natural product cyclooxygenase 2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Surface Amino Modification of SiO 2 Preparation of microspheres

[0056] (1) Activation of silica gel: Take 2.0 g of silica gel and boil it with 5% HCl for 45 minutes to remove organic matter adsorbed on the surface, then wash it with ultrapure water, and dry it at 80°C for 24 hours.

[0057] (2) Amino modified SiO 2 Preparation of microspheres (APS-Si): 0.50 g of activated SiO 2 The carrier was placed in 20 mL of toluene solution containing 5% silylating agent APS, and refluxed at 90°C for 24 hours. SiO bonded with APS 2 Wash with acetone several times to remove the unreacted silylating agent, volatilize excess acetone at room temperature, dry in an oven at 110°C for 6 hours, and store for future use.

[0058] (3) Take a small amount of activated SiO 2 , APS-Si powder is adhered to the sample holder, which is then placed in an ion sputtering apparatus for gold plating. After the gold plating is completed, it is transferred to the FESEM sample cell, and ...

Embodiment 2

[0059] The preparation of the APS-Si immobilized COX-2 enzyme of embodiment 2 surface amino groups

[0060] (1) Prepare surface amino-modified APS-Si according to Example 1.

[0061] (2) Preparation of PBS: weigh 4.56gK 2 HPO 4 Dissolve in 200mL ultra-pure water as liquid A, weigh 2.72g KH 2 PO 4 Dissolve in 200mL ultrapure water to form B solution. Mix A and B at a volume ratio of 80.2:19.8 to obtain a PBS buffer solution with a concentration of 0.1mol / L and a pH of 7.4.

[0062] (3) Precisely weigh 0.050g APS-Si and place it in 2.0mL PBS buffer solution to make it fully swell, then add 10µL GA solution with a concentration of 50% (w / w), shake at a constant temperature at 28°C for 6h, and then use Wash with ultrapure water until there is no GA in the cleaning solution, and filter until dry. Add 1mL PBS buffer and 20U COX-2 solution (20µL) to the cross-linked silica gel carrier, shake and fix at 20°C for 24h, after the reaction is completed, wash with 3.5% NaCl solution, ...

Embodiment 3

[0064] Example 3 Determination of immobilized COX-2 enzyme and free enzyme activity.

[0065] (1) Preparation of levoephrine solution: Weigh a certain amount of levoephrine into a brown volumetric flask, add Tris-HCl and dissolve with a small amount of HCl, and finally add Tris-HCl to the scale to make the final concentration 40mmol / L.

[0066] Preparation of heme solution: Weigh a certain amount of heme and place it in a brown volumetric flask, add DMSO to prepare a heme solution with a concentration of 1 mmol / L, and dilute it to 100 μmol / L with Tris-HCl.

[0067] (2) Take 200 µL of immobilized COX-2 enzyme solution, add 2 µL of heme and 10 µL of levoephrine, mix well, and incubate at room temperature (20°C) for 2 min. Afterwards, 20 µL of AA was added to the buffer to make the final concentration 2 mg / L, and reacted in a metal bath at 37 °C for 30 min. After the reaction, the solution was centrifuged for 75sec (1.2×10 4 rpm), take the supernatant and let it stand at room t...

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Abstract

The invention belongs to the technical field of marine drugs, enzyme engineering, and analysis of natural products, and discloses an immobilized enzyme based method for rapidly evaluating the inhibition of the activity of a marine natural product cyclooxygenase 2. The method comprises the following steps: 1, immobilizing a COX-2 enzyme; 2, measuring the enzyme activity of the COX-2 enzyme; 3, establishing an evaluating system for evaluating the inhibitory activity of the immobilized COX-2 enzyme through an inhibitor; 4, screening a natural product inhibitor of the COX-2; 5, evaluating the inhibiting effect of the natural product on the immobilized COX-2 enzyme; and 6, evaluating the reusability. In the technical scheme, the condition is mild, the method is simple and effective; the immobilized enzyme screens an actual sample in a complex matrix of a marine organism, can still keep more than 80% of the enzyme activity after being repeatedly used for 5 times, and can be rapidly conveniently recycled through centrifuging; the immobilized COX-2 enzyme is used to screen the potential inhibitor of a crude extract of the marine natural product, and sea anemone anthopleura xanthogrammica and styela clava are found to have the affinity binding capability with the COX-2 enzyme, and are potential sources of natural drugs.

Description

technical field [0001] The invention belongs to the technical fields of marine medicine, enzyme engineering and natural product analysis, and in particular relates to a method for rapidly screening COX-2 inhibitors in natural products, in particular, it involves the use of immobilized enzyme technology combined with chromatography and mass spectrometry A method for rapid screening of COX-2 enzyme inhibitors from natural product extracts. Background technique [0002] COX is a heme enzyme that catalyzes the generation of PGs from AA. PGs present in many tissues cause inflammation and pain. PGs produced by AA are mainly catalyzed by COX-2 enzyme, an inducible COX isoenzyme that plays an important role in inflammation and tumor formation. COX-2 enzyme can also act on the central nervous system to cause hyperalgesia, and is related to the occurrence of nephropathy. Since selective inhibition of the COX-2 enzyme not only avoids the major side effects associated with COX-1 enzy...

Claims

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Application Information

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IPC IPC(8): C12Q1/26C12N11/14
CPCC12N9/0083C12N11/14C12Q1/26C12Y114/99001
Inventor 史倩陈军辉郑立
Owner OCEANOGRAPHIC INSTR RES INST SHANDONG ACAD OF SCI
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